Urea Permeability Research Articles

Cloning and functional characterization of a rat urea transporter: expression in the brain

A cDNA coding for a rat urea transporter is described. The 1242 bp open reading frame codes for a 414 amino acids protein with 77.0% and 60.7% identity with the human UT11 and the rat UT2, respectively. When expressed in Xenopus oocytes, the protein induces a 10-fold rise in urea permeability, inhibited by mercurial reagents and phloretin. The rUT11 mRNA is massively expressed in the brain.

Permeability and reflection coefficients of urea and small amides in the human red cell.

Measurement of the transport parameters that govern the passage of urea and amides across the red cell membrane leads to important questions about transport of water. It had initially been thought that small protein channels, permeable to water and small solutes, traversed the membrane (see Solomon, 1987). Recently, however, very strong evidence has been presented that the 28 kDa protein, CHIP28, found in the red cell membrane, is the locus of the water channel (see Agre et al., 1993). CHIP28 transports water very rapidly but does not transport small nonelectrolytes such as urea. The irreversible thermodynamic parameter, sigma i, the reflection coefficient, is a measure of the relationship between the permeability of the solute and that of water. If a solute permeates by dissolution in the membrane, sigma i = 1.0; if it permeates by passage through an aqueous channel, sigma i < 1.0. For urea, Goldstein and Solomon (1960) found that sigma urea = 0.62 +/- 0.03 which meant that urea crosses the red cell membrane in a water-filled channel. This result and many subsequent observations that showed that sigma urea < 1.0 are at variance with the observation that CHIP28 is impermeable to urea. In view of this problem, we have made a new series of measurements of sigma i for urea and other small solutes by a different method, which obviates many of the criticisms Macey and Karan (1993) have made of our earlier method. The new method (Chen et al., 1988), which relies upon fluorescence of the intracellular dye, fluorescein sulfonate, leads to the corrected value, sigma urea,corr = 0.64 +/- 0.03 for ghosts, in good agreement with earlier data for red cells. Thus, the conclusion on irreversible thermodynamic and other grounds that urea and water share a common channel is in disagreement with the view that CHIP28 provides the sole channel for water entrance into the cell.

CDNA Cloning and Gene Structure of a Novel Water Channel Expressed Exclusively in Human Kidney: Evidence for a Gene Cluster of Aquaporins at Chromosome Locus 12q13

A 1.8-kb cDNA clone (designed hKID, gene symbol AQP2L) with homology to the aquaporins was isolated from a human kidney cDNA library. The longest open reading frame of 846 bp encoded a 282-amino-acid hydrophobic protein that contained the conserved NPA motifs of MIP family members. Cell-free translation produced a nonglycosylated protein migrating at 29 kDa. Amino acid alignment showed the greatest homology of hKID to human MIP (48% identity) and AQP-2 (52%), with lesser homology to human MIWC (AQP-4, 34%), CHIP28 (AQP-1, 38%), and GLIP (AQP-3, 22%). Northern blot analysis revealed a 2.2-kb transcript expressed only in human kidney. PCR/Southern blot analysis of human kidney cDNA using primers flanking the hKID coding sequence revealed expression of a full-length mRNA and short transcripts with partial exon 1 and partial exon 4 deletions. Expression of hKID cRNA inXenopusoocytes did not increase glycerol or urea permeability, but increased osmotic water permeability from (2.8 ± 0.5) × 10−4to (7.4 ± 0.7) × 10−4cm/s (10°C) in a mercurial-sensitive manner. Sequence comparison of hKID cDNA with a cloned 21-kb genomic DNA indicated three introns (lengths 0.7, 0.25, and 0.4 kb) separating four exons with boundaries at amino acids 121, 174, and 201. The hKID promoter was identified and contained TATA, SP1, E-box, and AP1 and AP2 elements; primer extension revealed hKID transcription initiation 654 bp upstream from the translational initiation site. Genomic Southern blot indicted a single-copy hKID gene. PCR analysis of a human/rodent somatic hybrid panel localized the hKID gene to chromosome 12. Chromosomal fluorescencein situhybridization mapped the hKID (AQP2L) gene to chromosome locus 12q13, the same location as the AQP-2 and MIP genes. The high sequence homology, similar genomic structure, and identical chromosomal loci of hKID, MIP, and AQP-2 suggest a MIP family gene cluster at chromosome locus 12q13. Further work is needed to establish the physiological significance of hKID.

