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.

Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C.

The hypothesis that feedback inhibition of the apical Na+ channels in the cortical collecting tubule (CCT) is mediated by activation of a Ca(2+)-dependent protein kinase was tested using the patch-clamp technique. Na+ channel activity was monitored in cell-attached patches in principal cells of split-open rat tubules. Mean number of open channels (NPo) and single-channel current (i) were measured at 37 degrees C during continuous tubule superfusion. Phorbol 12-myristate 13-acetate (PMA; 50 nM), an activator of protein kinase C (PKC), decreased NPo to 33% of the control value. Staurosporine (200 nM), an inhibitor of PKC and of Ca(2+)-calmodulin kinase II, practically abolished the effect of PMA. Ouabain (1 mM), reduced NPo to 29% of control values and decreased i. Ouabain did not downregulate the channels in tubules exposed to staurosporine, although it still reduced i. Incubation of the tubules with 10 microM KN-62, a specific cell membrane-permeable inhibitor of Ca(2+)-calmodulin kinase II, did not interfere with the ouabain-dependent downregulation of the channels. The results support the view that the downregulation caused by ouabain involves the Ca(2+)-dependent phosphorylation of the channel itself or of proteins regulating the channel.

Feedback regulation of Na channels in rat CCT. III. Response to cAMP.

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca2+i). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 microM), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NPo) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 microM), there was no effect of CPT-cAMP on either NPo or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca2+i from 104 to 198 nM. This increase in Ca2+i was abolished by benzamil (0.5 microM) or by low extracellular Ca2+. The cAMP-dependent reduction in NPo was still observed in Ca(2+)-free medium, indicating that a rise in Ca2+i was not essential for the feedback response. The decrease in NPo was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of AQP3, a water channel from the medullary collecting duct of rat kidney.

The terminal part of the inner medullary collecting duct exhibits a high degree of water permeability that is independent of increased intracellular cAMP and not accounted for by the activity of the known renal epithelial water channels CHIP28 (28-kDa channel-forming integral protein) and WCH-CD (collecting duct water channel protein). Starting with rat kidney papilla mRNA, reverse transcription PCR was performed with degenerate primers assuming that the putative channel would be a member of the major intrinsic protein (MIP) family of proteins. A cDNA fragment was identified and used to screen a rat kidney cDNA library. A 1.9-kb cDNA clone was isolated. The open reading frame of 876 bp coded for a protein of 292 amino acids (M(r), 31,431). Aquaporin 3 (AQP3; 31.4-kDa water channel protein) is a newly discovered member of the MIP family. Northern blot analysis showed a single transcript for AQP3 of approximately 1.9 kb present in the renal medulla, predominantly in the inner medulla. With in situ hybridization, abundant message was found in the cells of the medullary collecting ducts. Injection of the complementary RNA of AQP3 into Xenopus oocytes markedly increased the osmotic water permeability. This permeability had an energy of activation of 3.0 kcal/mol (1 cal = 4.184 J), it was fully blocked by 1 mM p-chloromercuriphenylsulfonate, and this inhibition was reversed by 5 mM dithiothreitol. cAMP did not increase this water permeability. AQP3 did not permit passage of monovalent ions (Na, K, Cl); however, it is slightly permeable to urea. The present study demonstrates the existence of an additional water channel, AQP3, in epithelial cells of the medullary collecting duct.

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.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump.

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry.

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and single-channel currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of amiloride (10 microM) or benzamil (0.5 microM) to the superfusate resulted in a twofold increase in the mean number of open channels (NPo) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NPo reversed within 3 min after removal of amiloride. The increase in NPo was accounted for, at least in part, by an increase in Po. Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NPo. No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel Po by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.

Expression of renal Na(+)-Ca2+ exchange activity in Xenopus laevis oocytes.

