Gustavo Frindt

Collecting duct–specific gene inactivation of αENaC in the mouse kidney does not impair sodium and potassium balance

Aldosterone controls the final sodium reabsorption and potassium secretion in the kidney by regulating the activity of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). ASDN consists of the last portion of the distal convoluted tubule (late DCT), the connecting tubule (CNT), and the collecting duct (CD) (i.e., the cortical CD [CCD] and the medullary CD [MCD]). It has been proposed that the control of sodium transport in the CCD is essential for achieving sodium and potassium balance. We have tested this hypothesis by inactivating the alpha subunit of ENaC in the CD but leaving ENaC expression in the late DCT and CNT intact. Under salt restriction or under aldosterone infusion, whole-cell voltage clamp of principal cells of CCD showed no detectable ENaC activity, whereas large amiloride-sensitive currents were observed in control littermates. The animals survive well and are able to maintain sodium and potassium balance, even when challenged by salt restriction, water deprivation, or potassium loading. We conclude that the expression of ENaC in the CD is not a prerequisite for achieving sodium and potassium balance in mice. This stresses the importance of more proximal nephron segments (late DCT/CNT) to achieve sodium and potassium balance.

Selectivity of the Renal Collecting Duct Water Channel Aquaporin-3

Aquaporin-3 (AQP3) is a water channel found in the basolateral cell membrane of principal cells of the renal collecting tubule as well as in other epithelia. To examine the selectivity of AQP3, the permeability to water (Pf), urea (Pur), and glycerol (Pgly) of Xenopus oocytes injected with cRNA encoding AQP3 was measured. Oocytes injected with cRNA encoding either human or rat aquaporin-1 (AQP1) were used as controls. Although both aquaporins permit water flow across the cell membrane, only AQP3 was permeable to glycerol and urea (Pgly > Pur). The uptake of glycerol into oocytes expressing AQP3 was linear up to 165 mM. For AQP3 the Arrhenius energy of activation for Pf was 3 kcal/mol, whereas for Pgly and Pur it was >12 kcal/mol. The sulfhydryl reagent p-chloromercuriphenylsulfonate (1 mM) abolished Pf of AQP3, whereas it did not affect Pgly. In addition, phloretin (0.1 mM) inhibited Pf of AQP3 by 35%, whereas it did not alter Pgly or Pur. We conclude that water does not share the same pathway with glycerol or urea in AQP3 and that this aquaporin, therefore, forms a water-selective channel.

Epithelial Na channel activation and processing in mice lacking SGK1

Amiloride-sensitive Na(+) channel activity was examined in the cortical collecting ducts of a mouse line (SGK1(-/-)) deficient in the serum- and glucocorticoid-dependent protein kinase SGK1. This activity was correlated with changes in renal Na handling and in the maturation of epithelial Na(+) channel (ENaC) protein. Neither SGK1(-/-) mice nor paired SGK1(+/+) animals expressed detectable channel activity, measured as amiloride-sensitive whole-cell current (I(Na)), under control conditions with standard chow. Administration of aldosterone (0.5 microg/h via osmotic minipump for 7 days) increased I(Na) to a similar extent in SGK1(+/+) (378 +/- 61 pA/cell at -100 mV) and in SGK1(-/-) (350 +/- 57 pA/cell) animals. However, the maturation of ENaC, assessed as the ratio of cleaved to full-length forms of gamma-ENaC, was more pronounced in SGK(+/+) mice. The SGK1(-/-) animals exhibited a salt-wasting phenotype when kept on a low-Na diet for up to 2 days, losing significantly more Na in the urine than wild-type mice. Under these conditions, I(Na) was enhanced more in SGK1(-/-) (94 +/- 14 pA/cell) than in SGK(+/+) (23 +/- 5 pA/cell) genotypes. Despite the larger currents, the ratio of cleaved to full-length gamma-ENaC was lower in the knockout animals. The mice also expressed a smaller amount of Na(+)-Cl(-) cotransporter protein under Na-depleted conditions. These results indicated that SGK1 is essential for optimal processing of ENaC but is not required for activation of the channel by aldosterone.

