Abstract P399: High Salt Induced Mitochondrial Uncoupling And Enhanced Respiratory Activity In Isolated Proximal Tubules From Dahl Salt-sensitive Rats

Abstract

The nephron actively reabsorbs ~99% of the Na + from the glomerular filtrate to maintain fluid and solute homeostasis, ~70% of which occurs in the proximal tubules (PT), where mitochondria consume a great amount of O 2 for ATP production to meet the energy demand. However, the effect of a high salt diet upon the PT energy metabolism and respiratory activity is not well-known. To determine this, the PT of Dahl salt-sensitive (SS) rats were isolated with the rats fed either 0.4% NaCl (LS) or 7, 14 and 21 days after feeding a 4.0% NaCl (HS) diet. Changes in O 2 consumption rate (OCR) of the PT were determined using a high-throughput Agilent Seahorse XF96 Extracellular Flux Analyzer and Mitochondrial Stress Test Protocol. Basal OCRs were assessed with only amino acids used as fuel for oxidative phosphorylation (OxPhos). Under these conditions, by day 7 of the HS diet, OCR increased from 9.2±0.5 to 17.2±0.8 pmol/min/μg, (p<0.05), which rose to 20.2±1.0 and 18.9±0.7 pmol/min/μg (p<0.05 vs LS), respectively, at HS day 14 and HS day 21. The Seahorse protocol was also modified to determine the OCR responses to representative circulating substrates. The OCR responses from baseline to glucose or pyruvate were unchanged by the HS diet compared to the PT of LS fed rats. However, a sustained increase in the proton leak OCR was observed for glucose (LS: 2.5±0.5 vs HS day 7: 7.3±1.1; p<0.05) and for pyruvate (LS: 2.3±0.3 vs HS day 7: 7.9±0.2; p<0.05). These high levels of proton leak were maintained throughout the 21 days of the HS diet. Since it is known that uncoupling proteins (UCP) and ADP/ATP translocase (ANT) enhance proton leak, gene expression was quantified from the isolated PT segments. Increased Ucp2 expression was found (p<0.05) in response to the HS diet (days 7, 14, and 21) compared to LS fed rats but no changes were observed in the expression of Slc25A4 (ANT1) or Slc25A5 (ANT2). In summary, the HS diet resulted in increased PT O 2 consumption and proton leak in SS rats which was associated with an increase of Ucp2 gene expression. Given recent evidence that this is associated with a decrease in renal blood flow (RBF/kidney wt), we propose that increases in the PT O 2 consumption contribute to the onset of regional chronic ischemia and exacerbates renal damage and hypertension.