Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb.

In vitro microperfusion experiments were performed to examine the effects of peptide hormones on bicarbonate and ammonium transport by the medullary thick ascending limb (MTAL) of the rat. Arginine vasopressin (AVP; 2.8 X 10(-10) M in the bath) reduced bicarbonate absorption by 50% (from 7.8 to 3.7 pmol/min per mm). AVP caused a similar reduction in bicarbonate absorption in tubules perfused with 10(-4) M furosemide to inhibit net NaCl absorption. Glucagon (2 X 10(-9) M in the bath) also reduced bicarbonate absorption (from 11.7 to 7.6 pmol/min per mm). The inhibition of bicarbonate absorption could be reproduced with either exogenous 8-bromo-cAMP or forskolin. With 8-bromo-cAMP (10(-3) M) in the bath, addition of vasopressin to the bath did not significantly affect bicarbonate absorption. PTH significantly inhibited bicarbonate absorption, but the extent of inhibition was less than that observed with either AVP or glucagon. Vasopressin had no effect on net ammonium absorption in MTAL perfused and bathed with 4 mM NH4Cl. These findings indicate that: (a) vasopressin, glucagon, and PTH directly inhibit bicarbonate absorption in the MTAL of the rat; (b) this inhibition occurs independent of effects on net NaCl absorption and appears to be mediated in part by cAMP; and (c) HCO3- and NH4+ absorption can be regulated independently in the MTAL.     

Hyposmolality stimulates apical membrane Na(+)/H(+) exchange and HCO(3)(-) absorption in renal thick ascending limb

The regulation of epithelial Na(+)/H(+) exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO(3)(-)) absorption is mediated by apical membrane Na(+)/H(+) exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO(3)(-) solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO(3)(-) absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO(3)(-) absorption. Hyposmolality increased apical Na(+)/H(+) exchange activity over the pH(i) range 6.5-7.5 due to an increase in V(max). Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO(3)(-) absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO(3)(-) absorption in the MTAL through a novel stimulation of apical membrane Na(+)/H(+) exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na(+)/H(+) exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.     

An independent effect of osmolality on urea transport in rat terminal inner medullary collecting ducts.

We have shown that urea transport across the terminal inner medullary collecting duct (terminal IMCD) is mediated by a vasopressin-stimulated, facilitated diffusion process exhibiting properties consistent with a transporter. To investigate whether hypertonic NaCl, as exists in vivo in the inner medulla, affects urea permeability, we studied isolated perfused rat terminal IMCD segments. Perfusate and bath osmolality were varied symmetrically by adding or removing NaCl or mannitol. Urea permeability rose progressively when osmolality was increased with NaCl or mannitol from 290 to 690 mOsm/kg H2O in the absence of vasopressin; there was no further increase at 890 mOsm/kg H2O. In the presence of 10(-8) M arginine vasopressin, urea permeability increased when NaCl was added to raise osmolality from 290 to 490 mOsm/kg H2O but there was no further increase at 690 mOsm/kg H2O. When 1 mM 8-bromo cyclic AMP was added to the bath, raising NaCl still increased urea permeability. These results suggest that urea transport across the rat terminal IMCD is regulated both by vasopressin and by osmolality at values present in the renal inner medulla. Osmolality seems to activate urea transport across the rat terminal IMCD by mechanisms distinct from those of vasopressin or cyclic AMP.     

Effects of osmolality and oxygen availability on soluble cyclic AMP-dependent protein kinase activity of rat renal inner medulla.

