Expression and role of sodium, potassium, chloride cotransport (NKCC1) in mouse inner medullary collecting duct (mIMCD-K2) epithelial cells

Abstract

Loop-diuretic-sensitive 86Rb+(K+) transmembrane fluxes were determined in cells of a mouse inner medullary collecting duct cell line (mIMCD-K2). The furosemide-sensitive (0.1 mM) influx was a substantial fraction of the total influx (0.39+/-0.04 or 0.42+/-0.03, n=5 in the presence or absence of ouabain, respectively). Furosemide also reduced 86Rb+(K+) efflux by a similar fraction (0.46). RT-PCR analysis revealed expression of mRNA for the Na+-K+-2Cl- cortransporter-1 (NKCC1), but not NKCC2. Loop-diuretic-sensitive 86Rb+(K+) influx was confined to the basolateral membrane, confirming its localisation there. The physiological properties of NKCC1 expressed in mIMCD-K2 cells, including the dependence upon medium Na+, K+ and Cl- and the relative sensitivity to loop diuretics as assessed by the concentration required for half-maximal inhibition (IC50) (bumetanide 3.3+/-1.4x10-7 M>piretanide 2.5+/-0.15x10-6 M>furosemide 2.3+/-1.2x10-5 M) were typical for NKCC1. Possible functions of NKCC1 were tested; furosemide did not inhibit the majority of forskolin-stimulated secretory short-circuit current (Isc) (83.5+/-5.3% of the maintained response at 5 min). Secondly, total 86Rb+(K+) influx was stimulated markedly when external osmolarity was increased to 600 mosmol/l by mannitol due to an increase via NKCC1 from 55+/-11 to 191+/-2 nmol/106 cells per 15 min, (both n=4, P<0.01). In contrast, 10-5 M forskolin did not stimulate total 86Rb+(K+) influx. Finally, the ability of both K+ and NH4+ to compete for ouabain-insensitive 86Rb+(K+) influx via NKCC1 was confirmed with similar concentrations for half-maximal influx reduction (K0.5). Apical exposure to NH4+ elicited rapid cytosolic alkalinisation in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded epithelial layers, consistent with selective permeability of the apical membrane to NH3. Conversely, NH4+ (5 mM) at the basal cell surface resulted in progressive acidification, the initial rate being reduced by 43% by furosemide. We conclude that NKCC1 participates in selective uptake of NH4+ at the basal surface, and that IMCD may function in direct NH4+ deposition to urine.

Bumetanide Hyperpolarizes Madin–Darby Canine Kidney Cells and Enhances Cellular Gentamicin Uptake by Elevating Cytosolic Ca2+ Thus Facilitating Intermediate Conductance Ca2+-Activated Potassium Channels

“…Finally, the EC 50 s of bumetanide and furosemide for GTTR uptake showed a much smaller difference between bumetanide and furosemide (Fig. 1) than that reported for NKCC1 inhibition (0.33 μM vs. 23 μM, [61]).…”

Molecular Mechanisms of Renal Ammonia Transport

“…Thus, it is unlikely that NKCC-1 contributes to OMCD ammonia secretion. Studies using cultured IMCD cells suggest that basolateral Na + -K + -2Cl − cotransport in the IMCD, presumably involving NKCC-1,contributes to basolateral NH 4 + uptake (47,48). However, other studies using in vitro-microperfused rat terminal IMCD segments have not confirmed this observation (49).…”

Both seawater acclimation and environmental ammonia exposure lead to increases in mRNA expression and protein abundance of Na+:K+:2Cl− cotransporter in the gills of the climbing perch, Anabas testudineus

“…It is present almost exclusively in the basolateral membrane of A-type intercalated cells in the outer and inner medullary collecting ducts of the kidney (Ginns et al 1996). Studies using cultured cells from the inner medullary collecting duct suggest that basolateral NKCC1 contributes to basolateral NH 4 + uptake (Wall et al 1995;Glanville et al 2001), although other studies using in vitro microperfused rat terminal inner medullary collecting duct segments have not conWrmed this observation (Wall 1997). On the other hand, NKCC2 is speciWcally expressed in the apical membrane of the thick ascending limb of the loop of Henle , where it is the major mechanism for NH 4 + reabsorption (Good 1994).…”