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.

Apical membrane permeability of MDCK cells.

The osmotic water permeability (Pf) and permeability to nonelectrolytes were determined for the apical membrane of clonal strain Madin-Darby canine kidney (MDCK) C12 cells cultured as cysts with the apical membrane facing the surrounding medium. Pf and solute permeabilities were calculated from the rate of volume change of cysts by digitizing images at 1-s intervals after instantaneous osmotic challenge. Image measurement was fully automated with the use of a program that separated the image of the cyst from the background by using adaptive intensity thresholding and shape analysis. Pf, calculated by curve fitting to the volume loss data, averaged 2.4 +/- 0.1 micron/s and was increased by addition of amphotericin B. The energy of activation for Pf was high (16.3 kcal/mol), and forskolin (50 microM) had no effect on Pf. Two populations of MDCK cysts were studied: those with two to three cells and those that appeared to be composed of only one cell. The Pf of multicell cysts was the same as single cell cysts, suggesting that paracellular water flow is not significant. Solute permeability was measured using paired osmotic challenges (sucrose and test solute) on the same cyst. Urea permeability was not different from zero, whereas the permeabilities of acetamide and formamide were consistent with their relative oil-water partition coefficients. Our data are similar to values from studies on the permeability properties of vesicles of water-tight epithelial apical membrane. The combination of the unique model of MDCK apical-out cysts and fully automated data analysis enabled determination of apical membrane permeability in intact epithelial cells with high precision.

Active urea transport in the rat inner medullary collecting duct: Functional characterization and initial expression cloning

Active transport of urea has been proposed to exist in the inner medullary collecting duct (IMCD) of low-protein fed mammals for over 30 years. We perfused IMCD subsegments from rats fed a standard (18%) or a low (8%) protein diet and tested for the presence of active urea transport. We found no active urea transport in terminal IMCDs, regardless of diet. In initial IMCDs from rats fed 18% protein or fed 8% protein for one to two weeks, we again found no active urea transport. However, in rats fed 8% protein for three to four weeks, we found significant net urea reabsorption. This active urea reabsorption was inhibited when Na+, K(+)-ATPase activity was inhibited by adding 1 mM ouabain or removing bath potassium, suggesting a secondary active transport process. Removing sodium from the perfusate completely inhibited net urea reabsorption, demonstrating that this active urea transport is dependent upon the presence of sodium in the tubule lumen. Unlike the facilitated urea transporter, the active urea transporter was not inhibited by phloretin nor stimulated by vasopressin, suggesting that it is a distinct transport protein. To test this hypothesis, we size-separated poly(A)(+)-RNA prepared from inner medullae of rats fed 8% protein for three weeks and injected it into Xenopus laevis oocytes. RNA from a 4.4 to 8.4 kb size fraction increased urea permeability fourfold compared to water-injected oocytes or injecting RNA from other size-fractions. We conclude that feeding rats a low-protein diet for three weeks induces the expression of an unique, secondary active, sodium-dependent urea transporter whose cDNA is between 4.4 and 8.4 kb in size. In addition, our results suggest that it will be possible to clone the cDNA for this sodium-urea cotransporter by expression in Xenopus laevis oocytes.

Permeability criteria for effective function of passive countercurrent multiplier.

The urine concentrating effect of the mammalian renal inner medulla has been attributed to countercurrent multiplication of a transepithelial osmotic difference arising from passive absorption of NaCl from thin ascending limbs of long loops of Henle. This study assesses, both mathematically and experimentally, whether the permeability criteria for effective function of this passive hypothesis are consistent with transport properties measured in long loops of Henle of chinchilla. Mathematical simulations incorporating loop of Henle transepithelial permeabilities idealized for the passive hypothesis generated a steep inner medullary osmotic gradient, confirming the fundamental feasibility of the passive hypothesis. However, when permeabilities measured in chinchilla were used, no inner medullary gradient was generated. A key parameter in the apparent failure of the passive hypothesis is the long-loop descending limb (LDL) urea permeability, which must be small to prevent significant transepithelial urea flux into inner medullary LDL. Consequently, experiments in isolated perfused thin LDL were conducted to determine whether the urea permeability may be lower under conditions more nearly resembling those in the inner medulla. LDL segments were dissected from 30-70% of the distance along the inner medullary axis of the chinchilla kidney. The factors tested were NaCl concentration (125-400 mM in perfusate and bath), urea concentration (5-500 mM in perfusate and bath), calcium concentration (2-8 mM in perfusate and bath), and protamine concentration (300 micrograms/ml in perfusate). None of these factors significantly altered the measured urea permeability, which exceeded 20 x 10(-5) cm/s for all conditions. Simulation results show that this moderately high urea permeability in LDL is an order of magnitude too high for effective operation of the passive countercurrent multiplier.

