Role of PGE(2) in alpha(2)-induced inhibition of AVP- and cAMP-stimulated H(2)O, Na(+), and urea transport in rat IMCD.

PGE(2) inhibits osmotic water permeability (P(f)) in the rat inner medullary collecting duct (IMCD) via cellular events occurring after the stimulation of cAMP, i.e., post-cAMP-dependent events. The alpha(2)-agonists also inhibit P(f) in the rat IMCD via post-cAMP-dependent events. The purpose of this study was to determine whether PGE(2) plays a role in alpha(2)-mediated inhibition of P(f), Na(+), and urea transport in the rat IMCD. Isolated terminal IMCDs from Wistar rats were perfused to measure, in separate experiments, P(f), lumen-to-bath (22)Na(+) transport (J(lb)), and urea permeability (P(u)). Transport was stimulated with 220 pM arginine vasopressin (AVP) or 0.1 mM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP). Indomethacin was used to inhibit endogenous prostaglandin synthesis, and the alpha(2)-agonists clonidine, oxymetazoline, and dexmedetomidine were used to test the role of PGE(2) in the alpha(2)-mediated mechanism that inhibits transport. All agents were added to the bath. Indomethacin at 5 microM significantly elevated CPT-cAMP-stimulated P(f), J(lb), and P(u), and subsequent addition of 100 nM PGE(2) reduced these transport parameters. Indomethacin reversed alpha(2) inhibition of CPT-cAMP-stimulated P(f), J(lb), and P(u), and subsequent addition of PGE(2) reduced transport in each case. Indomethacin partially reversed alpha(2) inhibition of AVP-stimulated P(f), J(lb), and P(u), and PGE(2) reduced transport back to the alpha(2)-inhibited level. These results indicate that PGE(2) is a second messenger involved in the mechanism of transport inhibition mediated by alpha(2)-adrenoceptors via post-cAMP-dependent events in the rat IMCD.

Inhibition of water permeability in the rat collecting duct: effect of imidazoline and alpha-2 compounds.

Arginine vasopressin (AVP) increases water permeability in the collecting duct of the nephron via activation of adenylyl cyclase. Alpha-2 (alpha2) agonists inhibit AVP-stimulated water permeability via binding to alpha2 adrenoceptors that have been divided into 3 subtypes- alpha2A, alpha2B, and alpha2C. Some biological effects mediated by alpha2 agonists result from nonadrenergic imidazoline receptors that exist in the rat kidney. Thus, alpha2-inhibition of AVP-stimulated water permeability in the rat collecting duct could be caused by imidazoline receptors. The purpose of this study was to test agonists and antagonists selective for alpha2 and imidazoline receptors on AVP-stimulated water permeability in the rat inner medullary collecting duct (IMCD). Some experiments were conducted where water permeability was stimulated by a nonhydrolyzable analog of adenosine 3', 5'-cyclic monophosphate (cAMP). Agonists included dexmedetomidine, clonidine, oxymetazoline, agmatine and rilmenidine. The latter two are selective imidazoline agonists. Antagonists included yohimbine, RX821002, atipamezole, prazosin, WB4101, idazoxan, and BU239. Prazosin and WB4101 demonstrate selectivity for the alpha2B and alpha2C subtypes, respectively, and oxymetazoline and RX821002 are selective for the alpha2A subtype. BU239 is selective for imidazoline receptors. Wistar rat terminal IMCDs were isolated and perfused to determine the osmotic water permeability coefficient (Pf). All agonists except agmatine inhibited AVP-stimulated Pf. Inhibition by rilmenidine indicated a different mechanism of action from other agonists. Dose-response data show dexmedetomidine to be the most potent inhibitor. Oxymetazoline and clonidine inhibited cAMP-stimulated Pf indicating that the mechanism involves postcAMP cellular events. It was reported previously that dexmedetomidine inhibits cAMP-stimulated Pf (1). All antagonists except prazosin and WB4101 reversed alpha2-inhibition of AVP-stimulated Pf. BU239 was effective at 1 microM but not at 100 nM. Results suggest that alpha2A adrenoceptors modulate water permeability in the IMCD. The involvement of imidazoline receptors is inconclusive.

