Podocyte Actin Research Articles

Excessive arachidonic acid induced actin bunching remodeling and podocyte injury via a PKA-c-Abl dependent pathway

Recent studies have shown that serum secretory phospholipase A2 group IB (sPLA2-IB) is associated with proteinuric kidney diseases and plays a pivotal role in podocyte injury via its natural receptor. Arachidonic acid (AA), as a major metabolite of sPLA2-IB, regulates the actin bungling remodeling and contributes to the podocyte injury. However, the underlying mechanism of AA in the regulation of podocyte actin remodeling and human podocyte injury is unclear. Here, we reported that AA induced F-actin cytoskeletal ring formation and promoted protein kinase A (PKA), nephrin and c-Abl phosphorylation. Moreover, AA promoted c-Abl translocation from the nucleus to the cytoplasm and increased the recruitment of c-Abl to p-nephrin by the interaction between them. H89 (PKA inhibitor) provided protection against AA-induced F-actin bunching remodeling, down-regulated nephrin phosphorylation, and suppressed the c-Abl translocation and activation. STI571 (c-Abl inhibitor) also improved the AA associated F-actin bunching remodeling. In addition, H89 and STI571 both alleviated apoptosis and adhesion damage of podocyte. These results indicate that an excess of AA treatment is detrimental to the podocyte actin cytoskeleton and promotes podocyte injury due to the activation of PKA-c-Abl signaling.

The role of vasodilator-stimulated phosphoprotein in podocyte functioning.

Vasodilator-stimulated phosphoprotein (VASP) is a 39-kDa protein belonging to the Ena/VASP protein family, which is involved in adhesion, migration, cell-cell interaction, and regulation of pathways connected with actin cytoskeleton remodeling. VASP is phosphorylated at Tyr39, Ser157, Ser239, Thr278, and Ser322 mainly by tyrosine kinase Abl, cAMP-dependent protein kinase, protein kinase G, AMP-activated protein kinase, and protein kinase D1, respectively. VASP phosphorylation, as a regulator of actin dynamics, may lead to impaired reorganization of the podocyte actin cytoskeleton not only by indirect interaction of VASP with actin but also by regulation of other signaling pathways. A few studies have shown that VASP participates in the development of renal diseases and mediates podocyte movement through its interaction with proteins of the slit diaphragm. VASP phosphorylation may cause reduced actin filament assembly in podocytes and mediate disturbances in regulation of filtration barrier permeability as a consequence of podocyte foot process effacement. In this paper, we describe the role of VASP in podocyte function, mainly in the context of actin dynamics and glomerular filtration barrier permeability. In addition, we discuss the involvement of VASP and its phosphorylated forms in the development of kidney diseases.

Disruption of podocyte cytoskeletal biomechanics by dasatinib leads to nephrotoxicity

Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.

Increased levels of serum pigment epithelium-derived factor aggravate proteinuria via induction of podocyte actin rearrangement

PurposeTo assess the role of serum pigment epithelium-derived factor (PEDF) in the occurrence and development of proteinuria and renal dysfunction and determine its relevant signaling pathway.MethodsWe analyzed serum PEDF, creatinine, the urinary albumin-to-creatinine ratio, and renal morphology of normal or streptozotocin (STZ)-induced diabetic mice, before and after treatment with PEDF. In vitro, podocytes were stimulated with PEDF under normal or high-glucose conditions; permeability was measured by the transwell assay with fluorescein isothiocyanate (FITC)-dextran; and F-actin cytoskeleton was analyzed by phalloidin staining. Apoptosis was assessed by flow cytometry. RhoA activity and ROCK1, ZO-1, nephrin, and podocin levels were detected by Western blotting.ResultsDiabetic mice exhibited a high serum PEDF level. In vivo, elevated serum PEDF led to proteinuria, increased serum creatinine, and podocyte foot process fusion in normal or diabetic mice. In vitro, both high-glucose and PEDF stimulation activated the RhoA/ROCK1 pathway in podocytes and promoted cell permeability, F-actin rearrangement, and apoptosis. Inhibition of RhoA/ROCK1 alleviated the damage from these effects.ConclusionsElevated serum PEDF aggravates the development of proteinuria and renal dysfunction by inducing F-actin arrangement, foot process fusion, and apoptosis of podocytes in both normal and diabetic mice, and this effect may be mediated by activation of the RhoA/ROCK1 pathway.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

SHROOM3-FYN Interaction Regulates Nephrin Phosphorylation and Affects Albuminuria in Allografts.

