NEW - Impressions from the 38th Congress of the German Society of Nephrology

Pathogenesis of FSGS – From Mouse to Human
Two Examples of How Patients Benefit from the Laboratory Mouse

Animal models have become indispensable both as experimental tools to study the pathophysiology of many human diseases, and for the generation of novel therapeutics.  Mice are particular suitable for these purposes mainly for two reasons: firstly, they are a mammalian model system sharing many similarities with the human both on the genome level, i.e. the DNA constituting the genetic code, and on the physiological level, i.e. how tissues and organs are organized to maintain the function of the whole organism.  Second, they are fairly easy to handle and match the practical requirements in daily laboratory practice: small size, high reproduction, and reasonable cost.

    In particular when it comes to studying the function of a particular gene, mouse models can be extremely useful.  The experimental approach of generating so-called transgenic mice has become a standard tool in biomedical research at large.  For example, there are established protocols which enable researchers to "knock-out" a single gene in mice, thereby creating knock-out mice, which are deficient of the corresponding protein.  By observing the effects in these mice that are caused by the knock-out, one is then able to conclude for the possible function of the lacking protein.

    A well known example for such a model are CD2AP-knockout mice.  These animals are missing the CD2AP gene encoding for the CD2-associating protein, CD2AP.  CD2AP’s general function is the one of an adapter, interconnecting different structures within a cell.  While CD2AP can be found in many cell types throughout the body, it appears to play a very distinct role in kidney podocytes, which represent the cell type most commonly affected in kidney diseases associated with proteinuria. While CD2AP-deficient mice develop severe glomerular kidney disease associated with profound podocyte damage resembling the human condition of focal segmental glomerulosclerosis (FSGS), other organs appear to be unaffected by the loss of CD2AP.  This observation in the mouse model, published in 1999 in Science, and numerous published studies since have greatly advanced our understanding of the role of CD2AP at the glomerular filtration barrier.  It is now generally accepted that in podocytes, CD2AP plays an important role in connecting the slit diaphragm to the podocyte actin cytoskeleton.

    Published findings relating CD2AP to glomerular disease in the mouse prompted researchers to look for mutations associated with a dysfunctional CD2AP protein in the human CD2AP gene in patients with FSGS.  Recently, a novel case of a patient carrying such a mutation was presented by Lowik and coworkers.  Interestingly, the identified mutation in this individual resulted in only a marginal alteration of the normal CD2AP protein in that the mutated variant was 4% shorter.  Yet, this marginal differences is apparently sufficient to cause FSGS, subtantiating the essential role of CD2AP in podocytes and for the maintenance of normal glomerular structure and kidney function.  The authors of this study also provided a possible explanation for the pathogenic effect of the mutated, short CD2AP; the latter appears to have problems binding actin, which is the main constituent of the podocyte actin cytoskeleton.  The compromised binding of the mutated CD2AP protein may compromise its fundamental function as a cytoskeletal adapterand thereby cause the disease.  In summary, the original discoveries in the mouse ultimately led to a refined understanding of FSGS pathogenesis.

Lowik MM, Groenen PJTA, Pronk I, Lilien MR, Goldschmeding R, Dijkman HB, Levtchenko EN, Monnens LA, van de Heuvel LP | Department of Pediatric Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands | Focal segmental glomerulosclerosis in a patient homozygous for a CD2AP mutation. | Kidney Int. 2007, 72:1198-1203.

    Most nephrotic syndrome patients of young age have kidney biopsy findings consistent with the diagnosis of minimal change disease.  Typically, these patients will have remission of clinical symptoms after treatment with high doses of the steroid prednisone and they rarely develop progressive disease that leads to end-stage renal disease (ESRD).  Because they respond to treatment with steroids, these patients are referred to as steroid-sensitive.  In contrast, a subgroup of children and young adults with nephrotic syndrome suffer from steroid-resistant nephrotic syndrome and their kidney biopsies show evidence of focal segmental glomerulosclerosis (FSGS).  Patients with this condition do not respond to treatment with the steroid prednisone.  In consequence, the long-term prognosis for this steroid-resistant subgroup is poor because no satisfactory treatment exists for their condition.

    Among the therapeutics that have recently received much attention with respect to their use in patients with nephrotic syndrome is the drug rituximab.  Rituximab is a genetically engineered monoclonal antibody, which is derived from genetically engineered mouse spleen cells, but can be administered to humans as a therapeutic.  There are several studies linking rituximab to a remission of proteinuria in patients with steroid-sensitive nephrotic syndrome and recurrent FSGS after transplantation. 

