OK, well maybe
we should get started. Sure more people will
be signing on.
In my role is the
director of the Yale O'Brien Center.
I'd like to welcome everyone to today's
symposium that's been organized
by Shooter. She be
before getting to
the symposium. I want to say a couple
of words about our O'Brien Center
and battle Brian Centers in general.
People may know or may not be
aware that IDK sponsors a number
of kidney disease
centers. These include
pediatric centers of excellence.
PKD centers in the O'Brien centers.
There are eight O'Brien centers
around the country and these are
largely set up to provide resources.
Core resources to be shared
by the kidney community. Our
own center has three cores, animal
Physiology and phenotyping core.
Pat Price IG a disease.
Models and mechanisms core led
by Steve Somlo and Lloyd Cantley, and
Human Genetics and clinical
research cores. Really three
cores in one led by Perry
Wilson, Chirag Parekh at Johns Hopkins,
Shrikant money and and Steve Somlo. What
we're here for
today is is part of the
enrichment program the the
director of which
is shoot as
she be who's organized today's
wonderful symposium. We also have a
pilot and feasibility
grant program. The
next cycle of applications will be
next winter, so let me just say a
couple of give a little more detail
about a couple of the about the core
facilities in case people want to
access any of these services. The
animal Physiology and phenotyping
core basically provides ways to
assay Physiology and
small animals, mainly mice. Services
include clearing studies, GFR
measurements, Histology, serum and
urine, electrolytes including
creatinine assays, blood gas
parameters, balance studies,
circadian rhythm studies,
blood pressure measurements,
and both by telemetry
and anesthetized mice.
The disease models and
mechanisms core provides access
to unique mouse models of
kidney disease, cell line
resources and advanced
human translational technologies.
It includes bacterial Archer
bacterial, artificial chromosome,
recombineering and transgenesis support.
For crisper CAS 9 genome editing, support
for kidney cell line
production using the Terminator,
Mac mice and development of imaging
mass cytometry for analysis of human
kidney biopsy samples. And then finally
the clinical and
and and Human Genetics research
core provides a series of
services with regard to genetics.
It provides high throughput,
snip, genotyping,
whole exomes sequencing,
additional sequencing technologies,
and relevant bioinformatics support
and the clinical Research Services
include protocol development,
patient recruitment,
sample processing, bio
and data banking. Assay
to archive samples from several NIH
studies, biomarker assays, extraction,
analysis of electronic health data,
machine learning and biostatistical
support, and Perry Wilson is an
extension of these core activities.
Actually provides a free course
on study design, research methods,
and statistical interpretation
which is available through Coursera.
People want to note this web address.
Now finally, I'd like to acknowledge the
help we received from an external Advisory
Board and chaired by Peter Igarashi,
the other members or Laura
Denver Ali Grabian, Mark Okusa.
They really played important role
in our center reviewing pilot grant
applications and also reviewing the
progress of the center on a yearly basis,
and we're very grateful for the time
that they donate to helping our center
run more efficiently and provide better
service to the community. So with
that we turn
it over to shoot a.
I should can't thank him
enough for organizing today's
wonderful symposium. He
was assisted by an prodigy who
provided really great administrative
support and we're receiving wonderful
Technical Support from Kyle Finn today.
So I really appreciate everyone's effort
to bring this symposium to fruition.
Thank you, Peter.
Thank God speakers were able to
attend as well as the participants,
but participants.
At the bottom of your zoom next to
your raised hand, there is a Q&A,
so I'll be monitoring the Q&A if
there's any questions during the talk,
but I could ask these questions to our
speakers after the conclusion of each talk.
So the first speaker is
Doctor Anupam Agrawal.
He's from the University of Alabama
at Birmingham School of Medicine and
today he's going to talk to us about
renal lymphangiogenesis in a Ki.
Thank you.
Thank
you, should I really appreciate the
invitation? Uh, and delighted that
y'all can have this symposium even
during this continuing pandemic.
Maybe there's some sign of relief
at at the end of the tunnel.
Again, thank you.
Can you all see my slides?
OK, thank you. So I wanted to
share some relatively new work
that we began looking at a real
lymphangiogenesis in acute kidney injury.
These are my disclosures.
None of these are relevant,
you know, to the talk today.
Uhm, it turns out you know the
study of lymphatics goes back.
You know, 2000 plus years when these
two you know Greek investigators here
at Phyllis and harasses Striders,
actually found a second vascular system.
You know, they've first thought there was,
you know, the arteries and the veins.
But these two you know Greek.
People actually found a second
vascular system,
so really along time ago subsequently eseli,
you know,
in the early 1600s described
what you know became known to be
lymphatics was Milky veins in the gut.
You know of dogs that had, you know,
just being fed and you can see that you
know picture here in the mid century.
These lymphatics you know,
all Milky,
and that's because of all these the
lymphatics that had high lipid content.
In those early days,
the studies were really limited because
it was hard to differentiate between
a blood vessel and a lymphatic vessel.
When a lot of work has been done,
as you can see on this slide,
summarizing the publications
on lymphangiogenesis,
you know very few papers going back
you know into the 1980s and so on,
and now you know you see about 250 or
so publications every year on this topic,
and so something really happened around.
You know that the mid 90s and
was really seminal work from.
Doctor Kori Ali tallow from Finland,
where he found the Veg FC Veg FD
veg of artery signaling in lymphatic
vessels and subsequent to that a lot
of work was developed to identify
markers for lymphatics and some of
them are shown here on this slide.
But the most prominent are highlighted here.
Veg FR3,
which is the vascular endothelial
growth factor receptor three?
It's a tyrosine kinase receptor for
VEG FC and Magic D Portal plane in
may not be very specific because
also you know expressed in Porter
sites also termed as D240 Live 1
which is lymphatic vessel endothelial
huyler on receptor one.
It's also sometimes expressed
in macrophages and then the
transcription factor prox one.
Prospero related homeobox one
and I'll refer to some of these.
In some of the studies that
we've done later on in the talk.
Also,
several growth factors that are
implicated in Lymphangiogenesis
and a few of them are, you know,
shown on this slide veg FC Veg
FD that I mentioned to you,
veg FA PDGF B hepatocyte growth Factor,
IGF one osteopontin and others and
the others are not very specific.
'cause as we know they have effects
on the vascular system as well.
So the characteristics of the lymphatics
are quite distinct in terms of when
you compare it to a blood vessel,
and this figure here in a the right is a
blood vessel and this is a small arterial.
So the purpose and you can see these
endothelial cells and the parasites.
You know that line the blood capillary and
the blood flow in these Kappel Aries is,
you know going downwards right
towards the end organ. In contrast,
lymphatics the floor is opposite.
And to facilitate,
there are actually valves and
you can see these valves here.
If you can see my pointer, but.
The lymphatics the flow is in the
opposite direction and they don't have
these parasites that support them.
They actually have.
Inflammatory cells that can't support them.
For example, macro pages and so on.
But they have lymphatic endothelial
cells and their tethering proteins.
Very complicated protein systems that hold
these lymphatics you know together and
you can see these staying here in green.
And then you can see this valve
here by intravital imaging that
how these valves are positioned to
enable this unidirectional flow,
and that there is no back.
Low per say.
I told you about some of the
ligands and some of the receptors,
but this is just to highlight
again the differences between
angiogenesis and lymphangiogenesis.
So the main mediators of angiogenesis are
veg FA badge FP and they worked through Veg
F1 and VEGF also worked through Veg F R2.
Veg FC and Magic D predominantly
worked through the VEG FR3 receptor,
which is very specific to lymphatics
and they mediate lymphangiogenesis.
So why? Why are these important?
So clearly they're critical for
maintaining fluid homeostasis,
and you can see in this cartoon here
in green the lymphatics are you know,
all over the body.
Clearly the skin has a very.
A robust network of lymphatics but
ending with the thoracic duct that I
missed and into the vascular system
so they maintain immune surveillance.
They are critical in resolution
exacerbation and inflammation,
and wound healing, fatty acid absorption,
and some of that Milky veins in the dog
that I showed you in the miss entry
was predominantly from the fatty acid
absorption and if you have no lymphatics,
it's actually embryonically
lethal and incompatible with life.
And patients that have dysfunctional
lymphatic vessels actually have chronic
edema and have impaired immune mechanisms.
There's been a lot of question
about lymphangiogenesis in
the setting of inflammation.
Is it beneficial, or is it detrimental?
So clearly you know Lymphangiogenesis
would help clear the debris.
It would remain remove.
You know you're pro inflammatory cytokines
help with migration of inflammatory,
you know,
T cells and drain excess fluid.
So this is clearly being shown
to be beneficial.
For example and Michael infarction
and polycystic kidney disease
and lung transplantation.
And I'll show you some work from
others in polycystic kidney disease.
Given the relevance of, you know,
GAIL, being a powerhouse,
you know for PKD research.
Also, the impending genesis,
on the other hand, could worsen outcomes.
You know there's increased migration
of leukocytes to the site of injury.
Could antigen presentation be increased and
therefore inflammation could be made worse?
Or could the inflammatory environment
be maintained by sick reading?
You know proving climate re
cytokines so it could be both.
There's again and a Yank here and
in some conditions you know renal
transplants and corneal transplants
in diabetes and tumor metastasis,
lymphatic or lymphangiogenesis.
Is clearly detrimental.
Uhm, here's the anatomy of the lymphatics.
And this when I saw this figure,
it really struck me that a
lot of work that we do.
You know whether it's acute
kidney injury or studying?
You know, renal pathobiology.
We focus on, you know,
the tubular epithelial cells,
the interstitial compartment,
or the blood vessels.
You know, the arteries,
and maybe less so the veins.
You know the predictable a
capillary network and so on.
But as you can see on this slide,
every blood vessel here has a
lymphatic that wraps around it.
You can see shown here in green.
And even in the Subcapsular
region there is robust.
You know lymphatics that are present,
so every.
Country in vain.
You know whether it's interlobar
or interlobular you have lymphatics
that are surrounding them and a
very robust network of lymphatics
around the renal chemical.
You know around the renal
artery and the vein.
So this is some work done
from a group in China.
It's published in science advances
a few years ago where they looked
at human patients with kidney
biopsies with chronic kidney disease.
So in health you can see here
very few lymphatics and this is
for staining for the D240 marker.
Very few lymphatics,
but even with minimal change disease or
lupus nephritis IGA nephropathy tubule,
interstitial nefritis,
or obstructive nephropathy,
you can see marked increase in
lymphangiogenesis, you know,
within the kidney, and these data
just quantified on this panel here.
The same thing here.
If you look at other markers,
you know veg FC,
veg FT and FGF 2 market increase
and again not in the lymphatics.
But in the tubular epithelial
cells and most of these approximate
tubular epithelial cells,
both VEGF C and vegetti,
or markedly increased in in these
human kidney biopsy samples.
On the other hand,
if you administer Veggie FC and this
was really nice work published in Jason.
Looking at effects of.
Actually modulating the lymphatics
in polycystic kidney disease.
So they use the PKD one wild type and
the PKD mutant and injected daily Veg
FC or vehicle into these animals.
And what they found that VEGF C
administration actually resulted
in phosphorylation of Fr.
3 And you can see that on these
Western blocks,
so suggesting that it was
activating the signal pathway.
And if you looked at the
effect on the phenotype.
That treatment with VEG FC.
You can see here the vehicle treated animals,
competitive edge of seat treated animals,
the cyst size and the kidney
size was dramatically reduced,
so treating with Veg FC actually
would reduce PKD cyst formation in
an animal model of polycystic kidney
disease and the quantification of
the kidney weight is shown here as well as.
The average size following Veg FC
administration and renal function
accordingly was preserved with
VEG FC treatment in this setting.
And multiple papers you know looking
at mouse models of unilateral
ureteral obstruction.
Again,
a lot of work in mice and rats on
this setting. Again, macrophage.
Being studied in this model,
so really a lot of work in PKD
as well as unilateral.
You know the obstructive that
properly modeled in in mouse models?
So our questions.
When we first began this work was
what's the role of lymphangiogenesis
in AK and potentially AKI to CKD?
Where in the kidney are these
ligands expressed?
You know,
after injury and one of the
dynamics of these ligands,
you know how does this progress
and is it really beneficial or
is it detrimental in the setting
of acute kidney injury?
So we use the classic ischaemia re
perfusion model. You use a clamp.
At the renal pedicle for 20 minutes
and then we looked at expression.
So this is just to show you serum creatinine.
It's a severe acute kidney injury and
day one and then the kidneys recovered.
So it's a reversible model and this model.
Starting at day seven, day 14,
you see significant you know
fibrosis in the setting.
And we looked at some of the markers.
You know you can see here Veg FC veg
of D as well as veg Fr three you know
marked increases in these ligands in
this model and this is in in the kidney.
You can also look at other
markers you know live 1,
Portal plane and prox one that I mentioned
to you that these are also increased.
You know within the kidney
after acute kidney injury,
in the scenery perfusion model
and here are some Western blasts.
Look at Veg Fr three approx.
One and magic D.
Interestingly,
Veg FC initially starts going down
and then it takes awhile for it
to come back to normal setting
at Day 28 and this is just.
Modification. Up those data.
We also looked at secretion of these
molecules both in the serum and in the urine,
and you can see here both VEG FC
and Veg FD increase in the serum.
You know,
in this ischaemia re perfusion injury
model and this is in the urine.
The levels you know dramatically
increased even higher than what
we see in the urine in the serum.
For both Veg FC and Veg FT.
So the question was where in
the kidney are these ligands?
You know, coming from.
So we stay in the kidney for,
you know,
Betsy,
shown in the upper panel in green and
Veg FD in the red panel shown in the
middle and the merge on the bottom panel.
And you can see you know at baseline
that is significant veg FC Vegetti
mostly in the proximal tubules
and an early following injury.
These expression levels actually go
down and then they start coming back.
As recovery occurs,
you know in the tubule,
and we think some of this is because
these molecules are being secreted
into the serum and because of the
damage and you can see the lumen,
there is abundant.
At least vegab teeth can be identified.
We think that is secreted by
jabbed into the tubular lumen.
In the setting.
And we stained for veg FR3 which is
the receptor for veg FC&D and you can
see here there is marked increase a day,
seven day,
14 day 28.
In Veg FR3 and these are lymphatics
that stain positive for the
receptor and this the bottom panel.
Here is a quantification that
you know as early as day one.
These veggie Fr three increases
and then it becomes significant
by day 714 and day 28.
So one of the questions that came up.
Actually I was presenting this at
grand rounds at Adrenal Grand Rapids
at Vanderbilt several years ago,
and Doctor Agnes Fogo,
you know,
who's a renal pathologist said you
know and open your clamping the
renal pedicle and you showed this.
Now that there is abundant lymphatics,
you know around the pedicle.
So is this just a consequence
of you clamping the
renal pedicle? Therefore you're
damaging the lymphatics and this is
an injury response to the lymphatics
and was a great question and.
It opened their eyes to look at other models,
so we looked at cisplatin.
Induced nephrotoxicity where you
give a single injection of cisplatin,
20 milligrams per kilo body weight
intraperitoneally and was the results were
identical to what we saw in the scheme.
Every perfusion injury model you can see.
With the tubular injury,
either using a high dosage pattern
or a repeated lordosis,
platina chronic model,
we could see the same finding that
there was an initial decrease in
terms of staining for veg FC&D,
but increases in the serum.
And in the urine in this model,
and you can see here the ligands
in the in the serum for both
T and as well as in the urine,
markedly increased in the cisplatin model.
And this again shows you veg Fr three
staining as the receptor on lymphatics.
Very similar to what we saw in the scheme.
Every perfusion injury model in
the upper middle and right panels
with the high doses flatten as
well as the repeated low doses.
Find ninja remodel.
And this is just the quantification
for that setting.
We also looked at another model that Ray
Harris is lab at Vanderbilt had developed,
so they developed diphtheria toxin
induced model of just damaging
the proximal tubule so mice do
not have are not sensitive to the
theory toxin so they engineered.
The human diphtheria toxin receptor
using the GT One Cree that way only
the proximal tubules have the DTR
receptor and if you give these mice
human diphtheria toxin only the cells
that contain the receptor damage
and this is a selective mechanism
of damaging the proximal tubules
and when we did this model again
we saw very similar results.
Initially there was a decrease
in both veg FC&D expression.
And veg FR3.
The receptor was markedly increased
in the lymphatics of following
diphtheria toxin administration.
In this model,
SO2 models that did not involve
a mechanical injury you know was
associated with marked increase
in lymphangiogenesis in in the.
In these in these two other models.
And this is just a quantification
of major part three in the setting.
So the sources of lymph angiogenic
veg of ligands,
we did some in vitro experiments
where we cultured primary proximal
tubular cells as well as bone
marrow derived macrophages.
So it turns out that.
The proximal tubules express high
levels of both VEG FC and Measure B,
but macrophages really express
only Veg FC but not Patch FD.
And you can see here staining both
in the kidney and in the spleen.
You can see for Veg FC you can detect
positive signal for Betsy in macrophages.
You know both in the kidney and the spleen,
but Veg FD is only in the tubular
epithelial cells but not in
inflammatory cells within the kidney.
And this is just to show that
it's a higher
power magnification of the splenic
macrophages staining positive for.
Betsy, in in the setting.
So, just to summarize this part, you know,
irrespective of the nature of the insult,
lymphangiogenesis is rapidly and
robustly induced in the kidney
that both VEG FC and badge of D
are quite abundant in tubules at
baseline and their expression you
know decreases following injury.
Both these ligands increase in the serum
and urine after injury and potentially
you know urinary biomarkers of AK I as
well as progression of kidney disease.
Potentially both macrophages and
tubular cells secrete VEGF C.
But better D is only produced
by tubular epithelial cells.
More recently, we've done some work.
This is not published,
we're just getting ready to
submit this hopefully next week,
where what is the functional effect?
You know, I showed you some work in the
polycystic kidney disease model where
you induced VEGF C When you give Veg FC,
you can alter the phenotype
in polycystic kidney disease.
So what happens if you block
lymphangiogenesis? And to do this,
we use the cisplatin injury model?
And we used a a veg FR3
receptor antagonist Maze 51.
And you can see here after cisplatin injury,
there's marking trees in serum
creatinine shown in these blue triangles.
But when you give this maze 51 alone,
there is no effect.
But when you give the combination
kidney function is much worse.
We also measure GFR using Fizzie
sinister in transcutaneous monitoring,
and you can see GF are much worse as well.
And Kim one as a kidney injury marker
is significantly increased following.
Inhibiting VEGF R3 in this setting.
And we also looked at
some inflammatory markers.
And interestingly,
we submassive upregulation of P-65,
which is a critical component of NF Kappa
BA Pro inflammatory transcription factor.
You can see here by Western blot
high levels of P-65 expression
phosphorylated P-65 after inhibition
of FR3 with this maze 51 along with
oil 6 and these data are quantified
down here high levels of IL 6.
As well as phosphorylated P-65.
When you block veg Fr three signaling
in the cisplatin injury model.
