Research Article
Jian Hua Li1, Chung-Lin Chou2, Bo Li1, Oksana Gavrilova3, Christoph Eisner4, Jrgen Schnermann4, Stasia A. Anderson5, Chu-Xia Deng6, Mark A. Knepper2 as good as Jrgen Wess1
1Molecular Signaling Section, National Institute of Diabetes as good as Digestive as good as Kidney Diseases, 2Laboratory of Kidney as good as Electrolyte Metabolism, National Heart, Lung, as good as Blood Institute, 3Mouse Metabolic Core Facility as good as 4Renal Function as good as Injury Section, National Institute of Diabetes as good as Digestive as good as Kidney Diseases, 5Animal MRI/Imaging Core, National Heart, Lung, as good as Blood Institute, as good as 6Mammalian Genetics Section, Genetics of Development as good as Diseases Branch, National Institute of Diabetes as good as Digestive as good as Kidney Diseases, NIH, Bethesda, Maryland, USA.
Address correspondence to: Jrgen Wess, Laboratory of Bioorganic Chemistry, Molecular Signaling Section, National Institute of Diabetes as good as Digestive as good as Kidney Diseases, Bldg. 8A, Room B1A-05, 8 Center Drive, Bethesda, Maryland 20892, USA. Phone: (301) 402-3589; Fax: (301) 480-3447; E-mail: jwess@helix.nih.gov.
Published September 1, 2009Received for announcement Apr 27, 2009, as good as supposed in revised form July 1, 2009.
X-linked nephrogenic diabetes insipidus (XNDI) is a serious kidney mildew caused by inactivating mutations in a V2 vasopressin receptor (V2R) gene which outcome in a detriment of renal urine-concentrating ability. At present, no specific pharmacological care has been grown for XNDI, radically due to a miss of befitting animal models. To develop what you hold to be a initial viable animal indication of XNDI, you generated mice in which a V2R gene could be conditionally deleted during adulthood by administration department department department of 4-OH-tamoxifen. Radioligand-binding studies reliable a miss of V2R-binding sites in kidneys following 4-OH-tamoxifen treatment, as good as offer research indicated which upon V2R deletion, adult mice displayed all characteristic symptoms of XNDI, together with polyuria, polydipsia, as good as insurgency to a antidiuretic actions of vasopressin. Gene countenance research referred to which activation of renal EP4 PGE2 receptors competence recompense for a miss of renal V2R wake up in XNDI mice. Strikingly, both strident as good as ongoing diagnosis of a mutant mice with a resourceful EP4 receptor agonist severely marked down all vital manifestations of XNDI, together with changes in renal morphology. These physiological improvements were many approaching due to a approach movement upon EP4 receptors voiced upon pciking up channel cells. These commentary spell out a utility of a newly generated V2R mutant mice for elucidating as good as contrariety brand brand brand new strategies for a intensity diagnosis of humans with XNDI.
In nephrogenic diabetes insipidus (NDI), a kidney is incompetent to preserve H2O despite normal or augmenting plasma levels of a antidiuretic hormone arginine vasopressin (AVP) (15). Most patients (>90%) with inborn NDI bay inactivating mutations in a V2 vasopressin receptor (V2R) gene, which is located upon a enlarged arm of a X chromosome (27). This form of NDI, which is ordinarily referred to as X-linked NDI (XNDI), thus roughly exclusively manifests itself in males.
After a release in to a bloodstream from a posterior pituitary gland, AVP binds to renal V2Rs, which have been voiced in a principal cells of a renal pciking up channel complement (27). AVP contracting to a V2R triggers a activation of a stimulatory G protein Gs, ensuing in elevated intracellular cAMP levels which, by multiform middle steps, in a future foster a insertion of aquaporin-2 (AQP2) H2O channels in to a luminal surface of a principal cells (8). This resource allows for a pacifist movement of H2O from a tubule lumen in to a kidney interstitium as good as in a future in to a bloodstream. Moreover, enlarged V2R wake up offer promotes osmotic reabsorption of H2O from a tubule lumen by triggering increases in AQP2 as good as aquaporin-3 (AQP3) countenance levels in renal pciking up channel cells (9, 10).
At present, some-more than 200 opposite disease-causing V2R mutations have been identified (11). Loss of V2R duty in XNDI patients interferes with H2O reabsorption in a renal pciking up channel system, ensuing in a prolongation of vast volumes (>30 ml/kg/d) of dilute urine (<250 mOsmol/kg) (polyuria) as good as extreme lust (polydipsia). Hypernatremia as good as dehydration, which have been among a many serious complications compared with XNDI, competence cause ongoing renal ineptitude as good as mental retardation, during slightest in a subgroup of XNDI patients (1, 2). Long-term complications include vast dilatations of a urinary tract as good as bladder as good as kidney disaster delegate to shared hydronephrosis (1, 2, 5). Newborns pang from XNDI mostly uncover poor weight gain as good as an altogether disaster to thrive, sometimes complicated by convulsions or death due to hypertonic dehydration, especially if XNDI is not diagnosed early sufficient (1, 2).
