Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome

The visceral glomerular epithelial cell, also known as the podocyte, plays an important role in the maintenance of renal glomerular function. This cell type is highly specialized and its foot processes together with the interposed slit diaphragm (SD) form the final barrier to urinary protein loss. Effacement of foot processes is associated with the development of proteinuria and—if not reversed in a certain time—with permanent deterioration of the glomerular filter. To maintain an intact glomerular filter barrier, podocyte-podocyte interactions and podocyte interactions with the glomerular basement membrane (GBM) are essential. Recent years have highlighted podocyte functions by unraveling the molecular composition of the SD, but have also clarified the important role of the podocyte actin cytoskeleton, and the podocyte-GBM interaction in the development of foot process (FP) effacement. This review provides an update of podocyte functions with respect to novel podocyte-specific proteins and also focuses on the dynamic interaction between the actin cytoskeleton of podocytes, their cell surface receptors and the GBM.     

Pdlim2 is a novel actin-regulating protein of podocyte foot processes

The slit diaphragm and the apical and basal membrane domains of podocytes are connected to each other by an actin-based cytoskeleton critical to the maintenance of the glomerular filtration barrier. In an effort to discover novel regulatory proteins of the podocyte foot process, we identified and characterized pdlim2, a member of the actin-associated LIM protein subfamily of cytosolic proteins typified by an N-terminal PDZ domain and a C-terminal LIM domain. In the kidney, the pdlim2 protein is highly specific for the glomerulus and podocyte foot processes as shown by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. In cultured podocytes, pdlim2 was associated with stress fibers and cortical actin. Pdlim2 seems to regulate actin dynamics in podocytes since stress fibers were stabilized in its presence. Mechanistically, pdlim2 interacts with two actin-associated podocyte proteins, α-actinin-4 and angiomotin-like-1, as shown by immunoprecipitation and yeast two-hybrid analyses. By semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with minimal change nephrotic syndrome and membranous nephropathy, whereas its expression was unchanged in patients with focal segmental glomerulosclerosis. Hence, pdlim2 is a novel actin-regulating protein of podocyte foot processes that may have a role in the pathogenesis of glomerular diseases.     

The role of podocytes in glomerular pathobiology

Podocytes are unique cells with a complex cellular organization. With respect to their cytoarchitecture, podocytes may be divided into three structurally and functionally different segments: cell body, major processes, and foot processes (FPs). The FPs of neighboring podocytes regularly interdigitate, leaving between them the filtration slits that are bridged by an extracellular structure, known as the slit diaphragm (SD). Podocytes cover the outer aspect of the glomerular basement membrane (GBM). They therefore form the final barrier to protein loss, which explains why podocyte injury is typically associated with marked proteinuria. Chronic podocyte injury may lead to podocyte detachment from the GBM. Our knowledge of the molecular structure of the SD has been remarkably improved in the past few years. Several molecules, including nephrin, CD2AP, FAT, ZO-1, P-cadherin, Podocin, and Neph 1-3 have all been shown to be associated with the SD complex, and some of these molecules are critical for its integrity. Podocytes are injured in many forms of human and experimental glomerular disease. The early events are characterized either by alterations in the molecular composition of the SD without visible changes in morphology or, more obviously, by a reorganization of FP structure with the fusion of filtration slits and the apical displacement of the SD. Based on recent insights into the molecular pathology of podocyte injury, at least four major causes have been identified that lead to the uniform reaction of FP effacement and proteinuria: (1) interference with the SD complex and its lipid rafts; (2) direct interference with the actin cytoskeleton; (3) interference with the GBM or with podocyte-GBM interaction; and (4) interference with the negative surface charge of podocytes. There is also evidence, in focal segmental glomerular sclerosis (FSGS) and in idiopathic nephrotic syndrome in humans and rats, that podocyte damage may be caused by circulating albuminuric factors. Ongoing studies in many laboratories are aiming at an understanding of the dynamic relationship between SD proteins, the actin cytoskeleton, and the dynamics of FP structure in nephrotic syndrome and FSGS. These studies should provide us with a better understanding of the biological mechanism underlying the podocyte response to injury. Such studies will potentially translate into more refined treatment and the prevention of proteinuria and progressive glomerular disease.     