You know we were having challenges in
trying to visualize the lymphatics,
you know in our models of injury.
So we reached out to Tarek.
Oscar, you know who is the Co
director for the Indiana O'Brien
Center for Advanced Microscopy?
And with him we actually just recently
published work where we've done quantitative
3D imaging and tissue cytometry,
so we can better visualize the
lymphatics in acute kidney injury,
and I'll just share with you some
of the slides from that work.
So here we used approx one which is a
transcription factor TD tomato mice.
So all the lymphatics in this.
Model will stain red and you can see
very few lymphatics that you know
near the major vessels renal artery
and vein that are stained positive.
And if you want to see if I can
play this movie.
Come and this is just showing
you the cortex and medulla prox.
One is in green lymphatics and F
actin and DAPI is you know stains
all the nuclei and you can see
just at a higher resolution.
Very few lymphatics but they are
all associated with the major blood
vessels you know within the kidney.
And as you zoom in.
You can see there you know
associated with the major blood
vessels you know within the kidney.
And you can see the effect and
you know concentrated.
You know in glomeruli as well,
you know in the cortical region.
So very isolated,
you know few lymphatics that you
see within the kidney at baseline.
And when you look at acute kidney injury,
the same format Prox one is in red.
F actin is in green and DAPI is blue.
For nuclei.
You can see significant dispersion of this
prox one staining throughout the kidney.
And I'll show you a better
image in a subsequent file.
So significantly dispersed.
Red staining across,
you know, within the kidney,
suggesting new lymphatics that are,
you know, being generated within the kidney.
Much more abundant and you can
actually quantify this using VTA
and it goes into the outer stripe.
Of the kidney,
as well as you know,
the inner cortex within the kidney
in the in this model setting.
And it's been a pleasure to work with
Tarek and his team at the Indian O'Brien
Center and we also stain for live one.
So this is not the prox one mouse.
This is just a wild type mouse that
has not been injured just in the
baseline and you can see a major renal
vessel here and lymphatics nicely.
You know you can see it in the video
nicely wrapping around just like in
that schematic I showed you from the
anatomy of the lymphatics that they are.
Wrapping around this.
Interlobar interlobular
artery within the kidney,
in the outer stripe.
The medulla as well as the
inner cortical regions.
So these are 50 to 60 Micron
sections that we used to delineate
these lymphatics which otherwise
if you just do a regular four
to five Micron section it will
look like this staining that I
showed you for the veg FR3.
So with that, I'd like to, you know,
acknowledge a lot of folks in my
lab who were part of this work.
Lawrence Black, who's currently a
postdoc in my lab abolfazl Sarju,
is a K-8 recipient,
Dennis Assistant professor in our division.
Several folks in a lab and
really want to acknowledge Tariq
from the Indian O'Brien Center.
We also collaborated with Ray Harrison,
Minxy Zhang from Vanderbilt
for the DTR mouse model.
The funding for this is from
a VA merit award that I have.
And our O'Brien Center as well.
So thank you again for the invitation.
I'm really open to any questions
from the group.
This is just a skyline of Birmingham.
Wanted to share it with you also thank you.
Wonderful wonderful talk on a new pump,
so there's some questions that
I can try to read out for you.
So for example, in human kidney
biopsies have people staying for
veg F receptor three to after,
for example that we biopsy ATN all the time,
but after a Ki in seen also
an increase? Yeah, I'm
not sure if anybody has
done a Ki person shooter.
We're actually doing that now,
but when people have done.
Single cell, you know, work in.
In some cases it has shown up not veg
about 3:00 but Veg FC and Veg FD.
You know the lymphatics if you do
single cell the population is so
small that you cannot really detect
it unless you enrich for those cells.
So, but that's a great question.
We we are actually doing that
right now we have a human bio
Bank of AI and non HI samples.
So we're doing that right now.
So thank you.
Great, another question they had
is where do the renal lymphatic
drain in the in the kidney?
So they drained to the para.
You know, I otic lymph nodes.
And if you see a lot of injury models,
you know actually those
lymphatics lymph nodes go.
You know almost double in size.
You know, it's hard to see them,
but if you look at them under
a dissecting microscope,
they really enlarge in size and
you know this work was actually
prompted by some work we were
doing with GFP transgenic mice,
where we saw that after we did a
cute teen injuring those settings.
A lot of the GFP positive cells from the
kidney were actually found in the para iotic.
You know, renal made the renal hilum
in those lymph nodes, so we said.
How are these cells entering
to the lymph nodes?
They have to come through the lymphatic,
so we need to look at what's
happening with the lymphatics and
that's what God is to do that.
So they drain into lymph lymph nodes
and then into the you know the thoracic
duct and into the right atrium so.
And which cells were the APP 65 increased in?
Is it the whole kidney or is it or certain
proxamol tubular cells or immune cells? Or
so we are currently
staining for that shooter?
To find out the precise localization,
it turns out that NF Kappa B
is very highly expressed in
lymphatic endothelial cells.
Uh so collaborator at Mount Sinai.
Was telling me he's a plastic surgeon
who's been studying lymphatics
for over a decade in terms of
lymphoedema that if you take an
organ and disdain it for NF Kappa B,
the lymphatics had the highest expression,
but in the kidney that's not the case.
You know, in inflammatory conditions
the proximal tubules and inflammatory
cells have a ton of NF Kappa B,
so my prediction in the kidney after injury.
It's probably the proximal tubule
potentially may be followed
by inflammatory cells.
OK, great, there's another question.
UM, is there a significant
difference in the absolute
levels of VEGF C&B in the urine,
since after a kin normalizing,
creatinine could have some bias due
to the increased level of reduced
urinary creatinine in your kidney.
Yes, so you know, we've done it
normalized and you know not normalized
and and we still see differences and
we actually went back and looked at
some of the work of, you know Prasad,
Devarajan and others that you
know worked on end gal and so on.
And it turns out you know both badge
FC and legibly show up in there.
You know urine studies as well.
So you know going back to the early 2000s so.
Wonder wonderful, some more questions.
Is there a analogous time sensitive,
effective inhibiting VEGF C on kidney injury?
Now we have not right now.
Our work has only been one time point,
and there are some you know
issues with all you know,
chemical inhibitors,
however specific they are.
So we are actually.
We're getting a flocks to veg FC
mouse model where we can selectively,
you know, delete veg FC in
proximal tubules and really look
at more specific crosstalk between
epithelial cells and the lymphatics,
and these are tamoxifen inducible
so we can really look at you know,
time course you know before and
after injury so we've not done,
you know,
a detailed time course with the veg FR3
inhibitor. Made 51 wonderful.
Do you think that the lymphatic
system helps cyst fluid reabsorption
with another question that was asked
by the group? Yes,
you know it's not being studied,
and one of the reviewers on my
initial V Ameritrade suggested
that maybe we should try to,
you know micro perfuse or do micro
perfusion of these lymphatics to
see what their tubule content is.
You know. Are there debris and so on.
It has not been done.
You know people have done it
then the tail vein which is a.
Abundance lymphatics, but,
uh, we're looking into it.
We're speaking with the vocal on,
you know, to see if that is even feasible,
but I would guess yes,
there's probably more cyst fluid,
you know, in the lymphatics,
and that's how they are trying to.
You know,
that's why they're increased in that model.
Uh, another question is, UM,
interacts beautiful images in the
normal it looked like lymphatics kind of
surrounded the Perry glomerular area.
Do you think lymphatics play a
role in normal glomerular function?
Was another question I don't know
should I? That's a
great question. I I I don't
know what the answer is,
but it's definitely a great question.
Uhm? Are the resident lymphatic
stem cells in the cortex?
So our hypothesis, just like you know,
tubular epithelial regeneration is that.
The lymphatic endothelial cells are
the precursors to new lymphatics.
OK, and that's the subject of my,
you know, via Merritt Renewal,
where we're going to actually do
fake mapping using the Prox one TD
tomato mouse and then see there's
some work from China that macrophages
are precursors to lymphatics and
some work has refuted that that
really macrophages actually secrete.
You know, chemo,
Kines and other growth factors
that may promote lymphangiogenesis.
But just like Ben,
Humphries worked with fate mapping of
proximal tubules we're proposing to do
fake mapping of lymphatics to see you know,
how do these lymphatics really
emerge in the heart it's been
shown that these lymphatics
emerged from the cardinal vein,
so the small veins actually end
up giving rise to lymphatics,
but nobody knows what happens in the kidney,
so that's a really an interesting question.
OK, uh, another question is, UM?
Are there any correlations to AI
to CKD in either juos or other
human biomarker data concerning the
Jesse or these lymphatic markers?
Yes, definitely veg FC veg FT
both show up in a lot of
the you know analysis,
at least in animal models.
I'm I have not looked at
the human models per say,
but in the animal models it
does definitely show up so.
And after like kidney
transplantation, another question,
does our lymphatics regenerate?
So you know our colleagues and
transplant mythology that take care
of patients are well recognized.
You know, lymphocele's, right?
So and a lot of that is because
the lymphatics do not connect.
You know right away.
But it it's not being studied really well,
but my presumption is over months.
You know,
these lymphatics established connection,
you know, with the peritoneal,
or depending on you,
know how the transplant is
done with the lymphatics.
You know in the peritoneal whether it's the
out of visceral or the parietal peritoneum,
and they establish connections.
Otherwise,
everybody would get lymphocele's,
you know,
because of that,
so I think they do establish
connections and they some work
in animal models of transplant
that that that is the case so.
OK, I think those are the major
questions that I saw in the Q&A.
So, uh, thank you Doctor Agarwal for
a wonderful presentation. Thank you.
So I think we can go
to our on. Next speaker.
Which is Doctor Yang, Maggie Chen,
MD, PhD from Washington University
School of Medicine and today she will
be talking to us about Podocyte and
Luke Plasmic reticulum homeostasis
and nephrotic syndrome mutations
by markers and novel treatment.
Thank you Maggie. Oh
oh OK. Thank you very much for the
invitation or I'm going to share my screen.
Looks good, OK. Thank you.
Good morning everyone.
Today is a great pleasure for me
to talk about my research here.
I'm going to talk about policy,
ER homeostasis and frantic syndrome.
This is the Mike disclosure.
So one of our major raised focus focus is
is ER stress media looking at disease.
This focus integrates three major things.
First is to investigate molecular
mechanisms of disease pathogenesis by
utilizing a combination of stable,
transfected cell lines,
genetically engineered mice as
well as human biospecimen second
is to discover your stress.
Buy markers in human patients 3rd to
identify novel ER stress modulators.
Ultimately,
we hope to develop highly targeted
clinical trials to treat ER disease.
Emerging evidence has demonstrated
that ER stress or the derangement
of ER proteostasis, contributes to
the development and progression of
both glomerular and tubular disease.
These are the transmission M and
scanning MO publicized the diaphragm
and the glomerular basement membrane.
So the this tool so the process flow diagram
and the GBM are the major components
of the glomerular filtration barrier.
Any defect in the glomerular
filtration barrier.
Will cause proteinuria.
So in this talk I will focus on policy
year stress and polymer disease.
The human genetic study
in the past two decades.
Have highlighted in our politics
syndrome as the primary holder site
opposite with major discoveries of
PODOCYTE gene mutations in human
approaching syndrome patients.
Compared to much more complicated
spirited nephrotic syndrome,
nephrotic syndrome caused by monogenic
gene mutations is conceptually
much more amenable to testing out.
The disease pathogenesis.
And to discovering magnetic treatments,
this is the power of genetic disease.
Who decides the diaphragm?
Proteins include products in our
friend city to EP and TRIPS.
6 Lebanon is a major component
of the mature GBM.
Laminant exists in basement membrance as
head tremors a lemon and had to trimmer.
Includes one A1 beta and one gamma chain
and lemon and trimerization occurs in the ER.
Lamina is is synthesized and
secreted by both portal sites and
glomerular and serious cells.
Pearson syndrome is autosomal
recessive disease caused by mutations
in human laminin vayner too.
It is characterized by severe congenital
nephrotic syndrome and diffuse
mesangial sclerosis with significant
ocular and logical manifestations,
including microcoria and muscular hypotonia.
Children affected by Pearson
syndrome usually die within days
or weeks after birth from renal
failure in the mouse model,
laminant Belton Alchemize recapitulate
all the features of Pearson syndrome.
The C 321 mutation is located
in the Audi domain of BR2.
This mutation causes nephrotic
syndrome, but with much milder,
actually no features.
To investigate the mechanisms whereby
this mutation causes proteinuria,
we generated a now called transgenic mouse
model resembling human necrotic syndrome.
Patients carrying this mutation.
Previously we have demonstrated that
expression of white habitat two in
polar sites via the policies specific
network promoter is sufficient to
prevent proteinuria and restore the
structure of the glomerular filtration
barrier in Lebanon Village Nokomis.
So this is a proof of.
So, so this this is just the UM?
Proof of principle that laminant beta.
Now my Seacrest expressed in the mutant
beta two can serve as useful models
or mild variants of Pearson syndrome.
So we generated the three independent
lines of the mutant transgenic
mice on Laminant banner two.
Now background,
with different levels of beta two
in the glomerular basement membrane.
All three lines died from renal
failure before one month.
So as a strong in this graph in
the first person Edelman's the
severe proteinuria in Lebanon built
in Apple mice was attenuated in
a dose dependent fashion by GBM
accumulation of 321 R laminin beta two.
We focus on the high expresser
which developed trace proteinuria
before three to four weeks of age.
We we performed his logical analysis
at the P 24 Lebanon financial.
Now my show the same year.
Glamorous Cairo sis.
Prominent renal tubular protein
casts and loss of brush borders
compared to the white tablet meds.
In contrast,
no obvious pathology was detected
in TC 320 more mice.
At P 24 foot proof sets of basement
was observed in the mutant line.
However,
compared to diffuse food processor basement,
they were still focal areas with
much less food process investment
in the transgenic mutant.
By six weeks of age transmission Ian
showed diffuse through the process
effacement and mild GBM thickening.
In addition,
there was interstitial fibrosis at
the shown by trichrome staining.
Gene missense mutation can cause
protein is folding disruption or
protein trafficking and ER retention.
Protein folding in the ER occurs
with the help of molecular
chaperones and enzymes such as bid.
The imbalance between the misfolded
proteins and folding capacity of the ER
causes ER stress and unfolded protein
response JPR There is also a key
sensor linked to the regulation of UPR.
We next in in investigated whether it
was induced in the mutant portal sites.
The double fluorescence staining
of bourbon policy nucleus marker
WT one showed a very level,
very low level of expression in the
portal sites from wild type and TWT minds.
In contrast, even at three
weeks one the 3:24.
Had developed only trace proteinuria.
TC 321 portal sites exhibited
upregulation of bib in our grammar line.
The Western blot confirms the increased
expression of in the isolated glomeruli
in cultured primary portal sites from
the mutants versus control minds.
The UPR is initiated by three
putting sensors IR in one perk and
activating transcription factor 6.
The air in one spliced XBP 1 pathway.
The park eukaryotic initiation factor
to Alpha ATM 4 pathway and P98M62P58L6
pathway are the major JPR pathways.
XBP one as ATM four and P58F6 are
the three master transcription
factors controlling the UPR.
Cosplay style, janky and Trump are ER
stress mediated in politic pathways.
The consequence of the UPR is too attenuated.
Translation of misfolded proteins
to promote transcription of the
JPL target genes to increase,
ER associated degradation,
erect and to activate in our
specific impulses.
We next investigated the molecular
mechanisms controlling the UPR
in the ER stress photo sites.
As an we utilized the passage one to
passage to prime apologize from TWTT,
C321R and Y timelines at P27 as shown here.
The F2 alpha ATM for an alpha
XBP one as pathway but not P58F6
pathway were selectively activated.
In response to ER stress.
Crowley and stay near your stress
can cause ER dysfunction and
morphological changes as the early
stage transmission Ian showed a
significant drop ER distension in
the mutant photo sites compare ways.
The control portal sites, moreover,
compared to the TWT proto sites,
there was Desmond expression
in some portal sites,
which is the indicator of policy
engineering in the mildly proteinuric
TC 321 are mice and much stronger
testament expression in the
policies of heavily proteinuric
lemon band out now comics.
So, uhm. Taken together,
all these results indicate that cosigner,
strength and associated pallasites injury
precede the occurrence of significant
proteinuria in the TC 321 are mutants.
We next look at which your stress
specific apart Optic pathway was activated
at the early stage of the disease.
His striking cleavage of Caspase 12 was.
Detected in the mutant portal sites.
As a consequence, the expression of
the executioner caspase three was
also increase in the mutant pallasites
compared with control portal sites.
It has been shown that Captain 2,
which is a cytosolic cysteine protease,
is activated by cytosolic calcium elevation
and cliffs and activates ER president.
Caspase 12.
It also mediates proteolytic degradation of
different proteins and cleaves policies.
Cytoskeletal proteins including
alpha to spectrum, and Taylor,
one leading to put aside from
the process effacement.
In the culture of the primary
portal sites at P.
27, the Western blot revealed
that the TC 320 hour portal sites
exhibited increased cleavage of
offer to spectrum and Taylor one.
Indicative of enhanced activity
of CO2 in the mutant podocytes.
To directly demonstrate the increase,
cytosolic calcium levels in
the mutant portal sites,
the primary portal sites were
stained with fluorescent cytosolic
calcium indicator floor floor.
The floor for intensity measured by
flow cytometry confirmed the cytosolic
calcium elevation in the mutant
pallasites compared with control policies.
To gain insight into the mechanisms
underlying the intracellular calcium
dysregulation, we did a resequencing
of passage zero prime photo sites.
Isolated from the mutant
and control mice at P.
27, the gene set enrichment analysis showed
that the expression of genes involved
in calcium signaling was are increased.
In the mutant portal sites, in addition,
heat map showed that are why are two
was upregulated in the mutant portal
sites at the early stage of disease?
So what is R2?
Calcium in the ER lumen is maintained at.
1000 to 10,000 foot higher.
At.
Compared to.
The calcium concentration in the
cytoplasma by circa the circle here
calcium 80 PS so circa is a pump
for the uphill transport of calcium
from the cytoplasma to the ER.
The majority of ER calcium influx is mediated
by rendering receptor R and IP3 receptor.
Three isoforms of RYR IP3R have been.
Identify.
So whether the XR calcium pumps and
channels undergo remodeling in ER stress
put on sites and and what's their
functional impact on the other side,
integrity and injury had not
been had not been studied.
So we we look at the yrr
expression as a protein level.
Up first. As you can see here,
the Western blot did not review
difference of yrr expression by
utilizing an antibody that can
recognize our isoforms of yrr.
We further examined phosphorylation of RY
R2 at 72808 in card in cardiac myocytes.
It has been shown that chronic
hyper phosphorylation of R2 S
2808 is critical in causing leaky
ry R2 channel by destabilizing
the close states of the channel.
Indeed.
You can see the increased
phosphorylation of RY R2 at
AS2808 in the mutant portal sites
compared with control panel sites.
