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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 Institute, National Institutes of Health, Bethesda, Maryland
ABSTRACT I. INTRODUCTION A. Transport Processes in Henle's Loops That Concentrate Solutes in the Medullary Interstitium B. Osmotic Equilibration of Collecting Duct Fluid by Vasopressin-Regulated Water Transport C. Urea Recycling II. MOUSE MODELS A. Transport Proteins Involved in Urinary Concentration and Dilution B. Sodium Transporters and Channels 1. NHE3 2. NKCC2 3. NCC and ENaC C. Potassium Channels 1. ROMK D. Chloride Channels 1. ClC-K1 E. Aquaporins 1. AQP-1 F. Collecting Duct Aquaporins 1. AQP-2 2. AQP-3 3. AQP-4 4. AQP-7 G. Urea Transporters 1. UT-A1 and UT-A3 2. UT-A2 3. UT-B H. Receptors and Signaling Molecules 1. V2R 2. Bradykinin B2 receptor 3. Integrin alpha1beta1 4. Heterotrimeric G protein subunit, Gsalpha 5. Protein kinase C 6. Serine/threonine phosphatase, calcineurin Aalpha 7. Nitric oxide synthase 8. Endothelin-1 9. Endothelin A receptor I. Prostaglandins 1. Group IV cytosolic phospholipase A2 2. PGE2 E-prostanoid receptors, EP1 and EP3 J. Renin-Angiotensin-Aldosterone System 1. Renin 1C (Ren1c) 2. Angiotensinogen 3. ACE 4. Type 1 angiotensin receptors 5. Aldosterone synthase K. Osmoprotective Genes 1. OREBP/TonEBP 2. Aldose reductase L. Miscellaneous 1. Foxa1 2. Urate oxidase 3. Tamm-Horsfall protein GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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A. Transport Processes in Henle's Loops That Concentrate Solutes in the Medullary Interstitium
It is generally accepted that NaCl is concentrated in the renal medullary interstitium through the process of countercurrent multiplication, originally proposed by Wirz, Kuhn, and colleagues more than 50 years ago (139, 282). This process requires special properties in both the ascending limb and descending limb of Henle (124). The ascending limb in the outer medulla (MTAL) is like the CTAL (described above); it actively transports NaCl, but water remains in the lumen owing to a lack of aquaporin expression and thus low water permeability (186). This dilutional effect generates a small transepithelial osmolality difference (the "single effect" or Einzeleffekt) that is "multiplied" by the counterflow between the two limbs of Henle's loops, resulting in an axially aligned osmotic gradient that is much larger than the single effect (see Ref. 70 for details). The properties of the descending limb of Henle are critical because, for countercurrent multiplication to work effectively, the luminal fluid must be close to osmotic equilibrium with the surrounding interstitium at every point along the descending limb. How this osmotic equilibration occurs is one point of controversy (see Fig. 2). Theoretically, the equilibration could occur by rapid water efflux from the descending limb, by rapid solute entry into the descending limb, or by a combination of both processes. Studies of isolated perfused thin descending limbs from the outer medulla revealed that they have high water permeabilities (43, 130), which are thought to be due to the presence of high levels of aquaporin-1 in the apical and basolateral plasma membranes of thin descending limb cells (188). However, micropuncture studies addressing the mechanism of osmotic equilibration in the descending limb concluded that a substantial element of osmotic equilibration is due to solute entry (106). This controversy has been addressed using aquaporin-1 (AQP-1) knockout mice (see below).
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B. Osmotic Equilibration of Collecting Duct Fluid by Vasopressin-Regulated Water Transport
It is generally accepted that vasopressin regulates water permeability in the renal collecting duct system by stimulating the insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane and by regulating AQP-2 gene expression (186). Furthermore, there has been evidence in the older physiological literature that the kidney may be capable of urine concentration in the absence of vasopressin's actions in the renal collecting duct (21), which, if true, raises hopes for more effective treatment of patients with X-linked nephrogenic diabetes insipidus (NDI). The development of mice in which the renal vasopressin V2 receptor (V2R) has been deleted have begun to address this possibility (see below). Another point of controversy derived from the "micropuncture era" (105) has been the relative importance of water transport in the connecting tubule and various subsegments of the renal collecting duct in the urinary concentrating mechanism, with regard to renal water conservation. Mouse models in which the V2R or AQP-2 is selectively deleted from the collecting ducts and not the connecting tubule are addressing this issue (see below).
Urea accumulation in the renal inner medulla depends on passive exit of urea from the IMCDs to the inner medullary interstitium. The retention of urea in the inner medullary interstitium depends on recycling processes, which return urea that would otherwise be lost to the general circulation to the inner medulla. One recycling pathway is classical countercurrent exchange, in which urea absorbed from the ascending vasa recta in the inner medulla enters the descending vasa recta (22). Additional recycling pathways have been subsequently proposed (Fig. 3) (123): 1) direct transfer of urea to the long loops of Henle in the inner medulla, 2) uptake of urea in the ascending vasa recta with transfer to the descending limbs of short loops of Henle in the vascular bundles of the renal outer medulla, and 3) transfer of urea from the thick ascending limbs of the outer stripe of the outer medulla and cortical medullary rays to the neighboring proximal straight tubules. Some aspects of these recycling pathways have been addressed recently in mice in which genes encoding the urea transporters UT-A2 and UT-B have been deleted (see below).
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II. MOUSE MODELS |
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Our understanding of renal physiology has accelerated in recent years as a result of the advent of the era of "molecular physiology," characterized by the development and application of tools to study proteins that can mediate renal function. These techniques have led to the cloning of multiple cDNAs and genes that are thought to be involved in urinary concentration and dilution. Figure 4 summarizes the renal tubule sites with abundant expression of aquaporins, urea transporters, and ion transporters/channels that are important to the urinary concentrating process. Several of these transporters are molecular targets for vasopressin action and are also expressed in low abundance in other renal tubule segments (Fig. 5).
