P0029GLUCOSYLCERAMIDE SYNTHASE INHIBITION PRESERVES MITOCHONDRIAL FUNCTION AND REDUCES REACTIVE OXYGEN DAMAGE IN THE JCK MOUSE MODEL OF POLYCYSTIC KIDNEY DISEASE

Abstract

Abstract Background and Aims Polycystic kidney disease (PKD) is characterized by the formation and progressive growth of renal cysts ultimately leading to end-stage renal disease (ESRD). Previous work has shown that treatment of PKD mouse models with a glucosylceramide synthase inhibitor (GCSi) inhibits glycosphingolipid metabolism and blocks disease progression. Clinical trials to determine the efficacy of GCSi treatment for ADPKD are currently ongoing. ADPKD patient kidneys, as well as kidneys from in vivo mouse models of PKD, display defects in mitochondrial morphology and function, leading to reactive oxygen species (ROS) generation. Functional defects include decreased fatty acid oxidation and increased glycolysis that can promote cellular proliferation. To determine if glucosylceramide synthase inhibition ameliorates mitochondrial dysfunction, we assessed the impact of GCSi treatment on mitochondrial formation and function in the jck mouse model of PKD. Method Twenty-six-day old WT or jck mice were treated with vehicle or 60 mg/kg GCSi (Genz667161) in feed for 38 days prior to tissue harvest. Kidney mRNA expression was measured using RT-PCR. Kidney protein levels were measured by western blot. Mitochondrial DNA content was measured using real-time PCR. Oxidized DNA was detected by immunohistochemistry using anti-8OHdG antibodies. Oxidized proteins were measured using the Oxyblot system (manufacturer). Results Reduced cystogenesis following GCSi treatment was associated with preserved mitochondrial number in jck mice as evidenced by increased levels of mitochondrial DNA, increased mitochondrial proteins (Tom20, VDAC, SirT3, and MT-Co1), and elevated mRNA expression (Nd3, Cox3, and Atp8). Stabilization of mitochondrial number was accompanied by activation of pathways that promote mitochondrial biogenesis, including increased PGC1α protein and Ppargc1a and Tfam1. Downregulation of several antioxidant genes (Gpx6, Gstk1, Prdx3, and Sod2) were observed in untreated jck samples. Consistent with this, levels of oxidized DNA and oxidized proteins were increased in untreated jck samples. Treatment with GCSi partially reversed the downregulation of antioxidant genes and decreased the levels of oxidized DNA and proteins to near WT levels in jck tissues. We then compared the jck model to tissues derived from ADPKD patients. Western blots demonstrate a reduction in electron transport chain (CxI-CxV) and mitochondrial outer-membrane markers in both untreated jck and ADPKD samples compared to controls. Similarly, the mitochondrial genes Nd1, Nd2, Cox2, and Atp6 were reduced in untreated jck kidneys and ADPKD samples relative to controls. As previously mentioned, we observed a generalized loss of antioxidant gene expression in untreated jck kidneys. Consistent with this, there were increased levels of both oxidized DNA and oxidized protein in untreated jck samples; this increase in DNA and protein oxidation was mirrored in ADPKD patient samples. Conclusion Mitochondrial number and function were reduced in both untreated jck mouse kidneys and human ADPKD samples. Increased oxidative stress, as evidenced by increased levels of oxidized DNA and proteins, is evident in both jck and ADPKD tissues. In vivo treatment with a GCSi effectively reversed the observed increase in oxidative stress and inhibited disease progression in the jck model of ADPKD. Our data demonstrate that reduced kidney cyst growth following GCSi treatment is associated with preserved mitochondrial formation and function.