Transmembrane collagen XVII is a novel component of the glomerular filtration barrier

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.     

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

Characterization of the interactions of the nephrin intracellular domain

Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.     

Nephrin--a unique structural and signaling protein of the kidney filter

Since the discovery of nephrin, the first integral component of the slit diaphragm to be identified, the podocyte slit pore has become a major focus in research concerning the glomerular filtration barrier. Nephrin is a central component of the glomerular ultrafilter, with both structural and signaling functions. The extracellular domain of nephrin and other components of the slit diaphragm seem to form a porous molecular sieve. The intracellular domain of nephrin is associated with linker proteins, such as CD2-associated protein and Nck proteins that can connect nephrin to the actin cytoskeleton. Alterations in nephrin interactions with other proteins during development or injury can lead to complex signaling reactions aimed at establishing or restoring the filter function.     

Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus

The human kidneys filter 180 l of blood every day via about 2.5 million glomeruli. The three layers of the glomerular filtration apparatus consist of fenestrated endothelium, specialized extracellular matrix known as the glomerular basement membrane (GBM) and the podocyte foot processes with their modified adherens junctions known as the slit diaphragm (SD). In this study we explored the contribution of podocyte β1 integrin signaling for normal glomerular function. Mice with podocyte specific deletion of integrin β1 (podocin-Cre β1-fl/fl mice) are born normal but cannot complete postnatal renal development. They exhibit detectable proteinuria on day 1 and die within a week. The kidneys of podocin-Cre β1-fl/fl mice exhibit normal glomerular endothelium but show severe GBM defects with multilaminations and splitting including podocyte foot process effacement. The integrin linked kinase (ILK) is a downstream mediator of integrin β1 activity in epithelial cells. To further explore whether integrin β1-mediated signaling facilitates proper glomerular filtration, we generated mice deficient of ILK in the podocytes (podocin-Cre ILK-fl/fl mice). These mice develop normally but exhibit postnatal proteinuria at birth and die within 15 weeks of age due to renal failure. Collectively, our studies demonstrate that podocyte β1 integrin and ILK signaling is critical for postnatal development and function of the glomerular filtration apparatus.     

The Structural and Functional Organization of the Podocyte Filtration Slits Is Regulated by Tjp1/ZO-1

Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood. In this study, we suggest that tight junction protein 1 (Tjp1 or ZO-1), which is encoded by Tjp1 gene, plays an essential role in establishing the podocyte filtration barrier. The podocyte-specific deletion of Tjp1 down-regulated the expression of podocyte membrane proteins, impaired the interdigitation of the foot processes and the formation of the slit diaphragm, resulting in glomerular dysfunction. We found the possibility that podocyte filtration barrier requires the integration of two independent units, the pre-existing epithelial junction components and the newly synthesized podocyte-specific components, at the final stage in glomerular morphogenesis, for which Tjp1 is indispensable. Together with previous findings that Tjp1 expression was decreased in glomerular diseases in human and animal models, our results indicate that the suppression of Tjp1 could directly aggravate glomerular disorders, highlights Tjp1 as a potential therapeutic target.     

Early glomerular filtration defect and severe renal disease in podocin-deficient mice

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.     

Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton

Podocytes of the renal glomerulus are unique cells with a complex cellular organization consisting of a cell body, major processes and foot processes. Podocyte foot processes form a characteristic interdigitating pattern with foot processes of neighboring podocytes, leaving in between the filtration slits that are bridged by the glomerular slit diaphragm. The highly dynamic foot processes contain an actin-based contractile apparatus comparable to that of smooth muscle cells or pericytes. Mutations affecting several podocyte proteins lead to rearrangement of the actin cytoskeleton, disruption of the filtration barrier and subsequent renal disease. The fact that the dynamic regulation of the podocyte cytoskeleton is vital to kidney function has led to podocytes emerging as an excellent model system for studying actin cytoskeleton dynamics in a physiological context.     

MAGI-1 Interacts with Nephrin to Maintain Slit Diaphragm Structure through Enhanced Rap1 Activation in Podocytes

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.     

