Surface Expression of Epithelial Na Channel Protein in Rat Kidney

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface β and γENaC subunits were assayed by immunoblot. Surface αENaC was assessed by membrane fractionation. Most of the γENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla

K+ secretion in the rat kidney: Na+ channel-dependent and -independent mechanisms

Renal Na+ and K+ excretion was measured in rats with varying dietary K+ intake. The requirement for channel-mediated distal nephron Na+ reabsorption was assessed by infusing the animals with the K+-sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, the concentration of amiloride in the urine was 38 μM, corresponding to concentrations of 9–23 μM in the distal tubular fluid, sufficient to block >98% of Na+ transport through apical Na+ channels (ENaC). With a control K+ intake (0.6% KCl), amiloride reduced K+ excretion rates (UKV) from 0.85 ± 0.15 to 0.05 ± 0.01 μmol/min during the first 2 h of infusion, suggesting that distal nephron K+ secretion was completely dependent on the activity of Na+ channels. When K+ intake was increased by feeding overnight with a diet containing 10% KCl, amiloride reduced UKV from 7.5 ± 0.7 to 1.3 ± 0.1 μmol/min despite an increased plasma K+ of 9 mM, again suggesting a major but not exclusive role for the Na+ channel-dependent pathway of K+ secretion. The maximal measured rates of amiloride-sensitive K+ excretion correspond well with estimates based on apical K+ channel activity in distal nephron segments. However, when the animals were adapted to the high-K+ diet for 7–9 days, the diuretic decreased UKV less, from 6.1 ± 0.6 to 3.0 ± 0.8 μmol/min, indicating an increasing fraction of K+ excretion that was independent of Na+ channels. This indicates the upregulation of a Na+ channel-independent mechanism for secreting K+

Regulation of maturation and processing of ENaC subunits in the rat kidney.

Antibodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7-9 days, the abundance of two forms of the alpha-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the beta-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the gamma-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in alpha- and gamma-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in alpha- and gamma- but not beta-subunits when animals were treated with exogenous aldosterone, and in beta- and gamma- but not alpha-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney

Na channel expression and activity in the medullary collecting duct of rat kidney.

The expression and activity of epithelial Na(+) channels (ENaC) in the medullary collecting duct of the rat kidney were examined using a combination of whole cell patch-clamp measurements of amiloride-sensitive currents (I(Na)) in split-open tubules and Western blot analysis of alpha-, beta-, and gamma-ENaC proteins. In the outer medullary collecting duct, amiloride-sensitive currents were undetectable in principal cells from control animals but were robust when rats were treated with aldosterone (I(Na) = 960 +/- 160 pA/cell) or fed a low-Na diet (I(Na) = 440 +/- 120 pA/cell). In both cases, the currents were similar to those measured in principal cells of the cortical collecting duct from the same animals. In the inner medullary collecting duct, currents were much lower, averaging 120 +/- 20 pA/cell in aldosterone-treated rats. Immunoblots showed that all three ENaC subunits were expressed in the cortex, outer medulla, and inner medulla of the rat kidney. When rats were fed a low-Na diet for 1 wk, similar changes in alpha- and gamma-ENaC occurred in all three regions of the kidney; the amounts of full-length as well as putative cleaved alpha-ENaC protein increased, and the fraction of gamma-ENaC protein in the cleaved state increased at the expense of the full-length protein. The appearance of a presumably fully glycosylated form of beta-ENaC in Na-depleted animals was observed mainly in the outer and inner medulla. These findings suggest that the capability of hormone-regulated, channel-mediated Na reabsorption by the nephron extends at least into the outer medullary collecting duct