Inhibition of TGF-β in Tubule Cells Increases Respiration via Upregulation of Respiratory Genes.

Abstract

<b><i>Background:</i></b> Renal tubule cells lose differentiated characteristics in artificial culture, limiting their application in medical research and cell therapy. We previously showed that adding inhibitors of transforming growth factor-β (TGF-β) signaling to cell culture media increased specific transport functions characteristic of differentiated tubule cells. Transport in proximal tubule cells is energetically demanding; <i>in vivo</i>, these cells rely primarily on oxidative phosphorylation of fatty acids for adenosine triphosphate (ATP) generation. We examined whether TGF-β inhibition, with or without metformin, altered glycolysis and oxidative phosphorylation compared with standard culture conditions. <b><i>Approach:</i></b> Primary renal tubule cells (PRTC) were cultured with or without an inhibitor of TGF-β receptor I and with or without metformin in a 2 × 2 factorial design. First, expression of proteins in fatty acid transport and the electron transport chain was compared between conditions. The relative contributions of glycolysis and oxidative phosphorylation to ATP generation were assessed by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). We also tested substrate-specific contributions using inhibitors of pyruvate, glutamine, and carnitine mitochondrial entry. Finally, OCR and transport were measured after 48 weeks in culture to determine durability of culture phenotype. <b><i>Results:</i></b> Metformin and SB431542 increased expression and phosphorylation of proteins in the electron transport chain and involved in fatty acid transport. Metformin and TGF-β inhibition increased oxidative phosphorylation. Metformin decreased glucose dependency, while combination with TGF-β inhibition increased fatty acid dependency. Differences in OCR and transport between treatment conditions persisted at 48 weeks in culture. <b><i>Discussion:</i></b> Renal tubule cell transport is energetically demanding, so cellular differentiation requires matching increases in energetic machinery. We found that metformin and inhibition of TGF-β increased oxygen consumption and utilization of fatty acids in cultured primary tubule cells. These data support the hypothesis that TGF-β inhibition <i>in vitro</i> not only increases expression of a broad array of transporters characteristic of the proximal tubule, as we previously showed, but also improves the supply of energy to support active transport.