Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, vasopressin, and deoxycorticosterone.

Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.     

Luminal vasopressin modulates transport in the rabbit cortical collecting duct.

We explored the action of luminal AVP in rabbit CCD perfused in vitro at 37 degrees C. Nanomolar concentrations of luminal AVP induced a sustained hyperpolarization of transepithelial voltage (Vt) in contrast to a transient hyperpolarization caused by basolateral AVP. 10 microM basolateral ouabain abolished the latter but not the former change in Vt. Despite a sustained hyperpolarization (from -20.7 +/- 2.9 to -34.1 +/- 4.7 mV; P less than 0.01), 10 nM luminal AVP only slightly altered net Na+ and K+ fluxes (7.6% stimulation and no significant change, respectively). Instead, luminal AVP appeared to modulate an acetazolamide-sensitive electrogenic ion transport because 200 microM basolateral acetazolamide suppressed the luminal AVP-induced hyperpolarization (percentage of Vt from -50.4 +/- 10.8 to -5.1 +/- 1.4; P less than 0.005). In terms of water transport, 10 nM luminal AVP did not change hydraulic conductivity (Lp, x 10(-7) cm/atm per s) (from 3.9 +/- 0.8 to 5.0 +/- 1.2), but suppressed the increase in Lp induced by 20 pM basolateral AVP (134.9 +/- 19.2 vs. 204.3 +/- 21.1 in control; P less than 0.05). These findings demonstrate distinct luminal action of AVP, suggesting amphilateral regulation of epithelial transport by AVP in the CCD.     

Prostaglandin E2 inhibits sodium transport in rabbit cortical collecting duct by increasing intracellular calcium.

The mechanism by which prostaglandin E2 (PGE2) inhibits sodium absorption (JNa) in the rabbit cortical collecting duct (CCD) was explored. PGE2 activates at least three signaling mechanisms in the CCD: (a) by itself PGE2 increases cAMP generation (b) PGE2 also inhibits vasopressin-stimulated cAMP accumulation, and (c) PGE2 raises intracellular calcium([Ca++]i). We tested the contribution of these signaling pathways to PGE2's effect on Na+ absorption, measuring 22Na flux (JNa) and [Ca++]i (using fura-2) in microperfused rabbit CCDs. In control studies PGE2 reduced JNa from 28.2 +/- 3.4 to 15.6 +/- 2.6 pmol.mm-1.min-1. Lowering bath calcium from 2.4 to 45 nM did not by itself alter JNa but in this setting PGE2 failed to inhibit JNa (28.6 +/- 5.4 to 38.5 +/- 4.0). In separate tubules, PGE2 raised [Ca++]i in a spike-like fashion followed by a sustained elevation. However, in 45 nM bath Ca++, PGE2 failed to produce a sustained [Ca++]i elevation. While pretreatment of CCDs with pertussis toxin blocked PGE2 inhibition of vasopressin-stimulated water permeability, it did not block the effect of PGE2 on JNa. To see if cAMP generation contributes to the effect of PGE2 on JNa, we tested the effect of exogenous cAMP, (8-chlorophenylthio(CPT)cAMP) on JNa. 0.1 mM 8-CPTcAMP reduced JNa from 35.75 +/- 2.3 to 21.6 +/- 2.2. However, the addition of PGE2 further blunted JNa to 15.9 +/- 1.3. In CCDs pretreated with indomethacin, 8-CPTcAMP did not significantly decrease JNa 33.6 +/- 2.8 vs. 28.4 +/- 2. However, superimposed PGE2 reduced JNa to 19.0 +/- 3.0. We conclude that PGE2 inhibits sodium transport predominantly by increasing intracellular calcium. This action is not mediated by a pertussis toxin-sensitive G protein. Finally, cAMP, through a cyclooxygenase-dependent mechanism, also inhibits CCD JNa and may contribute to the effects of PGE2 on JNa in the rabbit CCD.     

Inhibition of cortical collecting tubule chloride transport by organic acids.

