Role of the polarity protein Scribble for podocyte differentiation and maintenance

The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their complex foot process network. While deficiency of the polarity proteins Crumbs and aPKC result in impaired podocyte foot process architecture, the function of basolateral polarity proteins for podocyte differentiation and maintenance remained unclear. Here we report, that Scribble is expressed in developing podocytes, where it translocates from the lateral aspects of immature podocytes to the basal cell membrane and foot processes of mature podocytes. Immunogold electron microscopy reveals membrane associated localisation of Scribble predominantly at the basolateral site of foot processes. To further study the role of Scribble for podocyte differentiation Scribble(flox/flox) mice were generated by introducing loxP-sites into the Scribble introns 1 and 8 and these mice were crossed to NPHS2.Cre mice and Cre deleter mice. Podocyte-specific Scribble knockout mice develop normally and display no histological, ultrastructural or clinical abnormalities up to 12 months of age. In addition, no increased susceptibility to glomerular stress could be detected in these mice. In contrast, constitutive Scribble knockout animals die during embryonic development indicating the fundamental importance of Scribble for embryogenesis. Like in podocyte-specific Scribble knockout mice, the development of podocyte foot processes and the slit diaphragm was unaffected in kidney cultures from constitutive Scribble knockout animals. In summary these results indicate that basolateral polarity signaling via Scribble is dispensable for podocyte function, highlighting the unique feature of podocyte development with its significant apical membrane expansions being dominated by apical polarity complexes rather than by basolateral polarity signaling.     

Interaction with podocin facilitates nephrin signaling

Mutations of NPHS1 or NPHS2, the genes encoding for the glomerular podocyte proteins nephrin and podocin, cause steroid-resistant proteinuria. In addition, mice lacking CD2-associated protein (CD2AP) develop a nephrotic syndrome that resembles NPHS mutations suggesting that all three proteins are essential for the integrity of glomerular podocytes. Although the precise glomerular function of either protein remains unknown, it has been suggested that nephrin forms zipper-like interactions to maintain the structure of podocyte foot processes. We demonstrate now that nephrin is a signaling molecule, which stimulates mitogen-activated protein kinases. Nephrin-induced signaling is greatly enhanced by podocin, which binds to the cytoplasmic tail of nephrin. Mutational analysis suggests that abnormal or inefficient signaling through the nephrin-podocin complex contributes to the development of podocyte dysfunction and proteinuria.     

Growth hormone induces mitotic catastrophe of glomerular podocytes and contributes to proteinuria

Glomerular podocytes are integral members of the glomerular filtration barrier in the kidney and are crucial for glomerular permselectivity. These highly differentiated cells are vulnerable to an array of noxious stimuli that prevail in several glomerular diseases. Elevated circulating growth hormone (GH) levels are associated with podocyte injury and proteinuria in diabetes. However, the precise mechanism(s) by which excess GH elicits podocytopathy remains to be elucidated. Previous studies have shown that podocytes express GH receptor (GHR) and induce Notch signaling when exposed to GH. In the present study, we demonstrated that GH induces TGF-β1 signaling and provokes cell cycle reentry of otherwise quiescent podocytes. Though differentiated podocytes reenter the cell cycle in response to GH and TGF-β1, they cannot accomplish cytokinesis, despite karyokinesis. Owing to this aberrant cell cycle event, GH- or TGF-β1-treated cells remain binucleated and undergo mitotic catastrophe. Importantly, inhibition of JAK2, TGFBR1 (TGF-β receptor 1), or Notch prevented cell cycle reentry of podocytes and protected them from mitotic catastrophe associated with cell death. Inhibition of Notch activation prevents GH-dependent podocyte injury and proteinuria. Similarly, attenuation of GHR expression abated Notch activation in podocytes. Kidney biopsy sections from patients with diabetic nephropathy (DN) show activation of Notch signaling and binucleated podocytes. These data indicate that excess GH induced TGF-β1-dependent Notch1 signaling contributes to the mitotic catastrophe of podocytes. This study highlights the role of aberrant GH signaling in podocytopathy and the potential application of TGF-β1 or Notch inhibitors, as a therapeutic agent for DN.Subject terms: Podocytes, Diabetic nephropathy     

Mechanisms and consequences of TGF-? overexpression by podocytes in progressive podocyte disease

