THE NEPHCURE FOUNDATION

Proteinuria is often the first can progress to nephrotic syndrome and focal segmental glomeruloslerosis (FSGS) and result in loss of kidney function. Proteinuria is also a predictor of poorer outcomes and faster progression of renal insufficiency. In an attempt to slow down the progression of renal disease, the mainstay of therapy is blockade of the renin-angiotensin system. Other treatments, including immunomodulators such as steroids, cyclosporine and alkylating agents, are based on the individual clinical and histopathological diagnosis of a patient. Therapeutic intervention for proteinuric kidney diseases is limited by at least two critical issues: first, interventions are not curative and merely aimed to slow down the progression of glomerular disease; second, cell-specific therapies for proteinuria syndromes are presently not available.

In a recent article by Wei et al., it is shown that the urokinase receptor (uPAR), a molecule which is well known in the field of cancer biology, plays an unexpected role in the development of podocyte failure and proteinuria. Whereas the absence of uPAR in the kidney does not cause any immediate damage to podocytes, the authors present data demonstrating that uPAR induction in podocytes causes activation of alphavbeta3 integrin. The activation of this protein leads to increased podocyte motility, i.e. the ability of podocytes to move from A to B, foot process effacement and proteinuria. The blockade of alphavbeta3 integrin activation by a blocking antibody or by the small molecule cyclo-RGDfV inhibits the development of proteinuria in a mouse model. Moreover, already established proteinuria can be effectively resolved using cyclo-RGDfV injection. This compound, also called Cilengitide, is in phase II clinical trials for brain cancer.

A potential future clinical trial with Cilengitide in patients with proteinuria is a possible next step to study the effects of alphavbeta3 inhibition in humans. The study by Wei et al. serves a proof of principle study for targeting specific molecular pathways in podocytes to tackle nephrotic syndrome.

Oxygen Shortage Linked to Scarring

Novel roles for Hif-1 in promoting fibrosis of the kidney

Hypoxia, the medical term for a shortage of oxygen in the body, has been proposed as an important microenvironmental factor in the development of tissue scarring (fibrosis), however, the underlying mechanisms are not well defined. Furthermore, it has been shown that signaling through the hypoxia-inducible factor 1 (HIF-1) protein can have implications regarding kidney function.

The group of Volker Haase now shows that hypoxia and signaling through HIF-1 contribute to the development of interstitial fibrosis, a condition accompanying many cases of focal segmental glomerulosclerosis (FSGS) and nephrotic syndrome. These conditions can develop via the induction of a set of genes regulating the extracellular matrix including a gene encoding for lysyl oxidase, which is an important extracellular enzyme. All together, these changes triggered by HIF-1 lead to a characteristic transformation of certain kidney cells, called epithelial–to–mesenchymal transition (EMT), a process that leads to scarring and that thereby damages the kidney.

The presented findings highlight a need for careful evaluation of pharmacologic strategies that target molecular pathways involving the HIF-1 protein. This discovery also has clinical implications, as it strongly encourages therapies that aim at improving the balance between renal oxygen delivery and consumption to halt the progression of kidney scarring (fibrosis). Because the authors show that a lack of oxygen in the kidney leads to scarring of kidney tissue, delivering more oxygen to the kidney may be a way to lessen the outcome of kidney scarring.

Higgins DF, Kimura K, Bernhardt WM, Shrimanker N, Akai Y, Hohenstein B, Saito Y, Johnson RS, Kretzler M, Cohen CD, Eckhardt K, Iwano M, and Haase VH | Department of Medicine, Renal Electrolyte and Hypertension Division, University of Pennsylvania, Philadelphia, Pennsylvania, USA | Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition | J Clin Invest. 2007, 117:3810-3820.

Actin: Key to Filter Structure

Two Studies Reveal Novel Insights on the Regulation of Podocyte Structure

The glomerular slit diaphragm fills the space between neighboring podocyte foot processes and is thought to mainly contribute to the size-selectivity of the glomerular filtration barrier, i.e. the the ability of the glomerular filtration barrier to filter by size of a molecule. While long considered as merely serving as a molecular sieve, a number of studies now illustrate that the slit diapragm also serves as an important signaling platform, mediating signals coming from the environment surrounding the podocytes into the podocyte cell body. For example, it has been hypothesized that changes in the microenvironment of podocytes can trigger molecular pathways inside the cell leading to characteristic changes. The role of the slit diaphragm in this model would be to sense the changes in the microenvironment and together with other proteins at the slit diaphragm translate them in a way so that the molecular machinery inside the cells know how to adapt to the changes happening in the outside. Indeed, it has been shown previously that the integral slit diaphragm protein nephrin, which plays a key role during filtration, can mediate such signaling events at the slit diaphragm: nephrin interacts with Fyn, which is a protein that can chemically modify other proteins in a very specific fashion during a process that is called tyrosine phosphorylation. This event is a signature event frequently occuring in a number of signaling processes in most cell types. In podocytes, it has been shown previously that tyrosine phosphorylation of nephrin by Fyn triggers a cascade of subsequent intracellular events that eventually result in a characteristic reorganization of the podocyte actin cytoskeleton.

