In this study, we describe what we believe is the first viable mouse model of XNDI, the most common form of congenital NDI. In these newly developed mutant mice, the V2R gene can be deleted in a conditional (TMX-dependent) fashion in the kidneys of adult mice. The resulting V2R-KO mice showed all key symptoms of XNDI, including the production of large amounts of dilute urine (polyuria) and polydipsia. Following dDAVP administration, the V2R mutant mice were unable to increase their urine osmolality, consistent with the complete absence of renal V2R-binding sites observed in radioligand-binding assays.
Kidneys from V2R-KO mice also showed a distension of the renal pelvis, a characteristic morphological deficit that is generally observed with NDI mutant mice (12
). Moreover, immunoblotting studies showed that renal AQP2 and AQP3 expression levels were drastically reduced (by ~70%–80%) in V2R-KO mice. The kidney collecting duct AQP2 and AQP3 water channels are known to play a key role in mediating AVP-dependent water reabsorption (4
). These observations strongly suggest that human XNDI is also associated with pronounced decreases in AQP2 and AQP3 expression levels.
In addition, conscious V2R-KO mice showed an approximately 40% reduction in GFR (as compared with control littermates), despite having normal blood pressure. This observation is clearly of potential clinical interest, since no systematic GFR measurements have been carried out in XNDI patients (Daniel Bichet, unpublished observations). The precise mechanism underlying this phenotypic change remains unclear at present, but this effect may be secondary to chronic polyuria and dilatation of the renal pelvis with urinary reflux.
Current drug treatment strategies for XNDI are problematic and nonspecific in nature. They include the administration of thiazide diuretics (e.g., hydrochlorothiazide [HCT]) along with a reduction in salt intake, frequently in combination with prostaglandin synthesis inhibitors (e.g., indomethacin) or potassium-sparing diuretics (e.g., amiloride) (1
). However, these drugs only lead to a moderate reduction in urine production, and their use is often associated with severe side effects, including disturbances in electrolyte balance as well as renal and gastrointestinal complications (1
). Moreover, in XNDI, thiazides and indomethacin are predicted to reduce GFR (3
), thereby increasing the risk of nephropathy. Thus, there is a clear need for new classes of XNDI drugs endowed with increased efficacy and reduced side effects.
We speculated that renal collecting duct cells might express Gs
-coupled receptors (besides the V2R) that could serve as more specific targets for the pharmacotherapy of XNDI. Real-time qRT-PCR studies using mouse GPCR array plates demonstrated that mouse IMCD cells express another Gs
-coupled receptor (besides the V2R), the EP4 prostanoid receptor, at significant levels. This receptor subtype has also been shown to be expressed by rat IMCD cells (33
). The EP4 receptor is one of the 4 receptor subtypes through which PGE2
exerts its pharmacological actions (22
To test the potential usefulness of EP4 receptor agonists for the therapy of XNDI, we treated V2R-KO mice with ONO, a drug that is commonly used to study EP4 receptor function. Importantly, ONO is endowed with a very high degree of selectivity for the EP4 receptor subtype (13
). Remarkably, both acute and chronic treatment of V2R mutant mice with ONO greatly reduced all major manifestations of XNDI, leading to striking reductions in urine output and water intake and pronounced increases in urine osmolality.
Long-term complications of XNDI include large dilatations of the renal pelvic space as well as kidney failure secondary to bilateral hydronephrosis (1
). Interestingly, MRI studies showed that prolonged ONO treatment of V2R-KO mice could prevent the further progression of renal pelvic distension associated with the polyuric state caused by the disruption of the V2R
As mentioned above, V2R-KO mice displayed an approximately 40% reduction in GFR. Interestingly, prolonged treatment of V2R-KO mice with ONO restored normal GFR, excluding the possibility that the ONO-mediated decrease in urine output in V2R-KO mice is simply the consequence of reduced GFR. The ONO-mediated normalization of GFR in V2R-KO mice may provide an additional benefit for the potential treatment of XNDI, since it reduces the risk of nephropathy associated with the use of current XNDI drugs (see above). The ONO-mediated increase in GFR observed in the absence of functional V2Rs may be due to EP4 receptor–mediated vasodilator effects on afferent renal arterioles (35
Chronic ONO treatment had no effect on blood pressure, heart rate, and food intake, excluding the possibility that changes in cardiovascular parameters or general malaise contributed to the ONO-mediated decrease in urine output.