We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD.

Eucalyptol Inhibits Advanced Glycation End Products‐Induced Disruption of Podocyte Slit Junctions by Suppressing Rage‐Erk‐C‐Myc Signaling Pathway

The maintenance of interpodocyte slit diaphragm is critical in the sieving function of glomerular filtration barrier. Eucalyptol is a natural constituent in aromatic plants with antioxidant properties. This study investigates whether and how eucalyptol inhibits podocyte slit diaphragm malfunction in glucose-exposed podocytes and diabetic mouse kidneys. Podocytes were incubated in media containing 33mm glucose with 1-20μm eucalyptol. The in vivo model employed db/db mice orally administrated with 10mgkg-1 eucalyptol. Nontoxic eucalyptol enhanced podocyte expression of nephrin, podocin, FAT-1, CD2AP, and α-actinin-4 diminished by glucose. Oral administration of eucalyptol augmented the induction of the slit diaphragm proteins, α-actinin-4, and integrin β1 in diabetic kidneys, and ameliorated glomerular fibrosis and foot process effacement. Eucalyptol counteracted the receptor of advanced glycation end products (RAGE) induction in podocytes with glucose or AGE-BSA, and elevated the reduction of the slit diaphragm proteins by AGE-BSA. Eucalyptol attenuated the RAGE induction and AGE accumulation in diabetic kidneys. The blockade of ERK-c-Myc signaling enhanced the nephrin and CD2AP expression downregulated in AGE-exposed podocytes. These results indicate that eucalyptol blocked glucose-induced AGE-RAGE axis and podocyte injury through disturbing RAGE-ERK-c-Myc signaling. Eucalyptol may be a potent agent antagonizing diabetes-associated malformation of interpodocyte slit junction and podocyte actin cytoskeleton.

Loss of ezrin expression reduced the susceptibility to the glomerular injury in mice

Ezrin is highly expressed in glomerular podocytes and is reported to form a multi-protein complex with scaffold protein Na+/H+ exchanger regulatory factor 2 (NHERF2) and podocalyxin, a major sialoprotein. Podocalyxin-knockout mice died within 24 h of birth with anuric renal failure, whereas NHERF2-knockout mice show no apparent changes in the glomerular functions. However, the physiological roles of ezrin in glomerular podocytes remain unclear. Here, we investigated the importance of ezrin in the regulation of glomerular podocyte function using ezrin-knockdown mice (Vil2kd/kd). The Vil2kd/kd mice did not exhibit apparent glomerular dysfunction, morphological defects or abnormal localisation of podocalyxin and NHERF2 in podocytes. Thus, we investigated the influence of ezrin defects on Rho-GTPase activity, as ezrin interacts with the Rho-GTPase dissociation inhibitor (Rho-GDI), which plays a key role in the regulation of podocyte actin organisation. In Vil2kd/kd glomeruli, Rac1 activity was significantly reduced compared to wildtype (WT) glomeruli at baseline. Furthermore, Vil2kd/kd mice showed reduced susceptibility to glomerular injury. In WT glomeruli, Rac1 activity was enhanced in nephrotic conditions, but remained at baseline levels in Vil2kd/kd glomeruli, suggesting that loss of ezrin protects podocytes from injury-induced morphological changes by suppressing Rac1 activation.