A recent study conducted by the group of Larry Holzman at the University of Michigan Medical School now further supports the idea of the slit diaphragm serving as a signaling complex. The study involves neph1, which is a nephrin homologue, i.e. a protein belonging to the same class of proteins like nephrin. And just like nephrin, neph1 can be modified by tyrosine phosphorylation through the Fyn kinase, triggering similar events in podocytes as have been observed upon phosphorylation of nephrin in the previous studies. While being similar in nature, both processes of nephrin tyrosine phosphorylation and neph1 tyrosine phosphorylation appear to serve different roles in podocytes since the signaling cascades occuring downstream of tyrosine phosphorylation involve different molecules. For example, tyrosine phosphorylation of nephrin results in the recruitment of the protein Nck to the slit diaphragm, whereas tyrosine phosphorylation of neph1 results in the recruitment of the Nck homologue Grb2 instead.

Consequently, the authors suggest the presence of a carefully orchestrated signaling complex at the slit diaphragm including nephrin, neph1, Fyn, and other proteins. The authors show that an important function of this complex is the regulation of the highly dynamic podocyte actin cytoskeleton, which is the main structure enabling podocytes to react to changes in their microenvironment such as alterations in glomerular blood pressure or blood composition.

Garg P, Verma R, Nihalani D, Johnstone DB, Holzman LB | Division of Nephrology, University of Michigan Medical School, Ann Arbor, Michigan, USA | Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization | Mol Cell Biol. 2007, 27:8698-8712.

Mutations in the ACTN4 gene encoding the protein a-actinin-4 were among the first ones to be associated with late-onset focal segmental glomerulosclerosis (FSGS). a-actinin-4 is a regulator of the cellular actin cytoskeleton, whose maintenance is of particular significance in kidney podocytes. The molecular scaffold providing the structural stability of podocyte foot processes is mainly made up by highly organized polymers of a protein called actin. A single actin subunit is the basic element that forms higher-order structures by association with other actin subunits in a process called actin polymerizaion. Among the most prominent higher-order structures in podocyte foot process are stress fibers, relatively thick bundles of actin that serve as a main backbone and can be considered the middle axis of podocyte foot processes, similar to what would be the spine in the human body. In contrast, the so-called cortical actin cytoskeleton consists of less organized, shorter actin polymers that like a rigid wallpaper decorate the inner surface of podocyte foot processes. Both actin stress fibers, and the cortical actin cytoskeleton, contribute to the structural integrity of podocyte foot processes, and abnormalities in these structures are typical companions of both acute and chronic podocyte injury.

Since the originally described role of a-actinin-4 is to mediate the tight regulation of the actin cytoskeleton by serving as a crosslinker of actin-based structures, it comes to no surprise that mutations leading to an abnormal a-actinin-4 protein can affect the actin cytoskeleton. The observation that the primary outcome of a-actinin-4 mutations is failure of podocytes, eventually resulting in podocyte foot process effacement and FSGS, underscores that in podocytes the actin cytoskeleton plays a unique role that is likely much different from its role in any other cell type.

To date, five distinct mutations in ACTN4 have been identified. They share the common feature of an increased binding of a-actinin-4 to actin subunits. To determine a molecular mechanism by which this biochemical property can translate into the onset of FSGS, Astrid Weins from the Brigham and Women’s Hospital and Harvard Medical School and her coworkers initiated a number of in vitro and in vivo studies looking in more detail at one of the five mutants. Their data show that, as a direct result of the increasing actin-binding affinity of the mutant a-actinin-4, aggregates of actin form in podocytes of humans with ACTN4-related glomerular disease, and in a mouse model thereof. The authors were further able to provide an explanation for the increased actin-binding capacity of mutated a-actinin-4 by resolving the three-dimensional structures of normal and mutant a-actinin-4: the mutation in a-actinin-4 leads to a characteristic change in its three-dimensional structure, exposing an additional, high-affinity actin-binding motif that is not accessible in the normal a-actinin-4. Consequently, when this binding site was inactivated in the mutant a-actinin-4 protein, the mutant again essentially behaved just like the normal a-actinin-4.

This study is a prime example for the elucidation of molecular pathways associated with the pathogenesis in inherited forms of FSGS and nephrotic syndrome, in which mutations in important podocyte proteins are the culprits. The presented findings refine our understanding of the role of a-actinin-4 for podocyte cytoskeletal organization, and suggest that changes in the three-dimensional arrangement of podocytes may play an important regulatory role in health and disease.

Weins A, Schlondorff JS, Nakamura F, Denker BM, Hartwig JH, Stossel TP, Pollak MR | Renal and Translational Medicine Divisions, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA | Disease-associated mutant a-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity | Proc Natl Acad Sci U S A. 2007, 104:16080-16085.

Fighting Cell Death in the Kidney

A Cell Survival Factor and a Common Antibiotic Protect Against Renal Damage

A novel study by Isermann and coworkers demonstrates that one of the crucial signaling pathways in podocytes, which represent a vital component of the glomerular filtration barrier, is suppressed in patients with diabetes, because in the state of hyperglycemia (overload of glucose in the blood circulation), one of the key proteins for cell survival, called Protein C, is not sufficiently produced. The lack of Protein C subsequently leads to the programmed cell death (apoptosis) of podocytes. Since podocytes cannot be readily regenerated in the body, this detrimental process can compromise the glomerular filtration barrier and result in kidney failure and end-stage renal disease (ESRD).

In a genetically engineered mouse model, the authors of this study were able to show that when mice produced sufficient amounts of Protein C, they were protected from kidney damage mediated by diabetes. Another key finding of this study is that Minocycline, a common antibiotic usually used for the treatment of acne and gingival inflammation, achieves the same protective effect as Protein C when administered in the same mouse model of diabetic kidney disease.

The next step will be to apply these approaches to models of cell survival in other renal diseases such as focal segmental glomerulosclerosis (FSGS), in which podocyte apoptosis may play a major role during pathogenesis, and to check for the feasibility of specific therapeutic interventions involving Protein C or the Minocycline antibiotic.

