Quantitative Morphology in Kidney Research
February 13-14, 2012 Conference Videos

Breakout Session Reports
  • Glomerular Area and Volume
    John Bertram, Monash University, Australia and Wendy Hoy, The University of Queensland, Australia
  • Interstitial Capillaries
    Michael Mauer, University of Minnesota and Agnes Fogo, Vanderbilt University
  • Interstitial Fibrosis
    Kevin Lemley, University of Southern California and Cindy Nast, Cedars – Sinai
  • Digital Pathology
    Stephen Hewitt, National Cancer Institute and Laura Barisoni, New York University Langone Medical Center

Video Transcript

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JEFFREY KOPP: Okay. Let’s go ahead and get started with our reports in the breakout session. I know planes are departing, I know people are

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departing, so we’ll go ahead and do reports from the four sessions and then Kevin has a brief wrap-up, and we’ll try to get all of this done in the

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next 25 minutes or so. John is going to start.
JOHN BERTRAM: So, Wendy Hoy and I kind of

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chaired this little session looking at glomerular area and volume. I don’t know that we did everything we were asked to but this is where

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we got to. I think there was agreement that the reality is that not all of the quantitative morphology QM techniques that we’ve heard about here in

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the last day or so may be applicable to the renal biopsy. Where we’re dealing with a limited amount of tissue, the priority has to be the correct

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pathological diagnosis, but perhaps we can think about how best to utilize this precious resource, and maybe by talking to the pathologists more in

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terms of how sections are collected and stained and shared and so forth, we may be able to get more quantitative data out of those biopsies. I

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think the issue of how these techniques we’ve heard about, again, that the workshops hold up in pathology is an important point. We heard from

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Jennifer Weil today and her talk on the Pima project, the issue of cell identification and how that’s tough and as the pathology gets more and

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more advanced, this issue becomes more and more difficult. So, maybe some of these methods we’ve been talking about here some of them may

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do very well and be very useful there and others may start to fall down. The issue of specific markers and so forth for cells of glomeruli or

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whatever, again, may well change with disease. Our group was very interested in the emerging opportunities that we’ve heard about today to

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combine the structural and functional data with these new methods. The obvious one we heard today about doing the MR and getting the nephron

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count, getting the volume of every glomerulus; being able to combine that with estimates, non-invasive estimates, of GFR and even longitudinal

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studies is a very powerful new approach to be able to understand the functioning of the kidney. The point was also made that if glomerular

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hypertrophy is so important—I guess most of us assume it probably is because we spend a lot of time thinking about how to size glomeruli—then if

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we were able to identify the molecular markers that drive that hypertrophy—the growth factors, the cytokines, etc.—then to be able to measure

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those factors in blood or urine would be a big step forward. None of us assume that it’s going to be the same factors and the same mechanisms

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in every setting of glomerular hypertrophy; biology and medicine is never that simple. But as these drivers of hypertrophy emerge, we may

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well be able to measure those factors as well. There was a call for step-by-step protocols to be made available for these techniques to enable

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beginners or new users to get started, and rather then have to do a Ph.D. in stereology, if they got some easy step-by-step protocols they could get

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off to a quick start and hopefully avoid some of the pitfalls that come into these techniques and they come into all techniques, so why don’t we

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try to make it easier for new people? Also, there was a call for standardization of approaches and protocols. In terms of new approaches for

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measuring glomerular area and volume, that was our brief, in just a short amount of time six methods were mentioned, or five or six new

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methods were mentioned which made me think we could have probably kept going at this meeting for a little longer or do another one at

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some point in another way. Bob Bacallao told us about a method he’s developing and has had some experience with involving confocal

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microscopy where he’s taking kidney slices, he’s not embedding them in formalin or plastic or whatever, he’s simply immersing them in saline or

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a physiological solution, fluorescently labeling these slices with some of these markers here and then optical imaging through to a depth of

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about 150 micrometers to be able to measure some of the parameters we’ve been talking about. O course there’s a lot of interest in the MR