Tropomyosin‐related kinase C (TrkC) enhances podocyte migration by ERK‐mediated WAVE2 activation

Podocyte malfunction is central to glomerular diseases and is marked by defective podocyte intercellular junctions and actin cytoskeletal dynamics. Podocytes share many morphologic features with neurons, so that similar sets of proteins appear to regulate cell process formation. One such protein is the tropomyosin-related kinase C (TrkC). TrkC deficiency in mice leads to proteinuria as a surrogate of defective kidney filter function. Activation of endogenous TrkC by its ligand neurotrophin-3 resulted in increased podocyte migration-a surrogate of podocyte actin dynamics in vivo. Employing a mutagenesis approach, we found that the Src homologous and collagen-like (Shc) binding site Tyr516 within the TrkC cytoplasmic domain was necessary for TrkC-induced migration of podocytes. TrkC activation led to a mobility shift of Wiskott-Aldrich syndrome family verprolin-homologous protein (WAVE)-2 which is known to orchestrate Arp2/3 activation and actin polymerization. Chemical inactivation of Erk or mutagenesis of 2 of 4 known Erk target sites within WAVE2, Thr346 and Ser351, abolished the TrkC-induced WAVE2 mobility shift. Knockdown of WAVE2 by shRNA abolished TrkC-induced podocyte migration. In summary, TrkC signals to the podocyte actin cytoskeleton to induce migration by phosphorylating WAVE2 Erk dependently. This signaling mechanism may be important for TrkC-mediated cytoskeletal dynamics in podocyte disease.-Gromnitza, S., Lepa, C., Weide, T., Schwab, A., Pavenstädt, H., George, B. Tropomyosin-related kinase C (TrkC) enhances podocyte migration by ERK-mediated WAVE2 activation.

Transcriptomic analysis uncovers novel synergistic mechanisms in combination therapy for lupus nephritis

A recent clinical study showed that combination therapy consisting of mycophenolate mofetil, tacrolimus and steroids was shown to be more effective in achieving complete remission in patients with severe forms of lupus nephritis than conventional therapy consisting of intravenous cyclophosphamide and steroids. To explore the underlying molecular and cellular mechanisms of increased efficacy of the combination therapy regimen, we employed a mouse model of lupus nephritis, MRL/lpr mice, and treated them with monotherapies of prednisone, mycophenolate mofetil, or tacrolimus, or with their combination. Consistent with previous clinical findings, combination therapy markedly improved renal outcome compared to the monotherapies in mice with lupus nephritis. Transcriptomic analysis of their kidneys revealed distinct molecular pathways that were differentially regulated in combination therapy versus monotherapies. Combination therapy not only provided additive immunosuppressive effects, but also induced gene expression and molecular pathways to confer enhanced renoprotection. Specifically, combination therapy inhibited TLR7 expression in the kidneys of mice with lupus nephritis; combination of tacrolimus and mycophenolate mofetil led to better stabilization of the podocyte actin cytoskeleton through the reciprocal regulation of RhoA and Rac1 activities. Combination therapy strongly suppressed the IL-6/Stat3 pathway. These findings were further validated in renal biopsy samples from patients with lupus nephritis before and after treatments with mycophenolate mofetil, tacrolimus or combination therapy. Thus, our study further supports the earlier clinical finding and further provides insights into the molecular basis for increased efficacy of combination therapy.

Mutations in INF2 may be associated with renal histology other than focal segmental glomerulosclerosis

In 2010, INF2 mutations were associated with autosomal-dominant focal segmental glomerulosclerosis (FSGS), clinically presenting with moderate proteinuria in adolescence. However, in the meantime, cases with more severe clinical courses have been described, including progression to end-stage renal disease (ESRD) during childhood. INF2 mutations in patients with isolated FSGS are clustered in exons 2 to 4, encoding the diaphanous inhibitory domain, involved in the regulation of the podocyte actin cytoskeleton. We report a family with 14 affected individuals (autosomal-dominant mode of inheritance), most of whom presented with nephrotic-range proteinuria, hypertension, and progressive renal failure. Four members received a kidney transplant without disease recurrence. Two patients underwent renal biopsy with the result of minimal-change glomerulopathy and IgA nephropathy respectively. We performed mutational analysis of ACTN4, CD2AP, COQ6, INF2, LAMB2, NPHS1, NPHS2, PLCE1, TRPC6, and WT1 in the index patient by next-generation sequencing. Additionally, in 6 affected and 2 unaffected family members target diagnostics were performed. We identified a novel heterozygous mutation c.490G>C (p.(Ala164Pro) in exon 3 of the INF2 gene in the index patient and 6 additionally examined affected family members. In silico analysis predicted it as "probably damaging". Additionally, three patients and 2 unaffected relatives harbored a novel heterozygous variant in ACTN4 (c.1149C>G, p.(Ile383Met)) with uncertain pathogenicity. Mutations in INF2 are associated with familial proteinuric diseases - irrespective of the presence of FSGS and in the case of rapid disease progression. Therefore, mutational analysis should be considered in patients with renal histology other than FSGS and severe renal phenotype.