Isermann B, Vinnikov IA, Madhusudhan T, Herzog S, Kashif M, Blautzik J, Corat MAF, Zeier M, Blessing E, Oh J, Gerlitz B, Berg DT, Grinnell BW, Chavakis T, Esmon CT, Weiler H, Bierhaus A, Nawroth PP | Department of Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany | Activated protein C protects against diabetic nephropathy by inhbiting endothelial and podocyte apoptosis | Nat Med. 2007, 13: 1349-1358.

Novel View on Mutations in the NPHS2 gene

May also play a role in late-onset, sporadic FSGS

Mutations in the NPHS2 gene encoding the important slit diaphragm protein podocin are a well-established cause of both familial and sporadic steroid-resistant focal segmental glomerulosclerosis (FSGS) in children. In contrast, they have not been well-characterized in late-onset glomerular disease, which unlike kidney disease that presents at birth or in early childhood, appears later in life, for example in youths and adults. A novel study by McKenzie and colleagues investigated the role of NPHS2 in sporadic cases of late-onset FSGS, i.e. a form of FSGS that appears later on in life as an idiopathic disease.
To investigate the role of NPHS2 polymorphisms in sporadic cases of late-onset FSGS, the authors studied 377 individuals, in whom FSGS had been confirmed by a kidney biopsy, and 919 control patients. Among the significant differences on the DNA level observed between patients with FSGS and control patients was a mutation leading to the exchange of one amino acid in the podocin protein at position 138 (R138Q), found more frequently in patients with FSGS (4 individuals out of 377) than in control patients without FSGS (2 individuals out of 919). A statistical analysis of the patient data revealed that an individual carrying this mutation has a four- to five-fold higher risk of developing late-onset FSGS than another individual not carrying the R138Q mutation.

Furthermore, the authors found another genetic variation in the NPHS2 gene that seems to occur more frequently in African-Americans than in European-Americans. This specific variation was associated with a 50% reduction in risk for sporadic FSGS. Albeit the African-American population in general has been shown to be more affected by FSGS than the Caucasian population, this specific variation was associated with a 50% reduction in risk for sporadic FSGS.

While further studies will be necessary to elucidate a disease-causing mechanism for the R138Q mutation, the results of this study indicate that genetic variation or mutation of NPHS2 may play a role in late-onset sporadic FSGS.

McKenzie LM, Hendrickson SL, Briggs WA, Dart RA, Korbet SM, Mokrzycki MH, Kimmel PL, Ahuja TS, Berns JS, Simon EE, Smith MC, Trachtman H, Michel DM, Schelling JR, Cho M, Zhou YC, Binns-Roemer E, Kirk GD, Kopp JB, Winkler CA | Labortory of Genomic Diversity, SAIC-Frederick, National Cancer Institute, Frederick, Maryland, USA | NPHS2 variation in sporadic focal segmental glomerulosclerosis | J Am Soc Nephrol. 2007, 18:2987-2995.

Genetic Studies Unravel Cause for Proteinuria

Disruption of the Sialic Acid Biosynthesis Pathway Leads to Glomerular Disease

Enzymes are proteins which catalyze biochemical reactions that are vital for sustained body function. One important biochemical reaction is the generation of sialic acid. Sialic acid is an essential molecule in any cell type since it covalently binds to a plethora of different proteins in a chemical reaction called sialylation. Since sialylated proteins differ in their structural and functional properties from their non-sialylated forms, this process plays an important physiological role. In a cell, sialic acid is generated through a series of several biochemical reactions. Glucose serves as raw material which is converted into sialic acid in a number of steps involving different enzymes. One of the key enzymes in this process is a protein called ManNAc kinase (MNK).

Disruption of sialic acid production, for example due to genetic defects in the MNK enzyme, have been shown to be associated with certain human diseases. Because these diseases mainly affect muscle function, sialyation is thought to be particularly important in muscle cells.

A recent study featured in the Journal of Clinical Investigation (JCI) shows that decreased availability of sialic acid in mice due to a mutation in MNK leads to an unexpected observation. The mice with the mutated MNK enzyme died within 72 hours after birth from severe glomerular disease including podocyte foot process effacement, structural damages in the glomerular basement membrane, hematuria and proteinuria. Notably, the disease outcome was ameliorated when sialic acid was administered to mice in its free form. These mice did not exhibit muscle dysfunction.

The present study indicates a key role of sialic acid in kidney development and function in mice. Genetic mutation in the same enzyme MNK obviously results in different outcomes in humans compared to mice; in human patients there was no indication of renal abnormalities whereas mice died soon after birth from severe glomerular injury. This finding illustrates that there may be considerable differences in the organization of cellular functions between humans and widely applied animal models such as mice or rats. Therefore, one has to apply caution when trying to readily adapt experimental observations made in animal models to a human disease. More importantly though, the results of this study impressively demonstrate the importance of the sialic acid biosynthesis pathway for podocyte function; whereas the studied MNK mutation had no effect on kidney function in humans, enzymes other than MNK may well be a culprit for sialic acid biosynthesis disruption in the human kidney.

Molecular geneticists will most likely use this study as a starting point to identify other, still undiscovered mutations in the sialic acid biosynthesis pathway that are associated with human glomerular disease. In addition, the observation that the administration of sialic acid ameliorated the glomerular disease in mice will prompt researchers to further investigate this phenomenon as a possible therapeutic approach for glomerular diseases.