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work that Kevin Bennett and Norbert Gretz talked about. Kevin was in our group. He thought the biggest issue going forward will be the correcting

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for the RF artifacts, the radio frequency artifacts; the development of better coils, better surface coils. Perhaps he’s lucky enough to have a 19

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Tesla machine you saw was about the size of a small building but perhaps for many of our applications 3T or 7T will be adequate. Julie

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Ingelfinger reminded us of methods developed in the 70s or so using dextrans of different sizes and charges and so forth and sieving

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approaches to be able to estimate glomerular function, and I presume…I’m not an expert in this but perhaps the functional surface area or

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capacity of the filtration barrier and perhaps that should be something as we go forward with these technologies is maybe an approach that

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might be worth thinking about resurrecting in one form or another. Yu Chen told us about his work with optical coherence tomography; I hope this is

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the right words, I apologize for any mistakes. He’s using this approach in rats and in people as a clinical study in progress. He’s getting

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resolution of the 3-10 micrometer level, which is quite impressive, so I’m sure we’re going to hear a little bit more about that over the coming months

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and years. Ashraf El-Meanawy is using ultrasound to measure glomerular diameter in human kidneys and is about to launch a study or

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is involved in a study looking at glomerular size in control subjects versus subjects with glomerulomegaly. So there are a lot of new

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methods kind of in the pipeline, if you will. Some of them are already being applied to patients and the like, which hold great promise and who

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knows what we might be talking about in two or three years’ time? The point was made that, you know, with the techniques we’ve heard about in

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the last day but also with these new techniques coming along, that gives us even more meaning but also more opportunity to try to cross-validate

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these approaches and see how reproducible and similar they are in the kinds of data that, at the end of the day, they provide. I think the final task

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we were asked to do was look at opportunities and how using quantitative morphology might help us improve our understanding of disease. David

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Archer pointed out, and we’ve heard it plenty of times here at the workshop, we need something at least in the clinical setting that’s quick and

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effective. Time efficiency is paramount. We need a morphological marker, ideally, of progression or non-progression so we can start to do

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longitudinal studies and be able to measure things like glomerular drop-out; so, the safe non-invasive approach either in laboratory animals or

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in patients and so forth. The point was made “don’t forget the tubules and the interstitium,” which apparently are some part of the kidney as

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well. I guess we haven’t talked about them a lot here, but I was reminded of a paper I think it was in JASN last year by Inoue and colleagues who

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used MR to measure interstitial fibrosis in the kidney. I think that’s, again, something of great interest, so I recommend you have a look for that

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paper. Finally of course, there was a lot of interest in non-invasive quantification, quantitation of glomerular number and size in patients. This

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would assist us with issues of organ allocation, with risk stratification, being able to monitor disease progression, assess the effects of

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various treatments, assess glomerular endowment in premature kids and low birthweight babies and so forth and really give us

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a lot of new power and new opportunities in our research. So, that’s as far as we got. I hope that reflects what we talked out.

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KEVIN LEMLEY: I didn’t know I was the appointed speaker, so Laura’s getting…

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LAURA BARISONI: We didn’t decide.
KEVIN LEMLEY: Yeah, we didn’t decide. So the

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group, which was about eight of us, seven or eight of us, talked and discussed…primarily the focus that our group discussed was the

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challenges of the implementation of morphometric analysis within the context of clinical trial settings, and we spent actually far more time

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talking about the complexity of doing the clinical trials and collecting the pathology. This is something that has been an ongoing developing

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subject within NEPTUNE and is really the first large multi-center trial in nephrology to use virtual pathology or whole-slide imaging in this space.

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So, most of the discussion was the complexity and how one would implement that and some of the advantages of moving to an independent

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rather than consensus microscope review process. We also discussed the complexities of developing appropriate scoring systems that are

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piloted because, you know, you come in with a system and you need to test it a priori before you do this because it may not fit the data set that

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you’re collecting, either because the natural history of the disease is changing or the questions that you’re trying to evaluate are

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different. We also discussed slightly some of the technical challenges, especially multi-center environment, in collecting the imaging to make it

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adequate for morphometric analysis and other utilities, and these are ongoing developments.

