Metformin Inhibits Cyst Growth and Alters Cell Metabolism in Vitro and Improves Relevant Disease Parameters in a Hypomorphic ADPKD Mouse Model

Abstract

BackgroundAutosomal dominant polycystic kidney disease (ADPKD), caused by mutations in the Pkd1 or Pkd2 genes encoding polycystins, presents with progressive development of renal cysts and eventual end‐stage kidney disease and has limited treatment options. Previous work showed that metformin treatment reduces cyst growth in early, rapid ADPKD mouse models, potentially through AMPK‐dependent inhibition of CFTR‐mediated fluid secretion, mTOR signaling and cAMP production. Here we tested whether metformin treatment ameliorated ADPKD manifestations in vitro and in a relevant, slowly progressive ADPKD mouse model.MethodsImmortalized Pkd1‐null mouse epithelial cells were grown in Matrigel for 3D cyst growth assays to assess the effects of treatment with metformin ± other drugs (tolvaptan and the FXR agonist INT‐747) in vitro. These cells were also used for metabolic assays using the Seahorse XFp system and immunoblotting for key enzymes with these treatments. Using the slowly developing ADPKD mouse model with an R3277C knock‐in point mutation in both alleles of the Pkd1 gene (Pkd1RC/RC transgenic mice), male and female mice were treated ± metformin (300 mg/kg/day in drinking water) from 3 months through 9–12 months of age. During this treatment period, we periodically measured tail cuff blood pressures, glomerular filtration rates (GFR) by the FITC‐sinistrin technique, and blood studies by i‐Stat. At euthanasia, we assessed kidney histology (e.g., cystic index), total kidney weight/body weight ratio (TKW/BW), and mRNA and protein expression by qPCR and immunoblotting of key cell signaling, injury and inflammatory markers.ResultsMetformin treatment inhibited 3D cyst growth in Pkd1‐null cells in vitro and had added inhibition when used in combination with tolvaptan or INT‐747. Metformin promoted basal and compensatory glycolytic flux and inhibited oxidative metabolic flux in Pkd1‐null cells while reducing the activity of ERK1/2. As previously shown, Pkd1RC/RC females had a more severe disease phenotype as compared with males. Metformin treatment reduced TKW/BW relative to age‐ and sex‐matched controls at both 9 and 12 mos. of age. Metformin treatment also improved systolic blood pressures and increased GFR relative to controls at 9 mos. in both sexes. Moreover, metformin increased hematocrit and generally lowered blood urea nitrogen (BUN) levels relative to controls in both sexes. Finally, metformin treatment also reduced gene and protein expression levels of cyclin‐dependent kinase (CDK1) and gene expression of the key kidney injury markers kidney injury molecule‐1 (KIM‐1) and neutrophil gelatinase‐associated lipocalin (NGAL) and the inflammation markers tumor necrosis factor‐α and interleukin‐6 in these mice, along with protein expression of KIM‐1.ConclusionsMetformin inhibits cyst growth in a potentially additive manner with other ADPKD therapies and alters cellular metabolism in vitro and improves various key ADPKD disease parameters in a relevant, slowly progressive ADPKD model. Additional studies to examine the effects of metformin in PKD clinical trials are underway.Support or Funding InformationSupport by U.S. Dept. of Defense, UKRO and USC