The renal inner medulla is ordinarily exposed to osmolalities that are much higher and to O2 tensions that are lower than those in other tissues. The effects of media osmolality and O2 availability on basal and arginine vasopressin(AVP)-responsive soluble cyclic (c)AMP-dependent protein kinase activity were examined in slices of rat inner medulla. Increasing total media osmolality from 305 to 750 or 1,650 mosM by addition of urea plas NaCl to standard Krebs-Ringer bicarbonate buffer significantly reduced basal cAMP content and protein kinase activity ratios. This occurred in the presence or absence of O2. Incubation of slices in high osmolality buffer also blunted increases in inner medullary slice cAMP and protein kinase activity ratios induced by O2. These changes reflected predominantly an action of the urea rather than the NaCl content of high osmolality buffers. In contrast to effects on basal activity, high media osmolality significantly enhanced activation of inner medullary protein kinase by AVP. Conversely, increases in media O2 content suppressed AVP stimulation of enzyme activity. This inhibitory effect of O2 was best expressed at low osmolality. Naproxen and ibuprofen, inhibitors of prostaglandin biosynthesis, reduced basal kinase activity ratios and increased AVP responsiveness in the presence, but not in the absence, of O2. Exogenous prostaglandins (PG) modestly increased (PGE2 and PGE1) or did not change (PGF2alpha) cAMP and protein kinase activity ratios in O2-deprived inner medullary slices. Protein kinase activation by PGE2 was not observed in oxygenated inner medulla with high basal activity ratios. The stimulatory effects of PGE2 and PGE1 on protein kinase activity observed in O2-deprived slices were additive with those of submaximal or maximal AVP. PGE2, PGE1, and PGF2alpha all failed to suppress AVP activation of protein kinase. Thus, enhanced endogenous PGE production may contribute to the higher basal protein kinase activity ratios induced by O2. However, the results do not support a role for PGE2, PGE1, or PGF2alpha in O2-mediated inhibition of AVP responsiveness. The present data indicate that both solute content and O2 availability can alter the expression of AVP action on cAMP-dependent protein kinase activity in inner medulla. AVP activation of protein kinase is best expressed when osmolality is high and O2 availability is low, conditions that pertain in inner medulla during hydropenia.     

Anion dependence of rabbit medullary collecting duct acidification.

Rabbit medullary collecting duct (MCD) acidification has been demonstrated to occur by means of a sodium-independent, aldosterone-stimulated mechanism. We have examined the anionic dependence of this process by means of the isolated perfused tubule technique. Total replacement of perfusate chloride with gluconate enhanced tubular bicarbonate reabsorption (JHCO3), from a basal rate of 10.7 +/- 1.0 pmol X mm-1 X min-1 to a rate of 15.01 +/- 1.0 pmol X mm-1 X min-1. Removal of bath chloride, with and without removal of perfusate chloride completely abolished acidification. Bath, but not luminal 4-acetamido-4' isothiocyano-2,2'-disulfonic stilbene provoked a marked decrease in JHCO3 from 10.1 +/- 1.2 pmol X mm-1 X min-1 to 2.3 +/- 0.3 pmol X mm-1 X min-1. Measurement of chloride reabsorptive rate (JCl) revealed colinearity between JHCO3 (9.18 +/- 0.9 pmol X mm-1 X min-1) and JCl (9.75 +/- 1.18 pmol X mm-1 X min-1). We propose a model of mammalian distal nephron acidification in which (a) cellular base exit is effected by means of a basolateral membrane Cl-base exchanger and (b) net electroneutrality of electrogenic proton secretion is maintained by the parallel movement of an anionic species, functionally chloride.     

Electroneutral K+/HCO3- cotransport in cells of medullary thick ascending limb of rat kidney.

The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H+ secretion and basolateral HCO3- transport into the peritubular space. To characterize HCO3- transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3-/CO2-containing solutions and then abruptly diluted into HCO3-/CO2-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3-, was inhibited by 10(-4) M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl- or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl- removal from or addition of 2 mM barium into the external medium did not affect HCO3(-)-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3- transport was electroneutral. The HCO3(-)-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion. Finally, as measured by use of a K(+)-selective extracellular electrode, a component of K+ efflux out of the cells was HCO3- dependent and DIDS sensitive. The results provide evidence for an electroneutral K+/HCO3- cotransport in rat MTAL cells.     

