P0982PTEN-INDUCED KINASE 1 (PINK1) DEPLETION ALTERS MITOCHONDRIAL EFFICIENCY AND GLYCOLYSIS IN HUMAN PODOCYTES

Abstract

Abstract Background and Aims Podocytes are terminally differentiated cells, which constitute an inner layer of the renal filtration barrier. Podocytes are characterized by high metabolic activity, and their elevated energy requirements are met by maintaining the appropriate mitochondrial number and quality, which depend on mitochondrial biogenesis and mitophagy. Alteration of mitochondrial dynamics is linked to the development of insulin resistance and diabetes. The main goal of the research was to determine the role of mitophagy in podocytes bioenergetics and to elucidate the effects of hyperglycemia on mitochondrial dynamics. Method In order to inhibit mitophagy, we generated a human podocyte cell line stably expressing PINK1 shRNA through lentiviral transduction. Biochemical analyses were performed to assess the oxidative phosphorylation efficiency (by measurement of oxygen consumption rate; OCR) and glycolysis contribution to the total cell energy production (by measurement of extracellular acidification rate; ECAR) in the differentiated shPINK1 podocytes. The expression levels of mRNAs and proteins were evaluated in podocytes cultured in standard glucose (11 mM) and high glucose (30 mM) concentrations using real-time PCR and western blot. Intracellular mitochondrial network was visualized by MitoTrackertTM staining. The co-localization of proteins in podocytes was analysed by double immunofluorescence labelling and confocal microscopy. Results PINK1-deficiency resulted in the significant decrease of maximal respiration and spare respiratory capacity in podocytes (by 40% and 70%, respectively). Non-mitochondrial respiration was also decreased by 45% in shPINK1 cells. Interestingly, basal respiration and ATP production appeared similar in shPINK1 and control podocytes. PINK1 depletion increased glycolytic flux by 70%, in addition, the accompanying decline in glycolytic capacity and glycolytic reserve was observed (by 38% and 63%, respectively). Moreover, we observed accumulation of small and ring-shaped mitochondria in PINK1-deficient podocytes compared with the control cells. We showed a decreased expression of PINK1 and Parkin (mRNA and protein) in normal human podocytes cultured in hyperglycemic medium, which was associated with an elevated levels of mitochondrial fission markers (DRP1, FIS1) and with a decreased levels of PGC1α and TFAM, which play a role in mitochondrial biogenesis and mtDNA replication. Conclusion In this research, we demonstrated a novel role of mitophagy in podocyte bioenergetics. Moreover, we showed that high glucose inhibits mitophagy and promotes mitochondrial fission leading to the accumulation of damaged mitochondria and podocyte injury, which underlies the pathogenesis of diabetic nephropathy.