Podocyte Proteins Research Articles

Simvastatin Increases ADAMTS13 Expression in Podocytes

IntroductionADAMTS13 is a specific von Willebrand factor–cleaving protease. Severe deficiency of ADAMTS13 is the main cause of thrombotic thrombocytopenic purpura. ADAMTS13 is mainly synthesized and released from hepatic stellate cells and endothelial cells, but is also expressed in other cells, including kidney podocytes. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has a beneficial effect on atherosclerosis and also has anti-inflammatory and antithrombotic properties. A recent study indicates that ADAMTS13 reduces inflammatory plaque formation during early atherosclerosis in mice. In our study, we investigated the effects of simvastatin on inflammatory cytokines–induced ADAMTS13 expression in podocytes. Materials and MethodsA conditionally immortalized mouse podocyte cell line was utilized to study the expression of ADAMTS13 in podocytes. The influence of TNF-α, IL-4, IL-6 and simvastatin on ADAMTS13 was investigated. ADAMTS13 mRNA levels in podocytes were measured by using real-time PCR and protein levels were detected by Western blotting. ResultsSimvastatin significantly up-regulated the expression levels of ADAMTS13 mRNA and protein in podocytes. IL-6 decreased ADAMTS13 expression, and TNF-α had no significant effects on ADAMTS13 expression in podocytes. IL-4 reduced ADAMTS13 mRNA expression but not its protein level. Simvastatin was able also reversed the inhibitory effect of IL-6. ConclusionsWe demonstrate that simvastatin increases the expression of ADAMTS13 in a dose-dependent manner in podocytes, which likely contributes to the antithrombotic property of statin. Different inflammatory cytokines have different effects on the levels of ADAMTS13 mRNA expression and protein within podocytes.

Effects of leflunomide on podocytes exposed to high glucose condition and its mechanism

To explore the protective effects of leflunomide (A771726) on the expression of podocalyxin, NF-κB and matrix metalloproteinase-9 (MMP-9) in podocytes exposed to high glucose environment and elucidate its mechanism. Podocytes were cultured in high glucose. And the altered expressions of podocyte protein podocalyxin were detected by Western blotting at different timepoints. Then podocytes were divided into 4 groups of normal glucose control, leflunomide, high glucose and hypertonic control. The expression level of podocalyxin protein in each group was detected by Western blotting. And NF-κB p65 and phosphorylation of NF-κB p65 (P-NF-κBp65) in podocytes cultured in high glucose were detected at different timepoints. And then the podocytes were divided into 5 groups of normal glucose, mannitol, hypertonic control, high glucose, leflunomide and PDTC (NF-κB blocker). And the expressions of MMP-9 protein in these groups were also detected by Western blotting. In the high glucose environment, the expression of podocalyxin declined instantly. Compared with the high-glucose group, the podocalyxin expression of the leflunomide group was significantly higher than the high glucose group (0.46 ± 0.04 vs 0.13 ± 0.03, P < 0.05). After 30-minute stimulation by high glucose, the activation of NF-κB started and the expression of P-NF-κBp65 protein increased. Such activities peaked at 60 minutes and reverted to a basic level after 6 hours. Compared with the high glucose group, the expressions of MMP-9 in PDTC and leflunomide groups were significantly lower than the high glucose group. And the differences were statistically significant (0.71 ± 0.01, 0.64 ± 0.03 vs 1.64 ± 0.03, both P < 0.05). Leflunomide has protective effects on podocytes in high glucose. And its mechanism is possibly due to a lowered expression of MMP-9 through an inhibition of NF-κB activation.

Molecular fingerprinting of the podocyte reveals novel gene and protein regulatory networks

A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.

