Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC
Recommended Citation
Haque MZ, and Ortiz P. Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC. Am J Physiol Renal Physiol 2019; 317(1):F99-F106.
Document Type
Article
Publication Date
7-1-2019
Publication Title
American journal of physiology. Renal physiology
Abstract
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.
PubMed ID
31091128
Volume
317
Issue
1
First Page
F99
Last Page
F106