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.

Calcium and sodium transport by the distal convoluted tubule of the rat.

Calcium and sodium transport were studied in the distal convoluted tubule of the rat by micropuncture and microperfusion techniques. The animals received either control infusions, 0.9% saline at 0.02 ml/min, or chlorothiazide (CTZ), 0.5 mg/min-kg. In free-flow micropuncture, distal calcium reabsorption occurred against an electrochemical gradient; it was 9% of the filtered load in the controls and 13.8% in the CTZ-treated rats. The drug dissociated calcium and sodium transport along the distal tubule. Absolute reabsorptive rates for calcium and sodium, measured in pump perfusion experiments, were proportional to the distal loads. CTZ, acting from the lumen, enhanced net calcium (P less than 0.01) and reduced net sodium (P less than 0.01) reabsorption. In stationary microperfusion experiments, sodium backflux produced rapid establishment of a steady-state concentration of 50 mM. In contrast, net backflux of calcium was negligible. With calcium in the luminal perfusion fluid, there was a concentration-dependent reabsorptive efflux which was not saturated at a luminal calcium concentration of 9.5 mM and which was enhanced by CTZ.

Effects of PTH, ADH, and cyclic AMP on distal tubular Ca and Na reabsorption.

Tubular microperfusion experiments were performed in rats to examine the effects of thyroparathyroidectomy (TPTX), parathyroid hormone (PTH), antidiuretic hormone (ADH), and cyclic AMP (cAMP) on distal tubular Ca, Na, and water reabsorption. TPTX caused a significant decrease in the Ca reabsorptive rate as compared to intact animals. PTH (5 U/kg; 2 U x kg-1 x h-1) replacement in TPTX animals restored Ca transport to control levels. Application of either cAMP (10(-3) M) or 8-(p-chlorophenylthio)-cyclic 3',5'-adenosine monophosphate (10(-5) M) to the surface of the kidney caused a stimulation of Ca reabsorption similar to that produced by PTH. Neither TPTX nor PTH changed Na or water reabsorption significantly, whereas the cyclic nucleotides increased both of these parameters. These later actions of cAMP duplicated effects of ADH observed in these distal tubules.

Possible role of cytosolic calcium and Na-Ca exchange in regulation of transepithelial sodium transport.

Emerging evidence in a number of different epithelia suggests that changes in cytosolic calcium ion levels play a critical role in the regulation of transepithelial sodium transport. Maneuvers believed to raise cytosolic calcium ion activity lead to an inhibition of net sodium transport in toad urinary bladder, frog skin, and isolated perfused proximal renal tubules. Regulation of the level of ionized calcium in the cytosol of the epithelial cells appears to involve a process of coupled Na-Ca exchange across the basolateral plasma membrane, energized, at least in part, by the sodium gradient. It is suggested that changes in cytosolic calcium ion levels, secondary to changes in Na-Ca exchange, in turn dependent in part on the activity of the sodium pump, constitute a link in a negative feedback mechanism. Through such a feedback mechanism, the rate of entry of sodium into the cell across the apical surface may be kept in step with its rate of extrusion across the basolateral surface.

Estimate of relative thickness of peritubular interstitial space in Necturus kidney.

Electrophysiological techniques were used to evaluate volume changes of compartments interposed between the peritubular membrane of proximal tubue cells and capillary wall or peritoneal surface of Necturus kidney. The time courseof potassium-induced peritubular membrane depolarization was measured during a switch from 3- to 90-meq potassium sulfate solution in superfusion as well as in vascular perfusion experiments. When superfusion fluids contained 6, 2, and 0 g dextran/100 ml, mean half times of depolarization in normal kidneys were 4.1, 1.9, and 5.8 s. Decreasing the colloid osmotic pressure of superfusion fluids resulted in a significant increase in half-time values. Similar results were obtained in saline expansion. In controls the mean half time was 4.5s; in saline expansion it was 7.7 s. In vascular perfusions, mean half-time values were 225, 350, 355, and 450 s for dextran concentrations of 6, 4, 2, and 0 g/100 ml, respectively. These results indicate that the half time of depolarization increases significantly as the colloid osmotic pressure of perfusion fluids decreases. Estimates of effective unstirred-layer thickness on the peritubular side of proximal tubular epithelium indicate a decrease by about 25% when vascular colloid osmotic pressure is increased from 0 to 54 mmHg.

