Endogenous flow-induced superoxide stimulates Na/H exchange activity via PKC in thick ascending limbs

Abstract

Hong NJ, Garvin JL. Endogenous flow-induced superoxide stimulates Na/H exchange activity via PKC in thick ascending limbs. Am J Physiol Renal Physiol 307: F800 -F805, 2014. First published July 30, 2014 doi:10.1152/ajprenal.00260.2014.-Luminal flow stimulates Na reabsorption along the nephron and activates protein kinase C (PKC) which enhances endogenous superoxide (O 2 Ϫ ) production by thick ascending limbs (TALs). Exogenously-added O 2 Ϫ augments TAL Na reabsorption, a process also dependent on PKC. Luminal Na/H exchange (NHE) mediates NaHCO 3 reabsorption. However, whether flow-stimulated, endogenously-produced O 2 Ϫ enhances luminal NHE activity and the signaling pathway involved are unclear. We hypothesized that flow-induced production of endogenous O 2 Ϫ stimulates luminal NHE activity via PKC in TALs. Intracellular pH recovery was measured as an indicator of NHE activity in isolated, perfused rat TALs. Increasing luminal flow from 5 to 20 nl/min enhanced total NHE activity from 0.104 Ϯ 0.031 to 0.167 Ϯ 0.036 pH U/min, 81%. The O 2 Ϫ scavenger tempol decreased total NHE activity by 0.066 Ϯ 0.011 pH U/min at 20 nl/min but had no significant effect at 5 nl/min. With the NHE inhibitor EIPA in the bath to block basolateral NHE, tempol reduced flow-enhanced luminal NHE activity by 0.029 Ϯ 0.010 pH U/min, 30%. When experiments were repeated with staurosporine, a nonselective PKC inhibitor, tempol had no effect. Because PKC could mediate both induction of O 2 Ϫ by flow and the effect of O 2 Ϫ on luminal NHE activity, we used hypoxanthine/ xanthine oxidase to elevate O 2 Ϫ . Hypoxanthine/xanthine oxidase increased luminal NHE activity by 0.099 Ϯ 0.020 pH U/min, 137%. Staurosporine and the PKC␣/␤1-specific inhibitor Gö6976 blunted this effect. We conclude that flow-induced O 2 Ϫ stimulates luminal NHE activity in TALs via PKC␣/␤1. This accounts for part of flow-stimulated bicarbonate reabsorption by TALs. reactive oxygen species; luminal flow; protein kinases; sodium/hydrogen exchange LUMINAL FLOW IN THE NEPHRON is highly variable. Acutely it changes due to glomerular filtration rate (29), tubuloglomerular feedback (17, 28), peristalsis of the renal pelvis (8, 44), and fluid reabsorption (29, 50). Hypertension (4, 6), volume status (3, 42), and diabetes (40) can change luminal flow chronically. Increases in luminal flow enhance Na reabsorption in the proximal tubule (7, 46), thick ascending limb of the loop of Henle (39), and cortical collecting duct (10, 43). However, the mechanisms involved are not fully understood.The thick ascending limb is important in salt, water, and acid/base homeostasis (15,16,35). Na is reabsorbed by thick ascending limbs as NaCl and Na bicarbonate via Na-K-2Cl cotransport (35) and Na/H exchange (NHE) (15, 16), respectively. Luminal flow in this segment may be as high as 20 nl/min during conditions of diuresis (4, 30, 42) and may stop due to peristalsis of the renal pelvis (8,42,44 enhances Na reabsorption in thick ascending limbs. We showed that exogenously-added O 2 Ϫ stimulates net N...

Differential effects of superoxide and hydrogen peroxide on myogenic signaling, membrane potential, and contractions of mouse renal afferent arterioles

“…Our results confirm that PKC is required for myogenic contractions and further show that it mediates the membrane depolarization by perfusion pressure or paraquat. This is consistent with activation of PKC by O 2 •Ϫ in renal tubules (19,53), Moreover, the reduction in E m and enhanced myogenic contraction with paraquat depend in part on Cl Ϫ channels (CaCCs). The relationship between O A novel finding was that exogenous H 2 O 2 blunted myogenic contractions of afferent arterioles, but H 2 O 2 apparently was not generated with increased perfusion pressure in normal mouse afferent arterioles since myogenic contractions of normal vessels were unaffected by metabolizing H 2 O 2 with PEG-catalase.…”

“…Therefore, paraquat was used to generate intracellular O 2 •Ϫ (42) to study the graded effects of O 2 •Ϫ on myogenic tone. PKC is activated by O 2 •Ϫ (19) and can mediate myogenic contractions (18) by activating Cl Ϫ channels (30) that can reduce E m of VSMCs (3) and afferent arterioles (21). Ca 2ϩactivated Cl Ϫ channels (CaCCs) have been proposed recently to play a key role in the depolarization of E m of VSMCs during a myogenic contraction (10).…”

A radical approach to balancing the tides of tubular flow

“…Ϫ (demonstrated to be dependent on NOX4) produces essentially the same effect to promote NHE3 activity (5), suggesting the location of O 2…”

“…The current manuscript by Hong and Garvin addresses this issue and is an important step toward understanding flow-regulated Na/HCO 3 Ϫ reabsorption in the thick ascending limb, as it demonstrates for the first time that endogenous levels of O 2 Ϫ , stimulated by physiologically relevant increases in luminal flow rate, are capable of activating NHE3 activity via PKC␣/␤1 (5).…”

NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs

“…Chronically, urinary flow increases with high-salt (49) and high-protein (64) diets, diabetes (57), and hypertension (5,14). Flow of the forming urine alters the reabsorption and secretion of a number of ions and compounds in nearly all nephron segments (17,22,31,32,55,58,69). Increasing luminal flow in thick ascending limbs elevates NaCl reabsorption, a process dependent on NKCC2, above what would be predicted simply on the basis of increased ion delivery (54,68).…”