In patients with progressive podocyte disease, such as focal segmental glomerulosclerosis (FSGS) and membranous nephropathy, upregulation of transforming growth factor-? (TGF-?) is observed in podocytes. Mechanical pressure or biomechanical strain in podocytopathies may cause overexpression of TGF-? and angiotensin II (Ang II). Oxidative stress induced by Ang II may activate the latent TGF-?, which then activates Smads and Ras/extracellular signal-regulated kinase (ERK) signaling pathways in podocytes. Enhanced TGF-? activity in podocytes may lead to thickening of the glomerular basement membrane (GBM) by overproduction of GBM proteins and impaired GBM degradation in podocyte disease. It may also lead to podocyte apoptosis and detachment from the GBM, and epithelial-mesenchymal transition (EMT) of podocytes, initiating the development of glomerulosclerosis. Furthermore, activated TGF-?/Smad signaling by podocytes may induce connective tissue growth factor and vascular endothelial growth factor overexpression, which could act as a paracrine effector mechanism on mesangial cells to stimulate mesangial matrix synthesis. In proliferative podocytopathies, such as cellular or collapsing FSGS, TGF-?-induced ERK activation may play a role in podocyte proliferation, possibly via TGF-?-induced EMT of podocytes. Collectively, these data bring new mechanistic insights into our understanding of the TGF-? overexpression by podocytes in progressive podocyte disease.     

C3aR过度活化与糖尿病肾病肾组织损伤的关系

C3aR是补体C3裂解产物C3a的受体.最近的一些研究提示C3aR通路可能参与了糖尿病肾病(DN)的病理过程,但有关C3aR通路在DN中的确切病理作用及有关机制远未清楚.需要特别指出的是,现有的有关C3aR参与DN肾组织损伤的证据主要来自一些动物模型的研究,临床上尚缺乏较为系统全面的对DN患者肾组织C3aR通路与肾组织损伤关系的观察分析.为此,本文首次以较大的样本量分析了不同病理时期DN患者肾组织C3aR和C3a的表达变化情况及其与DN患者肾组织损伤的相关性.在此基础上,进而利用体外细胞模型,对高糖环境下C3aR活化致肾小球足细胞损伤的作用及机制进行了探讨.结果显示:a.与正常对照组相比,DN患者肾组织C3a和C3aR的表达水平随DN的进展而升高,C3aR在DN患者肾组织中的表达上调主要见于肾小管上皮细胞和肾小球足细胞;b.DN患者肾组织C3aR和C3a水平与患者肾组织损伤程度,特别是小管和小管间质损伤程度、肾小球足细胞损伤程度具有显著相关性;c.外加C3a激活C3aR可使高糖环境中的足细胞的细胞骨架发生明显改变、足细胞标记分子表达下调、足细胞通透性增加.这些结果说明:a.DN患者肾组织中确实存在C3a/C3aR轴过度活化的现象;b.C3a/C3aR轴的过度活化很可能在DN患者肾组织损伤,特别是小管和小管间质损伤、肾小球足细胞损伤中具有重要作用;c.可能通过破坏成熟足细胞特有的细胞骨架,改变足细胞标记分子表达,增加足细胞的通透性,C3a/C3aR轴过度活化参与DN足细胞损伤过程.本文不仅为C3a/C3aR通路参与DN病理过程提供了新的必不可少的临床证据,也增加了对C3a/C3aR通路过度活化致DN患者肾组织损伤机制,特别是肾小球足细胞损伤机制的了解,这对于拓展对DN病理机制的认识,发展DN防治新思路,无疑都是有益的.     

GAP-43 ameliorates Podocyte injury by decreasing nuclear NFATc1 expression

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

CD2AP/CIN85 balance determines receptor tyrosine kinase signaling response in podocytes

Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP(-/-)) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP(-/-) mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP(-/-) podocytes in vitro. Stimulation of CD2AP(-/-) podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP(-/-) mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/CIN85 protein balance in the normal signaling response of podocytes.     