Immunoblotting studies demonstrated that prolonged treatment of V2R-KO mice with ONO increased renal AQP2 levels by approximately 70%, probably due to EP4 receptor–mediated elevations of cAMP levels in kidney collecting duct cells (20
). Since AQP2 plays a key role in renal water reabsorption (4
), this observation is of considerable therapeutic relevance.
ONO treatment of collecting duct tubule preparations led to a pronounced increase in cAMP levels and enhanced water permeability. Taken together, these data support the concept that the beneficial effects of ONO in the treatment of XNDI mice are due to a direct action on EP4 receptors expressed by kidney collecting duct cells.
ONO is relatively unstable in aqueous solution (Takayuki Maruyama, unpublished observations). Moreover, due to the limited solubility of ONO, we could not deliver more than 55 μg of ONO per day (per mouse) in the chronic infusion experiments. These factors may explain why ONO only partially reversed the phenotypic changes displayed by the XNDI mutant mice. The development of more stable EP4 receptor agonists with improved pharmacokinetic properties is therefore likely to yield agents that exhibit increased efficacy in the treatment of XNDI. However, it should be noted that even a drug that shows only moderate activity in reducing urine flow is likely to significantly improve the quality of life of individuals suffering from XNDI.
Whereas ONO-mediated activation of renal EP4 receptors leads to reduced urine output, intrarenal PGE2
infusion is known to promote diuresis (40
), probably via activation of other PGE2
receptor subtypes mediating inhibition of salt and water absorption along the nephron (22
). Inhibition of these latter PGE2
effects by indomethacin, which reduces tissue PGE2
levels via nonselective inhibition of cyclooxygenase 1 and 2, is thought to contribute to the ability of this drug to reduce urine production in XNDI patients.
It should be noted that the EP4 receptor is not only expressed in the kidney, but also in several other organs and cell types (23
). Studies with EP4 receptor–deficient mice and EP4 receptor–selective agonists have shown that EP4 receptors play a role in facilitating the closure of the ductus arteriosus in newborns, promoting bone growth, protecting against inflammatory bowel disease, and facilitating the migration and maturation of Langerhans cells in the skin as well as in several other physiological functions (23
Interestingly, GPCR expression analysis demonstrated that mouse IMCD cells also express many orphan GPCR transcripts (Supplemental Table 2). The identification of activating ligands for these receptors might reveal novel Gs
-coupled receptors as potential targets for the treatment of XNDI. Moreover, in a recent review, Bouley et al. (39
) proposed a number of new strategies that might prove useful for the treatment of NDI by bypassing V2R signaling. The potential clinical usefulness of these various strategies can now be tested in the newly generated XNDI mutant mice.
Whereas most patients with congenital NDI harbor inactivating mutations in the V2R
gene, a small subpopulation of these patients (~10%) exhibit mutations in the AQP2
gene, causing NDI with either autosomal dominant or recessive inheritance (2
). As has been observed with V2R-KO mice, inactivation of the AQP2
gene in mice throughout development results in neonatal death (25
). However, several viable mouse models of the autosomal form of congenital NDI have been generated recently (16
In conclusion, we have generated what we believe is the first viable mouse model of human XNDI. Our data support the concept that selective EP4 receptor agonists may represent a new class of drugs useful for the treatment of XNDI due to their direct action on kidney collecting duct cells. The newly generated V2R mutant mice should prove generally useful for testing new strategies for the treatment of XNDI.