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B. Sodium Transporters and Channels
The Na+-H+ exchanger (NHE3) is the major Na absorptive pathway in the proximal tubule. In addition to the proximal tubule, NHE3 has also been immunolocalized to the outer medullary thin descending limb of Henle and the TAL (219, 220). In the MTAL, NHE3 activity is increased by hypotonicity via a phosphatidylinositol 3-kinase (PI-3-K)-dependent pathway, which is inhibited by vasopressin working though cAMP (81, 82). Thus vasopressin has the net effect to inhibit NHE3 activity in the MTAL.
NHE3 knockout mice have been developed and are viable (233) (in contrast to NKCC2 knockout mice, see below). Their renal phenotype is predominantly associated with the fact that NHE3 is the major Na entry pathway in the proximal tubule (Fig. 4). NHE3 null mice have a marked reduction in proximal tubule fluid absorption and a compensatory decrease in glomerular filtration rate (GFR). This decrease in GFR is the result of activation of the tubuloglomerular feedback (TGF) mechanism (153).
Metabolic cage studies revealed that with free access to water, NHE3 null mice manifest a moderate increase in water intake associated with lower urinary osmolalities (average of 1,737 mosmol/kgH2O), although maximal urinary osmolality was not evaluated (4). In addition, NHE3 knockout mice have a marked decrease in renal NKCC2 expression, despite elevated plasma vasopressin levels (36). In conclusion, NHE3 null mice have a mild urinary concentrating defect that may be associated with a reduction in NKCC2 expression.
The renal Na+-K+-2Cl– cotransporter (NKCC2; also known as "BSC1") is expressed in the TAL, where it is localized to the apical membrane of epithelial cells (110, 187). NKCC2 is also expressed in the macula densa (187). In rat, long-term increases in the circulating vasopressin concentration result in an increase in NKCC2 protein abundance in the TAL (118), which is associated with an increase in the maximal urinary concentrating capacity (119). In addition, NaCl absorption in the MTAL can be increased by acute vasopressin administration (86). This is thought to occur, in part, due to increased apical membrane expression of NKCC2 in association with phosphorylation of the NH2-terminal tail (76, 198).
NKCC2 knockout mice have been developed by standard gene targeting techniques (252). However, due to perinatal fluid wasting and dehydration, the animals are not viable and die prior to weaning. Treatment of these mice with indomethacin and the administration of fluid allowed some mice to survive until adulthood, although the extreme polyuria, hydronephrosis, and growth retardation could not be abrogated. Studies of NKCC2 heterozygous animals showed essentially no difference from wild-type mice (251).
Why does deletion of NKCC2 result in such a severe phenotype, whilst deletion of NHE3, a transporter responsible for the majority of Na reabsorption in the kidney, results in a viable mouse capable of maintaining extracellular fluid volume? The answer appears to be in the special role that NKCC2 plays in the macula densa in the mediation of TGF. An intact TGF system in NHE3 knockout mice allows them to maintain a relatively normal distal fluid delivery through a reduction in GFR. In contrast, NKCC2 mice cannot compensate in this manner since the transporter is necessary for the TGF response to occur. Thus, in NKCC2 knockout mice, the distal nephron will be exposed to a NaCl load that drastically exceeds its absorptive capacity, leading to massive salt wasting and osmotic diuresis.
The thiazide-sensitive Na+-Cl– cotransporter (NCC) and the amiloride-sensitive Na+ channel (ENaC) are important targets for the action of aldosterone in the regulation of sodium excretion. Due to their interdependent role, we discuss them together in this section. NCC has been immunolocalized to the distal convoluted tubule (DCT) (58, 206), whereas ENaC is predominantly expressed in the connecting tubule, initial collecting tubule, and the cortical collecting duct (CCD) (85, 151). An increase in circulating vasopressin levels results in increases in abundance of both NCC and the 
- and 
-subunits of ENaC (53, 184). In addition, vasopressin acutely increases Na absorption in the rat CCD by increasing apical Na entry via ENaC (214, 227, 266), which is proposed to be due to vasopressin-induced trafficking of ENaC-containing vesicles from intracellular stores to the apical plasma membrane (240).
NCC knockout mice have a mild phenotype, with a small decrease in blood pressure (234). On a normal diet, they appear to have a normal urinary concentrating ability, but upon dietary potassium restriction, the NCC null mice develop hypokalemia and a consequent polyuria that is associated with an apparent central defect in the regulation of vasopressin secretion (172). It is only after prolonged hypokalemia that the mice develop evidence of NDI, resulting from a suppression of AQP-2 expression in the collecting ducts.
Knockout of any of the three ENaC subunits results in a severe phenotype, with the mice suffering from neonatal death (15, 95, 165). In the 
-ENaC knockout mice (95), early death appears to be due to failure to adequately clear fluid from the pulmonary alveoli after birth, whereas knockout of the - and -subunits of ENaC results in mice that die from hyperkalemia and sodium chloride wasting (15, 165). Interestingly, when -ENaC expression was deleted selectively from the collecting ducts, leaving intact ENaC expression in the renal connecting tubule and nonrenal tissues, the mice were viable and exhibited only a very mild phenotype with little or no inability to maintain fluid homeostasis in the face of salt or water restriction (217). In these mice, after water restriction, urine osmolality was not different from wild-type controls; thus it appears that Na absorption from the renal collecting duct via ENaC does not appear to be necessary for urinary concentration.
Taken together, NCC deleted only from the DCT or ENaC deleted only from the collecting duct results in a very mild phenotype, presumably because one can compensate for the other with regard to sodium balance. In this respect, double knockout animals could be informative. At present, it remains unclear whether the severity of the phenotype seen when any ENaC subunit is deleted globally is due to the importance of ENaC in nonrenal tissues or is related to the role of ENaC in the connecting tubule, which is conserved in the collecting duct specific -ENaC knockout mice (217).
