Effects of Metformin on the AMPK Pathway, Cystogenesis and Metabolomic Biomarkers in ADPKD Kidney Epithelial Cells

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy characterized by the slow and continuous development of cysts derived from renal tubular epithelial cells and is caused by mutations in genes that encode polycystin‐1 (PKD1) and polycystin‐2 (PKD2). Cyst formation and growth results from dysregulated intracellular calcium homeostasis and increased cAMP generation, CFTR activity, and mammalian target of rapamycin (mTOR) pathway signaling. Recent studies in PKD1‐null cells also suggest that there is decreased activity of the metabolic sensor AMP‐activated protein kinase (AMPK) and an excessive reliance on aerobic glycolysis (the ‘Warburg effect’). Here we sought to determine the roles of AMPK and dysregulated metabolism in the pathophysiology of ADPKD, and how the AMPK activator metformin, a potential novel therapeutic for ADPKD, affects these parameters and cyst growth in vitro in both PKD1‐ and PKD2‐null cells. We used immortalized PKD1‐ and PKD2‐null kidney epithelial cells for immunoblotting to measure cell proliferation (mTOR and ERK) and AMPK pathway markers and the levels of key glycolytic enzymes. We also used 3‐D Matrigel cultures to assess cyst growth and Seahorse Extracellular Flux Analyzer assays to evaluate the metabolic phenotype of cells as a function of different treatments. Unlike in PKD1‐null cells, PKD2‐null cells displayed neither excessive activation of the mTOR pathway, inhibition of the AMPK pathway, nor apparent metabolic dysregulation under baseline conditions. Nevertheless, metformin effectively activated AMPK in polarized PKD2‐null cells and significantly inhibited cyst growth in 3‐D cultures, both in the presence and absence of cAMP agonists. PKD1‐null cells had significantly increased cAMP production, with increased expression of the key glycolytic pathway enzymes pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Metformin treatment dose‐dependently inhibited expression of these enzymes and ERK activation in PKD1‐null cells. Seahorse metabolic assays showed that PKD1‐null cells have significantly increased basal and compensatory glycolysis. Metformin inhibited oxidative metabolic flux in both PKD1‐ and PKD2‐null cells. We conclude that cystogenesis may occur through differing signaling pathways depending on mutation in ADPKD. Metformin may thus exert beneficial effects in PKD2 mutant cells by mechanisms distinct from those proposed in PKD1 mutant cells and may be in part AMPK‐independent. Additional studies in primary cells and in vivo will be applied to confirm these immortalized cell results.Support or Funding InformationSupported by NIH‐NIDDK and U.S. Dept. of DefenseThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.