Pathological correlations between podocyte injuries and renal functions in canine and feline chronic kidney diseases

Podocytes cover the glomerulus and their adjacent foot processes form a principal barrier called the slit diaphragm. Podocyte dysfunctions, including podocyte loss and slit diaphragm disruptions, induce chronic kidney diseases (CKD). In this study, we analyzed the correlations between podocyte injuries and renal dysfunctions in domestic carnivores. Dogs and cats were divided into normal and CKD groups according to renal histopathology and plasma creatinine values. Immunostaining results showed that linear reactions of slit diaphragm molecules, e.g., nephrin, podocin, and ACTN4, were parallel to glomerular capillaries in all animals. However, in dogs, reactions of nephrin and ACTN4 were changed to a granular pattern in the CKD group, and their intensities significantly decreased with the number of podocytes in the glomerulus. Moreover, the expression of nephrin and ACTN4 negatively correlated with creatinine. Real-time PCR analysis showed that nephrin mRNA expression in the kidneys of CKD dogs was significantly lower than that in normal animals, and negatively correlated with creatinine. Although no significant correlation between renal dysfunction and podocyte injury was detected in cats, histoplanimetric scores of tubulointerstitial lesions in CKD cats were higher than those in both normal cats and diseased dogs. Furthermore, mRNAs of WT1 and SD molecules were detected in urine from CKD animals. In conclusion, podocyte injuries such as podocytopenia and decreased expression of nephrin and ACTN4 in the glomerulus were more strongly correlated with renal dysfunction in dogs than in cats. These findings suggest that the CKD pathogenesis, especially susceptibilities to podocyte injuries, differed between dogs and cats.     

Neph-Nephrin proteins bind the Par3-Par6-atypical protein kinase C (aPKC) complex to regulate podocyte cell polarity

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their foot processes and the interposed slit diaphragm. So far, very little is known about the guidance cues and polarity signals required to regulate proper development and maintenance of the glomerular filtration barrier. We now identify Par3, Par6, and atypical protein kinase C (aPKC) polarity proteins as novel Neph1-Nephrin-associated proteins. The interaction was mediated through the PDZ domain of Par3 and conserved carboxyl terminal residues in Neph1 and Nephrin. Par3, Par6, and aPKC localized to the slit diaphragm as shown in immunofluorescence and immunoelectron microscopy. Consistent with a critical role for aPKC activity in podocytes, inhibition of glomerular aPKC activity with a pseudosubstrate inhibitor resulted in a loss of regular podocyte foot process architecture. These data provide an important link between cell recognition mediated through the Neph1-Nephrin complex and Par-dependent polarity signaling and suggest that this molecular interaction is essential for establishing the three-dimensional architecture of podocytes at the kidney filtration barrier.     

An update: the role of Nephrin inside and outside the kidney

Nephrin is a key molecule in podocytes to maintain normal slit diaphragm structure. Nephin interacts with many other podocyte and slit diaphragm protein and also mediates important cell signaling pathways in podocytes. Loss of nephrin during the development leads to the congenital nephrotic syndrome in children. Reduction of nephrin expression is often observed in adult kidney diseases including diabetic nephropathy and HIV-associated nephropathy. The critical role of nephrin has been confirmed by different animal models with nephrin knockout and knockdown. Recent studies demonstrate that knockdown of nephrin expression in adult mice aggravates the progression of unilateral nephrectomy and Adriamycin-induced kidney disease. In addition to its critical role in maintaining normal glomerular filtration unit in the kidney, nephrin is also expressed in other organs. However, the exact role of nephrin in kidney and extra-renal organs has not been well characterized. Future studies are required to determine whether nephrin could be developed as a drug target to treat patients with kidney disease.     

“Treasure your exceptions”: recent advances in molecular genetics of glomerular disease

The glomerular filtration barrier consists of endothelial cells, the glomerular basement membrane, and podocytes. The membrane is a highly crosslinked macromolecular meshwork composed of specific extracellular matrix proteins. The adjacent foot processes of podocytes are bridged along their basolateral surfaces by a slit diaphragm (a porous filter structure of nephrin molecules). Recent discoveries of mutations in the range of genes encoding proteins involved in the structure or function of the glomerular filtration barrier have provided new insights into mechanisms of glomerular diseases. In this review, we summarize recent progress in the elucidation of the genetic basis of some glomerulopathies in humans.     

Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers

Glomerular visceral epithelial cells (podocytes) appear to play a central role in maintaining the selective filtration barrier of the renal glomerulus. While the immunoglobulin superfamily member Nephrin was proposed to act as a cell adhesion molecule at the podocyte intercellular junction necessary for maintaining glomerular perm selectivity, the Nephrin ligand has not been identified. The existence of a new subfamily of Nephrin-like molecules including Neph1 was recently described. Genetic deletion of Nephrin or Neph1 resulted in similar phenotypes of podocyte foot process effacement and proteinuria. The subcellular localization of Neph1 and the possibility that Nephrin and Neph1 interact was investigated. Polyclonal antiserum for Neph1 was raised and characterized. Neph1 migrated as a 90-kDa protein on SDS-PAGE under reducing conditions. Neph1 was identified in a glomerular and podocyte-specific distribution in adult rat kidney. Like Nephrin and Podocin, Neph1 was enriched in Triton X-100 detergent-resistant membrane fractions. Consistent with this observation, immunogold electron microscopy demonstrated that Neph1 localized exclusively to lateral margins of podocyte foot processes at the insertion of the slit diaphragm. Neph1 and Nephrin participate in a direct cis-interaction involving their cytoplasmic domains. In addition, interactions between the extracellular domain of Nephrin and itself and between the extracellular domain of Nephrin and that of Neph1 were detected. Neph1 did not interact via a homophilic interaction. These observations suggest that Nephrin and Neph1 form a hetero-oligomeric receptor complex in the plane of the membrane that might interact across the foot process intercellular junction through interactions between Nephrin with itself and Neph1.     

