Superoxide stimulates NaCl absorption by the thick ascending limb

The thick ascending limb of the loop of Henle (THAL) plays an important role in the regulation of NaCl and water reabsorption. In vivo studies have shown that the free radical superoxide (O-2(-)) stimulates Na and water reabsorption by the kidney. However, it is not known whether O-2(-) regulates transport along the nephron in general or in the THAL specifically. We hypothesized that O-2(-) stimulates THAL NaCl reabsorption. Cl absorption was measured in isolated, perfused THALs from Sprague-Dawley rats. First, we tested whether extracellular O-2(-) stimulates Cl absorption. Addition of the O-2(-)-generating system xanthine oxidase/hypoxanthine increased Cl absorption from 112.7 +/- 12.0 to 146.2 +/- 13.9 pmol.mm(-1).min(-1), a 33% increase (P < 0.03). When superoxide dismutase (300 U/ml) was present in the bath, addition of xanthine oxidase/hypoxanthine did not significantly increase Cl absorption (116.9 +/- 13.8 vs. 102.5 +/- 8.5 pmol.mm(-1).min(-1)). Furthermore, adding 200 nM H2O2 to the bath did not significantly affect Cl absorption (from 130.3 +/- 13.7 to 125.3 +/- 19.6 pmol.mm(-1).min(-1)). Because extracellular O-2(-) stimulated Cl absorption, we next tested whether endogenously produced O-2(-) could stimulate transport. Under basal conditions, THALs produced detectable amounts of O-2(-), as measured by lucigenin-enhanced chemiluminescence. Adding the O-2(-) scavenger tempol to the bath decreased Cl absorption from 198.1 +/- 35.4 to 132.4 +/- 23.5 pmol.mm(-1)min(-1), a 31% decrease (P < 0.02). To make sure tempol was not exerting cytotoxic effects, we tested whether its effect was reversible. With tempol in the bath, Cl absorption was 117.2 +/- 9.3 pmol.mm(-1).min(-1). Sixty minutes after tempol was removed from the bath, Cl absorption had increased to 149.2 +/- 9.1 pmol.mm(-1).min(-1) (P < 0.05). We concluded that both exogenous and endogenous O-2(-) stimulate THAL NaCl absorption. To our knowledge, these are the first data showing a direct effect of O-2(-) on nephron transport.