1,25-Dihydroxyvitamin D(3) Inhibits Podocyte uPAR Expression and Reduces Proteinuria

BackgroundAccumulating studies have demonstrated that 1,25-Dihydroxyvitamin D(3) (1,25(OH)2D3) reduces proteinuria and protects podocytes from injury. Recently, urokinase receptor (uPAR) and its soluble form have been shown to cause podocyte injury and focal segmental glomerulosclerosis (FSGS). Here, our findings showed that 1,25(OH)2D3 did inhibit podocyte uPAR expression and attenuate proteinuria and podocyte injury.Methodology/Principal FindingsIn this study, the antiproteinuric effect of 1,25(OH)2D3 was examined in the lipopolysaccharide mice model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model(NTX rats). uPAR protein expression were tested by flow cytometry, immune cytochemistry and western blot analysis, and uPAR mRNA expression by real-time quantitative PCR in cultured podocytes and kidney glomeruli isolated from mice and rats. Podocyte motility was observed by transwell migration assay and wound healing assay. Podocyte foot processes effacement was identified by transmission electron microscopy. We found that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in LPS-treated podocytes, LPS mice and NTX rats, along with 1,25(OH)2D3 reducing proteinuria in NTX rats and LPS mice.1,25(OH)2D3 reduced glomerulosclerosis in NTX rats and alleviated podocyte foot processes effacement in LPS mice. Transwell migration assay and wound healing assay showed that LPS-induced podocyte motility, irrespective of random or directed motility, were substantially reduced by 1,25(OH)2D3.Conclusions/SignificanceOur results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR expression in vitro and in vivo, which may be an unanticipated off target effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria.

MiR-150 Promotes Renal Fibrosis in Lupus Nephritis by Downregulating SOCS1

MicroRNAs (miRs) seem to mediate renal fibrosis in several renal diseases, with some miRs having profibrotic effects and others having opposing effects. Although differential expression of certain miRs has been described in lupus nephritis, it is unknown whether miRs contribute to fibrosis or could serve as biomarkers of specific histologic manifestations of lupus nephritis. Here, we compared miR expression in kidney biopsies from patients with lupus nephritis and identified miR-150 as the most differentially expressed miR in kidneys with high chronicity (chronicity index [CI] ≥ 4); miR-150 positively correlated with chronicity scores and the expression of profibrotic proteins. Overexpression of miR-150 significantly reduced expression of the antifibrotic protein suppressor of cytokine signaling 1 (SOCS1) and upregulated profibrotic proteins in both proximal tubular and mesangial cells. Directly targeting SOCS1 with a small interfering RNA produced similar results. Furthermore, TGF-β1 induced miR-150 expression, decreased SOCS1, and increased profibrotic proteins in proximal tubular cells and podocytes; a miR-150 inhibitor reversed these changes, suggesting that the profibrotic effects of TGF-β1 are, at least in part, mediated by miR-150. Consistent with these in vitro observations, biopsies with high miR-150 and high CI exhibited substantial expression of TGF-β1, reduced SOCS1, and an increase in profibrotic proteins. In summary, miR-150 is a promising quantitative renal biomarker of kidney injury in lupus nephritis. Our results suggest that miR-150 promotes renal fibrosis by increasing profibrotic molecules through downregulation of SOCS1.

Development of Podocyte Injuries in Osborne–Mendel Rats is Accompanied by Reduced Expression of Podocyte Proteins

Osborne-Mendel (OM) rats spontaneously develop glomerulopathy with progressive podocyte injury. Changes in protein expression levels in the foot processes of podocytes have been suggested to play an important role in the development of renal disease. The aim of this study was to investigate the temporal relationship between the expression of five podocyte proteins (nephrin, podocin, synaptopodin, α-actinin-4 and α3-integrin) and the development of podocyte injuries, proteinuria and glomerulosclerosis in OM rats. Male OM rats 5-20 weeks of age and age-matched Fischer 344 rats were used. Semiquantitative analysis of expression of the five podocyte proteins was performed by immunofluorescence labelling. Nephrin mRNA expression was determined by quantitative real-time reverse transcriptase polymerase chain reaction and nephrin protein expression was determined by mass spectrometry. Progressive reduction in expression of the podocyte proteins correlated with the progression of podocyte injuries, the development of proteinuria and the subsequent development of glomerulosclerosis. Nephrin mRNA expression and nephrin concentration also showed temporal decreases in OM rats. Altered expression of podocyte proteins preceded the development of proteinuria and glomerulosclerosis, suggesting that this event contributes to podocyte dysfunction and progression to glomerulosclerosis.

