Diabetic nephropathy (DN) is primary cause of end-stage renal disease. A previous study has shown that miR-32-5p (miR-32) is highly expressed in kidney tissue during chronic allograft dysfunction with interstitial fibrosis and tubular atrophy. However, the role of miR-32-5p (miR-32) in DN is still unclear. In this study, streptozotocin-induced DN rat models and high glucose (HG)-incubated human kidney proximal tubular epithelial (HK-2) cells were established to investigate the role and underlying mechanisms of miR-32 in DN. Results of real-time PCR revealed that miR-32 levels were greatly increased in DN rats and HG-incubated HK-2 cells. Downregulation of miR-32 effectively relieved HG-induced autophagy suppression, fibrosis, epithelial-mesenchymal transition (EMT) and inflammation in HK-2 cells. Besides, miR-32 overexpression significantly down-regulated the expression of mothers against decapentaplegic homolog 7 (SMAD7), whereas knockdown of miR-32 markedly up-regulated the level of SMAD7. Dual-luciferase reporter gene assay confirmed that SMAD7 was a target of miR-32. Reintroduction of SMAD7 expression rescued miR-32-induced HK-2 cells autophagy suppression, EMT and renal fibrosis. Our findings indicate that miR-32 may play roles in the progression of EMT and fibrosis in DN.
Purpose To investigate the role and underlying mechanism of GDF11 on diabetic nephropathy (DN)-related mitochondrial dysfunction and apoptosis. Methods A DN model of rats was established in this study. Human Kidney-2 (HK-2) cells were cultured under high-glucose (HG) condition with or without recombinant GDF11 (rGDF11). Mitochondrial morphology of HK-2 cells was analyzed by transmission electron microscope and MitoTracker Red CMXRos staining. Mitochondrial membrane potential (MMP) and ROS production were monitored using JC-1 assay kit and MitoSOX staining, respectively. Cell apoptosis was detected by TUNEL or flow cytometry assays. Results Herein, we observed that GDF11 was down-regulated in renal cortex and serum of DN rats, which was accompanied by renal mitochondrial morphological abnormalities. In line with the findings in vivo, HK-2 cells exposed to HG presented with mitochondrial morphological alterations and further apoptosis accompanied by GDF11 reduction. In addition, HG promoted a decrease in MMP while an increase in mitochondrial ROS production. Conversely, rGDF11 treatment significantly alleviated these HG-induced mitochondrial defects in HK-2 cells. Meanwhile, HK-2 cell apoptosis induced by HG was simultaneously suppressed by rGDF11. Mechanistically, the decreased levels of p-AKT induced by HG were attenuated after rGDF11 administration. Inhibition of the PI3K/AKT pathway resisted the effects of rGDF11 on the MMP and apoptosis of HK-2 cells. In addition, we identified that GDF11 is a target of miR-32-5p. Up-regulation of miR-32-5p could inhibit the expression of GDF11. Conclusion rGDF11 treatment rescued HG-induced HK-2 cell mitochondrial dysfunction and apoptosis, which may be dependent on the activation of the PI3K/AKT pathway.
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