The Drosophila Nesprin-1 homolog MSP300 is required for muscle autophagy and proteostasis.

Nesprin proteins, which are components of the linker of nucleoskeleton and cytoskeleton (LINC) complex, are located within the nuclear envelope and play prominent roles in nuclear architecture. For example, LINC complex proteins interact with both chromatin and the cytoskeleton. Here, we report that the Drosophila Nesprin MSP300 has an additional function in autophagy within larval body wall muscles. RNAi-mediated MSP300 knockdown in larval body wall muscles resulted in defects in the contractile apparatus, muscle degeneration and defective autophagy. In particular, MSP300 knockdown caused accumulation of cytoplasmic aggregates that contained poly-ubiquitylated cargo, as well as the autophagy receptor ref(2)P (the fly homolog of p62 or SQSTM) and Atg8a. Furthermore, MSP300 knockdown larvae expressing an mCherry-GFP-tagged Atg8a transgene exhibited aberrant persistence of the GFP signal within these aggregates, indicating failure of autophagosome maturation. These autophagy deficits were similar to those exhibited by loss of the endoplasmic reticulum (ER) fusion protein Atlastin (Atl), raising the possibility that Atl and MSP300 might function in the same pathway. In support of this possibility, we found that a GFP-tagged MSP300 protein trap exhibited extensive localization to the ER. Alteration of ER-directed MSP300 might abrogate important cytoskeletal contacts necessary for autophagosome completion.

Regulation of Syntaxin3B-Mediated Membrane Fusion by T14, Munc18, and Complexin.

Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV.

Growth hormone induces transforming growth factor-ß1 in podocytes: Implications in podocytopathy and proteinuria.

Pituitary growth hormone (GH) is essential for growth, metabolism, and renal function. Overactive GH signaling is associated with impaired kidney function. Glomerular podocytes, a key kidney cell type, play an indispensable role in the renal filtration and express GH receptors (GHR), suggesting the direct action of GH on these cells. However, the precise mechanism and the downstream signaling events by which GH leads to diabetic nephropathy remain to be elucidated. Here we performed proteome analysis of the condition media from human podocytes and confirmed that GH-induces TGF-ß1. Inhibition of GH/GHR stimulated-JAK2 signaling abrogates GH-induced TGF-ß1 secretion. Mice administered with GH showed glomerular manifestations concomitant with proteinuria. Pharmacological inhibition of TGF-ßR1 in mice prevented GH-induced TGF-ß dependent SMAD signaling and proteinuria. Conditional deletion of GHR in podocytes protected mice from streptozotocin-induced diabetic nephropathy. GH and TGF-ß1 signaling components expression was elevated in the kidneys of human diabetic nephropathy patients. Our study identifies that GH induces TGF-ß1 in podocytes, contributing to diabetic nephropathy.

Podocyte derived TNF-α mediates monocyte differentiation and contributes to glomerular injury.

Diabetes shortens the life expectancy by more than a decade, and the excess mortality in diabetes is correlated with the incidence of kidney disease. Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. Macrophage accumulation predicts the severity of kidney injury in human biopsies and experimental models of DKD. However, the mechanism underlying macrophage recruitment in diabetes glomeruli is unclear. Elevated plasma growth hormone (GH) levels in type I diabetes and acromegalic individuals impaired glomerular biology. In this study, we examined whether GH-stimulated podocytes contribute to macrophage accumulation. RNA-seq analysis revealed elevated TNF-α signaling in GH-treated human podocytes. Conditioned media from GH-treated podocytes (GH-CM) induced differentiation of monocytes to macrophages. On the other hand, neutralization of GH-CM with the TNF-α antibody diminished GH-CM's action on monocytes. The treatment of mice with GH resulted in increased macrophage recruitment, podocyte injury, and proteinuria. Furthermore, we noticed the activation of TNF-α signaling, macrophage accumulation, and fibrosis in DKD patients' kidney biopsies. Our findings suggest that podocytes could secrete TNF-α and contribute to macrophage migration, resulting in DKD-related renal inflammation. Inhibition of either GH action or TNF-α expression in podocytes could be a novel therapeutic approach for DKD treatment.

Advanced-Glycation End-Products Induce Podocyte Injury and Contribute to Proteinuria.

The prevalence of diabetes reaches epidemic proportions. Diabetes is the leading cause of end-stage kidney disease (ESKD) since 30-40% of diabetic patients develop diabetic nephropathy. Albuminuria and glomerular filtration rate used to assess kidney function are considered surrogate outcomes of chronic kidney disease. The search for a biomarker that predicts progression to diabetic kidney disease is intense. We analyzed the association of serum advanced glycation end-products (AGEs) index (AGI) with impaired kidney function in poorly controlled type II diabetic patients. We observed an association between AGI and impaired kidney function in microalbuminuria patients with hyperglycemia. A significant association between AGEs, particularly carboxymethyl lysine (CML), and impaired kidney function were observed. Administration of AGEs to mice showed heavy proteinuria and glomerular abnormalities. Reduced podocyte number in mice administered with AGEs could be attributed to the epithelial-mesenchymal transition of podocytes. Our study suggests CML could be independently related to the podocyte injury and the risk of DN progression to ESKD in patients with microalbuminuria. AGEs in general or CML could be considered a prognostic marker to assess diabetic kidney disease.

