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.     

Chronic COX-2 inhibition reduces medullary HSP70 expression and induces papillary apoptosis in dehydrated rats

BACKGROUND: Papillary cells adapt to their hyperosmotic environment by accumulating organic osmolytes and by enhanced synthesis of heat shock protein 70 (HSP70), which protect against high-solute concentrations. Because cyclooxygenase-2 (COX-2) is expressed abundantly in the renal papilla and is induced by dehydration, and because HSP70 expression is stimulated by specific prostaglandins, COX-2 inhibition may interfere with cellular osmoadaptation. METHODS: In vivo, rats received rofecoxib before water deprivation. Medullary expression of several tonicity-responsive genes was analyzed and apoptosis was monitored by transferase-mediated dUTP nick-end labeled (TUNEL) staining and determination of papillary caspase-3 activity. In vitro, inner medullary collecting duct 3 (IMCD3) cells were exposed to hypertonic medium containing a COX-2-specific inhibitor. Thereafter, expression of tonicity-responsive genes was analyzed and resistance to high-solute concentrations was examined. Further, the effect of Delta 12-PGJ2, a urinary prostaglandin, and of HSP70 overexpression on resistance against high urea concentration, was evaluated. RESULTS: Rofecoxib treatment significantly increased urine osmolality due to higher urea concentrations, but reduced papillary HSP70 abundance by 50%. TUNEL staining showed numerous apoptotic cells in the papilla, associated with increased caspase-3 activity. These in vivo results were confirmed by experiments on cultured IMCD3 cells, in which COX-2 inhibition impaired the tonicity-induced up-regulation of HSP70 expression and rendered the cells susceptible to high urea concentrations. Furthermore, Delta 12-PGJ2 increased both HSP70 expression and resistance against high urea, which was causally linked to higher HSP70 levels. CONCLUSION: These observations support the view that chronic COX-2 inhibition reduces medullary HSP70 expression, thus rendering papillary cells susceptible to damage by high urea concentrations, especially when accompanied by dehydration.     

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.     

Urinary concentrating ability in patients with Jk(a-b-) blood type who lack carrier-mediated urea transport.

Water homeostasis is regulated in large part by the proper operation of the urinary concentrating mechanism. In the renal inner medulla, urea recycling from the inner medullary collecting duct to the inner medullary interstitium is thought to be essential for the production of a concentrated urine; however, it has not been possible to test this hypothesis in humans. Recently, a unique combination of genetic abnormalities has been described: absence of Kidd blood group antigens and absence of carrier-mediated urea transport in erythrocytes. Because animal studies indicate a similarity between urea transport in red blood cells and the nephron, it was postulated that patients without the Kidd antigen might lack facilitated urea transport in their kidneys. Hence, their ability to concentrate urine maximally was measured. Current models of nephron function would predict that in the complete absence of urea transport, the maximal concentrating ability would be around 800 to 900 mosM/kg H2O. Two homozygous patients had a moderate decrease in maximal concentrating ability (UosM,max = 819 mosM/kg H2O); a heterozygote also had some limitation. These studies raise the possibility that the erythrocyte urea transporter and the kidney urea transporter are encoded by a single gene (detected by the mutational loss of the Kidd antigen) and that a lack of facilitated urea transport impairs urea recycling in the kidney and, hence, maximal urinary concentrating ability.     

Cortical and papillary absorptive defects in gentamicin nephrotoxicity

Renal function was examined in rats given daily injections of gentamicin (100 to 150 mg/kg) for 10 to 14 days. Whole kidney inulin clearance fell and urine volume increased. Single nephron GFR of surface nephrons varied. Some nephrons had no filtration, some had low rates, and some had high rates. Abnormal renal tubular epithelial inulin permeability was demonstrated by microinjection. Micropuncture of individual nephrons early and later in their course demonstrated reduced fluid reabsorption along the proximal convoluted tubule of superficial nephrons. Rates of fluid delivery to the late proximal and distal tubule were elevated. The rate of fluid reabsorption in the superficial loop of Henle was increased. Maximal urine osmolality and papillary tissue content of urea was reduced. The polyuria, therefore, results from decreased fluid reabsorption by proximal tubules and, probably, by papillary collecting ducts. The decrease in proximal fluid reabsorption is probably secondary to impaired solute reabsorption. A decrease in collecting duct fluid absorption can be attributed to the observed decrease in papillary solute concentration.     

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.     

