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

Abstract

Luminal flow augments Na⁺ reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na⁺, playing a key role in its homeostasis. Whether flow elevations enhance Na⁺-K⁺-2Cl^- cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

Keywords: NADPH oxidase; kidney; sodium reabsorption; transport.

Fig. 1.

Representative trace of an experiment measuring Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) activity [expressed as increase in intracellular Na⁺ (${\text{Na}}_{\text{i}}^{+}$)/s] in isolated perfused thick ascending limbs. AFU, arbitrary fluorescent units.

Fig. 2.

A: NKCC2 activity in tubules perfused at 5 and 20 nl/min (n = 5 experiments). B: effect of time on NKCC2 activity in tubules perfused at 5 nl/min (n = 6).

Fig. 3.

A:) NKCC2 activity in tubules perfused at 20 nl/min with and without 30 µmol/l of the manganese superoxide (${\text{O}}_2^{-}$) dismuatse mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), n = 6. B: NKCC2 activity in tubules perfused at 5 nl/min with and without 30 µmol/l MnTBAP, n = 4.

Fig. 4.

Effect of time on NKCC2 activity in tubules perfused at 20 nl/min (n = 7).

Fig. 5.

A: NKCC2 activity in tubules perfused at 20 nl/min with and without 5 µmol/l NADPH oxidase (NOX) 1/4 specific inhibitor GKT-137831 (n = 6). B: NKCC2 activity in tubules perfused at 5 nl/min with and without 5 µmol/l NOX1/4 specific inhibitor GKT-137831 (n = 5).

Fig. 6.

NKCC2 activity in tubules perfused at 20 nl/min with and without 100 µmol/l xanthine oxidase inhibitor allopurinol (n = 4).

Fig. 7.

${\text{O}}_2^{-}$ production by thick ascending limbs without and with flow at 20 nl/min. A: without 5 µmol/l NOX1/4 inhibitor GKT-137831. B: with 5 µmol/l NOX1/4 inhibitor GKT-137831. C: GKT-137831 blunts flow-induced ${\text{O}}_2^{-}$ production by 50% (n = 5 for each group).

Fig. 8.

NKCC2 activity in tubules pretreated with 30 nmol/l tetanus toxin perfused at 5 and 20 nl/min (n = 4).