Indomethacin and staurosporine reverse alpha 2 inhibition of water transport in rat IMCD.

These studies were conducted to determine if the prostaglandin-synthesis inhibitor indomethacin or the protein kinase C (PKC) inhibitor staurosporine affect the inhibition of osmotic water permeability (Pf) by the alpha-2 (alpha 2) agonist dexmedetomidine in the rat inner medullary collecting duct (IMCD). Terminal IMCDs from Wistar rats were perfused and Pf was increased with either 220 pM arginine vasopressin (AVP) or 0.1 mM 8-chlorophenylthio cyclic adenosine monophosphate (8CPTcAMP). All agents were added to the bathing solution. Dexmedetomidine at 100 nM inhibited both AVP- and 8CPTcAMP-stimulated Pf. When Pf was increased by AVP, indomethacin at 0.1 mM or 5 microM reversed the dexmedetomidine-induced inhibition by 68% and 43%, respectively. When Pf was increased by 8CPTcAMP, indomethacin at 0.1 mM or 5 microM reversed inhibition by 83% and 70%, respectively. Indomethacin increased AVP and 8CPTcAMP-stimulated Pf by 20 to 30% and dexmedetomidine inhibited the AVP+ indomethacin-stimulated Pf. Staurosporine at 10 nM yielded similar results. Results suggest that PKC and prostaglandins are involved in the alpha 2 mediated mechanism, and staurosporine and indomethacin-sensitive cellular mediators modulate basal Pf.

Dexmedetomidine inhibits osmotic water permeability in the rat cortical collecting duct.

The purpose of this study was to determine whether the selective alpha-2 agonist dexmedetomidine inhibits basic transport properties in the rat cortical collecting duct (CCD). Sprague-Dawley rat CCDs were isolated and perfused to allow measurement of osmotic water permeability (Pf), transepithelial voltage (Vt) and resistance (Rt). Arginine vasopressin (AVP) increases Pf, hyperpolarizes Vt and decreases Rt in the CCD via stimulation of adenylyl cyclase. Dexmedetomidine at 100 nM added to the basolateral side of the CCD reduced AVP-stimulated Pf by 95% to 100%, and the alpha-2 antagonist atipamezole reversed the inhibition. In the presence of the protein kinase C inhibitor staurosporine, dexmedetomidine reduced AVP-stimulated Pf by 70% to 75% compared with the complete inhibition without staurosporine. When Pf was increased by the use of the non-hydrolyzable analog of cAMP, 8-chlorophenylthio-cAMP, in lieu of AVP, dexmedetomidine inhibited Pf by approximately 35%. This demonstrated alpha-2-mediated inhibition of Pf despite the presence of constant cellular cAMP levels. Dexmedetomidine reversed AVP-induced effects on Vt and Rt, indicating inhibition of Na+ transport. Results confirm an alpha-2-mediated mechanism that reduces Na+ and water transport in the CCD and suggest that a cellular messenger other than cAMP is involved. This messenger could be protein kinase C.

Alpha 2-adrenergic-mediated inhibition of water and urea permeability in the rat IMCD.

