Characterizing Lipid Metabolism in Diseased Kidneys and Understanding the Impact of Blood Contamination

Background: The kidney exhibits a high bioenergetic flux owing to constitutive filtration and absorption. Dysfunction of proximal tubule metabolism, specifically reduced mitochondrial fatty acid oxidation and increased lipid synthesis, are considered key drivers of chronic kidney disease (CKD) and are recapitulated in CKD mouse models. There are numerous ways to characterize these processes, including lipid profiling, enzyme activities and flux tracing of energy substrates. However, the kidney is enriched by blood, which itself contains lipids and may influence the measurement of endogenous lipids or synthesis byproducts. Methods: We characterized lipid abundance, mitochondrial markers and kidney functional readouts in two renal disease models: 1) AAV-renin, uninephrectomized db/db mice and 2) mice fed a 0.15% adenine diet for 6 weeks. Lipids were measured by HPLC, mtDNA by qPCR, and mitochondrial enzymes by absorbance kinetic assays. To isolate endogenous kidney lipids from the blood, we compared kidneys from C57BL6 mice with and without systemic PBS perfusion prior to collection. We also compared the effects of perfusion on de novo lipogenesis (DNL) by tracing D2O incorporation into tissue palmitate. All procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process. Results: In the db/db Unx Renin mice, we found reduced GFR and mitochondrial content and a significant elevation of plasma cholesterol and cholesterol esters relative to controls. However, we observed no impact on kidney cholesterol or triglyceride levels. Similarly, in the adenine model, we found reduced GFR, mitochondrial content, and kidney cholesterol content. There were no effects of perfusion on kidney triglyceride levels or rates of DNL, yet there was higher free cholesterol in perfused vs. non-perfused kidney. Conclusion: In two renal disease models we found reduced mitochondrial oxidative capacity, but no elevation in kidney lipids. Moreover, the lack of lipid changes in diseased kidney is unlikely to be due to contamination of kidney lipids by the blood, but further studies are warranted. Funding: Commercial Support - Pfizer, Inc