Consequences of potassium recycling in the renal medulla. Effects of ion transport by the medullary thick ascending limb of Henle''s loop.

The consequences of K recycling and accumulation in the renal medulla were examined by measuring the effect of elevated K concentration on ion transport by the medullary thick ascending limb of Henle's loop. Perfused and bathed in vitro, thick limbs from both mouse and rabbit displayed a graded, reversible reduction of transepithelial voltage after increasing K concentration from 5 to 10, 15, or 25 mM. The effect was reproducible whether osmolality was 328 or 445 mosmol/kg H2O, and whether K replaced Na or choline. Net chloride absorption and transepithelial voltage were reduced by almost 90% when ambient K concentration was 25 mM. When either lumen or bath K was increased to 25 mM, net Na absorption was reduced. There was spontaneous net K absorption when perfusate and bath K concentration was 5 mM. Analysis of transepithelial K transfer after imposition of chemical gradients demonstrated rectification in the absorptive direction. Absorption of K by this segment provides a means to maintain high medullary interstitial concentration. Accumulation of K in the outer medulla, by reducing NaCl absorption, would increase volume flow through the loop of Henle and increase Na and water delivery to the distal nephron. K recycling thus might provide optimum conditions for K secretion by the distal nephron.     

Bicarbonate secretion and chloride absorption by rabbit cortical collecting ducts. Role of chloride/bicarbonate exchange.

Cortical collecting ducts (CCD) from rabbits treated with deoxycorticosterone (DOC) actively secrete bicarbonate at high rates. To investigate the mechanism of bicarbonate secretion, we measured bicarbonate and chloride transport in CCD from rabbits treated with DOC for 9-24 d. Removal of chloride (replaced with gluconate) from both perfusate and bath inhibited bicarbonate secretion without changing transepithelial voltage. Removal of chloride only from the bath increased bicarbonate secretion, while removal of chloride only from the perfusate inhibited secretion. In contrast to the effect of removing chloride, removal of sodium from both the perfusate and bath (replacement with N-methyl-D-glucamine) did not change the rate of bicarbonate secretion. The rate of bicarbonate secretion equaled the rate of chloride absorption in tubules bathed with 0.1 mM ouabain to inhibit any cation-dependent chloride transport. Under these conditions, chloride absorption occurred against an electrochemical gradient. Removal of bicarbonate from both the perfusate and bath inhibited chloride absorption. Removal of bicarbonate only from the bath inhibited chloride absorption, while removal of bicarbonate from the lumen stimulated chloride absorption. We conclude that CCD from DOC-treated rabbits actively secrete bicarbonate and actively absorb chloride by an electroneutral mechanism involving 1:1 chloride/bicarbonate exchange. The process is independent of sodium.     

Bicarbonate transport along the loop of Henle. I. Microperfusion studies of load and inhibitor sensitivity.

We microperfused the loop of Henle (LOH) to assess its contribution to urine acidification in vivo. Under control conditions (Na HCO3- = 13 mM, perfusion rate approximately 17 nl/min-1) net bicarbonate transport (JHCO3-) was unsaturated, flow- and concentration-dependent, and increased linearly until a bicarbonate load of 1,400 pmol.min-1 was reached. Methazolamide (2 x 10(-4) M) reduced JHCO3 by 70%; the amiloride analogue ethylisopropylamiloride (EIPA) (2 x 10(-4) M) reduced JHCO3 by 40%; neither methazolamide nor EIPA affected net water flux (Jv). The H(+)-ATPase inhibitor bafilomycin A1 (10(-5) M) reduced JHCO3 by 20%; the Cl- channel inhibitor 5-nitro-2'-(3-phenylpropylamino)-benzoate (2 x 10(-4) M) and the Cl(-)-base exchange inhibitor diisothiocyanato-2,2'-stilbenedisulfonate (5 x 10(-5) M), had no effect on fractional bicarbonate reabsorption. Bumetanide (10(-6) M) stimulated bicarbonate transport (net and fractional JHCO3-) by 20%, whereas furosemide (10(-4) M) had no effect on bicarbonate reabsorption; both diuretics reduced Jv. In summary: (a) the LOH contributes significantly to urine acidification. It normally reabsorbs an amount equivalent to 15% of filtered bicarbonate; (b) bicarbonate reabsorption is not saturated; (c) Na(+)-H+ exchange and an ATP-dependent proton pump are largely responsible for the bulk of LOH bicarbonate transport.     