Currently, no specific, rarely in outcome pharmacological care has been grown for a diagnosis of XNDI (13), radically due to a miss of befitting animal models. We formerly created V2R-KO mice not in organic V2Rs around expansion (12). However, mutant XNDI pups died inside of a initial postnatal week due to massive dehydration (12).
We here report a era of viable V2R mutant mice in which a V2R gene can be deleted in a redeeming conform in adult mice. Using this brand brand brand new rodent model, you denote which a devalue which selectively activates a EP4 PGE2 receptor subtype (ONO-AE1-329 [ONO]; ref. 13) is rarely in outcome in ameliorating all vital manifestations of XNDI, together with disaster to combine urine, polyuria, as good as dilatation of a renal pelvis, many approaching by a approach movement upon EP4 receptors voiced by kidney pciking up channel cells. These commentary should stimulate a expansion of a brand brand brand new era of drug utilitarian for a diagnosis of tellurian XNDI.
Generation of redeeming V2R-KO mice. The gene-targeting plan used for generating an inducible rodent indication of XNDI is shown in Figure 1A. Importantly, a targeting erect contained a neo selection cassette, flanked by 2 loxP sites, which was introduced in to intron 2 of a V2R gene. A third loxP site was extrinsic in to intron 1. Heterozygous V2R mutant mice (females) in which 1 WT V2R allele was transposed with a loxPneo-modified chronicle of a gene (V2R+/flneo) were achieved by customary rodent genetargeting techniques (Figure 1, A as good as B). In a subsequent step, a neo cassette was removed by crossing V2R+/flneo mice with EIIa-Cre transgenic mice, which denote Cre recombinase in a early bud (14). Female mice heterozygous for a floxed V2R allele (V2Rfl/+) not in both a neo cassette as good as a EIIa-Cre transgene were afterwards crossed to Esr1-Cre transgenic mice, which denote a tamoxifen-inducible Cre recombinase/mutant estrogen receptor alloy protein underneath a transcriptional carry out of a clever ubiquitous promoter (chicken -actin promoter; refs. 15, 16). This mating schedule yielded mice of opposite genotypes together with masculine hemizygous V2R mutant pups harboring a Esr1-Cre transgene (V2Rfl/yEsr1-Cre mice). Male V2Rfl/y mice not in a Esr1-Cre transgene were used as carry out animals in many experiments. All genotypes were achieved during a approaching Mendelian frequency.
To interrupt a V2R gene in V2Rfl/yEsr1-Cre mice, 7- to 8-week-old masculine mice perceived every day injections of 4-OH-tamoxifen (TMX) (0.5 mg i.p./mouse) for 6 consecutive days. PCR research of genomic DNA rebuilt from kidneys of TMX-injected mice of opposite genotypes reliable which TMX diagnosis led to a erasure of coding exon 2 usually in V2Rfl/yEsr1-Cre mice (Figure 1C). To confirm a deficiency of organic V2Rs in kidneys of TMX-treated V2Rfl/yEsr1-Cre mice, you carried out [3H]-AVP saturation contracting studies regulating whole-kidney surface homogenates. Since a kidney additionally expresses low levels of V1a vasopressin receptors (17) as good as [3H]-AVP binds to all vasopressin receptor subtypes, all contracting studies were carried out in a presence of a resourceful V1a receptor antagonist, HO-LVA (2 nM; refs. 18, 19). This research showed which kidney preparations from TMX-treated carry out mice (V2Rfl/y mice) bound [3H]-AVP with tall affinity (Kd = 0.73 0.06 nM) as good as a Bmax worth (maximum receptor density) of 54 eleven fmol/mg surface protein (n = 3; Figure 1D). In distinguished contrast, kidney membranes rebuilt from TMX-treated V2Rfl/yEsr1-Cre mice were utterly abandoned of specific [3H]-AVPbinding wake up (n = 4; Figure 1D), confirming which TMX-induced activation of a Esr1-Cre alloy protein led to a in outcome intrusion of a V2R gene in V2R-expressing kidney cells. Thus, for a consequence of simplicity, TMX-treated V2Rfl/yEsr1-Cre mice have been referred to as V2R-KO mice in a following.