Cl self-exchange by the rabbit cortical collecting tubule (CCT) occurs via an apical anion exchanger in series with a basolateral Cl conductance. We studied the effects of organic acids on CCT Cl self-exchange. We found no evidence for transport of acid anions by the self-exchange system. Rather, Cl self-exchange was inhibited by a variety of organic acids. The degree of inhibition correlated with the chloroform/water partition coefficient and was enhanced by lowering pH, indicating inhibition by the lipid-soluble, protonated species. Inhibition by the representative acid iso-butyrate was dose-dependent and showed sidedness (basolateral greater than apical). Iso-butyrate also reversibly reduced transepithelial conductance without altering K permeability, suggesting inhibition of the principal cell basolateral Cl conductance. Because small organic compounds with similar lipid solubilities but no carboxyl group had no effect, both the carboxyl group and the lipid-solubility of organic acids appear to be important. The results are consistent with blockade of chloride channels by organic acids.     

Direct measurement of papillary collecting duct sodium transport in the rat. Evidence for heterogeneity of nephron function during Ringer loading.

It has been suggested that collecting duct sodium transport was inhibited by extracellular volume expansion. To directly evaluate this possibility, micropuncture of the papillary collecting duct of young rats was performed during hydropenia and Ringer loading. The possibility of heterogeneity of nephron function was evaluated during Ringer and hyperoncotic albumin loading by comparing the delivery of sodium to the end of the distal tubule of superficial nephrons with papillary base delivery. During hydropenia (n = 14), sodium delivery to the base averaged 0.95% of the filtered sodium load and reabsorption along the collecting duct was noted from base to tip in each collection pair averaging 0.80% of the filtered load. During Ringer loading, sodium delivery to the base was markedly greater than in hydropenia, 11.8 vs. 0.95% of the filtered load (P less than 0.001). Yet, sodium reabsorption was also much greater, 6 vs. 0.8% (P less than 0.001). In 13 paired collections, during Ringer loading, sodium delivery to the papillary base, 12.2% of the filtered load, was consistently greater than late distal tubular delivery from superficial nephrons. 8% (P less than 0.005). In contrast, reabsorption of sodium from late distal tubule to papillary base was found during albumin infusion, 6.2 vs. 3.1% (P less than 0.001). Therefore, these studies demonstrate that: (a) the delivery of sodium to and reabsorption along the papillary collecting duct were markedly greater during Ringer loading than in hydropenia; (b) the amount of sodium delivered to the papillary base was greater than the delivery to the end of the distal tubule of superficial nephrons during Ringer loading, suggesting that deeper nephrons deliver more sodium to the collecting duct in this setting; and (c) the difference in sodium excretion between Ringer loading and hyperoncotic albumin infusion is due to events occurring between the late distal tubule of superficial nephrons and the base of the papillary collecting duct.     

Expression of GTPase-deficient Ras inhibits vasopressin signaling in cultured cortical collecting duct cells.

Cross-talk between signaling pathways is increasingly recognized as integral to cellular function. We investigated whether the mitogen-activated protein kinase (MAPK) pathway alters vasopressin (AVP) stimulation of protein kinase A (PKA) by specifically studying the role of Ras. Mouse cortical collecting duct cells (M-1) were transfected with a cDNA encoding oncogenic Ras. Transfection was confirmed by Western blot analysis and functionally by enhanced basal MAPK activity. When compared with basal MAPK activity of 26.4 +/- 6.6 pmol/mg/min in controls, basal MAPK activity varied widely in Ras-transfected clones from 29.0 +/- 6.6 to 96.6 +/- 13.4 pmol/mg/min. Clones that functionally expressed activated Ras displayed complete abolition of AVP-stimulated PKA activity, whereas those that failed to express elevated basal MAPK activity showed intact AVP-stimulated PKA. The correlation between expression of high basal MAPK activity and inhibition of AVP-induced PKA yielded a correlation coefficient of -0.92 (P = 0.009). Exposure to 10 microM forskolin or 1 microgram/ml cholera toxin resulted in comparable activation of PKA in all clones. We found no correlation between PKC activity of the clones and PKA inhibition. To assess whether the observed effect was due to one known Ras target, cells were transfected with constitutively activated Raf. M-1 cells expressing activated Raf exhibited elevated MAPK activity. The Raf clones showed no impairment of AVP-stimulated PKA activity. We conclude that expression of activated Ras is inhibitory of AVP-induced PKA activation in the M-1 cortical collecting duct cell line at a site proximal to G alpha s protein. The failure of Raf to influence AVP signaling indicates that the action of Ras is through a pathway independent of this Ras target.     

Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct.

Vasopressin increases both the urea permeability and osmotic water permeability in the terminal part of the renal inner medullary collecting duct (terminal IMCD). To identify the second messengers that mediate these responses, we measured urea permeability, osmotic water permeability, intracellular calcium concentration, and cyclic AMP accumulation in isolated terminal IMCDs. After addition of vasopressin, a transient rise in intracellular calcium occurred that was coincident with increases in cyclic AMP accumulation and urea permeability. Half-maximal increases in urea permeability and osmotic water permeability occurred with 0.01 nM vasopressin. The threshold concentration for a measurable increase in cyclic AMP accumulation was approximately 0.01 nM, while measurable increases in intracellular calcium required much higher vasopressin concentrations (greater than 0.1 nM). Exogenous cyclic AMP (1 mM 8-Br-cAMP) mimicked the effect of vasopressin on urea permeability but did not produce a measurable change in intracellular calcium concentration. Conclusions: (a) Cyclic AMP is the second messenger that mediates the urea permeability response to vasopressin in the rat terminal IMCD. (b) Vasopressin increases the intracellular calcium concentration in the rat terminal IMCD, but the physiological role of this response is not yet known.     

Cytochrome P450 metabolites of arachidonic acid are potent inhibitors of vasopressin action on rabbit cortical collecting duct.

AA is metabolized by a cytochrome P450, NADPH-dependent epoxygenase to four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs are further hydrated enzymatically to their respective diols, vic-dihydroxyeicosatrienoic acids (DHETs). We studied the effect of pretreatment with DHETs on 10 microU/cm2 arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp) (Lp x 10(-7) cm/atm/s, mean +/- SE) in rabbit cortical collecting ducts (CCDs) perfused in vitro at 37 degrees C. At 10(-6) M all four DHETs were potent inhibitors of the hydroosmotic effect of AVP. 14,15-DHET was the most potent isomer; it reduced AVP-induced Lp from a control value of 234.75 +/- 11.7, n = 17, to a value of 95.2 +/- 8.39, n = 5, P less than 0.0001, a reduction of AVP-mediated water flow of 60%. The inhibitory effect of 14,15-DHET was dose dependent and significant to nanomolar concentrations. 14,15-DHET at 10(-7) M was as potent an inhibitor of AVP's activity as was 10(-7) M PGE2. AVP's hydroosmotic effect is mediated through its intracellular second messenger, cAMP. 8-p-Chlorophenylthio-cAMP (CcAMP) at 10(-4) M induced a peak Lp of 189.6 +/- 11.0, n = 8; pretreatment with 10(-6) M 14,15-DHET reduced CcAMP-peak Lp to 132.0 +/- 13.4, n = 5, P less than 0.01, demonstrating a post-cAMP effect. Gas chromatography/mass spectroscopy suggests that EETs are present in extracts purified from CCDs. We conclude that cytochrome P450 epoxygenase eicosanoids are potent inhibitors of the hydroosmotic effect of vasopressin and are endogenous constituents of normal CCDs, the major target tissue for AVP.     

Effects of uninephrectomy on electrical properties of the cortical collecting duct from rabbit remnant kidneys.

