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1Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland; and 2Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Submitted 25 August 2005 ; accepted in final form 2 February 2006
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ABSTRACT |
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6,000 µosmol/day. In the same titration experiments, when increasing amounts of NaCl were added to the diet, mice were unable to increase urinary NaCl concentrations to >420 mM, resulting in osmotic diuresis at NaCl excretion rates of 3,500 µosmol/day. Thus both urea and NaCl can induce osmotic diuresis when large amounts are given, supporting the conclusion that the decrease in water excretion with mixtures of urea and NaCl added to the diet (compared with pure NaCl or urea) is due to the separate abilities of urea and NaCl to induce osmotic diuresis, rather than to any specific interaction of urea transport and NaCl transport at an epithelial level. vasopressin; passive model; urinary concentrating mechanism
In recent years, two urea transporter genes have been identified, namely, UT-A and UT-B (reviewed in Refs. 14 and 15), which encode multiple facilitative urea transporter proteins. Both UT-A and UT-B proteins are expressed within the kidney. Splice variants of UT-A are expressed in renal tubule segments; UT-A1 and UT-A3 are localized to the inner medullary collecting duct (IMCD), whereas UT-A2 is localized to the thin descending limbs of Henle's loop (tDL). In contrast, UT-B is expressed in the blood vessels of the renal medulla, specifically the descending vasa recta.
Recently, we generated knockout mice in which the collecting duct isoforms of UT-A (UT-A1 and UT-A3) have been deleted (UT-A1/3–/– mice) (6). These animals manifest a failure to accumulate urea in the inner medulla and demonstrate increased renal water excretion. These findings are consistent with the proposal by Berliner and colleagues (1) that the accumulation of urea in the renal medulla is important to the urinary concentrating mechanism because the interstitial urea osmotically balances the luminal urea in the IMCD, allowing large amounts of urea to be excreted without creating an osmotic diuresis. In contrast to the reduced inner medullary urea content, UT-A1/3–/– mice did not exhibit any significant diminution of NaCl accumulation in the inner medulla (6, 7), indicating that the process that concentrates NaCl in the inner medulla does not depend on rapid urea exit from the IMCD.
The question that we raise in this manuscript is whether Gamble's "economy of water in renal function referable to urea" is owing to the role of UT-A proteins expressed in the IMCD. We addressed this question by repeating the Gamble experiments in both wild-type and UT-A1/3–/– mice.
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METHODS |
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Diets. The basal diet consisted of a gelled diet made up of (per 5 g total) 1 ml of deionized water, 4 g of special low-salt (NaCl) synthetic food [0.001% Na (wt/wt); Research Diets (New Brunswick, NJ)], and 25 mg of agar; the basal diet contained 4% protein by weight (as casein). As established previously (6), the basal diet was determined to be sufficient for nutritional maintenance throughout the duration of the study. The aim of the initial experiment was to measure the concentrations of NaCl and urea in the urine when these substances were added to the basal diet in isosmotic quantities, either singly or as a mixture. Thus for the first experimental period, urea alone was added to the diet. Then, in successive periods the urea was progressively replaced by NaCl (see Table 1), until the final period when it was replaced entirely. In all experiments, the total number of osmotically active particles added to the diet remained the same, 1.5 mosmol/g of food.
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Second metabolic cage study. Four male UT-A1/3–/– mice and 4 wild-type mice of the same background (C57/BL6J) and age (12 wk) were used for all studies. Studies were performed as outlined above, except the diets were as follows: diet 1, 1.5 mosmol NaCl/g food; diet 2, 1.0 mosmol NaCl/g food; and diet 3, 1.0 mosmol NaCl/g food plus 0.5 mosmol urea/g food. Mice had access to supplemental drinking water throughout the duration of the study.
Third metabolic cage study. Five male C57/BL6J wild-type mice 12 wk of age were housed in metabolic cages as outlined above. Mice received a fixed daily ration of 5 g of gelled diet containing 0, 1.0, 1.5, or 2.0 mosmol urea/g food or NaCl. Mice had access to supplemental drinking water throughout the duration of the study.
Urine analysis. Urine volume was measured gravimetrically assuming a density of one. Urine osmolality was determined using a vapor pressure osmometer (Wescor). Na+, urea, and K+ concentrations in the urine were determined using an autoanalyzer (Monarch 2000, Instrumentation Laboratories). Osmolality due to non-urea and non-NaCl solutes was calculated as follows: osmolality = total osmolality – [([urea] x 0.96) + ([Na] x 1.84)], where 0.96 and 1.84 are the osmotic coefficients for urea and Na, respectively (3), and brackets indicate concentration.
Statistical analysis. Unless otherwise indicated, all values are quoted as means ± SE. Initial statistical analysis was performed by ANOVA. Contrasts between a dietary condition and the previous dietary condition in the same group were done by Bonferroni's method. P values <0.025 were considered significant. Significant differences between two different groups of animals (knockout vs. wild-type) on the same dietary intake were made with the Student's unpaired t-test. P values <0.05 were considered significant.
