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Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes.

ABSTRACT: Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP-LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP-LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.

SUBMITTER: Xiong J

PROVIDER: S-EPMC3914646 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes.

Xiong Jing J Xia Min M Xu Ming M Zhang Yang Y Abais Justine M JM Li Guangbi G Riebling Christopher R CR Ritter Joseph K JK Boini Krishna M KM Li Pin-Lan PL

Journal of cellular and molecular medicine 20131117 12

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a rob ...[more]

PMID: 24238063

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Project description:BackgroundAs a fundamental process internalizing molecules from the plasma membrane, endocytosis plays a crucial role in podocyte biology. Our previous study has identified that overexpression of Myole may enhance podocyte endocytosis. However, its potential mechanism has been not well understand. Thus, we aimed to analyze whether albumin endocytosis by mouse glomerular podocytes is dependent on Myo1e expression. Also, we aimed to elucidate whether the underlying mechanism is mediated by Dynamin.MethodsFirstly, mouse podocyte cells (MPC5) were treated with different concentrations of FITC-bovine serum albumin (BSA). The fluorescence intensity and cell viability were detected by flow cytometry and MTT assays, respectively. Afterwards, the optimal concentration of FITC-BSA was determined. Secondly, MPC5 cells were treated with Myole overexpression or knockdown. Cell morphology was observed under microscope. Immunofluorescence assay was used to determine the expression of F-actin. The protein expression of nephrin and podocin was detected by western blot. Flow cytometry was used to detect MPC5 cell apoptosis with annexin V. Finally, MPC5 cells were treated with Myole overexpression and/or Dynasore (a GTPase inhibitor of Dynamin). The fluorescence intensity was detected using flow cytometry assay.ResultsMPC5 endocytosis BSA was elevated with a concentration-dependent manner. MTT results showed that MPC5 cell viability was inhibited with a concentration-dependent manner. Myo1e overexpression promoted podocyte endocytic FITC-BSA, which was contrary to its knockdown. Under microscope, after inhibition of Myo1e, podocyte foot process fusion was observed. Myo1e overexpression promoted the expression of cytoskeleton F-actin and podocyte-specific molecules (nephrin and podocin) in podocyte endocytic FITC-BSA. Furthermore, we found that Myo1e promoted the apoptosis of podocytes. Dynasore attenuated the increase in endocytosis of FITC-BSA induced by Myo1e overexpression, suggesting that podocytes might mediate albumin endocytosis via Myo1e-Dynamin-Albumin.ConclusionOur findings revealed that overexpression of Myo1e promotes albumin endocytosis in mouse glomerular podocyte endocytic albumin mediated by Dynamin.

Project description:Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg-1 · d-1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

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Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes.

Publications

Autophagy maturation associated with CD38-mediated regulation of lysosome function in mouse glomerular podocytes.

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