A Novel Approach for Assessing Mitochondrial Respiration in Freshly Isolated Glomerular and Tubular Fractions from Whole Mouse Kidney

Abstract

Intro: Understanding mitochondrial function within specific renal compartments is crucial for unraveling the intricate metabolic processes underlying kidney physiology and pathophysiology. In this study, we present a novel method for accurately measuring mitochondrial respiration in freshly isolated glomerular and tubular fractions derived from dissected kidneys. Methods: Employing a differential adhesive sieving technique, we isolated glomerular and tubular fractions from freshly harvested whole kidneys (n=2) and subsequently utilized a standard substrate-inhibitor titration protocol to assess mitochondrial respiration in the Oroboros Oxygraph O2K (Oroboros Instruments, Innsbruck AT). Results: Notably, our findings revealed distinct state 3 respiration rates in various fractions, with the 70 μm tubule fraction exhibiting a rate of 2133.029 pmol/sec/mgprotein, the 40 μm tubule fraction displaying a rate of 522.4116 pmol/sec/mgprotein, the first glomerular fraction demonstrating a rate of 407.7911 pmol/sec/mgprotein, and the second tubular fraction manifesting a rate of 362.3286 pmol/sec/mgprotein. Moreover, these fragments exhibited differential substrate preferences, as the addition of succinate increased state 3 respiration by ~90% in the 70 μm tubule fraction. In contrast, succinate increased respiration by ~30% in the 40 μm tubule and glomerular fractions. Discussion: These ongoing experiments provide compelling evidence supporting the feasibility and reliability of our approach in quantifying mitochondrial respiration within isolated renal fractions. Our methodology not only enables precise measurements of mitochondrial function in distinct nephron segments but also lays the groundwork for further investigations into the physiological and pathological implications of mitochondrial dynamics in kidney health and disease. This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.