Effects of potassium on ammonia transport by medullary thick ascending limb of the rat

Renal ammonium excretion is increased by potassium depletion and reduced by potassium loading. To determine whether changes in potassium concentration would alter ammonia transport in the medullary thick ascending limb (MAL), tubules from rats were perfused in vitro and effects of changes in K concentration within the physiological range (4-24 mM) were evaluated. Increasing K concentration from 4 to 24 mM in perfusate and bath inhibited total ammonia absorption by 50% and reduced the steady-state transepithelial NH+4 concentration gradient. The inhibition of total ammonia absorption was reversible and occurred when K replaced either Na or N-methyl-D-glucamine. Increasing K concentration in the luminal perfusate alone gave similar inhibition of total ammonia absorption. At 1-2 nl/min per mm perfusion rate, increasing K concentration in perfusion and bathing solutions had no significant effect on transepithelial voltage. With either 4 or 24 mM K in perfusate and bath, an increase in luminal perfusion rate markedly increased total ammonia absorption. Thus, both potassium concentration and luminal flow rate are important factors capable of regulating total ammonia transport by the MAL. Changes in systemic potassium balance may influence renal ammonium excretion by affecting NH+4 absorption in the MAL and altering the transfer of ammonia from loops of Henle to medullary collecting ducts.     

Effects of vasopressin and bradykinin on anion transport by the rat cortical collecting duct. Evidence for an electroneutral sodium chloride transport pathway.

Our previous studies in cortical collecting ducts isolated from rat kidneys have shown that vasopressin increases both sodium absorption and potassium secretion, while bradykinin inhibits sodium absorption without affecting potassium transport. To determine which anions are affected by these agents, we perfused cortical collecting ducts from rats treated with deoxycorticosterone and measured net chloride flux, net bicarbonate flux (measured as total CO2), transepithelial voltage, and the rate of fluid absorption. Arginine vasopressin (10(-10) M in the peritubular bath) caused a sustained sixfold increase in net chloride absorption and a two- to threefold increase in the magnitude of the lumen negative transepithelial voltage. Before addition of vasopressin, the tubules secreted bicarbonate. Vasopressin abolished the bicarbonate secretion, resulting in net bicarbonate absorption (presumably due to proton secretion) in many tubules. Bradykinin (10(-9) M added to the peritubular bath) caused a reversible 40% inhibition of net chloride absorption, but did not affect the transepithelial voltage or the bicarbonate flux. We concluded: (a) that arginine vasopressin stimulates absorption of chloride and inhibits bicarbonate secretion (or stimulates proton secretion) in the rat cortical collecting duct; and (b) that bradykinin inhibits net chloride absorption in the rat cortical collecting duct without affecting transepithelial voltage or bicarbonate flux. Combining these results with the previous observations on cation fluxes described above, we conclude that bradykinin inhibits electroneutral NaCl absorption (or stimulates electroneutral NaCl secretion) in the rat cortical collecting duct.     

Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle''s loop.

Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.     

Ammonia production by isolated mouse proximal tubules perfused in vitro. Effect of metabolic acidosis.