Galeano B, Klootwijk R, Manoli I, Sun M, Ciccone C, Darvish D, Starost MF, Zerfas PM, Hoffmann VJ, Hoogstraten-Miller S, Krasnewich DM, Gahl WA, Huizing M | National Human Genome Research Institute, NIH, Bethesda, Maryland, USA | Mutation in the key enzyme of sialic acid biosynthesis causes severe glomerular proteinuria and is rescued by N-acetylmannosamine | J Clin Invest. 2007, 117:1585-1594.

Proteinuria is a hallmark of glomerular disease and results from an increased permeability of the glomerular filtration barrier. In many forms of glomerular diseases, proteinuria is correlated with injury of podocytes and podocyte foot process effacement. If the underlying cause for podocyte injury and foot process effacement persists, severe and progressive glomerular damage is likely to develop. Eventually this can lead to detachment of podocytes from the glomerular basement membrane and podocyte cell death.

A recent study conducted by investigators from the Program in Glomerular Disease at the Nephrology Division from the Massachusetts General Hospital in Boston, Massachusetts, provides evidence for a novel mechanism leading to podocyte injury, proteinuria and nephrotic syndrome. In this report, which is published in the August issue of the Journal of Clinical Investigation, the authors show that a novel cytoplasmic variant of the enzyme cathepsin L (CatL) cleaves a protein called dynamin in podocytes and that this leads to foot process effacement and proteinuria.

Cathepsin L is an enzyme that is usually confined to lysosomes, which can be seen as the waste processing plants of the cell. In these structures, cathepsin L contributes to the destruction of defective or unwanted proteins by chopping them into smaller fragments which are then subject to further degradation. The authors of the new study found that cathepsin L was induced, i.e. more abundant, in podocytes from patients with glomerular kidney disease and in podocytes grown in the laboratory that were stressed with certain reagents known to damage podocytes. When the authors looked for the subcellular location of the induced cathepsin L in podocytes, they surprisingly found it not only in lysosomes, but throughout the cell body in the cytoplasm. Using a computer-based algorithm the protein dynamin was identified as a potential target for cytoplasmic cathepsin L activity, and biochemical studies confirmed that dynamin is indeed cleaved by cathepsin L in the cell body of podocytes. Cleavage of dynamin by cathepsin L has a devastating effect in podocytes, leading to foot process effacement and proteinuria. This led the authors to conclude that dynamin is a critical regulator of glomerular filtration that is specifically targeted by enzymatic cleavage under pathological conditions. The authors could also show that it was possible to successfully treat proteinuria in mice using podocyte-specific gene delivery of DNA encoding a cleavage-resistant form of dynamin into living animals.

This study identified two new players involved in the development of proteinuric kidney disease; cathepsin L and dynamin. The cleavage of dynamin by cathepsin L under pathological conditions represents a novel mechanism contributing to podocyte injury. Of note, this mechanism appears to be independent from genetic predisposition and is therefore of particular importance for acquired form of proteinuric kidney disease. In contrast to inherited, familial forms of glomerular disease, which develop due to a known genetic defect and occur only in a relatively small number of patients, acquired forms account for the major part of proteinuric diseases. Given that for most acquired glomerular diseases the molecular mechanisms leading to glomerular dysfunction remained yet to be identified, this study represents a major advance in the understanding of glomerular disease. The successful use of gene delivery to podocytes shown in this study opens the door to future studies investigating this finding as a possible therapeutic approach to ameliorate proteinuria in patients with nephrotic syndrome.

Sever S, Altintas MM, Nankoe SR, Möller CC, Ko D, Wei C, Henderson J, del Re E, Hsing L, Erickson A, Cohen CD, Kretzler M, Kerjaschki D, Rudensky A, Nikolic B, Reiser J | Nephrology Division, Massachusetts General Hospital, Boston, Massachusetts, USA | Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease. | J Clin Invest. 2007, 117 in press

+++The editor of these notes was one of the scientists who worked on this project.+++

The slit diaphragm is thought to be an essential structural part of the glomerular filtration barrier. It also is thought to serve important signaling functions for podocytes, mediating extracellular stimuli (for example, changes in fluid flow through the glomerular filtration barrier into the podocyte cell body). It has been hypothesized that under normal conditions slit diaphragm signaling contributes to the sustained structure and function of podocytes, whereas in disease states an altered slit diaphragm signaling induces changes in podocytes such as foot process effacement or apoptosis, the programmed death of podocytes.

A recent report by the group of Dr. Peter Mundel provides new evidence for this concept by demonstrating the involvement of the newly discovered slit diaphragm protein dendrin in podocyte signal cascades and apoptosis. The authors found that dendrin is a component of the slit diaphragm complex, which translocates to the nucleus of injured podocytes in a mouse model of glomerular kidney disease. Furthermore, they showed that the relocation of dendrin was functionally linked to podocyte apoptosis. When cells were treated with TGF-b, a signaling protein that induces apoptosis in cells, translocation of dendrin to the nucleus was promoted. Likewise, when more dendrin was present in the nucleus, TFG-b -induced apoptosis was amplified.

This study substantiates the role of the slit diaphragm as a signaling complex. If the relocation of dendrin proves to be a general mechanism occuring in a number of glomerular diseases, this protein may be an interesting target for therapeutic intervention.

Asanuma K, Campbell KN, Kim K, Faul C, Mundel P | Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA | Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes | Proc Natl Acad Sci U S A. 2007, 104:10134-10139.

Functioning kidneys are essential for life after birth and, although a fetus can survive without kidneys, development of the entire organism is severely compromised. Obviously, there are many reasons why the study of kidney development is important. For example, recently it has been proposed that many disease states of the adult, including elevated blood pressure and kidney disease, may be determined by events that occured during fetal development. Furthermore, through the study of kidney development we can learn more about the basic mechansims regulating kidney structure and function in the adult.