Injury-induced actin cytoskeleton reorganization in podocytes revealed by super-resolution microscopy.

The architectural integrity of tissues requires complex interactions, both between cells and between cells and the extracellular matrix. Fundamental to cell and tissue homeostasis are the specific mechanical forces conveyed by the actomyosin cytoskeleton. Here we used super-resolution imaging methods to visualize the actin cytoskeleton in the kidney glomerulus, an organized collection of capillaries that filters the blood to make the primary urine. Our analysis of both mouse and human glomeruli reveals a network of myosin IIA-containing contractile actin cables within podocyte cell bodies and major processes at the outer aspects of the glomerular tuft. These likely exert force on an underlying network of myosin IIA-negative, noncontractile actin fibers present within podocyte foot processes that function to both anchor the cells to the glomerular basement membrane and stabilize the slit diaphragm against the pressure of fluid flow. After injuries that disrupt the kidney filtration barrier and cause foot process effacement, the podocyte's contractile actomyosin network relocates to the basolateral surface of the cell, manifesting as sarcomere-like structures juxtaposed to the basement membrane. Our findings suggest a new model of the podocyte actin cytoskeleton in health and disease and suggest the existence of novel mechanisms that regulate podocyte architecture.

Podocytes and the quest for precision medicines for kidney diseases

In this review, I describe a 30-year journey in the quest for precision medicines for patients with kidney diseases. In 1987, when I started my reseach career, most scientists studying glomerular disease biology were focused on mesangial cells. The crucial role of the podocyte in many kidney diseases characterized by proteinuria, including focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, had not yet been recognized. We were not aware of genetic causes or drivers of kidney diseases nor of molecular markers and cell culture systems for mechanistic studies of podocyte biology. Tools for generating podocyte-specific knockout mice did not exist, and the key role of the podocyte actin cytoskeleton in the pathogenesis of proteinuria had not yet been identified. Clinically, treatment options for proteinuric kidney diseases were empiric, non-specific, and restricted to steroids and cyclosporine, without an understanding of their underlying mechanism of action. Since then, we have come a long way: a host of genetic causes for FSGS affecting podocytes has been identified, and with the advent of next generation sequencing approaches, the number of genetic causes continues to increase. Thinking "outside the box," empowered me to turn my attention to podocytes, develop the first differentiated podocyte cell culture system, and pioneer studies on the critical role of the podocyte actin cytoskeleton. Now, with the advent of iPSCs, we can build on these efforts by generating human podocytes and kidney organoids from patient cells, which, in combination with CRISPR-Cas9 gene editing and big data analyses, represent important next generation tools for bringing urgently needed precision medicines to patients with kidney disease. These new directions in kidney research should also increase the feasibility of much needed clinical trials in the kidney space. From Heidelberg to Boston, it has been an amazing scientific adventure. I will close with my thoughts about the path forward in making precision medicines for kidney diseases a reality.

A High-Content Screening Technology for Quantitatively Studying Podocyte Dynamics.