    A novel study now shows a beneficial effect for rituximab also in children with steroid-resistant nephrotic syndrome.  Five children ages 1 to 4, all treated unsucessfully with steroids previously, were administered rituximab by intravenous infusion at a dose of 375 mg per square meter of body-surface area once weekly for 4 weeks.  About 4 weeks after the last dose had been administered, four patients had a complete remission of proteinuria while one patient had a partial remission. 

    Obviously, a study involving only five patients alone does not allow conclusive results and cannot serve as a recommendation guideline for the treatment of steroid-resistant nephrotic syndrome at large.  Yet, the present results are very encouraging, and certainly may give rise to larger scale clinical trials validating these preliminary findings.  Likewise, it will be necessary to carefully evaluate the side-effects of rituximab treatment in children with steroid-resistant nephrotic syndrome before considering this treatment as an alternative approach to the current therapeutic regimens.

    In summary, this promising discovery illustrates the increasing significance of protein drugs such as monoclonal antibodies produced in mice for the treatment of human diseases.  The present advances are the result of more than two decades of research with the mouse model. These early benefits of the mouse model are but a glimpse of an expected panorama of future sucesses.

Bagga A, Sinha A, Moudgil A | All India Institute of Medical Sciences, New Delhi, India | Rituximab in Patients with the Steroid-Resistant Nephrotic Syndrome | N Engl J Med. 2007, 356:2751-2752.

NEW - Impressions from the 40th ASN Renal Week Annual Meeting and Scientific Exposition

Double Kidney Trouble
ASN Projections: End-Stage Renal Failure on the Rise

The American Society of Nephrology (ASN) recently presented an updated projection for the expected number of U.S. patients with end-stage renal disease (ESRD)  between 2005 and 2020.  ASN estimates that the number of patients with chronic kidney disease will double until 2020.  The annual increase of patients with ESRD will surpass 150,000, meaning that by 2020 around 785,000 individuals will depend on dialysis. 

    The age group 65 to  75 seems particularly affected by this trend.  Whereas decreasing kidney function due to aging partly accounts for this observation,  the alarming development appears to be the large increase of the so-called diseases of civilization including high blood pressure and type II-diabetes.  Twenty percent of all patients with high blood pressure die from kidney disease, and 40% of patients with type II-diabetes are diagnosed with diabetic nephropathy, a highly morbid state frequently resulting in ESRD.

And a Doubled War Chest to Carry the Fight
National Kidney Foundation (NKF) to Double Research  Funding

To meet the rising demand for novel cures targeting chronic kidney disease, the National Kidney Foundation (NKF) recently announced to double its funding of research grants and fellowships to promising young scientists over the next five years.  Beginning in 2008, the research budget of $2,800,000 will increase in five yearly increments until it reaches $5,600,000 annually.  Top areas of research focus include chronic kidney disease, hemodialysis, high blood pressure, cardiovascular disease, and pediatric kidney disease.

Two Podocyte Proteins and a European FSGS-Study
A View on This Year’s Outstanding Presentations

From the more than 4,600 abstracts submitted to this year’s ASN Renal Week meeting the organizing committee selected eight studies for a special communication session. Three dealt with the pathophysiology of the glomerular filtration barrier.

    The first study revealed that the Vascular Endothelial Growth Factor A (VEGF-A) is highly expressed in podocytes, which are a vital element of the glomerular filtration barrier.  VEGF-A belongs to an class of proteins known as signaling proteins, i.e. proteins enabling cells and tissues to talk to each other on a molecular level.  VEFG-A specifically plays an important role in promoting the growth of new blood vessels, a process referred to as angiogenesis, which is a deleterious event in cancer since it makes tumors grow and metastasize. 

    Treatment of cancer with drugs inhibiting VEGF-A is associated with the development of proteinuria in 10 to 30% of cancer patients. This is why the authors of this study decided to look for a role of VEGF in podocytes,  the cell type most often damaged in diseases associated with proteinuria,  including focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome.  It was shown that mice which are lacking VEGF in their podocytes all develop glomerular injury progressing to end-stage renal disease (ESRD).  This demonstrates that VEGF-A is required for maintaining the process of glomerular filtration, and explains why cancer therapy involving inhibition of VEGF-A is commonly associated with glomerular disease.

Eremina V, Jothy S, Kopp J, Ferrara N, Gerber H, Kabir G, Backs P, Quaggin SE | The Mount Sinai Hospital, Toronto, Ontario, Canada | Local VEGF-A production is required for the filtering glomerulus | Abstract presented at the 38th ASN Renal Week Annual Meeting and Scientific Exposition, October 31- November 5, 2007, San Francisco, California, USA.