Yes-associated Protein (YAP) Promotes Cell Survival by Inhibiting Proapoptotic Dendrin Signaling

Kidney podocytes are highly specialized terminally differentiated cells that form the final barrier to urinary protein loss. Podocytes are a target for injury by metabolic, autoimmune, hereditary, inflammatory, and other stressors. Persistence of podocyte injury leads to podocyte death and loss, which results in progressive kidney damage and ultimately kidney failure. Dendrin is a dual compartment protein with proapoptotic signaling properties. Nuclear relocation of dendrin in response to glomerular injury promotes podocyte apoptosis. Here we show that Yes-associated protein (YAP), a downstream target of Hippo kinases and an inhibitor of apoptosis, is expressed in the nucleus of podocytes. The WW domains of YAP mediate the interaction with the PPXY motifs of dendrin. This interaction is functionally relevant because YAP binding to dendrin reduces dendrin-dependent, staurosporine-induced apoptosis in co-transfected HEK293 cells. Moreover gene silencing of YAP in podocytes increases adriamycin-induced podocyte apoptosis. It also increases staurosporine-induced caspase-3/7 activity, which is rescued by dendrin depletion in YAP knockdown cells. Our findings elucidate YAP binding to dendrin as a prosurvival mechanism. The antiapoptotic signaling properties of YAP in podocytes could hold significance in the quest for targeted therapeutics aimed at preventing podocyte loss.     

Liver X receptor activation induces podocyte injury via inhibiting autophagic activity

Podocyte injury plays a key role in the occurrence and development of kidney diseases. Decreased autophagic activity in podocyte is closely related to its injury and the occurrence of proteinuria. Liver X receptors (LXRs), as metabolic nuclear receptors, participate in multiple pathophysiological processes and express in several tissues, including podocytes. Although the functional roles of LXRs in the liver, adipose tissue and intestine are well established; however, the effect of LXRs on podocytes function remains unclear. In this study, we used mouse podocytes cell line to investigate the effects of LXR activation on podocytes autophagy level and related signaling pathway by performing Western blotting, RT-PCR, GFP-mRFP-LC3 transfection, and immunofluorescence staining. Then, we tested this effect in STZ-induced diabetic mice. Transmission electron microscopy and immunohistochemistry were employed to explore the effects of LXR activation on podocytes function and autophagic activity. We found that LXR activation could inhibit autophagic flux through blocking the formation of autophagosome in podocytes in vitro which was possibly achieved by affecting AMPK, mTOR, and SIRT1 signaling pathways. Furthermore, LXR activation in vivo induced autophagy suppression in glomeruli, leading to aggravated podocyte injury. In summary, our findings indicated that activation of LXRs induced autophagy suppression, which in turn contributed to the podocyte injury.

     

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.     

Vascular endothelial growth factor receptor 2 direct interaction with nephrin links VEGF-A signals to actin in kidney podocytes

The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.     

Actin-associated Proteins in the Pathogenesis of Podocyte Injury

Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies.     

MAD2B promotes podocyte injury through regulating Numb-dependent Notch 1 pathway in diabetic nephropathy

Rationale: Recent studies have demonstrated that the loss of podocyte is a critical event in diabetic nephropathy (DN). Previously, our group have found that the mitotic arrest deficient protein MAD2B was involved in high glucose (HG)-induced podocyte injury by regulating APC/C activity. However, the exact mechanism of MAD2B implicated in podocyte injury is still lacking.Methods: The experiments were conducted by using kidney tissues from streptozotocin (STZ) induced diabetic mice with or without podocyte-specific deletion of MAD2B and the cultured podocytes exposed to different treatments. Glomerular pathological injury was evaluated by periodic acid-Schiff staining and transmission electron microscopy. The endogenous interaction between MAD2B and Numb was discovered by yeast two-hybrid analysis and co-immunoprecipitation assay. The expressions of MAD2B, Numb and related pathway were detected by western blot, immunochemistry and immunofluorescence.Results: The present study revealed that MAD2B was upregulated in diabetic glomeruli and cultured podocytes under hyperglycemic conditions. Podocyte-specific deletion of MAD2B alleviated podocyte injury and renal function deterioration in mice of diabetic nephropathy. Afterwards, MAD2B was found to interact with Numb, which was downregulated in diabetic glomeruli and HG-stimulated cultured podocytes. Interestingly, MAD2B genetic deletion could partly reverse the decline of Numb in podocytes exposed to HG and in diabetic mice, and the expressions of Numb downstream molecules such as NICD and Hes-1 were decreased accordingly. In addition, overexpression of Numb ameliorated HG-induced podocyte injury.Conclusions: The present findings suggest that upregulated MAD2B expression contributes to Numb depletion and activation of Notch 1 signaling pathway, which ultimately leads to podocyte injury during DN progression.     

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.     

High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN.     

Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.     

Podocyte Expression of Membrane Transporters Involved in Puromycin Aminonucleoside-Mediated Injury

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA).We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family.Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration.In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.