Calcium regulates podocyte actin dynamics

Ca(2+)-mediated remodeling of the actin cytoskeleton is a dynamic process that regulates cell motility through the modulation of rho guanosine triphosphatase (GTPase) signaling. Kidney podocytes are unique, pericyte-like cells with a complex cellular organization consisting of a cell body, major processes, and foot processes (FPs). The FPs form a characteristic interdigitating pattern with FPs of neighboring podocytes, leaving in between filtration slits that are covered by the slit diaphragm (SD). The actin-based FP and the SD form the final barrier to proteinuria. Mutations affecting several podocyte proteins cause disruption of the filtration barrier and rearrangement of the highly dynamic podocyte actin cytoskeleton. Proteins regulating the plasticity of the podocyte actin cytoskeleton are therefore of critical importance for sustained kidney barrier function. Dynamic regulation of the actin-based contractile apparatus in podocyte FPs is essential for sustained kidney filter function. Thus, the podocyte represents an excellent model system to study calcium signaling and actin dynamics in a physiologic context. Here, we discuss the regulation of podocyte actin dynamics by angiotensin or bradykinin-mediated calcium influx and downstream Rho GTPase signaling pathways and how these pathways are operative in other cells including fibroblasts and cancer cells.     

Biochemical composition of the glomerular extracellular matrix in patients with diabetic kidney disease

In the glomeruli, mesangial cells produce mesangial matrix while podocytes wrap glomerular capillaries with cellular extensions named foot processes and tether the glomerular basement membrane (GBM). The turnover of the mature GBM and the ability of adult podocytes to repair injured GBM are unclear. The actin cytoskeleton is a major cytoplasmic component of podocyte foot processes and links the cell to the GBM. Predominant components of the normal glomerular extracellular matrix (ECM) include glycosaminoglycans, proteoglycans, laminins, fibronectin-1, and several types of collagen. In patients with diabetes, multiorgan composition of extracellular tissues is anomalous, including the kidney, so that the constitution and arrangement of glomerular ECM is profoundly altered. In patients with diabetic kidney disease (DKD), the global quantity of glomerular ECM is increased. The level of sulfated proteoglycans is reduced while hyaluronic acid is augmented, compared to control subjects. The concentration of mesangial fibronectin-1 varies depending on the stage of DKD. Mesangial type III collagen is abundant in patients with DKD, unlike normal kidneys. The amount of type V and type VI collagens is higher in DKD and increases with the progression of the disease. The GBM contains lower amount of type IV collagen in DKD compared to normal tissue. Further, genetic variants in the α3 chain of type IV collagen may modulate susceptibility to DKD and end-stage kidney disease. Human cellular models of glomerular cells, analyses of human glomerular proteome, and improved microscopy procedures have been developed to investigate the molecular composition and organization of the human glomerular ECM.     

F-actin fiber distribution in glomerular cells: structural and functional implications

BACKGROUND: Glomerular distention is associated with cellular mechanical strain. In addition, glomerular distention/contraction is assumed to influence the filtration rate through changes in filtration surface area. A contractile cytoskeleton in podocytes and mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. In this study, the glomerular cell distribution of F-actin and vimentin filaments was studied in normal control and nine-month streptozotocin-diabetic rats. Results in situ were compared with observations in tissue culture. METHODS: Microdissected rat glomeruli were perfused to obtain a physiological 25% glomerular expansion over the basal value. Fixation was done without change in glomerular volume. Dual fluorescent labeling of F-actin and vimentin was carried out, and samples were examined by confocal laser scanning microscopy. RESULTS: The podocyte cell bodies and their cytoplasmic projections, including the foot processes, contained bundles of vimentin-containing fibers. Except for a thin layer at the base of foot processes, they did not demonstrate F-actin. While mesangial cells in culture had F-actin as long stress fibers resembling tense cables, mesangial cells stretched in situ contained a maze of short tortuous F-actin fibers organized in bundles that often encircled vascular spaces. This fibrillar organization was disrupted in diabetic glomeruli. CONCLUSION: Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.     

Podocyte directed therapy of nephrotic syndrome—can we bring the inside out?

