Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.     

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.     

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.     

Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex

Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism. [BMB Reports 2013; 46(4): 230-235]     

Detection of urinary podocytes and nephrin as markers for children with glomerular diseases

The purpose of this study was to detect the urinary podocytes and its related protein, nephrin, in the urine of the children with glomerular disease in order to analyze the relationship of the clinical testing with the significance of the glomerular disease. A total of 65 children with nephrotic syndrome were selected for this study. The podocytes and nephrin were detected in the urinary sediment by indirect immunofluorescence, enzyme-linked immunosorbent assay, and Western blotting. The urinary podocytes and nephrin positive rates were 53.8% and 50.8%, respectively, in the children with glomerular disease. The serum total protein and albumin decreased in the podocyte-positive children, while the urine total protein at 24 h, urinary albumin/creatinine ratio, blood urea nitrogen, and serum creatinine were significantly elevated as compared to those of the podocyte-negative patients. Furthermore, the results were the same in the patients with positive nephrin as compared to that of the patients with negative nephrin. The podocyte number and nephrin level were significantly higher in the lupus nephritis group as compared to those of the other groups. Likewise, the podocyte number and nephrin level dramatically increased in the focal segmental glomerulosclerosis group as compared to those of the mesangial proliferative glomerulonephritis and minimal change disease groups. In addition, the podocyte numbers and nephrin expression were significantly higher in severe proteinuria group as compared to those of the mild proteinuria group. The urinary nephrin expression was positively related to podocyte and urinary albumin/creatinine ratio. We concluded that the detection of the urinary podocytes and nephrin could be taken as markers for children with glomerular disease, reflecting the type of the disease. Therefore, this can be used as a noninvasive method to evaluate the severity of the kidney disease in children.     

Role of nephrin phosphorylation inducted by dexamethasone and angiotensin II in podocytes

The phosphorylation of nephrin plays an important role in maintaining the normal structure and function in podocytes. Dexamethasone (Dex) is usually used to treat glomerular diseases with proteinuria. In this study, we observated the effect of Dex and angiotensin II (AngII) on the change of nephrin phosphorylation in cultured podocytes. In vitro, cultured podocytes were exposed to AngII (10^?6 mol/L) pretreated with or without Dex (100 nM) for different time periods. Nck or Fyn were silenced by small interfering RNA (siRNA), nephrin and its phosphorylation expression were analyzed by Western blotting. In vitro, the phosphorylation of nephrin was significantly reduced after AngII stimulation (P < 0.05). Dex significantly resisted podocyte injury inducted by AngII via increasing the phosphorylation of nephrin (P < 0.05), siRNA silencing Nck can partially inhibited nephrin phosphorylation, siRNA silencing Fyn can completely inhibited nephrin phosphorylation. Phosphorylation of nephrin is important for the survival status of podocytes. Glucocorticoid treatment for human glomerulonephritis may exert its function by regulating Nck and Fyn complex to promote phosphorylation of nephrin. These results elucidate a novel mechanism of glucocorticoid treatment for glomerulonephritis.     

“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.     

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.     

Trans-interaction of nephrin and Neph1/Neph3 induces cell adhesion that associates with decreased tyrosine phosphorylation of nephrin

Slit diaphragms are specialized junctions between glomerular epithelial cells (podocytes) that are crucial for glomerular ultrafiltration. The Ig superfamily members nephrin and Neph1 are essential components of the slit diaphragm, whereas the role of Neph1 homologue Neph3 in the slit diaphragm is unknown. In the present paper we show that Neph3 homodimerizes and heterodimerizes with nephrin and Neph1. We further investigated whether these interactions play a role in cell adhesion by using mouse L fibroblasts that lack endogenous cell-adhesion activity and found that Neph1 and Neph3 are able to induce cell adhesion alone, whereas nephrin needs to trans-interact with Neph1 or Neph3 in order to promote formation of cell-cell contacts. Tyrosine phosphorylation of nephrin was down-regulated after nephrin trans-interacted with either Neph1 or Neph3 leading to formation of cell-cell contacts. We further found that the expression of Neph3 was increased in nephrin-deficient mouse podocytes. The findings of the present paper show that nephrin and Neph1 or Neph3 trans-interactions promote cell-contact formation, suggesting that they may also function together in slit diaphragm assembly.     

GAP-43 ameliorates Podocyte injury by decreasing nuclear NFATc1 expression

Podocyte injury is sufficient to cause glomerulosclerosis and proteinuria, eventually leading to kidney failure. Previous studies found that podocytes and neurons had similar biological characteristics. Growth-associated protein-43 (GAP-43) is a growth cone protein in neurons, and a marker of axonal and synaptic growth. However, it is not known whether GAP-43 is expressed in podocytes. Compared with normal glomerular podocytes, GAP-43 was significantly reduced in patients with glomerular diseases. GAP-43 also significantly reduced in lipopolysaccharide (LPS)-treated podocytes. We found that the decreased expression of nephrin, the cell marker of the podocyte, was significantly recovered with GAP-43 overexpression. In contrast, the migration ability in LPS-treated podocyte was reduction after GAP-43 overexpressing. Moreover, overexpression of GAP-43 attenuated podocyte apoptosis by up-regulating the ratio of Bcl-2/Bax with LPS treatment. Finally, Plaue and Rcan1 which are downstream target gene of NFATc1 decreased with overexpression of GAP-43 podocytes. We concluded that GAP-43 attenuated podocyte injury by inhibiting calcineurin/NFATc1 signaling. The findings may provide a promising treatment for podocyte injury-related diseases.     

Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes.     

A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier

The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.     

Integrin Ligation Results in Nephrin Tyrosine Phosphorylation In Vitro

Nephrin is expressed at the basolateral aspect of podocytes and is an important signaling protein at the glomerular slit diaphragm. In vitro studies have demonstrated that Nephrin phosphorylation-dependent signaling is able to assemble a protein complex that is able to polymerize actin. However, proximal signaling events that result in nephrin tyrosine phosphorylation are not well understood. Nephrin deletion in mice and human nephrin mutations result in developmental failure of the podocyte intercellular junction resutling in proteinuria. This has been presumed to be due to a failure to respond to an external polarized cue in the absence of nephrin or a failure to transduce an outside-in signal in patients with nephrin mutations. The nephrin extracellular domain binds to itself or neph1 across the foot process intercellular junction. Nephrin is tyrosine phosphorylation-silent in healthy glomeruli when presumably the nephrin extracellular domain is in an engaged state. These observations raise the possibility of an alternate proximal signaling mechanism that might be responsible for nephrin tyrosine phosphorylation. Here we present data showing that integrin engagement at the basal aspect of cultured podocytes results in nephrin tyrosine phosphorylation. This is abrogated by incubating podocytes with an antibody that prevents integrin β1 ligation and activation in response to binding to extracellular matrix. Furthermore, nephrin tyrosine phosphorylation was observed in podocytes expressing a membrane-targeted nephrin construct that lacks the extracellular domain. We propose, integrin-activation based signaling might be responsible for nephrin phosphorylation rather than engagment of the nephrin extracellular domain by a ligand.     

Effects of insulin and high glucose on mobilization of slo1 BKCa channels in podocytes

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Podocytes are affected in the earliest stages of diabetic nephropathy and insulin signaling to podocytes is essential for normal glomerular function. Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) encoded by the Slo1 gene are expressed in podocytes in a complex with multiple glomerular slit diaphragm proteins including nephrin, TRPC6 channels, and several different actin-binding proteins. Here we show that insulin increases cell surface expression of podocyte BK(Ca) channels, which is accompanied by a corresponding increase in the density of current flowing through these channels. Insulin stimulation of BK(Ca) channels was detectable in 15 min and required activation of both Erk and Akt signaling cascades. Exposure to high glucose (36.1 mM) for 24 h caused a marked reduction in the steady-state surface expression of BK(Ca) channels as well as of the slit diaphragm signaling molecule nephrin. High glucose treatment also abolished the stimulatory effects of insulin on BK(Ca) current density, although insulin continued to increase phosphorylation of Erk and Akt under those conditions. Therefore, in contrast to most other cell types, high glucose abrogates the effects of insulin in podocytes at relatively distal steps in its signaling pathway. Insulin stimulation of BK(Ca) channels in podocytes may prepare podocytes to adapt to changes in pressure gradients that occur during postprandial hyperfiltration.     

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.     

Rat nephrin modulates cell morphology via the adaptor protein Nck

Nephrin is a transmembrane molecule essential for morphology and function of kidney podocytes. We and others reported previously that the cytoplasmic domain of human and mouse nephrin interacts with the adaptor protein, Nck, in a tyrosine phosphorylation-dependent manner. In the current study, we characterized the interaction of rat nephrin with Nck and further addressed its impact on cell morphology. Rat nephrin expressed in Cos-1 cells co-immunoprecipitated with Nck in a manner dependent on the phosphorylation of Y1204 and Y1228. Nephrin from normal rat glomeruli was also tyrosine phosphorylated and associated with Nck. Overexpression of rat nephrin in HEK293T cells induced morphological changes resembling process formation, which became more distinct when the extracellular domain of nephrin was cross-linked by antibodies. The morphological changes were attenuated by expression of dominant negative constructs of Nck. In the rat model of podocyte injury and proteinuria, nephrin tyrosine phosphorylation and nephrin-Nck interaction were both reduced significantly. Taken together, we propose that Nck couples nephrin to the actin cytoskeleton in glomerular podocytes and contributes to the maintenance of normal morphology and function of podocytes.     

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.     

Vitamin D3 ameliorates podocyte injury through the nephrin signalling pathway

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D₃ through its receptor, VDR, provides renal protection in DN but limited data exist about its effect on podocytes. In this study, we used isolated rat glomeruli to assess podocalyxin and nephrin expression after treatment with the 1,25‐dihydroxyvitamin D₃ analogue paricalcitol in the presence of normal and diabetic glucose levels. The role of 1,25‐dihydroxyvitamin D₃ (calcitriol) and its analogue, paricalcitol, on podocyte morphology and survival was also investigated in the streptozotocin (STZ)‐diabetic animal model. In our ex vivo model, glomeruli exhibited high glucose‐mediated down‐regulation of podocalyxin, and nephrin, while paricalcitol reversed the high glucose‐induced decrease of nephrin and podocalyxin expression. Paricalcitol treatment enhanced VDR expression and promoted VDR and RXR co‐localization in the nucleus. Our data also indicated that hyperglycaemia impaired survival of cultured glomeruli and suggested that the implemented nephrin down‐regulation was reversed by paricalcitol treatment, initiating Akt signal transduction which may be involved in glomerular survival. Our findings were further verified in vivo, as in the STZ‐diabetic animal model, calcitriol and paricalcitol treatment resulted in significant amelioration of hyperglycaemia and restoration of nephrin signalling, suggesting that calcitriol and paricalcitol may provide molecular bases for protection against loss of the permselective renal barrier in DN.