Enzymatic Activity of Renal H-K-ATPase in the Outer Medullary Collecting Duct of Transgenic Mice

Abstract

The H-K-ATPase (HKA), a potassium-dependent proton transporter in the outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis. The OMCD contains two HKA isoforms; gastric (HKA α 1), dominant under normal dietary conditions (ND), and colonic (HKA α 2), induced under a K-free diet (KD). The enzymatic activity (EA) of HKA in the OMCD is incompletely understood. The focus of the present study is elucidating the EA of the HKA in HKA α 1 and HKA α 2 knockout (KO) mice under ND and KD. KO mice were subjected to ND or KD for 10 days. Ten OMCD tubules were extracted, half placed in potassium-free media (Solution 2), half in potassium-containing media (Solution 3). Fluorescence measurements are based on the hydrolysis of ATP to ADP, coupled with the oxidation of NADH. ADP is determined by a decrease in NADH fluorescence. In K presence, NADH fluorescence of HKA α 1 KO mice read 13.5 ± 0.7 ppm for ND and 10.3 ± 0.2 ppm for KD, indicating stimulation of the colonic isoform. HKA α 2 KO mice averaged 6.8 ± 0.3 ppm for ND and 5.4 ± 0.3 ppm for KD in solution 2 ( p p α 2 isoform. A significant difference in ATP production in HKA α 2 KO mice is likely due to enhanced EA of H-ATPase under potassium depletion.