Hyposmolality stimulates Na⁺/H⁺ exchange and HCO₃/^- absorption in thick ascending limb via PI 3-kinase

The signal transduction mechanisms that mediate osmotic regulation of Na⁺/H⁺ exchange are not understood. Recently we demonstrated that hyposmolality increases HCO₃/^- absorption in the renal medullary thick ascending limb (MTAL) through stimulation of the apical membrane Na⁺/H⁺ exchanger NHE3. To investigate the mechanism of this stimulation, MTALs from rats were isolated and perfused in vitro with 25 mM HCO₃/^- containing solutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (100 nM) and LY-294002 (20 μM) blocked completely the stimulation of HCO₃/^- absorption by hyposmolality. In tissue strips dissected from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs, hyposmolality increased PI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-induced PI 3-K activation. Further studies examined the interaction between hyposmolality and vasopressin, which inhibits HCO₃/^- absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO₃/^- absorption, due to an effect of cAMP to inhibit hyposmolality-induced activation of PI 3-K. In contrast to their effects to block stimulation by hyposmolality, PI 3-K inhibitors and vasopressin have no effect on inhibition of apical Na⁺/H⁺ exchange (NHE3) and HCO₃/^- absorption by hyperosmolality. These results indicate that hyposmolality increases NHE3 activity and HCO₃/^- absorption in the MTAL through activation of a PI 3-K-dependent pathway that is inhibited by vasopressin and cAMP. Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediated through different signal transduction mechanisms.