So in summary, our study has shown that
hyper phosphorylation of our two in ER
stress photo sites contributes to the
increase ER calcium efflux leading to
the downstream activation of helping
to tell optic pathway and polar side
injury at the early stage of disease.
So our two important sites may be a
therapeutic target. So all this work.
Has been found by Sanji Park,
a outstanding postdoc in the lab.
Our ultimate goal is to discover
novel ER stress modulators.
The key to conduct.
Truck High School project
screening is to establish.
High throughput functional assays.
So serve him. They created the ER calcium
monitoring protein is localized to
the ER lumen under normal conditions.
In our Council deletion induces
circum secretion which can be
detected by luciferase assay.
As you can see here,
circumcision was significantly
increased in response to ER.
Calcium depletion caused by subject guardian,
which is irreversible circle inhibitor,
compared to the control.
So this can be used as a useful tool to
perform high throughput drug screening.
Very few pharmaceutical company has
interest in developing drugs for rare
disease due to the risk of clinical
development and commercialization.
However, Uncast had very strong interest
in developing drugs for ER disease
due to unmet medical needs and the
profound impact that these underlying
mechanisms may have on broader biology.
So we are collaborating with
young cats and utilizing circamp.
As I see they have identified a
chemical compound key to one as the ER,
calcium stabilizer.
So this this chemical compound is also in
our computer and an investigational drug
in the treatment of cardiac arrhythmia.
So one control in TGC 321 R photo
sites were trading with Ketel
one individual for 24 hours.
The cytosolic calcium levels
were significantly decreased in
the traded mutant portal sites.
Which is consistent with reduced
phosphorylation of R2 S 2808.
In the treated mutant portal sites compared
with untreated mutant Apollo sites.
As a result,
the TC 321 are treated with K201.
Showed.
Inhabited activity of CO2,
as demonstrated by attenuated cleavage
of A2 spectrum and talent one.
As well as suppression of
cleaved active caspase trial
and caspase 3 compared to
untreated mutant portal sites.
Consequently, K21 treatment
substantially attenuated the early
property rates from 24% to 15%
at the early stage of disease.
As a proof of concept study,
our nephrotic central minds were
administered vehicle or Ketel one.
Once daily over a course of four weeks.
As shown here, the urinary calpin
activity was dramatically decreased in.
In the mutant mice treated with Kato,
one compared with mutant mice treated
with vehicle as early as two weeks
and continue to be surprised by P21
at three to four weeks post treatment.
This indicates that paperwork injection can.
Yard calcium block in the mutant.
Puddle size in vivo.
As the as a result,
the urinary proteinuria was
significantly decreased.
In the mice traded always Kato one
compared with the control group.
And the kidney function was
also significantly improved
at the end of treatment.
So in summary, we have
identified a new class of drugs.
We change the polar side here.
Calcium stabilizers in the
treatment of nephrotic syndrome.
So for early therapeutic intervention
and treatment response monitoring,
it is imperative to discover non
invasive urinary by markers for
detection of policy ER stress.
So for the first time we have
identified myself cephalic,
astrocyte derived neurotrophic factor
meant as a urinary youngsters by marker
in our nephrotic syndrome mouse model.
Samantha is a recently identified,
highly soluble 18 kilodalton protein
which is localized inside the ER.
It is induced.
To induce and secreted in response to stress.
We have also identified a second
year stress biomarker title to the
system rich with EGF like domain
to as an as you can see here,
this protein does not have the
classical year retention signal
KDEL at the state terminus,
which may explain why it's easily secreted.
In addition to its utility as
the ER stress by mercury in our
mouths and projects and your model,
we have also shown that cradle
two can serve as a ER stress
marker in AutoZone dominant cable.
Interstitial kidney disease ADT KD,
which is a stress induced.
Kidney disease, as shown in this graph.
A lifestyle shows that you're not required
on 2 levels in in patients with ADT,
PD was significantly increase
compared to controls.
So currently we are collaborating
with the nanoparticle lab.
Trying to develop a ultra sensitive
bioassay for the detection of
low abundant protein by markers.
This novel technology is based on a
newly invented ultra bright fluorescent
nano constructs named plasmonic floor.
Uhm so so I'm gonna stop here.
I would like to thank everyone in my
lab you want is my lab manager and she
has been leading the projects in the
development of your stress by markers.
Sanji has done amazing work in
bisecting poster years, trust,
signaling, cascade and and develop.
Chemical compound to treat
nephrotic syndrome.
A trunk is working on a DTKD.
I also would like to thank all of my
collaborators for their generous support.
I'm also very grateful for all these
funding agencies for their financial support.
So I'm going to stop here and I
will be happy to take any questions.
Right now the wonderful talk, Maggie.
So so I can start paying off a heavy
treated your mice like not pre treatment
but but after injury has occurred.
Actually, we treat the mice at three weeks.
At that time, the injury.
Has developed because the proteinuric yes,
but it's it's kind of pretty mild.
OK, and do you have like
histological like improvements in
in these mice after treatment or?
Uh, yes, we we did. I did now show here.
We did have histological evidence
besides biochemical evidence.
Have you also tested any mice besides
a laminant Mysore? Yeah, that's a
great question. We haven't tested it,
but I I would think this chemical
compound may be useful in any
nephrotic syndrome model,
cause bipolar stress now,
for example, airport disease.
And then odd.
You mentioned that that calcium
is coming out from the ER,
but what do you think about like?
Extracellular calcium,
like from like people have
mentioned trip C5 or trip see six.
So what are your thoughts
on that? Yeah,
I mean that's that's a great question.
In our model we did a lot of experiments.
We we demonstrate that in our model,
the increase, central solid calcium
level was due to ER calcium.
League now due to increased
transcellular calcium influx.
Uhm? So yeah, so we are just
a different model from like
hyperactivation of chips estates.
Sure, so like I've used inhibitors to block
that or is that happy dog determined that it
was more coming from the ER?
OK, so actually we we also use that circle.
You know we we construct the Lenten
virus content containing the circle.
Because circle this protein if we
can we can detect it in the cell
culture medium by luciferase assay.
That means the calcium leak. Is from the ER.
Not from the actual cellular.
Compartment sure sure yeah.
There's a question, do you think
that one would need a kidney target
delivery of this novel compound?
Uh, please see the question again. Do
you think one would need a kidney targeted
delivery of this novel compound?
Yeah, I mean, I actually we we are.
We are in the process.
We are collaborating with
nanoparticle lab in Northwestern.
And we're trying to design the
specific product specific Portal site
targeted nanoparticle for the precision
intracellular drug delivery now.
So you'd have to get it across.
The basement member would be
filtered in through and then
get it to the Podocyte right?
Mean? Yeah yes no, no,
this is not the issue because these
are very these are chemical compounds,
so they are very small. You know so.
So guiding through the basement
membrane is not an issue.
But but we want to increase the
delivery efficiency and avoid
the off target side effects.
And did they get like oh or
do they get taken up by
just regular endocytosis?
Or how does it get take?
Or do they not know or?
I I was just saying it just by the
endocytosis kind of nonspecific, you know,
and they and this drug may be and
and those titles by other cell types.
OK, but the point is only the
ER stress portal sites have
increased your calcium in flux,
not in other normal cells
also sure. So you will target those OK,
right? Yeah I'm looking for other questions.
No, I think that's great Maggie.
I appreciate your time giving
a sort of a nice review as well
as a nice data on ER stress and
laminin mediated diseases. Thank
you, thank you. So
I think, uh, this ends the first
two talks and we'll have a break
until 10:40 when we'll reconvene.
Right? So those who were able to rejoin us,
I think we can get started again,
and for all the attendees,
there's a question to answer right
beside the raised hand on the bottom
of your zoom panel if you need to ask
questions and I will be monitoring it,
as Doctor Segev speaks,
and we can provide him with questions
for to answer after his talk,
so the next speaker is Doctor
Dorie Segev from Johns Hopkins
University School of Medicine.
So today he's going to be talking
to us about kidney transplantation.
You know suppression and COVID
vaccination recipe for disaster?
Question mark.
Thank you, dori.
Thank you come. So thank
you for the invitation
to participate in this.
We've been through multiple
iterations of trying to plan this,
and originally I was going to talk
about living donation and then the
pandemic hit and then we started,
you know, dealing with the pandemic
and our transplant patients,
and so we we pivoted and this seemed like
a more important topic to talk about so.
Before I move forward in the talk,
I will ask that nobody share slides
of what I show on social media or
other public forums because a lot
of the data you're going to see
are unpublished and we have gotten
into trouble before with journals
reviewing something that then they
discovered on social media and were
very unhappy to publish something.
Has it already been?
Already been disseminated
so please don't do that.
I'm on the flip side,
I'm giving you lots of new cool,
unpublished data.
And updated data from things that
already are published, so I'm I'm.
I'm sure this doesn't come as
a big surprise to most people,
but there's a pandemic going on and
that pandemic has impacted transplant
patients particularly highly.
Transplant patients, obviously,
given their chronic state
of immunosuppression,
have a higher risk of disease acquisition
of any communicable disease have
higher prevalence of comorbidities,
which we know in and of
themselves are associated with.
With worse outcomes from COVID-19
and have a higher risk in general
of morbidity and mortality
from infectious disease.
So we've seen reports you know,
ranging up to 30 to 40% mortality rate in
transplant patients who've gotten COVID-19,
so the last year and a little bit has
been quite a challenge for our patients.
Now in December,
the first EUA for the vaccines
came on the scene and you know,
sort of the country celebrated
and continues to celebrate.
The uh, the.
Return possible return to normalcy
with vaccination and the unbelievable
efficacy of these new vaccines etc.
But transplant patients and those
in general on immunosuppression.
Are not quite to the point of
being able to celebrate yet,
because first of all immunosuppressed
patients were excluded from
all of the major trials,
so we don't really know,
or at least we didn't know from the trials
whether the effectiveness was going to
be the same for immunosuppressed people.
And when I say immunosuppressive,
I mean transplant patients,
but also in general people,
on immunosuppression for autoimmune diseases
and all the other stuff that we see.
And we know that vaccine response you know,
developing immunity to a vaccine
requires an intact immune system,
and so those on immunosuppression might have.
An inhibited response,
and we know in general that there's
lower vaccine responses in transplant
patients than in immunocompetent patients.
But obviously, we had really no
idea what the impact would be here.
We're talking about new technology.
We're talking about, you know,
a completely different.
A disease to vaccinate against,
etc so.
Back in December,
when the first EUA came out,
it occurred to us that there would be
transplant patients all over the country
who are also healthcare workers or
other people that would be at the very,
very top of the priority
list for getting vaccinated.
And that based on the experiences
that they had,
if we could capture this very early,
we could inform the entire
transplant community,
both in terms so you know.
Admittedly,
initially,
our concerns were or our focus
was safety because we we knew the
transplant patients were hesitant
to be vaccinated because they were
worried that it might impact their
allograft because transplant patients
are always worried that everything
will impact their allograft.
So.
And we initially just wanted to
be able to say look 1000 people
have gotten this in the last two
months and they're all fine,
so we should,
you know,
be able to encourage transplant
patients to get vaccinated.
But also obviously we wanted
to look at immunogenicity,
the impact of the vaccines
on antibody responses,
and then a little bit more deeply
on memory B cell and T cell
responses and things like that.
Knowing that it would take
us six months plus to get up.
To get you know,
IRB approval at 2530 different
transplant centers and data use
agreements and all of that stuff.
We turned instead to sort of a
more modern social media driven.
Type of research paradigm?
Uhm so.
A few things that made this unusually
challenging is one that this was in the
height of wave three of the pandemic,
so it was very dangerous for transplant
patients even to leave the house
let along to go into healthcare
settings and get blood drawn.
And things like that.
There was a prioritized rollout,
so again,
you know every transplant center would
only have a handful of patients who
were themselves healthcare workers or
other first line people,
and we wanted to capture them.
As quickly as possible so.
Rather than sort of your traditional,
either single center or
small multicenter design,
we did this all through social media
recruitment and this was a Hopkins study.
Even though the patients came from
all over the country from transplant
centers all over the country,
everything was done at Hopkins.
No other IRB's are required, etc.
And there was a very high volume,
so in the first week that
we announced the study,
we got over 1000 patients enrolled.
And another challenge was that we had
multiple visits for reactive unicity,
including the screening visit,
but also,
you know one week post dose,
11 week post dose two etc etc after
the vaccination and then also multiple
immunogenicity draws again at baseline.
A draw between dose one and two and then.
Post dose two and durability blood
draws as well and all of these you
know we we didn't want to have to
send transplant patients to blood
collection centers or anywhere integrated
into the healthcare community,
so we used a novel remote blood
collection device called the tap
two through a company called
7th Sense Biosystems,
which is basically a painless
capillary blood collection system.
I call it the mechanical leach.
You take this.
Thing that's illustrated here,
you stick it to your arm.
You push the plunger.
I'd must admit the first time I
pushed the plunger I kind of winced,
too,
in anticipation that there would
be some pain from it,
but there was no pain at all.
You cannot feel what is happening.
It injects a.
I mean it.
It introduces a bunch of tiny little
capillary needles into your arm,
and those cause a capillary blood collection
and you can get about 200 microliters.
Out of this,
so we mailed several devices to
each of our participants in case one
of them didn't work or something,
they would still have a backup device.
In order to get the blood on the day
that that it needed to be to be obtained,
we've sent them returned mailers.
Initially,
there was a little bit of confusion
on how to use the device,
so we made an instructional video which,
if you're interested in the device,
you can check out that video and
then we arranged for home phlebotomy
and as a backup,
and then when the pandemic started
to when wave three started to wane,
we also let people go into LabCorp if
they would prefer to do things that way.
Uhm,
the general timeline of the study
was that you know there would be
a questionnaire for eligibility
medical History Questionnaire and
to tell us their vaccine date and
then we got antibody testing right
before the first shot and then
they got their first shot in a
week later a safety questionnaire.
Then antibody testing right
before their second shot.
And then they got their second shot
and another safety questionnaire and
then 136 and 12 months later we would
do. Antibody testing and then
three 612 months also continued
to do questionnaires with them.
We used two platforms for antibody testing
and this is looking at circulating IgG.
We use the immune platform on the
ones that came to us from the
mechanical leach and those that
we had phlebotomy or LabCorp.
We used the Roche assays.
Both of these assays in our lab we.
Correlated to neutralizing assays so.
Both of those we had very,
very good correlation with the level
of antibody through these assays and
the ability to to neutralize the virus.
As of today,
we have 3700 people enrolled in the study.
We capped enrollment very very
quickly and now we're only doing
selective enrollment for the
different types of subgroups that
were that we lack in the study.
2000 have already gotten dose one and
1000 have made it to one month or two,
three weeks after dose one to get
us there antibodies for testing
the drop from 2000 to 1000.
Here is not a participation drop off or.
Something like that we get nearly 100%
engagement through the entire process.
It just means that 2000 got their
dose less than three weeks ago,
and we're still waiting for 1000
of them to be antibody tested.
1400 got dose two and.
We got antibody testing on
970 of them already.
You'll see sort of an increase
from this 1000 to 1400,
which is attributable to late
entries because in some of the
target populations were allowing late
entries from the demographics look.
As you might expect,
the majority are kidney transplant
patients with a smattering of
the other organ types that kind
of correlate with the number of
transplants that we do in this
country median of six years since.
Transplant very few of them are very fresh.
Post transplant a lot because most
transplant centers are recommending
that their patients wait three
months after transplantation for
vaccination and now at this point most
people that were transplanting were
already vaccinated pre transplant.
Uhm, median age 58.
These are the the demographics
of the people participating.
A handful had a prior COVID diagnosis
which I'll talk about later,
but most of what I'm going to show
you is in COVID naive patients
and this is how we found them.
We recently published the REACTOGENICITY
in people who received both doses of
the M RNA vaccines as 740 patients.
All COVID naive.
We had one report of an acute.
Rejection after dose 2.
Hard to ascertain whether it was
directly related to the vaccine,
but that's pretty rare and in a
group of 742 patients you would
expect that there would be one
rejection somewhere in that group.
No major infections,
and obviously those who were
younger had higher reacted unicity.
This sort of follows what we saw.
You know what was published in the the
big randomized trials and you know
typical increase in reactogenicity
from dose one to dose two.
Again just like in immuno competent people.
Very very few severe reaction,
so you know we can conclude that it's
quite safe to to give these vaccines to
transplant patients back in March 15th,
we had enough data on 436 people who we
had antibodies after their first dose
that we published a brief report in JAMA
to talk about antibody detectability.
This was a group of COVID naive patients.
I'm showing you now updated data
on more than 1000. Of them.
And the sort of very important,
surprising news was that only 20% of
participants after one dose of the vaccine
had any detectable antibody whatsoever.
Meaning 80% had absolutely
zero antibody detectable.
This is in stark contrast
to the randomized trials,
where you see already at 21 days or 15 days.
After the two major M RNA vaccines,
immuno competent people already have
dramatically high antibody responses.
So 100% of immunocompetent people
have antibody responses to the first
dose of the M RNA series compared to
only 20% of transplant recipients.
And even of those transplant
recipients who had antibody detectable,
the antibody levels were far lower
than that of immuno competent people.
So you know, we thought.
Sure,
vaccines are not going to be
as good in transplant patients,
but we had no idea there would be
this bad in transplant patients now.
If you were on an antimetabolite,
things were far worse for you,
so you had 1/3 the chance of
getting antibodies after dose one.
If you were on an anti metabolite.
So just to put this in numbers,
only 10% of people on Antimetabolites
developed antibodies compared to 30% of
people who are not on antimetabolites.
Obviously,
being older gave you a bit of a hit as well,
but being further out from transplantation,
you were more likely.
To get antibodies and actually having
received the Moderna versus the Pfizer,
you were also more likely to get antibodies,
so you know some of the early questions
were dissecting out the impact.
The direct impact of the medications
themselves versus the impact of being
a transplant patient and having been
immunosuppressed on such a high level
of immunity suppression or whatever
else is going on in the immune system
of our transplant patients in parallel.
I won't get too much into detail.
On this,
but we did run a parallel study
of patients who with autoimmune
diseases and our safety and vaccine
response data have been reported in
the annals of rheumatic diseases,
which is sort of their big journal.
And we found that indeed,
even among people taking immunosuppression
for reasons other than transplantation,
there was a decrease in immunogenicity
after the first dose,
although compared to the 20%
of transplant patients,
75% of patients with autoimmune
diseases already showed a antibody
response to vaccination and the
ones that took the biggest hits
were the ones that were on tack.
And Mycophenolate and Rituxan the kind
of things that transplant patients take.
So there is definitely some
medication specific impact.
But also I think there's some
impact of being on
a broad cocktail of very aggressive
immunosuppression modulation.
Uhm yesterday,
so you're really getting up to date data.
Here are actually two days
ago we published in JAMA.
The dose two data on the paper
has 658 patients already.
Today we have an update 873 patients.
This stuff moves unbelievably quickly
and by dose two we are now seeing
antibody detectable in slightly
more than half of participants.
