Molecular approaches to urea transporters

Urea plays a critical role in the urine-concentrating mechanism in the inner medulla. Physiologic data provided evidence that urea transport in red blood cells and kidney inner medulla was mediated by specific urea transporter proteins. Molecular approaches during the past decade resulted in the cloning of two gene families for facilitated urea transporters, UT-A and UT-B, encoding several urea transporter cDNA isoforms in humans, rodents, and several nonmammalian species. Polyclonal antibodies have been generated to the cloned urea transporter proteins, and the use of these antibodies in integrative animal studies has resulted in several novel findings, including: (1) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; (2) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (3) UT-A protein abundance is upregulated in uremia in both liver and heart; and (4) UT-B is expressed in many nonrenal tissues and endothelial cells. This review will summarize the knowledge gained from using molecular approaches to perform integrative studies into urea transporter protein regulation, both in normal animals and in animal models of human diseases, including studies of uremic rats in which urea transporter protein is upregulated in liver and heart.     

Aldosterone decreases UT-A1 urea transporter expression via the mineralocorticoid receptor

Adrenalectomy in rats is associated with urinary concentrating and diluting defects. This study tested the effect of adrenal steroids on the UT-A1 urea transporter because it is involved in the urine-concentrating mechanism. Rats were adrenalectomized and given normal saline for 14 d, after which they received (1) vehicle, (2) aldosterone, or (3) spironolactone plus aldosterone. Adrenalectomy alone significantly increased UT-A1 protein in the inner medullary tip after 7 d, whereas aldosterone repletion reversed the effect. Spironolactone blocked the aldosterone-induced decrease in UT-A1, indicating that aldosterone was working via the mineralocorticoid receptor. For verifying that glucocorticoids downregulate UT-A1 protein through a different receptor, three groups of adrenalectomized rats were prepared: (1) vehicle, (2) adrenalectomy plus dexamethasone, and (3) adrenalectomy plus dexamethasone and spironolactone. Dexamethasone significantly reversed UT-A1 protein abundance increase in the inner medullary tip of adrenalectomized rats. When spironolactone was given with dexamethasone, it did not affect the dexamethasone-induced decrease in UT-A1. There was no significant change in serum vasopressin level, aquaporin 2, or Na(+)-K(+)-2Cl(-) co-transporter NKCC2/BSC1 protein abundances or UT-A1 mRNA abundance in any of the groups. In conclusion, either mineralocorticoids or glucocorticoids can downregulate UT-A1 protein. The decrease in UT-A1 does not require both steroid hormones, and each works through a different receptor.     

Regulation of renal urea transporters

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.     

Urea and renal function in the 21st century: insights from knockout mice

Since the turn of the 21st century, gene knockout mice have been created for all major urea transporters that are expressed in the kidney: the collecting duct urea transporters UT-A1 and UT-A3, the descending thin limb isoform UT-A2, and the descending vasa recta isoform UT-B. This article discusses the new insights that the results from studies in these mice have produced in the understanding of the role of urea in the urinary concentrating mechanism and kidney function. Following is a summary of the major findings: (1) Urea accumulation in the inner medullary interstitium depends on rapid transport of urea from the inner medullary 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 is to prevent a urea-induced osmotic diuresis; (3) the absence of IMCD urea transport does not prevent the concentration of NaCl in the inner medulla, contrary to what would be predicted from the passive countercurrent multiplier mechanism in the form proposed by Kokko and Rector and Stephenson; (4) deletion of UT-B (vasa recta isoform) has a much greater effect on urinary concentration than deletion of UT-A2 (descending limb isoform), suggesting that the recycling of urea between the vasa recta and the renal tubules quantitatively is less important than classic countercurrent exchange; and (5) urea reabsorption from the IMCD and the process of urea recycling are not important elements 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 target collecting duct urea transporters have the potential for clinical use as potassium-sparing diuretics that function by creation of urea-dependent osmotic diuresis.     

Urea transporters and renal function: lessons from knockout mice

PURPOSE OF REVIEW: Gene knockout mice have been created for the collecting duct urea transporters UT-A1 and UT-A3, the descending thin-limb urea transporter UT-A2 and the descending vasa recta isoform, UT-B. In this brief review, the new insights in our understanding of the role of urea in the urinary concentrating mechanism and kidney function resulting from studies in these mice are discussed. RECENT FINDINGS: The major findings in studies on urea transporter knockout mice are as follows: rapid transport of urea from the inner medulla collecting duct lumen via UT-A1 or UT-A3 is essential for urea accumulation in the inner medullary interstitium; inner medulla collecting duct urea transporters are essential in water conservation by preventing urea-induced osmotic diuresis; an absence of inner medulla collecting duct urea transport does not prevent the concentration of sodium chloride in the inner medulla interstitium; deletion of the vasa recta isoform UT-B has a much greater effect on urinary concentration than deleting the descending limb isoform UT-A2. SUMMARY: Multiple urea transport mechanisms within the kidney are essential for producing maximally concentrated urine.     

Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4

Urea transport in the kidney is important for the production of concentrated urine and is mediated by a family of transporter proteins, identified from erythropoietic tissue (UT-B) and from kidney (UT-A). Two isoforms of the renal urea transporter (UT-A) have been cloned so far: UT-A1 and UT-A2. We used rapid amplification of cDNA ends to clone two new isoforms of the rat UT-A transporter: UT-A3 and UT-A4. UT-A3 and UT-A4 are 87% homologous. The UT-A3 cDNA encodes a peptide of 460 amino acids, which corresponds to the amino-terminal half of the UT-A1 peptide and is 62% identical to UT-A2. The UT-A4 cDNA encodes a peptide of 466 amino acids, which is 84% identical to UT-A2. Transient transfection of HEK-293 cells with the UT-A3 or UT-A4 cDNA results in phloretin-inhibitable urea uptake, which is increased by forskolin. Thus, both new isoforms encode functional urea transporters that may be vasopressin-regulated. UT-A3 and UT-A4 mRNA are expressed in the renal outer and inner medulla but not in the cortex; unidentified UT-A isoforms similar to UT-A3 may also be expressed in the testis. It is concluded that there are at least four different rat UT-A urea transporters.     

Down-regulation of urea transporters in the renal inner medulla of lithium-fed rats

BACKGROUND: Lithium is commonly used to treat bipolar psychiatric disorders but can cause reduced urine concentrating ability. METHODS: To test whether lithium alters UT-A1 or UT-B urea transporter protein abundance or UT-A1 phosphorylation, rats were fed a standard diet supplemented with LiCl for 10 or 25 days, and then compared to pair-fed control rats. To investigate another potential mechanism for decreased urea transport, inner medullary collecting duct (IMCD) suspensions from lithium-fed or control rats were incubated with 32P-orthophosphate to measure the phosphorylation of UT-A1. RESULTS: In lithium-fed rats (25 days), UT-A1 abundance was reduced to 50% of control rats in IM tip and to 25% in IM base, and UT-B abundance was reduced to 40% in IM base. Aquaporin-2 (AQP2) protein abundance was reduced in both IM regions. Vasopressin (100 pmol/L) increased UT-A1 phosphorylation in IMCD suspensions from control but not from lithium-fed rats; a higher vasopressin concentration (100 nmol/L) increased UT-A1 phosphorylation in control and lithium-fed rats. CONCLUSIONS: Decreases in UT-A1, UT-B, and AQP2 protein abundance, and/or vasopressin-stimulated phosphorylation of UT-A1, can contribute to the reduced urine concentrating ability that occurs in lithium-treated rats.     

Vasopressin increases plasma membrane accumulation of urea transporter UT-A1 in rat inner medullary collecting ducts

Urea transport, mediated by the urea transporter A1 (UT-A1) and/or UT-A3, is important for the production of concentrated urine. Vasopressin rapidly increases urea transport in rat terminal inner medullary collecting ducts (IMCD). A previous study showed that one mechanism for rapid regulation of urea transport is a vasopressin-induced increase in UT-A1 phosphorylation. This study tests whether vasopressin or directly activating adenylyl cyclase with forskolin also increases UT-A1 accumulation in the plasma membrane of rat IMCD. Inner medullas were harvested from rats 45 min after injection with vasopressin or vehicle. UT-A1 abundance in the plasma membrane was significantly increased in the membrane fraction after differential centrifugation and in the biotinylated protein population. Vasopressin and forskolin each increased the amount of biotinylated UT-A1 in rat IMCD suspensions that were treated ex vivo. The observed changes in the plasma membrane are specific, as the amount of biotinylated UT-A1 but not the calcium-sensing receptor was increased by forskolin. Next, whether forskolin or the V(2)-selective agonist dDAVP would increase apical membrane expression of UT-A1 in MDCK cells that were stably transfected with UT-A1 (UT-A1-MDCK cells) was tested. Forskolin and dDAVP significantly increased UT-A1 abundance in the apical membrane in UT-A1-MDCK cells. It is concluded that vasopressin and forskolin increase UT-A1 accumulation in the plasma membrane in rat IMCD and in the apical plasma membrane of UT-A1-MDCK cells. These findings suggest that vasopressin regulates urea transport by increasing UT-A1 accumulation in the plasma membrane and/or UT-A1 phosphorylation.     