The expression of a renal Na(+)-Ca2+ exchanger by Xenopus oocytes has been investigated. Each oocyte was injected with 50 ng of poly(A)+ RNA from either rat or rabbit kidney or with an equivalent volume of water. Na(+)-Ca2+ exchange was determined 3 days after injection, by measuring Ca2+ uptake by oocytes in the presence or absence of an outwardly directed Na+ concentration gradient. To manipulate Na+ concentration gradients, oocytes were first loaded with Na+ in Ca(2+)-free medium containing 90 mM Na+ and nystatin. They were then exposed to medium containing 45Ca and either 90 mM or 0 Na+. Na(+)-free media contained (in mM) either 90 K+, 90 choline or 85 choline plus 5 K+. Oocytes injected with rat kidney poly(A)+ RNA showed a Na+ gradient-dependent Ca2+ uptake of 6.6 +/- 0.8 (SE, n = 5) pmol.oocyte-1.30 min-1. This is significantly higher than the value of 3.4 +/- 0.5 (SE, n = 5) pmol.oocyte-1.30 min-1 obtained in water-injected oocytes (P less than 0.001). Similar results were obtained using poly(A)+ RNA from rabbit kidney cortex. Neither 10 microM nifedipine nor 0.5 mM D 600 significantly affected this Ca2+ uptake. However, 90% of the Ca2+ uptake was inhibited in the presence of 0.1 mM La3+. The poly(A)+ RNA-induced Na(+)-Ca2+ exchange activity was stimulated by the presence of 5 mM K+ in the extracellular choline solution compared with choline alone. Fractionation experiments indicate that the rat kidney Na(+)-Ca2+ exchanger was encoded by poly(A)+ RNA of 3-4 kb.

Ca2(+)-dependent inhibition of sodium transport in rabbit cortical collecting tubules.

Experiments were carried out to test whether maneuvers believed to increase intracellular Ca2+ concentration [( Ca2+]cell) inhibit Na transport in cortical collecting tubules (CCTs). Unidirectional Na efflux (JNa1----b) and Na influx (JNab----1) were measured isotopically in isolated perfused renal CCTs of rabbits. The animals were either untreated or pretreated with deoxycorticosterone (DOC) for 1-3 wk. To raise [Ca2+]cell, ionomycin or quinidine were added to, or [Na] reduced in, pertubular fluid. In control DOC-pretreated CCTs JNa1----b tended to saturate as luminal Na concentration was increased, reaching 22.9 +/- 1.2 pmol.cm-1.s-1 at 145 mM. In addition, in these CCTs, in contrast to non-DOC-treated tubules, the apical cell membrane was not found to be rate limiting for Na reabsorption as neither amphotericin B nor vasopressin further enhanced JNa1----b. In non-DOC-treated CCTs 10(-6) M ionomycin inhibited JNa1----b by 44.7%. When DOC-pretreated CCTs were exposed to either 10(-6)M ionomycin or 10(-4)M quinidine, JNa1----b was inhibited by 27 and 26%, respectively, while JNab----1 remained unchanged. This ionomycin-induced inhibition was Ca dependent. Exposure of DOC-pretreated CCTs to 5 mM Na-Ringer solution (Na replaced by choline or N-methyl-D-glucamine) for 30 min reduced JNa1----b by 18-30%. The inhibition of JNa1----b caused by any of the three maneuvers was fully reversed upon addition of amphotericin B to the luminal fluid. The results are consistent with the view that a sustained increase in [Ca2+]cell reduces Na transport by inhibition of the rate of Na+ entry across the apical cell membrane.

Effect of Ca on Na-D-glucose cotransport across isolated renal brush-border membranes.

Brush-border membrane vesicles were prepared from rat kidney cortex by Mg precipitation. Using quin2 (free acid), intravesicular [Ca2+] was found to be 44 microM and less than 300 nM when vesicles were incubated in 0.2 mM CaCl2 or Ca-free buffer, respectively. In Ca-loaded vesicles, the initial D-glucose uptake, measured in the presence of 150 mM Na+ and 0.1 mM D-glucose inward gradients, was reduced to 30% of the control uptake. This reduction persisted when the extra-vesicular Ca2+ was chelated by ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid but was abolished in the presence of saturating concentrations of D-glucose. Whereas KG0.5 (g) for D-glucose at constant [Na] in the Ca-loaded membranes increased by approximately 50% of the control value (0.5 +/- 0.1 mM), no significant change in Jmax was observed. In contrast, both Jmax and KG0.5 (Na) for glucose, measured as a function of [Na] in the extravesicular fluid, were found to be significantly reduced. Na uptake, determined in the presence of 0.5 mM amiloride, was found to increase by approximately 30% of the value for control membrane. This increase was abolished when vesicles were preincubated with 0.5 mM neomycin or 0.5 mM phlorizin. The results suggest that the effect of Ca2+ on Na entry may be mediated in part by activation of phospholipase C and are consistent with a model of cotransport in which Ca2+ increases the mobility of the binary Na-sugar-translocator complex, thus leading to uncoupling of Na transport from glucose uptake ("slipping") and in part with Ca-induced Na entry by nonmediated leakage.