Several of the drugs currently used for the treatment of glomerular diseases are prescribed for their immunotherapeutic or anti-inflammatory properties, based on the current understanding that glomerular diseases are mediated by immune responses. In recent years our understanding of podocytic signalling pathways and the crucial role of genetic predispositions in the pathology of glomerular diseases has broadened. Delineation of those signalling pathways supports the hypothesis that several of the medications and immunosuppressive agents used to treat glomerular diseases directly target glomerular podocytes. Several central downstream signalling pathways merge into regulatory pathways of the podocytic actin cytoskeleton and its connection to the slit diaphragm. The slit diaphragm and the cytoskeleton of the foot process represent a functional unit. A breakdown of the cytoskeletal backbone of the foot processes leads to internalization of slit diaphragm molecules, and internalization of slit diaphragm components in turn negatively affects cytoskeletal signalling pathways. Podocytes display a remarkable ability to recover from complete effacement and to re-form interdigitating foot processes and intact slit diaphragms after pharmacological intervention. This ability indicates an active inside-out signalling machinery which stabilizes integrin complex formations and triggers the recycling of slit diaphragm molecules from intracellular compartments to the cell surface. In this review we summarize current evidence from patient studies and model organisms on the direct impact of immunosuppressive and supportive drugs on podocyte signalling pathways. We highlight new therapeutic targets that may open novel opportunities to enhance and stabilize inside-out pathways in podocytes.     

Activation of adenosine 2A receptors preserves structure and function of podocytes

Adenosine 2A receptor (A(2A)R) activation was recently shown to be renoprotective in diabetic nephropathy. A(2A)R are found in glomeruli and have been shown to associate with the podocyte cytoskeletal protein alpha-actinin-4, but the effect of their activation on podocyte structure and function is unknown. Podocyte injury was induced in C57BL/6 mice with puromycin aminonucleoside, and the selective A(2A)R agonist ATL313 was found to attenuate the resulting albuminuria and foot process fusion. The selective A(2A)R antagonist ZM241385 reversed the effects of ATL313. In vitro, A(2A)R mRNA and protein were expressed in a conditionally immortalized podocyte cell line, and A(2A)R-like immunoreactivity co-localized with the actin cytoskeleton. Treatment with ATL313 also blocked the increased podocyte permeability to albumin and disruption of the actin cytoskeleton that accompanied puromycin aminonucleoside-induced injury in vitro. ATL313 was ineffective, however, in the presence of the A(2A)R antagonist and in A(2A)R-deficient podocytes. It was concluded that A(2A)R activation reduces glomerular proteinuria, at least in part, by preserving the normal structure of podocyte foot processes, slit diaphragms, and actin cytoskeleton.     

Podocyte-specific loss of cdc42 leads to congenital nephropathy

Rho family GTPases are molecular switches best known for their pivotal role in dynamic regulation of the actin cytoskeleton. The prototypic members of this family are Cdc42, Rac1, and RhoA; these GTPases contribute to the breakdown of glomerular filtration and the resultant proteinuria, but their functions in normal podocyte physiology remain poorly understood. Here, mice lacking Cdc42 in podocytes developed congenital nephropathy and died as a result of renal failure within 2 weeks after birth. In contrast, mice lacking Rac1 or RhoA in podocytes were overtly normal and lived to adulthood. Kidneys from Cdc42-mutant mice exhibited protein-filled microcysts with hallmarks of collapsing glomerulopathy, as well as extensive effacement of podocyte foot processes with abnormal junctional complexes. Furthermore, we observed aberrant expression of several podocyte markers and cell polarity proteins in the absence of Cdc42, indicating a disruption of the slit diaphragm. Kidneys from Rac1- and RhoA-mutant mice, however, had normal glomerular morphology and intact foot processes. A nephrin clustering assay suggested that Cdc42 deficiency, but not Rac1 or RhoA deficiency, impairs the polymerization of actin at sites of nephrin aggregates. Taken together, these data highlight the physiological importance of Cdc42, but not Rac1 or RhoA, in establishing podocyte architecture and glomerular function.     

