Urea and Renal Function in the 21st Century: Insights from Knockout Mice
Robert A. Fenton* and
Mark A. Knepper
* Water and Salt Research Center, Institute of Anatomy, University of Aarhus, Aarhus, Denmark; and Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institutes, National Institutes of Health, Bethesda, Maryland
Address correspondence to: Dr. Robert A. Fenton, Water and Salt Research Center, Institute of Anatomy, Building 233/234, University of Aarhus, DK-8000 Aarhus, Denmark. Phone: +45-894-23008; Fax: +45-861-98664; E-mail: rofe{at}ana.au.dk
Since the turn of the 21st century, gene knockout mice havebeen created for all major urea transporters that are expressedin the kidney: the collecting duct urea transporters UT-A1 andUT-A3, the descending thin limb isoform UT-A2, and the descendingvasa recta isoform UT-B. This article discusses the new insightsthat the results from studies in these mice have produced inthe understanding of the role of urea in the urinary concentratingmechanism and kidney function. Following is a summary of themajor findings: (1) Urea accumulation in the inner medullaryinterstitium depends on rapid transport of urea from the innermedullary collecting duct (IMCD) lumen via UT-A1 and/or UT-A3;(2) as proposed by Robert Berliner and colleagues in the 1950s,the role of IMCD urea transporters in water conservation isto prevent a urea-induced osmotic diuresis; (3) the absenceof IMCD urea transport does not prevent the concentration ofNaCl in the inner medulla, contrary to what would be predictedfrom the passive countercurrent multiplier mechanism in theform proposed by Kokko and Rector and Stephenson; (4) deletionof UT-B (vasa recta isoform) has a much greater effect on urinaryconcentration than deletion of UT-A2 (descending limb isoform),suggesting that the recycling of urea between the vasa rectaand the renal tubules quantitatively is less important thanclassic countercurrent exchange; and (5) urea reabsorption fromthe IMCD and the process of urea recycling are not importantelements of the mechanism of protein-induced increases in GFR.In addition, the clinical relevance of these studies is discussed,and it is suggested that inhibitors that specifically targetcollecting duct urea transporters have the potential for clinicaluse as potassium-sparing diuretics that function by creationof urea-dependent osmotic diuresis.
Urea is a small molecule, only 60 Da, that constitutes the majorform of waste nitrogen that is excreted in mammals. Its extraordinarilyhigh solubility in water (saturation at >6 M) makes it idealfor excretion by the concentrating kidney. Studies by JamesShannon in the 1930s demonstrated that the rate of urea excretionis determined chiefly by its rate of filtration through theglomerulus and tubular reabsorption (1,2). Despite this seeminglysimple view of renal urea handling, multiple hypotheses thatascribe more complex roles to urea have accrued during the courseof the 20th century. The molecular era, shepherded by genomeprojects in various species, has opened the door for new toolsthat are capable of addressing these hypotheses, namely, mouselines in which various molecular urea carriers have been geneticallydeleted. Here we review the new light that has been shed onrenal urea handling through experimentation in these knockoutmouse lines.
In mammals, approximately 90% of waste nitrogen is excretedby the kidney as urea. The majority of this urea is generatedin the liver as a byproduct of protein metabolism. Under mostcircumstances, the dietary protein intake of humans and animalsgreatly exceeds that necessary for the support of anabolic processes;therefore, a large excess of urea is generated (Figure 1). Excretionof this urea constitutes a large osmotic load to the kidney.Most solutes that are excreted in such large amounts, for examplemannitol (3), would obligate large amounts of water excretionby causing an osmotic diuresis. However, as first determinedby Gamble and colleagues (4,5) in the 1930s, the kidney possessesspecialized mechanisms that allow large amounts of urea to beexcreted without obligating water excretion.
Figure 1. Urea handling in mammals. The majority of mammals consume diets that are high in protein. Under most circumstances, this dietary protein intake greatly exceeds that which is necessary for the support of anabolic processes. Excess protein is catabolized by the liver, which results in the formation of large amounts of urea by the ornithine-urea cycle. Urea is freely filterable by the kidney and the excretion of this urea constitutes a large osmotic load to the kidney. Most solutes excreted in such large amounts would obligate large amounts of water excretion by causing an osmotic diuresis. However, along the nephron, the specialized urea transporters UT-A1, UT-A2, UT-A3, and UT-B are involved in complex urea reabsorption and recycling pathways that allow large amounts of urea to be excreted without obligating water excretion. Illustration by Josh Gramling—Gramling Medical Illustration.
Evidence for specialized urea transport in the kidney, mediatedby molecular urea carriers, has been provided in numerous studies.It is not the purpose of this article to review all of these,but an overview is informative. Urea reabsorption by the mammaliannephron occurs by two different mechanisms: A constitutive processthat occurs in the proximal nephron and accounts for reabsorptionof nearly 40% of the filtered load of urea and a regulated processthat occurs in the distal nephron and depends on the level ofantidiuresis (1,2) among other factors. In addition, the useof the isolated perfused tubule technique has determined that(1) the cortical collecting duct has a very low urea permeabilitythat is not increased by vasopressin (AVP) (6); (2) the terminalpart but not the initial part of the inner medullary collectingduct (IMCD) possesses extraordinarily high urea permeability(7); (3) AVP increases urea permeability only in the terminalIMCD (8); (4) urea transport in the IMCD is inhibited by phloretinand urea analogues and is a saturable process, consistent witha transporter-mediated (facilitated) mechanism (9,10); and (5)urea and water transport across the IMCD occur by two distinctpathways, but the time course of increased urea and water permeabilitychanges in the IMCD in response to AVP are virtually indistinguishable(11–14). Combined, this rich body of descriptive physiologicobservations facilitated the cloning of urea transporters fromthe kidney as detailed in what follows.