These studies were conducted to determine whether the alpha 2-agonists epinephrine and dexmedetomidine inhibit osmotic water permeability (Pf) and urea permeability (Pu) in the rat inner medullary collecting duct (IMCD). Wistar rat IMCD segments were perfused via standard methods, and Pf and Pu were determined in separate studies. The control period was followed by adding 220 pM arginine vasopressin (AVP) or 10(-4) M dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) to the bath. Epinephrine or dexmedetomidine, both at 1 microM, was then added to the bath, and this period was followed by adding 1 microM atipamezole, a selective alpha 2-antagonist. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine was present in all experiments with DBcAMP. Epinephrine inhibited AVP- and DBcAMP-stimulated Pf by 90% and 80%, respectively. Dexmedetomidine inhibited AVP- and DBcAMP-stimulated Pf by 98% and 97%, respectively. Epinephrine inhibited AVP- and DBcAMP-stimulated Pu by 70% and 60%, respectively. Dexmedetomidine failed to affect Pu. Atipamezole reversed all inhibitory effects. These data confirm an alpha 2-mediated mechanism in the IMCD that modulates Pf and Pu, and they indicate that inhibition occurs via post-cAMP cellular events.

Sodium and water transport in cortical collecting duct of Dahl salt-resistant rat.

Studies were conducted to determine whether the cortical collecting duct (CCD) of the Dahl salt-resistant rat (inbred Rapp strain; R/Jr) exhibits the same responses to deoxycorticosterone (DOC; 2.5 mg as a depot injection in vivo, 3-8 days before experimentation) and arginine vasopressin (AVP, 220 pM in vitro) as the Sprague-Dawley (SD) [L. Chen, S.K. Williams, and J.A. Schafer. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F147-F156, 1990] and Dahl salt-sensitive (inbred Rapp strain, S/Jr) [C.T. Hawk and J.A. Schafer. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F471-F478, 1991] CCD. Qualitatively, the R/Jr CCD responded as in the other two strains: AVP elevated the osmotic water permeability (Pf, micron/s) from 0 to approximately 1,200; either AVP or DOC, when used alone, increased the lumen-to-bath 22Na+ flux (Jl-->b, pmol.min-1.mm-1) from the control range of 20-25 to approximately 40 and hyperpolarized the transepithelial voltage. AVP and DOC effects were synergistic, elevating Jl-->b to 90 +/- 5 (mean +/- SE) with both hormones, but this value was significantly lower than observed previously in both the SD and the S/Jr CCD, 125 +/- 6 and 140 +/- 6, respectively. However, bath-to-lumen fluxes (Jb--l) were also significantly lower than observed in the SD and S/Jr CCD. Because net fluxes (Jnet) in these experiments can be determined only as nonpaired differences between unidirectional fluxes, it is uncertain whether Jnet values in the R/Jr CCD are significantly lower than in the SD or S/Jr CCD.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Ca2+ and PKC activation do not inhibit Na+ and water transport in rat CCD.

Experiments examined the effects of elevation of intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC) on Na+ and water transport in the rat cortical collecting duct (CCD). We measured the lumen-to-bath 22Na+ flux (J1-->b), transepithelial voltage (VT), and water permeability (Pf) in CCD from deoxycorticosterone (DOC)-treated rats. Ionomycin (0.5 and 1 microM) and thapsigargin (1 and 2 microM) were used to increase [Ca2+]i. Phorbol 12-myristate 13-acetate (PMA; 0.3 and 1 microM) and oleoyl-acetyl-glycerol (OAG; 100 microM) were used as activators of PKC. [Ca2+]i was measured in isolated perfused tubules using the fluorescent dye fura 2. When added to the bathing solution, 220 pM arginine vasopressin (AVP) failed to affect [Ca2+]i, whereas 1 microM ionomycin increased [Ca2+]i by 103 +/- 15% and 2 microM thapsigargin increased [Ca2+]i by 24 +/- 4%. In flux studies, neither ionomycin nor thapsigargin affected J1-->b or Pf, although ionomycin caused marked morphological changes. Ionomycin also failed to alter either parameter in tubules from non-DOC-treated rats. Neither 100 microM OAG nor 1 microM PMA affected J1-->b or Pf. OAG at 50 microM had no effect on VT or transepithelial resistance, indicating no inhibition of conductive Na+ transport. We conclude that increased [Ca2+]i and PKC activation do not affect J1--b or Pf in the rat CCD. These findings may account for the sustained increase in J1--b produced in the rat CCD by AVP.