Effects of dietary K on cell-surface expression of renal ion channels and transporters

Changes in apical surface expression of ion channels and transporters in the superficial rat renal cortex were assessed using biotinylation and immunoblotting during alterations in dietary K intake. A high-K diet increased, and a low-K diet decreased, both the overall and surface abundance of the β- and γ-subunits of the epithelial Na channel (ENaC). In the case of γ-ENaC, the effect was specific for the 65-kDa cleaved form of the protein. The overall amount of α-ENAC was also increased with increasing K intake. The total expression of the secretory K(+) channels (ROMK) increased with a high-K diet and decreased with a low-K diet. The surface expression of ROMK increased with high K intake but was not significantly altered by a low-K diet. In contrast, the amounts of total and surface protein representing the thiazide-sensitive NaCl cotransporter (NCC) decreased with increasing K intake. We conclude that modulation of K(+) secretion in response to changes in dietary K intake involves changes in apical K(+) permeability through regulation of K(+) channels and in driving force subsequent to alterations in both Na delivery to the distal nephron and Na(+) uptake across the apical membrane of the K(+) secretory cells.

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.

Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) beta-subunit (beta-ENaC) and gamma-subunit (gamma-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of beta-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of gamma-ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of gamma-ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

Regulation of apical K channels in rat cortical collecting tubule during changes in dietary K intake

Long-term adaptation to a high-K diet is known to increase the density of conducting secretory K (SK) channels in the luminal membrane of the rat cortical collecting tubule (CCT). To examine whether these channels are involved in the short-term, day-to-day regulation of K secretion, we examined the density of K channels in animals fed a high-K diet for 6 or 48 h. CCTs were isolated and split open to provide access to the luminal membrane. Cell-attached patches were formed on principal cells with 140 mM KCl in the patch-clamp pipette. SK channels were recognized from their characteristic single-channel conductance (40-50 pS) and gating patterns. Animals fed a control diet had SK channel densities of 0.40 channels/micrometer(2). When the diet was changed for one containing 10% KCl for 6 h, the channel density increased to 1.51 channels/micrometer(2). Maintaining the animals on a high-K diet for 48 h resulted in a further increase in SK channels to 2.29 channels/micrometer(2). Animals fed a low-K diet for 5 days or longer had SK densities of 0.53 channels/micrometer(2), not significantly different from control values. The presence of conducting Na channels in the luminal membrane will also affect K secretion by the CCT by altering the electrical driving force through the K channels. The density of Na channels, measured with LiCl in the pipette, was 0. 08 for controls and 1.00 and 1.08 channels/micrometer(2) after 6 h and 48 h on a high-K diet. Plasma aldosterone increased from 15 +/- 4 ng/dl (controls ) to 36 +/- 8 and 98 +/- 23 ng/dl after 6 and 48 h of K loading, respectively. The increase in K channel density could not be reproduced by infusion of the animals with aldosterone. We conclude that regulation of the density of conducting Na and K channels may contribute to day-to-day variation in the rate of renal K secretion and to the short-term maintenance of K balance.Long-term adaptation to a high-K diet is known to increase the density of conducting secretory K (SK) channels in the luminal membrane of the rat cortical collecting tubule (CCT). To examine whether these channels are involved in the short-term, day-to-day regulation of K secretion, we examined the density of K channels in animals fed a high-K diet for 6 or 48 h. CCTs were isolated and split open to provide access to the luminal membrane. Cell-attached patches were formed on principal cells with 140 mM KCl in the patch-clamp pipette. SK channels were recognized from their characteristic single-channel conductance (40-50 pS) and gating patterns. Animals fed a control diet had SK channel densities of 0.40 channels/μm2. When the diet was changed for one containing 10% KCl for 6 h, the channel density increased to 1.51 channels/μm2. Maintaining the animals on a high-K diet for 48 h resulted in a further increase in SK channels to 2.29 channels/μm2. Animals fed a low-K diet for 5 days or longer had SK densities of 0.53 channels/μm2, not significantly different from control values. The presence of conducting Na channels in the luminal membrane will also affect K secretion by the CCT by altering the electrical driving force through the K channels. The density of Na channels, measured with LiCl in the pipette, was 0.08 for controls and 1.00 and 1.08 channels/μm2 after 6 h and 48 h on a high-K diet. Plasma aldosterone increased from 15 ± 4 ng/dl (controls ) to 36 ± 8 and 98 ± 23 ng/dl after 6 and 48 h of K loading, respectively. The increase in K channel density could not be reproduced by infusion of the animals with aldosterone. We conclude that regulation of the density of conducting Na and K channels may contribute to day-to-day variation in the rate of renal K secretion and to the short-term maintenance of K balance.

Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule

1 The idea that luminal Na+ can regulate epithelial Na+ channels was tested in the cortical collecting tubule of the rat using whole‐cell and single‐channel recordings. Here we report results consistent with the idea of Na+ self‐inhibition. 2 Macroscopic amiloride‐sensitive currents (INa) were measured by conventional whole‐cell clamp. INa was a saturable function of external Na+ concentration ([Na+]o) with an apparent Km of 9 mm. Single channel currents (iNa) were measured in cell‐attached patches. iNa increased with pipette Na+ concentration with an apparent Km of 48 mm. Since INa= (iNa)NPo, the different Km values imply that the channel density (N) and/or open probability (Po) increase as [Na+]o decreases. Reduction of [Na+]o after increasing intracellular Na+ concentration also increased the outward amiloride‐sensitive conductance, consistent with activation of the Na+ channels. 3 The underlying mechanism was studied by changing pipette Na+ concentration while recording from cell‐attached patches. No increase in NPo was observed, suggesting that the effect is not a direct interaction between [Na+]o and the channel. 4 [Na+]o was varied outside the patch‐clamp pipette while recording from cell‐attached patches. When amiloride was in the bath to prevent Na+ entry, no change in NPo was observed. 5 Activation of the channels by hyperpolarization was observed with 140 mm Na+o but not with 14 mm Na+o. 6 The results are consistent with the concept of self‐inhibition of Na+ channels by luminal Na+. Activation of the channels by lowering [Na+]o is not additive with that achieved by hyperpolarization.

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 microM, corresponding to concentrations of 9-23 microM 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 (U(K)V) from 0.85 +/- 0.15 to 0.05 +/- 0.01 micromol/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 U(K)V from 7.5 +/- 0.7 to 1.3 +/- 0.1 micromol/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 U(K)V less, from 6.1 +/- 0.6 to 3.0 +/- 0.8 micromol/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(+).

Dietary K regulates ROMK channels in connecting tubule and cortical collecting duct of rat kidney

The activity of ROMK channels in rat kidney tubule cells was assessed as tertiapin-Q (TPNQ)-sensitive current under whole cell clamp conditions. With an external K(+) concentration of 5 mM and an internal K(+) concentration of 140 mM and the membrane potential clamped to 0 mV, TPNQ blocked outward currents in principal cells of the cortical collecting duct (CCD) outer medullary collecting duct and connecting tubule (CNT). The apparent K(i) was 5.0 nM, consistent with its interaction with ROMK. The TPNQ-sensitive current reversed at voltages close to the equilibrium potential for K(+). The currents were reduced when the pipette solution contained ATP. In the CCD, the average TPNQ-sensitive outward current (I(SK)) was 476 +/- 48 pA/cell in control animals on a 1% KCl diet. I(SK) increased to 1,255 +/- 140 pA when animals were maintained on a high-K (10% KCl) diet for 7 days and decreased to 314 +/- 46 pA after 7 days on a low-K (0.1% KCl) diet. In the CNT, I(SK) was 360 +/- 30 pA on control, 1,160 +/- 110 on high-K, and 166 +/- 16 pA on low-K diets. The results indicate that ROMK channel activity is highly regulated by dietary K in both the CCD and the CNT.