    The second study showed that a novel podocyte protein called podoplextrin is required for the organization of podocyte foot processes and normal kidney function in zebrafish.  The zebrafish, danio rerio, is an emerging animal model used for the study of kidney development and disease.  One of the experimental advantages that this model offers is that kidney function can be studied by injecting variants of the sugar molecular dextran into the fish, which vary in size.  If kidney filtration is normal, only very small dextran molecules can pass through the glomerular filtration barrier, whereas larger dextran molecules are retained in the bloodstream and consequently cannot be detected in the fish renal tubules.  In contrast, during glomerular injury the glomerular filtration barrier gets leaky, allowing variants of dextran that are larger in size to pass through the glomerular filtration barrier and be detected in the primary filtrate filling the renal tubules. 

    Using this dextran injection approach, Mataleena Parikka and coworkers were able to show that when podoplextrin function was disrupted in zebrafish larvae these fish developed proteinuria and renal failure, revealed by the passage of large dextran molecules through the glomerular filter to the renal tubules.  When the authors explored the structure of glomeruli in these fish under the electron microscope they found the structural organization of the glomeruli severely compromised with heavy podocyte foot process effacement.  In summary, these results point to an essential role of the podoplextrin protein in the organization of the glomerular filtration barrier.

Parikka M, Nishibori Y, Hultenby K, Oddsson A, Ebarasi L, Wernerson A, Perisic L, Patrakka J, Takemoto M, Uhlen M, Betsholtz C, Majumdar A, Tryggvason K | Karolinska Institute, Stockholm, Sweden | A novel podocyte protein, podoplextrin, is required for the organization of foot processes and normal kidney function in zebrafish | Abstract presented at the 38th ASN Renal Week Annual Meeting and Scientific Exposition, October 31- November 5, 2007, San Francisco, California, USA.

    The third abstract presented the results from the European Collaborative FSGS Transplantation Study (ECoFTS) examining the pathogenesis of focal segmental glomerulosclerosis (FSGS), especially with regards to the therapy after transplantation.  While generally seen as a rare disorder, FSGS represents the third most frequent cause for end-stage renal disease (ESRD) in children. 

    The primary purpose of this multicentric clinical study with hospitals participating in Austria, Germany, France, Switzerland, Norway, the Czech Republic, and the U.S., was to investigate the causal relationship between FSGS-causing genetic mutations, course of the disease, and relapse after transplantation.  Currently, more than 80 patients have been enrolled in the study, 60 of whom have already been genetically tested for mutations in either of the two known FSGS-causing genes, NPHS2 encoding the protein podocin (53 patients genetically tested), or TRPC6 encoding the transient receptor potential canonical 6 (TRPC6) ion channel (7 patients tested).  None of the children with a mutation in the NPHS2 gene developed a relapse of FSGS in the transplated kidney, whereas around 40% of children without mutations in NPHS2 did so.  None of the 7 patients tested for mutations in the TRPC6 gene was carrying a mutation, allowing no conclusions about the role of TRPC6 in the relapse of FSGS at this point.

    This finding has a great significance for the clinical practice, perhaps suggesting that all children with FSGS awaiting a kidney transplant should be genetically screened for mutations in the NPHS2 gene in order to determine the best therapy post transplantation. 

    A secondary objective of the ECoFTS study, which is coordinated by Professor Dr. Lothar Zimmerhackl from the University Hospital in Innsbruck, Austria, will be to implement an electronic database that will enable tracking the course of disease in patients with FSGS throughout Europe.

    More information on the European Collaborative FSGS Transplantation Study can be found on the ECoFTS website under http://ecofts.uki.at .

Jungraithmayr TC, Cochat P, Fargue S, Hofer K, Knueppel T, Cortina G, Neuhaus T, Seeman T, Toenshoff B, Winn MP, Zimmerhackl LB | Medical University, Innsbruck, Austria | Role of NPHS2 and TRPC6 in recurring focal segmental glomerulosclerosis (FSGS): Results from the Ecofts (European Collaborative FSGS Transplantation Study) | Abstract presented at the 38th ASN Renal Week Annual Meeting and Scientific Exposition, October 31- November 5, 2007, San Francisco, California, USA.

Regeneration of the Kidney Filter
Mission Impossible?

Podocytes are part of the glomerular filtration barrier. They are located on top of the glomerular basement membrane on the outer surface of the blood vessels in the glomerulus. Podocyte injury can eventually lead to the detachment of podocytes from the glomerular blood vessels.  The damage and loss of single podocytes in increasing numbers over time leads to podocyte depletion, which is a major factor in the development of FSGS and chronic kidney failure.

