Extracellular Flux Analysis Reveals that Transport Activity Regulates Mitochondrial Bioenergetics in Thick Ascending Limbs and Distal Convoluted Tubules
- Fluid, Electrolyte, Acid-Base Disorders: Basic
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
- Lin, Meng-Hsuan, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
- Chen, Jen-Chi, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
- Huang, Chou-Long, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
- Cheng, Chih-Jen, University of Iowa Hospitals and Clinics, Iowa City, Iowa, United States
Alterations of mitochondrial functions and substrate oxidation have been proven pathogenic in many renal tubulopathies. Traditional assays using kidney slices or cultured cells do not provide tubule-specific resolution because of cell heterogeneity and rapid dedifferentiation in vitro. Understanding mitochondrial respiration and energy metabolism in renal tubules offers deeper insights into the metabolic adaptations of renal tubules to stimuli.
We applied extracellular flux analysis (EFA) to investigate mitochondrial respiration and energy metabolism in isolated renal tubules. Briefly, after mice were sacrificed and perfused, renal tubules were microdissected and incubated at 37°C for 1 hour. The dynamic oxygen consumption and extracellular acidification rates (OCR/ECAR) following the sequential injection of mitochondrial/metabolic inhibitors were measured by Seahorse XFp analyzer.
Isolated renal tubules remained metabolically active and energetically dependent on mitochondrial oxidative phosphorylation (OXPHOS) during the assay. EFA detected significant OCRs/ECARs linearly correlated with sample sizes in millimeter-length renal tubules. Mitochondrial electron transport chain inhibitors suppressed OCRs and induced a simultaneous increase of ECARs in thick ascending limbs (TALs) and distal convoluted tubules (DCTs), indicating compensatory glycolysis. However, the proximal tubules (PTs) lacked noticeable compensatory glycolysis when OXPHOS was shut off. TALs and DCTs relied on glucose/pyruvate oxidation for ATP production in the basal state, and stimulation of oxygen consumption by mitochondrial uncoupler did not provoke the utilization of glutamine or long-chain fatty acids. Two-hour ouabain treatment reduced basal and ATP-linked mitochondrial respiration in isolated TALs. Besides, two-week furosemide treatment or deletion of with-no-lysine (WNK) kinase 4 further decreased the maximal and spare capacity of mitochondrial respiration in TALs via downregulating mitochondrial biogenesis.
EFA successfully provides tubule-specific metabolic features, showing differential glycolysis capacity and substrate utilization in PTs, TALs and DCTs. The transport activity can acutely regulate ATP turnover rate and chronically modulate mitochondrial biogenesis in TALs and DCTs.
- NIDDK Support