Csk regulates angiotensin II-induced podocyte apoptosis

Increasing data have shown that angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. The mechanism underlying Ang II-induced podocyte apoptosis has not been established. C-terminal Src kinase (Csk) is a cytoplasmic kinase that interacts with scaffolding proteins involved in cell growth, adhesion, and polarization, and the role of Csk in regulating cellular apoptosis has gradually attracted attention. This study evaluates the role of Csk in Ang II-induced podocyte apoptosis. In vivo, Wistar rats were randomly subjected to a normal saline or Ang II infusion. In vitro, we exposed differentiated mouse podocytes to Ang II. Ang II increased Csk expression and induced podocyte apoptosis, stimulated Csk translocation and binding to Caveolin-1, and stimulated decreased Fyn pY416, increased Fyn pY529, and nephrin dephosphorylation. Csk knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn, accompanied by a reduced interaction between Csk and Caveolin-1. These findings indicate that Ang II induces podocyte injury via a Csk-dependent pathway.     

Angiotensin II down-regulates nephrin–Akt signaling and induces podocyte injury: roleof c-Abl

Recent studies have shown that nephrin plays a vital role in angiotensin II (Ang II)–induced podocyte injury and thus contributes to the onset of proteinuria and the progression of renal diseases, but its specific mechanism remains unclear. c-Abl is an SH2/SH3 domain–containing nonreceptor tyrosine kinase that is involved in cell survival and regulation of the cytoskeleton. Phosphorylated nephrin is able to interact with molecules containing SH2/SH3 domains, suggesting that c-Abl may be a downstream molecule of nephrin signaling. Here we report that Ang II–infused rats developed proteinuria and podocyte damage accompanied by nephrin dephosphorylation and minimal interaction between nephrin and c-Abl. In vitro, Ang II induced podocyte injury and nephrin and Akt dephosphorylation, which occurred in tandem with minimal interaction between nephrin and c-Abl. Moreover, Ang II promoted c-Abl phosphorylation and interaction between c-Abl and SH2 domain–containing 5′-inositol phosphatase 2 (SHIP2). c-Abl small interfering RNA (siRNA) and STI571 (c-Abl inhibitor) provided protection against Ang II–induced podocyte injury, suppressed the Ang II-induced c-Abl–SHIP2 interaction and SHIP2 phosphorylation, and maintained a stable level of nephrin phosphorylation. These results indicate that c-Abl is a molecular chaperone of nephrin signaling and the SHIP2-Akt pathway and that the released c-Abl contributes to Ang II–induced podocyte injury.     

Calmodulin-dependent Protein Kinase II/cAMP Response Element-binding Protein/Wnt/β-Catenin Signaling Cascade Regulates Angiotensin II-induced Podocyte Injury and Albuminuria

Angiotensin II (Ang II) plays a pivotal role in promoting podocyte dysfunction and albuminuria, however, the underlying mechanisms have not been fully delineated. In this study, we found that Ang II induced Wnt1 expression and β-catenin nuclear translocation in cultured mouse podocytes. Blocking Wnt signaling with Dickkopf-1 (Dkk1) or β-catenin siRNA attenuated Ang II-induced podocyte injury. Ang II could also induce the phosphorylation of calmodulin-dependent protein kinase (CaMK) II and cAMP response element-binding protein (CREB) in cultured podocytes. Blockade of this pathway with CK59 or CREB siRNA could significantly inhibit Ang II-induced Wnt/β-catenin signaling and podocyte injury. In in vivo studies, administration of Ang II promoted Wnt/β-catenin signaling, aggregated podocyte damage, and albuminuria in mice. CK59 could remarkably ameliorate Ang II-induced podocyte injury and albuminuria. Furthermore, ectopic expression of exogenous Dkk1 also attenuated Ang II-induced podocytopathy in mice. Taken together, this study demonstrates that the CaMK II/CREB/Wnt/β-catenin signaling cascade plays an important role in regulating Ang II-induced podocytopathy. Targeting this signaling pathway may offer renal protection against the development of proteinuric kidney diseases.     