The ATP-sensitive, inward rectifier ROMK potassium channel (Kir1.1) is expressed in the TAL, DCT, connecting tubule, and collecting duct system, predominantly in the apical plasma membrane (147, 166, 287). In the TAL, ROMK plays a critical role in the process of active NaCl transport and thus the urinary concentrating and diluting mechanism. The abundance of ROMK in the TAL is increased with chronic vasopressin administration (52). In the connecting tubule and collecting duct, ROMK is responsible for the process of potassium secretion, thus regulating urinary potassium excretion and systemic potassium balance. The later process is strongly regulated by vasopressin (266). This secretory process may be an indirect consequence of vasopressin's action to increase Na entry via ENaC, which results in apical plasma membrane depolarization and an increase in the electrochemical driving force for K+ movement through ROMK (226). Alternatively, vasopressin may regulate the open probability of ROMK, in a process mediated by the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP responsive protein (136, 154).
ROMK knockout mice, developed by Lorenz et al. (152), manifest early death associated with hydronephrosis and severe dehydration, consistent with the known role of ROMK in active NaCl absorption in the TAL. Approximately 5% of these mice survive the perinatal period, but suffer from metabolic acidosis, hypernatremia, reduced blood pressure, polydipsia, polyuria, and an impaired urinary concentrating ability. Furthermore, whole kidney GFR is reduced, apparently as a result of hydronephrosis, and the fractional excretion of electrolytes is elevated. Micropuncture analysis revealed that the single-nephron GFR was relatively normal, absorption of NaCl in the TAL was reduced and the TGF mechanism was severely impaired. From these animals, a line of mice has been derived that has a greater survival rate and no hydronephrosis, although adults have higher water excretion rates (155). Interestingly, these mice do not exhibit hyperkalemia, indicating that the connecting tubule and/or collecting duct principal cells must be capable of secreting K via some other pathway, presumably flow-dependent, Ca2+-activated K channels referred to as "maxi-K" channels (283).
ClC-K1 is a kidney-specific chloride channel that is localized to the apical and basolateral plasma membranes of the thin ascending limb of Henle's loop (tAL) (271). ClC-K1 expression in both the apical and basolateral plasma membranes, and examination of its transport properties, could explain why the tAL possesses an extremely high transepithelial chloride permeability compared with the TAL.
In 1999, Matsumura et al. (163) generated ClC-K1 null mice (Clcnk1–/–) and have made use of this model to examine the role of ClC-K1 in the urinary concentrating mechanism. Microperfusion studies determined that there was drastically reduced transepithelial chloride transport in the tAL of knockout mice. Importantly however, Clcnk1–/– mice had no significant differences in the plasma concentrations of Na+, K+, Cl–, and HCO3– and pH values compared with controls, indicating that a loss-of-function mutation of ClC-K1 does not result in hypokalemic alkalosis (unlike CLCNKB mutations, type III Bartter syndrome). Physiological studies revealed that Clcnk1–/– mice had significantly greater urine volume and lower urine osmolality compared with controls and that even after a 24-h water deprivation, the knockout mice were unable to concentrate their urine. This observed polyuria was insensitive to [deamino-Cys1, D-Arg8]-vasopressin (dDAVP) administration, indicating NDI. Clearance studies have shown that the fractional excretion of sodium, chloride, and urea are not different in knockout mice. Taken together, these data indicate that the polyuria observed in ClC-K1 null mice is water diuresis and not osmotic diuresis. Solute analysis of the inner medulla of Clcnk1–/– mice determined that the concentrations of urea, Na+, and Cl– were approximately half those of controls, resulting in a significantly reduced osmolality of the papilla (2). Unlike wild-type mice, the accumulation of these solutes was not increased by water deprivation.
In conclusion, Clcnk1–/– mice showed that rapid chloride exit from the tAL in the inner medulla is essential for generating a hypertonic inner medullary interstitium. As is pointed out by Knepper et al. (124), rapid solute exit from the tAL is theoretically critical to any process that concentrates solutes in the inner medulla because of a need for avoidance of dissipation of solutes from the inner medulla. Similarly, rapid Na+ and urea exit from the descending limb is a necessity for any inner medullary concentrating process (124), and the mechanisms involved in the exit of these entities will be an important area of focus for future research.
In the kidney, to date, eight aquaporins have been localized to various segments of the renal tubule. In this review we focus only on the mouse models where gene deletion results in either a urinary concentrating defect or a reduction in transepithelial water transport (Fig. 6).
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AQP-1 is localized to the apical and basolateral plasma membrane of epithelial cells in the proximal tubule (189), where the majority of fluid filtered by the glomerulus is reabsorbed by an active near-isosmolar transport mechanism. AQP-1 is also expressed in the thin descending limb of Henle's loop (tDL) and the epithelium of the descending vasa recta (DVR) (186, 188), nephron segments thought to be involved in countercurrent multiplication and exchange. The constitutively high water permeability of nephron segments expressing AQP-1 is consistent with a lack of regulation of AQP-1 by vasopressin (259).
Verkman and colleagues (158) generated a knockout mouse model of AQP-1 by targeted gene deletion. Compared with wild-type littermates, AQP-1 knockout mice have a reduced urinary osmolality that is not increased in response to water deprivation. Indeed, the urinary concentrating defect is so severe in these mice that after 36 h of water deprivation, the average body weight decreased by 35% and serum osmolality increased to greater that 500 mosmol/kgH2O. The urinary concentrating defect observed in these mice is likely due to a combination of different mechanisms. Schnermann et al. (229) used a combination of isolated tubule microperfusion and free-flow micropuncture to define the role of AQP-1 in proximal tubule water transport and fluid reabsorption. These studies determined that the transepithelial osmotic water permeability (Pf) of isolated microperfused proximal tubule S2 segments was fivefold less in AQP-1 knockout mice, indicating that the major pathway for osmotically driven transepithelial water transport is through AQP-1. In addition, the proximal fluid reabsorption rate in AQP-1 knockout mice was approximately half that observed in control animals. Additional studies in AQP-1 knockout mice (273) demonstrated that active transport in the absence of a high water permeability increased the transepithelial osmotic gradient in the proximal tubule to 
40 mosmol/kgH2O in contrast to the usual gradient of 5 mosmol/kgH2O. However, micropuncture of distal nephron segments revealed that the single-nephron glomerular filtration rate (snGFR) is reduced in AQP-1 mice, thus reducing distal fluid delivery to approximately the same level as in wild-type mice (229).