Inhibition by epinephrine of AVP- and cAMP-stimulated Na+ and water transport in Dahl rat CCD.

We examined the effects of epinephrine in perfused cortical collecting ducts (CCD) isolated from inbred Dahl-Rapp salt-sensitive (SS) and salt-resistant (SR) rats and from Sprague-Dawley (SD) rats. Rats were treated with 2.5 mg deoxycorticosterone pivalate (DOC; depot injection 4-9 days before study), and the CCD were treated with 220 pM vasopressin (AVP) to maximize Na+ transport. In CCD from all three strains 10 microM epinephrine in the bathing solution completely inhibited net Na+ transport, osmotic water permeability (Pf), and transepithelial voltage. In the SS CCD, epinephrine increased the fractional resistance of the luminal membrane to the same extent as 10 microM amiloride, indicating that it blocked the amiloride-sensitive conductance of the luminal membrane. Even at 100 nM epinephrine inhibited 80-100% of Na+ and water transport, and 1 microM yohimbine reversed or prevented these effects. In SS CCD, 0.1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) plus 0.1 mM 3-isobutyl-1-methylxanthine in place of AVP increased lumen-to-bath Na+ flux (J1-->b) from 56 +/- 5 to 143 +/- 3 pmol.min-1 x mm-1 and Pf from 6 +/- 12 to 1067 +/- 152 microns/s, but 100 mM epinephrine still significantly inhibited cAMP-stimulated J1-->b and Pf by 40 +/- 5% and 31 +/- 9%, respectively. Similar results were observed in the SR and SD rat CCD; however, the ability of yohimbine to reverse the epinephrine effect on cAMP-dependent transport was variable among the rat strains. We conclude that epinephrine acts via an alpha 2-receptor to inhibit adenylate cyclase but that at least one additional intracellular second messenger system may be involved.

Phosphate transport in isolated rat inner medullary collecting duct.

Phosphate transport by the inner medullary collecting duct of normal rats was studied using an in vitro microperfusion technique. Net (Jnet), lumen-to-bath (Jlb) and bath-to-lumen (Jbl) phosphate fluxes were measured using 32PO4 as tracer, in the absence of net water absorption. A net absorption of phosphate (22.3 +/- 3.3 pmol cm-2 s-1) was observed by direct determination, and was similar to the difference between the Jlb and Jbl (57.7 +/- 8.2 and 32.2 +/- 1.5 pmol cm-2 s-1 respectively). The addition of amiloride (10 microM) to the perfusate did not change the Jlb of phosphate but blocked the efflux of sodium. Also, the withdrawal of sodium from the bath and perfusion solution did not change the Jlb of phosphate. In parallel, the addition of ouabain (10 mM) to the bath fluid decreased the Jlb of sodium more (37%) than the Jlb of phosphate (12%) and did not change the Jbl of phosphate. The addition of arsenate (10 microM) to the perfusate both in the presence and in the absence of sodium caused a decrease in Jlb, but Jbl remained unchanged, and parathyroid hormone (10 U) added to the bath did not change the Jlb. The increase in pH of the bath and perfusion fluid was associated with an increase in the Jlb of phosphate, and the decrease in pH was similarly followed by a decrease in phosphate efflux. The Jbl did not change with the pH alterations. These data demonstrate that a net phosphate absorption takes place in rat inner medullary collecting duct perfused in vitro and that this transport appears to be independent of sodium absorption and the action of parathyroid hormone. Moreover, a decrease in luminal and bath pH induces a decrease in phosphate efflux.

Effect of peritubular hypertonicity on water and urea transport of inner medullary collecting duct.