There are two distinct but closely related urea transportergenes: UT-A (Slc14a2) and UT-B (Slc14a1) (15–17). Severalurea transporter isoforms are derived from the UT-A gene viaalternative splicing and alternative promotors (Figure 2) (15).Multiple cDNAs (Figure 2) that encode urea transporters havebeen isolated and characterized (18–25). UT-A1 is expressedexclusively in IMCD cells (Figure 3) (19,26). UT-A1 activityis regulated acutely by AVP (27), but the long-term effectsof AVP on UT-A1 abundance are still a subject of controversy.UT-A2 is expressed in the inner stripe of the outer medulla,where it is localized to the lower portions of the thin descendinglimbs (tDL) of short loops of Henle (Figures 1 and 3) (19,28,29)and, under prolonged antidiuretic conditions, in the inner medulla,where it is localized to the tDL of long loops of Henle (29).AVP increases UT-A2 abundance (19,29,30), and recent studieshave determined that UT-A2–mediated urea transport canbe regulated acutely by cAMP (31). In a similar manner to UT-A1,expression of UT-A3 is restricted to the terminal IMCD (Figures 1and 3), where, in mouse, it is both intracellular and in thebasolateral membrane domains (32). UT-A3 mRNA abundance canbe upregulated by the prolonged action of AVP (15). In contrastto the multiple UT-A isoforms, the mouse UT-B gene encodes onlya single protein that is expressed throughout the kidney medullain the basolateral and apical regions of the descending vasarecta (DVR) endothelial cells (Figures 1 and 3) (33–35).Long-term treatment with the type II vasopressin receptor agonistdDAVP causes downregulation of UT-B protein abundance (36).
Figure 2. Urea transporters derived from mouse UT-A gene. At the top is a schematic representation of the largest isoform, UT-A1, with putative membrane-spanning domains represented as barrels. Below, H1 through H4 represent hydrophobic domains, with amino acid numbers indicated. UT-A1 and UT-A3 are driven by the same promoter and are identical through amino acid 459. Use of an alternative exon inserts a stop codon that terminates UT-A3 after amino acid 460 (an aspartic acid). UT-A2 is identical to the terminal 397 amino acids of UT-A1 and is driven by an alternative promoter in intron 13 of the mouse gene (15). Illustration by Josh Gramling—Gramling Medical Illustration.
Figure 3. Localization of UT-A urea transporters. UT-A1 is localized to the terminal portion of the inner medullary collecting duct (IMCD), whereas UT-A2 is localized to the thin descending limbs of Henle’s loop in the inner stripe of the outer medulla (A). Higher magnification shows that both UT-A2 (B) and UT-A1 (C) predominantly are intracellular. UT-A3 is localized to the terminal portion of the IMCD (D) and is both intracellular and in the basolateral membrane domains (F). UT-B is expressed in the descending vasa recta (G), where it is localized to the basolateral and apical regions (E).
Recently, several mouse models with selective deletion of differenturea transporter isoforms have been created. The remainder ofthis article summarizes the studies from these mice and discussesthe conclusions of these studies with respect to the role ofurea in the urinary concentrating mechanism.
The two UT-A isoforms that are expressed in the IMCD are UT-A1and UT-A3—the so-called "collecting-duct urea transporters."Recently, by knocking out both collecting duct urea transportersin tandem, we developed a mouse model that allowed us to assessspecifically the role of IMCD urea transport in kidney function(37). These mice (termed UT-A1/3–/– mice) were generatedby replacing 3 kb of the UT-A gene, which contains a single148-bp exon (exon 10), with a neomycin selection cassette. Exon10 of the UT-A gene codes for amino acids 291 to 339 of UT-A1or UT-A3 and is situated in a large, hydrophobic, membrane-spanningregion (38); therefore, it is thought to be of functional significance.Immunoblotting and immunocytochemistry with several isoform-selectivepolyclonal antibodies demonstrated successful deletion of thetransporters from the IMCD. Importantly, a functional assessmentof UT-A1/3–/– mice was performed using isolatedperfused tubule studies and showed a complete absence of phloretin-sensitiveand AVP-regulated urea transport in IMCD segments, whereas AVP-stimulatedwater permeability was unaffected. We concluded from these studiesthat UT-A1 and/or UT-A3 is responsible for the high urea permeabilityof the IMCD that was observed previously and that water andurea are transported by different mechanisms.
Role of IMCD Urea Transporters in the Urinary Concentrating Mechanism
Much of our fundamental understanding of the contribution ofurea transporters to the urinary concentrating mechanism isbased on a model of urea handling proposed in the 1950s by Berlineret al. (39). The following is a brief explanation undated withsubsequent observations. The concentration of urea in the tubulefluid that enters the collecting duct system in the renal cortexis relatively low. During antidiuresis, water is osmoticallyabsorbed from the urea-impermeable parts of the collecting ductsystem via aquaporin water channels, causing a progressive increasein luminal urea concentration along the collecting duct system.Subsequently, when the tubule fluid reaches the highly urea-permeableterminal IMCD, urea can exit rapidly from the lumen to the innermedullary interstitium. This urea is trapped in the inner medullaryinterstitium because the effective blood flow is very low owingto countercurrent exchange by the vasa recta (40,41). In thepresence of AVP, the urea permeability of the terminal IMCDis extremely high, and, under steady-state conditions, ureaaccumulates to very high concentrations in the interstitiumand nearly equilibrates across the IMCD epithelium. This allowsurea in the interstitium to almost completely balance osmoticallythe high urea concentration in the collecting duct lumen, preventingthe osmotic diuresis that would otherwise occur in associationwith the large amounts of urea that are present in the urine.