Expression of salt and urea transporters in rat kidney during cisplatin-induced polyuria.

BACKGROUND: Cisplatin (CP) induced polyuria in rats is associated with a reduction in medullary hypertonicity, normally generated by the thick ascending limb (TAL) salt transporters, and the collecting duct urea transporters (UT). To investigate the molecular basis of this abnormality, we determined the protein abundance of major salt and UT isoforms in rat kidney during CP-induced polyuria. METHODS: Male Sprague-Dawley rats received either a single injection of CP (5 mg/kg, N = 6) or saline (N = 6) intraperitoneally five days before sacrifice. Urine, blood, and kidneys were collected and analyzed. RESULTS: CP-treated rats developed polyuric acute renal failure as assessed by increased blood urea nitrogen (BUN), urine volume and decreased urine osmolality. Western analysis of kidney homogenates revealed a marked reduction in band density of the bumetanide-sensitive Na-K-2Cl cotransporter in cortex (60% of control values, P < 0.05), but not in outer medulla (OM) (106% of control values). There were no differences in band densities for the renal outer medullary potassium channel (ROMK), the type III Na-H exchanger (NHE3), the alpha-subunit of Na,K-ATPase in the OM; or for UT-A1, UT-A2 or UT-A4 in outer or inner medulla. However, the band pattern of UT-A2 and UT-A4 proteins in the OM of CP-treated rats was different from the control rats, suggesting a qualitative modification of these proteins. CONCLUSIONS: Changes in the abundance of outer or inner medullary salt or urea transporters are unlikely to play a role in the CP-induced reduction in medullary hypertonicity. However, qualitative changes in UT proteins may affect their functionality and thus may have a role.     

Renal phenotype of UT-A urea transporter knockout mice

The urea transporters UT-A1 and UT-A3 mediate rapid transepithelial urea transport across the inner medullary collecting duct (IMCD). In a previous study, using a new mouse model in which both UT-A1 and UT-A3 were genetically deleted from the IMCD (UT-A1/3(-/-) mice), we investigated the role of these transporters in the function of the renal inner medulla. Here the authors report a new series of studies investigating more generally the renal phenotype of UT-A1/3(-/-) mice. Pathologic screening of 33 tissues revealed abnormalities in both the testis (increased size) and kidney (decreased size and vascular congestion) of UT-A1/3(-/-) mice. Total urinary nitrate and nitrite (NOx) excretion rates in UT-A1/3(-/-) mice were more than double those in wild-type mice. Total renal blood flow was not different between UT-A1/3(-/-) and wild-type mice but underwent a greater percentage decrease in response to NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME) infusion. Whole kidney GFR (FITC-inulin clearance) was not different in UT-A1/3(-/-) mice compared with controls and underwent a similar increase in response to a greater dietary protein intake. Fractional urea excretion was markedly elevated in UT-A1/3(-/-) mice on a 40% protein diet, reaching 102.4 +/- 8.8% of the filtered load, suggesting that there may be active urea secretion somewhere along the renal tubule. Although there was a marked urinary concentrating defect in UT-A1/3(-/-) mice, there was no decrease in aquaporin 2 or aquaporin 3 expression. Furthermore, although urea accumulation in the inner medulla was markedly attenuated, there was no decrease in sodium ion concentration in tissue from outer medulla or two levels of the inner medulla. These results support our conclusion that the urinary concentrating defect in UT-A1/3(-/-) mice is caused by a failure of urea transport from the IMCD lumen to the inner medullary interstitium, resulting in osmotic diuresis.     

Acidosis mediates the upregulation of UT-A protein in livers from uremic rats

Liver expresses a 49-kD UT-A protein whose abundance is increased by uremia. Chronic renal failure causes acidosis; therefore, the role of acidosis in increasing UT-A abundance was tested. Rats underwent 5/6 nephrectomy, and half were given bicarbonate mixed in their food. Bicarbonate administration significantly increased blood pH. Compared with sham-operated rats, UT-A protein abundance was significantly increased by 50% in livers from uremic, acidotic rats; bicarbonate administration prevented the increase in UT-A protein. To determine whether acidosis alone would increase UT-A protein in liver, rats were made acidotic, but not uremic, by feeding them HCl. HCl-feeding significantly lowered blood pH, increased urea excretion, and increased the abundance of the 49-kD liver UT-A protein by 36% compared with pair-fed nonacidotic rats. HCl-feeding significantly increased the abundance of the 117-kD UT-A1 protein in kidney inner medulla but did not change aquaporin-2 protein. Next, rats were fed urea to determine whether elevated blood urea would increase UT-A protein. However, urea feeding had no effect on UT-A in liver or kidney inner medulla. It was, therefore, concluded that acidosis, either directly or through a change in ammonium concentration, rather than other dietary components, stimulates the upregulation of UT-A protein in liver and kidney inner medulla.     

