The mitochondria rich epithelial cells of the renal medullary thick ascending limb (mTAL) reabsorb nearly 25% of filtered sodium (Na+) and are a major source of cellular reactive oxygen species (ROS). Although we have shown that delivery of Na+ to the mTAL of rats increases superoxide (O2•-) production in mTAL, little is known about H2O2 production given the lack of robust and selective fluorescent indicators for determining changes within the whole cell, specifically in the mitochondria. The present study determined the effect of increased tubular flow and Na+ delivery to mTAL on the production of mitochondrial H2O2 in mTAL. H2O2 responses were determined in isolated perfused mTAL of Sprague Dawley rats using a novel mitochondrial selective fluorescent H2O2 indicator, MitoPY1, and a novel, highly sensitive and stable cytosolic-localized H2O2 indicator, PF6-AM. The results showed that mitochondrial H2O2 and cellular fluorescent signals increased progressively over a period of 30 minutes following increased tubular perfusion (5 to 20 nL/min) reaching levels of statistical significance at ~ 10 to 12 minutes. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), PEG-catalase and by reducing Na+ transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H2O2 production. Cytoplasmic H2O2 (PF6-AM) increased in parallel with mitochondrial H2O2 (MitoPY1) and was partially attenuated (~65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H2O2 is produced in the mitochondria in response to increased flow and delivery of Na+ to the mTAL and that whole cell H2O2 levels are triggered by the mitochondrial ROS production. The mitochondrial production of H2O2 may represent an important target for development of more effective antioxidant therapies.
- reactive oxygen species
- medullary thick ascending limb
- sodium delivery and flow
- Copyright © 2011, American Journal of Physiology - Renal Physiology