On the basis of this model, one would predict that the deletionof specialized urea transporters from the IMCD should resultin an impaired capacity to conserve water, owing to urea-dependentosmotic diuresis. For testing of this hypothesis, the urinaryconcentrating function of UT-A1/3–/– mice on threedifferent levels of dietary protein intake was examined in aseries of metabolic cage studies (Figure 4) (37,42). With freeaccess to water, UT-A1/3–/– mice that were fed eithera standard-protein (20% protein by weight) or high-protein (40%)diet had a significantly greater fluid intake and urine flowthan wild-type controls, resulting in a decreased urine osmolality.However, on a low-protein diet (4% protein) UT-A1/3–/–mice did not show a substantial degree of polyuria. With thislow protein intake, hepatic urea production is low and ureadelivery to the IMCD is low, thereby preventing urea-inducedosmotic diuresis. Further studies examined the maximal concentratingability of UT-A1/3–/– mice after 18 h of water restriction(Figure 4). Knockout mice on a 20 or 40% protein intake wereunable to reduce their urine flow to levels below those thatwere observed under basal conditions, resulting in severe volumedepletion and loss of body weight. In contrast, UT-A1/3–/–mice that were on a low-protein diet were able to maintain fluidbalance without a marked loss of body weight.
Figure 4. Water conservation and urinary concentrating ability of UT-A1/3–/– mice. For all graphs, data are means ± SEM; wild-type mice are indicated by solid lines, and knockout mice are represented by dashed lines. Mice received 4, 20, or 40% protein intake for 7 d before and throughout the duration of the study. Graphs show either the urine output under basal conditions (free access to drinking water) for 3 consecutive days, followed by a 24-h water restriction on a 4% (A), 20% (B) or 40% (C) protein diet or the corresponding urine osmolality on a 4% (A), 20% (B), or 40% (C) protein diet. The conclusion from these data is that the role of IMCD urea transporters in water conservation is to prevent a urea-induced osmotic diuresis. Adapted from data in references (37,42).
In accordance with the Berliner model, these studies showedthat the concentrating defect in UT-A1/3–/– micelargely is a result of urea-dependent osmotic diuresis. However,collecting duct urea transport has been proposed to play anadditional role in the urinary concentrating mechanism, actingas the first step of the so-called "passive model" in whichaccumulation of NaCl in the inner medullary interstitium dependsindirectly on urea reabsorption from the IMCD (43,44). As discussedin the next section, studies in UT-A1/3–/– miceprovided a direct test of the passive model.
Accumulation of NaCl in the Inner Medulla: Role of IMCD Urea Transporters
Early experiments that used tissue slice analysis determinedthat a corticomedullary osmolality gradient exists in the kidneywith maximum osmolality at the tip of the inner medulla (45).This gradient was found to be due mainly to accumulation ofNaCl in the outer medulla and urea in the inner medulla. Thecause of the gradient in the outer medulla is well understoodon the basis of the classical countercurrent multiplier model(46). This model relies on active NaCl reabsorption in the water-impermeablethick ascending limb of the loop of Henle (TAL) (47,48) foran energy source. In the inner medulla, the urea gradient isdue to passive urea reabsorption from the IMCD via UT-A ureatransporters, aided by countercurrent exchange (see Urea Recycling).In addition to the urea gradient, the inner medulla generatesa NaCl gradient that is not as steep as the NaCl gradient inthe outer medulla but nevertheless is important in the productionof a concentrated urine. The mechanism that is responsible forthis NaCl gradient in the inner medullary interstitium has beenan important focus of research in the past 35 yr, and no clearconsensus has emerged. Repeated studies of thin ascending limbshave failed to show evidence for an active NaCl transport processin the inner medulla (49,50). Therefore, another process presumablyis responsible for the energy that is needed to concentrateNaCl in the inner medulla. One hypothesis was the "passive model"that was introduced in the previous section. This model wasproposed independently by Stephenson (44) and by Kokko and Rectorin 1972 (43) and also is referred to as the "passive countercurrentmultiplier mechanism." In this mechanism, rapid urea reabsorptionfrom the IMCD generates and maintains a high urea concentrationin the inner medullary interstitium and causes the osmotic withdrawalof water from the thin descending limb, concentrating NaCl inthe lumen. This highly concentrated NaCl then is proposed toexit passively from the thin ascending limb. If the urea permeabilityof the ascending limbs is extremely low, then any NaCl thathas been reabsorbed from the ascending thin limb will not bereplaced by urea and the ascending limb fluid will become diluterelative to the surrounding interstitial fluid. This dilutionalprocess is proposed to constitute a "single effect" for countercurrentmultiplication, similar to that in the outer medulla but generatedby passive transport processes within the inner medulla.
The passive countercurrent multiplier mechanism in the innermedulla relies on rapid urea transport from the IMCD, facilitatedby the urea transporters UT-A1 and UT-A3. If the passive modelis correct, then we would predict that in UT-A1/3–/–mice, the lack of urea transport across the epithelium of theIMCD would impair the ability to concentrate NaCl in the innermedulla. Direct tests of this prediction were made by measurementof inner medullary solute concentrations in inner medullarytissue in two different studies (37,42). In one study, the meanurea, Na+, Cl–, and K+ concentrations were measured inwhole inner medullary tissue isolated from UT-A1/3–/–mice and wild-type littermates (37). UT-A1/3–/–mice displayed a marked decrease in inner medullary urea concentration,but there was no reduction in the mean Na+, Cl–, or K+concentrations. In a separate study, the osmolality and ureaand Na+ concentrations were measured in the cortex, outer medulla,and two levels of the inner medulla from UT-A1/3–/–and wild-type mice that were fed either a low-protein (4%) ora high-protein (40%) diet (42). In UT-A1/3–/– micethat were on either protein intake, there was a substantiallyreduced corticomedullary osmolality gradient and no urea gradient.However, the observed corticomedullary sodium gradients in wild-typeand knockout mice that were on either level of dietary proteinintake were indistinguishable. Furthermore, in wild-type mice,decreasing the dietary protein intake resulted in a decreasein tissue osmolality that was caused solely by reduced ureaaccumulation in the inner medulla. That is, sodium concentrationsalong the corticomedullary axis were unchanged by the changein protein intake and the resulting change in medullary ureaaccumulation. Therefore, a marked depletion in medullary ureaconcentration had no effect on the ability of the medulla toaccumulate NaCl in the inner medulla, whether the depletionwas caused by dietary protein restriction or by deletion ofIMCD facilitative urea transporters. On the basis of these twostudies, it seems that NaCl accumulation in the inner medulladoes not depend on either IMCD urea transport or the accumulationof urea in the medullary interstitium. These findings, therefore,contradict an essential prediction of the passive concentratingmodel, namely that elimination of passive urea absorption fromthe IMCD and the resulting depletion of inner medullary ureawould decrease inner medullary NaCl accumulation. These resultsin UT-A1/3–/– mice and their wild-type controlsseem to refute the passive countercurrent multiplier hypothesisfor inner medullary NaCl accumulation. That is, the passivemodel is not the chief mechanism by which NaCl is concentratedin the inner medulla. It should be emphasized that previousobjections have been raised to the validity of the passive modellargely on the basis of the high urea permeabilities that havebeen measured in thin descending and ascending limbs (summarizedin reference [51]), but these studies in UT-A1/3–/–mice provide the most direct test of the hypothesis to date.