MicroRNAs in Idiopathic Childhood Nephrotic Syndrome

Childhood nephrotic syndrome (NS)3 is associated with an increase in the permeability across the glomerular filtration barrier due to processes that affect the dynamics and permselectivity of the glomerular filtration barrier. This increased permeability leads to an inability to restrict the loss of protein to 50 mg/kg per day), hypoalbuminemia (<3 g/dL), edema, and hyperlipidemia (1). Severe complications include bacterial infections due to immunoglobulin losses and thromboembolisms related to a relative increase in the hematocrit (1). Children with NS have been classified into different categories. Patients with primary NS, in which a systemic disease cannot be identified, include children with idiopathic NS (absence of glomerular inflammation on renal biopsy) and children with primary glomerulonephritis. Secondary NS is related to a systemic disease. Less commonly, NS is caused by an inherited renal disease (congenital or infantile NS). This form occurs in children younger than 1 year and is associated with a poor outcome (2). Primary idiopathic NS may be associated with at least 3 different histologic abnormalities: minimal-change disease (MCD), focal segmental glomerulosclerosis (FSGS), and membranous nephropathy (1). Pathogenetically, MCD and FSGS have long been considered to be caused by a circulating permeability factor (3). Recently, circulating soluble urokinase plasminogen activator receptor has been shown to cause FSGS (4). Congenital or infantile NS is caused by mutations in podocyte or slit diaphragm proteins \[e.g., CD2AP (CD2-associated protein), podocin, the Wilms tumor-suppressor gene, and nephrin\] (1). Idiopathic NS has been identified as …

Alteration of Podocyte Protein Expression and Localization in the Early Stage of Various Hemodynamic Conditions

Given that podocalyxin (PCX) and nestin play important roles in podocyte morphogenesis and the maintenance of structural integrity, we examined whether the expression and localization of these two podocyte proteins were influenced in the early stage of various hemodynamic conditions. Mice kidney tissues were prepared by in vivo cryotechnique (IVCT). The distribution of glomeruli and podocyte proteins was visualized with DAB staining, confocal laser scanning microscopy and immunoelectron microscopy. The mRNA levels were examined by real-time quantitative PCR. The results showed the following: Under the normal condition, PCX stained intensely along glomerular epithelial cells, whereas nestin was clearly staining in the endothelial cells and appeared only weakly in the podocytes. Under the acute hypertensive and cardiac arrest conditions, PCX and nestin staining was not clear, with a disarranged distribution, but the colocalization of PCX and nestin was apparent under this condition. In addition, under the acute hypertensive and cardiac arrest conditions, the mRNA levels of PCX and nestin were significantly decreased. Collectively, the abnormal redistribution and decreased mRNA expressions of PCX and nestin are important molecular events at the early stage of podocyte injury during hemodynamic disorders. IVCT may have more advantages for morphological analysis when researching renal diseases.

Podocyte repopulation by renal progenitor cells following glucocorticoids treatment in experimental FSGS

Prednisone is a mainstay of treatment for patients with focal segmental glomerulosclerosis (FSGS), a disease characterized by reduced podocyte number and glomerulosclerosis. Although the systemic immune-modulatory effects of prednisone are well-known, direct tissue effects on glomerular cells are poorly understood. Experimental FSGS was induced in mice with a cytotoxic anti-podocyte antibody, resulting in an abrupt decrease in podocyte number by day 3, proteinuria, and the development of glomerulosclerosis. Administering daily prednisone to mice with FSGS, beginning at day 3, significantly increased podocyte number at weeks 2 and 4. Podocyte number did not increase in control mice with FSGS given DMSO. The increase in podocyte number in prednisone-treated mice correlated significantly with reduced glomerulosclerosis. Prednisone reduced podocyte apoptosis measured by synaptopodin⁺/caspase-3⁺ double staining. Additionally, the number of podocyte progenitors, defined as cells expressing both a parietal epithelial cell protein and a podocyte protein, was significantly increased in prednisone-treated mice with FSGS at weeks 2 and 4. This was associated with increased phospho-ERK staining in both parietal epithelial cells (PAX2⁺/p-ERK⁺) and in podocyte progenitors (WT-1⁺/p-ERK⁺ lining Bowman's capsule). These data show that in this model of experimental FSGS, prednisone augments glomerular repair by increasing podocyte number through direct effects on both glomerular epithelial cells. Prednisone limits podocyte loss by reducing apoptosis, and it increases regeneration by augmenting the number of podocyte progenitors. The data support a direct glomerular cell action for prednisone in improving outcomes in FSGS.

