Tug, Ali

Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD. Methods We investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1 HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy. Results Serum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling. Conclusion In PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression. Support or Funding Information R25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Introduction Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive development and enlargement of bilateral renal cysts, and the majority of renal complications including organ failure arise mainly as a consequence of the cystic burden along with associated inflammation and fibrosis. Abnormal epithelial cell proliferation, along with the inability to maintain planar cell polarity, underlies cyst formation and enlargement. Therefore, processes that stimulate renal cell proliferation have the potential to generate the cystic phenotype. Interestingly, an increased nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) response has been shown to direct cancer cells into an anabolic mode that favors cellular proliferation, and has been associated with renal cyst formation in experimental and human fumarate hydratase deficiency. However, the Nrf2 response has not been described in ADPKD. We hypothesized that early ADPKD presents with an elevation in the Nrf2 response that favors cellular proliferation and contributes to cystogenesis. Methods We sought to longitudinally characterize the Nrf2 response and association with cystogenesis and fibrosis in a slow progressive mouse model of ADPKD ( Pkd1 RC/RC ) and its wildtype controls (n=6 males, 6 females per group). Urine and plasma samples were collected at 30, 60, 120, and 180 days for chemistries and metabolic profiles, and cystic index (CI), and total kidney volume (TKV) were determined from abdominal MRI. Nrf2 levels and related response enzymes, as well as cell proliferation and fibrosis were analyzed using western‐blots, immunofluorescence, and assay kits. Results At 30 days, Pkd1 RC/RC mice presented increased CI (Figure ) and TKV/BW. However, serum creatinine and fibrotic markers were not different compared to controls. Pkd1 RC/RC mice exhibited elevated Nrf2 expression and immunoreactivity early on that declined as ADPKD progressed from 30 to 180 days (Figure ) and correlated directly with cell proliferation (R 2 =0.693, p<0.05) and inversely with fibrotic markers (R 2 =0.672, p<0.05). Conclusions Our study shows longitudinal changes in the Nrf2 response in Pkd1 RC/RC mice that are associated with cystogenesis early on and renal fibrosis at later stages of the disease. These findings have significant implications for the treatment of human ADPKD, and suggest that Nrf2 modulators might represent an advantageous intervention for the disease Support or Funding Information NIDDK R21 DK118391, R25‐DK101405, NIDDK P30 DK090728 A) Representative H&E‐stained kidney mid‐sections from Wild Type (WT) and Pkd1 RC/RC mice at 1 month. Pkd1 RC/RC mice presented increased cystic index (CI) from early stages of the disease. CI is calculated as % of cyst area within the total kidney section. B) Longitudinal levels of Nrf2 activation in WT and Pkd1 RC/RC mice. Pkd1 RC/RC mice exhibited elevated Nrf2 levels early on that declined as the disease progressed. Nrf2 activation is calculated from nuclear extracts by colorimetric assay. Figure 1