If AQP-1 knockout mice have relatively normal distal fluid delivery (despite defective proximal tubule water reabsorption), why do they have much greater urinary flow? It is likely that the answer to this lies in the role of AQP-1 in countercurrent multiplication. As described earlier, AQP-1 is abundantly expressed in the tDL. The osmotic water permeability of tDLs from AQP-1 knockout mice is 10-fold reduced compared with control animals (44), thus confirming the hypothesis that AQP-1 is the major water channel in tDL and pointing to the possibility that the countercurrent multiplier mechanism is impaired as a result of deletion of AQP-1 and diminished water absorption in the tDL. In addition, in isolated microperfused outer medullary DVRs from AQP-1 knockout mice, osmotic water permeability was reduced by greater than 50-fold compared with controls (201). The impairment of water transport in the vasa recta presumably results in a defect in countercurrent exchange, with concomitant depletion of medullary solutes. Taken together, these results indicate that the diminished urinary concentrating ability observed in AQP-1 knockout mice is likely due to a reduced ability to generate and maintain a hypertonic medullary interstitium. This conclusion is supported by the finding in AQP-1 null mice, based on tissue slice analysis, that there is a profound decrease in inner medullary solute accumulation (M. A. Knepper and T. Pisitkun, unpublished data). Also consistent with this conclusion is the finding that there is an almost complete lack of an increase in urine osmolality in AQP-1 null mice after the administration of vasopressin (158).
In humans, AQP-1 encodes the Colton blood group antigen (239). Consistent with the finding in knockout mice, it was observed that Colton-null individuals are unable to concentrate their urine as effectively as "normal" subjects when challenged by water deprivation (120).
The production of concentrated urine requires high collecting duct water permeability, allowing for the osmotically driven movement of water from the lumen to the interstitium. Transepithelial water transport across the collecting duct epithelium is generally believed to occur by a transcellular route with serial passage across the apical and basolateral plasma membranes. The water channels responsible for this transport appear to be AQP-2 in the apical plasma membrane and a combination of aquaporin-3 (AQP-3) and aquaporin-4 (AQP-4) in the basolateral plasma membrane (186). Knockout models have been developed to investigate the roles of these aquaporins in the urinary concentrating mechanism.
AQP-2 is abundantly expressed in all renal tubule segments beyond the DCT, including the connecting tubule (connecting tubule cells), the cortical and outer medullary collecting duct (principal cells), and the IMCD (IMCD cells) (185, 186). In these cell types, AQP-2 is found in the apical plasma membrane, subapical vesicles, and (especially in IMCD cells) in the basolateral plasma membrane. AQP-2 can be regulated by vasopressin (short-term and long-term) both by changes in transporter abundance (long-term regulation) and trafficking (short-term regulation) (reviewed in Ref. 186). The mechanisms behind arginine vasopressin (AVP)-mediated trafficking of AQP-2 have been discussed in detail elsewhere (reviewed in Refs. 37, 180), but for the purposes of this review are summarized as follows (see Fig. 7). Vasopressin binds to the V2R in the basolateral plasma membrane, resulting in activation of adenylate cyclase through V2R-coupled GTP-binding protein Gs. This increases intracellular cAMP levels and leads to activation of protein kinase A (PKA), intracellular Ca2+ oscillations (297), activation of myosin light-chain kinase (42) as well as cAMP-dependent, PKA-independent activation of the Rap-GEF Epac (298). Through multiple (largely unknown) mechanisms, V2R-mediated signaling leads to the exocytic insertion of AQP-2-bearing intracellular vesicles into the apical plasma membrane. These vesicles appear to be recycling endosomes rather than secretory vesicles (14). In addition, long-term exposure to vasopressin leads to an increase in AQP-2 synthesis via increased gene transcription, increasing the total abundance of AQP-2 in the cells. These mechanisms ultimately allow an increase in water reabsorption in the collecting duct.
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Despite the strong evidence implicating an essential role of AQP-2 in the urinary concentrating mechanism, a number of suitable mouse models to examine its function have only recently been developed/discovered. In 2001, a mouse knock-in model of AQP-2-dependent NDI was generated by inserting a T126M mutation into the mouse AQP-2 gene by a Cre-loxP strategy (292). This mutation has been shown to result in a failure of delivery of mature AQP-2 protein to the apical plasma membrane. Although the mutant mice appeared normal at birth, they failed to thrive and generally died within 1 wk. Analysis of the urine and serum revealed serum hyperosmolality and low urine osmolality, trademark characteristics of a defective urinary concentrating mechanism. Forward genetic screening of ethylnitrosourea-mutagenized mice isolated another mouse model of NDI with a F204V mutation (150). These mice survive beyond the neonatal period and have a much milder form of NDI. Examination of these mice could result in the identification of molecular chaperones that can correct similar forms of human NDI.
Two other mouse models have been developed that allow the role of AQP-2 in the adult mouse to be examined. One model, developed by Rojek et al. (216), makes use of the Cre-loxP system of gene disruption to create a collecting duct specific deletion of AQP-2, leaving relatively normal levels of expression in the connecting tubule. Another model, developed by Yang et al. (294), with inducible AQP-2 protein deletion in the kidney, was accomplished by tamoxifen-inducible Cre-recombinase expression in homozygous mice in which loxP sites were introduced in introns of the mouse AQP-2 gene. The major phenotype in both of these mice is severe polyuria, with average basal daily urine volumes approximately equivalent to body weight (216, 294). The high urine output observed resulted in very low urine osmolality, which was not increased after water restriction. However, despite the polyuria, with free access to water, plasma concentrations of electrolytes, urea, and creatinine are not different in knockout mice compared with controls, and neither was the estimated GFR. Thus, despite having normal renal function (presumably normal active Na+ transport), there is a major defect in the urinary concentrating mechanism in these mice. This defect confirms that AQP-2 is responsible for the majority of transcellular water reabsorption at the rate-limiting luminal membrane of the collecting duct.