If the passive countercurrent multiplier model is not the explanationfor the NaCl gradient in the renal inner medulla, then whatis? A full answer to this question is beyond the scope of thisshort review, because the proposed alternatives do not involveurea transporters. The reader is referred to either a chapterby Gamba and Knepper (51) or a recent review article (52) fora discussion of alternative models. Fundamentally, it is safeto conclude at this point that more research is needed to developa full understanding of the concentrating function of the renalinner medulla.
The Gamble phenomenon (described more than 70 yr ago as "aneconomy of water in renal function referable to urea" [4]) pointedto a special role for urea in the urinary concentrating mechanismand provided part of the original support for the Kokko-Rector-Stephensonpassive model. The general features of the Gamble phenomenonare that (1) the water requirement for the excretion of ureais less than for the excretion of an osmotically equivalentamount of NaCl, and (2) when fed various mixtures of urea andsalt in the diet, less water is required for the excretion ofthe two substances together than the water needed to excretean osmotically equivalent amount of either urea or NaCl alone.The latter finding suggests that the concentrating mechanismdepends in some complex way on an interaction between NaCl andurea. Indeed, the latter finding is what would be predictedby the passive model; therefore, the Gamble phenomenon was viewedas providing support for the validity of the passive model (38).UT-A1/3–/– mice were used to investigate the roleof collecting duct urea transport in the Gamble phenomenon (53).Indeed, in UT-A1/3–/– mice, both elements of theGamble phenomenon were absent, indicating that IMCD urea transportersplay a critical role. A titration study in which wild-type micewere given progressively increasing amounts of urea or NaClshowed that both substances can induce osmotic diuresis at highenough levels of excretion (6000 µosmol/d for urea; 3500µosmol/d for NaCl). It is interesting that mice were unableto increase urinary NaCl concentrations to beyond approximately420 mM. The second component of the Gamble phenomenon derivesfrom the fact that both urea and NaCl excretion are saturable,presumably a result of the ability to exceed the respectivereabsorptive capacity for urea and NaCl. Thus, conservationof water with mixtures of NaCl and urea versus pure NaCl orurea occurs simply as a result of lowering the concentrationof each to levels that avoid osmotic diuresis, rather than toany specific interaction of urea transport and NaCl transportat an epithelial level.
In addition to countercurrent exchange, urea recycling is believedto provide an important means of maintaining a high level ofurea in the renal inner medulla (54). Recycling occurs whenurea that is reabsorbed from the IMCD is re-secreted into theloop of Henle, causing it to be returned to the collecting ductlumen with the flow of tubule fluid (Figure 1). Recycling firstwas demonstrated by Lassiter et al. (55), who showed that themass flow rate of urea in the superficial tubule exceeds thefiltered load of urea. It has been proposed (54) that a majorelement of urea secretion into Henle’s loop is via transferfrom the vasa recta to the thin descending limbs of short-loopnephrons in the vascular bundles of the outer medulla, wherethese two structures are in close apposition (56,57). The chiefurea transporter of the vasa recta is UT-B, whereas the chiefurea transporter in the thin descending limbs is UT-A2 (Figures 1and 3). Therefore, it would be predicted that the deletion ofeither of these transporters would impair significantly ureaaccumulation in the inner medulla, resulting in increased waterexcretion via urea-induced osmotic diuresis. However, it cameas a surprise when Uchida et al. (58) showed that deletion ofUT-A2 did not affect substantially the concentrating abilityor water excretion with a normal level of protein intake. Onlywhen urea excretion was diminished by administration of a low-proteindiet was there a decrease in medullary urea accumulation relativeto wild-type control mice. Therefore, urea secretion into thethin descending limb of Henle’s loop does not seem tobe as important in medullary urea accumulation as previouslybelieved.
In contrast to the results that were observed with UT-A2 knockoutmice, deletion of UT-B, the major urea transporter of the DVRand of erythrocytes, resulted in a substantial impairment inrenal water conservation. UT-B knockout mice were developedin 2002 (59), and their physiology recently was discussed extensivelyelsewhere (60). On a normal-protein diet, UT-B null mice havea significantly higher daily urine output, resulting in lowerurine osmolality, compared with wild-type mice. However, whenUT-B knockout mice are subjected to water deprivation for 36h, they are able to concentrate their urine, although to a lesserextent than controls. Knockout mice have a significantly higherplasma urea, and their urine-to-plasma urea ratio is reducedmore severely than that of other solutes, indicating that theUT-B null mice have a "urea-selective" urinary concentratingdefect (61). This diminished ability to concentrate urea ishighlighted by a lower inner medullary urea concentration comparedwith other solutes.
