211 - Mitophagy confers the nephroprotective effect of Berberine in hyperglycemic NRK-52E cells via Sirt1-FoxO3a-Bnip3 axis

Abstract

Mitochondria appear as a critical determinant for progressive loss of renal function during hyperglycemia. However, there is limited understanding about mitophagy mediated renoprotection against hyperglycemia. Unabated growth in the incidence of high mortality and morbidity, underpinned by diabetic nephropathy has made it essential to find out new strategies for its management. The present study was focussed to explore the intervention of Berberine (isoquinoline alkaloid) in inducing mitophagy via Sirt1-FoxO3a-Bnip3 axis in hyperglycemic NRK-52E cells. Hyperglycemia induced intracellular and mitochondrial superoxide generation was found to be significantly reduced in Berberine treated cells, as assessed by Dihydroethidium and MitoSOX respectively. Pre-treatment with N-acetyl-L-cysteine showed the crucial role of ROS generation in hyperglycemia induced nephrotoxicity. Berberine also improved diminished level of cellular glutathione (GSH), mitochondrial membrane potential, mitochondrial dynamics and mitochondrial mass. Treatment of NRK-52E cells with Berberine caused differential expression of autophagy and mitophagy promoting genes like Beclin1, LC3B, Bnip3, Sirt1, FoxO3a, PINK1 and Parkin at mRNA level and protein level. Rapamycin, an autophagy inducer negated hyperglycemia induced suppression of autophagy and strengthened the protective effect of Berberine while Bafilomycin A1, an autophagy inhibitor showed reverse effect, suggesting the involvement of autophagy in protection accorded to hyperglycemic NRK-52E cells. Further, autophagolysosome resident mitochondria shown by transmission electron microscopy and localization of autophagy machinery in mitochondria also confirmed improved mitophagy in Berberine treated cells. The improved autophagy, attenuated mitochondrial abnormalities and decreased apoptosis after SRT-1720 (Sirt1 activator) pre-treatment and reversal exhibited by the pre-treatment with Nicotinamide (Sirt1 inhibitor) evidenced the crucial role of Sirt1 in Berberine mediated renoprotection against hyperglycemia. Upregulated transcriptional activity of FoxO was ascertained by GFP reporter assay. FoxO3a knock-down increased the disruption of autophagy and resulted in increased apoptosis. Thus, our findings suggest that the nephroprotective activity of Berberine involved autophagy mediated mitochondrial turnover and activation of Sirt1-FoxO3a-Bnip3 axis.