Massive reduction of urea transporters in remnant kidney and brain of uremic rats

BACKGROUND: The facilitated urea transporters (UT), UT-A1, UT-A2, and UT-B1, are involved in intrarenal recycling of urea, an essential feature of the urinary concentrating mechanism, which is impaired in chronic renal failure (CRF). In this study, the expression of these UTs was examined in experimentally induced CRF. METHODS: The abundance of mRNA was measured by Northern analysis and that of corresponding proteins by Western blotting in rats one and five weeks after 5/6 nephrectomy (Nx). RESULTS: At five weeks, urine output was enhanced threefold with a concomitant decrease in urine osmolality. The marked rise in plasma urea concentration and fall in urinary urea concentration resulted in a 30-fold decrease in the urine/plasma (U/P) urea concentration ratio, while the U/P osmoles ratio fell only fourfold. A dramatic decrease in mRNA abundance for the three UTs was observed, bringing their level at five weeks to 1/10th or less of control values. Immunoblotting showed complete disappearance of the 97 and 117 kD bands of UT-A1, and considerable reduction of UT-A2 and UT-B1 in the renal medulla. Similar, but less intense, changes were observed at one-week post-Nx. In addition to the kidney, UT-B1 is also normally expressed in brain and testis. In the brain, its mRNA expression remained normal one-week post-Nx, but decreased to about 30% of normal at five-weeks post-Nx, whereas no change was seen in testis. CONCLUSIONS: (1) The decline in urinary concentrating ability seen in CRF is largely due to a major reduction of UTs involved in the process of urea concentration in the urine, while factors enabling the concentration of other solutes are less intensely affected. (2) The marked reduction of brain UT expression in CRF may be responsible for brain edema of dialysis disequilibrium syndrome observed in some patients after fast dialysis.     

Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

The urea transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC-α regulates UT-A1 urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC-α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC-α–deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC-α–induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.     

Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.

This study tested whether glucocorticoids regulate tubular urea transport. Urea permeability was measured in perfused inner medullary collecting duct (IMCD) subsegments from rats that underwent adrenalectomy, adrenalectomy plus replacement with a physiologic dose of glucocorticoid (dexamethasone), or sham operation. Compared with sham rats, basal urea permeability in terminal IMCD was significantly increased in adrenalectomized rats and reduced in dexamethasone-treated rats. Vasopressin significantly increased urea permeability in all three groups. In contrast, there was no difference in basal or vasopressin-stimulated urea permeability in initial IMCD between the three groups. Next, membrane and vesicle fraction proteins were isolated from inner medullary tip or base and Western analysis was performed by use of an antibody to the rat vasopressin-regulated urea transporter. Vasopressin-regulated urea transporter protein was significantly increased in both membrane and vesicle fractions from the inner medullary tip of adrenalectomized rats. There was no change in vasopressin-regulated urea transporter protein in the inner medullary base, and Northern analysis showed no change in urea transporter mRNA abundance in either inner medullary region. It was concluded that glucocorticoids can downregulate function and expression of the vasopressin-regulated urea transporter in rat terminal IMCD.     

Triazolothienopyrimidine inhibitors of urea transporter UT-B reduce urine concentration

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.     

Molecular mechanisms of impaired urinary concentrating ability in glucocorticoid-deficient rats