A mouse model has also been "discovered" that implicates an essential role of AQP-2 phosphorylation on transporter function (164). This mouse model has a single base change in codon 256 of AQP-2, resulting in a serine to leucine amino acid substitution and loss of AQP-2 phosphorylation at amino acid 256. This mutation results in an absence of AQP-2 accumulation in the apical plasma membrane. Phenotypically, the mutant mice have no response to vasopressin and produce large quantities of hypotonic urine, characteristic of NDI. This mouse model provides direct genetic evidence that phosphorylation of AQP-2 at S256 is essential for its apical membrane accumulation and maximal water reabsorption in the collecting duct.
The latest mouse model to study the role of AQP-2 has helped us to understand the molecular mechanisms behind AQP-2-dependent autosomal-dominant nephrogenic diabetes (AD-NDI) insipidus (AD-NDI). Using a "knockin" strategy, and based on previous studies identifying three frameshift mutations in the COOH terminus of AQP-2 that result in AD-NDI (140), Sohara et al. (242) have created a mouse model that expresses 76 amino acids of the COOH terminus of a human AD-NDI mutant AQP-2 (763–772del), fused to the "wild-type" 254 NH2-terminal amino acids of mouse AQP-2. Mice that are heterozygous for the mutation exhibited a severely impaired urinary concentrating ability, but after dehydration were able to moderately increase their urine osmolality, a milder phenotype indicative of AD-NDI compared with autosomal-recessive NDI. Furthermore, the mutant AQP-2 was missorted to the basolateral plasma membrane and formed heteroligomers with wild-type AQP2, resulting in a dominant-negative effect on the normal apical sorting of wild-type AQP-2. Additional studies in this mouse model determined that the phosphodiesterase 4 inhibitor rolipram was partially able to restore concentrating ability, indicating that phosphodiesterase inhibitors may be useful drugs for the treatment of AD-NDI.
Xenopus oocyte expression studies have shown that AQP-3 is not only permeable to water but functions efficiently as a glycerol transporter, thus making it a member of the so-called aquaglyceroporins (56, 103, 293). In the kidney, AQP-3 is localized to the basolateral plasma membranes of the connecting tubule cells and collecting duct principal cells in cortex and outer medulla (48, 54, 104). AQP-3 is thought to mediate basolateral exit of water that enters via AQP-2. Interestingly, AQP-3 is not abundant in the cytoplasm, and there is no evidence for the short-term regulation of AQP-3 by vasopressin-induced trafficking. However, a marked increase in the abundance of AQP-3 mRNA and protein is observed during long-term vasopressin stimulation, such as seen during water deprivation or vasopressin infusion in Brattleboro rats (54, 104, 176, 259).
AQP-3 knockout mice have been generated by targeted gene deletion and found to have a greater than threefold reduction in osmotic water permeability of the basolateral membrane of the CCD compared with wild-type control mice (156). AQP-3 null mice are markedly polyuric (10-fold greater daily urine volume than controls), with an average urine osmolality of <300 mosmol/kgH2O. However, unlike AQP-1 or AQP-2 null mice, AQP-3 knockout mice are able to partially raise their urine osmolality after either water deprivation or the administration of dDAVP. Serum electrolyte concentrations from AQP-3 null mice are not significantly different from controls, although plasma osmolality is mildly elevated. In contrast to AQP-1 null mice, with a defective countercurrent exchange mechanism, the countercurrent exchange in AQP-3 knockout mice is virtually intact.
What is the basis for the defective urinary concentrating mechanism in these mice? It is likely that when AQP-3 is deleted, the reduced osmotic water permeability of the basolateral membrane results in a decrease in transepithelial water permeability. Since the majority of vasopressin-dependent fluid reabsorption in the antidiuretic kidney is in the connecting tubule and collecting duct, reduced water permeability is predicted to result in a hyposmolar urine. However, AQP-3 null mice are able to partially raise their urine osmolality after water deprivation or vasopressin stimulation despite a relatively water-impermeable collecting duct (156). This response is likely due to the fact that AQP-4 and not AQP-3 is the predominant basolateral water channel in the medullary parts of the collecting duct system, allowing a normal response to vasopressin in the most distal portions of the collecting duct system.
Another factor leading to increased urinary flow rates in adult AQP-3 knockout mice is the striking distortion of medullary architecture that progressively destroys the inner medulla and replaces it with a dilated renal pelvis (hydronephrosis) (156). The mechanism of hydronephrosis in this model is unknown.
AQP-4 is localized to the basolateral plasma membrane of outer medullary collecting duct (OMCD) principal cells and IMCD cells (258). AQP-4 is most abundant in the inner medulla, with a gradual decrease in expression towards the cortex (258). In mice, AQP-4 has also been reported to be expressed in the basolateral plasma membrane of proximal tubule S3 segments (274). Renal medullary AQP-4 mRNA abundance was found to be increased in response to water restriction (176) or vasopressin infusion (34), whereas there was no change in protein expression in response to either manipulation (259).
AQP-4 null mice have been generated by standard gene deletion methods (157). Isolated perfused tubule studies determined that there is a fourfold decrease in IMCD osmotic water permeability in AQP-4 null mice relative to wild-type mice, indicating that AQP-4 is responsible for most of the basolateral membrane water movement in this segment (45). Despite this reduced water permeability in the IMCD, in hydrated mice, there was no difference in urine osmolality compared with controls and no difference in serum electrolyte concentrations. However, there was a significant reduction in maximal urine osmolality in AQP-4 null mice after a 36-h water deprivation, and this reduced urine osmolality could not be further increased by vasopressin administration, indicating a mild urinary concentrating defect (157). Why does deletion of AQP-4 have such a profound effect on water permeability of the IMCD, with only a modest decrease in urinary concentrating ability? The answer to this is based on the normal distribution of water transport along the collecting duct. Micropuncture studies performed under antidiuretic conditions demonstrated that the amount of water reabsorbed osmotically in the late distal tubule (connecting tubule plus initial collecting tubule) is much greater than that absorbed in the medullary nephron segments (145). Thus, since AQP-4 in mouse is expressed predominantly in the medullary collecting ducts, deletion of AQP-4 results in only a mild defect in kidney water absorption.