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RESULTS |
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Figure 1 shows water excretion (Fig. 1A), urinary osmolality (Fig. 1B), and osmolar excretion (Fig. 1C). Both cardinal observations made by Gamble et al. in rats were confirmed in wild-type mice (solid lines in Fig. 1). 1) The water requirement for the excretion of 6.6 mmol urea/day (4.7 ml/day, Fig. 1A, left) was much less than the requirement for the excretion of an equivalent osmolar quantity of NaCl (9.9 ml/day, Fig. 1A, right). 2) When mixtures of urea and NaCl were given in the diet, urinary osmolality was increased and water excretion was decreased relative to levels seen for the excretion of the osmolar equivalent amounts of either urea or NaCl alone. In agreement with the Gamble study, a maximal urinary osmolality was seen at a urea:NaCl osmolar ratio somewhat >1 (Fig. 1B).
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6.6 mmol urea/day was virtually identical to that needed to excrete an osmotically equivalent amount of NaCl and was substantially greater than the water excretion seen in the wild-type mice (Fig. 1A). Second, the ability of the kidneys to increase urinary osmolality to a higher level with mixtures of urea and NaCl, compared with urea or NaCl alone added to the diet, was not seen in UT-A1/3–/– mice (Fig. 1B). Thus Gamble's "economy of water referable to urea" is dependent on facilitated urea transport in the IMCD by UT-A1, UT-A3, or both. The excretion of urea in the same experiments is shown in Fig. 2. Urea excretion was fixed by dietary intake and therefore was virtually identical in wild-type (solid lines) and UT-A1/3–/– (dashed lines) mice (Fig. 2A). The reduction in daily urea excretion in both groups as one traverses from the left-hand side to the right-hand side of the graph reflects the replacement of urea in the diet with NaCl. In contrast, urinary urea concentration was much lower in UT-A1/3–/– mice than in wild-type mice (Fig. 2B), reflecting the higher water excretion, presumably due to the urea-induced osmotic diuresis seen in the UT-A1/3–/– mice. However, urine urea concentration decreases monotonically with reduced dietary urea intake in both groups.
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DISCUSSION |
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One such tool is the development of mouse models, in which a single gene, or part of a gene, has been "knocked out." Recently, we generated UT-A1/3–/– mice, in which the facilitative urea transporters expressed in the IMCD were deleted. In contrast to wild-type mice, the IMCDs from UT-A1/3–/– mice have a very low urea permeability that does not increase with vasopressin, and these mice excrete much more water, unless the rate of urea excretion is diminished by dietary protein restriction. We concluded that in the absence of the urea transporters that are normally expressed in the IMCD, water excretion is increased as a result of urea-dependent osmotic diuresis (6, 7). Furthermore, UT-A1/3–/– mice failed to accumulate urea in their inner medullas, but the accumulation of NaCl was not attenuated. Thus the absence of IMCD urea transport does not prevent the concentration of NaCl in the inner medulla, contrary to what would be predicted from passive concentrating models (12, 16). These findings in UT-A1/3–/– mice added to the substantial volume of evidence from other studies (10, 11, 13) that the passive model in the form proposed by Kokko and Rector (12) and Stephenson (16) is not the explanation for the accumulation of NaCl in the renal inner medulla.
The observations by Gamble and colleagues (8), summarized at the beginning of this paper and confirmed in wild-type mice in this study, provided early evidence for a special role of urea in the renal handling of water. Indeed, the appeal of the passive model derived, in part, from the fact that it appeared to provide a plausible explanation for the Gamble findings. If the special role of urea is not attributable to an involvement of urea in the passive concentration of NaCl in the inner medulla, then what is the special role? Our studies show that the Gamble phenomenon (namely, higher urinary osmolality with urea supplementation of the diet rather than with NaCl supplementation and also higher urinary osmolality with mixtures of urea and NaCl added to the diet than with either NaCl or urea alone) is not seen in UT-A1/3–/– mice. Our data suggest that the Gamble phenomenon is largely a manifestation of facilitated urea transport in the IMCD by UT-A1 and UT-A3.
The first element of the Gamble phenomenon (a higher urinary osmolality with urea supplementation of the diet than with NaCl supplementation) can be explained directly by what is known about the role of UT-A1 and UT-A3 in the IMCD. These transporters allow rapid passive reabsorption of urea from the IMCD and accumulation of urea in the inner medullary interstitium. The high concentration of urea in the interstitium osmotically balances urea in the IMCD lumen, allowing large amounts of urea to be excreted without obligating water (1). In contrast, excretion of equivalent amounts of NaCl causes an osmotic diuresis, increasing water excretion through its osmotic effect.