Puerarin Attenuated Early Diabetic Kidney Injury through Down-Regulation of Matrix Metalloproteinase 9 in Streptozotocin-Induced Diabetic Rats

Radix puerariae, a traditional Chinese herbal medication, has been used successfully to treat patients with early stage of diabetic nephropathy. However, the underlined mechanism of this renal protective effect has not been determined. In the current study, we investigated the effects and the mechanism of puerarin in Streptozotocin (STZ)-induced diabetic rats. We treated STZ-rats with either puerarin or losartan, an angiotensin II receptor blocker, as compared to those treated with vehicle. We found that both puerarin and losartan attenuated kidney hypertrophy, mesangial expansion, proteinuria, and podocyte foot process effacement in STZ rats. In addition, both puerarin and losartan increased expression of podocyte slit diaphragm proteins such as nephrin and podocin. Interestingly, we found that puerarin treatment induced a more pronounced suppression of oxidative stress production and S-nitrosylation of proteins in the diabetic kidneys as compared to losartan treatment. Furthermore, we found that matrix metalloproteinase-9 (MMP-9), which is known to be activated by oxidative stress and S-nitrosylation of proteins, was also suppressed more extensively by puerarin than losartan. In conclusion, these data provide for the first time the potential mechanism to support the use of puerarin in the treatment of early diabetic nephropathy.     

Angiopoietin-like protein 3 regulates the motility and permeability of podocytes by altering nephrin expression in vitro

It is well known that podocyte injury plays a vital role in massive proteinuria. The increase of podocyte motility results in podocyte foot process (FP) effacement, a typical form of podocyte injury. Our previous studies demonstrated that glomerular podocytes can express angiopoietin-like protein 3 (ANGPTL3) and that the increase of ANGPTL3 in dysfunctional glomerulus is correlated with podocyte FP effacement. Little is known, however, about the role of ANGPTL3 in podocyte injury. In this study, we investigated ANGPTL3’s effect on the motility and permeability of podocytes and on the expression of nephrin, a key molecule in podocytes. By scrape-wound and transwell migration assay, we found that ANGPTL3 over-expression significantly increased podocyte motility, whereas after ANGPTL3 knockdown by RNA interference, motility remained the same as that of the control group. Adriamycin (ADR) treatment significantly promoted podocyte motility. However, the same dose of ADR treatment could not promote motility after the knockdown of ANGPTL3. In addition, we assayed the diffusion of FITC-BSA across the podocytes’ monolayer to investigate whether ANGPTL3 could promote protein loss by means of an increase in podocyte motility. The results showed that the changes in the FITC-BSA permeability of the podocytes corresponded to changes in motility. Furthermore, we found that ANGPTL3 over-expression dramatically increased the expression of nephrin but that the up-regulation of nephrin induced by ADR was significantly inhibited when ANGPTL3 was diminished by RNAi. In conclusion, we found ANGPTL3 to be capable of regulating the motility and permeability of podocytes and that the mechanism of ANGPTL3’s regulation could be associated with the altered expression of nephrin.     

POROUS SUBSTRUCTURE OF THE GLOMERULAR SLIT DIAPHRAGM IN THE RAT AND MOUSE

The highly ordered, isoporous substructure of the glomerular slit diaphragm was revealed in rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde. The slit diaphragm was similar in both animal species and appeared as a continuous junctional band, 300–450 Å wide, consistently present within all slits formed by the epithelial foot processes. The diaphragm exhibited a zipper-like substructure with alternating, periodic cross bridges extending from the podocyte plasma membranes to a central filament which ran parallel to and equidistant from the cell membranes. The dimensions and spacing of the cross bridges defined a uniform population of rectangular pores approximately 40 by 140 Å in cross section and 70 Å in length. The total area of the pores was calculated to be about 2–3% of the total surface area of the glomerular capillaries. Physiological data indicate that the glomerular filter functions as if it were an isoporous membrane which excludes proteins larger than serum albumin. The similarity between the dimensions of the pores in the slit diaphragm and estimates for the size and shape of serum albumin supports the conclusion from tracer experiments that the slit diaphragm may serve as the principal filtration barrier to plasma proteins in the kidney.