AQP-7 is a member of the aquaglyceroporins and effectively transports both glycerol and water (102). In the kidney, AQP-7 is abundantly expressed on the apical plasma membrane of the proximal straight tubules (101, 181). AQP-7 knockout mice have reduced water permeability in the proximal tubule brush-border membrane (241). However, AQP-7 null mice do not exhibit a urinary concentrating defect or water balance abnormality. Because AQP-1 has been shown to be the major water transport pathway in the proximal tubule, it is not surprising that there is no abnormality in water excretion in AQP-7 knockout mice. However, AQP-1/AQP-7 double-knockout mice have been generated and showed a significantly greater urine output compared with AQP-1 null mice, leading the authors to speculate that the amount of water reabsorbed through AQP-7 in the proximal straight tubules is physiologically substantial, although direct measurements of proximal tubule water transport were not reported (241). The greater water excretion observed in the AQP-1/AQP-7 double-knockout mice compared with AQP-1 solo-knockout mice was accompanied by a proportional decrease in urine osmolality, suggesting that the total excretion of osmolar substances was not altered in these mice.
In mammals, 90% of waste nitrogen is normally excreted by the kidney as urea, the balance being attributable to ammonium and uric acid. The majority of this urea is generated in the liver via the urea-ornithine cycle. In humans and animals, under most circumstances, dietary protein intake greatly exceeds that necessary for the support of anabolic processes; thus excess quantities of urea are generated that need to be excreted. This urea constitutes a large osmotic load to the kidney. Most solutes excreted in such large amounts, for example, mannitol (7), would obligate large amounts of water excretion by causing an osmotic diuresis. However, under normal circumstances, urea does not induce an osmotic diuresis. Studies in the 1930s by Gamble et al. (71) demonstrated "An economy of water in renal function referable to urea," which provided early evidence for a unique role of urea in the urinary concentrating mechanism.
The process of urea accumulation in the medulla has been thoroughly studied, and it is generally accepted that urea accumulation is dependent on facilitated urea transport across the epithelium of the IMCD (125). This urea transport process and regulation have been reviewed extensively (65, 221, 222) and thus are not covered here. Central to urea transport within the kidney are UT-A and UT-B facilitative urea transporters. Recently, three mouse models have been created with selective deletion of different urea transporter isoforms. These mouse models have shed new light on the role of urea in the urinary concentrating mechanism.
UT-A1 and UT-A3 are expressed exclusively in IMCD cells. Immunochemical methods have localized UT-A1 to the cytoplasm and apical region of the IMCD (66, 190), whereas UT-A3 is localized both intracellularly and in the basolateral membrane (248, 260) (Fig. 8). In 2004, we developed a mouse model that allowed us to specifically assess the role of inner medullary urea transport in kidney function (63) by deleting both UT-A1 and UT-A3 by standard gene targeting techniques (UT-A1/3–/– mice).
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B) ROLE OF IMCD UREA TRANSPORTERS IN SODIUM CHLORIDE ACCUMULATION IN THE INNER MEDULLA. In 1959, Kuhn and Ramel (139) proposed the classical countercurrent multiplier model, the basis for the urinary concentrating mechanism (see sect. I). The gradient that drives the countercurrent multiplier in the outer medulla, thus concentrating the urine, is dependent on the active reabsorption of NaCl in the water-impermeable TAL. However, the mechanism that concentrates NaCl in the inner medulla interstitium and thus water reabsorption from the collecting ducts remains controversial, as the tAL is apparently incapable of measurable rates of active NaCl transport (97, 132). Various mechanisms have been offered to explain NaCl accumulation in the inner medulla (121, 124, 228, 261), with the most influential model independently proposed by Stephenson (246) and by Kokko and Rector in 1972. In this mechanism, known as the "passive countercurrent multiplier mechanism," the rapid reabsorption of urea from the IMCD generates a high urea concentration in the inner medullary interstitium, resulting in urea-induced water absorption in the descending limb of Henle generating a transepithelial gradient for the passive reabsorption of NaCl from the tAL. In addition, if tAL urea permeability is extremely low (almost zero), then NaCl that has been reabsorbed from the tAL will not be replaced by urea and the ascending limb fluid will be dilute relative to other nephron segments. This dilution process, analogous to that observed in the outer medulla, is proposed to constitute a "single effect" that can be multiplied by the counterflow between the ascending and descending limbs of Henle's loops.
If the passive model of NaCl accumulation in the inner medulla functions as proposed, abolition of rapid passive urea absorption in the IMCD as seen in UT-A1/3–/– mice would be expected to eliminate NaCl accumulation in the inner medulla. However, two independent experiments in UT-A1/3–/– mice failed to corroborate the view that inner medullary NaCl accumulation depends on facilitated urea transport in the IMCD. In an initial experiment, the mean urea, Na+, Cl–, and K+ concentrations were measured in whole inner medulla tissue isolated from water-restricted UT-A1/3–/– mice and wild-type littermates (63). In UT-A1/3–/– mice there was a significantly reduced inner medullary urea concentration. However, there were no reductions in the mean Na+, Cl–, or K+ concentrations. In a second study, osmolality, urea, and Na+ concentrations were measured in the cortex, outer medulla, and two levels of the inner medulla from UT-A1/3–/– and wild-type mice (64) fed either a low- (4%) or high-protein (40%) diet. In wild-type mice, changing the dietary protein intake from 4 to 40% resulted in a greater tissue osmolality that was attributable solely to a greater accumulation of urea in the inner medulla. However, at all levels of the corticomedullary axis, sodium concentrations were unaffected by changes in dietary protein intake. Furthermore, UT-A1/3–/– mice had a substantially attenuated corticomedullary osmolality gradient and no urea gradient on either diet. Despite this difference in urea concentration, the corticomedullary sodium gradients of UT-A1/3–/– mice were virtually identical to wild-type mice. Thus marked medullary urea depletion resulting from either dietary protein restriction or deletion of collecting duct urea transporters does not affect the ability of the kidney to form a corticomedullary sodium chloride gradient. We conclude from these studies in UT-A1/3–/– mice that NaCl accumulation in the inner medulla is not reliant on either IMCD urea transport or the accumulation of urea in the IMCD interstitium. Thus the passive concentrating model in the form originally proposed by Stephenson and by Kokko and Rector, where NaCl reabsorption from Henle's loop depends on a high IMCD urea permeability, is apparently not the mechanism by which NaCl is concentrated in the inner medulla.