Podocytes form the visceral layer of a kidney glomerulus and express a characteristic octopus-like cellular architecture specialized for the ultrafiltration of blood. The cytoskeletal dynamics and structural elasticity of podocytes rely on the self-organization of highly interconnected actin bundles, and the maintenance of these features is important for the intact glomerular filtration. Development of more differentiated podocytes in culture has dramatically increased our understanding of the molecular mechanisms regulating podocyte actin dynamics. Podocytes are damaged in a variety of kidney diseases, and therapies targeting podocytes are being investigated with increasing efforts. Association between podocyte damage and disease severity-or between podocyte recovery and the performance of therapeutic molecules-have been the venues of research for years. In this perspective, more standardized high--content screening has emerged as a powerful tool for visualization and analysis of podocyte morphology. This high-throughput fluorescence microscopy technique is based on an automated image analysis with simultaneous detection of various phenotypes (multiplexing) across multiple phenotypic parameters (multiparametric). Here, we review the principles of high-content screening technology and summarize efforts to carry out small compound screen using podocytes.

Non-muscle myosin-IIA is critical for podocyte f-actin organization, contractility, and attenuation of cell motility.

Several glomerular pathologies resulting from podocyte injury are linked to genetic variation involving the MYH9 gene, which encodes the heavy chain of non-muscle myosin-IIA (NM-IIA). However, the functional role of NM-IIA has not been studied extensively in podocytes. We hypothesized that NM-IIA is critical for maintenance of podocyte structure and mechanical function. To test this hypothesis, we studied murine podocytes in vitro subjected to blebbistatin inhibition of NM-II activity, or RNA interference-mediated, isoform-specific ablation of Myh9 gene and protein (NM-IIA) or its paralog Myh10 gene and protein (NM-IIB). Using quantitative immunofluorescence microscopy, traction force microscopy, and attachment and "wound healing" assays, we found that NM-IIA ablation altered podocyte actin cytoskeletal structure and focal adhesion distribution, decreased cell attachment and contractility, and increased cell motility. Blebbistatin treatment had similar effects. NM-IIB ablation produced cells that exhibited poor attachment, but cytoskeletal structural organization, contractility and motility were maintained. These findings indicate that NM-IIA is essential for maintenance of podocyte cytoskeletal structure and mechanical function in vitro, and NM-IIB does not replace it in this role when NM-IIA expression is altered. We conclude that critical podocyte functions may be affected by MYH9 mutations or disease-associated haplotypes. © 2016 Wiley Periodicals, Inc.

Targeting the podocyte cytoskeleton: from pathogenesis to therapy in proteinuric kidney disease.

Glomerular injury often incites a progression to chronic kidney disease, which affects millions of patients worldwide. Despite our current understanding of this disease's pathogenesis, there is still a lack of therapy available to curtail its progression. However, exciting new data strongly suggest the podocyte-an actin-rich, terminally differentiated epithelial cell that lines the outside of the glomerular filtration barrier-as a therapeutic target. The importance of podocytes in the pathogenesis of human nephrotic syndrome is best characterized by identification of genetic mutations, many of which regulate the actin cytoskeleton. The intricate regulation of the podocyte actin cytoskeleton is fundamental in preserving an intact glomerular filtration barrier, and this knowledge has inspired new research targeting actin-regulating proteins in these cells. This review will shed light on recent findings, which have furthered our understanding of the molecular mechanisms regulating podocyte actin dynamics, as well as discoveries that have therapeutic implications in the treatment of proteinuric kidney disease.

The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury

Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A alsostimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria wasgreater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

Editorial: Podocyte Pathology and Nephropathy.

Editorial: Podocyte Pathology and Nephropathy.

Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

BackgroundGlomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte actin cytoskeleton. In contrast, a potential stabilization of microtubules by everolimus has not been studied so far.MethodsTo elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury.ResultsUpon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involved in cytoskeletal reorganization, cell adhesion, migration and extracellular matrix composition to be affected. Everolimus was capable of protecting podocytes from injury, both on transcriptional and protein level. Rescued genes included tubulin beta 2B class IIb (TUBB2B) and doublecortin domain containing 2 (DCDC2), both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Validating gene expression data, Western-blot analysis in cultured podocytes demonstrated an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Interestingly, Tubb2b brdp/brdp mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm’s tumour 1, Podocin, Nephrin and Synaptopodin.ConclusionsTaken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development.