So the cup is kind of half empty, half full.
The good news is.
That antibody that you know with
whatever priming came from dose one,
we are seeing people with detective
more people with detectable antibody
after dose to the bad news is that
still half the people have nothing and
of the ones who have antibodies again
they are not anywhere near the levels
that you would see in immunocompetent people.
Let's walk through this a little
bit in detail.
This is on the left.
The lab core assay on the right,
the your immune assay.
We have more people who had the Roche assay,
so I'm going to stick with this.
Come and kind of walk you
through what this means.
The the blue lines are the ones who had any
response to dose one and then these pink,
orange,
whatever color they are dots are the
ones who had no response to dose one.
And you see the dose one response on the
left and the dose two response on the right.
So remember 20% had a response to
dose one and the overwhelming majority
of people who had some response
to dose one boosted that to a very
very high response to dose too.
So 250 is sort of the.
Reporting cap for the Roche
assay by LabCorp and at 2:50.
That's where you would expect
immuno competent people to be.
So if you had a response to
dose one by dose two,
you're basically like the
immuno competent people.
If you had no response to dose one again,
50% had no response to dose
to either another 25% or so.
Had some response.
But again,
not as robust as immuno competent
people and then another 25%.
Again of the whole cohort,
25% are ultimately lucky enough to
have that sort of greater than 250
on the the Rocha antibody assay,
but the other 25% have something
and another 50% have nothing and
this distinction between the people
who have something and the people
have nothing might play a role.
Later when we start to think about well now,
what do we do with these people?
Again this is unpublished,
was not in the JAMA Paper.
But I'm sharing with you the
the regression model of.
You know the predictors of getting
an antibody response after dose two,
again being on an anti metabolite hurts you,
but not nearly as much as it hurt
you for dose one being further out
from your transplant is helpful.
Age no longer makes a difference,
and while the manufacturer of
the platform makes a slight
difference, it doesn't make
anywhere near a difference as much
of a difference after dose 2.
There is a group of transplant
patients taking Blat Escept
who have not done well at all.
We have 24 of them in our.
In our study, remember,
Bella blocks the helper T
cell B cell communication.
Those who had only one dose,
we saw zero percent who had
any detectable antibody.
We matched them to people who looked
like them in our M RNA transplant
population and of the ones who
were matched 14% in our match
population had detectable antibodies,
so even after dose one we
already saw a problem.
But after those two we saw a major problem.
Still almost nobody with detectable antibody.
Who was on Bella versus 50% of
people who looked like them?
Who got the M RNA?
I mean who who got who were not on?
Bella were on other immunosuppression and.
And and had an antibody response
and the the the the people had
detectable antibody had really
pathetic levels of detectable
antibody to the point where again,
if you match people up,
Bella was associated with basically
94% decreased odds of having
a positive dose to response.
And another thing we've been looking at.
It obviously is the new platform.
So you know,
the the early participants in our
study all received the M RNA platform.
But very recently mid February Ish
the the Janssen, Johnson and Johnson.
Platform came out and started
to become available and some
transplant patients started to
to take this vaccine as well.
And we're just now starting
to get results from them,
so we're looking at one month post
vaccination of the the JANJAY
vaccine and we're actually,
you know,
there was some hope that maybe the
J&J would work better than the M RNA
is less reliance on cellular machinery,
you know,
sort of a more well established vaccine,
platform, etc.
But it actually looks quite the opposite.
The only 20% of our JANJAY recipients
had an antibody response versus
more than half of M RNA recipients.
Again,
in a matched model,
the odds ratio was .16 for J&J and then
here you see the entire antibody level map,
and then you see the remember that
group that we talked about that had the
M RNA is that had a robust response.
You see those here.
Here the people had kind of.
I'm I'm moderate,
response somewhere,
and then here the people like no
response and the Janssen is very,
very heavily shifted to the left there so.
This unfortunately at least early
on in preliminary data does not
seem like it's going to be the
answer for our patients.
I'll talk for a second about convalescent
patients so everybody I've told you
about so far has been COVID naive.
There's two pieces of information
that's worth mentioning in
convalescent patients.
One is when we measure immune
responses in people in transplant
patients who had COVID-19.
When we measure natural immunity in them,
they do develop natural immunity.
About 90% of transplant patients
who have COVID-19, you know,
sort of over the last year when
we measured their antibodies.
Three to six months after their disease,
we were able to detect natural
immunity in those patients now.
One thing that's important to keep in
mind is when they had that disease.
They also got their immunosuppression
reduced because the first thing we
do on a transplant patient when we
find out that their COVID positive
is we dropped their immunosuppression
so it could be either that natural
immunity is stronger than vaccination
immunity in transplant patients and
or that by dropping their immuno
suppression we gave their immune
system an opportunity to respond
to the natural infection that they
had of those who had COVID-19 and
then went on to get vaccinated.
Very similar to what we're seeing
in immunocompetent people after the
first dose of the M RNA vaccine,
their antibody levels were sky high,
with most of them being in that
sort of closer to 250 range that
was seen in immunocompetent people.
And if you throw everybody together
in the mix,
if you've had COVID-19 in the past,
then you're more than six times
more likely to have an antibody
response to dose one.
Then if you if you are COVID naive, so.
Some optimism that it's not like their
entire immune system is completely shot,
but there's something they have the
capacity to develop immune system.
We just have to figure out what
the right conditions are.
For them to do that.
When
we started talking about like, what do
we do with people who had very no or
little antibody response after one month,
should we wait to see?
Maybe they just have?
A sluggish immune response and
transplant patients will be fine.
We just have to wait longer,
which we've seen in some
other instances in the past,
so we've been checking
people at month 3 as well,
and we have about 100 patients.
We have three month data on
and this is what we're finding,
so this is a
histogram of the change
from month 1/2 month three.
So if you're positive you've
increased your antibodies.
If you're negative,
you've decreased, and if you're 0,
you've really had no change,
and the overwhelming
conclusion that we can make.
Here is that there's minimal
change from one month aftershock
two to three months after shot.
2 here you see a tiny tiny group increasing.
Actually, some people even decreasing.
But the overwhelming majority have no change.
So I think that the thought of Oh well,
we'll just wait from month one.
Maybe wait a couple of more months
to see if things get better.
That's not going to happen.
Things aren't going to get better.
We need to figure out something else to do.
Uhm, you know,
sort of the next natural conclusion is,
well, maybe they need a third dose.
This has happened in other vaccines and
transplant patients where everybody
else needs two or three doses.
Transplant patients need three or four doses,
and you know.
So giving people a third dose is something
that we've been very interested in.
Unfortunately, it's not allowable by the EU,
as the French government is allowed it,
and so we may get some data
coming in from France.
But we're also getting data.
Coming in from very crafty transplant
patients who have learned to work the system,
learn to navigate health care,
and have somehow managed to
get themselves a third dose.
And we already have eight who are
one month out from a third dose,
and obviously this is changing every day.
We're getting more and more data on
people and it's starting to look as follows.
It's starting to look like if
you had nothing after dose 2.
You'll probably not boost with a third
dose if everything else stays the same,
but if you had something borderline
low positive with dose two,
you're going to get a major boost
or you are much more likely to get
a major boost with a third dose.
So obviously we're not encouraging
anybody to do anything.
The third dose could activate the
immune system caused somebody to get
a rejection or something like that,
but certainly people are going
out and doing it.
They're talking to their transplant
doctors or transplant doctors,
or supporting doing this.
They go out,
they get themselves at Third Ghost,
and we're learning more and more
about what those third doses mean.
If I were to make a guess about
what I would tell you six months
from now after we finish big trials
on 3rd dose is my guess would be
that if you had some evidence
of response after dose 2,
the answer for you is to get a third
dose and you'll be fine or even to boost
with a fourth dose
if your third dose gets you there
but not all the way up. And
I would say that for people who had
no response whatsoever to two doses,
we're gonna have to do something like
immunosuppression modulation reduce their MF.
Something particularly in people
who look like that who have come.
Who are safe enough to reduce their amino
suppression are relatively low risk, etc.
I think there will be some sort of
immunomodulation happening there just
to tell you off the record I've been
working with NIH and FDA and Operation
Warp speed for the last month or so and
we're hoping to get a trial launched.
That will be a single center pilot and
then ultimately it a national randomized
trial to try to help figure out what to
do with the 75% of transplant recipients.
Who did not have a robust antibody
response to?
To the more traditional EUA
approved immunization now.
Everything we've been talking about
thus far as antibodies, as we all know,
antibodies are kind of the tip
of the immunological iceberg.
There's memory B cell responses,
T cell repertoire,
T cell activation.
There's a whole bunch of immunology
happening under the surface were in
the process of studying that right now,
but we can't say anything other than what
we can say based on antibody response.
There's also things that we have
in transplant patients that we've
not managed to collect,
like drug levels we don't have on them.
Use of Lymphodepletion early on
post transplant or for rejection.
We're starting to gather more detailed
data on that in our transplant patients.
And uhm.
Obviously the the ultimate outcome in
all of these things is clinical efficacy,
which means reduction of actual infections.
I don't think we'll ever get those data.
There's no way that right now will
be able to do those kind of major.
You know,
30,000 patient efficacy studies
in transplant patients.
However,
we are starting to explore correlation
of protection assays and some
of our labs as well as the NIH.
Labs are proposing CRP assays,
correlation protection as states to the FDA,
and hopefully we will be able to
correlate antibody levels with
protective immunity in the near future.
So you know we're seeing that the
safety profile and transplant
recipients is fine.
Transplant with recipients should
be encouraged to get vaccinated,
however,
the immune response is not anywhere near
what we're seeing in immunocompetent people,
and will probably.
Require some boosting in some.
Former or another for the majority
of transplant patients we are seeing
we have now a case series of about 40
patients at Hopkins who've developed
COVID after full vaccination with
two doses of the M RNA vaccine.
Some of them have been hospitalized.
One of them have has died.
We're hearing these reports from
across the country,
so we do feel like this lack
of antibody response is highly
likely correlated with the lack
of protective immunity as well.
As I said,
we're looking at full deep analysis.
Of the B cell and T cell.
Components of the immune response
to much better understand what's
going on with these patients and how
better to improve their their immunity.
And I will just end by
acknowledging our epidemiology,
research group and organ
transplantation and all of the people.
The many, many people who have
been working crazy hard in the last
few months to to get these data to
get these data better understood
and out to everyone.
So thank you.
It was a wonderful condori awesome
questions on what is the cost of
the seventh sense of bias system
collections and any feedback from
patients if they had problems with it.
Yeah, it's a great question and
they're not expensive at all.
I don't know the exact dollar amounts,
but they're they're very cheap.
We might have gotten some deal with
the company or something like that,
but they are not prohibitively expensive.
I can tell you that they're much cheaper
than sending home phlebotomy to somebody.
House there are a handful of patients
that it just doesn't seem to work for.
I would say there is a larger handful
of patients that it doesn't work for
until they give properly trained on it.
But even with everything we can possibly do,
there is a handful of patients
that it doesn't work for.
For them you have to have a backup option
like home phlebotomy or something like that,
but it is surprising how well it has
worked for lots of patients and obviously
has us thinking in giant directions.
For other research studies or even
clinical applications where we need
frequent blood monitoring on somebody,
and we want to be able to do that in
the most logistically easy way possible.
On so solid organ transplant
recipients had like no antibodies
against the JANJAY vaccine,
but what do you think of
these patients progressed?
If they want to get the M RNA vaccine,
would you suggest that or no?
So most of what I showed you is transplant
patients getting the M RNA vaccine,
so most of what I showed you is,
you know 20% antibody
response after the first dose,
50% antibody response after the second dose.
Among those who have antibody responses,
the majority have less than you would
see in immunocompetent people, etc etc.
We were hoping that JJ would be better,
but the JJ has not proved
to be better at all.
And what do you tell these patients
to continue to wear their masks and,
uh, if they, when they come
to your office? Yeah, so you know the the.
The clinical interpretation of these
data is quite challenging, right?
Because if somebody if a transplant
patient gets, you know greater than
250 antibody level after dose two.
Can I really tell them that they're
as safe as an immunocompetent patient?
I really can't, because I don't know what's
going on with their T cell repertoire.
I don't know what's going on
with their memory B cells.
I really don't know what's going on
with their immune system enough to say,
you know, your immune response is going
to lead to protective immunity, right?
Because they are also on medications.
Block T cell responses and things like that.
So I think until we really understand
what's going on under the surface of
the big the deeper immune response.
It is not wise to tell transplant
patients that they should act
vaccinated and basically what I'm
telling everybody is this whatever the
CDC says is safe for an unvaccinated
patient should be the most that you do
as a vaccinated transplant patient.
You should not do anything
that the CDC says is.
Unsafe for an unvaccinated
patient because as far as we know,
until we have more evidence,
we don't know that you have
protective immunity.
Even if your antibody titers are high
and for the overwhelming majority
their antibody titers are quite low
and then they have this visceral
knowledge they see the LabCorp slip
that says no detectable antibodies,
and they're like, OK.
Well I guess I'm kind of in
trouble right now.
Do you think all transplant patients
should have their antibodies
measured? So that's a really good question.
It's a tough answer because I
don't know exactly what to do
with that result yet, right?
So it's a data point of course.
I mean, obviously if you have
greater than 250 IgG after dose two,
you probably have more protection
than if you have less than .8
undetectable antibody, right?
I mean, you know we'd have to be out
of our minds to think that there isn't
a difference between those two things,
but I can't tell you if
you have greater than 250.
That you have protective immunity
and I can certainly tell you
if you have less than .8,
you're unlikely to have protective immunity,
so I think if people get tested,
it's really important for them to
remember that the test result
can't really be directly applied
to clinical practice yet.
I do think that you know.
So, for example, we have some
people whose whose job is forcing
them to come back in person,
and we say, OK, well, look.
If you get your antibodies tested and
you show that there's no antibodies,
you can write a very,
very compelling justification
to your work that you have
no protective immunity,
and you're probably you know host,
but if it
comes back greater than 250, we can still
write something, but it'll be less. Sort of.
You know it'll be less impactful, so I think.
The impact of a negative test is very useful.
The impact of a positive test is probably
less useful, right?
Another question upon the attendees
is has there been analogous
detailed studies like yours in other
vaccines for transplant patients?
Like for example flu or Shingrix or etc?
Yeah so. In general,
we know that transplant patients
don't have as good of a response
to vaccines period like we.
We know that in general,
but usually like if one were to quantify it.
It would be like they have 25% less efficacy,
25% less immune response to the vaccine,
not anything close to what we're seeing.
For these COVID vaccines which you know,
and for this particular vaccine,
it also becomes is,
you know, very proximal.
In terms of of,
you know existent prevalent
risk two more questions
I can try to get to is given the
effect of baila on the vaccine
responsiveness, which wasn't good.
Should we reconsider using Bella
and transplant protocols? I
mean, Bella is an amazing drug
for the right patient and
some people have, you know,
sort of amazing responses with Bella.
What we're going to do
to get people on Bella?
Immunized is going to be challenging I'd.
I suspect. You know,
maybe so the Nova VAX platform is
going to come out soon I hope,
and maybe the Nova VAX platform,
which does not require all this cellular
machinery to produce the protein.
Maybe that one will be better,
maybe will understand the Bella
patients have no antibodies but
have a good T cell repertoire
or something like that.
But if it turns out that they
don't have a good response,
it's going to be challenging with like
immuno temporary immunomodulation trials.
To try to get them
vaccinated and was there any data with
cyclosporin versus tack or not, no
difference. I mean, cyclosporin
is not that commonly used in the US,
so we grouped together with
tech as just see and eyes OK.
Wonderful thank you for a great talk Dorie.
Great, thanks again for the
invitation. Really nice to see
everyone right. So we'll move
on or next speaker is Doctor Kathleen
Lu from the University of California,
San Francisco School of Medicine.
She'll be talking about COVID-19 clinical
trials experience in lessons learn.
Thank you Kathleen. Let me
get my screen shared.
Good morning or good afternoon
depending on what time zone you're in,
or I guess it's late morning for you.
So you know. So I'm really thank you
so much shooter for the invitation
to speak to your to the to this
really exciting symposium. You know.
I think my talk is very timely.
Kind of after Dory stock,
which is really just a fascinating overview
of what happens in our transplant patients.
As many of you know,
I'm both enough Rolla, just an intensivist,
so COVID-19 is very much been
part of my life for the last year.
Uhm, I thought by way of a road
map as I put this talk together I
thought no no nephrologists can
talk about areas without talking a
little bit about acute kidney injury.
I thought I would just spend a few
minutes talking about why some
of our API trials have failed,
giving just a couple of relevant examples.
And then I'm going to spend the bulk
of my time talking about lessons that
we can learn from Air DS in general
and from COVID-19 over the past year,
focused on both how we can identify
patients for clinical trials.
And I'm going to tell you about platform
design trial that we have executed.
Over the past 15 months or so.
To the number of reasons that,
AKA clinical trials have failed,
one of them is really the importance
of timing of delivering interventions.
This is an oldie but goodie,
as I like to say this is a beautiful
representative study now published
over 20 years ago.
But this is a study of a trial match
rettich peptide that many of you
are familiar with.
This is a study that had extremely
reasonable exclusion criteria
that we would probably use.
We probably would think about using
something similar as we designed a
clinical trial for a Ki even in 2021,
and the inclusion criteria for this
study where a clinical diagnosis.
Toxic or ischemic ATN with a 1 milligram
per deciliter rising craton over 48 hours,
so maybe our KD GO stage 2 ish stage,
3 ish disease.
Sort of a moderate to severe
phenotype of AK I patients who
had sort of advanced CKD defined.
In this case it's a baseline credit
of greater than three works clue
did and what you all know is that
this study was resoundingly negative
with an approximately equivalent
number of patients in the amp and
placebo arms achieving the.
Primary endpoint of dialysis free survival.
So why might that be?
I told you that this was a very reasonable
definition of acute kidney injury.
When you dive into the study itself,
you can see that despite this
very reasonable definition of AK,
I designed to identify patients
relatively early patients who were
enrolled in the study actually
had relatively severe.
AKA,
you can see here with a mean craton
in this sort of four to five
range across the oliguric and non
oliguric subgroups of the study.
So despite sort of a definition that would.
Identify patients early.
It's tremendously difficult
in clinical practice or
in fact, identify and enroll these
people as early as you might need to.
When you use serum creatinine based
definitions for acute kidney injury,
and again you know another way
to think about biomarkers.
Just think about the presence or
absence of oliguria and this study
also nicely represents that in this
study you can see that there's a trend
towards differences in the group of
patients who had Algeria favoring amp.
This was obviously not
statistically significant.
So that sort of is less than one,
so a key clinical trials may have failed
because timing is really quite challenging.
This may be improved by novel biomarkers.
That's an entirely separate talk
that I'm not going to touch today.
I'm going to just talk briefly
about patient selection,
again using a very nice example
here from the Yale group,
led by Shroud Creek and this sort of
highlights the differences in sort
of when we think about the clinical
entity of acute kidney injury,
that there are really many different
entities within that entity,
and so this is focused on the.
