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

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 (HKAα1), dominant under normal dietary conditions (ND), and colonic (HKAα2), 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 HKAα1 and HKAα2 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 HKAα1 KO mice read 13.5 ± 0.7 ppm for ND and 10.3 ± 0.2 ppm for KD, indicating stimulation of the colonic isoform. HKAα2 KO mice averaged 6.8 ± 0.3 ppm for ND and 5.4 ± 0.3 ppm for KD in solution 2 (p < 0.002). Solution 3 readings were 6.0 ± 0.3 ppm for ND and 4.6 ± 0.2 ppm for KD (p < 0.0005). K addition produced significant changes in NADH fluorescence of ND and KD KO mice. The results demonstrated potassium depletion’s association with increased EA of H-K-ATPase in OMCD, consistent with the activation of HKAα2 isoform. A significant difference in ATP production in HKAα2 KO mice is likely due to enhanced EA of H-ATPase under potassium depletion.