Sodium transport in rat renal papillary collecting tubule cells in culture

Abstract

Sodium transport in the papillary collecting duct (PCD) is poorly understood because of the inaccessibility of the distal nephron to micropuncture. Cultured rat renal papillary collecting tubule (RPCT) cells were investigated as a model for the PCD. RPCT cells have the morphologic appearance and hormonal responsiveness of the papillary collecting tubule. Sodium transport was studied using 22Na+ uptake measurements. Sodium uptake, measured at 23 degrees C in the absence of K+ and in the presence of 0.5 mM ouabain, was saturable at 100 mM extracellular NaCl, and half-maximal uptake occurred at 40 mM NaCl. The accumulation of 22Na+ appeared to be intracellular and was regulated by (Na+,K+)-ATPase activity, since activation of the Na+/K+ pump with K+ reduced 22Na+ accumulation by 90%. The time course for uptake was linear, showed only a single component, and followed first order kinetics with a t1/2 of 16 min. Amiloride and lithium inhibited 22Na+ influx, and a Dixon plot was linear, with a Ki of 16 microM amiloride. Chloride replacement of 1 mM furosemide, with or without K+, reduced uptake by only 20%. Sodium efflux from RPCT cells in the presence of ouabain showed a similar time course (t1/2, 15 min) and was also inhibited by amiloride (IC50 = 20 microM). Increased extracellular pH stimulated 22Na+ uptake and inhibited 22Na+ efflux. Addition of permeable organic acids, acetate, and bicarbonate, enhanced 22Na+ uptake. These results are consistent with Na+/H+ and Na+/Na+ exchange as mechanisms of 22Na+ uptake in the RPCT cell. This exchanger may be important in regulation of transepithelial sodium flux, maintenance of intracellular pH and cell volume, and hormonal stimulation of the papillary collecting duct.

Relationship between intracellular ionic strength and expression of tonicity‐responsive genes in rat papillary collecting duct cells

“…During the incubation, the suspension was passed repeatedly through the tip of a plastic pipette every 15–20 min. Then, two volumes of distilled water were added (Konieczkowski & Dunn, 1988) and the suspension was centrifuged at 4 °C (28 g , 2 min). The pellet was resuspended in modified Ringer solution containing DNase and the separation procedure repeated twice.…”

“…The experiments were performed on primary cultures of papillary collecting duct (PCD) cells. This approach offers the possibility of controlling precisely the extracellular solute concentrations of cells that are very similar to PCD cells of the intact tissue with respect to a variety of functional and biochemical properties (Stokes et al 1987; Konieczkowski & Dunn, 1988; Burger‐Kentischer et al 1999). In addition to ar, bgt1 and smit mRNA expression, we also examined the effect of altered extracellular tonicity, and hence intracellular ionic strength, on the abundance of several mRNAs encoding several other proteins, the expression of which is influenced by tonicity: inducible nitric oxide synthase (iNOS), heat shock protein 70 (HSP70) and osmotic stress protein 94 (OSP94).…”

A bumetanide-sensitive, apically localized Na + 2Cl - K + cotransport in the rat inner medullary collecting duct

“…We corrected for 22 Na + or 86 Rb + bound to the cell surface by subtracting radioactivity measured at time point 0 min from counts obtained after the indicated incubation time; the difference was considered to represent uptake into the cells (as reported in [17,31]). We corrected for 22 Na + or 86 Rb + bound to the cell surface by subtracting radioactivity measured at time point 0 min from counts obtained after the indicated incubation time; the difference was considered to represent uptake into the cells (as reported in [17,31]).…”

“…RNA (1 µg) was denaturated at 70 °C for 10 min in the presence of 0.5 µg oligo-(dT) [12][13][14][15][16][17][18] (Gibco BRL, Eggenstein, Germany) and cooled on ice for 1 min. RNA (1 µg) was denaturated at 70 °C for 10 min in the presence of 0.5 µg oligo-(dT) [12][13][14][15][16][17][18] (Gibco BRL, Eggenstein, Germany) and cooled on ice for 1 min.…”

K+ fluxes mediated by Na+‐K+‐Cl− cotransport and Na+‐K+‐ATPase pumps in renal tubule cell lines transformed by wild‐type and temperature‐sensitive strains of simian virus 40

“…Sodium influx into RC.SV,,A,, cells was determined under culture conditions similar to those of the "Rb+ uptake studies. Experiments were performed on sodium-depleted cells (Konieczkowski and Dunn, 1988). Cells were rinsed twice with choline medium (in mM: 140 choline chloride, 2 glutamine, 1.7 MgCl,, 1.8 CaCI,, 5 glucose, 10 HEPES, pH 7.2) and incubated in the same medium for 60 min a t 37°C.…”