Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC

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American journal of physiology. Renal physiology


The apical Na/K/2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limbs (TAL). The free radical superoxide (O2(-)) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, NO scavenges O2(-) and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that O2(-) stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot. Treatment of TALs with O2(-) produced by exogenous xanthine oxidase (1 mU/mL) and hypoxanthine (500 microM) stimulated surface NKCC2 expression by ~18+/-5% (p<0.05). O2(-) stimulated surface NKCC2 expression was blocked by the O2(-) scavenger tempol (50 microM). Scavenging H2O2 with 100 U/mL catalase did not block the stimulatory effect of xanthine oxidase/hypoxanthine (22+/-8% increase from control, p<0.05). Inhibition of endogenous NO production with L-nitro-methyl arginine (LNAME) enhanced surface NKCC2 expression by 21+/-6 and when added together with xanthine oxidase/hypoxanthine increased surface NKCC2 by 41+/-10% (p<0.05). Scavenging O2(-), with superoxide dismutase (300 U/mL) decreased this stimulatory effect by 60% (39+/-4 to 15+/-10%, p<0.05). Protein kinase C inhibition with Go6976 (100 nM) blocked O2(-) stimulated surface NKCC2 expression (p<0.05). O2(-) did not affect NKCC2 phosphorylation at threonines 96/101 nor its upstream kinases SPAK/OSR1. We conclude that O2(-) increases surface NKCC2 expression by stimulating PKC and this effect is blunted by endogenous NO. O2(-) stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

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