We examined the effects of metabolic acidosis in vivo and reduced bath and luminal pH in vitro on total NH3 (NH3 + NH+4) production rates by isolated mouse proximal tubule segments. Midproximal tubule segments were obtained from mice with NH4Cl-induced metabolic acidosis and from nonacidotic controls. The segments were perfused with modified Krebs-Ringer bicarbonate (KRB) buffer, incubated in KRB buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, and gassed with 95% O2 and 5% CO2. Isolated unperfused and perfused proximal tubules from acidotic mice produced total NH3 at higher rates than corresponding tubules from nonacidotic mice. Perfusion of the tubular lumen stimulated total NH3 production by tubules from both acidotic and nonacidotic mice. In contrast, lowering the bath pH to 7.0 by lowering the HCO3- concentration increased total NH3 production rates by tubules from nonacidotic mice but not by tubules from acidotic mice. Reducing the HCO3- concentration of the bath buffer to 10 mM while maintaining a pH of 7.4 had no significant effect on total NH3 production by tubules from nonacidotic mice. Lowering the luminal fluid pH by reducing the perfusate HCO-3 from 25 mM to 10, 5, or 1.2 mM while maintaining a bath pH of 7.4 lowered collected luminal fluid pH but had no effect on total NH3 production by proximal tubules from nonacidotic mice. These observations demonstrated that metabolic acidosis in vivo stimulated total NH3 production in isolated mouse proximal tubule segments and that low peritubular pH and HCO-3 stimulated total NH3 production by proximal tubule segments from nonacidotic mice in vitro.     

Effects of osmolality on phosphoinositide hydrolysis in renal medulla

We have reported previously that carbachol stimulates hydrolysis of phosphoinositides (PIs) in the renomedullary slices when incubated in a buffer of 300 mOsm/kg of H2O. However, the mammalian renal medulla has a hypertonic environment that changes with the state of hydration of the animal. In order to determine if the change in renal osmolality produces a change in the response of the inner medulla to hormones and neurotransmitters, we determined the effects of osmolality on carbachol-stimulated hydrolysis of PIs in the inner medullary slices of the rabbit kidney. The hydrolysis was determined by incorporation of [3H]inositol into PIs and the release of [3H]inositol phosphates in the presence and absence of 1 mM carbachol. The osmolality of the incubation media was increased from 300 to 1200 mOsm/kg of H2O in increments of 300 mOsm/kg of H2O by addition of either urea, NaCl, mannitol or an equiosmolar mixture of urea and NaCl. Increasing the osmolality of the incubation media by any one of these solutes decreased carbachol-stimulated release of inositol phosphates in the inner medullary slices of the rabbit kidney. Our results suggest that the effect of carbachol on PI messenger system in the renal medulla in vivo will depend on the tissue osmolality that itself depends on the state of hydration of the animal.     

Bicarbonate absorption stimulates active calcium absorption in the rat proximal tubule.

To evaluate the effect of luminal bicarbonate on calcium reabsorption, rat proximal tubules were perfused in vivo. Perfusion solution contained mannitol to reduce water flux to zero. Total Ca concentration was measured by atomic absorption spectrometry, Ca ion concentration in the tubule lumen (CaL2+) and the peritubular capillary (CaP2+), and luminal pH (pHL) with ion-selective microelectrodes and transepithelial voltage (VTE) with conventional microelectrodes. When tubules were perfused with buffer-free Cl-containing solution, net Ca absorption (JCa) averaged 3.33 pmol/min. Even though VTE was 1.64 mV lumen-positive, CaL2+, 1.05 mM, did not fall below the concentration in the capillary blood, 1.07 mM. When 27 mM of Cl was replaced with HCO3, there was luminal fluid acidification. Despite a decrease in VTE and CaL2+, JCa increased to 7.13 pmol/min, indicating that the enhanced JCa could not be accounted for by the reduced electrochemical gradient, delta CCa. When acetazolamide or an analogue of amiloride was added to the HCO3 solution, JCa was not different from the buffer-free solution, suggesting that HCO3-stimulated JCa may be linked to acidification. To further test this hypothesis, we used 27 mM Hepes as the luminal buffer. With Hepes there was luminal fluid acidification and JCa was not different from the buffer-free solution but delta CCa was significantly reduced, indicating enhanced active calcium transport. We conclude from the results of the present study that HCO3 stimulates active Ca absorption, a process that may be linked to acidification-mediated HCO3 absorption.     