Clinical problem of identifying
ATM versus proving Lisa Timia.
This is a prospective subgroup of
the tribe AKA I study and this
study was designed to adjudicate
pre-release at EMEA versus ATN using
a very reasonable consensus process.
As you can see here at the bottom
kind of standardized case report
forms that you would have the same
information about each patient.
Nephrologists independently reviewed
the case report form in hospital
course and Judicators had to
talk amongst themselves.
Until they came to agreement.
I think the results of this
study were very striking to me,
so they examined about 67%
of patients they examined.
Excuse me,
67 patients,
and what you can see here is that
there was tremendous variation in the
adjudicators and their interpretation
of ATN versus non ATM right.
So you can see here that
at one extreme adjudicator,
one thought that almost 75% of patients
in this cohort had ATN in marked
contrast adjudicator three who thought
that only 20% of patients had ATN.
Despite looking at sort of the same clinical
data and in fact when you look at the.
Other tables in this.
In this paper you you'll see that full
consensus was only achieved about 20%
of the time in this patient population,
so only 20% of the time could you
get those three nephrologists to
say three out of three think this
is pre-release atenia with three
out of three things,
this is ATM.
So again, this highlights and it's
just sort of one more comment.
This study,
I think also highlights that
these patients can be tremendously
difficult to sort of detect
clinical differences between.
Obviously there are differences with
regards to outcomes for patients who
have what we did identify as potentially
prerenal is at EMEA versus ATN right.
But again,
not a lot of differences that are,
you know,
some differences in terms of craton,
but not a lot of differences that would be
so easy to detect early on in the course.
Disease,
and this paper went on to demonstrate
the biomarkers at least early on are
generally not that different either.
So again, another reason that patient
these clinical trials may have failed
is that defining disease in and of
itself not all a key is the same.
It may not respond to therapies the same way,
and clearly more work will be needed
to define biomarker profiles to
differentiate different types of disease.
Sort of with that brief segue
into sort of some of the reasons
AKA clinical trials have failed.
I'm going to turn now to Air DS that,
unfortunately, in 2021,
is a condition that needs no
introduction to all of you.
After the COVID-19 pandemic,
I'll just remind you that even before
COVID-19 Air DS was considered the leading
cause of hypoxemic respiratory failure,
known to develop following
certain clinical conditions with a
mortality rate of at least 30 to
40% and without specific therapies,
we really have focused more
traditional management.
Of Air DS on ventilator and fluid
management strategies that is
not for lack of trying though,
so this is a failed list of
therapies for air DS.
It looks very I could show you an
almost identical slide or failed
therapies for acute kidney injury.
People have tried numerous pharmacotherapies.
Numerous supportive care strategies
in trials that really spent thousands
of patients.
And so the first question is,
can we in fact split patients more
effectively and identify sort of
precision medicine strategies right?
And and this is been done very successfully.
As you know in the oncology
literature where people have taken
numerous forms of breast cancer and
you can take sort of this big lump
of sort of heterogeneous disease.
In fact, split it into a variety of
different sub phenotypes of disease.
For example, ER versus PR,
positive versus negative and now sort of,
you know, and.
Ongoing list of sub phenotypes
have been developed and identified
and we'll talk more about that
on some of the next slides.
So, Carolyn Calfee,
who's one of my good friends
and colleagues at UCSF,
has been thinking about this
for the last several years,
focused on Air DS,
and she's used the approach of latent
class modeling to examine this approach.
I have my friendly picture of a crab here,
so this is actually, you know,
not a new statistical technique.
This was developed over 100 years
ago to study the development
to study crab evolution, right?
Biologists were examining crab populations.
On a single island and the
crabs looked different,
but they wondered genetically
how similar they were.
And this month,
this modeling strategy is based
on the hypothesis that you can
track identify a number of classes
within a broad group of patients,
and basically the method asks
serial questions of is,
is this data best described
by one patient by one class?
Excuse me?
Is the state of best described by two
classes or this data described best by
three classes for classes and so on.
So you can see here in this
density figure that right?
You can imagine that you have a population
that looks heterogeneous like this,
and in fact using.
This method you can identify
three different subclasses.
Let's say of crabs in this case.
So Caroline first applied this strategy to
some of our AREDIUS network clinical trials,
so these are,
as you know,
large multicenter NHLBI based
clinical trials networks,
and what she demonstrated she demonstrated is
that using biomarkers defined in this cohort,
she could in fact identify
two different sub phenotypes,
which we now call the hyper inflammatory
and hypo inflammatory phenotypes.
The hyper inflammatory phenotype
makes up about.
Up about 1/3 of patients is
characterized by very high
levels of what we would consider
proinflammatory biomarkers,
including interleukin 6,
and are looking 8 soluble TNF receptor.
Low levels, not surprisingly then,
of bicarbonate lower blood pressure.
For example, the hypo inflammatory
phenotype, in contrast,
makes up about 2/3 of patients,
and this character is sort of
an opposite biomarker profile.
These I've highlighted some of the important
differences between these phenotypes,
gratifyingly, as she's done these analysis,
you can see that sort of many of the
parameters that we would think about.
As you know, as critical care physicians.
When we think about severe aredius like
respiratory rate, plateau, pressure,
which is a marker of lung stiffness
and positive and expert Tori pressure.
Again a marker of area severity kind of fall
in the middle of these sub phenotypes, right?
So again, for Air DS you can't
classify the disease based on markers
that we would conventionally.
Think of as markers of aredius
severity may be akin to our serum
creatinine in urine output, right?
Rather you have to look at other types
of biomarkers in this population to
identify these two sub phenotypes.
So Caroline has gone on with our
group to look at these bio to look
at these sub phenotypes in a number
of different clinical trials.
So a couple of important points.
Every time you look every time you
look for subpeona types using this
method in a different population,
you actually run the models.
So gratifyingly I think one important
take home point is that in all of
these analysis she's consistently
found two sub phenotypes not.
Not, they're not stage where we see two
sub phenotypes and then others where
we see 3-4 or five subpoena types.
You can see here that in all cases
the hypo inflammatory sub phenotype
has a lower mortality rate compared to
the hyper inflammatory phenotype and
those mortality rates are relatively
similar across these populations
so they vary a little bit depending
on the clinical trial design.
One of the nice things about doing this
work in the framework of a clinical
trial is that you can in fact then go
on to examine whether or not these
the sub phenotypes have different
responses to the therapy sort of delivered.
So you will call that ARMA,
which is the study in which
we first conducted this work.
As a is the is a study of a low
tidal volume ventilation strategy.
This study demonstrated significant
mortality benefit to the low tidal
volume ventilation strategy.
In contrast,
alveolae.
Which is the study of positive and
expert Tori pressure and the two
statin trials were resoundingly
negative clinical trials.
Fact is maybe the more controversial
issue of these studies where there
was a trend towards mortality benefit
and an increase in the number of
ventilator free days in patients
treated with the fluid conservative
intervention.
But since the primary endpoint of
the study was in fact mortality,
the study is not typically considered
a positive study.
So, but in particular, using negative,
resoundingly negative studies like
the people and statin trials.
You can then go on to to ask the
question of do these phenotypes have
differential responses to therapy?
And in fact, you can see that the
you in these secondary analysis,
these two phenotypes do respond
differently to these therapies.
So first in the hype versus low peep trial,
patients in the hyper inflammatory
phenotype have a better response
to the high peep strategy potential
because they have more severe disease.
Similarly, you see similar effects
with the fluid conservative
strategy being more beneficial in
the hyper inflammatory phenotype.
Same results with a statin trials.
Now that you know now to really apply
these phenotypes in clinical practice,
you're not going to have that sort of
wide spectrum of biomarkers, and in fact.
You can develop classifier models
that identify these sub phenotypes
using just a few biomarkers.
These are not all markers that are
ready for primetime at the bedside,
but that's obviously an area of
active investigation, and you know.
So the next question, I think,
really then, is how can we proceed
to precision clinical trials?
And I think this is again an example where
it important lesson learned is that you
need to rely on other experts in fields,
and here the sort of classic
example is oncology.
So I'm going to take this a moment
to introduce the I spy two trial.
This is a this is really,
I think, the paradigm.
One of the very earliest adaptive
platform trials led by Laura Esterman,
who is a surgical oncologist at UCSF,
and this is a study that enrolls
Hyatt Breast breast cancer patients.
It's a phase two clinical trial of novel
agents as we'll talk about in detail.
It's a platform trial so you can
rapidly move new drugs in and out.
It has an adaptive design,
so the flex of the sample size is flexible
and there's as I'll show you response.
Adaptive randomization.
As your population changes,
and as you're more informed
about your population,
you can change the randomization engine
and this these trials all use Bayesian
statistics for their analytic plan.
So there are some pros and cons.
The platform designed so the pros are
really that you can seamlessly add
and remove agents without rebuilding
the entire trial infrastructure.
This is very efficient response.
Adaptive randomization as I mentioned,
sort of allude to the fact that
if a if a given agent that's in
the platform looks more promising,
you can change the randomization strategy.
You may start randomizing 1:00 to 1:00
to 1:00 to 1:00 when you can change
that randomization engine to that you
accrue more patients in arms that.
Seem to be more favorable,
more quickly.
You can also learn as you go and the
obviously the oncology population
is really thought about this.
You can use biomarker signatures to
identify subpopulations within your trial.
There are some cons out of
the strategy as well.
You need relatively uniform
eligibility requirements across agents,
so you can't have you really need a
population that you can compare or
sort of across the whole population,
and this may lead to some tradeoffs
for a specific agents.
The standard of care may evolve over time,
though,
and this is you can imagine for COVID.
Clinical trial is a big issue, right?
But one of the benefits is that
with contemporaneous controls
and a COVID population, you can.
Control into some except for that
evolving standard of care overtime.
Obviously one of the challenges for
these studies is also funding, right?
This is not a study where one
pharma company
or you write 1 grant focused on one agent
developing and maintaining this pipeline
takes quite a bit of effort and work.
So as I told you,
I spy 2 is really sort of the paradigm.
This is a biomarker stratified trial.
At this point focused on
estrogen receptor status.
Her two status as well as a multi gene
array patients a new participant.
As you can see here is
randomized into the trial.
And randomized to the
experimental or control arm.
The patient outcome is then,
you know, is then the patient is
followed for their outcome overtime.
You can then in real time a dip
update the predicted probabilities.
This is the adaptive portion,
the response adaptive portion of
the study and then you can graduate
agents or which means that you
think that there's these agents
look good and you either you know
in a in a in an oncology platform
might go directly for FDA approval.
In the COVID space,
we would consider larger phase three trials,
or you can drop things for futility
and then as new agents come in,
you can continue to update the randomization.
Probabilities given,
sort of what you know about the
population and how the backbone therapy
for or standard of care may have changed.
And this is again a little
bit of a busy slide,
but this is really to just show you sort
of how rapidly adaptive this platform
has been sort of for breast cancer,
so over the past ten years the
I spy two network has conducted
a new number of clinical trials,
and you can see has rapidly graduated.
A number of agents and combinations
of agents.
And as you can see there,
you can also see that the trial
is very seamless, right?
There's never a period where things are
not sort of in the randomization engine.
You're always evolving and changing
the standard of care for these.
Patient
So what does this have to do with their DS?
Right? Again, I think serendipity can
be a really important part of science.
Unfortunately or fortunately for us,
and I want to take this opportunity
to reduce so Caroline Calfee,
as I've mentioned, is one of my
good friends and colleagues at UCSF.
We've collaborated on a number of
these RDS analysis and Carolyn,
actually before COVID hit spent a
year with Laura Esterman in her
laboratory learning about this adaptive
platform design and thinking about,
could we apply this?
Just sort of air DS in general and
the thought at the time was that we
really didn't necessarily have sort of
the right clinical trial design to.
We didn't have sort of the right
infrastructure or timing to really
apply this to RDS,
and so COVID came along and really
presented us with a unique opportunity
to design an adaptive platform
trial for COVID Air DS, right?
So there were a number of opportunities
that presented to itself plus to ourselves.
I think Dory stock really alludes to this.
There's been unprecedented focus and energy.
Devoted to COVID-19 treatment,
this comes from clinicians.
It comes from pharma.
It comes from the FDA.
Dori alluded to this as well.
It also comes from NIH.
NIH is really focused on developing
a pipeline for clinical trials,
in particular for phase three trials.
There's less of a phase two sort
of trial platform as well.
Talk about as well as patients and families.
As I've told you already had sort of
an established connection to sort of
oncology expertise for this trial,
and that team brought.
In that team,
brought to brought with it experience
with the adaptive platform design and
obviously over the past year there
have been sort of ample eligible patients.
There are also a number of challenges
to designing such a study,
which I allude to.
So first there's sort of no established
network to take this on,
and no established funding.
We've been extremely fortunate that
Barta and other agencies have recognized
the importance of rapid deployment
of agents in this time frame and how
we've been able to secure funding.
We,
you know,
similar to the way Dorit alluded
to the fact that he endured Rule
3000 patients within a few months,
we had an extremely compressed
timeline for trial. Set up and launch.
We wrote.
We started writing this Ind in April
of last year and launched the first
patient in approximately July.
Similarly compressed timeline for
patient enrollment and stratification
with a lack of real time biomarker
availability to stratify patients.
So we hope to learn a lot from this
study through biomarker analysis.
Obviously clinicians caring for
these patients are tremendously
busy and under stress,
which makes clinical trial
enrollment more challenging.
And I alluded to a little bit
the changing standards of care,
which are fortunately in our platform,
design fairly well accounted for.
So our eyes by COVID trial
was designed as follows,
so it focuses on we focus on patients who
are WHO COVID level 5 or higher, right?
So patients are screened at
the time of hospital admission.
We essentially identifying
anybody who is on high flow nasal
cannula or on the ventilator.
These patients are then randomized to either
receive backbone therapy which currently
is rim disappear and dexamethasone,
though can adapt overtime.
Whatever the appropriate standard of
care is at the time we have up to
four agents in the trial of the time,
so the master protocol is approved
by the FDA,
and each drug has its own amendment.
This gives us tremendous efficiency
because we can have multiple amendments
that are approved and reviewed by the FDA,
but those agents aren't active,
and so if something drops,
we can very rapidly activate the next
agent to put it into clinical trial.
We have a real world observation
alarm to this study,
so patients who are ineligible
because they've exclusion criteria.
Patients who decline enrollment.
Are all set.
We also do some real world evidence
data collection on these patients
to understand importantly how our
clinical trial population compares to
sort of the world of patients with COVID-19.
I will say transplantation is not
an exclusion to participation in
this study and we have a number
of organ transplant patients.
We are obtaining biomarkers specimens
for future studies that will allow us
to sub phenotype these population.
This population and this is a
phase two trial.
So really our goal is to identify
big signals are stopping rules.
They really are written to detect
a big signal into graduate and
agent to a phase three.
Clinical trial I should come in for a
variety of reasons that we could talk about.
This is an open label trial,
so again to graduate to potentially
blinded phase three trial.
Our primary endpoint is time to
durable COVID level 4 which is nasal
cannula oxygen for greater than
48 hours and we with our Bayesian
statistical designer enrolling
approximately 50 to 125 patients
per arm before the arm is either
graduated for success AKA Face based.
Three design or dropped for a futility.
Uhm,
this is just to show you this is
The Who COVID a scale.
You know,
these scales have now again rapidly involved,
but kind of the key distinction
is that this is a very patient
centered scale focused on sort of
the need for oxygen therapy to use
fairly widely across COVID trials,
and I think importantly in this
disease where resources can be
potentially limited at any given center.
It this is a patient centered outcome
of not being on oxygen again,
we do not use really ICU admission
as a criteria for admission because
as you all know,
in pandemic surge the location of
where you deliver critical care may
vary widely and so this is really
designed to identify patients with a
certain disease severity regardless
of
where they're cared for in an institution.
So one of the big efficiencies for the
ice by COVID clinical trial was the I spy,
two breast cancer network.
The first sites that we enrolled out.
The first sites that we identified to
be part of this network where all sites
that were already participating in I
spy two this gave us an infrastructure
of important things for clinical trials
like contract ING and IRB familiarity.
I just want to highlight that Yale
has been one of our early enrolling
centers and the John Cough who many of
you know has been the leader of that.
Group and has been the chaperone for now.
A number of our agents that I'll talk
to you about anything again this I just
want to highlight a little bit John
and sort of the joys of working in sort
of a multidisciplinary collaborative
Group One of our currently active
arms is PULMOZYME, which is, you know,
is FDA approved for the treatment
of cystic fibrosis?
It's thought that inhaled DNS may also
breakdown entities that are formed
in this in the case of COVID-19,
and this is.
Hypothesis that John really
championed designed the arm and now
is really leading for us.
As part of this clinical trial.
So this is a network of investigators
who are bringing forth their own
ideas and able to execute them in
phase two trials through this network.
So as of March 2021, we have over.
This is a little bit and also with
18 active sites we focus as I said
on our original academic partners,
we're working now to bring more
community partners into the network.
So more recently we've identified.
Sites like Hogan, Southern California,
which is a private hospital and
are bringing them along.
As of this week,
we've enrolled over 1200 participants in
the study, 650 to interventional arms.
The first four agents that we put into
this trial were agents that we could rapidly.
Agents that we identified for this
clinical trial where things that could
be rapidly scalable and were readily
available so of our first four agents.
Two of these agents you may recognize are FDA
approved drugs with different indications.
Those would be a Prem Alastor PDE,
four inhibitor used for
some autoimmune conditions,
and a cat abanto Brady Kainin
antagonist used for the treatment
of hereditary angioedema.
We also.
Tested Seneca River AKA CR25 inhibitor
and Reza Pro Fitbit I two agonist
in our first four are in our first
four agents rather protofab was
dropped for reasons of feasibility
and the other three agents have
were have been dropped and with
the first two drop for futility the
Academy at analysis is still ongoing.
We now have four additional
agents and rolling.
I told you the plumbers I'm story,
we have an anti CD 14 antibody
that we're enrolling in.
Or sopla mab is our third is our
third currently active agent,
which, as you all know,
functions via complement inhibition.
There's some,
there's some interesting preliminary
data focused on this mechanism in
patients with COVID and then promoting
celecoxib is an arm brought forth by Detroit,
which is part of the Defense
Department of Defense,
with some interesting
preclinical data focused there.
We have focused on real
time data entry using us,
a streamlined CRF.
Because we are in because we
were trying to rapidly deploy,
these were trying to rapidly deploy
and analyze these agents or data
monitoring our data safety monitoring
board meets every two weeks.
The data has to be really active and cleaned.
The data has to be entered really in
real time so that we can see signals
for these agents in real time and
we are working to further streamline
our data capture from the HR.
At our sites I'm using sort of
one source single source type
ways to pull data from the HR.
We have a pipeline of agents
evaluated every two weeks.
We've identified,
as I alluded to,
a number of different funding
mechanisms in addition to sort of
our to our government partnerships.