Osmotically induced changes in electrical resistance of distal tubules of rat kidney.

Changes in peritubular oncotic pressure do not alter the electrical resistance of distal convoluted tubules. Transverse specific electrical resistance decreases along the distal convoluted tubule in nondiuretic rats. This drop in resistance can be at least in part accounted for by the relative increae in osmolality of late distal fluid. Osmotic gradients influence the magnitude of intercellular rather than transcellular conductance. Hyposmolality of tubular fluid leads to reduced shunting, whereas increased tubular osmolality has the opposite effect. The osmotic effect forms part of an intraepithelial positive feedback loop for sodium reabsorption in the diluting segment of the renal nephron.

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.

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.

pH effect on osmotic response of collecting tubules to vasopressin and 8-CPT-cAMP.

The effect of peritubular and luminal pH changes on hydraulic conductance, (Lp, 10(-7) cm X s-1 X atm-1) in the isolated perfused rabbit cortical collecting tubule (CCT) was tested at 37 degrees C before and after administration of 20 microU/ml vasopressin or 10(-4) M 8-[p-chlorophenylthio]-adenosine cyclic monophosphate (8-CPT-cAMP). In vasopressin experiments when bath pH was changed from 7.58 to 7.16 or from 7.58 to 6.70, mean Lp decreased from 249 +/- 32 to 199 +/- 23 (n = 5; P less than 0.01) and from 231 +/- 38 to 201 +/- 36 (n = 5; NS), respectively. In contrast, in 8-CPT-cAMP experiments when bath [HCO3] was kept constant while CO2 was elevated the hydroosmotic response was increased. Using 2.5 mM HCO3, Lp at 0.4% CO2 was 275 +/- 15 and at 6% CO2 it was 352 +/- 50 (n = 4; paired t test; P less than 0.05). At 8.5 and 21.5 mM HCO3 raising CO2 from 2 to 13% and from 4 to 32% increased Lp from 237 +/- 71 to 410 +/- 32 (n = 4; paired t test; P less than 0.05) and from 282 +/- 45 to 449 +/- 63 (n = 6; paired t test; P less than 0.001), respectively. Reducing luminal pH from 7.40 to 5.40 had no effect on either vasopressin- or cAMP-induced changes in Lp. Accordingly, lowering the bath pH by increasing the PCO2 at constant [HCO3] markedly stimulates the response to 8-CPT-cAMP, whereas lowering the bath pH by reducing [HCO3] inhibits the vasopressin response.

Hydroosmotic response of collecting tubules to ADH or cAMP at reduced peritubular sodium.

Changes in cytosolic Ca2+ activity have been implicated in the hydroosmotic response to vasopressin (VP) in amphibian urinary bladder; the level of cytosolic free Ca2+ may be regulated, in part, by a process of Na-Ca exchange across the basolateral cell membrane. To assess whether similar mechanisms operate in the mammalian nephron, the effect of low peritubular [NA] on the hydraulic conductivity (Lp) of the isolated perfused rabbit collecting tubule (CT) exposed to either 20 microunits/ml VP or 5 X 10(-5) M 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate (ClPheS-cAMP) was studied. Low peritubular [Na] had no effect on the basal water permeability of the CT. After exposure to VP, CTs bathed in 145 mM Na Ringer solution developed an Lp of 324 +/- 27 X 10(-7) cm.s-1.atm-1, while tubules bathed in 4 mM Na and 141 mM tetramethylammonium Ringer solution achieved an Lp of only 112 +/- 13 X 10(-7) cm.s-1.atm-1 (P less than 0.001). Inhibition of the VP response was estimated to be half-maximal when peritubular Na was 120 mM. The hydroosmotic response to ClPheS-cAMP was diminished by 44% of the control values when CTs were bathed in a 4 mM Na medium; this inhibition was greatly attenuated when the peritubular Ca concentration was reduced to 0.05 mM. These results are consistent with the view that 1) a Na-Ca exchange process operates at the basolateral surface of the mammalian cortical collecting tubule cells, and 2) elevated cytosolic Ca ion activity inhibits the increase in water permeability elicited by VP or cAMP in this nephron segment.

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.

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.

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.

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.

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.