Unlike other cell types in the body, which can be routinely replaced by the body, e.g. skin cells or bowel epithelial cells, the highly-specialized podocytes have no known regeneration mechanism.  It is commonly believed that these cells cannot be readily regenerated, which makes their damage or loss so devastating.

The irreversible consequences of long-term podocyte injury have long prompted researchers to
seek possible mechanisms for podocyte  regeneration in the glomerulus.  At the 2007 World Congress of Nephrology from April 21-25 in Rio de Janeiro, Brazil, two researchers presented interesting novel concepts of how podocytes might be regenerated in vivo, using mice as an animal models.

Raghu Kalluri, Associate Professor at Harvard Medical School in Boston, Massachusetts, illustrated that stem cells may hold the potential to replace podocytes lost due to disease.  Stem cells represent a unique type of cells that can self-renew and furthermore have the ability to differentiate into any other cell type.  When Kalluri cultured such cells in the laboratory, growing them under conditions similar to those in the glomerulus, he found that they started to show typical podocyte features, that is, the expression of certain slit diaphragm proteins.  Moreover, when stem cells were injected into mice carrying a known genetic defect that causes them to suffer from severe foot process effacement and proteinuria, a portion of these stem cells apparently differentiated to become new podocytes.  Furthermore, the kidney function in the injected mice significantly improved, lessening proteinuria and improving the survival rate.  While these studies still are in early stages, they provide notable evidence for the long-term potential of stem cell therapy in the glomerulus.

The group of Marcus J. Moeller from Aachen, Germany, proposed an entirely different mechanism of how podocytes might be replaced.  In contrast to the previous approach requiring the injection of stem cells into mice, they provide evidence for a mechanism that might naturally occur in the glomerulus without treatment from the outside.  Specifically, Moeller and his colleagues demonstrated that so-called parietal epithelial cells were able to regenerate podocytes in mice.  In addition to podocytes, endothelial and mesangial cells, the parietal epithelial cells represent another cell type in the glomerulus.  They are thin cells located on the inner surface of Bowman’s Capsule.  Because these cells exhibit certain features of podocytes, it has been long hypothesized that parietal epithelial cells and podocytes might actually be very similar. Moreover, it is been hypothesized that, like the aforementioned stem cells, the parietal epithelial cells might even be able to differentiate into podocytes, Moeller and his group tested this hypothesis by labelling the parietal epithelial cells in a trangenic mouse model specifically generated for this task.  When these labelled parietal epithelial cells were observed over time, it became evident that in some cases these cells migrated from their original location at the inner surface of Bowman’s Capsule onto the surface of the glomerular basement membrane, where podocytes usually reside.  Morever, they changed their shape, differentiating from thin cells with almost no cell body to branched cells resembling normal podocytes.

The discovery that parietal epithelial cells are able to move to the glomerular filtration barrier, potentially assuming the role of podocytes, supports the long-term possibility of therapies leading to the regeneration of podocytes. This could bcome a valuable weapon against kidney diseases such as FSGS that are characterized by a damage and loss of podocytes.

Exploring the Kidney on the Grand Scale
Identification of 300 Novel Proteins Specific for the Glomerulus

Until only years ago, the molecular nature of the kidney filtration system was unknown and our understanding of the proteins making up the glomerular filtration barrier was very limited. Since then, researchers have been able to identify several components of the kidney filter such as the nephrin protein, which is a key element of the slit diaphragm.

Despite the progress that has been made, many aspects of the glomerulus and particularly the development of different types of kidney diseases are still poorly understood. A new project pursued by Karl Tryggvason from the Karolinska Institute in Stockholm, Sweden, and his coworkers now aims to providing new information into the biology and pathology of the renal glomerulus. This is an extensive project on the functional genomics of the renal glomerulus and its diseases.

So far, over 300 novel proteins highly specific for the glomerulus have been identified. This pool of previously unknown proteins will likely lead to the discovery of new important players in kidney function. In the long-term, these new proteins might be targeted for drug therapy when the glomerulus malfunctions. The next immediate step will be the systematic characterization of the 300 new proteins to determine what their function in the kidney is.

Among the tools created by Tryggvason and his collaborators are transgenic knock-out mice. These animals are produced so they lack one of the 300 discovered genes, which leads to the absence of the corresponding protein in these mice. Studying the effect of this loss of a specific protein may lead to conclusions about the function of this protein in a healthy animal. Researchers also are generating antibodies against each of the discovered proteins, which will make it easier to detect the exact locations of these proteins in the glomerulus.