Quantitatively, the most important loss of urea from the innermedullary interstitium is thought to occur via the vasa recta(54); therefore, the greater concentrating defect in UT-B nullmice compared with UT-A2 null mice may not be surprising. UT-Bpotentially is important for both countercurrent exchange ofurea between ascending vasa recta (AVR) and DVR and for transferof urea from the vasa recta to the thin descending limb. A comparisonof the results in UT-B and UT-A2 knockout mice suggests thatthe former may be more important to the overall process thattraps urea in the inner medulla. That is, at face value, theresults suggest that the recycling of urea between the DVR andAVR is more important quantitatively than recycling of ureabetween the AVR and the renal tubules with regard to net effecton water conservation. A key element of data that needs to beprovided for full acceptance of this conclusion is micropunctureof the distal tubule to verify that UT-A2 deletion actuallyeliminates urea recycling. Specifically, it would be of valueto repeat the measurements that were made originally by Lassiteret al. (55,62) in wild-type and UT-A2 knockout mice to addressthe hypothesis that UT-A2 is necessary for urea recycling.
In addition to the DVR, UT-B protein is expressed in red bloodcells (RBC) (63) and contributes to their high urea permeability.This high urea permeability is thought to have a physiologicalrole; rapid urea transport may help to preserve the osmoticstability and deformability of the RBC (64) and thereby helpto prevent dissipation of the urea gradient in the renal medulla(discussed in reference [65]) and overall concentrating ability.Because erythrocytes from UT-B knockout mice have an approximately45-fold lower urea permeability compared with those from controls,it is important to recognize that the concentrating defectsthat are observed in the UT-B knockout mice could be due tothe loss of urea transport in the vasa recta, in RBC, or both.Furthermore, the loss of UT-B from both the vasculature andRBC also may help to explain the difference in concentratingability between the UT-A2 and UT-B knockout mice.
Urea Recycling and Regulation of GFR by High-Protein Diets
Consumption of diets that are rich in protein results in increasesin whole-kidney GFR (66,67). Microperfusion studies by Seneyet al. (68) determined that protein-induced increases in GFRresult from changes in the tubuloglomerular feedback (TGF) system.Their studies found that the sensing mechanism of the TGF systemwas rendered less responsive by high protein intake and thatthe diminished TGF was caused, at least in part, by a reducedearly distal NaCl concentration, without a change in early distaltubule osmolality (69). However, the cause of the reduced luminalNaCl concentration remains unknown. One model for this reducedearly distal NaCl concentration and the subsequently reducedGFR was proposed by Bankir et al. (70,71) and depends on changesin urea concentration in the fluid that is delivered to theTAL. This hypothesis posits that increased luminal concentrationsof urea, consequent to a high-protein diet, causes increasesin osmotic water secretion in the TAL, thereby lowering luminalNaCl concentration in the fluid that is delivered to the maculadensa. A lower NaCl generally is recognized to be a signal thatcan increase GFR via the TGF mechanism.
Increases in urea concentration in the TAL with dietary proteinexcess are believed to depend on both an increase in the ureaconcentration of the glomerular filtrate and an increase inthe extent of urea recycling. In UT-A1/3–/– mice,the drastically reduced urea reabsorption from the collectingduct and the consequent reduction in inner medullary interstitialurea concentration is likely to virtually eliminate urea recyclingand therefore is a useful model to address whether the mechanismthat was proposed by Bankir et al. is correct. In UT-A1/3–/–mice, one would predict that the increase in GFR in responseto high protein feeding would be attenuated markedly. For examinationof this, a series of clearance studies in conscious UT-A1/3–/–and wild-type control mice that were fed either a low-protein(4%) or a high-protein (40%) diet were performed. Increasingthe protein content of the diet approximately doubled the GFRin both UT-A1/3–/– mice and controls. Furthermore,under both dietary conditions, no significant differences wereobserved in the FITC-inulin clearance between UT-A1/3–/–and wild-type mice. The conclusion from these studies is thaturea reabsorption from the IMCD and, more specific, the processof urea recycling are not necessary elements of the overallprocess that is responsible for protein-induced increases inGFR.
Possible Role of Urea Transporters in Regulation of Extracellular Fluid Volume
As discussed, deletion of the two collecting duct urea transportersUT-A1 and UT-A3 in mice results in a urea-induced osmotic diuresis.It therefore seems plausible that regulating the activity orexpression of IMCD urea transporters could regulate indirectlywater and NaCl excretion by modulation of the extent of urea-inducedosmotic diuresis. Such a regulatory process would require feedbackmechanisms that alter collecting duct urea transport in responseto changes in extracellular fluid (ECF) volume. Therefore, itis pertinent to ask whether changes in ECF volume or tonicityare associated with regulation of urea transporters. Indeed,urea transporter expression was found to be downregulated inaldosterone-induced ECF volume expansion (72), in ECF volumeexpansion that is associated with nephrotic syndrome (73), inobese Zucker rats with type 2 diabetes and hypertension (74),in response to hypertension induced by angiotensin II or norepinephrine(75), and in an animal model of the syndrome of inappropriateantidiuresis (76). These decreases in urea transporter expressionpotentially could be a homeostatic response to ECF volume expansionor hypertension, increasing NaCl and water excretion via urea-inducedosmotic diuresis. Conversely, in the salt-sensitive Dahl rat,which is another model with ECF volume expansion and hypertension,the higher expression of urea transporters may be responsiblein part for the hypertension by reduction of urea-induced osmoticdiuresis (77). In this regard, it would be informative to addresswhether the hypertension that is seen in the salt-sensitiveDahl rat is altered by changes in dietary protein. Beyond this,an important goal for future research is to address the extentto which urea transporter regulation can affect ECF fluid volumeand BP by measuring these variables in knockout and wild-typemice on different levels of protein intake.
One of the limitations of knockout mouse studies is that othergenes/proteins potentially may compensate for the loss of thetargeted protein, thereby rendering the observed phenotype lessthan anticipated. This may be the case in both UT-A1/3–/–mice and UT-B knockout mice, in which targeted proteomic studiesusing an ensemble of antibodies have shown upregulation of otherproteins that are involved in the urinary concentrating mechanism.For example, UT-A2 is upregulated in UT-B knockout mice (78),potentially as a means of compensating for the diminished urearecycling, and both aquaporin-2 and aquaporin-3 are upregulatedin UT-A1/3–/– mice (42). These compensatory mechanismshave to be considered when addressing the role of the deletedgene in biological mechanisms.