Preoperative Endogenous Ouabain Predicts Acute Kidney Injury in Cardiac Surgery Patients*

Acute kidney injury is a frequent complication of cardiac surgery and increases morbidity and mortality. As preoperative biomarkers predicting the development of acute kidney injury are not available, we have tested the hypothesis that preoperative plasma levels of endogenous ouabain may function as this type of biomarker. Endogenous ouabain is an adrenal stress hormone associated with adverse cardiovascular outcomes. Its involvement in acute kidney injury is unknown. With studies in patients and animal settings, including isolated podocytes, we tested the above mentioned hypothesis. Preoperative endogenous ouabain was measured in 407 patients admitted for elective cardiac surgery and in a validation population of 219 other patients. We also studied the effect of prolonged elevations of circulating exogenous ouabain on renal parameters in rats and the influence of ouabain on podocyte proteins both "in vivo" and "in vitro." In the first group of patients, acute kidney injury (2.8%, 8.3%, 20.3%, p < 0.001) and ICU stay (1.4±0.38, 1.7±0.41, 2.4±0.59 days, p = 0.014) increased with each incremental preoperative endogenous ouabain tertile. In a linear regression analysis, the circulating endogenous ouabain value before surgery was the strongest predictor of acute kidney injury. In the validation cohort, acute kidney injury (0%, 5.9%, 8.2%, p < 0.0001) and ICU stay (1.2±0.09, 1.4±0.23, 2.2±0.77 days, p = 0.003) increased with the preoperative endogenous ouabain tertile. Values for preoperative endogenous ouabain significantly improved (area under curve: 0.85) risk prediction over the clinical score alone as measured by integrate discrimination improvement and net reclassification improvement. Finally, in the rat model, elevated circulating ouabain reduced creatinine clearance (-18%, p < 0.05), increased urinary protein excretion (+ 54%, p < 0.05), and reduced expression of podocyte nephrin (-29%, p < 0.01). This last finding was replicated ex vivo by incubating podocyte primary cell cultures with low-dose ouabain. Preoperative plasma endogenous ouabain levels are powerful biomarkers of acute kidney injury and postoperative complications and may be a direct cause of podocyte damage.

Reduced podocin expression in minimal change disease and focal segmental glomerulosclerosis is related to the level of proteinuria

Glomerular podocyte molecules are involved in the pathogenesis of congenital nephrotic syndrome. However, their role in primary nephrotic syndrome is not clear. This study investigated the expression of nephrin, podocin and synaptopodin in primary nephrotic syndrome. Eighty-seven patients with primary nephrotic syndrome including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and membranoproliferative glomerulonephritis Type I (MPGN) were included in the study. Glomerular expression of nephrin, podocin and synaptopodin was studied in renal biopsies by immunofluorescence and immunohistochemistry. Correlation of expression with clinical and biochemical parameters was performed. The pattern of expression for all podocyte proteins in controls was uniform fine granular along the capillary walls towards the visceral epithelial cell aspect. Glomerular expression of nephrin was present in all renal biopsies and was similar to that in controls. Glomerular synaptopodin expression was seen in all MN and MPGN patients, while it was seen in 74% (17/23) MCD and 93.5% (29/31) FSGS. Reduced synaptopodin expression showed no correlation with clinical and biochemical factors. Podocin expression was present in 5/23 MCD (22%), 3/31 FSGS (9.6%), 13/17 MN (76.4%) and 13/16 MPGN (81%) patients. The reduced expression of podocin significantly correlated with the degree of proteinuria (p=0.032). No correlation with age, gender and serum creatinine level was observed. Reduction of glomerular podocin expression found in MCD and FSGS is related to the amount of proteinuria. Our findings suggest that alteration in podocyte phenotype may not be a primary event and may reflect the degree of podocyte injury in primary nephrotic syndrome.

