E SMITH1,2, T HEWITSON1,2
1Royal Melbourne Hospital, Melbourne, Australia, 2University of Melbourne, Melbourne, Australia
Aim: To define the mechanisms of metabolic reprogramming that occur in fibroblast activation
Background: TGF-β1 reprograms metabolism in renal fibroblasts, inducing a switch from oxidative phosphorylation to aerobic glycolysis and a reduction in intracellular acetyl-CoA. Our recent transcriptome analysis implicates the pyruvate dehydrogenase complex (PDC) in this process.
Methods: Fibroblasts from normal and injured rat kidneys (3-days post unilateral ureteric obstruction) were treated with 1ng/ml TGF-β1. Flow cytometry (FC) for α-smooth muscle actin (SMA) and pro-collagen I were used as markers of myofibroblast differentiation and activation. PDC activity was measured by PDH-E1α subunit phosphorylation (FC, western blotting) and acetyl-CoA generation. Enzymatic regulation of PDC was assessed through expression of activating kinases and inactivating phosphatases. Overexpression of mutant PDH-E1α (resistant to phosphorylation) and chemical inhibitors of PDC (CPI-613) activity and PDH kinase 1 (PDK1) (sodium dichloroacetate; DCA) activity were used to confirm significance.
Results: TGF-β1 downregulates acetyl-CoA biosynthesis via regulation of the PDC. TGF-β1 reduced the PDC subunit PDH-E1α in injured but not normal fibroblasts. An increase in expression of PDK1, and reduction in the phosphatase PDP1, were commensurate with net phosphorylation and inactivation of PDC. Over-expression of mutant PDH-E1α ameliorated effects of TGF-β1, while CPI-613 induced SMA and pro-collagen I expression. While TGF-β1 decreased mitochondrial PDH-E1α, there was a parallel increase in the nucleus, although nuclear activity remained substantially less than mitochondrial activity. The effect of TGF-β1 on PDC activity, acetyl-CoA, SMA and pro-collagen I was ameliorated by DCA. A reduction in acetyl-CoA, and therefore acetylation substrate, also resulted in a generalised loss of protein acetylation with TGF-β1.
Conclusion: TGF-β1 in part regulates fibroblast activation in primed cells via effects on PDC activity.
A/Prof. Tim Hewitson is a basic scientist in the Department of Nephrology at The Royal Melbourne Hospital and a Principal Fellow in the University of Melbourne, Department of Medicine. His long standing research interests are focused on how AKI transitions to CKD, and the mechanisms of its subsequent progression.