The effect of bath fluid hypertonicity on hydraulic conductivity (Lp) and [14C]urea permeability (Pu) of the distal inner medullary collecting duct (IMCD) was studied in the absence and in the presence of vasopressin (VP) using the in vitro microperfusion technique of rat IMCD. In the first three groups of IMCD, we observed that in the absence of VP the Lp was not different from zero when the osmotic gradient was created by hypotonic perfusate and isotonic bath fluid, but it was significantly greater than 1.0 x 10(-6) cm.atm-1.s-1 when the osmotic gradient was created by hypertonic bath and isotonic perfusion fluid. The increase in Lp was observed when the hypertonicity of the bath fluid was produced by the addition of NaCl or raffinose, but no such effect was observed with urea. The stimulated effect of bath fluid hypertonicity on Lp was also observed in the IMCD obtained from Brattleboro homozygous rats in which VP is absent. The NaCl hypertonic bath increased the Pu in the absence of VP. In another series of experiments with VP (10(-10) M) we observed that the hypertonic bath fluid increased in a reversible manner the VP-stimulated Lp of distal IMCD. However, the NaCl hypertonicity of the bath fluid was not able to increase dibutyryladenosine 3',5'-cyclic monophosphate-stimulated Lp. The Pu stimulated by VP (10(-10) M) increased twofold when the bath fluid was hypertonic. Therefore hypertonicity of the peritubular fluid produced by the addition of NaCl or raffinose increases the Lp and Pu in the absence and in the presence of VP. No such effect was noted with the addition of urea.

Renal involvement in leptospirosis: a pathophysiologic study.

The kidney involvement in leptospirosis appears to be a special form of acute renal failure due to a higher frequency of polyuric forms and the presence of hypokalemia with an elevated urinary fractional excretion of potassium. Using a clearance technique, we detected higher fractional urinary potassium excretion in leptospirotic guinea pigs (26.5 +/- 4.7%) than in normal animals (14.1 +/- 2.8%, p < 0.05). After blocking distal NaCl reabsorption with furosemide, it was observed that in leptospirotic animals both fractional sodium excretion (40.0 +/- 7.4%) and fractional potassium excretion (136.3 +/- 32.7%) were higher than in normal animals (20.4 +/- 3.8%, p < 0.05, and 43.6 +/- 9.0%, p < 0.05, respectively). Microperfusion studies showed that the normal and leptospirotic medullary thick ascending limb had both identical transepithelial potential difference (+3.7 +/- 0.4 vs. 3.9 +/- 0.2 mV) and relative sodium-to-chloride permeability. The same technique showed that the osmotic water permeability (Posm; 0.9 +/- 0.4 x 10(-5) cm/s.atm) and diffusional permeability (34.7 +/- 6.6 x 10(-5) cm/s) observed in the leptospirotic inner medullary collecting duct (IMCD) in the presence of vasopressin were unchanged, as was also the case for urea permeability (3.74 +/- 0.7 x 10(-5) cm/s). These data show that acute renal failure in leptospirosis is characterized by tubular changes leading to potassium secretion probably due to a decrease in proximal sodium reabsorption. Furthermore, the inability to concentrate urine evidenced by the low P(o)sm present in leptospirotic animals is due, at least in part, to IMCD resistance to vasopressin.

Renal concentrating defect in protein malnutrition: the role of the thick ascending limb of Henle and inner medullary collecting duct.