Optimal transport parameters of the inner medullary collecting duct in interaction between urine concentrating and urea excreting mechanisms: a computer simulation study.

Although the accumulation of urea in the renal medulla is essential for the formation of concentrated urine, it is also necessary for the kidney to excrete considerable amounts of urea into the urine as a waste product of protein degradation. Thus, the urine concentrating capacity is attained by the interaction with the efficiency of urea excretion. To seek the best condition for this phenomenon, we developed an objective function for evaluating urea excreting capacity relative to urine concentrating capacity by using a mathematical model consisting of components of the countercurrent multiplication system: the ascending thin limb, capillary network, and inner medullary collecting duct. The values of the objective functions were calculated as three-dimensional functions of transport parameters for the inner medullary collecting duct, including hydraulic conductivity, urea permeability, and reflection coefficient for urea. The results of the computer analysis revealed that the maximum value of the objective function was attained when values for transport parameters of the inner medullary collecting duct corresponded to those experimentally obtained values reported previously. We conclude that the maximum urine concentrating capacity is limited by the efficiency of urea excreting capacity of the kidney, and vice versa.

Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts.

We investigated immunohistochemical localization of V2 vasopressin receptor along the nephron using a specific polyclonal antibody. Staining was observed in some of thick ascending limbs and all of principal and inner medullary collecting duct (IMCD) cells. Not only basolateral but also luminal membrane was stained in collecting ducts, especially in terminal IMCD (tIMCD). To learn the functional role of luminal V2 receptor in tIMCD, we studied the luminal effects of arginine vasopressin (AVP) on osmotic water permeability (Pf), urea permeability (Pu), and cAMP accumulation using isolated perfused rat tIMCD. In the absence of bath AVP, luminal AVP caused a small increase in cAMP accumulation, Pf and Pu, confirming the presence of V2 receptor in the lumen of tIMCD. In contrast, luminal AVP inhibited Pf and Pu by 30-65% in the presence of bath AVP by decreasing cAMP accumulation via V1a or oxytocin receptors and by an unknown mechanism via V2 receptors in the luminal membrane of tIMCD. These data show that V2 receptors are localized not only in the basolateral membrane but also in the luminal membrane of the distal nephron. Luminal AVP acts as a negative feedback system upon the basolateral action of AVP in tIMCD.

The effect of temperature upon the permeation of polar and ionic solutes through human epidermal membrane

The temperature dependence of in vitro permeation through human epidermal membrane (HEM) was determined for urea, mannitol, tetraethylammonium ion (TEA), and corticosterone. The effect of temperature upon HEM electrical resistance was also measured. The majority of the experiments involved measuring the permeability coefficients of a specific permeant at 27°C and 39°C for a given HEM sample, the electrical resistance was also measured at each temperature. Similar experiments were also conducted with a model synthetic porous membrane. The effect of temperature was quantitated as the ratio of the permeability at 39°C to the permeability at 27°C for each permeant. These ratios observed for HEM with urea, mannitol, and TEA as the permeants were 1.66 ± 0.05, 1.76 ± 0.14, and 1.71 ± 0.11, respectively. The change in temperature was shown to have a similar effect upon the electrical conductance of the HEM samples. The observed ratio for corticosterone permeation was 4.5 ± 0.4. The experimental ratios observed for the three polar/ionic permeants were shown to approach those obtained from the model porous membrane and differed greatly from the ratio observed for the more lipophilic corticosterone, indicating differences in the effective transport mechanism/pathway for these classes of permeants. The permeability of urea was also observed to be inversely proportional to the electrical resistance of the HEM samples; this relationship was shown to be independent of temperature over the temperature range studied. The temperature dependence data and the observed relationship between urea permeability and electrical resistance strongly support the existence of a porous permeation pathway through the HEM as an operative diffusional route for polar/ionic permeants.