Under normal conditions, UT-A2 mRNA and protein are expressed in the inner stripe of the outer medulla (Fig. 8), where it is localized to the lower portions of the tDL of short loops of Henle (66, 237, 278). Under prolonged vasopressin action, UT-A2 mRNA and protein can also be detected in the inner medulla, being localized to the tDL of long loops of Henle. Multiple studies have shown that UT-A2 mRNA and protein can be regulated in response to changes in circulating vasopressin concentration (34, 66, 278), and recent studies have determined that UT-A2-mediated urea transport can be acutely regulated by cAMP (207). The presence of vasopressin receptors has not been reported in the descending limb of Henle's loop, raising the possibility that the effects of vasopressin are indirect, e.g., mediated by autacoids released by vasopressin-responsive cells.
What is the predicted role of UT-A2 in the urinary concentrating mechanism and why is the thin limb localization of UT-A2 important for the kidneys concentrating ability? The large amount of urea that is reabsorbed from the IMCD (via UT-A1 and UT-A3) is trapped in the inner medullary interstitium by countercurrent exchange processes. Any urea that "escapes" the inner medulla via the ascending vasa recta (AVR) can exit via the fenestrated endothelium of the AVR. However, the close proximity of the AVR and short-loop descending limbs in the vascular bundles of the inner stripe theoretically can permit transfer of this urea from the AVR into the descending limbs via UT-A2 (Fig. 3). This mechanism is predicted to be important for the concentrating mechanism by ensuring efficient recycling of urea, thus making urea available to distal sites of short-looped nephrons. Evidence for urea recycling was obtained from the micropuncture studies of Lassiter et al. (145, 146), showing that there is a large amount of net urea secretion into the short loops of Henle, resulting in the delivery of large amounts of urea to the superficial distal tubule that often exceeds the filtered load. UT-A2 in the inner medullary portion of the tDL may also recycle urea absorbed from the ascending thin limb of Henle [which is highly permeable to urea (43) despite the absence of any known urea transporter] and from the AVR. The enhanced countercurrent exchange between ascending and descending structures would help maintain a high urea concentration in the inner medullary interstitium and thus the driving force for the osmotic extraction of water.
UT-A2 knockout mice have recently been developed, and some aspects of their renal phenotype have been described (272). On a normal level of protein intake (20% protein), the UT-A2 null mice do not have significant differences in daily urine output compared with control mice and, even after a 36-h period of water deprivation, differences in urine output and urine osmolality are not observed. Furthermore, UT-A2 knockout mice do not have an impairment of urea or chloride accumulation in the inner medulla (272). These results are surprising, considering the role that UT-A2 has been proposed to play in maintaining a high inner medullary urea concentration. However, on a low-protein diet (4% protein), UT-A2 null mice have a mildly reduced maximal urinary concentrating capacity compared with wild-type controls and a significant reduction in urea accumulation in the inner medulla.
The findings from these initial studies in UT-A2 knockout mice are surprising, especially considering the strongly induced upregulation of UT-A2 under antidiuretic conditions. Clearly from these experiments, under basal conditions, UT-A2 makes a minimal contribution to urea accumulation in the inner medullary interstitium and does not play a major role in the formation of concentrated urine. However, when urea supply to the kidney is limited, UT-A2 may be important for maintaining a high concentration of urea in the inner medulla and thus maximal urinary concentrating ability.
In contrast to the multiple UT-A isoforms described above, the mouse UT-B gene encodes only a single protein. UT-B is expressed exclusively throughout the kidney medulla in the basolateral and apical (luminal) regions of the DVR endothelial cells (209, 268, 288). In humans, UT-B protein carries the Kidd blood group antigen, and subjects lacking this antigen [JK (a–,b–) individuals] have a dramatically reduced urea permeability of red blood cells and a mild urinary concentrating defect (224). UT-B is thought to contribute to the urinary concentrating mechanism by allowing any urea that "escapes" the inner medullary interstitium via the venous AVR, to be recycled back into the DVR, thus not allowing this urea to return to the general circulation. This complex intrarenal urea recycling between AVR and DVR, in combination with recycling of urea between UT-B and UT-A2 (in thin descending limbs of Henle's loop), is thought to maintain the high inner medullary interstitium urea concentration and thus the driving force for passive water reabsorption.
In 2002, Yang et al. (291) developed a mouse model with genetic deletion of UT-B that has enabled the role of UT-B in the urinary concentrating mechanism to be thoroughly investigated. However, since the physiology of the UT-B knockout mice has been recently discussed very extensively elsewhere (290), for the purposes of this article only a brief overview is given. Erythrocytes from UT-B knockout mice have an 45-fold lower urea permeability compared with those from controls. Under basal conditions (normal protein diet), daily urine output in UT-B null mice is significantly higher and urine osmolality significantly lower compared with wild-type mice. However, when UT-B knockout mice are subjected to water deprivation for 36 h, they are able to concentrate their urine, although to a lesser extent than controls. Knockout mice have a significantly lower urine urea concentration (although in same proportion to other solutes) and significantly higher plasma urea; thus their urine-to-plasma urea ratio (U/P) is more severely reduced than that of other solutes. This reduced capacity to concentrate urea compared with other solutes indicates that the UT-B null mice have a "urea-selective" urinary concentrating defect (13). This diminished ability to concentrate urea is highlighted by a lower inner medullary urea concentration compared with other solutes. Taken together, these data indicate that the urinary concentrating defect observed in UT-B null mice is due to impaired urea recycling in the vasa recta, although at present, it is unclear whether the loss of urea transport in red blood cells also contributes to this phenomenon. The latter could occur if urea can enter erythrocytes via non-UT-B pathways as they enter the medulla, but could not exit fast enough to equilibrate with the surrounding interstitium as the erythrocytes are carried back to the general circulation.
