Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress, and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 in the generation of H2O2 and O2·− in the renal medullary thick ascending limb of Henle (mTAL) of SS rats in response to increasing luminal flow (from 5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the mTAL of SS rats. Responses of SS rats were compared with those of SS rats with knockout of Nox4 (SSNox4−/−) or functional mutation of p67phox (SSp67phox−/−). Nox4 was the dominant source of increased intracellular H2O2 production in response to increased luminal flow as determined using the fluorescent dye peroxyfluor 6-AM (PF6-AM). The rate of mitochondrial H2O2 production [as determined by mitochondria peroxy yellow 1 (mitoPY1)] was also significantly reduced in SSNox4−/− compared with SS rats, but not in SSp67phox−/− rats. In contrast, intracellular superoxide (O2·−) production (the ratio of ethidium to dihydroethidium) in the mTAL of SSNox4−/− rats was nearly identical to that of SS rats in response to luminal flow, indicating that Nox4 made no measurable contribution. mTAL O2·− production was reduced in SSp67phox−/− compared with SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases in intracellular and mitochondrial H2O2, which are dependent on the presence of Nox4, and that p67phox/Nox2 accounts solely for increases in O2·− production.
- Dahl salt-sensitive rat
- medullary thick ascending limb
- luminal flow
- mitochondrial and cellular H2O2
- Copyright © 2016 the American Physiological Society
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