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]     

c-Abl mediates angiotensin II-induced apoptosis in podocytes

Angiotensin II (Ang II) has been reported to cause podocyte apoptosis in rats both in vivo and in vitro studies. However, the underlying mechanisms are poorly understood. In the present study, we investigated the role of the nonreceptor tyrosine kinase c-Abl in Ang II-induced podocyte apoptosis. Male Sprague–Dawley rats in groups of 12 were administered either Ang II (400 kg/kg/min) or Ang II + STI-571 (50 mg/kg/day) by osmotic minipumps. In addition, 12 rats-receiving normal saline served as the control. Glomeruli c-Abl expression was carried out by real time PCR, Western blotting and immunolabeled, and occurrence of apoptosis was carried out by TUNEL staining and transmission electron microscopic analysis. In vitro studies, conditionally immortalized mouse podocytes were treated with Ang II (10^?9–10^?6 M) in the presence or absence of either c-Abl inhibitor, Src-I1, specific c-Abl siRNA, or c-Abl plasmid alone. Quantification of podocyte c-Abl expression and c-Abl phosphorylation at Y245 and Y412 was carried out by real time PCR, Western blotting and immunofluorescence imaging. The nuclear c-Abl and p53 were quantified by co-immunoprecipitation and Western blotting studies. Podocyte apoptosis was analysed by flow cytometry and Hoechst-33342 staining. c-Abl expression was demonstrated in rat kidney podocytes in vivo and cultured mouse podocytes in vitro. Ang II-receiving rats displayed enhanced podocyte c-Abl expression. And Ang II significantly stimulated c-Abl expression in cultured podocytes. Furthermore Ang II upregulated podocyte c-Abl phosphorylation at Y245 and Y412. Ang II also induced an increase of nuclear p53 protein and nuclear c-Abl-p53 complexes in podocytes and podocyte apoptosis. Down-regulation of c-Abl expression by c-Abl inhibitor (Src-I1) as well as specific siRNA inhibited Ang II-induced podocyte apoptosis; conversely, podoctyes transfected with c-Abl plasmid displayed enhanced apoptosis. These findings indicate that c-Abl may mediates Ang II-induced podocyte apoptosis, and inhibition of c-Abl expression can protect podocytes from Ang II-induced injury.     

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.     

Role of nephrin in podocyte injury induced by angiotension II

Objective: To investigate the function of nephrin in podocytes and its relation to proteinuria in kidney diseases, and to study more clearly theoretical basis for the molecular mechanism of losartan anti-proteinuria and the special beneficial effects of losartan on podocyte injury. Methods: Experiment set up control, Ang II and losartan group. Cell morphology was observed perturbation, and using image processing software to analyze the cell body of cell morphology and size of the difference after 8?h, 24?h and 48?h. Detecting nephrin mRNA and protein expression changes by real time PCR (RT-PCR) and western blotting at different time points. Results: Podocyte cell bodies were significantly reduced after Ang II injury (p?<?0.01), losartan directly reduces the rate of apoptotic podocytes induced by Ang. Apoptotic podocytes may related to the decrease of nephrin mRNA and protein expressions, losartan reduced the apoptosis and proteinuria by declining nephrin mRNA and protein expressions. Conclusion: Ang II induced podocyte injury caused abnormal expression and distribution of nephrin in podocytes, losartan maybe maintain the stability of nephrin expression and the integrity of hole diaphragm (SD) structure and function by blocking the signal path, playing a important role in protection mechanisms of anti-proteinuria. Our findings provide some possible clues for further exploring the pharmacological targets to the proteinuria. These novel findings provide new insights into the beneficial effects of losartan on podocytes directly.     

Increased SHP-1 Protein Expression by High Glucose Levels Reduces Nephrin Phosphorylation in Podocytes

Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with nephrin and whether its elevated expression affects the nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with nephrin. Coexpression of nephrin-CD16 and SHP-1 reduced nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat nephrin Tyr¹¹²⁷ and Tyr¹¹⁵² are required to allow SHP-1 interaction with nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2^+/C96Y) compared with control littermate mice (Ins2^+/+), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with nephrin, causing decreased nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.     