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FEMALE: [inaudible question]
LAURA BARISONI: So, do we have it? So, we

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talked about the difficulties in the regulations, that’s more Steve’s job, and my part is actually to say why we use it and which are the

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advantages, and if you think about classic clinical trials as we normally do it, we have to ship the slides, we have to meet to have consensus

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meetings, pathologists look at the same slide, not necessarily the same structure, etc., and so I just want to show a couple of things that we have

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done. Okay. So basically, this is the diagram if we use the conventional light microscopy. The slide is stained with…the stain of the slide’s fate with

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time. We have description errors if we use [---] sheet. We have to look at multiple slides, multiple structures, etc. So, the possibility of error is high

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and these are our consensus meeting, but if we use the virtual microscopy methodology we can cut down on some of these issues and we have

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can two processes. We can have digital imaging created in a data center, so slides are indeed mailed but once and don’t’ go around the world

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between the different pathologists; or we can have local scanning. Of course, this brings up a lot of QC issues and regulations and protocols

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that need to be defined up front so we have a system that is consistent among biocenters if we use that. The other thing that virtual microscopy

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can allow you, because now we’re talking about imaging, we can select the structures that we want to score, for example, measure, whatever

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we want to do. So for example, NEPTUNE introduced the annotator step, so the annotator selects, for example, the glomeruli that need to be

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scored by the other pathologists and that decrees tremendously the intra- and inter-reader viability so there is a chance that there is more

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reproducibility of whatever we want to do. I want to go fast. These are the digital slides as they can appear to you and this is what we do when we

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do the annotation. Each glomerulus, for example, for NEPTUNE receives a number and the same number is maintained throughout all the sections,

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so glomerulus number one would be glomerulus number one throughout all the sections of the biopsy. That allows us to have a multi-level

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reconstruction of the glomerulus and have a better sense of what happens to that given glomerulus. Of course, we are working with

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renal biopsies, so we don’t have a complete 3D reconstruction, but as in this example, we can see how we can follow what happens to any

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given annotated glomerulus. Multiple pathologists or multiple investigators can view the digital images at the same time on the phone and

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discuss each of these features in a consensus meeting or whatever it is. I don’t want to go into this core. I have a disclosure. I am consulting with

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Amicus and Protalix for Fabry disease and digital pathology, but this is the summary of the application of the system with advantages. So,

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multiple users; you can avoid mailing system; you can create a library of the images that can be maintained permanently with the data linked to it

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permanently; full transparency for regulatory agencies to think about a clinical trial for drug development; same slides can be used for

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multiple studies simultaneously and for future studies; the cost becomes low in the long time, the more you use it, of course; you can annotate

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the slides; you can have multi-level construction of renal biopsies; you can do morphometric analysis of certain structures, at least; and you

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can measure the density of certain structures in the renal parameter such as the case of peritubular capilliaries, and this is where my

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disclosure comes. For example, here we counted 300 capillaries per biopsy and you can do that in a precise way only if you use digital pathology

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because if you’re trying to do that using light microscopy you end up counting the same capillary twice, you get interrupted, you have to

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start again, etc. So, we mark each capillary and we can count the number of GL3 inclusion per endothelial cells in this specific example and you

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can see that appears as a number next to the arrow that identifies the capillary. This is a specific study for Fabry disease, but if you think

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about reproducibility of these data, of course now we’re looking at the same exact capillaries. So, two pathologists can disagree on a certain

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number of those but at least they are the same, and you can do a reconciliation process just pulling out those capillaries you disagree upon

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and you don’t have to review the entire biopsy, recount 300 capillaries as you would do by light microscopy, and we have shown that there is an

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increased reproducibility if you use virtual microscopy versus light microscopy using this scoring system. So, the applications are multiple

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and we are just at the beginning. I don’t know of any other studies on renal biopsy beside the Fabry and the NEPTUNE, but certainly there are

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advantages and the key, as Steve pointed out, is just very rigorous protocols, a lot of training, that is critical, and training of multiple personnel, from

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the study coordinator to the pathologist to all the elements that are involved in this process. I don’t know, Steve, if you want to add anything.