Microelectrode techniques were used to determine the Na+ and K+ transport properties of the collecting duct cell in the isolated cortical collecting duct (CCD) from rabbits 14 d after uninephrectomy (UNX); results were compared with those from sham-operated rabbits (control). UNX had no effects on plasma aldosterone levels. The CCDs from UNX rabbits exhibited structural hypertrophy. The lumen negative transepithelial voltage and the basolateral membrane voltage (VB) were elevated in the UNX group. Although the transepithelial conductance (GT) and the fractional apical membrane resistance (fRA) were not different between the two groups, the conductances of the apical and the basolateral membranes were increased, and the tight junction conductance was decreased in the UNX group. The amiloride-sensitive changes in apical membrane voltage (VA), fRA, and GT were greater in the UNX group. The changes in VA upon raising the perfusate K+ concentration and the changes in VA and GT upon addition of Ba2+ to the perfusate were elevated in the UNX group. Upon raising K+ in the bath, a large depolarization of VB was observed in the UNX group. Lowering the bath Cl- resulted in a small depolarization of VB in the UNX group. Addition of Ba2+ to the bath in the UNX group caused the VB to hyperpolarize in parallel with decreases in GT and fRA whereas in the control group it had no effect on VB. Addition of ouabain to the bath resulted in a large depolarization of VB in the UNX group. We conclude that (a) UNX stimulates conductances of Na+ and K+ in the apical membrane, active Na(+)-K+ pump activity, and K+ conductance in the basolateral membrane, independently of plasma aldosterone; (b) The basolateral membrane in the tubules of UNX rabbits is more selective to K+; and (c) the hyperpolarization of VB upon UNX may increase passive K+ entry into the cell across the basolateral membrane.     

Adaptation of rabbit cortical collecting duct HCO3- transport to metabolic acidosis in vitro.

Net HCO3- transport in the rabbit kidney cortical collecting duct (CCD) is mediated by simultaneous H+ secretion and HCO3- secretion, most likely occurring in a alpha- and beta-intercalated cells (ICs), respectively. The polarity of net HCO3- transport is shifted from secretion to absorption after metabolic acidosis or acid incubation of the CCD. We investigated this adaptation by measuring net HCO3- flux before and after incubating CCDs 1 h at pH 6.8 followed by 2 h at pH 7.4. Acid incubation always reversed HCO3- flux from net secretion to absorption, whereas incubation for 3 h at pH 7.4 did not. Inhibition of alpha-IC function (bath CL- removal or DIDS, luminal bafilomycin) stimulated net HCO3- secretion by approximately 2 pmol/min per mm before acid incubation, whereas after incubation these agents inhibited net HCO3- absorption by approximately 5 pmol/min per mm. Inhibition of beta-IC function (luminal Cl- removal) inhibited HCO3- secretion by approximately 9 pmol/min per mm before incubation, whereas after incubation HCO3- absorption by only 3 pmol/min per mm. After acid incubation, luminal SCH28080 inhibited HCO3- absorption by only 5-15% vs the circa 90% inhibitory effect of bafilomycin. In outer CCDs, which contain fewer alpha-ICs than midcortical segments, the reversal in polarity of HCO3- flux was blunted after acid incubation. We conclude that the CCD adapts to low pH in vitro by downregulation HCO3- secretion in beta-ICs via decreased apical CL-/base exchang activity and upregulating HCO3- absorption in alpha-ICs via increased apical H+ -ATPase and basolateral CL-/base exchange activities. Whether or not there is a reversal of IC polarity or recruitment of gamma-ICs in this adaptation remains to be established.     

Enhancement of electrogenic Na+ transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones.

We have investigated the effect of steroid hormones on Na+ transport by rat renal inner medullary collecting duct (IMCD) cells. These cells, grown on permeable supports in primary culture, grow to confluence and develop a transmonolayer voltage oriented such that the apical surface is negative with respect to the basal surface. The results of these experiments demonstrate that this voltage is predominantly (or exclusively) the result of electrogenic Na+ absorption. Na+ transport can be stimulated two- to fourfold by exposure to either dexamethasone or aldosterone (100 nM). Experiments using specific antagonists of the glucocorticoid and mineralocorticoid receptors indicate that activation of either receptor stimulates electrogenic Na+ transport; electroneutral Na+ transport is undetectable. Two other features of the IMCD emerge from these studies. (a) These cells appear to have the capacity to metabolize the naturally occurring glucocorticoid hormone corticosterone. (b) The capacity for K+ secretion is minimal and steroid hormones do not induce or stimulate conductive K+ secretion as they do in the cortical collecting duct.     

Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells.