From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy

Nephropathy is a major complication of diabetes. Alterations of mesangial cells have traditionally been the focus of research in deciphering molecular mechanisms of diabetic nephropathy. Injury of podocytes, if recognized at all, has been considered a late consequence caused by increasing proteinuria rather than an event inciting diabetic nephropathy. However, recent biopsy studies in humans have provided evidence that podocytes are functionally and structurally injured very early in the natural history of diabetic nephropathy. The diabetic milieu, represented by hyperglycemia, nonenzymatically glycated proteins, and mechanical stress associated with hypertension, causes downregulation of nephrin, an important protein of the slit diaphragm with antiapoptotic signaling properties. The loss of nephrin leads to foot process effacement of podocytes and increased proteinuria. A key mediator of nephrin suppression is angiotensin II (ANG II), which can activate other cytokine pathways such as transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) systems. TGF-beta1 causes an increase in mesangial matrix deposition and glomerular basement membrane (GBM) thickening and may promote podocyte apoptosis or detachment. As a result, the denuded GBM adheres to Bowman's capsule, initiating the development of glomerulosclerosis. VEGF is both produced by and acts upon the podocyte in an autocrine manner to modulate podocyte function, including the synthesis of GBM components. Through its effects on podocyte biology, glomerular hemodynamics, and capillary endothelial permeability, VEGF likely plays an important role in diabetic albuminuria. The mainstays of therapy, glycemic control and inhibition of ANG II, are key measures to prevent early podocyte injury and the subsequent development of diabetic nephropathy.     

α‐Actinin‐4 is involved in the process by which dexamethasone protects actin cytoskeleton stabilization from adriamycin‐induced podocyte injury

Aim: Glucocorticoid therapy has been used in childhood nephrotic syndrome since the 1950s, where the characteristic change is effacement of the actin‐rich foot process of glomerular podocytes. Recent studies have shown that glucocorticoids, in addition to their general immunosuppressive and anti‐inflammatory effects, have a direct effect on podocytes, regulate some apoptotic factors, and increase the stability of actin filaments. However, the precise mechanism(s) underlying the protective effects of glucocorticoids on podocytes remain unclear. It is known that adriamycin (ADR) can induce podocyte foot process effacement and trigger massive proteinuria in rodent models. However, few reports have examined the direct role of ADR in podocyte actin rearrangement in vitro. In this study, we investigated how ADR directly induced podocyte actin cytoskeleton rearrangement and further analyzed how dexamethasone prevented such injury. Methods: We used confocal microscopy to assess podocyte actin rearrangement. Western blot analysis and real‐time polymerase chain reaction were performed to measure the protein and mRNA levels of α‐actinin‐4. Results: We demonstrated that there was a time‐dependent ADR‐induced podocyte actin rearrangement with less than 12 h of ADR treatment in cultured podocytes. Dexamethasone could protect podocytes from ADR‐induced injury and also stabilize the expression of α‐actinin‐4. Conclusion: This study showed that dexamethasone had direct effects on podocytes: α‐actinin‐4 may be one of the potential target molecules.     

The glomerular epithelial cell anti-adhesin podocalyxin associates with the actin cytoskeleton through interactions with ezrin.

During development, renal glomerular epithelial cells (podocytes) undergo extensive morphologic changes necessary for creation of the glomerular filtration apparatus. These changes include formation of interdigitating foot processes, replacement of tight junctions with slit diaphragms, and the concomitant opening of intercellular urinary spaces. It was postulated previously and confirmed recently that podocalyxin, a sialomucin, plays a major role in maintaining the urinary space open by virtue of the physicochemical properties of its highly negatively charged ectodomain. This study examined whether the highly conserved cytoplasmic tail of podocalyxin also contributes to the unique organization of podocytes by interacting with the cytoskeletal network found in their cell bodies and foot processes. By immunocytochemistry, it was shown that podocalyxin and the actin binding protein ezrin are co-expressed in podocytes and co-localize along the apical plasma membrane, where they form a co-immunoprecipitable complex. Selective detergent extraction followed by differential centrifugation revealed that some of the podocalyxin cosediments with actin filaments. Moreover, its sedimentation is dependent on polymerized actin and is mediated by complex formation with ezrin. Once formed, podocalyxin/ezrin complexes are very stable, because they are insensitive to actin depolymerization or inactivation of Rho kinase, which is known to be necessary for regulation of ezrin and to mediate Rho-dependent actin organization. These data indicate that in podocytes, podocalyxin is complexed with ezrin, which mediates its link to the actin cytoskeleton. Thus, in addition to its ectodomain, the cytoplasmic tail of podocalyxin also likely contributes to maintaining the unique podocyte morphology.     