Vasopressin stimulates Cl- transport in ascending thin limb of Henle's loop in hamster.

The effect of arginine vasopressin (AVP) on NaCl transport was investigated in the isolated microperfused hamster ascending thin limb of Henle's loop by measuring transepithelial voltage (Vt) and transmural 22Na+ and 36Cl- fluxes. In the presence of a transmural NaCl concentration gradient (100 mM higher in the lumen), Vt was 8.4 +/- 0.4 mV. Addition of 1 nM AVP to the basolateral solution increased Vt to 9.6 +/- 0.4 mV, which corresponds to an increase in the Cl- to Na+ permselectivity ratio (PCl/PNa) from 2.8 +/- 0.2 to 3.4 +/- 0.2. AVP at physiological concentrations increased Vt in a dose-dependent manner with an ED50 of 5 pM. AVP increased the Cl- efflux coefficient from 99.6 +/- 6.3 to 131.4 +/- 10.6 x 10(-7) cm2/s without affecting the Na+ efflux coefficient. 5-Nitro-2-(3-phenyl-propylamino)-benzoate (0.2 mM), a Cl- channel inhibitor, in the perfusate decreased the basal Cl- efflux coefficient and inhibited the AVP-induced increase in this parameter. The AVP-induced increase in Vt was not affected by [d(CH2)5(1),O-Me-Tyr2,Arg8] vasopressin, a V1 receptor antagonist, but was abolished by [d(CH2)5,D-Ile2,Ile4,Arg8] vasopressin, a V2 receptor antagonist. The selective V2 agonist dDAVP in 1 nM also increased Vt from 8.6 +/- 0.7 to 9.5 +/- 0.6 mV. Dibutyryl cAMP and forskolin both increased Vt, whereas H89, an inhibitor of cAMP-dependent protein kinase, abolished the AVP-induced increase in Vt. These results demonstrate that AVP stimulates Cl- transport in the ascending thin limb of Henle's loop by activating Cl- channels via a signal transduction cascade comprising V2 receptors, adenylate cyclase, and cAMP-dependent protein kinase. The ascending thin limb of Henle's loop thus participates in the formation of concentrated urine as one of the target renal tubular segments of AVP.     

Sodium-dependent net urea transport in rat initial inner medullary collecting ducts.

We reported that feeding rats 8% protein for 3 wk induces net urea transport and morphologic changes in initial inner medullary collecting ducts (IMCDs) which are not present in rats fed 18% protein. In this study, we measured net urea transport in microperfused initial IMCDs from rats fed 8% protein for > or = 3 wk and tested the effect of inhibiting Na+/K(+)-ATPase activity and found that adding 1 mM ouabain to the bath reversibly inhibited net urea transport from 14 +/- 3 to 6 +/- 2 pmol/mm per min (P < 0.01), and that replacing potassium (with sodium) in the bath reversibly inhibited net urea transport from 18 +/- 3 to 5 +/- 0 pmol/mm per min (P < 0.01). Replacing perfusate sodium with N-methyl-D-glucamine reversibly inhibited net urea transport from 12 +/- 2 to 0 +/- 1 pmol/mm per min (P < 0.01), whereas replacing bath sodium had no significant effect on net urea transport. Adding 10 nM vasopressin to the bath exerted no significant effect on net urea transport. Finally, we measured Na+/K(+)-ATPase activity in initial and terminal IMCDs from rats fed 18% or 8% protein and found no significant difference in either subsegment. Thus, net urea transport in initial IMCDs from rats fed 8% protein for > or = 3 wk requires sodium in the lumen, is reduced by inhibiting Na+/K(+)-ATPase, and is unchanged by vasopressin or phloretin. These results suggest that net urea transport may occur via a novel, secondary active, sodium-urea cotransporter.     