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-ß1 signaling and provokes cell cycle reentry of otherwise quiescent podocytes. Though differentiated podocytes reenter the cell cycle in response to GH and TGF-ß1, they cannot accomplish cytokinesis, despite karyokinesis. Owing to this aberrant cell cycle event, GH- or TGF-ß1-treated cells remain binucleated and undergo mitotic catastrophe. Importantly, inhibition of JAK2, TGFBR1 (TGF-ß 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-ß1-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-ß1 or Notch inhibitors, as a therapeutic agent for DN.

Activation of Notch1 signaling in podocytes by glucose-derived AGEs contributes to proteinuria.

INTRODUCTION: Advanced glycation end-products (AGEs) are implicated in the pathogenesis of diabetic nephropathy (DN). Previous studies have shown that AGEs contribute to glomerulosclerosis and proteinuria. Podocytes, terminally differentiated epithelial cells of the glomerulus and the critical component of the glomerular filtration barrier, express the receptor for AGEs (RAGE). Podocytes are susceptible to severe injury during DN. In this study, we investigated the mechanism by which AGEs contribute to podocyte injury. RESEARCH DESIGN AND METHODS: Glucose-derived AGEs were prepared in vitro. Reactivation of Notch signaling was examined in AGE-treated human podocytes (in vitro) and glomeruli from AGE-injected mice (in vivo) by quantitative reverse transcription-PCR, western blot analysis, ELISA and immunohistochemical staining. Further, the effects of AGEs on epithelial to mesenchymal transition (EMT) of podocytes and expression of fibrotic markers were evaluated. RESULTS: Using human podocytes and a mouse model, we demonstrated that AGEs activate Notch1 signaling in podocytes and provoke EMT. Inhibition of RAGE and Notch1 by FPS-ZM1 (N-Benzyl-4-chloro-N-cyclohexylbenzamide) and DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenyl glycine t-butylester), respectively, abrogates AGE-induced Notch activation and EMT. Inhibition of RAGE and Notch1 prevents AGE-induced glomerular fibrosis, thickening of the glomerular basement membrane, foot process effacement, and proteinuria. Furthermore, kidney biopsy sections from people with DN revealed the accumulation of AGEs in the glomerulus with elevated RAGE expression and activated Notch signaling. CONCLUSION: The data suggest that AGEs activate Notch signaling in the glomerular podocytes. Pharmacological inhibition of Notch signaling by DAPT ameliorates AGE-induced podocytopathy and fibrosis. Our observations suggest that AGE-induced Notch reactivation in mature podocytes could be a novel mechanism in glomerular disease and thus could represent a novel therapeutic target.

Cerebral ischemia induces TRPC6 via HIF1α/ZEB2 axis in the glomerular podocytes and contributes to proteinuria.

Podocytes are specialized cells of the glomerulus and key component of the glomerular filtration apparatus (GFA). GFA regulates the permselectivity and ultrafiltration of blood. The mechanism by which the integrity of the GFA is compromised and manifest in proteinuria during ischemic stroke remains enigmatic. We investigated the mechanism of ischemic hypoxia-induced proteinuria in a middle cerebral artery occlusion (MCAO) model. Ischemic hypoxia resulted in the accumulation of HIF1α in the podocytes that resulted in the increased expression of ZEB2 (Zinc finger E-box-binding homeobox 2). ZEB2, in turn, induced TRPC6 (transient receptor potential cation channel, subfamily C, member 6), which has increased selectivity for calcium. Elevated expression of TRPC6 elicited increased calcium influx and aberrant activation of focal adhesion kinase (FAK) in podocytes. FAK activation resulted in the stress fibers reorganization and podocyte foot process effacement. Our study suggests overactive HIF1α/ZEB2 axis during ischemic-hypoxia raises intracellular calcium levels via TRPC6 and consequently altered podocyte structure and function thus contributes to proteinuria.

Growth hormone induces Notch1 signaling in podocytes and contributes to proteinuria in diabetic nephropathy.