Partial rescue of glomerular laminin alpha5 mutations by wild-type endothelia produce hybrid glomeruli

Both endothelial cells and podocytes are sources for laminin alpha1 at the inception of glomerulogenesis and then for laminin alpha5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin alpha5, laminin alpha5beta2gamma1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin alpha5 mutants, alpha5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: alpha5 was found only on the inner endothelial half of GBM, whereas alpha1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin alpha5 were quantified: Hybrid GBM contained approximately 50% the normal alpha5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin alpha5.     

Glomerular expression of dystroglycans is reduced in minimal change nephrosis but not in focal segmental glomerulosclerosis

Extensive flattening of podocyte foot processes and increased permeability of the glomerular capillary filter are the major pathologic features of minimal change nephrosis (MCN) and focal segmental glomerulosclerosis (FSGS). Adhesion proteins anchor and stabilize podocytes on the glomerular basement membrane (GBM), and presumably are involved in the pathogenesis of foot process flattening. Thus far, ao3 P,-integrin was localized to basal cell membrane domains. In this report, ao- and 3-dystroglycan (DG) were detected at precisely the sa-ne location by immunoelectron microscopy. and the presence of ac- and /-DG chains was confirmed by immunoblotting on isolated human glomeruli. Because the major DG binding partners in the GBM (laminin, agrin, perlecan), and the intracellular dystrophin analogue utrophin are also present in glomeruli, it appears that podocytes adhere to the GBM via DG complexes, similar to muscle fibers in which actin is linked via dystrophin and DG to the extracellular matrix. As with muscle cells, it is therefore plausible that podocytes use precisely actin-guided DG complexes at their "soles" to actively govern the topography of GBM matrix proteins. Expression of the a//3-DG complex was reported to be reduced in muscular dystrophies. and therefore a search for similar pathologic alterations in archival kidney biopsies from patients with MCN (it = 16) and FSGS (ni = 8) was conducted by quantitative immunoelectron microscopy. The density of a-DG on the podocyte's soles was significantly reduced to 25% in MCN, whereas it was not different in normal kidneys and FSGS. The expression of 3-DG was reduced to >50% in MCN, and was slightly increased in FSGS. Levels of DG expression returned to normal in MCN after steroid treatment (7 = 4). Expression of /3-integrin remained at normal levels in all conditions. These findings point to different potentially pathogenic mechanisms of foot process flattening in MCN and FSGS.     

FSGS-associated alpha-actinin-4 (K256E) impairs cytoskeletal dynamics in podocytes

Mutations in the ACTN4 gene, encoding the actin crosslinking protein alpha-actinin-4, are associated with a familial form of focal segmental glomerulosclerosis (FSGS). Mice with podocyte-specific expression of K256E alpha-actinin-4 develop foot process effacement and glomerulosclerosis, highlighting the importance of the cytoskeleton in podocyte structure and function. K256E alpha-actinin-4 exhibits increased affinity for F-actin. However, the downstream effects of this aberrant binding on podocyte dynamics remain unclear. Wild-type and K256E alpha-actinin-4 were expressed in cultured podocytes via adenoviral infection to determine the effect of the mutation on alpha-actinin-4 subcellular localization and on cytoskeletal-dependent processes such as adhesion, spreading, migration, and formation of foot process-like peripheral projections. Wild-type alpha-actinin-4 was detected primarily in the Triton-soluble fraction of podocyte lysates and localized to membrane-associated cortical actin and focal adhesions, with some expression along stress fibers. Conversely, K256E alpha-actinin-4 was detected predominantly in the Triton-insoluble fraction, was excluded from cortical actin, and localized almost exclusively along stress fibers. Both wild-type and K256E alpha-actinin-4-expressing podocytes adhered equally to an extracellular matrix (collagen-I). However, podocytes expressing K256E alpha-actinin-4 showed a reduced ability to spread and migrate on collagen-I. Lastly, K256E alpha-actinin-4 expression reduced the mean number of actin-rich peripheral projections. Our data suggest that aberrant sequestering of K256E alpha-actinin-4 impairs podocyte spreading, motility, and reduces the number of peripheral projections. Such intrinsic cytoskeletal derangements may underlie initial podocyte damage and foot process effacement encountered in ACTN4-associated FSGS.     

