Assessment of Mitochondrial Respiratory Function in Isolated Nephron Segments of Dahl SS Rats in Salt Induced Hypertension

Abstract

RationaleKidney nephrons actively reabsorb 99% of the filtrate to maintain body fluid and solute homeostasis. A large amount of ATP synthesis and O2 consumption must work coordinal to support this demand. We hypothesized that mitochondrial function of proximal tubules (PT) and medullary thick ascending limbs (mTAL) is altered during the development of salt induced hypertension in the Dahl salt‐sensitive (SS) rat.MethodThe mitochondrial oxygen consumption rates (OCR) were determined in freshly bulk isolated PT and mTAL segments from SS rats fed 0.4% NaCl diet (low salt; LS) or 4% NaCl diet (high salt; HS) for 7 days. A protocol was developed to determine basal levels of OCR and the effects of various key circulating substrates (glucose, lactate, pyruvate, glutamine, and palmitate) upon mitochondrial respiration (OCR) using the Agilent Seahorse XF96 Extracellular Flux Analyzer combined with the Agilent Seahorse XF Cell Mito Stress Test protocol.ResultsAnalysis of the renal tubular mitochondrial OCR data for five different circulating substrates confirmed that there were clear differences in the substrate preference between the PT and mTAL which were altered by HS feeding. It was found that PT preferred lactate and glutamine while mTAL preferred glucose and pyruvate and that both segments could effectively utilize palmitate as a mitochondrial substrate. PT isolated from SS rats fed HS displayed higher OCR levels compared to PT from LS fed rats, but HS diet did not result in significant differences in individual substrate effects. In contrast, isolated mTAL from HS fed rats exhibited increased OCR in response to glutamine and palmitate compared to mTAL from LS fed rats although no basal differences in OCR were observed.ConclusionThese observations demonstrated that renal tubular mitochondrial respiratory function is altered in SS rats during the development of salt‐induced hypertension and that both PT and mTAL exhibit an increased respiratory capacity in response to HS feeding. These changes in metabolism appear necessary to meet the increased tubular workloads and need for greater ATP production and O2 utilization in both PT and mTAL. We conclude, however, that different metabolic strategies are utilized by each of these tubular segments to meet these increased metabolic requirements.