FP424FIBROSIS-RELATED MICRORNA ARE DYSREGULATED BY HYPERGLYCAEMIA IN HUMAN PROXIMAL TUBULAR EPITHELIAL CELLS

Abstract

Introduction and Aims: Renal tubulointerstitial fibrosis (TIF) is the hallmark of chronic kidney disease and drives its progression to end-stage renal disease. Excessive deposition of extracellular matrix (ECM) in TIF causes thickening of basement membranes, disruption of normal cell-to-cell interactions and loss of tissue elasticity, thus leading to the destruction of the renal tubules. Diabetes can cause diabetic nephropathy, which is characterised by ECM accumulation in the glomerular mesan-gium and tubulointerstitium mediated by the key pro-fibrotic cytokine TGF-b1. The aim of this study is to identify fibrosis-related microRNAs (miRNAs) as potential therapeutic targets for prevention of TIF in diabetes. Methods: Immortalised human renal proximal tubular epithelial cells (HK-2 cells) were exposed to glucose (5, 25, 30mM D-glucoseor the osmotic control 5mM D-glucose+25mM L-glucose) for up to 72h. Glucose consumption, scratch wound healing, cell viability/proliferation, mitochondrial function analyses and TGF-B1 ELISA tests were undertaken. The expression of 84 fibrosis-related miRNAs was evaluated using qPCR arrays (Qiagen, UK) in HK-2 cells after 24h, 48h or 72h incubation with different concentrations of glucose. Results: The results demonstrated that the cell viability/proliferation was significantly decreased following exposureto30mM D-glucose compared with 5mM D-glu-cose. Furthermore, reduced glucose consumption and wound healing were observed in response to 30mM D-glucose, whereas 25mM D-glucoseaccelerated wound healing. In contrast to the effect of 25mM D-glucose, TGF-B1 levels in media from cells exposed to 30mM D-glucose were significantly elevated. Although the mitochondrial energy (ATP) generation was decreased in cells exposed to either 25 or 30mM D-glucose, this effect was significant at 30mM D-glucose. qPCR array experiments demonstrated dys-regulated expression of over 15 fibrosis-related miRNAs in response to hyperglycaemia. Upregulated miRNAs included Mir-150-5pand Mir-216a-5p, and amongst the down-regulated miRNAs were Mir-211-5p and Mir-122-5p. Conclusions: Our data suggest that a critical threshold level of glucose (between 25mM and 30mM) increases TGF-B1 in HK-2 cells and is detrimental to cell proliferation, migration and metabolism. Furthermore, a number of fibrosis-related miRNAs are dysregulated by hyperglycaemia in HK-2 cells. The effects of these miRNAs need to be investigated further to identify potential therapeutic targets for the prevention of proximal tubular damage in diabetes.