Mechanisms of the proteinuria induced by Rho GTPases

Podocytes are highly differentiated cells that play an important role in maintaining glomerular filtration barrier integrity; a function regulated by small GTPase proteins of the Rho family. To investigate the role of Rho A in podocyte biology, we created transgenic mice expressing doxycycline-inducible constitutively active (V14 Rho) or dominant-negative Rho A (N19 Rho) in podocytes. Specific induction of either Rho A construct in podocytes caused albuminuria and foot process effacement along with disruption of the actin cytoskeleton as evidenced by decreased expression of the actin-associated protein synaptopodin. The mechanisms of these adverse effects, however, appeared to be different. Active V14 Rho enhanced actin polymerization, caused a reduction in nephrin mRNA and protein levels, promoted podocyte apoptosis, and decreased endogenous Rho A levels. In contrast, the dominant-negative N19 Rho caused a loss of podocyte stress fibers, did not alter the expression of either nephrin or Rho A, and did not cause podocyte apoptosis. Thus, our findings suggest that Rho A plays an important role in maintaining the integrity of the glomerular filtration barrier under basal conditions, but enhancement of Rho A activity above basal levels promotes podocyte injury.     

Sensitizing the Slit Diaphragm with TRPC6 ion channels

Physiologic permeability of the glomerular capillary depends on the normal structure of podocyte foot processes forming a functioning slit diaphragm in between. Mutations in several podocyte genes as well as specific molecular pathways have been identified as the cause for progressive kidney failure with urinary protein loss. Podocyte injury is a hallmark of glomerular disease, which is generally displayed by the rearrangement of the podocyte slit diaphragm and the actin cytoskeleton. Recent studies demonstrate a unique role for the Ca(2+)-permeable ion channel protein TRPC6 as a regulator of glomerular ultrafiltration. In both genetic and acquired forms of proteinuric kidney disease, dysregulation of podocyte TRPC6 plays a pathogenic role. This article illustrates how recent findings add to emerging concepts in podocyte biology, particularly mechanosensation and signaling at the slit diaphragm.     

The role of podocytes in proteinuria

SUMMARY:   Glomerular visceral epithelial cells, also known as podocytes, are highly specialized epithelial cells that cover the outer layer of the glomerular basement membrane. Podocytes consist of cell bodies, major processes and foot processes (FP) of neighbouring cells, with the filtration slits bridged by the slit membrane between them. The function of podocytes is largely based on their specialized cell architecture and functions such as stabilization of glomerular capillaries and participation in the barrier function of the glomerular filter. Therefore, they form the final barrier to protein loss, which explains why podocyte injury is typically associated with marked proteinuria. Under pathological conditions, podocytes exhibit various changes. Among these changes, FP effacement represents the most characteristic change in cell shape of podocytes. FP effacement is dependent on disruption of the actin cytoskeletal network in the podocytes, The mechanisms of organization and re-organization of actin in the FP of podocytes are discussed in this review.     

Angiopoietin-like protein 3 induces podocytes actin rearrangement via integrin beta 3

Objective To explore whether Angiopoietin-like protein 3 (ANGPTL3) is involved in podocyte actin rearrangement, and to analyze whether integrin β3 signal pathway is a key in ANGPTL3 inducing actin rearrangement. Methods The cultured podocytes were divided into six groups: wild type, ADR treated, ADR+Dex, MOCK, ANGPTL3- cDNA, miRNA, and AD +miRNA group. (1) We observed actin cytoskeleton using Invitrogen reagents with confocal microscopy; (2) Actin cytoskeleton after blocking β3 on podocytes was; (3) The expression of total FAK and p-FAK was through Western blotting. Results (1) The wild type podocyte's cytoskeleton is arranged orderly. After ADR treatment, podocyte's actin are rearranged and weaken (P﹤0.05). There was no significant difference in actin arrangement between knock-down and MOCK group. In ANGPTL3-cDNA group the podocyte actin was also significantly rearranged; on the contrary, in miRNA+ADR group, the actin rearrangement never obviously happened (P﹤0.05). (2) Over-expression of ANGPTL3 podocytes blocked integrin β3 did not happen actin rearrangement. (3) The expression of p-FAK significantly increased in over-expression ANGPTL3 podocytes. Conclusion ANGPTL3 is a key in inducing actin rearrangement. Intergrin β3 maybe a central pathway in ANGPTL3's role with podocytes.