Renoprotective Effects of 4‐Phenylbutyrate Against Palmitate‐Induced Renal Cell Injury

Abstract

BackgroundIncreased dietary intake of fats rich in saturated fatty acids and dyslipidemia have been shown to significantly aggravate the risk of cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Although multiple mechanisms including endoplasmic reticulum (ER) stress and oxidative stress are implicated in the pathophysiology of CKD, the effects of elevated free fatty acid (FFA) levels per se on renal cells is still unclear. Hence, we investigated the effects of palmitic acid (a saturated fatty acid) on rat renal proximal tubular cells in vitro, and tested the renoprotective effects of 4‐phenylbutyrate (4‐PBA), a chemical chaperone shown to inhibit ER stress in vitro and in vivo, in our model.MethodsSubconfluent rat renal proximal tubular (NRK‐52E) cells were exposed to 100 mM palmitic acid in presence and absence of 5 mM 4‐PBA for 24 hours. Cells were then assessed for alterations in cell viability (using MTT assay), oxidative stress (via Dichlorofluorescein (DCF) staining) and the expression of ER stress markers (i.e., glucose‐regulated protein, GRP78 and C/EBP homologous protein, CHOP) and apoptosis (caspase‐3 and ‐12) using western blotting. Flow cytometry for cell cycle analysis and immunoblotting for p27 (an inhibitor of cell cycle) were performed to determine the effects of palmitate and 4‐PBA on the cell cycle in renal cells.ResultsRenal cells exposed to palmitate for 24 hours showed decreased viability (76.9% to that of control), increased DCF staining (1.3‐fold increase over control), and marked upregulation of GRP78 and CHOP. Similarly, the expression of active forms of caspase‐3 (1.8‐fold increase over control) and caspase‐12 (3‐fold increase over control) were also increased in renal cells treated with palmitate. These changes were significantly attenuated in cells co‐treated with 4‐PBA. Intriguingly, exposure to palmitate did not alter the cell cycle in renal cells, whereas treatment with 4‐PBA caused downregulation of p27 (54% to that of control) and increased the transition of cells from G1 to S‐phase and G2/M phase.ConclusionsOur findings indicate that exposure to palmitate induces ER stress as well as oxidative stress in renal cells, and results in renal cell apoptosis. Co‐treatment with 4‐PBA protects renal cells against the perturbations induced by palmitate and improves renal cell viability, which underscores the renoprotective effects of 4‐PBA against fatty acid‐induced renal cell injury. Further studies are required to understand the mechanisms by which 4‐PBA prevents oxidative stress and alters cell cycle in renal cells.Support or Funding InformationSupported by student research grants # QUST‐CPH‐FALL‐12/13‐6 and QUST‐CPH‐SPR‐12/13‐3 from the Office of Academic Research, Qatar University.