Although our results clearly support a connection between IMCD urea transporters and the second element of the Gamble phenomenon (namely, higher urinary osmolality with mixtures of urea and NaCl added to the diet than with either NaCl or urea alone), it cannot be explained so readily from what is known about the role of UT-A1 and UT-A3 in the IMCD. However, one explanation can be derived from a consideration of the two ends of the Gamble water excretion curve (Fig. 1A). At the high-urea end, we speculated that the increased water excretion with greater urea excretion is owing to saturation of the urea transporters in the IMCD. Previous reports have shown that urea transport across the IMCD is indeed a saturable phenomenon, as expected from the properties of carrier proteins in general (2). In this study, we determined that saturation of IMCD urea transporters in vivo results in osmotic diuresis (Fig. 7). Thus as urea saturates the transporters, the osmotic effect of urea in the lumen will increase, resulting in a higher rate of water excretion. At the high-NaCl end of the Gamble water excretion curve (Fig. 1A), we propose that the increase in water excretion with a greater NaCl intake can be explained by the urine NaCl concentration reaching a maximum above which NaCl concentration cannot be raised further. In the present study, the average maximum NaCl concentration is 420 mM, after which the addition of more NaCl in the diet results in an increase in urine output. Indeed, when a small amount of NaCl was removed from the diet (and not replaced with urea), there was a reduction in urine volume that was not affected by the addition of urea, suggesting that the NaCl concentration itself in the urine had reached a maximum (Fig. 6A), essentially due to saturation of NaCl-absorptive mechanisms along the nephron. Indeed, direct titration studies in which dietary NaCl alone was progressively increased until a maximum urinary NaCl concentration was reached (Fig. 7) showed that above daily excretion rates of 3,500 µosmol, NaCl excretion could only occur via increases in water excretion, i.e., by osmotic diuresis.
Another possible explanation can be drawn from considering the regulation of thirst and drinking behavior. In our study, water intake was on an ad libitum basis. Thus any variations in water excretion were paralleled by variations in water intake. In general, differences in water intake are driven by differences in thirst. As originally described by Verney (17), NaCl and other salts are effective solutes with regard to their ability to stimulate hypothalamic osmoreceptors, whereas urea is ineffective. The stimulation of thirst by rising osmolality is a threshold phenomenon, requiring an increase in plasma osmolality above a threshold value as seen also for the regulation of vasopressin secretion. Thus one might expect that as NaCl is added to the diet, thirst would increase, although possibly only at the highest levels of salt intake. Indeed, we observed in wild-type mice that increasing NaCl intake from 1.0 to 1.5 mosmol/g food (Fig. 1), there was a marked increase in water excretion. Thus the second element of the Gamble phenomenon may be dependent on three factors: saturation of UT-A- mediated urea transport in the IMCD (at the high-urea end of the curve), saturation of NaCl absorption along the nephron (at the low-urea end), and enhancement of thirst (at the low-urea end). In general, the overall data are consistent with the view that the decreased water excretion with mixtures of urea and NaCl added to the diet is due to the separate abilities of urea and NaCl to induce osmotic diuresis at high concentrations, rather than to any specific interaction of urea transport and NaCl transport at an epithelial level.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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urea = 1 in rat terminal collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1173–F1180, 1990.This article has been cited by other articles:
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  Y. Wang, J. D. Klein, M. A. Blount, C. F. Martin, K. J. Kent, V. Pech, S. M. Wall, and J. M. Sands Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts J. Am. Soc. Nephrol., September 1, 2009; 20(9): 2018 - 2024. [Abstract] [Full Text] [PDF]
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 B. Hartman Bakken, L. G. Herrera M., R. M. Carroll, J. Ayala-Berdon, J. E. Schondube, and C. Martinez del Rio A nectar-feeding mammal avoids body fluid disturbances by varying renal function Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1855 - F1863. [Abstract] [Full Text] [PDF]
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T. L. Pannabecker, W. H. Dantzler, H. E. Layton, and A. T. Layton Role of three-dimensional architecture in the urine concentrating mechanism of the rat renal inner medulla Am J Physiol Renal Physiol, November 1, 2008; 295(5): F1271 - F1285. [Abstract] [Full Text] [PDF]
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R. A. Fenton and M. A. Knepper Urea and Renal Function in the 21st Century: Insights from Knockout Mice J. Am. Soc. Nephrol., March 1, 2007; 18(3): 679 - 688. [Abstract] [Full Text] [PDF]
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J. M. Sands Critical Role of Urea in the Urine-Concentrating Mechanism J. Am. Soc. Nephrol., March 1, 2007; 18(3): 670 - 671. [Full Text] [PDF]
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, solid lines) and UT-A1/3–/– (
, dashed lines) mice are shown. #Statistically significant difference within an animal group compared with the previous dietary condition (P < 0.025). *Statistically significant difference between wild-type and UT-A1/3–/– mice on any particular diet (P < 0.05).