Mechanisms to concentrate the urine: an opinion

PURPOSE OF REVIEW: Our goal is to suggest how the renal concentrating mechanism is regulated in vivo. RECENT FINDINGS: The majority of descending thin limbs of the loop of Henle lack aquaporin-1 water channels, and loops of Henle in the inner medulla lack urea transporters. SUMMARY: Lack of water permeability in the descending thin limbs of the loop of Henle offers several advantages. First, since much less water is added to the outer medullary interstitial compartment, inhibitory control mechanisms on sodium and chloride reabsorption from the medullary thick ascending of loop of Henle initiated by water addition from the medullary collecting duct can be effective. Second, recycling of urea is efficient, as little urea will be washed out of the medulla. Third, delivery of a larger volume of filtrate to the medullary thick ascending limb of the loop of Henle permits both an appreciable reabsorption of sodium along with only a small fall in the luminal concentration of sodium in each of these liters. Hence there need be only a small lumen positive voltage in the medullary thick ascending limb of the loop of Henle. The absence of urea transporters in the loop of Henle in the inner medulla is required for a passive mechanism of sodium and chloride reabsorption in the inner medulla. Control of urea reabsorption from the medullary collecting duct is needed to prevent excessive oliguria in electrolyte-poor urine.     

Renal concentration defect following nonoliguric acute renal failure in the rat

The mechanism of impaired renal concentrating ability following nonoliguric ischemic acute renal failure was studied in the rat. Fifty min of complete occlusion of the renal artery and vein with contralateral nephrectomy resulted in reversible, nonoliguric acute renal failure. Eight days following induction of acute renal failure, a defect in 30 hr dehydration urine osmolality was present when experimental animals were compared with uninephrectomized controls (1,425 +/- 166 versus 2,267 +/- 127 mOsm/kg water respectively, P less than 0.001). Comparable postdehydration plasma vasopressin levels in experimental and control animals and an impaired hydro-osmotic response to exogenous vasopressin in experimental animals documented a nephrogenic origin of the defect in urine concentration. Lower urinary excretion of prostaglandin E2 in experimental animals and a failure of cyclo-oxygenase inhibition with 10 mg/kg of indomethacin to improve dehydration urine osmolality suggested that prostaglandin E2 antagonism of vasopressin action did not contribute to the concentration defect. Postdehydration inner medullary (papillary) interstitial tonicity was significantly reduced in experimental animals versus controls (870 +/- 85 versus 1,499 +/- 87 mOsm/kg water respectively, P less than 0.001). To determine if this decreased interstitial tonicity was due to vascular mechanisms, papillary plasma flow was measured and found to be equivalent in experimental and control animals. To examine a role for biochemical factors in the renal concentration defect, cyclic nucleotide levels were measured in cytosol and membrane fragments. A decrease in vasopressin and sodium fluoride-stimulated adenylate cyclase was found in outer medullary tissue of experimental animals. In contrast, vasopressin-stimulated adenylate cyclase activity was comparable in the inner medullary tissue of control and experimental animals. Our study suggests a defect in generation of renal inner medullary interstitial solute as a mechanism of the impaired urinary concentration observed in this model of acute renal failure.     

Urinary concentrating defect in hypothyroid rats: role of sodium,potassium, 2-chloride co-transporter,and aquaporins

Hypothyroidism is associated with impaired urinary concentrating ability in humans and animals. The purpose of this study was to examine protein expression of renal sodium chloride and urea transporters and aquaporins in hypothyroid rats (HT) with diminished urinary concentration as compared with euthyroid controls (CTL) and hypothyroid rats replaced with L-thyroxine (HT+T). Hypothyroidism was induced by aminotriazole administration. Body weight, water intake, urine output, solute and urea excretion, serum and urine osmolality, serum creatinine, 24-h creatinine clearance, and fractional excretion of sodium were comparable among the three groups. However, with 36 h of water deprivation, HT rats demonstrated significantly greater urine flow rates and decreased urine and medullary osmolality as compared with CTL and HT+T rats at comparable plasma vasopressin concentrations. Western blot analyses revealed decreased renal protein abundance of transporters, including Na-K-2Cl, Na-K-ATPase, and NHE3, in HT rats as compared with CTL and HT+T rats. Protein abundance of renal AQP1 and urea transporters UTA(1) and UTA(2) did not differ significantly among study groups. There was however a significant decrease in protein abundance of AQP2, AQP3, and AQP4 in HT rats as compared with CTL and HT+T rats. These findings demonstrate a decrease in the medullary osmotic gradient secondary to impaired countercurrent multiplication and downregulation of aquaporins 2, 3, and 4 as contributors to the urinary concentrating defect in the hypothyroid rat.     