H. Receptors and Signaling Molecules
AVP is essential in the regulation of body fluid homeostasis. In response to small increases in plasma osmolality or a reduction in the effective circulating blood volume, AVP is released by the posterior pituitary gland and promotes water reabsorption in the kidney collecting duct, enhanced urinary concentration in the TAL, and vasoconstriction via four subtypes of receptors (93). The antidiuretic effects of AVP result from a cascade of events, initialized by the binding of AVP to the V2R in the collecting duct and TAL (reviewed in Refs. 37, 186). In the collecting duct, this binding causes activation of G-coupled proteins, a rise in intracellular cAMP, and eventually, through several mechanisms, the exocytic insertion of AQP-2 water channels into the apical plasma membrane (Fig. 7). This dramatically increases water permeability (typically 8- to 10-fold) and allows the osmotically driven movement of water from the kidney tubule lumen into the kidney interstitium, promoting water retention and thereby lowering plasma osmolality. Physiological studies, receptor binding, and radioactive tracer studies have shown that V2R is subject to internalization in response to binding of vasopressin (28, 92, 98–100). Internalization may also be involved in the vasopressin escape phenomenon, a physiological adaptation to prevent water intoxication with prolonged vasopressin action (262).
Inactivating V2R mutations in humans cause a rare kidney disease known as X-linked nephrogenic diabetes insipidus (XNDI), an AVP-insensitive form of diabetes insipidus that is inherited in an X-linked manner (223). XNDI patients produce large volumes of dilute urine, are polydipsic, and in the case of an inadequate water supply, can become severely hypernatremic. Failure to recognize the disease in affected boys soon after birth can result in abnormalities of the central nervous system, owing to severe dehydration.
In 2000, Yun et al. (300) created a mouse model of XNDI by introducing a nonsense mutation (Glu242stop) known to cause XNDI in humans into the mouse genome. This particular mutation was chosen as it has been shown that the encoded mutant receptor is retained intracellularly and completely lacks functional activity (231), thus mimicking the functional properties of many other disease-causing V2R mutants.
Male V2R mutant mice (V2R–/y) typically died within 7 days after birth (300). Urine osmolalities, collected from the bladders of 3-day-old pups, were significantly lower than controls. Serum electrolyte analysis revealed that V2R–/y pups have increased Na+ and Cl– levels, indicative of a severe state of hypernatremia. In control mice, an intraperitoneal injection of dDAVP resulted in a significant increase in urine osmolality, whereas no effect was observed in V2R–/y mice. Analysis of adult female V2R+/– mice revealed that the mice have polyuria, polydipsia, and a reduced urinary concentrating ability, clear symptoms of XNDI, although milder than in hemizygous males. Furthermore, females have an 50% decrease in total AVP binding capacity, resulting in an 50% decrease in vasopressin-induced intracellular cAMP levels. Taken together, the results obtained from this loss-of-function mutation in the V2R clearly show that the V2R is necessary for normal regulation of water excretion.
Kinins are produced by proteolytic cleavage of a protein precursor, kininogen, by the enzyme kallikrein. They act as "local hormones" by activating the release of endothelium-derived relaxing factor and prostaglandins (39). Kinins act through a family of G protein-coupled receptors, the B1 and B2 receptors, with the B2 receptor mediating vasodilation, diuresis, and natriuresis (213). In the kidney, kallikrein is localized to the distal nephron including the DCT, connecting tubule, and initial collecting tubule (210). It has been proposed that kinins are formed chiefly in the lumen of the collecting ducts (276). However, in isolated perfused tubule experiments, bradykinin is only effective in decreasing vasopressin-stimulated NaCl and water transport when added to the peritubular environment and not when added to the lumen (265, 266).
It has been proposed that renal kinins counteract the hydrosmotic effect of AVP, and tubule perfusion studies have shown that kinins added to the peritubular bath can decrease AVP-stimulated water reabsorption in the cortical collecting duct (235). Furthermore, in Brattleboro rats, the kallikrein inhibitor aprotinin augments the renal response to AVP (113, 114).
Bradykinin B2 receptor knockout mice have been developed and their renal phenotype partially characterized (3, 27). During basal conditions there is no significant difference in either urine volume or urine osmolality between knockout mice and controls, suggesting that renal kinins play little role in regulation of water excretion under normal conditions. However, after a 24-h fluid restriction, urine volume was significantly lower and urine osmolality significantly higher in knockout mice. Furthermore, subcutaneous administration of dDAVP results in a significantly greater increase in urine osmolality in knockout mice compared with controls. Taken together, these results suggest that water restriction or V2-receptor stimulation has a greater urinary concentrating effect in B2 receptor knockout mice than in controls, suggesting that endogenous kinins acting through B2 receptors oppose the antidiuretic effect of AVP in vivo (33).
Integrins are transmembrane receptors for extracellular matrix components and are thought to play an important role in regulating cytoskeletal organization (208), as well as in the src-dependent activation of mitogen-activated protein (MAP) kinases (84). One member of the integrin family, integrin 11, acts as a collagen binding receptor and is highly expressed in the glomerulus and along the renal tubule (137, 277). Studies of integrin 1 null mice have shown that integrin 11 plays a regulatory role in the hypertonicity-mediated accumulation of osmolytes in the kidney inner medulla (168). Integrin 1 knockout mice have reduced osmo
