Adaptation of rabbit cortical collecting duct to in vitro acid incubation

Cortical collecting ducts (CCDs) isolated from acid-loaded rabbits and perfused in vitro absorb HCO₃^-, whereas CCDs from normal animals secrete HCO₃^-. We have previously shown that CCDs incubated in vitro for 3 h at pH 6.9 show a reduction in net (baseline and stimulated) HCO₃^- secretion. In this study we ascertained the minimum duration of an acidic stimulus necessary to induce adaptive changes in stimulated HCO₃^- secretion (determined in the absence of basolateral Cl^-) and the roles of protein synthesis and cytoskeletal function in this process. CCDs incubated in acid (pH 6.8, HCO₃^- 6 mM) for 1 h followed by incubation at pH 7.4 (HCO₃^- 25 mM) for 2 h showed a 41% reduction in stimulated HCO₃^- secretion (P < 0.001), similar to that observed after 3 h of incubation at pH 6.8. However, this incubation protocol failed to enhance stimulated HCO₃^- absorption (determined in the absence of luminal Cl^-). Addition of 10 μM anisomycin, a reversible inhibitor of protein synthesis, throughout the entire period of incubation (1 h at pH 6.8 plus 2 h at pH 7.4) blocked adaptive reduction in HCO₃^- secretion, as did exposure to anisomycin only during the initial 1 h of acid incubation. In contrast, anisomycin application during the 2-h incubation at pH 7.4 failed to block this adaptation of HCO₃^- secretion. Application of 4 μM actinomycin D, an inhibitor of DNA transcription, during the acid incubation also prevented the adaptive response, as did application during the total or during the 2-h pH 7.4 incubation period of 0.2 μM cytochalasin D, an inhibitor of actin filament function. Thus, HCO₃^- secretion by the CCD is inhibited by incubation at low pH in vitro; this adaptation is initiated within 1 h of exposure to low pH, but requires an additional 2 h for significant expression. The decrease in HCO₃^- secretion requires de novo protein synthesis and integrity of the microfilaments. Because the decrement in HCO₃^- secretion was not associated with an increase in HCO₃^- absorption, it is unlikely that β-intercalated cells reverse their functional polarity in this model of metabolic acidosis.