Stimulation of the release of endothelium-derived relaxing factor (EDRF) in the kidney has been shown to result in natriuresis without affecting glomerular filtration rate. This may be due to EDRF directly regulating solute transport in the cortical collecting duct (CCD). To test this hypothesis, we measured the effect of bradykinin (Bk) or acetylcholine (Ach) on short-circuit current (Isc; a measure of active transport) in a CCD cell line (M-1), in the presence or absence of cow pulmonary artery endothelial (CPAE) cells. 10(-9) M Bk or 10(-7) M Ach had no effect on M-1 Isc in which CPAE cells were absent. The addition of CPAE cells to M-1 cells also did not affect M-1 Isc. On the other hand, when 10(-9) M Bk or 10(-7) M Ach were added to M-1 cells in the presence of CPAE cells, Isc decreased from 43 +/- 4.5 to 26 +/- 4 and 64 +/- 9 to 33 +/- 4 microA/cm2, respectively (P less than 0.001). Nitroarginine (N-Arg, 10(-4) M), a competitive inhibitor of EDRF production, blocked the inhibition in M-1 Isc due to both agonists. Since cGMP is the second messenger of EDRF in vascular smooth muscle, we measured the effects of Bk on cGMP production in M-1 cells in the presence and absence of CPAE cells. Bk increased cGMP content in M-1 cells in the presence of CPAE cells from 33 +/- 3.4 to 132 +/- 11.7 fmol/10(6) M-1 cells (P less than 0.001). When cultures of M-1 and CPAE cells were treated with N-Arg and challenged with Bk, Bk's effect on cGMP was partially blocked (61.4 +/- 12 fmol/10(-6) M-1 cells; NS). These data suggest that EDRF inhibits transport and increases cGMP content in M-1 cells.     

Evidence for sodium-dependent active urea secretion in the deepest subsegment of the rat inner medullary collecting duct.

Active reabsorption of urea appears in the initial IMCD (IMCD1) of rats fed a low-protein diet. To determine whether active urea transport also occurs in the deepest IMCD subsegment, the IMCD3, we isolated IMCDs from the base (IMCD1), middle (IMCD2), and tip (IMCD3) regions of the inner medulla from rats fed a normal protein diet and water ad libitum. IMCDs were perfused with identical perfusate and bath solutions. A significant rate of net urea secretion was present only in IMCD3s. Replacing perfusate Na+ with NMDG+ reversibly inhibited net urea secretion but replacing bath Na+ with NMDG+ or perfusate Cl- with gluconate- had no effect. Net urea secretion was significantly inhibited by: (a) 250 microM phloretin (perfusate); (b) 100 nM triamterene (perfusate); (c) 1 mM ouabain (bath); and (d) cooling the tubule to 23 degrees C. Net urea secretion was significantly stimulated by 10 nM vasopressin (bath). Next, we perfused IMCD3s from water diuretic rats (given food ad libitum) and found a significant, fivefold increase in net urea secretion. In summary, we identified a secondary active, secretory urea transport process in IMCD3s of normal rats which is upregulated in water diuretic rats. This new urea transporter may be a sodium- urea antiporter.     

Modulation of the hydro-osmotic effect of vasopressin on the rabbit cortical collecting tubule by adrenergic agents

