P0023RNA-BINDING PROTEIN HUR REGULATES HYPOXIA-INDUCED PARKIN/BNIP3L EXPRESSIONS AND MITOPHAGY IN RENAL TUBULAR CELLS

Abstract

Abstract Background and Aims Mitochondrial dysfunction contributes to the pathophysiology of acute kidney injury (AKI). Mitophagy selectively degrades damaged mitochondria and thereby regulates cellular function and homeostasis. RNA-binding proteins (RBPs) regulate RNA processing at multiple levels and thereby control cellular function. In this study, we report that RBP, human antigen R (HuR) regulates mitophagy in renal tubular cells (HK-2) under hypoxia condition. Method Human kidney proximal tubular 2 (HK-2) were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen, GmbH, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, GmbH, Germany), 1% penicillin/streptomycin (Gibco, Invitrogen, GmbH, Germany) at 37°C and 5% CO2. The cells were exposed to normoxia (37°C; 5% CO2) or hypoxia (serum and glucose deprived RPMI medium, 37°C, 1% O2) conditions. Mitophagy marker expressions (Parkin, p-Parkin, PINK1, BNIP3L, BNIP3, LC3) were determined by western blot analysis. ROS and mitochondrial membrane potential were determined using DCF-DA and JC-1 dye. Parkin and LC3 localization to mitochondria were performed by confocal laser scanning microscopy. HuR bound Parkin/BNIP3L mRNA expressions were determined by RNA-immunoprecipitation analysis. Results In this study, up-regulated mitophagy markers such as Parkin, PINK1, BNIP3 and BNIP3L protein expressions and mitophagosome formation were found in HK-2 cells under hypoxia condition. Stable HuR knockdown studies revealed that, HuR regulates mitophagy by mitophagosome (LC3 co-localization with mitochondria) and mitolysosome (mitochondria co-localization with lysosome) formation under hypoxia. Further, we show that HuR significantly regulated hypoxia-induced mitophagy by regulating Parkin/BNIP3L protein expressions and their mitochondrial localization. Bioinformatics studies showed that, HuR specifically binds to the AU-rich region in 3’UTR and CDS of Parkin and BNIP3L mRNA sequence. HuR was significantly bound to Parkin and BNIP3L mRNA and thereby regulated their mRNA stability under hypoxic conditions. Conclusion Altogether our data highlight on the functional importance of HuR in regulating mitophagy under hypoxic conditions in renal tubular cells through Parkin/BNIP3L expressions.