Among the organs in the human body, the kidney is perhaps one of the most complex ones. Its function in regulating the composition of the blood requires the integrated activity of at least 12 different cell types, precisely arranged in the form of the nephron and supporting tissue. The complex nature of the kidney renders its study on a molecular level a challenging task. In addition, studying the human kidney directly is hampered by the fact that it is not possible for obvious reasons to experimentally challenge patients in order to study the effect of a certain experimental regimen on the kidney. Historically, in an attempt to understand the complex functions of the kidney, investigators 999993have taken advantage of animal model systems. In contrast to the human, animal models often have a simpler organization of the kidney filtration apparatus and furthermore allow the study of experimental treatments within the ethical limits set by law.

Well-known animal models include rodents such as mice and rats. Since these mammals share human characteristics such as genes, proteins, and organ organization, experimental results obtained in these animals are often of high relevance for the human. Another important group of animal models is represented by lower vertebrates such as the frog, Xenopus laevis, and the zebrafish, Danio rerio. In particular the latter organism is an emerging model system that will be more and more widely used in basic life science research.

The zebrafish offers a number of experimental advantages that makes it a very attractive system for studies of kidney developments and disease. First, zebrafish embryos develop in freshwater outside of the mother and are transparent, allowing observation of internal organs without dissection. Development occurs rapidly and embryos progress from fertilized eggs to free-swimming fish larvae in roughy 2.5 days. Second, zebrafish can be bred in very high numbers, which makes high-throughput approaches such as large-scale screening studies possible. Third, the sequencing of the zebrafish genome is nearing completion and has already yielded a huge body of genetic information that can be directly exploited in experiments, for example, by targeted disruption of single genes in order to assess their function. The last point is crucial in that targeted gene disruption by so-called gene knock-out or knock-down has become the gold standard to study newly discovered genes and get an idea for their roles in health and disease. While gene knock-out in mice is a tedious and expensive task, zebrafish genes can be rapidly disrupted by the so-called morpholino anti-sense knock-down approach. Together, these features make the zebrafish a useful model for studying organ development and progression of disease, as well as the role of particular genes in these processes.

Several laboratories have started to use the zebrafish as an animal model to study the kidney. The simplicity of the kidney in developing zebrafish larvae, the so-called zebrafish pronephros, makes the study of this organ particularly interesting. In contract to human kidneys, which each contain up to one million nephrons organized in a very complex fashion, the zebrafish pronephros consists of only two nephrons with glomeruli fused at the midline, pronephic tubules connecting directly to the glomeruli via a neck segment, and paired bilateral pronephric ducts that convey the altered blood filtrate outside the animal. While the functional pronepros is remarkably simple, it represents a fully-functioning organ and, in free-swimming larvae of fish and amphibians, it performs the essential kidney functions of blood filtration and regulation of blood pressure. This feature allows efficient mechanistic studies of the kidney, looking on single nephrons as under a reading-glass. An example of how the zebrafish model can help advance our understanding of the kidney filtration apparatus can be found in a recent study that was conducted at the University of Michigan: in this study, which was also featured in the last update of the NephCure Research Newsticker, it was found that mutations in the PLCE1 gene cause nephrotic syndrome. To demonstrate the functional importance of this gene for the maintenance of the glomerular filtration barrier across species, the authors inactivated PLCE1 in zebrafish by the aforementioned morpholino anti-sense knock-down approach, which led to characteristic pathologic features of nephrotic syndrome in the fish. From this result, the authors were able to conclude for the functional conservation of the PLCE1 gene and its importance in the glomerulus.

While the experimental advantages of the zebrafish system are compelling, there are questions about the relevance of such zebrafish studies to the development of vertebrates and mammalian species in general. Obviously, fish organ shape and size is different from the human and other mammalian model systems. Yet, there is a high degree of similarity of organ cell types and tissue substructures between zebrafish and higher vertebrates. For example, in zebrafish pronephros one can find a full range of cell types typical of kidneys of higher vertebrates. The strength of lower vertebrate model systems is not their capacity to exactly model the development of the human kidney but the ease with which they can be manipulated to rapidly determine the function of genes and the importance of cell-cell interactions that underly the development of all kidney forms.

Free oxygen radicals represent a highly reactive form of oxygen. When these radicals come into contact with proteins, they oxidize these proteins, potentially compromising their native structure and function. The mechanism of increased oxidation due to the presence of free oxygen radicals is well-known for its adverse effects in many different diseases. Conversely, it also explains the favorable effect of so-called anti-oxidants. The latter are a class of certain proteins that can act as scavengers and are able to intercept free oxygen radicals in the body before the radicals can cause any damage.

Two recent publications from the group of Gian Marco Ghiggheri and colleagues now provide new evidence that free oxygen radicals also may play a role in the pathogenesis of FSGS when it occurs as an idiopathic disease. The authors found that the important plasma protein albumin is heavily oxidized in a set of patients with active FSGS. But in patients whose FSGS was in remission or who were FSGS-free after transplantation, plasma albumin was normal, they found.

The involvement of free radicals represents a novel potential mechanism in the development of idiopathic FSGS and may lead to specific therapeutic interventions.

Musante L, Bruschi M, Candiano G, Petretto A, Dimasi N, Del Boccio P, Urbani A, Rialdi G, Ghiggeri GM | Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Genoa, Italy | Characterization of oxidation end product of plasma albumin ‘in vivo’ | Biochem Biophys Res Commun. 2006, 349:668-673.

