Metformin Protects Renal Tubular Cells Against Albumin‐Induced Alterations and Cellular Injury

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Introduction Proteinuria (or increased albumin excretion), a hallmark of chronic kidney disease (CKD), plays a critical role in the development of CKD. Elevated albumin levels are thought to induce endoplasmic reticulum (ER) stress and subsequently activate the AKT pathway, and lead to inactivation of AMP‐activated kinase (AMPK). AMPK inactivation has been shown to activate mTOR (mammalian target of Rapamycin), which inhibits autophagy and induces epithelial‐to‐mesenchymal transition (EMT), ultimately resulting in accelerated renal cell apoptosis. Thus, the objective of this study is to investigate the effect of AMPK activation on ER stress, AKT, mTOR, EMT, autophagy and apoptosis that are thought to mediate renal cell injury during proteinuria using an in vitro model of albumin‐induced renal cell injury. Methods Normal rat kidney proximal tubular (NRK‐52E) cells grown to 60% confluency were exposed to albumin at concentrations ranging from 1 to 30 mg/ml for 24 to 72 hours. Following optimization of the albumin‐induced renal injury model, studies were performed in the presence and absence of AMPK activator metformin (1 mM) for 24 to 72 hours. Cells were then assessed for alterations in AMPK, AKT and mTOR pathway, and the markers of ER stress, EMT, autophagy and apoptosis. Results Exposure to albumin for 72 hours caused a 4‐fold induction in ER stress marker CHOP and EMT marker α‐SMA. Similarly, increased phosphorylation of AKT and P70S6K (a downstream target of mTOR) were noted in cells subjected to albumin treatment. In addition, albumin treatment caused a dose‐dependent reduction in AMPK phosphorylation and about 66% decrease in the expression of autophagy marker LC3‐II. The above changes were observed in conjunction with prominent induction of apoptotic markers ‐ caspase‐3 and caspase‐12 ‐ in cells exposed to albumin. In contrast, metformin treatment induced phosphorylation of AMPK, and inhibited AKT and P70S6K phosphorylation in NRK‐52E cells exposed to albumin. Notably, metformin also prevented albumin‐induced EMT; this was marked by a 50% decrease in α‐SMA and a 60% increase in E‐cadherin expression. In addition, 2.5‐fold increase in LC3‐II expression was noted. Intriguingly, the pro‐apoptotic protein CHOP was induced following treatment with metformin; nonetheless, the expression of apoptotic markers caspase‐12 and caspase‐3 was reduced indicating that metformin protected the cells against albumin‐induced apoptosis. Conclusion Albumin treatment induces ER stress, and activates AKT, EMT and apoptosis, with concomitant decreases in autophagy and AMPK phosphorylation in renal tubular cells. Activation of AMPK via metformin treatment suppresses AKT and mTOR activation, and prevents EMT and apoptosis, but increases autophagy and ER stress in renal tubular cells. Further studies are required to understand the mechanisms by which metformin differentially modulates ER stress and apoptosis in renal cells under proteinuria. Support or Funding Information This study is funded by a student research grant (# QUST‐CPH‐SPR‐14/15‐15) from the Office of Academic Research, Qatar University.