Regulation of cytosolic free calcium in isolated perfused proximal tubules of Necturus.

To study the role of intracellular Ca2+ in regulating renal tubular transport of ions and water, cytosolic calcium ion activity (aiCa), cytosolic sodium ion activity (aiNa), and intracellular pH (pHi) in cells of isolated perfused proximal tubules of Necturus kidney were measured with Ca2+-, Na+-, and H+-selective microelectrodes, respectively. In control conditions, i.e., HCO3-Ringer solution on both sides of the epithelium, aiCa was 82 +/- 7 (SE) nM (n = 54), aiNa averaged 12.8 +/- 0.4 mM (n = 53), and pHi was 7.33 +/- 0.03 (n = 27). When the Na-K pump was inhibited by nominally K-free Ringer circumfusion, aiCa increased from a control level of 75 +/- 13 to 237 +/- 40 nM (paired t test; n = 16; P less than 0.001); in a different set of tubules, aiNa rose from 11.3 +/- 0.6 to 51.5 +/- 5.8 mM (n = 11; P less than 0.001). When organic solutes were deleted in the luminal perfusate, aiCa decreased from 73 +/- 11 to 61 +/- 11 nM (n = 9; P less than 0.001) and aiNa decreased from 14.6 +/- 0.6 to 8.3 +/- 0.7 mM (n = 9; P less than 0.001). Depolarization of the peritubular cell membrane with high-K, low-Na Ringer decreased aiCa from 90 +/- 12 to 55 +/- 9 nM (n = 13; P less than 0.001) and reduced aiNa from 13.1 +/- 1.0 to 7.5 +/- 0.6 mM (n = 16; P less than 0.001). Ionomycin (2 X 10(-6) M) increased aiCa from 67 +/- 10 to 158 +/- 26 nM (n = 10; P less than 0.001) and pHi from 7.33 +/- 0.03 to 7.39 +/- 0.03 (n = 27; P less than 0.001) but reduced aiNa from 11.8 +/- 0.9 to 10.3 +/- 0.7 mM (n = 11; P less than 0.001). The data are consistent with the view that aiCa is determined, in part, by the magnitude of the electrochemical potential gradient for Na ions across the basolateral cell membrane.

Effect of peritubular [Ca] or ionomycin on hydrosmotic response of CCTs to ADH or cAMP.

To evaluate in the mammalian kidney the effect of maneuvers thought to alter intracellular [Ca2+] on the hydraulic conductivity (Lp) response to vasopressin (VP) or 8-(p-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (ClPheS-cAMP), water flow was measured in isolated perfused rabbit cortical collecting tubules (CCTs) exposed to either varying bath [Ca] or ionomycin or quin 2-acetoxymethyl ester (AM). The development of the response to VP (20 microU/ml) was enhanced 28% by lowering Ca from 1.0 to 0.1 mM, but was not altered by increasing Ca from 1.0 to 3.75 mM. When measured in the same tubule, the maintenance phase of a previously established hormone response was inhibited by acutely raising peritubular Ca from 1.0 to 3.75 mM. Exposing the tubules to 0.1 mM Ca and 65 microM quin 2-AM, inhibited by 68% the development of the response to VP compared with that observed in tubules bathed in 1.0 mM Ca Ringer without quin 2-AM. In contrast, quin 2-AM and low peritubular Ca added during the maintenance phase enhanced the VP response by 50%. The Ca ionophore ionomycin (1.0 microM) reduced the development of the VP-elicited Lp by 65% and reversibly decreased by 42% the maintenance phase of the VP-stimulated Lp in a Ca-dependent manner. A longer exposure to the ionophore was required to inhibit the development and maintenance phases of the response to 10(-4) M ClPheS-cAMP. These results are consistent with the view that transient changes in intracellular [Ca2+] may be required for the development of the hormone response but that sustained increases in cytoplasmic Ca2+ levels inhibit the development as well as the maintenance phase of the hydrosmotic response to VP or cAMP in CCTs.

Potential role of cytoplasmic calcium ions in the regulation of sodium transport in renal tubules.

