TGF-BETA1 IS A REGULATOR OF THE PYRUVATE DEHYDROGENASE COMPLEX IN PRIMED RENAL FIBROBLASTS

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.


Biography:
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.

Recent Comments
    Categories