Mechanism of impaired urinary concentration in chronic primary glomerulonephritis

To define the role of medullary damage and the influence of solute load and blood pressure (BP) in impairing urinary concentration, patients with chronic glomerulonephritis were investigated by histological and functional studies. In 59 biopsy specimens, the degree of medullary fibrosis was correlated inversely with urinary specific gravity and was significantly greater in hypertensive than in normotensive subjects. The following clearance studies were carried out in patients with a GFR of 15 to 40 ml/min in maximal antidiuresis: (1) Eight patients were studied while receiving a high sodium and protein diet and then after 1 week of low sodium, low protein diet; (2) ten patients were loaded with hypertonic saline (3%) to increase urine volume up to 25 to 30% of GFR; (3) the concentrating ability was compared in 15 normotensives and 15 hypertensives with comparable GFR; (4) the concentrating ability was studied in nine hypertensive patients before and after drug-induced normalization of BP. In (1) no change occurred in maximal urine osmolality (UOsm) even if fractional sodium excretion and filtered load of urea were reduced. In (2), values of UOsm fell below those of plasma osmolality. In (3), UOsm and negative free-water generation were lower in hypertensive than in normotensive subjects. In (4), normalization of BP was not associated with any change in UOsm. These results indicate that osmotic diuresis does not play a critical role in reducing urinary concentration. This defect is better accounted for by an intrinsic medullary damage, enhanced in hypertensive patients, which may impair the permeability of collecting ducts to water.     

Reverse pharmacological effect of loop diuretics and altered rBSC1 expression in rats with lithium nephropathy

BACKGROUND: Renal urinary concentration is associated with enhanced expression of rBSC1, a rat sodium cotransporter, in the thick ascending limb of Henle. Increased expression of rBSC1 was reported recently in nephrogenic diabetes insipidus induced by lithium chloride (Li nephropathy). However, the pathophysiological implication of altered rBSC1 expression has not yet been investigated. METHODS: Li nephropathy was induced in rats by an oral administration of 40 mmol lithium/kg dry food. In rats with reduced urinary osmolality to less than 300 mOsm/kg H2O, we examined the expression of rBSC1 mRNA and protein, plasma arginine vasopressin (AVP) and RNA expression of kidney-specific water channel, aquaporin-2 (AQP2), of collecting ducts. Rats with Li nephropathy were treated with furosemide (3 mg/kg body weight), which blocks the activity of rBSC1, and changes in urine concentration, plasma AVP, medullary accumulation of Li ions, and apical AQP2 expression were determined. RESULTS: Rats with Li nephropathy showed increased rBSC1 RNA and protein expression and reduced AQP2 RNA. In these rats, furosemide, which induces dilution of urine and polyuria in normal rats, resulted in a progressive and significant rise in urine osmolality from 167 +/- 11 (mean +/- SD) at baseline to 450 +/- 45 mOsm/kg H2O at three hours after administration, and significant oliguria. In the same rats, plasma AVP decreased significantly from 5.7 to 3.0 pg/mL. In addition, recovery of apical AQP2 expression was noted in a proportion of epithelial cells of the collecting ducts. Although Li+ in the renal medulla was slightly lower in rats with Li nephropathy treated with furosemide, statistical significance was not achieved. CONCLUSIONS: Our results suggest that dehydration or high plasma AVP results in an enhanced rBSC1 expression in Li nephropathy, and that rBSC1 expression is closely associated with the adverse effects of Li ions on collecting duct function.     

Renal concentrating and diluting function in deficiency of specific aquaporin genes

Aquaporins (AQP) are water-transporting proteins expressed in many fluid-transporting epithelia and endothelia. In kidney, AQP1 is expressed in plasma membranes of proximal tubule, thin descending limb of Henle and descending vasa recta, AQP2 in collecting duct luminal membrane, AQP3 and AQP4 in collecting duct basolateral membrane, AQP6 in intercalated cells, and AQP7 in the S3 segment of proximal tubule. Human mutations in AQP2 cause hereditary non-X-linked nephrogenic diabetes insipidus. Transgenic mice lacking the renal aquaporins have been useful in defining their role. Mice deficient in AQP1 are polyuric and unable to form a concentrated urine because of defective proximal tubule fluid absorption and countercurrent multiplication. Mice lacking AQP3 are markedly polyuric due to low water permeability across the cortical and outer medullary collecting duct. However, mice lacking AQP4, which is expressed mainly in inner medullary collecting duct, manifest only a mild defect in maximum urinary concentrating ability. The aquaporin null mice have normal urinary diluting ability. From many renal and extrarenal phenotype studies of aquaporin null mice, we conclude that aquaporins are important for rapid near-isosmolar transepithelial fluid absorption/secretion and for rapid vectorial water movement driven by osmotic gradients. The renal phenotype in aquaporin null mice suggests the utility of aquaporin blockers as novel aquaretic-diuretic agents.     

