Inhibition of Transforming Growth Factor-β Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Abstract

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its <i>in vivo</i> counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo <i>in vitro</i> thereby losing important cell-type specific functions. We previously identified transforming growth factor-β (TGF-β) as a signaling pathway necessary for <i>in vitro</i> differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells <i>in vitro</i>. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-β receptors possibly responsible for the improved differentiation, evaluate whether TGF-β inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-β signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-β inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-β signaling pathway governs multiple features of differentiation in renal proximal tubule cells <i>in vitro</i>. Inhibition of TGF-β by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells <i>in vitro</i>. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between <i>in vitro</i> and <i>in vivo</i> phenotypes, which has been elusive. We show that control of transforming growth factor-β signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.