Growth hormone (GH) plays a significant role in normal renal function and overactive GH signaling has been implicated in proteinuria in diabetes and acromegaly. Previous results have shown that the glomerular podocytes, which play an essential role in renal filtration, express the GH receptor, suggesting the direct action of GH on these cells. However, the exact mechanism and the downstream pathways by which excess GH leads to diabetic nephropathy is not established. In the present article, using immortalized human podocytes in vitro and a mouse model in vivo, we show that excess GH activates Notch1 signaling in a γ-secretase-dependent manner. Pharmacological inhibition of Notch1 by γ-secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenyl glycine t-butylester) abrogates GH-induced epithelial to mesenchymal transition (EMT) and is associated with a reduction in podocyte loss. More importantly, our results show that DAPT treatment blocks cytokine release and prevents glomerular fibrosis, all of which are induced by excess GH. Furthermore, DAPT prevented glomerular basement membrane thickening and proteinuria induced by excess GH. Finally, using kidney biopsy sections from people with diabetic nephropathy, we show that Notch signaling is indeed up-regulated in such settings. All these results confirm that excess GH induces Notch1 signaling in podocytes, which contributes to proteinuria through EMT as well as renal fibrosis. Our studies highlight the potential application of γ-secretase inhibitors as a therapeutic target in people with diabetic nephropathy.

Hypoxia induces ZEB2 in podocytes: Implications in the pathogenesis of proteinuria.

The glomerular filtration barrier (GFB) plays a critical role in ensuing protein free urine. The integrity of the GFB is compromised during hypoxia that prevails during extreme physiological conditions. However, the mechanism by which glomerular permselectivity is compromised during hypoxia remains enigmatic. Rats exposed to hypoxia showed a decreased glomerular filtration rate, podocyte foot-processes effacement, and proteinuria. Accumulation of hypoxia-inducible factor-1α (HIF1α) in podocytes resulted in elevated expression of zinc finger E-box binding homeobox 2 (ZEB2) and decreased expression of E- and P-cadherin. We also demonstrated that HIF1α binds to hypoxia response element localized in the ZEB2 promoter. Furthermore, HIF1α also induced the expression of ZEB2-natural antisense transcript, which is known to increase the efficiency of ZEB2 translation. Ectopic expression of ZEB2 induced loss of E- and P-cadherin and is associated with enhanced motility of podocytes during hypoxic conditions. ZEB2 knockdown abrogated hypoxia-induced decrease in podocyte permselectivity. This study suggests that hypoxia leads to activation of HIF1α-ZEB2 axis, resulting in podocyte injury and poor renal outcome.

Novel Actions of Growth Hormone in Podocytes: Implications for Diabetic Nephropathy.

The kidney regulates water, electrolyte, and acid-base balance and thus maintains body homeostasis. The kidney's potential to ensure ultrafiltered and almost protein-free urine is compromised in various metabolic and hormonal disorders such as diabetes mellitus (DM). Diabetic nephropathy (DN) accounts for ~20-40% of mortality in DM. Proteinuria, a hallmark of renal glomerular diseases, indicates injury to the glomerular filtration barrier (GFB). The GFB is composed of glomerular endothelium, basement membrane, and podocytes. Podocytes are terminally differentiated epithelial cells with limited ability to replicate. Podocyte shape and number are both critical for the integrity and function of the GFB. Podocytes are vulnerable to various noxious stimuli prevalent in a diabetic milieu that could provoke podocytes to undergo changes to their unique architecture and function. Effacement of podocyte foot process is a typical morphological alteration associated with proteinuria. The dedifferentiation of podocytes from epithelial-to-mesenchymal phenotype and consequential loss results in proteinuria. Poorly controlled type 1 DM is associated with elevated levels of circulating growth hormone (GH), which is implicated in the pathophysiology of various diabetic complications including DN. Recent studies demonstrate that functional GH receptors are expressed in podocytes and that GH may exert detrimental effects on the podocyte. In this review, we summarize recent advances that shed light on actions of GH on the podocyte that could play a role in the pathogenesis of DN.

In silico Structural characterization of podocin and assessment of nephrotic syndrome-associated podocin mutants.

Nephrotic syndrome (NS) is manifested by hyperproteinuria, hypoalbuminemia, and edema. NPHS2 that encodes podocin was found to have most mutations among the genes that are involved in the pathophysiology of NS. Podocin, an integral membrane protein belonging to stomatin family, is expressed exclusively in podocytes and is localized to slit-diaphragm (SD). Mutations in podocin are known to be associated with steroid-resistant NS and rapid progression to end-stage renal disease, thus signifying its role in maintaining SD integrity and podocyte function. The structural insights of podocin are not known, and the precise mechanism by which podocin contributes to the architecture of SD is yet to be elucidated. In this study, we deduced a model for human podocin, discussed the details of transmembrane localization and intrinsically unstructured regions, and provide an understanding of how podocin interacts with other SD components. Intraprotein interactions were assessed in wild-type podocin and in some of its mutants that are associated with idiopathic NS. Mutations in podocin alter the innate intraprotein interactions affecting the native structure of podocin and its ability to form critical complex with subpodocyte proteins. © 2016 IUBMB Life, 68(7):578-588, 2016.