We also have been fortunate to
receive funding through the
fast grants and for our first
agents in the pharmaceutical,
companies have been very willing
partners in our analysis and
in some sense in some cases.
They've been very happy to get
early preliminary data as it's
driven and inform their decisions
about larger phase three trials,
and this is largely through the bar
to funding one of the phase two trial
engines for the Operation Warp speed,
which is sort of the large and IID
infrastructure for these studies.
As you know, platform trials have
been very popular in COVID-19.
These have taken a number of different
forms we could talk about some of these
recovery as you know is the UK based
clinical platform and re map cap is the
University of Pittsburgh based platform.
Is enrolling sort of across the world.
We are focused much more than these
two platforms on phase two agents
novel therapies that could then
be adapted to these platforms.
There's a little bit of overlap with Rimap
cap in a couple of the agents selected.
So to summarize what lessons we could
learn from Air DS and COVID-19,
I think I've shown you that
selective enrichment strategies
are ready to close for prime time.
So area said phenotypes can define
populations with differential
response to treatments,
and in fact you can define
those sub phenotypes based on
a small number of biomarkers.
Adaptive platform trials are inefficient
means of evaluating multiple different
therapies for a common problem.
There is considerable complexity
of logistics,
statistics and regulatory issues.
It is really.
It's been instrumental to us to have
expertise and experience to guide
implementation of these studies.
This is likely the wave of
the future in critical care,
and I would argue many other disease states.
Again, let's say lessons learned
from other fields are critical.
And finally I spy COVID is in fact
enrolling in an ICU near you at yeah,
so I'll stop there.
Thank you for your attention.
Take any questions you might have
and I just want to thank the really.
This is really team science at its best,
and I'd want to thank my wide.
Variety of collaborators on this project,
in particular Caroline and Laura,
with whom I copii this study.
So I'll stop there.
Thank you.
And Kathleen was a wonderful talk on.
So question is. In this,
a RDS group, is it similar with
also with people who get a K,
either hyper inflammatory
versus non plumpers?
There are a lot more people who
develop AKI in those people who have
the biomarkers that are inflammatory.
That's a great question. So
when we've looked at this in some
of the areas network studies,
I mean part of the issue that has
been how we define AKI, right?
So if you look for small changes
in craton and you see there's
really not a big difference in the
rates of AK I across the groups.
Come and you know one of the
challenges is that we have.
We don't have a ton of data on
more severe phenotypes of a Ki
like dialysis, right?
And I think the other challenge with
analyzing AKA in these populations is is,
you know, in patients with severe
aredius is dialysis provided because
kidney injury is so bad or because
fluid overload is so bad, right?
And where do those two issues
kind of play into each other?
So I think those questions are
really tough to tease out and
we have not looked as well.
For preliminary analysis,
we've not seen big differences in
ECR rates in those populations,
but we we have not looked for example
in the in the Staten populations.
How do y'all
choose the like the the PDAY four inhibitors
going to be used on the CR2 inhibitors
and those drugs that gonna be tested
it from previous red perspective data?
Or is it from mouth data? That's just that?
This might be a salutary or as well.
Yeah, I mean yeah, I mean it's. I mean I
would say it's really, uh, right?
It's really a process as you sort of
identify as you identify a pipeline.
I'd say right early on right in the pandemic.
We really needed agents where
we had somebody who could.
Provide the agent rapidly was
invested in in the problem right,
and so we actually started.
There's a COVID pharma.
There was a COVID pharma sort of working
group and they sort of put together a list
of 10 agents that they were considering
across a wide variety of inpatients,
outpatients, and again. This is, I think,
where some partnerships with people who've
done this work before really instrumental.
So you know, Laura has partnered with
industry for 10 years doing this, right?
I'm a I'm an, I'm an.
I'm an academic right?
Who doesn't think about like how
do you talk to pharma when you
want something from them, right?
And so we rapidly were able to identify.
Agents that we thought were
scientifically interesting and where
the companies were engaged in the
process of pushing those agents forward,
and so that's kind of how we came to.
Kind of our first four agents right things
we could get into the clinic yesterday,
right?
As time has gone on,
and as we have the bar to funding,
which allows us to analyze
things that we may think are
more scientifically interesting,
we literally like meet every two weeks
and we talk about what's in the pipeline,
what could be new?
This is a shout out to this
group if you know somebody who
has some interesting therapy.
We're always welcoming.
Presentations because sucat may,
for example,
came and gave us a really wonderful
presentation on a novel agent
that is a generic drug, right?
And we have the opportunity to repurpose
these agents for that purpose.
And it's really kind of at
the scientific premise as we.
I mean,
I think one of the challenges
for COVID is right.
Our population is going to evolve overtime,
right?
We're already seeing the people who
are getting severe COVID now are
different than the people who had
severe COVID you know years ago.
Unfortunately I worry that our
transplant population will be part of
that kind of ongoing tale of severe COVID,
and so we're trying to think
now about as the pandemic wanes.
What are the some of the specific you know?
Maybe COVID specific antibodies
that we could test in that would
work in severe disease,
and so we really are kind
of actively tweaking.
Our pipeline and again one of the
benefits of the strategy right,
is that I have four arms that are active.
Now we have two arms that are,
you know, have been reviewed by the FDA,
but I can always, you know,
let's say you come along,
shoot it tomorrow with like the
greatest thing since sliced bread, right?
I can put in an appendix,
you know, get the FDA to review it,
and then if your thing has higher
priority because it has a really
compelling mechanism of action,
we do let things jump the queue.
And that's a shared decision making
process between the investor we have we.
Between the investigators and
sort of the you know,
there's always a funding priority,
but those things work together fairly well.
And when you
like, start publishing the
results after you have it.
Is it years from now or is it within
months or where this is always
a work in progress, right?
You know, I think and and as you probably,
as you can appreciate one of the real
challenges in the COVID space has been right.
The rapid proliferation of art, right?
Like it feels like every day.
There's another 25 articles on COVID,
so we're active.
We actually have several manuscripts
that were actually working on.
We probably are going to focus on.
Again, this is phase two, right?
So always a little harder to package,
but part of the first phase
two manuscript will focus,
I think on the first couple of
agents and just sort of the
rapid deployment of the platform.
OK.
One thing that that those are the questions,
not thank you so much Kathleen.
For a riveting talk. Thank you.
They will take a break for about
an hour and reconvene at 1:00 PM.
So how about nice lunch break for
those were on the East Coast?
Hi Arlene, how are you
good? How are you?
Do you want me to put my slides up
now so that they're ready to go?
Sure, that'd be great.
Do that just so that, UM.
I have to change it to.
There we go perfect.
Things are good in Chicago.
House House of COVID in Chicago.
Right now it's going down down trend. It's
still it's still busy but
it's not as bad as Michigan.
Or or Wisconsin at all.
I think the case rate in the
city right now is about 3 to 4%.
The hospitals been average.
It was a lot worse last
year around this time.
It's been under control.
Is that Mount Fuji behind you?
It is, it is. Yeah,
it's probably about I don't know.
8-9 years ago when I took
a picture, there's not as much
snow anymore. Yeah, exactly.
Peter, thanks for doing this.
Yeah, absolutely.
I've always wanted to come to Yale,
and every time the opportunity comes up,
something gets in the way.
It's actually not the easiest
place to get to.
Right, you have to either
take a train from New York or.
Everyone staying safe.
I think so. We had we had one faculty
member from Chennai who had to go
home because of the COVID illness.
And they had all received the
adenoviral vaccine that was
manufactured in India. Problematic.
Vaccinations have been good in Chicago.
Yeah, very good Donald,
more complexly than it needed to be,
but I think that's also par
for the course in Chicago,
so it was it was done by a
lottery system by ZIP code. OK.
So the the ZIP codes with the highest
case rates got the vaccinations first.
And I'm assuming your
cases are pretty low now.
Yeah, they're they're definitely
on the down down trend.
I think a peak last year was
400 beds were COVID patients,
but now it's probably 75
somewhere 80 in that range.
So definitely down.
Also from February where
it was in the two hundreds.
So so yeah, I understand all
of the Uber and Lyft companies are making
a lot of money at the Canadian border.
Yeah, 'cause if you fly into
Canada you have to go into
quarantine, but if you drive you
do not have to. Ah oh, that's interesting.
It looks like the conference
is nicely attended.
Yes, yes. We've had anywhere
from 85 to 100 in that range.
It's not, it's it's great,
it's more than the usual when we
have our O'Brien at not not virtual,
but in person.
It's hard to get people coming in
from all different cities regularly.
It's basically all limited in New
York and Boston area at the farthest,
but today seems like people are online,
so it's great.
Yep. We pulled our faculty to
see if they wanted to go back
to in person conferencing again,
'cause it that's a possibility
and overwhelming majority want to
keep it at zoom. Is that right?
I'll just point out that in honor
of the peak portion, Steve Sam
has been elevated to a panelist here.
Oh no.
Hi Steve.
Can you hear him? No no.
He must be muted. Yeah, he's mute.
Doesn't look like it's muted, but, uh.
I could hear him. You
could hear him OK.
Hey panelists, for me.
All your powers to you.
Can you hear me now?
Oh yeah, we can hear you.
I wasn't expecting so I don't
have a microphone setup because
I wasn't expecting to be heard.
Uhm so. Yeah.
I actually said to you,
Arlene. Welcome to
virtual Yale. I
know, right? I could
never seem to be able
to get there physically.
As Peter pointed out, it's probably
not the only you're not the
only person that's that
problem. New Haven's off
the beaten path, but.
I think we can resume.
Hope we have ever had a good break up.
Our next speaker is Doctor Arlene
Chapman from the University of Chicago
or Pritzker School of Medicine and
she's going to be talking to us about
amino acid metabolism in a DPKD.
And of course for the attendees.
Again, if you have any questions,
you can write them in the
Q&A section at the bottom.
Which is right next to the.
Raise hand so it's all years early.
Thanks shooter.
Thanks so much for having me too.
I really appreciate it.
First, I'd like to share my
disclosures with you before we begin.
Uhm, and my objective over the next 40
minutes is to provide human data with
support for the potential contribution
of amino acid metabolism to renal
cystic disease severity in autosomal
dominant polycystic kidney disease.
So is all of you are well aware?
ADP KD is the 4th leading cause of
renal failure in the United States.
There's no racial predilection,
it's a dominantly inherited
disorder and more than 3,000,000
individuals are affected worldwide.
Cysts are found in the kidney,
liver, pancreas, spleen,
brain and other organs.
Although the name comes from the kidney
itself and we typically see patients present
in their second or third decade of life,
but this disease.
Really begins in neutral.
So this is an end stage kidney.
It weighs approximately 10 kilograms.
You can see cysts have replaced
the renal parenchyma throughout,
but it's important to note that
only 5% of the nephrons are
actually cystic in this disorder,
and it's a focal cystic outpouching
along different segments of the nephron
that result in this kidney phenotype.
To initiate cyst formation,
it is felt that there needs to be a
second hit or haploinsufficiency in
a subset of cells that results in a
clonal outpouching and hyperproliferation
of a single clonal population of cells.
As the cyst grows and expands,
their alterations in the extra
cystic regions resulting from
into fibrosis from a pop ptosis
resulting in collapsing of cysts.
And also infiltration of inflammatory cells.
There are two peak jeans that
account for the majority of ATP,
ADP,
KD one and PKD 2 which are shown here
and they result in polycystin complex
integrating both polycystin one and
polycystin two and a heterotrimeric
fashion on the carboxy terminus.
So clinically,
we do not differentiate between
PKD one and PKD 2 individuals.
Although PKD 2 patients typically have
a milder form of disease initiating
dialysis approximately 20 years.
Later and again we can see
the two genotypes here.
PKD,
one accounts for approximately 85%
with an end stage renal disease.
Average age of 55 years,
and PKD 2,
accounting for approximately 15%
with an ESRD age of approximately 72.
So the cystic phenotype resulting
from a reduction in the Poly systems
has been very well documented
over the last three decades.
The epithelial cells become differentiated,
they have polarization defects.
They integrate incorrectly with
other cells with changes in
integrins and cadherins.
They have a very high rate of division
and a pop ptosis and when cells are
placed in college and rather than
forming branching tubules they create
cysts and they have a secretory.
Rather than an absorptive phenotype,
and they also lose their planar polarity.
So ADPKD is a tubular epithelial
hyperplastic disorder and and
there's loss of function of the
Poly systems that results in
this compensatory proliferation.
And then this results in a
tumor like microenvironment with
hypoxia, cytokines,
reactive oxygen species,
and alterations in nutrients.
So recently there had been
demonstrated by Rosetta Baloda that
there is a Warburg effect in ADP KD,
where glucose is rapidly changed
into pyruvate, and rather than
going through mitochondrial TCA,
cycling results in pyruvate going to lactate,
and this has been shown in a number
of animal models as well as cell
types that if you block this process,
you can actually limit cyst
formation and a good example of
this is when two deoxy glucose.
Is used.
This will inhibit cyst formation and this
has been established and another proximal,
tubular cystic disorder.
The hand SPRD rat,
so does this apply to human disease,
so we sought to evaluate the metabolic
pathways using nontargeted metabolomics
to determine what differs between ADP,
KD and controls and also to
determine what could associate
with disease severity and for this
we used an imaging biomarker.
Height corrected total kidney
volume using magnetic resonance
imaging measurements and techniques.
So our study subjects in design
included 3680 PKD patients who had
normal kidney function without any
other known medical conditions
and without medications.
These patients were not easy to
find as most ADP KD patients take at
least antihypertensive medications.
There were 18 age and gender matched
controls and they were placed on
a strict diet for three days and
studied in a clinical research
environment and blood in urine.
Samples were obtained at the same time.
On the same dates for each of the subjects.
So these individuals were admitted
to the Emory University Clinical
Research Center.
They were placed on a three
day isocaloric diet,
2500 kilocalories per kilogram per day,
and a 1.1 kilogram gram per kilogram per day.
Protein intake and they were
asked to consume all foods
within a specified time period.
Women were also studied in the
first half of the menstrual cycle,
during the mid follicular phase.
Subjects provided plasma and urine
samples at specific times of day,
both before and after meals and fasting,
urine and plasma samples from the
second day of study were used for the
analysis that I'm going to show you.
Samples were handled immediately and
frozen at minus 80 degrees until study.
We used a non targeted high
resolution metabolomic analysis
using Thermo cubes active HV R.
We had a C18 column and ionization
mode was positive and we used three
replicates for each sample and we
were able to identify over 11111
thousand features in urine and
over 13,000 features in plasma.
For our data processing approach,
we use features that were filtered
with at least 70% of the samples
having a non zero intensity.
Technical replicates were averaged,
data was performed,
log transformed and quantified
for normalization and we use the
linear models from microarray data
analysis to determine differences
in metabolites between ADP Katie,
patients and controls.
We then went on to use an inhouse
developed pathway program.
Called Mummichog for pathway
enrichment analysis and we used linear
regression to determine which metabolites
associated either with kidney function
measured as estimated glomerular
filtration rate or height corrected TKV
after adjusting for age and gender.
We also used SMS panda classification
analysis to look at visual representations
of differentially expressed metabolites
in arpkd and control subjects and we
use mummichog within metabol analyst
for pathway mapping to look at
differentially expressed metabolites.
We also used regressional analysis
to correlate the metabolites in
ADPKD with our clinical data.
So here are our demographic and
clinical characteristics of our
ADP KD subjects and controls,
and you can see that they were very
well matched with regard to age,
gender distribution, ethnicity,
their body mass index and height
and weight and their fasting.
Serum glucose is were similar as
well as their serum creatinine.
Total kidney volume in the ADP.
KD subjects was actually relatively low at.
Just over a leader and the height corrected,
TKV was less than 600.
We were first concerned that would
there would be high variability,
either due to meals or to
individual characteristics,
and so the first thing we did was to
look at day to day and time of day and
meal contributions for variability of
our urine and plasma metabolomic features.
And nicely enough there was really
very little impact of either meal
or patient and our highest level of
variability was really introduced by
the differences in different runs for.
The metabolomics analysis on our equipment.
So we chose the second day of study.
Fasting just to reduce any variability
at all in the measurements that
we were going to make.
So here's our principal component
analysis of our ADP card and controls
in both plasma and urine and there
were 165 differentially expressed
metabolites in plasma and there
were 93 differentially expressed
metabolites in urine.
We annotated significant metabolites
using different databases,
including mbed and keg,
and among 93 significant metabolites
in urine 43 were found to have matches
in RH MDB database with a confidence
level of at least two and using SMS
sanitater at 38 were found to have
matches in the KEGG database with
a confidence level of at least 2.
And among the 165 metabolites in plasma,
we found 56 to have a match in the
HMDB database and at least at the
level of two and 41 in the keg database
with a level of confidence of two.
So here are the Manhattan and
Volcano plots for plasma on the
top and urine on the bottom,
showing the metabolites that
are differentially expressed
in PKD versus controls,
with red being in a positive direction
and blue being in a negative.
And here is our heat map looking at
the urine and plasma metabolites,
where PKD patients are shown
in green and the yes and the
control patients are shown in red.
So we can see that there is quite a
difference in the distribution of the
metabolites between cases and controls.
So when we looked at the distribution
of the metabolites and looked at it
both by case control as well as by
the features of disease severity and
polycystic kidney disease. A number of.
Things were identified and so in this table,
as you look at the case control
in plasma in the first column and
and the 4th column for the urine,
you can see that there are many more
features in urine and plasma in a pathway
manner that showed to be different in ADP.
KD versus the controls.
The other thing that's quite
noticeable is that if we look at
the types of metabolites that.
Were differentially expressed.
There was a much larger clustering in
the amino acid metabolism pathways than
in the other vitamin nitrogenous bases.
Lipids, carbohydrates and drug.
Lipid metabolism is also highly expressed
as being different between PKD and controls,
but I'm going to focus on the amino acid
metabolism identities that were found.
So here are the pathways that
were associated with 80P KD.
Specifically,
and urine is shown in the top and
plasma is shown in green on the bottom,
and you can see that histidine metabolism
was the most highly expressed pathway
that was different between PKD and controls.
If we look at the urine pathways
a little further,
you can see that there were a number
of differentially expressed amino acid
pathways with again histidine most common,
followed by glutamate, alanine and aspart,
8 arginine and proline,
lysine and methionine assisting metabolism.
So to circle back to amino
acid biochemistry in general.
This is what happens for distribution and
usage in humans we have approximately
250 to 300 grams per day of protein,
70 to 80% of that is used from.
Muscle,
there is some endogenous amino
acid synthesis.
There's also dietary sources,
amino acids,
and really there is approximately 5 grams
of plasma free amino acids in the body,
and this is then utilized for
intermediates for synthesis of
creatine DNA and RNA polyamines.
It's also used for energy
gluconeogenesis and for fatty acids,
and we excrete approximately 12.
15 grams per day of urea nitrogen
from the dietary protein that we have.
And so amino acids can be used in a
number of ways, either as a glucogenic,
ketogenic, or both.