Although "science for science’s sake" is a broadly heldconcept among basic scientists, the ultimate objective of studiesin basic physiology is to improve medical care. In this section,we discuss the potential relevance of the new findings thathave been obtained in urea transporter knockout mice with regardto clinical nephrology. First and foremost, the results presentedhere predict that drugs that are designed to inhibit urea transportershave considerable promise in the treatment of water and saltimbalance disorders. In the absence of urea transport in therenal collecting duct, urea is rendered an osmotic diureticwith the potential to increase the excretion of both water andNaCl. Therefore, a drug that could inhibit urea reabsorptioneffectively in the IMCD potentially could be used as an aquareticand diuretic agent. Such drugs potentially would be useful inthe treatment of hypertension or ECF volume–expanded disorderssuch as congestive heart failure and hepatic cirrhosis. Theunique aspect of urea transporter antagonists (unlike thiazides,loop diuretics, epithelial sodium channel blockers, or mineralocorticoidreceptor blockers) are that they are unlikely to cause secondarypotassium balance and acid-base disorders, owing to the locationof the IMCD downstream from the nephron sites where potassiumand acid-base transport are regulated. Existing urea transporterinhibitors such as phloretin and mercurial agents are too toxicto be clinically useful, although mercurials once were usedclinically as diuretics (79), presumably because of actionsat multiple sites to inhibit NaCl reabsorption. Amiloride hasbeen reported to inhibit urea transport in toad bladder (80).However, we have found that amiloride is ineffective as a ureatransport inhibitor in isolated perfused IMCD tubules whetheradded to the luminal perfusate or the peritubular bath (9).Accordingly, we suggest that investigation of potential ureatransporter inhibitors would be an appropriate target for drugdiscovery studies in which automated assays are used to screenthousands of compounds that are present in chemical libraries(81).
As yet, few examples of clinical abnormalities have arisen fromgenetic defects in urea transporters. Patients with mutationsin the UT-B gene have been identified by the absence of theKidd blood group antigen that normally is present on the surfaceof erythrocytes (82,83). Clinically, these UT-B null patientsare overtly normal, although they exhibit a mild concentratingdefect (84), possibly as a result of the absence of urea transporteractivity in their vasa recta and/or RBC. The abnormal concentratingdefect in UT-B null patients is similar to what is observedin UT-B knockout mice.
No mutations of the UT-A gene have been defined explicitly,although families with "familial azotemia" have been describedin Europe (85) and the United States (86), with the disorderpotentially due to abnormal activation of collecting duct ureatransport. Affected members of the US family exhibited highserum urea concentrations in the setting of normal serum creatininelevels and had normal urinary concentrating and diluting abilities.The high serum urea concentrations are associated with low ureaclearances, suggesting an abnormally high rate of urea absorptionat some point along the nephron. To our knowledge, none of thesepatients have undergone genetic testing to identify possiblemutations in the UT-A gene that could account for possible hyperactivationof UT-A1 and/or UT-A3 in the collecting duct.
Finally, a number of single-nucleotide polymorphisms that pointto a possible relationship between polymorphisms in the UT-Agene and BP regulation have been identified in the UT-A gene(87). Specifically, it was found that two of the seven identifiedpolymorphisms in UT-A1 or UT-A2 (Val/Ile at position 227 andAla/Thr at position 357) were associated with decreased diastolicBP in men but not women. These findings add credence to theidea that pharmacologic manipulation of UT-A proteins potentiallycan be effective in treatment of high BP.
The work described in this article was supported in part bythe Intramural Budget of the National Heart, Lung, and BloodInstitute (Project ZO1-HL-01285-KE) to M.A.K. The Water andSalt Research Center at the University of Aarhus is establishedand supported by the Danish National Research Foundation (DanmarksGrundforskningsfond). R.A.F. is supported by the Carlsberg Foundation(Carlsbergfondet), the Nordic Council (the Nordic Centre ofExcellence Programme in Molecular Medicine), and the DanishNational Research Foundation.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
See the related editorial, "Critical Role of Urea in the Urine-ConcentratingMechanism," on pages 670–671.
Shannon JA: Glomerular filtration and urea excretion in relation to urine flow in the dog.
Am J Physiol 117
: 206
–225, 1936[Free Full Text]
Shannon JA: Urea excretion in the normal dog during forced diuresis.
Am J Physiol 122
: 782
–787, 1938[Free Full Text]
Atherton JC, Hai MA, Thomas S: Effects of water diuresis and osmotic (mannitol) diuresis on urinary solute excretion by the conscious rat.
J Physiol 197
: 395
–410, 1968[Abstract/Free Full Text]
Gamble JL, McKhann CF, Butler AM, Tuthill E: An economy of water in renal function referable to urea.
Am J Physiol 109
: 139
–154, 1934[Free Full Text]
Gamble JL, Putnam MC, McKhann CF: The optimal water requirement in renal function: I. Measurements of water drinking by rats according to increments of urea and of several salts in the food.
Am J Physiol 88
: 571
–580, 1929[Free Full Text]
Grantham JJ, Burg MB: Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules.
Am J Physiol 211
: 255
–259, 1966[Free Full Text]
Sands JM, Knepper MA: Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium.
J Clin Invest 79
: 138
–147, 1987[Medline]
Sands JM, Nonoguchi H, Knepper MA: Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments.
Am J Physiol 253
: F823
–F832, 1987
Chou CL, Knepper MA: Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues.
Am J Physiol 257
: F359
–F365, 1989
Chou CL, Sands JM, Nonoguchi H, Knepper MA: Concentration dependence of urea and thiourea transport in rat inner medullary collecting duct.
Am J Physiol 258
: F486
–F494, 1990
Chou CL, Sands JM, Nonoguchi H, Knepper MA: Urea gradient-associated fluid absorption with sigma urea = 1 in rat terminal collecting duct.
Am J Physiol 258
: F1173
–F1180, 1990
Knepper MA, Sands JM, Chou CL: Independence of urea and water transport in rat inner medullary collecting duct.