The effects of catecholamines on antidiuretic hormone ([Arg(8)]-vasopressin [AVP])-induced water absorption were evaluated in cortical collecting tubules isolated from the rabbit kidney and perfused in vitro. In the presence of AVP (100 muU/ml), net fluid volume absorption (J(v), nanoliters per minute per millimeter) was 1.14+/-0.12 and osmotic water permeability coefficient (P(f), X 10(-4) centimeters per second) was 217.3+/-39.9. The addition of the alpha-adrenergic agonist, phenylephrine (PE), in a concentration of 10(-6) M resulted in a significant decrease in J(v) and P(f) to 0.83+/-0.13 (P < 0.001) and 148.8+/-41.8 (P < 0.02), respectively. Increasing the concentration of PE to 10(-5) M resulted in a further decrease in J(v) and P(f) to 0.53+/-0.05 (P < 0.05 vs. PE 10(-6) M) and 88.5+/-9.0 (P 0.05 vs. PE 10(-6) M), respectively. In a separate group of tubules, in the presence of AVP (100 muU/ml) and PE (10(-5) M), J(v) and P(f) were 0.35+/-0.07 and 66.0+/-17.3, respectively. The addition of the alpha-adrenergic antagonist, phentolamine (PH), in a concentration of 10(-6) M resulted in a significant increase in J(v) to 1.07+/-0.19 (P < 0.001) and P(f) to 193.3+/-35.9 (P < 0.005). PH (10(-5) M) alone did not significantly affect J(v) and P(f) in the presence of AVP (100 muU/ml) nor in the presence of 8-bromo adenosine 3',5' cyclic monophosphate (8-BrcAMP). J(v) and P(f) were 1.20+/-0.21 and 174.0+/-25.8, respectively, in the presence of 8-BrcAMP (10(-4) M).We next examined the effect of the beta-adrenergic agonist, isoproterenol (ISO), on J(v) and P(f) in the presence of AVP. J(v) and P(f) were 1.04+/-0.10 and 202.6+/-17.2, respectively, in the presence of AVP (100 muU/ml) and 1.06+/-0.18 and 193.4+/-27.7, respectively, in the presence of AVP (10muU/ml). However, in the presence of AVP in a concentration of 2.5 muU/ml, J(v) was 0.60+/-0.07 and P(f) was 100.7+/-24.7. ISO (10(-6) and 10(-5) M) did not have any significant effect in the presence of the above maximal and submaximal concentrations of AVP. In the absence of AVP, control J(v) was 0.01+/-0.12 and P(f) was 4.6+/-11.0. The addition of ISO at 25 or 37 degrees C did not result in any significant change in J(v) or P(f).These studies indicate that alpha-adrenergic agonists directly inhibit AVP-mediated water absorption at the level of the tubule, an effect that can be blocked by a specific alpha-adrenergic antagonist. This effect appears to be exerted at the level of activation of adenylate cyclase since it is absent in the presence of cAMP. The beta-adrenergic agonists do not directly inhibit or enhance AVP-mediated water absorption at the level of the renal tubule.     

Bicarbonate transport by rabbit cortical collecting tubules. Effect of acid and alkali loads in vivo on transport in vitro.

Rabbit cortical collecting tubules were perfused in vitro to investigate the control of bicarbonate transport. Bicarbonate was measured by microcalorimetry as total CO2. The perfusate and bath were identical solutions containing 25 mM bicarbonate at pH 7.4. The mean pH of the urine in the bladders of untreated rabbits at the time they were killed was 7.4. Their individual tubules, studied in vitro, either absorbed or secreted bicarbonate, and, combining the results, there was on the average no significant net transport. When the rabbits were treated with NH4Cl the day before the experiment, their urine was acidic and their tubules studied in vitro absorbed bicarbonate (i.e., there was net lumen-to-bath transport). In contrast, when the rabbits were treated with NaHCO3, their urine was significantly more alkaline, and their tubules studied in vitro generally secreted bicarbonate (i.e., net bath-to-lumen transport). Thus, the direction of bicarbonate transport by cortical collecting tubules studied under standard conditions in vitro correlated with the urine pH and was determined by the preceding treatment of the animals in vivo with acidifying or alkalinizing salts. These results demonstrate a previously unrecognized mechanism which contributes to the control of urinary bicarbonate excretion.     

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.     

Osmolar regulation of endothelin-1 production by rat inner medullary collecting duct.

Recent evidence has implicated endothelin-1 (ET-1) as an autocrine inhibitor of inner medullary collecting duct (IMCD) sodium and water transport. The regulators of IMCD ET-1 production are, however, largely unknown. Because of the unique hypertonic environment of the IMCD, the effect of varying extracellular tonicity on IMCD ET-1 production was evaluated. Increasing media osmolality from 300 to 450 mosmol with NaCl or mannitol but not urea caused a marked dose- and time-dependent reduction in ET-1 release by and ET-1 mRNA in cultured rat IMCD cells. In contrast, increasing osmolality had no effect on ET-1 production by rat endothelial or mesangial cells. To see if ET-1 varies in a similar manner in vivo, ET-1 production was assessed in volume expanded (lower medullary tonicity) or volume depleted (high medullary tonicity) rats. Urinary ET-1 excretion and inner medulla ET-1 mRNA were significantly reduced in volume depleted as compared to volume expanded animals. These results indicate that extracellular sodium concentration inhibits ET-1 production specifically in IMCD cells. We speculate that extracellular sodium concentration, via regulation of ET-1 production, provides a link between volume status and IMCD sodium and water reabsorption.     