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.     

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.     

CD2-associated protein/phosphoinositide 3-kinase signaling has a preventive role in angiotensin II-induced podocyte apoptosis

Angiotensin II (Ang II) works as a paracrine or autocrine cytokine agent to regulate renal functions and promotes podocytes dysfunction directly or indirectly, causing proteinuria. The glomerular slit diaphragm (SD) serves as a size-selective barrier and is linked to the actin-based cytoskeleton by adaptor proteins, including CD2-associated protein (CD2AP). Therefore, damages to CD2AP affect not only the function of the SD, but also directly disrupt the podocyte cytoskeleton, leading to proteinuria. In addition, CD2AP can facilitate the nephrin-induced phosphoinositide 3-kinase (PI3-K)/Akt signaling, which protects podocytes from apoptosis. Here we found that CD2AP staining was located diffusely but predominantly in the peripheral cytoplasm and CD2AP co-localized with nephrin in mouse podocytes; however, Ang II decreased CD2AP staining diffusely and induced a separation from concentrated nephrin. Ang II notably reduced CD2AP expression in time- and concentration-dependent manners, and this was significantly recovered by losartan. Ang II induced podocyte apoptosis in time- and concentration-dependent manners in TUNEL and FACS assays. LY294002, a PI3-K inhibitor, further reduced CD2AP expression and increased podocyte apoptosis, which was augmented by siRNA for CD2AP. Thus, Ang II induces the relocalization and reduction of CD2AP via AT1R, which would cause podocyte apoptosis by the suppression of CD2AP/PI3-K signaling.     

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.     

SIRPα interacts with nephrin at the podocyte slit diaphragm

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in?vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury. Structured digital abstract ? Sirp-alpha?physically interacts?with?Nephrin?by?anti bait coimmunoprecipitation?(View interaction) ? Sirp-alpha?physically interacts?with?Nephrin?by?anti tag coimmunoprecipitation?(View interaction).     

Downregulation of CD2-associated protein impaired the physiological functions of podocytes

Emerging evidences show that CD2-associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P < 0.05). At the same time, cell cycle was arrested in G2/M phase (P < 0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA-transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P < 0.05). Further study revealed disordered distributions of F-actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin-CD2AP signaling pathway.     

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.     

A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase.

Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.     

Actin-depolymerizing Factor Cofilin-1 Is Necessary in Maintaining Mature Podocyte Architecture

Actin dynamics determines podocyte morphology during development and in response to podocyte injury and might be necessary for maintaining normal podocyte morphology. Because podocyte intercellular junction receptor Nephrin plays a role in regulating actin dynamics, and given the described role of cofilin in actin filament polymerization and severing, we hypothesized that cofilin-1 activity is regulated by Nephrin and is necessary in normal podocyte actin dynamics. Nephrin activation induced cofilin dephosphorylation via intermediaries that include phosphatidylinositol 3-kinase, SSH1, 14-3-3, and LIMK in a cell culture model. This Nephrin-induced cofilin activation required a direct interaction between Nephrin and the p85 subunit of phosphatidylinositol 3-kinase. In a similar fashion, cofilin-1 dephosphorylation was observed in a rat model of podocyte injury at a time when foot process spreading is initially observed. To investigate the necessity of cofilin-1 in the glomerulus, podocyte-specific Cfl1 null mice were generated. Cfl1 null podocytes developed normally. However, these mice developed persistent proteinuria by 3 months of age, although they did not exhibit foot process spreading until 8 months, when the rate of urinary protein excretion became more exaggerated. In a mouse model of podocyte injury, protamine sulfate perfusion of the Cfl1 mutant mouse induced a broadened and flattened foot process morphology that was distinct from that observed following perfusion of control kidneys, and mutant podocytes did not recover normal structure following additional perfusion with heparin sulfate. We conclude that cofilin-1 is necessary for maintenance of normal podocyte architecture and for actin structural changes that occur during induction and recovery from podocyte injury.