Am J Physiol 256
: F610
–F621, 1989
Wall SM, Han JS, Chou CL, Knepper MA: Kinetics of urea and water permeability activation by vasopressin in rat terminal IMCD.
Am J Physiol 262
: F989
–F998, 1992
Nielsen S, Knepper MA: Vasopressin activates collecting duct urea transporters and water channels by distinct physical processes.
Am J Physiol 265
: F204
–F213, 1993
Fenton RA, Cottingham CA, Stewart GS, Howorth A, Hewitt JA, Smith CP: Structure and characterization of the mouse UT-A gene (Slc14a2).
Am J Physiol Renal Physiol 282
: F630
–F638, 2002[Abstract/Free Full Text]
Lucien N, Sidoux-Walter F, Olives B, Moulds J, Le Pennec PY, Cartron JP, Bailly P: Characterization of the gene encoding the human Kidd blood group/urea transporter protein. Evidence for splice site mutations in Jknull individuals.
J Biol Chem 273
: 12973
–12980, 1998[Abstract/Free Full Text]
Nakayama Y, Naruse M, Karakashian A, Peng T, Sands JM, Bagnasco SM: Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter.
Biochim Biophys Acta 1518
: 19
–26, 2001[Medline]
Fenton RA, Howorth A, Cooper GJ, Meccariello R, Morris ID, Smith CP: Molecular characterization of a novel UT-A urea transporter isoform (UT- A5) in testis.
Am J Physiol Cell Physiol 279
: C1425
–C1431, 2000[Abstract/Free Full Text]
Fenton RA, Stewart GS, Carpenter B, Howorth A, Potter EA, Cooper GJ, Smith CP: Characterization of mouse urea transporters UT-A1 and UT-A2.
Am J Physiol Renal Physiol 283
: F817
–F825, 2002[Abstract/Free Full Text]
Karakashian A, Timmer RT, Klein JD, Gunn RB, Sands JM, Bagnasco SM: Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4.
J Am Soc Nephrol 10
: 230
–237, 1999[Abstract/Free Full Text]
Shayakul C, Steel A, Hediger MA: Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts.
J Clin Invest 98
: 2580
–2587, 1996[Medline]
Smith CP, Lee WS, Martial S, Knepper MA, You G, Sands JM, Hediger MA: Cloning and regulation of expression of the rat kidney urea transporter (rUT2).
J Clin Invest 96
: 1556
–1563, 1995[Medline]
Smith CP, Potter EA, Fenton RA, Stewart GS: Characterization of a human colonic cDNA encoding a structurally novel urea transporter, hUT-A6.
Am J Physiol Cell Physiol 287
: C1087
–C1093, 2004[Abstract/Free Full Text]
Olives B, Neau P, Bailly P, Hediger MA, Rousselet G, Cartron JP, Ripoche P: Cloning and functional expression of a urea transporter from human bone marrow cells.
J Biol Chem 269
: 31649
–31652, 1994[Abstract/Free Full Text]
You G, Smith CP, Kanai Y, Lee WS, Stelzner M, Hediger MA: Cloning and characterization of the vasopressin-regulated urea transporter.
Nature 365
: 844
–847, 1993[CrossRef][Medline]
Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, Knepper MA: Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney.
Proc Natl Acad Sci U S A 93
: 5495
–5500, 1996[Abstract/Free Full Text]
Frohlich O, Klein JD, Smith PM, Sands JM, Gunn RB: Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells.
Am J Physiol Cell Physiol 291
: C600
–C606, 2006[Abstract/Free Full Text]
Shayakul C, Knepper MA, Smith CP, DiGiovanni SR, Hediger MA: Segmental localization of urea transporter mRNAs in rat kidney.
Am J Physiol 272
: F654
–F660, 1997
Wade JB, Lee AJ, Liu J, Ecelbarger CA, Mitchell C, Bradford AD, Terris J, Kim GH, Knepper MA: UT-A2: A 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin.
Am J Physiol Renal Physiol 278
: F52
–F62, 2000[Abstract/Free Full Text]
Brooks HL, Ageloff S, Kwon TH, Brandt W, Terris JM, Seth A, Michea L, Nielsen S, Fenton R, Knepper MA: cDNA array identification of genes regulated in rat renal medulla in response to vasopressin infusion.
Am J Physiol Renal Physiol 284
: F218
–F228, 2003[Abstract/Free Full Text]
Potter EA, Stewert G, Smith CP: Urea flux across MDCK-mUT-A2 monolayers is acutely sensitive to AVP, cAMP and [Ca2+]i.
Am J Physiol Renal Physiol 291
: F122
–F128, 2006[Abstract/Free Full Text]
Stewart GS, Fenton RA, Wang W, Kwon TH, White SJ, Collins VM, Cooper G, Nielsen S, Smith CP: The basolateral expression of mUT-A3 in the mouse kidney.
Am J Physiol Renal Physiol 286
: F979
–F987, 2004[Abstract/Free Full Text]
Tsukaguchi H, Shayakul C, Berger UV, Tokui T, Brown D, Hediger MA: Cloning and characterization of the urea transporter UT3: Localization in rat kidney and testis.
J Clin Invest 99
: 1506
–1515, 1997
Xu Y, Olives B, Bailly P, Fischer E, Ripoche P, Ronco P, Cartron JP, Rondeau E: Endothelial cells of the kidney vasa recta express the urea transporter HUT11.
Kidney Int 51
: 138
–146, 1997[Medline]
Promeneur D, Rousselet G, Bankir L, Bailly P, Cartron JP, Ripoche P, Trinh-Trang-Tan MM: Evidence for distinct vascular and tubular urea transporters in the rat kidney.
J Am Soc Nephrol 7
: 852
–860, 1996[Abstract]
Trinh-Trang-Tan MM, Lasbennes F, Gane P, Roudier N, Ripoche P, Cartron JP, Bailly P: UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney.
Am J Physiol Renal Physiol 283
: F912
–F922, 2002[Abstract/Free Full Text]
Fenton RA, Chou CL, Stewart GS, Smith CP, Knepper MA: Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct.