Experimental maneuvers that increase intracellular calcium ion levels inhibit sodium transport by renal tubules. In the isolated perfused renal tubule, intracellular calcium ion activity (aiCa) changes in response to alterations in the magnitude of the electrochemical potential gradient for sodium ions across the basolateral cell membrane. However, a potassium-induced depolarization of this cell boundary does not cause a rise but rather a fall in intracellular calcium ion levels. Ionomycin raises aiCa without causing intracellular acidification. This observation does not support the view that high cytosolic calcium produces intracellular acidification. At least in the case of ionomycin, the inhibition of sodium transport appears to be due to ionophore-induced increases in aiCa. The changes in intracellular calcium ion concentration found in the different experimental conditions studied were consistent with the notion that cytosolic calcium ions may mediate a feedback mechanism that links the luminal entry to the peritubular extrusion of sodium ions. The mechanisms by which cytosolic calcium alters entry is not yet clear but recent experiments suggest an indirect effect on sodium channel activity.

Quinidine effect on hydrosmotic response of collecting tubules to vasopressin and cAMP.

Quinidine, a compound thought to increase cytosolic calcium ion activity, has been found to inhibit the hydrosmotic response to vasopressin (VP) and adenosine 3',5'-cyclic monophosphate (cAMP) in the toad urinary bladder. To test whether this drug has a similar action in the mammalian nephron, the effect of quinidine on the hydraulic conductivity of the isolated perfused rabbit cortical collecting tubule (CCT) exposed to either 20 microU/ml VP or 10(-4) M 8-(p-chlorophenylthio) - adenosine 3',5' - cyclic monophosphate (8-CPT-cAMP) was studied. Quinidine had no effect on the basal water permeability of the CCT. Quinidine sulfate (10(-4) M) reduced the VP-stimulated water permeability from 280 +/- 50 X 10(-7) to 115 +/- 41 X 10(-7) cm X s-1 X atm-1 (P less than 0.05). The hydrosmotic response to 8-CPTcAMP was likewise reduced following exposure to quinidine. This effect was shown to be dose dependent. In paired experiments, inhibition of the response to 10(-4) M 8-CPTcAMP averaged 11% at 10(-6) M, 27% at 5 X 10(-6) M, 53% at 5 X 10(-5) M, and 50% at 10(-4) M quinidine. Inhibition of the response to 8-CPTcAMP was estimated to be half maximal at approximately 5 X 10(-6) M quinidine. Tubules were protected against the quinidine-induced inhibition by the addition of 6.5 X 10(-5) M quin 2-acetoxymethylester in the presence of low peritubular Ca concentration. These results are consistent with the view that elevated cytosolic Ca ion levels inhibit the increase in water permeability elicited by VP or exogenous cAMP in the mammalian CCT.

Cytosolic Ca2+ and Na+ activities in perfused proximal tubules of Necturus kidney.

To study the role of intracellular calcium in the regulation of epithelial transport of ions and water, cytosolic calcium ion activity (aiCa) and cytosolic sodium ion activity (aiNa) were measured in cells of isolated perfused proximal tubules of Necturus kidney. aiCa was measured with Ca2+-selective microelectrodes, aiNa with Na+-selective microelectrodes. Under control conditions, i.e., Ringer solution on both sides of the epithelium, aiCa averaged 71 +/- 7 (SE) nM (n = 21) and aiNa was 12.9 +/- 0.6 mmol (n = 56). When peritubular bath sodium was reduced from 100 to 10 mM by choline substitution, aiCa increased from 73 +/- 14 to 382 +/- 69 nmol (paired t test; P less than 0.001; n = 4); in different tubules, aiNa decreased from 12.8 +/- 1.9 to 8.2 +/- 1.8 mM (P less than 0.001; n = 12). Quinidine (10(-4) M) increased aiCa from 87 +/- 19 to 556 +/- 121 nM (P less than 0.02; n = 5) but reduced aiNa from 15.1 +/- 1.2 to 11.8 +/- 0.8 mM (P less than 0.003; n = 8). In contrast, 10(-4) M ouabain increased both aiCa and aiNa; aiCa rose from 71 +/- 9 to 546 +/- 121 nmol (P less than 0.005; n = 9) and aiNa from 15.1 +/- 1.8 to 70.1 +/- 6.3 mM (P less than 0.001; n = 9). The results are consistent with the existence of a Na-Ca exchange process within the contraluminal cell membrane and with the view that increased aiCa inhibits the tubular transport of sodium by decreasing the sodium permeability of the luminal cell membrane.