Exploring the kidney glomerulus systematically on a large scale will likely lead to extensive new knowledge on the kidney filter system and its diseases. The envisioned studies may serve as an important basis for the development on diagnostics and drug development.

Some patients with FSGS suffer from recurrence of proteinuria and podocyte damage within 24 hours or less after kidney transplant. It has been suggested that this is due to the presence of one or more “factors” in the blood that are able to directly affect the integrity of the glomerular filtration barrier. The suspicion is that once the new, healthy transplant is exposed to the recipient’s blood, some type of “FSGS factor” circulating in the bloodstream of the recipient rapidly causes the development of FSGS in the new transplant.

The identification of this circulating factor – or the group of factors – is a doggedly persistent theory in Nephrology that puzzles clinicians and researchers alike. The factor, if it exists. Is a complete unknown. Scientists can only guess to which class of substances it may belong, that is, whether it is a protein, an antibody, a lipid, or something else.

Currently, researchers carry out several lines of investigation to better understand the mystery of the FSGS factor. Most of them are based on either of the following assumptions: The first assumption is that “normal” plasma (the blood portion that carries cells) contains the components which are necessary for regulated podocyte signals. In the affected patients, these components would be missing and that is what would cause the recurrence of FSGS upon a transplant. The second assumption is that “nephrotic” plasma from patients with recurrent FSGS possesses a component which can directly result in podocyte damage and thus proteinuria.

In line with the first hypothesis, novel data presented at the 8th International Podocyte Congress in Helsinki, Finland, suggests that normal plasma is necessary for deriving key signals from the slit diaphragm into the podocyte, and that these signals are lacking in patients with recurrent FSGS.

While the condition of recurrent FSGS may be treated with various medicines including steroids, cyclosporine and cyclophosphamide, the search for the mysterious circulating factor(s) constitutes an important area of research, bearing the potential of a better understanding of FSGS and new drug therapy.

The glomerular epithelial cells, or podocytes, are unique cells with an unusual octopus-like structure and a characteristic inner structure, or actin cytoskeleton. The layer formed by podocytes and the unique junctional slit diaphragm complexes between them represent an important part of the glomerular filtration barrier. In detail, it is thought that the network of podocytes and the interposed slit diaphragms contributes to the charge and size selectivity of the glomerular filtration barrier, meaning that a molecule can only penetrate the kidney filter when it meets certain criteria with regard to electrical charge and size. For example, molecules that are positively charged may pass the filter, while negatively charged molecules cannot pass and are retained. Likewise, small proteins may pass the filter, while proteins larger than albumin cannot pass and are retained.

Until fairly recently, the small size, location, structure, and high degree of specialization of podocytes hampered the comprehensive analysis of this cell type. For example, it was not possible to explain the morphology of these cells and to discover the existence of foot processes before electron microscopy became available – foot processes are very small structures that require the high magnification of an electron microscope to be visualized. Yet, in the past decade, there has been considerable progress in understanding the biology of these cells and their contribution to human disease. For example, cultured podocytes, i.e. cells that have first been extracted from mouse or human glomeruli and then are grown and maintained in cell culture dishes in the laboratory, have become available. These podocytes behave similarly to podocytes in an actual kidney, and are a very useful tool to better study this cell type in the laboratory. Many laboratories around the world now work with such rodent and human podocyte cell lines.

Subsequently, several research groups demonstrated that mutations in slit diaphragm protein genes led to disease phenotypes in humans and animal models, some leading to severe, nephrosis, others to slowly progressive proteinuria and renal insufficiency.

Since then, the podocyte has attracted considerable attention but many aspects of the biology of these cells remain puzzling. There has been substantial progress in understanding patterns of podocyte damage and foot process effacement and the now well-accepted notion that glomerular function fails without functional podocytes. But scientists have found no clearly defined common pathway leading to podocyte failure. Descriptions of histopathological observations, i.e., changes in the appearance of kidney tissue obtained by a kidney biopsy, therefore remain the standard method for classifying “podocytopathies,” as opposed to describing altered genetic and biochemical pathways

Nevertheless, identification of inherited gene defects affecting podocyte structure and function have proven helpful in understanding podocyte function as well as podocyte disease. The growing interest in podocyte research is also displayed in the amount of abstracts and presentations at national and international meetings. Over the past decade, the number of podocyte related topics has increased by around 10-fold. Many molecular pathways have been unraveled which carry possibilities for renal drug development. We may therefore see novel drugs approaching clinical trials in the near future. Since so far there is no pharmacologic substance available that specifically targets the podocyte, the development of podocyte-specific drug would be a major milestone in the treatment of podocyte-related kidney disease.