Inner medullary external osmotic driving force in a 3-D model of the renal concentrating mechanism

The mechanism by which the renal medulla establishes and maintains a gradient of osmolarity along the corticomedullary axis, especially in the inner medulla, where there is no active transmural flux out of the ascending limbs of Henle, remains a source of controversy. We show here that, if realistic values of urea permeability in the inner medullary descending limbs and water permeability in the upper inner medullary section of the collecting ducts are taken into account, even a model including the three-dimensional vascular bundle structures [A. S. Wexler, R. E. Kalaba, and D. J. Marsh. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F368-F383, 1991] fails to explain the experimentally observed inner medullary osmolality gradient. We show here that this failure can be overcome by application of an external osmotic driving force, an idea recently revived by J. F. Jen and J. L. Stephenson (Bull. Math. Biol. 56: 491-514, 1994) in the context of a single-solute, single-loop central core model. We show that inclusion of such an external driving force with a value equivalent to at least 100 mosM of inner medullary interstitial osmolytes in the three-dimensional model of Wexler et al. accounts for a physiological osmolality gradient, even in the face of realistic permeability values. Furthermore, inclusion of the external driving force makes the model less dependent on the positions of descending and ascending limbs of Henle with respect to the collecting ducts. In an effort to assess whether there is any experimental basis for osmolytes, we show that a significant amount of extra inner medullary interstitial osmolytes is plausible, based on extrapolation from existing experimental data.

Renal medullary microcirculation: architecture and exchange.

The architecture of the renal medullary microcirculation is highly specialized. Consistent with their role in countercurrent exchange, the vessels (the vasa recta and the intervening capillaries) have high permeabilities to fluid and small hydrophilic solutes. The urea permeability of the continuous endothelium of the descending vasa recta (DVR) in the outer medulla is greatly enhanced by a urea transporter. Aquaporin channels have also been identified in these vessels. In spite of the absence of lymphatics from the inner medulla, fluid uptake from the interstitial fluid (ISF) through the fenestrated endothelium of the ascending vasa recta (AVR) appears to be driven by differences in hydrostatic and oncotic pressure. Because the AVR have high Lp's [10(-5) cm s-1 (cm H2O)-1] and are mechanically linked to surrounding structures, small increments of ISF pressures above the pressure within the AVR can drive significant volumes of fluid into AVR if ISF volume expands. The lower reflection coefficients to serum albumin of the AVR as compared with the DVR may be important in the clearance of interstitial plasma protein. Recent work on isolated DVR from the outer medulla has revealed that these vessels are capable of vasoconstriction and thus of regulating medullary blood flow.

Ammonia and urea metabolism in relation to gill function and acid-base balance in a marine elasmobranch, the spiny dogfish (Squalus acanthias)

Nitrogenous waste excretion in resting dogfish occurred largely (>90 %) as urea-nitrogen (urea-N) efflux across the gills, with a very small urea efflux via the kidney. Ammonia excretion, almost entirely at the gills, accounted for less than 3 % of total nitrogen excretion. Given the extremely high blood urea levels (approximately 640 mmol-N l-1) 'retained' for osmoregulation, and blood ammonia levels (approximately 80 &micro;mol-N l-1) comparable to those of teleosts, the gills of resting dogfish were exceptionally impermeable to both urea and ammonia. Experiments investigated the origins of these low permeabilities and the responses of urea-N and ammonia-N excretion and acid&shy;base status to 6 h infusions with iso-osmotic solutions of NaCl (control), NH4Cl, NaHCO3, urea and its analogues thiourea and acetamide. NaCl had no effects, whereas NH4Cl loading caused intense acidosis and marked elevation of acidic equivalent, ammonia-N and urea-N excretion rates, the latter despite unchanged blood levels of urea-N. Apparent branchial ammonia permeability increased greatly. Acidosis resulted from both stimulated urea production and branchial NH3 loss, the former making the larger contribution. NaHCO3 loading caused intense alkalosis, a marked elevation of basic equivalent excretion and a moderate stimulation of urea-N excretion. Blood urea-N levels were again unchanged. Infusion of urea itself raised blood urea-N levels, but initially reduced branchial urea-N excretion. Acetamide and thiourea infusions both moderately elevated branchial urea-N excretion. We suggest that the low ammonia permeability may arise metabolically from an ammonia scavenging system in the gills, that a 'back-transport' mechanism in the gills may contribute to the low urea permeability, and that the dissociation between blood urea-N levels and excretion rates may reflect urea production at extrahepatic sites. These studies demonstrate that urea synthesis in the dogfish is linked more to nitrogen availability than to acid&shy;base status.