Musante L, Candiano G, Petretto A, Bruschi M, Dimasi N, Caridi G, Pavone B, Del Boccio P, Galliano M, Urbani A, Scolari F, Vincenti F, Ghiggeri GM | Laboratory on Pathophysiology of Uremia, G. Gaslini Children Hospital, Genoa, Italy | Active Focal Segmental Glomerulosclerosis Is Associated with Massive Oxidation of Plasma Albumin | J Am Soc Nephrol. 2007, 18:799-810.

Steroid-resistant forms of nephrotic syndrome are frequently associated with FSGS and commonly progress to end-stage kidney disease. Mutations in a number of genes have been identified as causing steroid-resistant nephrotic syndrome, including the genes NPHS1 (coding for the protein nephrin), NPHS2 (coding for podocin) and ACTN4 (coding for alpha-actinin-4). Of note, the majority of genes identified so far are coding for proteins that serve important functions in podocytes, which are key cells in the process of kidney filtration.

A group from the University of Michigan now has discovered another genetic defect that is associated with the development of steroid-resistant nephrotic syndrome. The researchers working with investigator Friedhelm Hildebrandt reported seven different mutations in the gene PLCE1, coding for the protein phospholipase C epsilon 1 (PLCe1), that were present in individuals with early-onset nephrotic syndrome but absent in healthy control individuals.

PLCe1 belongs to a family of proteins involved in the generation of certain intracellular messengers that are important for maintaining proper cell function. When the authors of this study looked at the localization of normal PLCe1 protein in the kidney, they found it located in kidney glomeruli and particularly in podocytes. This finding adds PLCe1 to the list of proteins present in the podocyte which can cause glomerular kidney disease when rendered dysfunctional by genetic mutation. Since the identified mutations seem to have halted normal glomerular development, the authors speculate that PLCe1 may play an important role in the early developmental stages of formation of the glomerulus.

It appears that the development of severe kidney disease may have been prevented in two of the observed individuals carrying mutations in PLCE1 gene. One child, who presented with the disease at 2 months of age, responded to an initial 4-month course cyclosporin A treatment, which was extended to 2.5 years. He remains free of proteinuria at his current age of 13 years under treatment with an The other child presented with the disease at 12 months and responded to an 8-month course of steroid therapy. He has been virtually free of symptoms then.

Since infantile nephrotic syndrome is traditionally regarded as treatment-resistant, this is a notable discovery. Furthermore, it provides further evidence that PLCe1 is particularly important for glomerular development and potentially less important for maintenance of the glomerulus once it has become mature.

In summary, the identification of mutations in the PLCE1 gene represents the first known molecular cause of a form of nephrotic syndrome that could be resolved after therapy in two individuals. Since the molecular cause of over 70% of all steroid-resistant nephrotic syndrome remains unknown and because treatment options have yet to be identified, this discovery is an important milestone in determining the molecular causes of nephrotic syndrome and FSGS.

Hinkes B, Wiggins RC, Gbadegesin R, et al. | Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA | Positional cloning uncovers mutations in PLCE1 responsible for a nephritic syndrome variant that may be reversible | Nat Gen. 2006, 38:1360-1361.

TRPC6 – Ion Channels and FSGS

Play Role in both Genetic and Acquired Forms of Kidney Disease

Injury to podocytes and their slit diaphragms typically leads to marked proteinuria. Mutations in the TRPC6 gene coding for the Transient Receptor Potential Canonical 6 (TRPC6) ion channel have recently been shown to be associated with FSGS.

Ion channels are important protein complexes located in the membrane of cells. They allow for the transport of negatively charged ions (so-called anions; e.g. the chloride ion Cl-) or positively charged ions (so-called cations; e.g. the calcium ion Ca2+) through the cell membrane, thereby mediating and regulating a large number of cellular functions.

It was found two years ago that TRPC6 ion channels are present in podocytes suggesting that it is the dysfunction of TRPC6 in this cell type that leads to foot process effacement and FSGS in individuals carrying one of the identified mutations in TRPC6. Some of the identified mutations resulted in in hyperactive TRPC6 channels; in other words, a single mutated TRPC6 channel was more active than a single normal TRPC6 channel, which may represent a possible disease-causing mechanism.

The knowledge about TRPC6 channels was advanced recently by the discovery that TRPC6 plays a role not only in genetic forms of FSGS but also in acquired forms of kidney disease such as idiopathic FSGS or Membranous Glomerulonephritis. The group of Jochen Reiser in Boston, Massachusetts, was able to show that normal TRPC6 channels were more abundant in podocytes in patients with FSGS, Minimal Change Disease, and Membranous Glomerulonephritis than in healthy control individuals. Furthermore, it was demonstrated that the treatment of rats with agents inducing kidney damage led to more TRPC6 in podocytes in these animals. Vice versa, when the authors induced higher levels of TRPC6 in formerly healthy mice, these mice developed proteinuria. Together, these results provide evidence that not only single mutated, hyperactive TRPC6 channels, but also the presence of too many normal TRPC6 channels can cause kidney disease.

In summary, this study provides further evidence for an important role for TRPC6 in podocytes and in the development of glomerular disease. While genetic mutations in TRPC6 resulted in more active ion channels and FSGS, the presence of too many normal ion channels obviously has adverse effects for the structure and function of the glomerular filtration barrier.

