Ion and water transport in a new rat cortical collecting duct cell line (RCCD1): regulation by AVP.

CAPURRO, CLAUDIA; RIVAROLA, VALERIA; FORD, PAULA; KIERBEL, ARLINET; FARMAN, NICOLETTE; BLOT-CHABAUD, MARCEL; PARISI, MARIO

Goteborg - Suecia

Congreso; 3rd International Conference on Molecular Biology and Physiology of Water and Solute Transport; 2000

The purpose of this work was to characterize water and ion coupling and its regulation by arginine vasopressin (AVP) in a high transepithelial resistance cell line established from the rat cortical collecting duct (RCCD1) [1]. Transepithelial net water fluxes (Jw) and short-circuit current (Isc were minute by minute recorded in RCCD1 monolayers cultivated on permeable supports. A net water secretion was observed in the absence of any osmotic or chemical gradient and even against a hydrostatic pressure difference, which is characteristic of energy-requiring mechanisms for the transport of fluid. Moreover, when transport stopped in response to temperature or inhibitors, the fluid movement reversed its direction. Since it is well established that different types of aquaporins are present in the kidney [2], the first candidates as water pathways in RCCD, were water channels. However, RT-PCR and irnmunoblotting experiments demonstrated that the until present cloned renal aquaporins were not expressed in RCCD] cells. The inhibitory effects of chloride and potassium transporter blockers on the Isc and Jw observed, is consistent with specific coupling of KC1 and water in the here reported secretion. It was previously reported that AVP stimulates cAMP production and sodium reabsorption in RCCD, cells [3]. We have now observed that the amiloride-sensitive AVP-induced increase in Isc was paralleled by a simultaneous modification of the observed Jw. Both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modified the osmotically driven Jw induced by serosal hypertonicity. These results led us to conclude that the natriferic response to AVP, preserved in RCCD, cells, was associated to a "net water absorptive component" observed even in the absence of AQP2, AQP3 or AQP4 but the hydrosmotic response to AVP was completely lost. As RCCD, cells also conserve feature of intercalated cells (secretoty cells), responsible of acid-base transport in the native tubule, we have studied intracellular pH regulation in these cells. Proton transport was determined using fluorescent pH-sensitive BCECF following an acid load. Our results show that pHi recovery kinetics is quite different if acidification is undertake from serosal or mucosal bath. AVP induce an immediate alkalization sensitive to 10-3 M apical amiloride. However, pH¡ recovery kinetics af ter serosal acidification is sensitive to basolateral, but not apical, amiloride. Taking in account these results, we propose the existence of different isoforms of the Na+-H+ transporter in the basolateral membrane (confirmed by RT-PCR and immunolocalization studies) which may be involved in cell volume regulation and in consequence in fluid secretion. 1. Blot-Chabaud, M., Laplace, M., Cluzeaud, F., Capurro, C., Cassingena, R., Vandewalle, A., Farman, N., Bonvalet, J.P. 1996. Kidney Int. 60: 367-376. 2. Nielsen, S., Agre, P, 1995. Kidney Int. 48: 1057-1068. 3. Djelidi. S., Fay, M., Cluzeaud, F., Escoubet, B., Eugene, E., Capurro, C., Bonvalet, J.P., Farman, N., Blot-Chabaud, M. 1997. /. Biol. Chem. 272: 32919-32924.