Oxytocin as an antidiuretic hormone. II. Role of V2 vasopressin receptor

We conducted this study to determine what receptor mediates the effect of oxytocin to increase osmotic water permeability (Pf) in the rat inner medullary collecting duct (IMCD). Reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated that mRNA for both the oxytocin receptor and the V2 receptor is present in the rat terminal IMCD. In isolated perfused IMCD segments, we found that the V2 vasopressin receptor antagonist [d(CH2)5(1),D-Ile2,Ile4,Arg8]vasopressin, but not oxytocin receptor antagonists, blocked the hydrosmotic response to 200 pM oxytocin. The selective oxytocin receptor agonist [Thr4,Gly7]oxytocin did not increase water permeability. Oxytocin also increased urea permeability in IMCD segments. Studies in IMCD suspensions showed that oxytocin increases adenosine 3',5'-cyclic monophosphate production in a dose-dependent fashion with a half-maximal (EC50) response at 5.2 nM. The dose-response curves were virtually identical for IMCD suspensions from Sprague-Dawley rats and Brattleboro rats. The oxytocin dose-response curve was displaced to the right of the vasopressin dose-response curve (EC50, 0.44 nM). From these results, we conclude that the V2 receptor mediates the hydrosmotic action of oxytocin in rat IMCD.

Urea transport in initial IMCD of rats fed a low-protein diet: functional properties and mRNA abundance.

Feeding rats a low-protein (8%) diet (LPD) for 2 wk induces a facilitated urea transporter in rat initial inner medullary collecting ducts (IMCDs). To determine whether this is preceded by an increase in mRNA abundance, we designed degenerate polymerase chain reaction primers to the rabbit facilitated urea transporter (UT2; G. You, C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger. Nature Lond. 365: 844-847, 1993) and amplified a 716-bp cDNA fragment to perform Northern analysis of the base or tip of rat inner medulla. In the base, the predominant transcript was a 2.9-kb band, which increased 55% after 1 wk on an LPD; there was no change in a 4-kb band. In the tip, the 4-kb band predominated, but neither band varied with an LPD. Next, we functionally characterized the induced urea transporter using microperfused initial IMCDs from rats fed an LPD for 2 wk. First, 100 pM arginine vasopressin (AVP) stimulated urea permeability (Purea); 10 nM AVP increased Purea further. Second, raising perfusate and bath osmolality to 690 mosmol/kgH2O (NaCl added) stimulated Purea; adding AVP (10 nM) increased Purea further. Third, thiourea reversibly inhibited AVP-stimulated Purea.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell volume measured by total internal reflection microfluorimetry: application to water and solute transport in cells transfected with water channel homologs

Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (approximately 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, Pf, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2-sensitive, threefold increase in Pf compared to nontransfected cells (Pf = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5) cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.

Gradation of microcapsule wall porosity by deposition of polymer mixtures (Eudragit RL and Eudragit RS). Phase separation of polymer mixtures and effects of external media and conditions on release.

With the aim of increasing flexibility in controlling release from microcapsules, mixtures of wall polymers varying in porosity were investigated by phase separation. Eudragit RL and RS (polymethylmethacrylate linear backbone polymers) mixtures differing in polar substituent content and porosity were used as the wall material and were deposited using a non-solvent addition method. Release rates increased with polar group content of the mixtures, using theophylline, potassium dichromate or sodium chloride as model core materials. Theophylline release rate had the same relationship to polar group content as found earlier for urea permeation of cast mixed-polymer films. Release was generally accelerated in these systems when the external medium contained sodium lauryl sulphate as a wetting agent but not consistently, decreasing unexpectedly for RL-theophylline microcapsules. Localized dissolution of core substance was visible microscopically during release from single microcapsules. The release rate was sensitive to agitation intensity only at low wall to core ratios. Temperature change revealed only a single release mechanism for sodium chloride by Arrhenius equation treatment. Buffer ions penetrated coatings readily, changing theophylline release rates and providing clear evidence of diffusion via a pore-capillary mechanism.

