Changes in mitochondrial NAD redox state drive acidosis induced acute kidney injury

Abstract

The kidney has an important role in maintaining normal blood pH. Mitochondria in the proximal tubule (PT) produce ammonia and bicarbonate from glutamine, and this pathway (ammoniagenesis) is acutely upregulated in PTs during metabolic acidosis (MA). MA is associated with an accelerated renal function decline in patients with chronic kidney disease (CKD). MA is also frequently found in patients with acute kidney injury (AKI) and correction of acid‐base status improves outcome, suggesting a direct relationship between MA and AKI. However, there is a surprising lack of experimental knowledge on the molecular mechanism responsible for cellular dysfunction associated with MA. Using live cell imaging and other complementary techniques we aimed to increase understanding of how PTs respond acutely to MA.Acute exposure of mouse kidney cortical slices to acidic extracellular pH caused a decrease in mitochondrial NADH fluorescence signal specifically in PTs, without changing total NADH content, consistent with a switch to a more oxidized state. Mitochondria remained energized and baseline oxygen consumption rate (OCR) was unchanged. Electron entry into the respiratory chain (RC) is thought to be predominantly via complex II (CII) in the kidney. However, under acidosis maximal OCR in isolated PTs was decreased and response to rotenone was exaggerated, suggesting a switch to complex I, which oxidizes NADH. Large vacuole like structures subsequently appeared in S2 PT segments, which were identified as lipid filled multi‐lamellar bodies (MLBs) on electron microscopy. Supplementation with nicotinamide or reducing NAD with lactate both inhibited the appearance of vacuoles. Moreover, increasing pH back to 7.4 reversed changes in NADH signal and led to disappearance of vacuoles.MA was induced in vivo using an established protocol (0.8 g/kg BW NH4Cl). Blood pH decreased to 7.0–7.1, which is comparable to patients with MA and AKI. Histological analysis showed thinning of PTs and shedding of debris, consistent with an acute metabolic insult. Large vacuoles were observed in S2 PT segments. Intravital imaging revealed that mitochondria in PTs remained energized, but uptake of fluorescently labeled dextrans by endocytosis was severely inhibited. Glomerular filtration rate (GFR) and tubular flow were unaltered during MA, excluding major hemodynamic effects.In summary, we have found evidence that MA induces an acute alteration in mitochondrial NAD redox state and lipid metabolism in PTs, which is explained by a switch to increased complex I activity. While these changes are potentially beneficial for ammoniagenesis, they are associated with major defects in PT cell structure and transport function. Thus, we show that MA directly causes AKI, and experiments are ongoing to assess whether bicarbonate supplementation has beneficial effects.Support or Funding InformationSwiss National Science Foundation and The Swiss National Centre for Competence in Research (NCCR) Kidney Control of Homeostasis. JRM is a IKPP2‐fellow funded by the EU 7th Framework Programme for research, technological development and demonstration under grant agreement 608847.