Proteomic analysis of podocyte exosome-enriched fraction from normal human urine

Urine results from a coordinated activity of glomerular and tubular compartments of the kidney. As a footprint of these cellular functional processes, urinary exosomes, and 40-80 nm membrane vesicles released after fusion with the plasma membrane into the extracellular environment by renal epithelial cells, are a source for identification of proteins and investigation of their role in the kidney. The aim of the present study was the identification of podocyte exosome proteins based on urine immunoabsorption using podocyte-specific CR1-immunocoated beads followed by proteomic analysis using LC MS/MS techniques. This methodology allowed the identification of 1195 proteins. By using a bioinformatic approach, 27 brain-expressed proteins were identified, in which 14 out of them were newly demonstrated to be expressed in the kidney at a mRNA level, and, one of them, the COMT protein, was demonstrated to be expressed in podocytes at a protein level. These results, attesting the reliability of the methodology to identify podocyte proteins, need now to be completed by further experiments to analyze more precisely their biological function(s) in the podocytes.

New TRPC6 gain-of-function mutation in a non-consanguineous Dutch family with late-onset focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a leading cause of steroid-resistant nephrotic syndrome. Hereditary FSGS is frequently caused by mutations in important structural podocyte proteins, including the slit diaphragm-associated transient receptor potential channel C6 (TRPC6). In five patients with biopsy-proven autosomal-dominant FSGS from five different Dutch families, all 13 exons of TRPC6 were sequenced. Upon identification of a novel TRPC6 sequence variant, the resultant amino acid change was introduced in the wild-type TRPC6 protein and functionally tested using patch-clamp analyses and cell-surface biotinylation experiments. None of the previously described TRPC6 mutations were found in our cohort. In one family, we identified a novel c.524G>A sequence variant resulting in a p.Arg175Gln (R175Q) substitution in the TRPC6 protein. This sequence variant was absent in 449 control subjects and from public SNP databases. The mutation was located in the third ankyrin repeat domain (ANK3) in the cytoplasmic N-tail of TRPC6, important for protein-protein interaction and regulation of ion channel activity. Patch-clamp analyses of the mutant channel indeed showed an increased TRPC6 channel-mediated current. However, cell-surface expression of the mutant channel was not increased. We identified a novel TRPC6 p.Arg175Gln gain-of-function mutation that shows increased TRPC6-mediated current, which is not due to altered cell-surface expression. This is the first mutation identified in ANK3 of the TRPC6 N-tail and is most likely responsible for the late-onset autosomal dominant FSGS in this family.

Roles of adaptor proteins in podocyte biology

Podocytes covering the glomerular basement membrane over the glomerular capillary consist of three morphologically and functionally different segments, the cell body, major processes and extending finger-like foot processes (FPs). The FPs of neighboring podocytes are connected by a continuous adherent junction structure named the slit diaphragm (SD). The extracellular SD is linked to the intracellular, a highly dynamic, cytoskeleton through adaptor proteins. These adaptor proteins, such as CD2-associated protein, zonula occludens 1, β-catenin, Nck and p130Cas, located at the intracellular SD insertion area near lipid rafts, have important structural and functional roles. Adaptor proteins in podocytes play important roles as a structural component of the podocyte structure, linking the SD to the cytoskeletal structure and as a signaling platform sending signals from the SD to the actin cytoskeleton. This review discusses the roles of adaptor proteins in the podocyte cytoskeletal structure and signaling from the SD to the actin cytoskeleton.