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

The clinical usefulness of amphotericin B (AMP-B) is limited by its nephrotoxicity, as characterized by decreased RPF, decreased GFR, impaired urinary acidification, and potassium excretion defects. Defects of renal concentrating ability have been noted, but the mechanisms responsible for them have not been investigated. The chief objective of this research was to analyze directly the effect of AMP-B on arginine-vasopressin (AVP)- or dibutyrl cAMP (DcAMP)-stimulated water and urea transport of the inner medullary collecting duct (IMCD) obtained from rats by the in vitro microperfusion technique. AMP-B (10(-5) M) added to the bath fluid in the absence of AVP did not impair the hydraulic conductivity (Lp) and the urea permeability (Pu) of rat IMCD. AMP-B (10(-5) M) added to the bath fluid decreased the AVP-stimulated Lp (x 10(-6) cm/s.atm) of rat IMCD from 19.41 +/- 2.19 to 10.00 +/- 1.39 (P < 0.001), and the reversibility of its action was observed during a third period when Lp increased to 19.80 +/- 2.19 (P < 0.001) after the initial conditions were restored. In addition, AMP-B reduced DcAMP-stimulated Lp from 20.95 +/- 1.75 to 10.52 +/- 0.71 (P < 0.01) in a reversible manner when the drug was withdrawn from the bath. AMP-B also decreased AVP-stimulated Pu (x 10(-5) cm/s) when added to the bath fluid from 36.60 +/- 2.05 to 29.88 +/- 1.36 (P < 0.001), and this effect was reversible after AMP-B was withdrawn from the bath (37.40 +/- 1.36; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the urea transporter in outer medullary descending vasa recta

Partially because of facilitated transport of urea, urea permeability (Pu) of the outer medullary descending vasa recta (OMDVR) frequently exceeds sodium permeability by more than an order of magnitude. This study characterizes the OMDVR urea transporter. Application of the urea analogue thiourea (200 mM) to the abluminal surface of microperfused OMDVR inhibited Pu by 33%. When osmolarity due to thiourea was balanced by addition of mannitol or thiourea, similar results were obtained. Thiourea produced graded inhibition of Pu from 343 +/- 54 (SE) to 191 +/- 43 x 10(-5) cm/s as concentration was increased from 0 to 100 mM. The thiourea concentration needed for half-maximal inhibition was 19 mM. The abilities of urea analogues to reduce Pu were compared by addition of 50 mM concentrations to the bath and perfusate. Thiourea and methylurea produced 32 and 34% inhibition of Pu, respectively, whereas urea and acetamide produced only 3 and 11% inhibition, respectively. The transporter showed negligible saturation as the transmural urea gradient was increased from 0 to 200 mM. Phloretin and p-chloromercuribenzenesulfonate inhibited Pu in a concentration-dependent fashion. It is concluded that a transporter confers high Pu to OMDVR. Pu is equally high when measured by urea influx or efflux. Properties of the transporter are similar to those expressed by the inner medullary collecting duct.

Protein restriction sequentially induces new urea transport processes in rat initial IMCD

We reported that feeding rats 8% protein for 4 wk induces two new urea transport processes in initial inner medullary collecting ducts (IMCD); neither is present in rats fed 18% protein. In this study, we measured the time course of induction of these transporters in perfused initial IMCD segments from rats fed 8% protein. Net urea flux was induced after 3 wk, whereas vasopressin-stimulated passive urea permeability (P(urea)) was induced after 2 wk. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) significantly increased P(urea)); adding vasopressin did not increase P(urea) further. In fact, there was no difference in vasopressin-stimulated cAMP production in initial or terminal IMCD segments from rats fed 18% or 8% protein, suggesting that the adaptive response was not due to increased cAMP production. Glucagon did not change cAMP production or P(urea). Specificity of the response was suggested because neither aldose reductase nor sorbitol dehydrogenase activity changed with feeding 8% protein. Thus 1) in initial IMCD segments, vasopressin-stimulated P(urea) is induced after 2 wk, but net urea flux requires 3 wk of feeding 8% protein; 2) this adaptation is not solely due to a higher rate of cAMP production; and 3) specificity of the adaptive response is suggested because activities of enzymes responding to decreases in concentrating ability are unchanged. These results suggest that two distinct urea transporters may be involved in the adaptation to a low-protein diet.