Mechanism of angiotensin II action on proximal tubular transport

Our previous studies have shown that a low dose of angiotensin II (Ang II) stimulates fluid and bicarbonate absorption from the apical side of rat proximal tubule (PCT). These effects can be blocked by Ang II antagonist, (Sar1, Ile8)Ang II. The cellular mechanism underlying Ang II action, however, remained unclear. Thus, this study was designed to investigate the possible role of phosphoinositide turnover in mediating this action of angiotensin. Rat PCT was perfused in vivo with Ringer's solution containing [3H]inulin as a volume marker. Bicarbonate flux (JHCO3) was determined by total CO2 changes between the collected fluid and the original perfusate as analyzed by microcalorimetry. Luminal perfusion of 10(-11) M Ang II stimulated both JHCO3 and fluid reabsorption. These effects could be blocked by 10(-3) M amiloride and 10(-5) M ethylisopropylamiloride. Luminal perfusion of 4 beta-phorbol-12-myristate-13-acetate (10(-8) M) also stimulated both fluid reabsorption and JHCO3. These effects could also be blocked by amiloride and ethylisopropylamiloride. When both Ang II (10(-11) M) and 4 beta-phorbol-12-myristate-13-acetate (10(-8) M) were perfused together, the stimulatory effects on fluid reabsorption and JHCO3 were not additive. These results suggest that Ang II interacts with its receptors on the apical membrane leading to modulation of the Na-H exchange mechanism in PCT. The phosphoinositide turnover may play an important role in the Ang II action on PCT transport.     

Modulation of phosphate absorption by calcium in the rabbit proximal convoluted tubule.

Proximal convoluted (S2) and straight (S3) renal tubule segments were studied to determine the effect of Ca on lumen-to-bath phosphate flux (JlbPO4). Increasing bath and perfusate Ca from 1.8 to 3.6 mM enhanced JlbPO4 from 3.3 +/- 0.7 to 6.6 +/- 0.6 pmol/mm per min in S2 segments (P less than 0.001) but had no effect in S3 segments. Decreasing bath and perfusate Ca from 1.8 to 0.2 mM reduced JlbPO4 from 3.7 +/- 0.6 to 2.2 +/- 0.6 in S2 segments. These effects were unrelated to changes in fluid absorption and transepithelial potential difference. Increasing cytosolic Ca with a Ca ionophore, inhibiting the Ca-calmodulin complex with trifluoperazine, or applying the Ca channel blocker nifedipine had no effect on JlBPO4 in S2 segments. Increasing only bath Ca from 1.8 to 3.6 mM did not significantly affect JlbPO4. However, increasing only perfusate Ca enhanced JlbPO4 from 3.4 +/- 0.7 to 6.1 +/- 0.7 pmol/mm per min (P less than 0.005). Inhibition of hydrogen ion secretion, by using a low bicarbonate, low pH perfusate, both depressed base-line JlbPO4 and abolished the stimulatory effect of raising perfusate Ca. Net phosphate efflux (JnetPO4) also increased after ambient calcium levels were raised, ruling out a significant increase in PO4 backflux. When net sodium transport was abolished by reducing the bath temperature to 24 degrees C, JnetPO4 at normal ambient calcium was reduced and increasing ambient calcium failed to increase it, ruling out a simple physicochemical reaction wherein phosphate precipitates out of solution with calcium. The present studies provide direct evidence for a stimulatory effect of Ca on sodium-dependent PO4 absorption in the proximal convoluted tubule, exerted at the luminal membrane. It is postulated that Ca modulates the affinity of the PO4 transporter for the anion.