TY - JOUR
T1 - Increase of sodium delivery stimulates the mitochondrial respiratory chain H 2O 2 production in rat renal medullary thick ascending limb
AU - Ohsaki, Yusuke
AU - O'Connor, Paul
AU - Mori, Takefumi
AU - Ryan, Robert P.
AU - Dickinson, Bryan C.
AU - Chang, Christopher J.
AU - Lu, Yi
AU - Ito, Sadayoshi
AU - Cowley, Allen W.
PY - 2012/1
Y1 - 2012/1
N2 - 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. Although we have shown that delivery of Na + to the mTAL of rats increases superoxide (O2 -) production in mTAL, little is known about H 2O 2 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 H 2O 2 in mTAL. H 2O 2 responses were determined in isolated, perfused mTAL of Sprague-Dawley rats using a novel mitochondrial selective fluorescent H 2O 2 indicator, mitochondria peroxy yellow 1, and a novel, highly sensitive and stable cytosolic-localized H 2O 2 indicator, peroxyfluor- 6 acetoxymethyl ester. The results showed that mitochondrial H 2O 2 and cellular fluorescent signals increased progressively over a period of 30 min following increased tubular perfusion (5-20 nl/min), reaching levels of statistical significance at ∼10-12 min. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), polyethylene glycol-catalase and by reducing Na + transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H 2O 2 production. Cytoplasmic H 2O 2 (peroxyfluor-6 acetoxymethyl ester) increased in parallel with mitochondrial H 2O 2 (mitochondria peroxy yellow 1) and was partially attenuated (∼65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H 2O 2 is produced in the mitochondria in response to increased flow and delivery of Na_ to the mTAL, and that whole cell H 2O 2 levels are triggered by the mitochondrial reactive oxygen species production. The mitochondrial production of H 2O 2 may represent an important target for development of more effective antioxidant therapies.
AB - 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. Although we have shown that delivery of Na + to the mTAL of rats increases superoxide (O2 -) production in mTAL, little is known about H 2O 2 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 H 2O 2 in mTAL. H 2O 2 responses were determined in isolated, perfused mTAL of Sprague-Dawley rats using a novel mitochondrial selective fluorescent H 2O 2 indicator, mitochondria peroxy yellow 1, and a novel, highly sensitive and stable cytosolic-localized H 2O 2 indicator, peroxyfluor- 6 acetoxymethyl ester. The results showed that mitochondrial H 2O 2 and cellular fluorescent signals increased progressively over a period of 30 min following increased tubular perfusion (5-20 nl/min), reaching levels of statistical significance at ∼10-12 min. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), polyethylene glycol-catalase and by reducing Na + transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H 2O 2 production. Cytoplasmic H 2O 2 (peroxyfluor-6 acetoxymethyl ester) increased in parallel with mitochondrial H 2O 2 (mitochondria peroxy yellow 1) and was partially attenuated (∼65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H 2O 2 is produced in the mitochondria in response to increased flow and delivery of Na_ to the mTAL, and that whole cell H 2O 2 levels are triggered by the mitochondrial reactive oxygen species production. The mitochondrial production of H 2O 2 may represent an important target for development of more effective antioxidant therapies.
KW - Flow
KW - Kidney
KW - Mitochondria
KW - Reactive oxygen species
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U2 - 10.1152/ajprenal.00469.2011
DO - 10.1152/ajprenal.00469.2011
M3 - Article
C2 - 21975873
AN - SCOPUS:83455242850
VL - 302
SP - 95
EP - 102
JO - American Journal of Physiology - Renal Physiology
JF - American Journal of Physiology - Renal Physiology
SN - 1931-857X
IS - 1
ER -