Rapamycin Ameliorates Proteinuria and Restores Nephrin and Podocin Expression in Experimental Membranous Nephropathy

Objective. Recent studies have shown a beneficial effect of rapamycin in passive and active Heymann Nephritis (HN). However, the mechanisms underlying this beneficial effect have not been elucidated. Methods. Passive Heymann Nephritis (PHN) was induced by a single intravenous infusion of anti-Fx1 in 12 Sprague-Dawley male rats. One week later, six of these rats were commenced on daily treatment with subcutaneous rapamycin 0.5 mgr/kg (PHN-Rapa). The remaining six rats were used as the proteinuric control group (PHN) while six more rats without PHN were given the rapamycin solvent and served as the healthy control group (HC). All rats were sacrificed at the end of the 7th week. Results. Rapamycin significantly reduced proteinuria during the autologous phase of PHN. Histological lesions were markedly improved by rapamycin. Immunofluorescence revealed attenuated deposits of autologous alloantibodies in treated rats. Untreated rats showed decreased glomerular content of both nephrin and podocin whereas rapamycin restored their expression. Conclusions. Rapamycin monotherapy significantly improves proteinuria and histological lesions in experimental membranous nephropathy. This beneficial effect may be mediated by inhibition of the alloimmune response during the autologous phase of PHN and by restoration of the normal expression of the podocyte proteins nephrin and podocin.

Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66 Shc

The coagulation protease activated protein C (aPC) confers cytoprotective effects in various in vitro and in vivo disease models, including diabetic nephropathy. The nephroprotective effect may be related to antioxidant effects of aPC. However, the mechanism through which aPC may convey these antioxidant effects and the functional relevance of these properties remain unknown. Here, we show that endogenous and exogenous aPC prevents glomerular accumulation of oxidative stress markers and of the redox-regulating protein p66(Shc) in experimental diabetic nephropathy. These effects were predominately observed in podocytes. In vitro, aPC inhibited glucose-induced expression of p66(Shc) mRNA and protein in podocytes (via PAR-1 and PAR-3) and various endothelial cell lines, but not in glomerular endothelial cells. Treatment with aPC reversed glucose-induced hypomethylation and hyperacetylation of the p66(Shc) promoter in podocytes. The hyperacetylating agent sodium butyrate abolished the suppressive effect of aPC on p66(Shc) expression both in vitro and in vivo. Moreover, sodium butyrate abolished the beneficial effects of aPC in experimental diabetic nephropathy. Inhibition of p66(Shc) expression and mitochondrial translocation by aPC normalized mitochondrial ROS production and the mitochondrial membrane potential in glucose-treated podocytes. Genetic ablation of p66(Shc) compensated for the loss of protein C activation in vivo, normalizing markers of diabetic nephropathy and oxidative stress. These studies identify a unique mechanism underlying the cytoprotective effect of aPC. Activated PC epigenetically controls expression of the redox-regulating protein p66(Shc), thus linking the extracellular protease aPC to mitochondrial function in diabetic nephropathy.

Animal models of nephrotic syndrome

Animal models of proteinuria and nephrotic syndrome are essential tools for studying the mechanisms of action of abnormalities in individual components of the podocyte and glomerular basement membrane. In recent years a variety of in vivo models have been developed to elucidate the function of specific podocyte proteins and their role in the pathogenesis of proteinuria and glomerulosclerosis. In this overview of the animal models currently available we discuss their contribution to our mechanistic understanding and their potential use in screening for novel targeted therapies of steroid-resistant nephrotic syndrome.

Inhibition of nephrin activation by c-mip through Csk–Cbp–Fyn axis plays a critical role in Angiotensin II-induced podocyte damage