Glucogenic and ketogenic amino acids,
and I want to point out here 'cause we're
going to come back to this in a minute.
These four amino acids here that lead
onto glutamate for entry into the TCA
cycle through alpha ketoglutarate.
So there's a significant inter
organ amino acid homeostasis that
goes on all of amino acids that
are ingested through the intestine
or immediately go into the liver.
There is some alterations in the
liver that lead through urea cycling
to the generation of urea and
also to the generation of energy.
There is a contributions of muscle
also into liver for similar utilization
and also for the regeneration.
Of branched chain amino acids,
it's important to note that the kidney
is not just an innocent bystander here
and is actually very very involved
in generating new amino acids.
For example arginine,
and also for degrading a number
of amino acids such as searing.
So the kidney is a very active organ
with regard to amino acid metabolism.
It recovers about 70 grams per day
of amino acids and if one infuses
intravenously amino acid solutions,
it will remove approximately 40% of
what is infused and they're transferred
to the kidney both through arterial
luminal and basal lateral surfaces,
and they're involved in degradation
of reabsorbed proteins,
usually those with molecular
weights less than 50 kilodaltons,
so most of the amino acids.
That are delivered to the kidneys
are actually metabolised.
This is done primarily in
the proximal tubule,
which is rich and peptidases.
It's a major site for the
catabolism of both circulating
and urinary peptides and proteins,
and when amino acids are not available,
for example, in a protein free diet.
The kidney can lose almost 20%
of its protein in seven days.
In order to provide amino acids
for new protein synthesis and
also for gluconeogenesis.
And 80% of gluconeogenesis comes from
glutamine and can account for up to
25% of glucose in the body overall.
So this is a modified KEGG
pathway for histidine.
From the study that I showed you
and what's shown here is which
of the metabolites were increased
in the urine or decreased in the
urine as well as increased in
plasma or decreased in plasma,
and so you can see here that plasma
histidine was elevated and that
there were a number of urinary
compounds that were also elevated and
serene and three methyl histidine.
Both components of the reactive
oxygen species and scavengers that
are a part of the histidine pathway.
There were also a number of urinary
metabolites related to the breakdown
of histamine and you can see these
highlighted in yellow and encircle
and you can see that histidine
will also make its way through
urocanic acid down to glutamate,
which then can be used for
energy substrate utilization.
In the TCA or Krebs cycle and I
don't have it highlighted here,
but in its own pathway,
glutamate is also elevated in the
PKD patients compared to controls.
So we thought that we weren't
sure whether or not histidine was
elevated for any one of the three
components of its pathway,
so we used primary human proximal
tubular epithelial cells as well
as primary human renal cystic
epithelial cells and used
histidine decarboxylase inhibitor alpha
FMH at increasing doses to see if we would
impact cell proliferation and turnover.
And we did not see much of an impact in
the proximal tubular epithelial cells,
but we saw a marked increase in
proliferation in the primary cystic
epithelial cells at all of the doses,
so that suggested to us that when histamine
was not being derived from histidine,
there was another route that Histidine
was going through to help with
increases in cell proliferation,
possibly through urocanic acid,
down to glutamate.
So we used nitromethane here,
shown on the last two bars to see what
would happen if we actually blocked
urocanic acid in the setting of alpha
FMH and no impact was really seen again
in the proximal tubular anthelio cells,
but there was a marked reduction in the
cystic epithelial cells down to close
to basal levels when urocanic acid.
Was not formed from histidine,
so this also suggested perhaps that this
was one of the rules that histidine was
playing in cystic epithelial proliferation.
Cyst expansion,
and increases in total kidney volume.
So we looked at two different
measures of disease severity with
measures from the histidine pathway.
One was glutamine and looked at height
corrected TCV and we found a modest
but significant inverse relationship
with decreasing levels of algemene
with increasing size of the kidneys.
We also looked at one of the
breakdown products for a Midas alone
5 propanoate and also found again.
A modest but a significantly inverse
relationship between declining
GFR and increases in this product.
So we went back and we measured
fluid histidine concentrations in
a patient where we had been able
to obtain at the time of surgery,
some cyst fluid and we found that,
surprisingly,
the cyst fluid concentrations were
almost six fold higher in cyst
fluid as compared to plasma and
urine also ended up having a higher
concentration of histidine than plasma.
So taking going back to our epithelial
cells and here we looked at both
proximal and distal primary human cells
and and self pre controlled media and
our primary cystic epithelial cells.
We can't remove the condition medium
after 8 and 24 hours and tested these
media for metabolism metabolites
very similar to what we did with
the patients and you can see that
in this hierarchical clustering.
There was a very nice separation of
the cystic epithelial cells from
the other three groups of of cells.
And two important amino acids
were found to be increased in the
condition media from these cells.
Both Aspart 8 and glutamate was
much higher in the primary cystic
cells than it was from either the
proximal or distal human epithelial
cells or the cell free media.
So going back to our patients again,
we looked at we found that there were four
amino acids that were elevated and L,
arginine, L glutamine, and L histidine,
and we also found that there were one in
the urine that was also elevated proline
and this gives you an idea of the boxplot
with whiskers for plasma arginine on the
top as well as proline in the urine.
So we did the same thing for these pathways
that we did with the histidine pathway,
and this is a modified KEGG pathway for
the arginine and proline metabolism
pathway and you can see here.
In plasma arginine is elevated with a
significantly lower urinary ornithine and
there's also an increase in glutamine
in the plasma of the PKD patients.
Both Sam or ESA dennisville L methionine
was found to be increased in the urine,
which is very important for regulation
of sustain and methionine metabolism
and proline levels,
as well as ornithine levels were
found to be elevated in the urine
in ADP Kitty patients.
And finally,
an acetyl purchase scene was found
to be decreased in the plasma in ADP.
KDS compared to controls.
So we also looked at the modified KEGG
pathway for Argentine by biosynthesis.
And again,
glutamine is shown here being elevated
and acetyl glutamate semi aldehyde
is elevated and we have increases in
citrulline and arginine in the blood
with an important reduction in El
Argentino succinate which is related
to ask one and the generation of arginine.
So again,
going back to disease severity,
we looked at metabolites in the plasma as
well as how they could correlate with HT TKV.
And here we have a somewhat stronger
inverse relationship of **** citrulline,
which we had just shown was also associated
with an increase in citrulline in ADP.
KD versus control patients,
and there were a total of 38
metabolites that were significantly
correlated with height TCV.
After adjusting for age,
gender and kidney function.
So we did have eight cysts
from a single patient,
where we were able to do metabolomic
analysis and you can see that the
cysts themselves differentiate from
each other and this is the principal
component analysis of the metabolites
of the eight cysts from a single patient
who had normal kidney function and
was known to have a PKD one mutation.
And if we look at the hierarchical
clustering of the metabolites
in these eight separate cysts,
they again differentiate from
each other quite nicely,
suggesting that there's heterogeneity
in the metabolomic profiles of
the cysts within an individual.
And we went ahead and measured amino
acid concentrations in these cysts,
and you can see that there's a significant
increase not just in histidine,
but markedly so for arginine, alanine,
glutamine, glutamate and lysine.
So overall significant increase
in almost all of the amino acids
were found in cyst fluid.
And if we looked at metabolomic
pathways that could potentially
associate with arginine and we compare
them to control urine and PKD,
urine and cyst fluid,
we get a very different relationship
between the different metabolism pathways.
So in this figure here I show you
the relationships with arginine
in control yarn where the urea
cycle is predominantly important.
Followed by beta alanine and if
we look at ADP KD urine we now see
that arginine and proline metabolism
is highly significant.
Followed by lysine and Aspart 8 and
asparagi mean as well as the glycine
searing and alanine and threonine metabolism.
So a very different relationship in
the urine of ADP. Kenny individuals.
And similarly we have in cyst fluid
similar amino acid pathways and now
histidine becomes quite important
in its relationship to arginine.
So in summary,
there is an increase in plasma arginine
and a decrease in plasma and acetyl
putrescine in PKD patients versus controls,
and this suggests that ornithine
is being utilized to form proline
activating the TCA cycle and the glycine
searing and threonine metabolism pathway.
And from the arginine and proline pathway,
arginine could be utilized in bio
mass generating metabolite pathways
as well as ATP forming pathways
such as the TCA cycle.
So formation of amino acids could
also be contributing to the TCA cycle
and could be an alternative energy
source particularly utilized by
cystic epithelial cells in ADP KD.
Downregulation of urinary L arginine
succinate with increased citrulline
and arginine levels suggests that
asked one depletion is impacting
arginine synthesis,
and I know that you and your
colleagues at Yale have looked at
this in a number of models and
arginine reprogramming in ADP.
KD has been reported by
Bob Weiss and Jose Trot,
and so a decrease in Sister Genesis
through arginine depletion may
yield arginine reprogramming.
In a DPKD to become a potentially
new and novel treatment.
So with that,
I'd like to say thank you to my colleagues,
Doctor, Paley, Chen,
Sharon Wang and Corinne Cochran,
as well as Doctor Dean Jones
and doctor Karen Heupel
at Emory University. And of course,
all of the ADP Kitty participants
who helped with this study.
Thank you very much.
Thank you Arlene. I was
wonderful so we can open up
for some questions.
So there are some questions
are from the audience.
You think that interstitial
inflammatory cells are contributing
to the metabolic profile in
the urine and the plasma of
PD patients?
Yeah, so that's a great question.
You know, as you know,
resident macrophages appear to play a
big role in cyst expansion and growth,
and we've looked at some of
the transporter functions.
Not in Picd resident macrophages,
but in healthy control macrophages and
and they do actually utilize many of
these amino acids in their function.
Whether that's changed in
the interstitial cells that
are have a germline mutation,
we we don't know the answer to that yet.
Great, what do you think is
the effect of dietary protein
restriction in these pikuni patients?
So we had. So the MDRD study was
done many decades ago and and this
is a modification of diet and renal
disease study and and it was highly
over recruited for ADP KD individuals.
22% of the MDRD study population
had polycystic kidney disease
and when that group was looked
at separately from the others,
there was no impact of low dietary protein.
On disease progression now,
when that was done,
it was a much more advanced disease.
Population in GFR measurements were
done directly and to be eligible
to be in the study it they had to
be less than 55 mil per minute.
So it's quite possible that it was
too late in the course of a DPKD
for there to be any effectiveness
of dietary protein restriction.
I think Thomas Weems has generated
a lot of interest looking at
dietary calorie restriction.
Not specifying protein per say
and has some very nice data in
animal models to suggest that if
there's alternative fasting or
keto base to the diet that assist
burden is actually quite low.
And there are currently some clinical
trials going on in humans with this
regard in at the University of Colorado.
So we still have to learn whether
or not it has any impact.
OK, so there's a follow-up question
also of like calorie restriction.
You think that would be
potentially beneficial?
So I think you know this
is what Tom has shown.
He hasn't specifically
looked at protein alone,
although you are reducing protein
intake when you do intermittent fasting,
you know, and so and the keto diet
really does focus on on more of
a protein balance to the diet.
So it's possible either through
pure calorie restriction or
specifically protein intake reduction.
There could be an impact.
We would expect that some of
the metabolic differences.
Most related to PKD itself would be
found in the urine more than the plasma.
Since plasma has greater input
from metabolism and other organs.
So I think plasma you know,
just as it is, you know, so I think
there's a couple of things I think,
UM, plasma is a very tightly
regulated compartment, right?
And so I don't think that it actually
gives us a a a a view into what's
going on at the organ level very well.
So I think urine is actually a
much richer source of information
when it comes to looking at
alterations in kidney metabolism.
Uhm, that said,
you know if you look at the
osmotic activity of amino acids,
they count for about 5,000,000
awesome in the circulation and
we actually thought that perhaps
the osmotic activity of the
amino acids has somewhat of a
role in altered utilization,
particularly in the cyst fluid,
and so we're actively looking
at that relationship right now.
Uh, are you able to measure the
flocks of the these pathways or?
We we can't. I don't have that
data to show you today and and and
that's actually ongoing right now.
OK. Wonderful thank God. See, So
what is driving though
the war mark effect?
So I think there's been a number of
different studies where the microenvironment
has been altered in experimental
settings both by using hypoxic chambers,
which would be consistent with
the intrarenal environment,
reducing oxygen tension levels
down to 1 to 2%, and this,
in part is probably part of how the Warburg.
Changes taking place?
Uhm, I don't know if there's
anything related primarily just
to the PKD mutations themselves
that change how energy substrate
utilization is is occurring.
But that also is a possibility.
It's one last question from the audience is.
There is an increase in amino
acids and assists, but, uh,
I was wondering if there's other
factors such as B, UN and creatinine
is also elevated in the cyst fluid.
Yeah and so on.
This slide where I showed the
amino acid concentrations,
the creatinine levels in
the cysts were also shown.
So there were two different
populations of cysts.
Some of the cysts had
creatinine identical to plasma,
so this in very old terms from
the PKD literature would be
consistent with a non gradient cyst.
And there were three or four cysts
that actually had elevated soon
cratons compared to the patients
serum creatinine and so those
looked to be more like gradient
cysts and actually in the gradient
cyst the amino acid concentrations
were significantly higher.
Other wonderful talk.
Arlene, thank you so much.
Yeah, and I think we'll
move on to our last speaker.
Which is a doctor regime horizontech
from Harvard Medical School and
he'll talk to us about kidney
organoids for modeling AutoZone,
more recessive PKD.
Thank you Reggie. Yes,
thank you for the kind introduction.
Uhm so. OK, yes we can see it.
So yeah, first I'd like to thank
the organizers for the kind
invitation to present our work today.
My name is Eugene Morrison and
I'm currently working for a
Massachusetts General Hospital,
and today I'd like to share
our research about kidney,
organoids and vascularization of
organoids and molding modeling AARP.
So. Since the disclosure slides.
So in 2000, 2007 Doctor Yamanaka
killed University has has invented
a new technology by which we can
convert the somatic cells into the
preboarding stem cells named IPS cells.
So these I PS cells are has
capacity to proliferate and also
differentiate into any type of tissues.
So at that time I finished my residency
at Keio University in Japan and when
I went into the Graduate School at
Cal University and started to work on
this IP S research to differentiate
them into kidney cells and tissues so.
We initially focused on kidney
keyboard translation. We used mouse.
Yes, I PS cells as well as human esip SLS.
So at that time we focused on
kidney specific protein,
which is expressed in the
entire kidney cubells.
So we we created a monoclonal
antibody against extracellular domain,
of this case P protein.
And, uh, we isolated the case,
be expressing cells from the
differentiated mouse and human cell human.
I PS cells and we cultured them in
the in the matrix to form those
three dimensional tubular structures.
So on the lights on the left side
we have a mouse ESI, PSD, live,
two blocks cells and on the right side
we have a human derived case be positive.
So after after that we we wanted to
make more complex skimmate issues,
which is the nephron.
So the next one is the functional unit
of the kidney and it consists of many
different cell types including photo sites,
proxy material robo handy
to the distal tables.
So to create such kind of complex
tissues we focused on the generation
of the progenitor cells of the nephron.
So that is the nephron progenitor cells.
So the network projector cells was
identified by occupancy in the lab.
So they identify these progenitor
populations in the mouse embryonic
kidney and these cells expressing
transcription factors 6/2.
So this 6/2 positive cells has ability
to differentiate into the segmented
nephrons from photo site to the distal tools.
So to create to differentiate the human ears,
I PS cells into the six two
positive movement progenitor cells.
Uh, we we designs directed
differentiation approach which which
is the essentially to follow the in
vivo kidney development step by step.
So we particularly focused on the
differentiation into the posterior
intermediate measurement cells.
Because we wanted to minimize
the contamination of the Pro net
flows or Miss Networks.
Well, we you know we specifically wanted
to generate the metanephric mesenchyme,
which is the functional kidney in the human.
So from the frozen missile networks are
derived from until your intermated wisdom,
which expresses some of the markers,
including parks,
to an electric swan.
But in the posterior region of
intermediate mesoderm cells,
those packs to electrics one
are not expressed but some other
specific markers were expressed,
so specifically induce the posterior
intermediate vision themselves.
We we first induced the late stage
primitive streak cells by activating
the wing signals and in some of
the lines we inhibited the BMP for
signal and then after the screening
of the small molecules and growth
factors we found that active in.
Can activate some of the
posterior Internet madness genes,
including hopes.
Even so, by this two step approach.
We first generated the posterior
specific intermediate Muslim cells
which express WG one and Hawks Dillon,
but not the park still and energex one.
So from this posterior intermittent
miss themselves.
We further differentiated into the
60 positive maximum project itself.
By activating the FGF 9 signal,
so these are the images of the front
projector sales English from human,
and I PS cells.
So with this three step protocols,
we induced the six to sell one WT one
tax two IL 1 positive maximum projected
sales from both human ES&IPS sales.
And on the right side we have the
quantification of these positive cells.
But we were able to generate the magenta
cells at 80 to 90% of the efficiency.
And then these different projectile
cells are actually was able to define
just spoke spontaneously differentiate
into electric compost, leaner risk.
Uh, that's that's, uh,
that's that's shown here.
But you know,
if when we simulated the signals
from the erratic,
but in this case wind four and F nine,
we were able to increase the
percentage of the electrics one
positive renal vascular cells.
So these are the higher magnification
image showing the round paralyzed.
Curio structures,
which resemble the original rascals,
so these are in a way still like
structures or expressing attacks.
8 rejects one the markers for the
individuals and also our surrounded
by the basement membrane lining.
Should we summarize?
So then you know these renal vessel cells?
Uh, you know why they differentiated
into the segmented networks.
And from that stage we did not
need to add any growth factors.
So these those winner vesicles were
just able to self differentiate
into the segmented methods.
So this is just one cross section
of the nephron organized so as
you can see there are multiple
natural structures induced in in
one electron organoid and if you
look at each structures it has some.
Clearly, segmented structures from
grooming from glomerulus to the tables.
And within these kind of bone
chops like structures,
we can identify politics in positive
photo site like cells and that's
that is connected to the LTL positive
proxy materials and also the CD term
positive distal tools and overtime
these structures elongate and forms
this kind of loop like structures.
And in each compartment,
for example,
in the policy IT clusters expressed
in publication that these cells
are also positive for other parts.
Site markers including TT One
and also never end.
And also it clearly shows the
similarity of each outside and
basement side because nepheline
is expressed on the base outside.
But the public action is expressed
on the outer side,
so the end of the age downside.
And in the tubular segment.
Positive proxy material segments.
We can also detect some of the
voter channels are covering one
marked by red color here.
And by the electron microscopy analysis
we can recognize the Bowmans jobs.
So like structures resembling the groomer
line and within that capsule like structures,
we can see some kind of paralyzed
structures which is photos,
right?
And if you look very closely
in some of the regions,
we can recognize these food
process like structures on the
very mature basement membrane.
And in the two black compartments,
we can clearly detect the
tubular lumen formation and.