The present study was carried out to examine the effect of chronic dietary protein restriction on renal water handling in the rat. During hypotonic saline infusion, the malnourished rats showed a lower free-water clearance, corrected by inulin clearance (7.2 +/- 0.4%), than normal rats (13.6 +/- 2.5%, p less than 0.051), although the fractional distal delivery of sodium did not differ from normal. Throughout hypertonic saline diuresis the free-water reabsorption (TcCH20) corrected by inulin clearance was lower in malnourished rats (6.62 +/- 0.64%) than in control animals (9.25 +/- 0.62, p less than 0.05). Moreover, when TcH20 was referred to the osmolar clearance, malnourished animals showed lower values than normal. These results suggest a defect in NaCl transport in the thick ascending limb of Henle. In vitro measurements of diffusional water permeability (PDW) in the inner medullary collecting duct (IMCD) obtained from malnourished rats showed an increase from 40.0 +/- 5.4 x 10(5) cm/s to 71.3 +/- 5.4 x 10(5) cm/s by adding maximum effective concentration (50 microU/ml) of arginine vasopressin (VP) to the bath. These values were not different from the PDW observed in the IMCD of normal rats. In another series of microperfusion experiments, the hydraulic conductivity in IMCD of malnourished rats measured also in the presence of maximum effective concentration of VP was 29.7 +/- 3.4 x 10(-6) cm/atm/s, a mean value not significantly different from that observed in the IMCD of normal rats (35.2 +/- 4.3 x 10(-6) cm/atm/s).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chlorpropamide on water and urea transport in the inner medullary collecting duct.

The present in vitro microperfusion study examined whether chlorpropamide (CPM) has a direct effect on hydraulic conductivity (Lp x 10(-6) cm/atm.sec) and 14C-urea permeability (Pu x 10(-5) cm/sec) in the middle and distal inner medullary collecting duct (IMCD) obtained from acutely water-loaded Wistar rats and rats homozygous for diabetes insipidus (DI). CPM (10(-4) M) added to the bath fluid increased the Lp in the water-loaded Wistar rats from -0.05 +/- 0.13 to 6.25 +/- 0.74 (p less than 0.01) and in the DI rats from 0.05 +/- 0.01 to 5.95 +/- 0.84 (p less than 0.01), but had no effect when it was added to the perfusate. CPM stimulated Lp in a dose-dependent manner with the threshold effect at 10(-6) M. However, the addition of CPM (10(-4) M) to submaximal concentration of VP in the bath fluid did not increase the Lp. Furthermore, CPM was unable to block the inhibitory action of PGE2 on the vasopressin (VP)-stimulated Lp. On the contrary, PGE2 blocked the CPM-stimulated Lp. CPM (10(-4) M) in the peritubular fluid was able to cause a significant rise of the Pu from 13.5 +/- 0.8 to 17.3 +/- 1.0 reversibly, which represented 16% of maximum stimulated effect produced by 50 microU/ml of VP. Thus, pharmacological doses of CPM added to the peritubular side have a direct effect on terminal IMCD increasing water and urea permeability in the absence of VP, but this drug does not potentiate the VP-stimulated water transport in the IMCD. Our results were unable to confirm the hypothesis that CPM potentiates the VP-antidiuresis by the inhibition of PGE2 action in the rat IMCD.

Effect of atrial natriuretic factor and cyclic guanosine monophosphate on water and urea transport in the inner medullary collecting duct.

We examined the action of high (2 x 10(-8)M) and low (6 x 10(-9)M) concentrations of atrial natriuretic factor (ANF) on water and urea transport in the rat inner medullary collecting duct (IMCD) using the in vitro microperfusion technique. We measured the hydraulic conductivity (Lp x 10(-6) cm/atm per second) and both lumen-to-bath (Pu(lb] and bath-to-lumen (Pu(bl)) 14C-urea permeabilities (Pu x 10(-5) cm/s) in the absence and in the presence of vasopressin (VP). High concentrations of ANF were able to inhibit the maximum activity of (50 microU/ml) VP-stimulated Lp but physiological concentration of ANF inhibit only submaximum activity (10 microU/ml) of VP-stimulated Lp. The hydrosomotic effect of dibutyryl-cyclic 3.5 adenosine monophosphate (cAMP) (10(-4)M) was unchanged by high concentrations of ANF (2 x 10(-8)M). Also we found that high (10(-4)M) and low (10(-6)M) concentrations of exogenous cyclic 3,5-guanosine monophosphate (GMP) while unable to change the Lp in the absence of VP, decreased the maximum activity of VP-stimulated Lp significantly. We also found that ANF inhibits partially and in a reversible manner the VP-stimulated Pu(lg) but not the VP-stimulated Pu(bl). These results demonstrated that plasma concentrations of ANF observed during volume expansion (10(-10)M) are able to inhibit submaximum activity of VP-stimulated (10 microU/ml) Lp in the rat IMCD, this effect seems to occur before cAMP formation and it appears to be mediated by cGMP. ANF (6 x 10(-9)M) also reduced the VP-stimulated urea outflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Atrial peptide and cGMP effects on NaCl transport in inner medullary collecting duct.