In vivo inhibition of renal 11beta-hydroxysteroid dehydrogenase in the rat stimulates collecting duct sodium reabsorption

In order to test the proposal that the aldosterone specificity of mineralocorticoid receptors in the collecting duct depends on inactivation of glucocorticoids by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD), we have assessed the effect of pharmacological inhibition of 11beta-HSD on collecting duct Na+ reabsorption in vivo. Adrenalectomized rats (n=14) were infused intravenously with high-dose corticosterone, and late-distal tubules were perfused orthogradely with artificial tubular fluid containing [14C]inulin and 22Na; urinary recoveries of the radioisotopes were monitored. Half of the rats received intravenous carbenoxolone to inhibit renal 11beta-HSD activity. The urinary recovery of [14C]inulin was complete in both groups of animals (101+/-2% versus 101+/-3%), but the recovery of 22Na was lower in carbenoxolone-treated rats (34+/-5%) than in the corticosterone-alone group (54+/-4%, P<0.01). These data, which provide the first demonstration of enhanced Na+ reabsorption in the distal nephron during inhibition of renal 11beta-HSD in vivo, strongly support the proposal that 11beta-HSD normally prevents endogenous glucocorticoid from exerting mineralocorticoid-like effects.     

Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the action of CHA. Stimulation of A1 adenosine receptors also increased the production of diacylglycerol, an activator of PKC. Exogenous PKC added to the cytoplasmic face of inside-out patches also stimulated the Cl- channel. Alkaline phosphatase reversed PKC activation. These results show that stimulation of A1 adenosine receptors activates a 305-pS Cl-channel in the apical membrane by a phosphorylation-dependent pathway involving PKC. In previous studies, we showed that the protein G alpha i-3 activated the 305-pS Cl- channel (Schwiebert et al. 1990. J. Biol. Chem. 265:7725-7728). We, therefore, tested the hypothesis that PKC activates the channel by a G protein-dependent pathway. In inside-out patches, pertussis toxin blocked PKC activation of the channel. In contrast, H7 did not prevent G protein activation of the channel. We conclude that adenosine activates a 305-pS Cl- channel in the apical membrane of RCCT-28A cells by a membrane-delimited pathway involving an A1 adenosine receptor, phospholipase C, diacylglycerol, PKC, and a G protein. Because we have shown, in previous studies, that this Cl- channel participates in the regulatory volume decrease subsequent to cell swelling, adenosine release during ischemic cell swelling may activate the Cl-channel and restore cell volume.     

Effects of atrial natriuretic peptide and vasopressin on chloride transport in long- and short-looped medullary thick ascending limbs.

Recent studies have suggested a selective effect of atrial natriuretic peptide (ANP) in regulating NaCl reabsorption in juxtamedullary nephrons. We examined (a) functional differences between medullary thick ascending limbs from long and short loops of Henle (lMAL and sMAL, respectively) and (b) the interaction of ANP and arginine vasopressin (AVP) on Cl- transport (JCl) in these two segments. AVP-, glucagon-, and calcitonin-stimulated cAMP accumulation was higher in lMAL than in sMAL. 10(-10) M AVP increased JCl in lMAL but not in sMAL. ANP-stimulated cGMP production was higher in lMAL than in sMAL. 10(-10) and 10(-8) M ANP inhibited AVP-stimulated JCl in lMAL by 26-30% (from 70.3 +/- 11.4 to 51.7 +/- 13.6 pmol/mm per min and from 88.1 +/- 10.1 to 61.8 +/- 11.7 pmol/mm per min, respectively), and this effect was mimicked by 10(-5) to 10(-4) M cGMP. This effect of ANP in lMAL could account for a large part of the ANP-induced natriuresis and diuresis in vivo, in that the rate of NaCl reabsorption in MAL is the largest among distal nephron segments, providing the chemical potential energy for the renal countercurrent multiplication system.