Proc Natl Acad Sci U S A 101
: 7469
–7474, 2004[Abstract/Free Full Text]
Berliner RW, Levinsky NG, Davidson DG, Eden M: Dilution and concentration of the urine and the action of antidiuretic hormone.
Am J Med 24
: 730
–744, 1958[CrossRef][Medline]
Berliner RW, Bennett CM: Concentration of urine in the mammalian kidney.
Am J Med 42
: 777
–789, 1967[CrossRef][Medline]
Jamison RL, Bennett CM, Berliner RW: Countercurrent multiplication by the thin loops of Henle.
Am J Physiol 212
: 357
–366, 1967[Free Full Text]
Fenton RA, Flynn A, Shodeinde A, Smith CP, Schnermann J, Knepper MA: Renal phenotype of UT-A urea transporter knockout mice.
J Am Soc Nephrol 16
: 1583
–1592, 2005[Abstract/Free Full Text]
Kokko JP, Rector FC: Countercurrent multiplication system without active transport in inner medulla.
Kidney Int 2
: 214
–223, 1972[Medline]
Stephenson JL: Concentration of urine in a central core model of the renal counterflow system.
Kidney Int 2
: 85
–94, 1972[Medline]
Ullrich KJ, Jarausch KH: Studies on the problem of urine concentration and dilution; distribution of electrolytes (sodium, potassium, calcium, magnesium, anorganic phosphate), urea amino acids and exogenous creatinine in the cortex and medulla of dog kidney in various diuretic conditions [in German].
Pflugers Arch 262
: 537
–550, 1956[CrossRef][Medline]
Kuhn W, Ramel A: Activer Salztransport als moeglicher (und wahrscheinlicher) Einzeleffekt bei der Harnkonzentrierung in der Niere.
Helv Chim Acta 42
: 628
–660, 1959[CrossRef]
Burg MB, Green N: Function of the thick ascending limb of Henle’s loop.
Am J Physiol 224
: 659
–668, 1973[Free Full Text]
Rocha AS, Kokko JP: Sodium chloride and water transport in the medullary thick ascending limb of Henle.
J Clin Invest 52
: 612
–624, 1973[Medline]
Imai M, Kokko JP: Sodium chloride, urea, and water transport in the thin ascending limb of Henle. Generation of osmotic gradients by passive diffusion of solutes.
J Clin Invest 53
: 393
–402, 1974[Medline]
Kondo Y, Abe K, Igarashi Y, Kudo K, Tada K, Yoshinaga K: Direct evidence for the absence of active Na+ reabsorption in hamster ascending thin limb of Henle’s loop.
J Clin Invest 91
: 5
–11, 1993[Medline]
Gamba G, Knepper MA: Urine concentration and dilution. In:
Brenner and Rector’s The Kidney, 7th Ed., edited by Brenner BM, Philadelphia, W.B. Saunders, 2004
, pp 599
–636
Knepper MA, Saidel GM, Hascall VC, Dwyer T: Concentration of solutes in the renal inner medulla: Interstitial hyaluronan as a mechano-osmotic transducer.
Am J Physiol Renal Physiol 284
: F433
–F446, 2003[Abstract/Free Full Text]
Fenton RA, Chou CL, Sowersby H, Smith CP, Knepper MA: Gamble’s "economy of water" revisited: Studies in urea transporter knockout mice.
Am J Physiol Renal Physiol 291
: F148
–F154, 2006[Abstract/Free Full Text]
Knepper MA, Roch-Ramel F: Pathways of urea transport in the mammalian kidney.
Kidney Int 31
: 629
–633, 1987[Medline]
Lassiter WE, Gottschalk CW, Mylle M: Micropuncture study of net transtubular movement of water and urea in nondiuretic mammalian kidney.
Am J Physiol 200
: 1139
–1147, 1961[Abstract/Free Full Text]
Lemley KV, Kriz W: Cycles and separations: The histotopography of the urinary concentrating process.
Kidney Int 31
: 538
–548, 1987[Medline]
Zhai XY, Thomsen JS, Birn H, Kristoffersen IB, Andreasen A, Christensen EI: Three-dimensional reconstruction of the mouse nephron.
J Am Soc Nephrol 17
: 77
–88, 2006[Abstract/Free Full Text]
Uchida S, Sohara E, Rai T, Ikawa M, Okabe M, Sasaki S: Impaired urea accumulation in the inner medulla of mice lacking the urea transporter UT-A2.
Mol Cell Biol 25
: 7357
–7363, 2005[Abstract/Free Full Text]
Yang B, Bankir L, Gillespie A, Epstein CJ, Verkman AS: Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.
J Biol Chem 277
: 10633
–10637, 2002[Abstract/Free Full Text]
Yang B, Bankir L: Urea and urine concentrating ability: New insights from studies in mice.
Am J Physiol Renal Physiol 288
: F881
–F896, 2005[Abstract/Free Full Text]
Bankir L, Chen K, Yang B: Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement of urinary concentrating ability.
Am J Physiol Renal Physiol 286
: F144
–F151, 2004[Abstract/Free Full Text]
Lassiter WE, Mylle M, Gottschalk CW: Micropuncture study of urea transport in rat renal medulla.
Am J Physiol 210
: 965
–970, 1966[Free Full Text]
Timmer RT, Klein JD, Bagnasco SM, Doran JJ, Verlander JW, Gunn RB, Sands JM: Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues.
Am J Physiol Cell Physiol 281
: C1318
–C1325, 2001[Abstract/Free Full Text]
Macey RI, Yousef LW: Osmotic stability of red cells in renal circulation requires rapid urea transport.
Am J Physiol 254
: C669
–C674, 1988
Macey RI: Transport of water and urea in red blood cells.
Am J Physiol 246
: C195
–C203, 1984
Dicker SE: Effect of the protein content of the diet on the glomerular filtration rate of young and adult rats.
J Physiol 108
: 197
–202, 1949
Top
Abstract
Introduction