Moller CC, Wei C, Altintas MM, Li J, Greka A, Ohse T, Pippin JW, Rastaldi MP, Wawersik S, Schiavi S, Henger A, Kretzler M, Shankland SJ, Reiser J | Nephrology Division, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA | Induction of TRPC6 Channel in Acquired Forms of Proteinuric Kidney Disease| J Am Soc Nephrol. 2007, 18:29-36.

New Grants for Research in Glomerular Diseases
NIH Announces Program to Foster the Understanding of the Kidney Glomerulus

On April 24, 2007, the National Institutes of Health (NIH) released a new program announcement inviting new or established researchers to pursue basic exploratory investigations in glomerular disease. The new program, which will be coordinated by NIH’s Division of Kidney, Urologic and Hematologic Diseases (DKUHD) of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), aims to foster the development of new ideas enhancing the understanding of disease detection, pathogenesis, pre-emption and treatment.

The new program stems from a two-day 2005 workshop organized by the NIDDK. This workshop assembled both basic scientists and clinical investigators from the international research community to focus on glomerular disease research advances as well as possibilities for future studies. Participants in the workshop compiled a report summarizing the current status of glomerular disease research and identifying the need for more funding to support promising avenues of future research.

The new program announcement will help meet that need with the NIH Research Project Grant award mechanism (called an RO1) to provide funding to investigators with outstanding applications. The RO1 award mechanism is among the most significant funding opportunities the NIH has to offer and as a rule of thumb provides a principal investigator enough support to run a small research laboratory including personnel for up to five years.

Relevant fields of study within the scope of the program announcement cover topics such as the understanding of basic glomerular cell biology, understanding the pathogenic mechansims of specific forms of glomerular disease such as FSGS, the development of new animal models for specific forms of glomerular disease, and the identification and characterization of glomerular disease biomarkers.

The new NIH program announcement will likely contribute significantly to the multiplication of research efforts in this area.

Two recent reports indicate there is a benefit to adding the diuretic Spironolactone (marketed as Aldactone®, Novo-Spiroton®, Spiractin®, Spirotone®, or Berlactone®) to the agiontensin-converting enzyme (ACE) inhibitor therapy which is common to many patients with FSGS. ACE inhibitors often are used in patients whose kidney filters leak valuable protein (albumin) into the urine, resulting in loss of albumin in the blood (albuminuria). The inhibitors are thought to reduce the kidney filter workload, protect the kidney from further degradation and help fight protein loss. The therapy also is used for Type 2 diabetes patients who suffer from protein loss in the urine (proteinuria).

Researcher Michael B. Davidson and coworkers report that patients with type 2 diabetes mellitus currently being treated with ACE -inhibitors benefit from an additional low dose of the diuretic spironolactone. At the annual meeting of the American Association of Clinical Endocrinologists (AACE), the researchers said spironolactone was an effective and safe method of decreasing albuminuria by a mechanism that is not fully understood. These findings may immediately be applied to patients with albuminuria.

Davidson MB, Wong A, Stevens M, Hamrahian A, Siraj ES | Addition of spironolactone reduces albuminuria in patients with type 2 diabetes who are on ACE inhibitors | AACE 15th Annual Meeting & Clinical Congress, April 26-30, 2006, Chicago, IL

A recent Australian study demonstrates that a dual therapy with ACE inhibitors and spironolactone signficantly reduced proteinuria in a group of 41 patients. This finding by Anastasia Chrysostomou and coworkers is a second indicator that spironolactone administration may offer a valuable adjuvant treatment when used with ACE inhibitor therapy fo the reduction of proteinuria.

Chrysostomou A, Pedagogos E, MacGregor L, Becker GJ | Royal Melbourne Hospital, Parkville, Victoria, Australia | Double-blind, placebo-controlled study on the effect of the aldosterone receptor antagonist spironolactone in patients who have persistent proteinuria and are on long-term angiotensin-converting enzyme ( ACE ) inhibitor therapy, with or without an angiotensin II receptor blocker | Clin J Am Soc Nephrol. 2006, 1:256-262

A mouse study from St. Louis deals with the genetic mutations known to cause problems in the podocyte and create kidney disease.

Oftentimes, a single mutation is sufficient for the onset of the disease. Yet, a recent study by Tobias B. Huber and coworkers reveals that certain mutations, which alone do not result in kidney disease, can lead to kidney disease only when they occur in presence of other mutations.

In detail, the authors found that certain defects in the podocyte gene CD2AP only lead to kidney disease when combined with defects in either of the two podocyte genes Synpo or Fyn. This finding demonstrates that genetic kidney diseases such as inherited forms of FSGS may originate not only from defects in single genes but also from an interplay of multiple gene defects in key regulatory genes of the podocyte.

This finding has broad implications for future efforts with regard to genetic testing of patients with kidney disease of unkown origin. The first commercial genetic test for nephrotic syndrome is now on the market. It screens for mutations in the podocyte proteins nephrin and podocin.

Huber TB, Kwoh C, Wu H, Asanuma K, Godel M, Hartleben B, Blumer KJ, Miner JH, Mundel P, Shaw AS | Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA | Bigenic mouse models of focal segmental glomerulosclerosis involving pairwise interaction of CD2AP, Fyn, and synaptopodin | J Clin Invest. 2006, 116:1337-1345

In 1998, genetic mutations in the NPHS1 gene that encodes the protein nephrin were shown to be associated with congenital nephrotic syndrome of the Finnish type. Nephrin can be found in podocytes, where it is part of the slit diaphragm. Since the nephrin protein is a key component of the glomerular filtration barrier, it was no surprise that mutations resulting in defective nephrin are causing severe renal damage.