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That’s it.
BEHZAD NAJAFIAN: So, I just summarized the

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highlights of our breakout session on peritubular capillary estimations on behalf of Agnes Fogo and Mike Mauer, who left earlier, so I apologize. I don’t

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have any slides to show; that was not my plan to summarize this session. So, basically we discussed different parameters that can be

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quantitated in peritubular capillaries, including length, density, volume density, surface density, per volume or surface density per surface of

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tubules, and number densities. These are different parameters that can be estimated. The importance of appropriate sampling was

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discussed in that we need to do a systematic uniform random sampling in order to reduce the variability, as it was discussed in other sessions

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during today and yesterday. Different information we can obtain by looking at the light microscopy and electron microscopy samples. By electron

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microscopy, Mike Mauer discussed the breakdown of different components of interstitium, including the volume fraction of the

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capillaries, the surface of those, and how much of the interstitium is composed of collagen fibers and a space. In light microscopy, Agnes Fogo

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discussed the huge amount of variability that we can get in relation to interstitial fibrosis and also peritubular capillary densities. Therefore, it will be

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very important to define which areas we are interested in for quantitation of peritubular capillaries in interstitial fibrosis. Do we want to

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include areas with scarring or do we want to avoid areas with scarring where we are interested to obtain an average of those areas?

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Combination of electron microscopy and light microscopy findings can be useful, however, the pitfall would be that the areas that we choose for

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electron microscopy studies should correspond to the same areas in light microscopy if we want to correlate those findings together. Adding of

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immunohistochemical studies in order to visualize peritubular capillaries for quantitation, such as CD31 or elected markers was discussed that

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would facilitate quantitation, as well as using cell markers to study other cells that increase in the interstitium—inflammatory cells, fibroblasts, or

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pericytes—will be also informative. We do not have much information about importance of peritubular capillaries in relation to progression of

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the disease. Performing longitudinal studies would be especially helpful to see if a reduction in peritubular capillary density comes first or just

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accompanies fibrosis. One of the important challenges, of course, because of the variability, would be that basically we don’t know what is

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going on by looking at the biopsy; we may not necessarily come to a conclusion about what is going on in the whole kidney. So, a reference trap

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will be a very important issue in studying the peritubular capillaries and therefore non-invasive methods to basically study the vasculature of the

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kidney hypoxia would be very important. Jeff Kopp mentioned about the usefulness, potential usefulness, of markers of hypoxia in the biopsies

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that together, with the quantitation of peritubular capillaries, could provide important information on which one comes first in areas that there is not

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significant scarring. The importance to do additional studies in clinical trials, doing clinical studies in areas that basically contribute a lot in

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end-stage kidney disease, diabetic nephropathy, especially Type II, hypertensive nephrosclerosis and focal and segmental glomerular sclerosis, the

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importance of APO1, those were discussed and the limitations that we have so far for the availability of the biopsies for those clinical trials

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and also the limitations that we’re facing for availability of control tissue. So, the importance of doing collaborative studies, sharing the biopsy

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materials, and the control material was discussed in our session. Also, the differences between the autopsy material and biopsies obtained from living

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donors or living controls was discussed. Some of the parameters, perhaps, can be studied, can be learned, from looking at the autopsy material such

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as glomerular scarring, interstitial fibrosis, maybe those things, while some other parameters could be significant or different. So depending on the

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study, those materials become handy or there might be more limitations. That was basically the wrap-up of what we discussed. Any questions

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or comments?

Date Last Updated: 10/5/2012

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