The purpose of this study was to examine urinary concentrating ability and protein expression of renal aquaporins and ion transporters in glucocorticoid-deficient (GD) rats in response to water deprivation as compared with control rats. Rats underwent bilateral adrenalectomies, followed only by aldosterone replacement (GD) or both aldosterone and dexamethasone replacement (control). As compared with control rats, the GD rats demonstrated a decrease in cardiac output and mean arterial pressure. In response to 36-h water deprivation, GD rats demonstrated significantly greater urine flow rate and decreased urine osmolality as compared with control rats at comparable serum osmolality and plasma vasopressin concentrations. The initiator of the countercurrent concentrating mechanism, the sodium-potassium-2 chloride co-transporter, was significantly decreased, as was the medullary osmolality in the GD rats versus control rats. There was also a decrease in inner medulla aquaporin-2 (AQP2) and urea transporter A1 (UT-A1) in GD rats as compared with control rats. There was a decrease in outer medulla Gsalpha protein, an important factor in vasopressin-mediated regulation of AQP2. Immunohistochemistry studies confirmed the decreased expression of AQP2 and UT-A1 in kidneys of GD rats as compared with control. In summary, impairment in the urinary concentrating mechanism was documented in GD rats in association with impaired countercurrent multiplication, diminished osmotic equilibration via AQP2, and diminished urea equilibration via UT-A1. These events occurred primarily in the relatively oxygen-deficient medulla and may have been initiated, at least in part, by the decrease in mean arterial pressure and thus renal perfusion pressure in this area of the kidney.     

"Avian-type" renal medullary tubule organization causes immaturity of urine-concentrating ability in neonates

BACKGROUND: While neonatal kidneys are not powerful in concentrating urine, they already dilute urine as efficiently as adult kidneys. To elucidate the basis for this paradoxical immaturity in urine-concentrating ability, we investigated the function of Henle's loop and collecting ducts (IMCDs) in the inner medulla of neonatal rat kidneys. METHODS: Analyses of individual renal tubules in the inner medulla of neonatal and adult rat kidneys were performed by measuring mRNA expression of membrane transporters, transepithelial voltages, and isotopic water and ion fluxes. Immunofluorescent identification of the rCCC2 and rCLC-K1 using polyclonal antibodies was also performed in neonatal and adult kidney slices. RESULTS: On day 1, the transepithelial voltages (V(Ts)) in the thin ascending limbs (tALs) and IMCDs were 14.6 +/- 1.1 mV (N = 27) and -42.7 +/- 6.1 mV (N = 14), respectively. The V(Ts) in the thin descending limbs (tDLs) were zero on day 1. The V(Ts) in the tALs were strongly inhibited by luminal bumetanide or basolateral ouabain, suggesting the presence of a NaCl reabsorption mechanism similar to that in the thick ascending limb (TAL). The diffusional voltage (V(D)) of the tAL due to transepithelial NaCl gradient was almost insensitive to a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The V(Ts) in the IMCDs were strongly inhibited by luminal amiloride. On day 1, both the tDL and tAL were impermeable to water, indicating the water impermeability of the entire loop. Diffusional water permeability (P(dw)) and urea permeabilities (P(urea)) in the IMCDs indicated virtual impermeability to water and urea on day 1. Stimulation by vasopressin (1 nmol/L) revealed that only P(dw) was sensitive to vasopressin by day 14. A partial isoosmolar replacement of luminal urea by NaCl evoked negligible water flux across the neonatal IMCDs, indicating the absence of urea-dependent volume flux in the neonatal IMCD. These transport characteristics in each neonatal tubule are similar to those in quail kidneys. Identification of mRNAs and immunofluorescent studies for specific transporters, including rAQP-1, rCCC2, rCLC-K1, rENaC beta subunit, rAQP-2, and rUT-A1, supported these findings. CONCLUSION: We hypothesize that the renal medullary tubule organization of neonatal rats shares a tremendous similarity with avian renal medulla. The qualitative changes in the organization of medullary tubules may be primarily responsible for the immature urine-concentrating ability in mammalian neonates.     

New insights into urea and glucose handling by the kidney, and the urine concentrating mechanism

The mechanism by which urine is concentrated in the mammalian kidney remains incompletely understood. Urea is the dominant urinary osmole in most mammals and may be concentrated a 100-fold above its plasma level in humans and even more in rodents. Several facilitated urea transporters have been cloned. The phenotypes of mice with deletion of the transporters expressed in the kidney have challenged two previously well-accepted paradigms regarding urea and sodium handling in the renal medulla but have provided no alternative explanation for the accumulation of solutes that occurs in the inner medulla. In this review, we present evidence supporting the existence of an active urea secretion in the pars recta of the proximal tubule and explain how it changes our views regarding intrarenal urea handling and UT-A2 function. The transporter responsible for this secretion could be SGLT1, a sodium-glucose cotransporter that also transports urea. Glucagon may have a role in the regulation of this secretion. Further, we describe a possible transfer of osmotic energy from the outer to the inner medulla via an intrarenal Cori cycle converting glucose to lactate and back. Finally, we propose that an active urea transporter, expressed in the urothelium, may continuously reclaim urea that diffuses out of the ureter and bladder. These hypotheses are all based on published findings. They may not all be confirmed later on, but we hope they will stimulate further research in new directions.