Abstract 92: Hvcn1 Contributes to Superoxide Production in the Medullary Thick Ascending Limb
We have previously reported that H+ efflux stimulates superoxide (O2-) production in the medullary thick ascending limb of the loop of Henle (mTAL) in rats by activation of an as yet unidentified H+ transport pathway. As HVCN1, a voltage gated H+ channel associated with NADPH oxidase in immune cells, is also expressed in mTAL, we hypothesized that HVCN1 is responsible for H+ efflux induced production of O2- in mTAL. In order to test this hypothesis we utilized HVCN1-/- mice and wild type litter mates obtained from KOMP. To confirm that HVCN1 was functionally knocked out in HVCN1-/- mice we stimulated the respiratory burst using PMA (100uM) in peritoneal macrophages extracted from HVCN1-/- and WT mice and determined maximal O2- production using L-012 luminescence. Maximal O2- production during the respiratory burst was significantly lower in macrophages from HVCN1-/- mice (23±4AU; n=6) compared to WT mice (92±3AU; n=5; p<0.05), consistent with loss of HVCN1 function. mTAL were isolated from the inner stripe of the outer-medulla by micro-dissection and loaded for 60 min with the O2- sensitive dye dihydroethidium (DHE). Live mTAL were then imaged on a heated chamber attached to a fluorescent microscope and the ratio of ethidium (Eth) to DHE quantified using metafluor imaging software as an index of O2- production. O2- producing H+ currents were isolated in 0 Na+, 100uM BaCl2 media and cellular H+ efflux stimulated using an NH4Cl (20mM) prepulse. Following removal of NH4Cl from the bath there was a significant increase in O2- production in mTAL from WT mice (Delta slope Eth/DHE 15.9±3.6 AU/sec; p<0.02 (n=5)) but not HVCN1-/- mice (delta slope 4.7±9.6; P=0.64 (n=6)). The response of WT mice was significantly greater than that of HVCN1-/- mice (P<0.001). We conclude that HVCN1 is required for maximal O2- production in mTAL in response to H+ efflux. These data suggest that HVCN1 may be a novel target to prevent renal oxidative stress.
- © 2012 by American Heart Association, Inc.