In 2000, genetic mutations in another important podocyte protein, podocin, was shown to be associated with steroid-resistant nephrotic syndrome. Patients with this condition do not respond to standard treatment with the steroid prednisone. In consequence, the long-term prognosis for this steroid-resistant subgroup is poor because no satisfactory treatment exists. Like nephrin, podocin plays an important role in regulating kidney filtration at the slit diaphragm.

In the wake of these two milestone discoveries in renal basic research, an assay has been developed that can be used to screen patients for the presence of these two mutations in a reliable and standardized fashion. This test has now become commercially available and doctors may decide to send from candidate patients for genetic screening. The test results can detect the presence of the curently known mutations in either nephrin or podocin and therefore provide clarity about the cause of the disease and a better starting point for treating the patient. The company offering this test is Athena Diagnostics, which can be found at this web site: http://www.athenadiagnostics.com.

Scientists are learning more about complex activities that can cause drastic changes within the structure (cytoskeleton) of a key cell in the kidney filter – the podocyte – and how those changes may cause problems with the glomerular filtration barrier and kidney function. Two recent studies deal with changes in the actin cytoskeleton within the podocyte, the octopus-shaped cell that is so vital to kidney filtration. The podocyte cytoskeleton, much like the human skeleton, provides stability but must also be able to move and respond to the ennvironment. Since changes of the actin cytoskeleton are a key event during foot process effacement, the reported studies may have implications for the elucidation of the mechanisms underlying podocyte injury as seen in FSGS and nephrotic syndrome.

A recent University of Michigan study described how actions involving the protein nephrin can result in the significant changes in the podocyte structure. One of numerous proteins that make up the slit diaphragm component of the kidney filter, nephrin is viewed as a major player in kidney filtration. The study by Rakesh Verma and coworkers shows nephrin can be chemically modified by yet another protein that is associated to the slit diaphragm, Fyn. When Fyn acts upon nephrin, the change then triggers a cascade of events within the cell, eventually resulting in the polymerization of actin filaments, i.e. the formation of larger structures of the actin cytoskeleton by assembly of smaller subunits. This finding substantiates the role of nephrin as an important podocyte-signaling molecule mediating slit diaphragm-derived signals from the extracellular space into the podocyte cell body.

Verma R, Kovari I, Soofi A, Nihalani D, Patrie K, Holzman LB | Division of Nephrology, University of Michigan Medical School, Ann Arbor, Michigan, USA | Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization | J Clin Invest. 2006, 116:1346-1359

A recent New York study by Katsuhito Asanuma in the group of Peter Mundel reports that a protein called synaptopodin plays a role in the activities of the podocyte structure (cytoskeleton). Synaptopodin regulates the function of the podocyte structure by protecting certain other proteins within the cell from degradation. The authors show that defective synaptopodin leads to an abnormal actin cytoskeleton and impairs the podocyte’s capability to move. This finding substantiates the role of synaptopodin as an important regulator of podocyte structure and function and is an important step towards the understanding of the plasticity of the dynamic podocyte actin cytoskeleton.

Asanuma K, Yanagida-Asanuma E, Faul C, Tomino Y, Kim K, Mundel P | Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA | Synaptopodin orchestrates actin organization and cell motility via regulation of RhoA signalling | Nat Cell Biol. 2006, 8:485-491

In a publication from 2003 by Gaétan Mayer and coworkers, it was demonstrated that it is possible to deliver genes to podocytes by injection of into the tail veins of mice. This is achieved using a commercially available set of chemical reagents specifically designed to transfer into mice via intravenous injection. Such a transfer to into a living organism is termed in vivo gene delivery, with the latin term “in vivo” indicating that something goes into or happens “in the living thing”.

Once the has been injected, it reaches organs and tissues throughout the mouse body while circulating in the bloodstream. After a certain time, usually 8-24 hours after gene delivery, the injection of the DNA leads to the expression of the corresponding protein in these tissues and organs, for example the liver and the kidney.

The great advantage of this technology is that researchers are now able to study whether the expression of certain proteins has a direct effect on kidney function. This can be done by measuring proteinuria or other determinants of kidney function in gene-delivered mice, for example.

In this context, the novel tool of in vivo gene delivery may serve as an useful alternative to established experimental methods such as the so-called transgenic mouse models. The generation of transgenic mice involves the breeding of animals carrying a genetic code that is different from the normal genetic code. This can be achieved by using certain techniques of molecular biology. For example, in a so-called knock-out mouse, a gene that is normally present in every mouse has been deliberately deleted to study the effect of the lack of this gene in the animal.

While transgenic mouse models are invaluable experimental tools, their generation generally takes a long time and comes with significant costs. This is why, for some applications, the in vivo gene delivery method may help to carry out certain experiments faster, on a bigger scale, and with less effort. For example, one potential use of the in vivo gene delivery approach is to rapidly test the effect of the expression of mutated proteins on kidney function – something that could be done with transgenic mice only in a very tedious and costly fashion. In addition, the gene delivery technology can be refined in a way that protein expression restricted to podocytes can be studied.

The near future will likely bring the publication of a number of studies in which the in vivo gene delivery methods has been applied to study the pathogenic mechanisms of FSGS and nephrotic syndrome. While this approach still constitutes a novel item within the nephrologist’s experimental toolbox and will need further characterization and optimization, the current results seem to be highly promising for upcoming research efforts.

Mayer G, Boileau G, Bendayan M | Université de Montréal, Montréal, Québéc, Canada | Furin interacts with proMT1-MMP and integrin alphaV at specialized domains of renal cell plasma membrane | J Cell Sci. 2003, 116:1763-1773.