STANDARDIZATION OF AN IN VITRO MODEL OF DIABETIC NEPHROPATHY IN RENAL TUBULAR CELLS AND INVESTIGATION OF THE ROLE OF ALDOSE REDUCTASE PATHWAY IN HIGH GLUCOSE-INDUCED RENAL CELL INJURY

Abstract

Diabetic nephropathy (DN) is the leading cause of end stage renal disease, and one of the most serious microvascular complications of diabetes mellitus. Increase in the shift of glucose into the aldose reductase pathway during diabetes leads to accumulation of sorbitol and fructose in the cells, and causes an imbalance in the associated cofactors, which in turn cause deleterious events such as oxidative stress, endoplasmic reticulum (ER) stress and cell death in the kidney. The objective of this study was to investigate the effects of high glucose on renal tubular cells cultured in vitro and evaluate the protective effects of inhibiting the aldose reductase pathway. Normal rat kidney (NRK-52E) cells were exposed to high glucose (30 mM) or normal glucose (5 mM) media for 24 to 72 hours, and then assessed for changes in cell viability using MTT assay. The expression of aldose reductase, markers of ER stress such as GRP78 and CHOP, and activation of Akt and ERK1/2 signaling has been measured using western blotting. In addition, mitochondrial membrane potential has been assessed using JC-1 assay. Exposing NRK-52E cells to 30 mM glucose containing media decreased cell viability after 48 h (84%) with further decline in viability at 72 h (64%). Aldose reductase expression was increased at 48 h, and this was associated with slight depolarization in mitochondrial membrane potential. In addition, high glucose exposure caused acute activation of both Akt and ERK pathways. In contrast, no induction of the ER stress markers has been identified in the model. Interestingly, co-incubating cells with 1 μM of an aldose reductase inhibitor epalrestat has reversed the cellular injury and signaling changes induced by high glucose. These findings suggest that high glucose conditions trigger cell death and depolarize mitochondrial membrane in renal tubular cells. Moreover, hyperglycemia was able to induce Akt and ERK pathways, which are actively involved in the mechanisms contributing to DN. Inhibition of the aldose reductase pathway has reversed the hyperglycemia-induced deleterious effects on renal cells, and hence, represents a potential therapeutic strategy to improve renal cell function during diabetes.