We examined the action of atrial natriuretic factor (ANF) on Na+ and Cl- transport in in vitro microperfused inner medullary collecting ducts (IMCD) isolated from rat kidneys. First we studied the isotopic fluxes at low perfusion rates (7 nl/min). The results showed that ANF added to bath decreased lumen-to-bath flux (Jl----b) of Na+ and increased Na+ bath-to-lumen flux (Jb----l). This was substantiated by a direct demonstration that ANF reduces net Na+ and Cl- absorption. The effect of ANF on Jl----b and Jb----l of Na+ was also observed at high perfusion rates (25 nl/min). The inhibitory effect of ANF was observed even when Na+ Jl----b was stimulated by vasopressin (VP). ANF (6 x 10(-11) M) added to bath increased Cl- Jb----l and generated a negative lumen potential difference (PD). These two effects were inhibited by furosemide and by the replacement of Na+ by choline and Cl- by SO4(2-) in the bath fluid. These observations are compatible with the existence of a Na(+)-Cl(-)-K+ cotransport mechanism stimulated by ANF. Moreover, the effects of guanosine 3',5'-cyclic monophosphate (cGMP) added to the bath on PD, Jl----b, and Jb----l of Na+ were similar to those observed with ANF. Thus, physiological concentrations of ANF inhibit directly Na+ and Cl- absorption in IMCD by two mechanisms, 1) by increasing cotransport Na(+)-Cl(-)-K+ secretion and 2) by inhibiting NaCl absorption both in the absence and in the presence of VP. These effects on NaCl transport appear to be mediated by cGMP.

Effect of furosemide on water and urea transport in cortical and inner medullary collecting duct.

The present in vitro microperfusion study examined whether furosemide has an effect on hydraulic conductivity (Lp X 10(-6) cm/sec.atm) and 14C-urea permeability (Pu X 10(-5) cm/sec) in inner medullary collecting ducts (IMCD) and cortical collecting tubules (CCT) isolated from rat and rabbit kidneys. Furosemide added to the bath fluid decreased arginine-vasopressin (AVP)-stimulated Lp of rat IMCD in a dose-dependent manner, with the threshold effect at 10(-6) M. Furosemide (10(-4) M) reduced Lp from 20.5 +/- 2.3 to 12.1 +/- 1.2 (P less than 0.01) reversibility, but had no effect when added to the perfusate. In addition, furosemide reduced dibutyryl cyclic AMP-stimulated Lp from 20.3 +/- 1.1 to 11.2 +/- 1.6 (P less than 0.01). This effect of furosemide was also observed with indomethacin, a PGE2 synthesis inhibitor. The addition of indomethacin (10(-4) M) to AVP (50 microU/ml) increased Lp from 24.7 +/- 2.3 to 29.7 +/- 2.8 (P less than 0.001), which was reduced to 20.3 +/- 2.6 (P less than 0.001) when furosemide was added to indomethacin in the bath. The inhibitory effect of furosemide on AVP-stimulated Lp was also observed in rabbit IMCD (Lp decreased from 12.8 +/- 0.8 to 5.15 +/- 1.46, P less than 0.02), but it was not observed in the CCT isolated from rabbit kidneys (7.96 +/- 1.87 with AVP vs. 7.94 +/- 1.41 with AVP + furosemide). Furthermore, in rat IMCD the stimulatory effect of AVP on Pu from 7.7 +/- 0.4 to 26.8 +/- 1.3 was reduced by furosemide to 19.7 +/- 1.2 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of vasopressin on sodium transport across inner medullary collecting duct.