That we can see the tight
junction between the two groups,
period cells and in some of the
cubicle structures we can also
see brush border like structures.
So by the M RNA analysis,
if we compare the kidney organoids.
To the human kidney specific data set.
Many of the kidney specific genes are
expressed in the kidney organize.
So now we can generate the kidney organoids
in the in the high throughput format in.
In our case we use the 96 well culture plate,
but you can see one kidney organoid
formation in the one where each
layer of the 96 well culture plate.
And the immunostaining shows the
multiple natural structures which
has the segmented features from the
album LY to the distal tubules.
So there are now a lot of applications
using this kidney organized,
but today I'd like to talk about the
vascularization of the kidney overnight
and also the modeling for the ARPD.
So, uh, what one of the, UM,
the the challenge in the static kidney
organoid was the the vascularization?
So by the electron microscopy analysis,
you know, we we, we saw those gromiller
like structures but we did not see
the capillary loop formation which
is necessary for a blood filtration.
So we thought that's the next critical step
to further make more mature community issues.
So we started the collaboration with
Louis Lab at Harvard University. So.
At that time we thought the the mechanical
stress is might be the critical mid
year of pasteurization and also the
maturation of the kidney tissues.
So we wanted to apply afraid,
just stress to the kidney organoids
so at least lab has fabricated a
Frederick chip which are shown here.
So essentially this is a very
simple chip system where we can just
circulate the culture medium through
this in depth and outlet.
And based on the analysis on the 3D profile.
Uh, in this specific region we we can,
we can apply a higher of
shear stress in this chip.
So we we decided to put the whole organize
onto onto this region in this chip.
So. This is just schematic,
these illustration showing the the
chip culture or licking the overnight.
So we just essentially,
you know,
take the organoid from the 96 well
plate and put onto this free **** chip
so they organized is attached on the
surface of the bottom of the chip.
And then we basically follow the
same differentiation protocol.
But we made a minor modification to
facilitate the endothelial proliferation,
which was the very low low
concentration of FVS.
And we tested a different ECM's to
support the adhesion on the override.
At the same time,
we found some of the ECM can actually
facilitate the vascular formation,
so that's that's shown here.
So as you can see,
when the organoid attached
to the other here and ECM.
There are some vascular formation on the ECM.
And then we applied the Free
Digest list from a very early time
point of organoid differentiation.
So this is a more like a
quantitative assessment.
Of the vascular structures.
So in in, in, in the red color Restained
CD 31 and one of the endothelial marker.
And in the in the UL means this
is just 96 through culture grade.
There are some CD's that you want
positive cells but not a lot.
But mainly applied the shear stress
at high highest level.
Uh, we we saw much more vascular structures.
As you can see here.
And then, uh,
we quantified those visit passing area.
And,
uh,
the junction of density means the
branching structures of the vessels,
and we also quantified the length
of each vessel.
So in high flow condition in all
of the past cents or junction
density and the length or increased.
And by the M RNA analysis.
This is 31.
Expression was also up regulated,
so suggesting the pasture
formation was enhanced on the high
floor conditions.
So this is just one representative image,
but you can see they are not.
They are now much more vascular
structures when we culture organoid
under high flow conditions.
So then we looked at the endless
video projector populations.
Which is a KDR posting sales one
of their industry project marker.
So on the left side we did
the flow cytometry analysis,
showing approximately 4%
of KTAR positive sales.
In the in the controller organoid,
but we applied the high floor chest list.
This population was increased up to 11%.
And the transcript form analysis
for the CVR and CD 146 or also
increased in the hyper conversion.
And we also capitalize the maturation
status of these induced vessels,
so the one of the characteristics of
the material vessel is the mural sales.
So those vascular structures is
supported by the neural cells,
so we immunostain for the PDG from
better in the yellow color here.
But you can see these posts to flask later.
Is supported by the PGA bedpost Paisley?
And we also looked at the roomier formation.
These are the the confocal imaging.
But, uh, you know,
we we did the electron microscopy analysis.
We were able to see the clear
vascular lumen formation.
As marked by by this red color here.
And then we evaluate it.
If these vessels are perfused
by the cultural media.
So to do that we used
immunofluorescence beads.
So we added the bees into the
culture media and we took the
image to visualize the media flow.
So on the left side there's a CD,
static and positive vascular structures.
And on the right side you can see
in the yellow color these bees
are actually within the vessels,
so suggesting these fast feature
or perfused by culture media.
And also, interestingly,
you know when we put multiple
organoids onto the one same chip.
We saw the connection between
the over noise by the strangers.
And this is a movie showing
the beast perfusion.
So the bees is in the cyan color and the
vessels are visualized in the red color.
So on the right side you
can see one best sales,
but also you can see the Scion positive bees.
Andy,
you can actually see that the
bees are coming out from this
vessels at this exit here,
so suggesting you know these best sales
are perfused by the culture media.
So at the same time we thought
these pasteurization as well
as the mechanical stress might
facilitate the chiller actuations.
So in this slide we evaluated the chiller
compartment in the vascular is overweight.
So on the left side we have studied
control showing the expression of LTL.
So LTL is approximately marker.
But it's supposed to be
expressed on the luminal side,
so HL side.
But in the static conditions,
its expression is more like diffused
to even at the base lateral side,
but maybe facilitated the pasteurization.
In the under the high floor.
Those are FDA expression was
concentrated at HL side,
suggesting the kibra exterior priority
was enhanced by the high floor culture.
And on the right side,
we evaluated the primary CDF
formation by the staining for
the associated to bring in the red color.
So on the light side you can see
there are much more ciated tubular
cells and under high flow conditions.
And this graph shows the
conjugation of the ciated jewels.
But you can see a significant
increase in the CC information.
And we also evaluated the M
RNA expression of the CD genes,
including PKD 1, PKD 2, pH D1,
and which are involved in the
polycystic kidney disease.
And DC engines are also up regulated.
And also we evaluate it.
The transporter expression,
such as aquaporin one.
But as you can see,
you know under the high flow
conditions these transport expression
was were also are enhanced.
So collectively suggesting the
tubes are became more mature
on the high floor conditions.
So then next we looked at the grow
Miller vascularization 'cause this
is very critical for the kidney
function to generate the primitive.
Umm so on the left top side we have
one representative image showing the
last feature and the whole side.
But you can see in this green
positive photo site cluster the
same bring 1 gram Li agree mill.
As we can see.
The CD 31 was CD 146 positive vascular
structures within this group mills,
so we quantified the vasculitis gloomy
light on the lights at top panel here,
but essentially under the
high flow conditions.
These vasculitis permalight
were found at around 50 to 60%.
While in the static it was very rare
to find such virtualized grumel.
At the same time,
we saw the upregulation of the VEGF.
So suggesting those endogenous
production of the GF.
See dates the vascularization of
the Carmelite and we also looked
at some pallasite jeans like NPHS,
one left wing or snapped pulling.
Those are also up regulated.
And this image shows the multiple
stages of the vascular single
line from his shaped boy.
So at Arias Arias stage in a state
body and the serial cells migrate
into these structures and on the
right side we have control samples
from mouse embryonic kidney.
But you can see very similar structures here,
showing the the interaction of the
endothelial cells to be these shapely bodies.
But after that we can see the Lumiere
formation within the gloomy light.
So this is an electron microscope image,
but you can see a single room information
in this one are really gross.
And after that we actually saw more
advanced chapiro like structures.
As you can see here.
And by the electron microscopy we can
recognize the multiple luminal formations.
And this woman steps like structures.
So suggesting the ground mill bastardization.
So then we also looked at the electron
microscope to see the room information
as well as the food process formations.
So in this image you can see
the food process formations.
Looks like surroundings and
vascular structures.
So after that you know we we also,
UM,
started to work on some of the disease
models to to study the kidney disease.
So one of the major work we
are doing is the ERP KD study.
So this is also the collaboration
with Waste Lab at Harvard,
but the three members are
working on this project,
so we initially made the patient denied
by peer cells from the RPG patients.
But we found that the.
There is a quite a difference,
you know when we differentiate those
patient client into the organoid,
so to find some disease mechanism.
We thought it would be better to have
the crisper mutant so that we can
use the parental line as a control.
When we analyze the gene expression profile.
So we generate it's the pH D1 crisper mutant.
Uh, with the with just one guide RNA.
Targeting the excellent 3 here.
So then we picked up the Crohn's which had.
Frameshift mutation.
He told tigers and he looks like.
So we made the heterozygotes and
sigusr frameshift mutant line.
And then, uh,
we differentiated those patient line
and crystal mutant lines into the 60
positive from projector serves as shown here.
Then these cells differentiate into
3 dimensional kidney organoids which
contains the segmented networks.
So from both Kholm tigers,
Newton Newton we were able to
make the kidney organoids.
And then we confirmed the
protein knockout of the pH.
D1 here.
As you can see in the control line, pH.
D1 is expressed in the cereal
cereal instructions.
But in the homozygous knockout
line we did not detect the protein,
so confirming the protein knocked out.
Then uh,
we stimulated the cyst formation
by activating the cyclic amp
E with the full screen.
So, uhm.
In the Tigers mutant line.
Even when we add for spring we did
not see the cyst formation.
But in the Homocidal senior tag
line on the right side here.
With the full screen treatments
we found the LTL positive cystic
structures suggesting the formation
of the cyst from the proximal tubal.
And on the right side we have
quantification in a in a,
in CLTL positive or CDs
from positive segments.
Well,
this left graph shows the
LTL positive structures.
So in the pH D1 home trailer
smoker over noyd maybe added
false claim to the culture media.
We found a significant diversion.
In in, in, in diameter,
but not in the CDH one, positive to Brussels.
So,
but in any case we did the
transcriptomic analysis to validate
the model of this crisper mutant line.
So we found a mini signals are
changed in the in the Home Tigers
new timeline when we compare
to the head of sales Milton.
So these samples are treated
with full screen.
But we did see many signal changes which were
already published by the previous studies.
So for example,
like site Skelton changes or
impulse signal pathways,
why ECM remodeling?
But kindness preparation
with CD4 CD 42 passwords?
So we we thought the model system
itself seems working but we you
know if we look at the patient
samples the cysts are delight
from more distal tear walls.
But not the proxy materials,
so we thought maybe you know we can
improve the model system by using the
vasculitis keeping organized system
because we can apply the mechanical stress.
So, uhm, what one of the
mechanism of the cyst formation?
Is the primary ciliary signals,
so when there is a urinary flow.
It activates it stimulates the primary Syria
and activates the intracellular signals.
So in the static 96, perform at.
It's difficult to expect such kind of
mechanical stress on the primary series.
So we decided to use the
vasculitis kidney organ system.
So basically, you know we differentiate it.
The mutant lines to the organoid
and cultured them on the chip
and applied with the Free Digest.
So in this conditions,
the primacy of formation is enhanced.
And then.
So these are just brightfield image
but showing you know the chip,
the power fusion chip and the
organoid on the chip.
So these are the organize when
we start the culture on the chip.
But overtime after starting the perfusion.
We were able to see some kind
of cystic like structures.
As you can see here.
In the pH D1 home flag as new clients
but not in the hillside is milk.
So then we confirmed the vascular
formation by CD one forty 60 minutes
training and the yellow color here.
But it is a slow flow conditions.
You can see there are more vascular
formation and also there are many
LTL positive geographic structures
in this overnight.
But on the right side,
in the control without the flow.
There are much fewer vascular structures.
Uh,
but you can see a lot of different
structures and then two, uh,
we confirmed the cyclic AMP
activation by the fluidic culture.
So these are the organoids made from the pH.
D1 homozygous mutant,
cultured on the fluidic chip.
But you can see some die rotation of the CDA.
Trump posted tables.
And we confirmed the cyclic amp
P activation on the light side.
So when we apply the fluidic flow,
the cyclic amp level was actually activated,
which suggests the CD signal is stimulated.
And then this is another image showing the.
The Max projection of the entire
organoid from top to bottom.
So in the green color we stay
inside each one.
A marker for the distal reference.
But in the homozygous mutant line,
when we applied the fluidic flow.
There are,
in a large dotation and CDH
one post distal segment.
But if you look at the LDL positive
proxy material settlements.
There is no cystic formation,
so this is more consistent with
the patient distancing.
And we also looked at the CFTR
and the red color.
But essentially the this AARP Katie model,
the CFTR,
was not expressed in the CDH one post exist.
I think this is consistent with
the previous finding.
That you know,
in the ADP KD the safety all is involved.
You know by secreting
provide into the room and to
facilitate the system rules,
but not in a RPG. Then we quantified.
The the diameter of these children
were still around the structures.
So these are again,
this is the image of the CDH on
positive cystic structures in
homocidal smoke outline under the flow.
And these cysts was not observed
in the hittle Cyrus Newtons.
And also we don't flow so on the right
side we have the quantification in the LTL,
positive proximate tables or CDH,
one positive distal reference.
So essentially,
in the CDM positive this reference,
we may apply the fluidic
stress through the shutters.
The dietitian was significantly increased,
but in the Lt positive segment
there was no change by the floor,
so this is consistent with the
patient finding and in the last
slide this is just last slide.
But this is very preliminary results,
but I wanted to share because I
thought this is quite exciting results.
So we tested several compounds to
to interpret the system formation.
In this free **** chip model system,
so in the control sample. So this is.
Uhm hypnotizes neutron with under the floor,
so the CDH one positive distal.
I did not form cyst but in
the homozygous mutant.
May be applied fluidics justice.
Mercy John positive segment performed
the system so we found this compound.
123 decreased the size of the distal segment.
But other compounds five and
six did not have effects.
Compound four had some effects
on marginal effect, but was not.
Uh,
effective enough,
but on the light side we have
quantification showing the some statistical
significance in some of the compound
to suppress the system formation.
So this is the summary slide,
so the differentiation protocols to
generate kidney tissues have been
developed and those organized contain the
multi segmented networks from other sites.
Do this to the distal nephron.
And the free did flow using the
the the the chip enhanced the
maturation and vascularization
of the kidney organize in vitro.
And the fluidic model of the kidney
organized the recaptcha late the
sister Genesis in the pH D1 mutants.
And those drug screening can
identify the therapeutic candidates
who they are beginning.
So lastly,
I'd like to thank the all of my lab members,
but particularly for the.
Pasteurization work I'd
like to thank Naveen Gupta,
who was the Co first author of the paper.
And also I'd like to thank Ken
here at Scott and Tomoya Yoshi.
For the PKD PhD one work.
And also I'd like to thank you guys.
Thank Jennifer, always.
For a generous support and
collaboration for the multiple project.
And also I'd like to thank our Chief
Eugene V at MGH Arena division
for the start up support.
And also I like this time.
Entry at Brigham and Women's.
For, uh,
the collaboration work for making
the organoids.
And lastly,
I'd like to hear his time the
funding from NIH.
A particular amenity DJ and cats and NIBIB.
And also I'd like to thank HCI.
How much steps to Institute and Brigham
and Women's Hospital for the seed funding?
So thank you very much for your kind of
patient and I'm happy to take any questions.
Of the great talk on Reggie Allen
just are asked some questions that's
already popping up from the audience.
I think you've shown that these nice
individual cells form on or specific
receptors also found like for example
by Jeff Perceptor or Efrain or Type 2.
And do you ever see from my question is like
fenestrated endothelial cells on your images?
Yeah, that's great point.
So we did see some finished rated
structures by electron microscopy
and we also did the mechanistic
analysis for the vascular formation.
So what we did was the because when
we apply the free Nick just list,
the expression was increased.
So we thought those are
produced by the neck materials,
particularly for the group.
Will have a situation.
Now there has to be some local
gradient to attract sales,
so we disrupted the gradient
by adding the VEGF.
So we added a lot of easier to
to to to really remove those
gradients from the indigenous.
Section and then it would be fine.
Was the vascular amount you know
what's same with additional VGA,
but association with the groom
LY was was completely decreased.
So at the same time we quantified
the distance from the proxy materials
to the vascular vascular structures.
And distance was also increased
by adding the PDF.
So I think those are not indigenous
production or the PDF from the nephron.
Epithelial cells is critical to to
facilitate the interaction of the nephron.
Bacterial cells and pacification.
Great, there's another
question from the audience.
Is there evidence of filtration in
these vascular organoids, and if not,
what drives the improved tubular
differentiation under higher
flow? So that's a really great point,
so we're trying to find out.
The mechanism you know there there
there has to be some mechanical stress,
but it may not be the luminal flow.
You know it can be the floor
outside the tables outside.
And also because the you know,
because we are using pretty static punk,
it's more like a policy file.
You know, stress so it could be
several stretching mechanism. So
that's how with her style, big a pump.
That's how you kind of set
high versus low flow rates or.
Yeah, so we are
using the static part for both
in high flow and low flow,
but the if we you know maybe maybe
increase the speed of the pump.
Ah, you know, we evaluated
the doctor flow on the chip.
The the wave of the floor
became more positive at the
higher in high high production.
How do you determine
what is high flow and what is low flow?
So this is just our very subjective division,
so the high flow means the maximum you
know speed that we were able to apply
because we feel pride too much stress.
The organized will just detach and
just crash. That way you know it's
just attached on the grid, so the high
means the highest that we could apply.
So the question is,
you know how different from
the physiological shear stress?
It's quite low,
so we have to improve the culture
system so that we can apply the
physiological level of stress.
Attendees is on the end.
We've been putting compounds were added,
well they added at the time of seeding
or after the cysts have formed.
Oh yes, so that's a yeah.
That's a really good point.
So in that in a pilot study we
started before the seat forms.
OK, and another question is PK HD one
was found in the Sicilian organoids
only underflow and wildtype organoids
without flow lack cilia which should be
similar to KV-1 mutants underflow but
the former do not form cysts so why?
So so the. So the the PhD want,
you know we used both.
Hit their tigers and the homozygous.
So in the hetero Cyrus pH.
D1 is expressed and the C is formed and
the formation was preceded by floor.
And we use that condition as a control.
So we did the same condition,
but we used homozygous snorkeled.
So the only difference was the pH.
D1 mutation in another order.
OK, last question is.
In have you made a model with
the 80 TKD so to Blender?
So can you disease,
umod associated kidney disease
models with this organoid? Yeah,
so that's also a good point.
So we we we're trying to do you
know more newtons, but for now we
we are focused on the RPD and.
We also worked on NPHS one,
then if nothing under producing
and beaches still.
And I really want to expand
our work for to, you know,
more genetic kidney disease.
Also, peak 81 and possibly
peak 82 also yes.
Alright. OK thank, thank you for
a great talk reje. I'll just turn
it over to Doctor Harrison. Well,
I just wanted to thank the speakers
for really a spectacular symposium.
All the talks were great
and everybody was perfectly on time.
Even finished early in some cases.
Really appreciate everyone's
participation. Really, on behalf of
the O'Brien Center, are very
grateful to shoot up for organizing
such a spectacular symposium and
for the assistance
you received from
an Prodotti and Kyle
Finn and we thank the audience
for all the great questions.
And I hope everyone has a
great weekend thinking about
all the wonderful research.
They've heard about today.