Urinary aquaporin-2 in children with acute pyelonephritis

Children with acute pyelonephritis develop polyuria and have reduced maximum urinary concentration capacity. We studied whether these abnormalities are associated with altered urinary excretion of the water channel aquaporin-2 (AQP2) in the renal collecting duct. AQP2 is the main target for antidiuretic action of arginine vasopressin (AVP), and the urinary excretion of this protein is believed to be an index of AVP signaling activity in the kidney. Children with acute pyelonephritis, aged 5–14 years, were examined for urinary flow rate, creatinine clearance, unchallenged urine osmolality, and urinary ion excretion. Urinary excretion of AQP2 was measured by dot immunoblotting technique. Studies were performed in the acute phase of pyelonephritis, in the same children after treatment, and in control patients. At the onset of pyelonephritis, urinary flow rate and solute excretion were increased, but the urinary osmolality was unchanged. The urinary level and urinary excretion of AQP2 was increased in acute pyelonephritis and decreased after treatment. Excretion of aquaporin-3 was unchanged, suggesting that the increase in AQP2 urinary excretion was not due to a shedding of collecting duct cells. The results suggest that a mechanism proximal to the collecting duct may be responsible for the polyuria observed in children with acute pyelonephritis. Increased urinary AQP2 levels suggest that a compensatory activation of apical plasma membrane targeting of AQP2 may occur in pyelonephritis.     

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.     

Osmoregulatory betaine uptake by rat renal medullary slices.

Betaine is an osmolyte present in high concentrations in renal medullary cells. Betaine and other organic osmolytes, such as glycerophosphorylcholine, myo-inositol, and sorbitol, have been shown to increase in concentration during antidiuresis when the inner medullary extracellular osmolality rises. Its concentration may increase in renal cells either by betaine uptake or by choline metabolism to betaine. These studies measured the uptake of (14C)betaine into cortical, outer medullary and inner medullary slices from rat kidney. The tissue-to-medium ratio of (14C) betaine increased with increasing osmolality up to 450 mosmol/kg in outer medullary and inner medullary slices, but not in cortical slices. Betaine uptake increased when the osmolality was raised with NaCl or mannitol, but not with urea. When LiCl was substituted for NaCl in a medium of 300 mosmol/kg, there was significant inhibition of betaine uptake, although the tissue-to-medium ratios remained greater then unity. Thus, increases in osmolality stimulate betaine uptake in rat renal medullary slices and this uptake occurs by both sodium-dependent and sodium-independent betaine transport.     

AT1 receptors in the collecting duct directly modulate the concentration of urine

Mice lacking AT(1) angiotensin receptors have an impaired capacity to concentrate the urine, but the underlying mechanism is unknown. To determine whether direct actions of AT(1) receptors in epithelial cells of the collecting duct regulate water reabsorption, we used Cre-Loxp technology to specifically eliminate AT(1A) receptors from the collecting duct in mice (CD-KOs). Although levels of AT(1A) receptor mRNA in the inner medulla of CD-KO mice were significantly reduced, their kidneys appeared structurally normal. Under basal conditions, plasma and urine osmolalities and urine volumes were similar between CD-KO mice and controls. The increase in urine osmolality in response to water deprivation or vasopressin administration, however, was consistently attenuated in CD-KO mice. Similarly, levels of aquaporin-2 protein in inner and outer medulla after water deprivation were significantly lower in CD-KO mice compared with controls, despite its normal localization to the apical membrane. In summary, these results demonstrate that AT(1A) receptors in epithelial cells of the collecting duct directly modulate aquaporin-2 levels and contribute to the concentration of urine.     

Urinary aquaporin-2 levels in healthy volunteers

SUMMARY:   Renal water handling is regulated by the release of arginine vasopressin (AVP) and the subsequent insertion of aquaporin 2 (AQP2) in the apical membrane of collecting duct cells. This in turn increases the membrane permeability to water and the passive reabsorption of water down the concentration gradient present in the medulla. Aquaporin 2 can be detected in the urine under conditions of antidiuresis. We wish to validate an assay for urinary AQP2. Fourteen volunteers participated in studies of water loading and water deprivation followed by the administration of 1-deamino-8- d -arginine vasopressin (dDAVP). Urine osmolality was measured by vapour pressure osmometry. Urinary AQP2 was measured by using a chemiluminescent assay. Baseline correlations between serum AVP levels, urinary osmolality and urinary AQP2 levels were not significant. Following the administration of dDAVP, a positive correlation between urine osmolality and urinary AQP2 was evident ( r  = 0.762). For specific conditions where renal water retention is stimulated via AVP, urinary AQP2 measurements provide a reproducible measurement of the renal actions of AVP.