β-hydroxybutyrate-induced growth inhibition and collagen production in HK-2 cells are dependent on TGF-β and Smad3

Abstract

Background. Ketonuria is common in diabetes. The major form of ketone body is β-hydroxybutyrate (β-HB), which is metabolized by the proximal tubule. Transforming growth factor β (TGF-β) and tubulopathy are important in diabetic nephropathy. Thus, the role of TGF-β and the downstream Smad3 in β-HB-induced effects in the human proximal tubule (HK-2 cell) was studied. Methods. Effects of β-HB (0.1 to 10 mmol/L) on HK-2 cells were determined for: proliferation, cell cycle distribution, collagen production, tubular transdifferentiation [expression of α-smooth muscle actin (α-SMA) protein], TGF-β, Smad2/3, p21WAF1, and p27kip1. Results. β-HB (0.1 to 10 mmol/L) dose dependently decreased proliferation, arrested the cells in G0/G1 phase of the cell cycle, and increased p21WAF1/p27kip1 protein expression at 48 hours (without affecting p21WAF1/p27 kip1 mRNA and transcription). β-HB (1 mmol/L) increased p21 WAF1/p27kip1 protein half-lives. β-HB (1 mmol/L) increased TGF-β transcription at 24 hours and TGF-β1 mRNA/bioactivity at 48 hours. β-HB (1 mmol/L) increased nuclear Smad2/3 protein expression and increased collagen production (without affecting tubular transdifferentiation), which were reversed by Smad7, dominant-negative Smad3, and N-acetylcysteine. Dominant-negative Smad3 reversed β-HB-induced TGF-β transcription at 24 hours, and reversed TGF-β1 bioactivity at 48 hours. Dominant-negative Smad3 reversed β-HB-induced p21 WAF1/p27kip1 protein expression at 48 hours. Finally, N-acetylcysteine, TGF-β antibody, Smad7, and dominant-negative Smad3 reversed β-HB (1 mmol/L)-induced growth inhibition at 48 hours. Conclusion. β-HB activated Smad 2/3 by oxidative stress. TGF-β and Smad3 mediate β-HB-induced cell cycle-dependent growth inhibition while Smad3 mediate β-HB-induced collagen production and p21WAF1/p27kip1 protein expression in HK-2 cells. Moreover, β-HB increased p21 WAF1/ p27kip1 protein expression by increasing p21 WAF1/p27kip1 protein stability.