CFTR mediated chloride secretion in the avian renal proximal tubule

In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl^--dependent short circuit current (I_SC) response, consistent with net transepithelial Cl^- secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl^- secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I_SC responses in filter grown cultured monolayers. Bumetanide (10μM), a specific blocker of NKCC, inhibited forskolin activated I_SC by about 40%, suggesting that basolateral uptake of Cl^- is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl^- conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl^- gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl^- current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl^- channel to mediate cAMP-activated Cl^- secretion.