It has been demonstrated that nephrin inactivation plays a critical role in Angiotensin II (AngII)-induced podocyte damage both in in vitro and in vivo, but the underlying molecular mechanisms are still unclear. Recently, c-maf inducing protein (c-mip) has been identified as a key component in the molecular pathogenesis of acquired podocyte diseases. In this study, the role of c-mip on AngII-induced nephrin inactivation and podocyte damage was explored in a mouse podocyte cell line. AngII stimulation caused podocyte damage, presenting with a time and dose dependent cell apoptosis increment, and obvious reorganization of actin cytoskeleton, both of which was remarkably prevented by knockdown of c-mip (siCmip). In AngII stimulated podocyte, c-mip and Csk expressions increased obviously at protein level, and nephrin phosphorylation decreased while Cbp phosphorylation increased. AngII-induced Csk increment and nephrin inactivation was remarkably inhibited by siCmip treatment. AngII stimulation increased the interaction of c-mip and Csk, as well as Csk and Cbp. Notably, the binding of Csk to active form pY418 decreased while the binding of Csk to inactive form pY530 of Src kinase Fyn increased in AngII-stimulated podocyte. Nevertheless, c-mip knockdown prevented AngII-induced reduction of pY418 and increase of pY530. In addition, AngII stimulation significantly decreased the expression of phosphor-Akt (Ser473) and antiapoptotic protein Bcl-2, whereas increased the expression of apoptotic proteins caspase-3 and BAD, all of which were prevented by siCmip treatment. Taken together, our results demonstrated that AngII induced nephrin inactivation and podocyte damage by the novel podocyte protein c-mip through Csk–Cbp–Fyn signaling pathway.

Plekhh2, a novel podocyte protein downregulated in human focal segmental glomerulosclerosis, is involved in matrix adhesion and actin dynamics

Pleckstrin homology domain-containing, family H (with MyTH4 domain), member 2 (Plekhh2) is a 1491-residue intracellular protein highly enriched in renal glomerular podocytes for which no function has been ascribed. Analysis of renal biopsies from patients with focal segmental glomerulosclerosis revealed a significant reduction in total podocyte Plekhh2 expression compared to controls. Sequence analysis indicated a putative α-helical coiled-coil segment as the only recognizable domain within the N-terminal half of the polypeptide, while the C-terminal half contains two PH, a MyTH4, and a FERM domain. We identified a phosphatidylinositol-3-phosphate consensus-binding site in the PH1 domain required for Plekhh2 localization to peripheral regions of cell lamellipodia. The N-terminal half of Plekkh2 is not necessary for lamellipodial targeting but mediates self-association. Yeast two-hybrid screening showed that Plekhh2 directly interacts through its FERM domain with the focal adhesion protein Hic-5 and actin. Plekhh2 and Hic-5 coprecipitated and colocalized at the soles of podocyte foot processes in situ and Hic-5 partially relocated from focal adhesions to lamellipodia in Plekhh2-expressing podocytes. In addition, Plekhh2 stabilizes the cortical actin cytoskeleton by attenuating actin depolymerization. Our findings suggest a structural and functional role for Plekhh2 in the podocyte foot processes.

Retinoids Augment the Expression of Podocyte Proteins by Glomerular Parietal Epithelial Cells in Experimental Glomerular Disease

Background/Aims: A decrease in glomerular podocyte number in membranous nephropathy and focal segmental glomerulosclerosis (FSGS) ultimately underlines glomerulosclerosis and the decrease in kidney function. Recent studies have shown that in these diseases, glomerular parietal epithelial cells begin to express proteins considered unique to podocytes, and that these glomerular epithelial transition cells might serve as podocyte progenitors. Because retinoids improve many forms of experimental glomerular disease characterized by podocyte injury and loss, we asked if all-trans retinoic acid (ATRA) induces parietal epithelial cells to express podocyte proteins. Methods: ATRA or vehicle was administered to rats with experimental membranous nephropathy (passive Heymann nephritis model) and mice with experimental FSGS (anti-glomerular antibody model) following the onset of proteinuria. Immunohistochemistry staining of PAX2 (parietal epithelial cell marker), WT-1 (podocyte cell marker), and Ki-67 (proliferation marker) were performed on kidney tissues. Results: Compared to diseased animals receiving vehicle, ATRA statistically significantly increased the number of glomerular transition cells, defined as cells double-staining for PAX2 and WT-1, in membranous nephropathy at weeks 2, 5 and 16, and in FSGS at weeks 1 and 2. This was accompanied by an increase in the number of podocytes compared to diseased controls receiving vehicle. Conclusion: ATRA increases the number of glomerular epithelial transition cells in experimental proteinuric glomerular diseases. Thus, ATRA may provide a useful pharmacologic approach to decipher the mechanisms underlying the possible progenitor role of parietal epithelial cells.