We have used rat inner medullary collecting ducts (IMCD) perfused "in vitro" to study the effect of vasopressin (VP) on the unidirectional Na+ flux (in nmol.cm-2.s-1). We found that, at a high perfusion rate in the basal state, 24Na lumen-to-bath flux (Jl----b) was greater than the bath-to-lumen flux (Jb----l) (4.88 +/- 0.15 vs. 2.57 +/- 0.21), resulting in a significant net flux (Jnet) (P less than 0.001). Addition of 10 microU/ml of lysine vasopressin (LVP) to the bath produced a stable increase in Jl----b to 6.66 +/- 0.35 (P less than 0.001) without significant effect on Jb----l. Measuring directly the net flux absorption at lower perfusion rate (8 nl/min), we observed that LVP (10 microU/ml) produced a reversible stimulation on Jnet from 1.39 +/- 0.14 to 2.79 +/- 0.23 (P less than 0.01). The transtubular potential difference (PD) measured in the middle and final third of IMCD showed a small but significant PD (0.30 +/- 0.02 mV lumen positive) that increased significantly to 0.60 +/- 0.04 mV in the presence of LVP. However, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 10(-4) M) added to the bath fluid did not change the JNa+l----b, nor was 1-desamino-8-D-arginine vasopressin (50 microU/ml), a specific V2-agonist, able to increase the Na+. We also demonstrated that JNa+l----b stimulated by LVP from 4.70 +/- 0.08 to 6.33 +/- 0.26 (P less than 0.01) was completely and reversibly inhibited by V1-antagonist, d(CH2)Tyr(Me)AVP, to 4.79 +/- 0.05. On the other hand, the absence of Ca2+ in the bath or the addition of amiloride to the lumen fluid or ouabain to the bath fluid completely inhibited AVP-stimulated JNa+l----b. Therefore, AVP and LVP increase Na+ absorption in the rat IMCD by increasing the Na+ outflux, probably generated by an increase of luminal membrane Na+ permeability modulated by extracellular Ca2+ and mediated through V1-receptors and independent of cAMP cascade.

Renal concentration defect induced by cisplatin. The role of thick ascending limb and papillary collecting duct.

The effects of cisplatin (5 mg/kg BW given intraperitoneally) on renal concentration mechanism were evaluated initially by clearance studies in rats 5-7 days after cisplatin administration and compared to normal rats. During hypotonic saline infusion, cisplatin rats showed a lower inulin clearance (0.56 +/- 0.07 vs. 1.12 +/- 0.09 ml/min/100 g BW, p less than 0.01), a higher fractional distal delivery (CNa + CH2O/Cin) (36.3 +/- 4.4 vs. 22.8 +/- 4.5%, p less than 0.05), and lower CH2O/CNa + CH2O (33.6 +/- 5.8 vs. 56.5 +/- 5.0%, p less than 0.01). During hypertonic saline infusion the TcH2O/Cosm was lower in cisplatin (18.3 +/- 1.1%) than in normal rats (33.4 +/- 3.5%, p less than 0.01). These results suggest a defect in NaCl transport in the thick ascending limb of Henle and proximal tubule. In order to characterize these tubular defects, we measured Na-K-ATPase activity (microM Pi/mg protein/h). In the renal cortex of cisplatin rats the ATPase activity was lower (18.1 +/- 3.2) than in normal rats (33.4 +/- 6.4, p less than 0.05), also in the inner strip of the outer medulla of cisplatin rats Na-K-ATPase was reduced (26.0 +/- 5.7) when compared with normal rats (67.3 +/- 9.2, p less than 0.01), presumably representing a decrease in enzyme activity in the thick ascending limb.(ABSTRACT TRUNCATED AT 250 WORDS)