Overexpression of Myo1e promotes albumin endocytosis by mouse glomerular podocytes mediated by Dynamin.

Huijun Shen,Yu Bao,Jianhua Mao,Haidong Fu,Chunyue Feng

doi:10.7717/peerj.8599

Abstract

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.

Highlights

Albuminuria is a hallmark of nephropathy, usually caused by a deterioration in the integrity of the glomerular filtration barrier (Schiessl et al, 2016)
The results showed that bovine serum albumin (BSA)-FITC (250 ug/ml, 500 ug/ml and 1 mg/ml) significantly inhibited MPC5 cell apoptosis (p < 0.001; Fig. 1C)
Immunofluorescence results showed that the content of BSA phagocytized by MPC-5 was increasing depending on the concentration of BSA, which was consistent with flow cytometry results (Fig. 1D)

Summary

Introduction

Albuminuria is a hallmark of nephropathy, usually caused by a deterioration in the integrity of the glomerular filtration barrier (Schiessl et al, 2016). Down-regulation of Cullin-5 expression in UPS as a cytoskeletal protein of E3 ligase in UPS can cause edema, proteinuria and glomerular structural abnormalities in zebrafish, and endoplasmic reticulum stress in podocytes (Mao et al, 2013). These finding reveal that it is necessary to explore the mechanisms of abnormally expressed genes in the podocyte cells in the development of albuminuria (Tryggvason, Patrakka & Wartiovaara, 2006). Myo1e overexpression promoted the expression of cytoskeleton F-actin and podocyte-specific molecules (nephrin and podocin) in podocyte endocytic FITC-BSA. Our findings revealed that overexpression of Myo1e promotes albumin endocytosis in mouse glomerular podocyte endocytic albumin mediated by Dynamin

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Conclusion