Conditional V2R-KO mice vaunt sustained excretion of vast amounts of hypotonic urine. Male V2Rfl/yEsr1-Cre mice as good as carry out littermates (V2Rfl/y mice) (mouse age, 7 to 8 weeks) were placed in to metabolic cages, as good as 24-hour urine prolongation as good as H2O money coming in were monitored over 10 days. Each rodent perceived a singular every day injection of TMX (0.5 mg i.p./mouse) for a 6-day period. Prior to TMX administration, urine outlay as good as H2O expenditure were not statistically opposite in between a 2 groups of mice (however, V2Rfl/yEsr1-Cre showed a slight trend to an augmenting urine outlay as good as H2O intake; P > 0.05; Figure 2, A as good as B). The TMX injections had no poignant outcome upon these 2 variables in carry out mice (Figure 2, A as good as B). In contrast, TMX diagnosis of V2Rfl/yEsr1-Cre mice led to a time-dependent, thespian enlarge in urine prolongation as good as H2O money coming in (Figure 2, A as good as B). At a finish of a 10-day regard period, TMX-treated V2Rfl/yEsr1-Cre mice displayed conspicuous polyuria as good as polydipsia, producing you estimate 2530 g of urine as good as immoderate about a same amount of H2O per day (Figure 2, A as good as B). Figure 2C shows which a urine produced by TMX-treated V2Rfl/yEsr1-Cre mice had a really low osmolality (175 eighteen mOsmol/kg; n = 5). These interpretation have been in agreement with a regard which TMX diagnosis of V2Rfl/yEsr1-Cre mice leads to a finish detriment of renal V2R-binding wake up (Figure 1D).
We additionally remarkable which a urine osmolality of a V2Rfl/yEsr1-Cre mice was significantly reduce (by ~25%) than which of a carry out mice even prior to to TMX diagnosis (day 50, P < 0.01; Figure 2C). One possible reason for this materialisation is which a Esrl-Cre transgene is slightly leaky, displaying a tiny grade of TMX-independent Cre wake up (15).
Consistent with a urine osmolality measurements, TMX-mediated erasure of a V2R gene resulted in thespian reductions in urine urea, creatinine, as good as Na+ as good as K+ concentrations (Supplemental Table 1; supplemental material accessible online with this article; doi:10.1172/JCI39680DS1). However, a serum Na+ levels of V2R-KO mice remained in a physiological operation (serum Na+ is a vital determinant of serum osmolality). The serum Na+ levels of V2Rfl/yEsr1-Cre mice totalled 2 weeks prior to as good as 2 weeks after a TMX injection duration amounted to 146 1 as good as 149 1 mEq/l, respectively (mean SEM; n = 7).
V2R-KO mice additionally showed marked down weight gain, as compared with their TMX-treated carry out littermates. Figure 2D shows which 160-day-old V2R-KO mice weighed you estimate 25% less than a carry out animals, substantially due to a physiological stress caused by a XNDI phenotype. Most importantly, TMX-induced erasure of a V2R gene resulted in viable V2R-KO mice, in contrariety with mutant V2R mice not in organic V2Rs around expansion (12).
We subsequent totalled urine osmolalities in reply to a singular injection (0.4 g/kg, i.p.) of 1-desamino-8-D-AVP (dDAVP), a resourceful V2R agonist. Prior to TMX treatment, V2Rfl/yEsr1-Cre mice as good as carry out littermates (V2Rfl/y mice) were able to combine their urine to a identical extent in reply to dDAVP (Figure 2, EG). Similarly, dDAVP administration department department department led to a poignant enlarge in urine osmolality in TMX-treated carry out mice (Figure 2, H as good as J). In distinguished contrast, dDAVP diagnosis was incompetent to enlarge urine osmolality in TMX-treated V2Rfl/yEsr1-Cre mice (Figure 2, we as good as J), unchanging with a miss of renal V2Rs (Figure 1D).
Conditional V2R-KO mice uncover changes in renal morphology as good as marked down renal AQP2 as good as AQP3 countenance levels. To establish possibly a inducible erasure of a V2R gene led to changes in kidney morphology, right kidneys from TMX-treated V2Rfl/yEsr1-Cre mice (n = 5) as good as carry out littermates (V2Rfl/y mice; n = 7) were resected as good as photographed (mouse age, 8 months). We found which all kidneys rebuilt from V2R-KO mice, though none of a kidneys achieved from a carry out mice, displayed a substantial distension of a renal pelvis (representative photographs have been shown in Figure 3A).
V2R wake up is good good known to regulate a countenance levels of assorted renal proteins critical for urine concentration, together with opposite sodium channels as good as a AQP2 as good as AQP3 H2O channels (for a recent review, see ref. 20). Immunoblotting studies showed which AQP2 as good as AQP3 countenance levels were drastically marked down (by ~70%80%) in V2R-KO mice as compared with carry out mice (mouse age, 8 months; Figure 3B). In contrast, redeeming erasure of a V2R gene had no poignant outcome upon a protein countenance levels of a Na-K-2Cl cotransporter (NKCC2), a Na-Cl cotransporter (NCC), as good as a epithelial sodium channel subunits -ENaC as good as -ENaC (Supplemental Figure 1).
Mouse middle medullary pciking up channel cells denote a Gs-coupled EP4 PGE2 receptor. The V2R is a Gs-coupled receptor, as good as Gs-mediated cAMP prolongation is good good known to be essential for a antidiuretic wake up mediated by this receptor subtype. We speculated which renal pciking up channel cells competence denote alternative Gs-coupled receptors which could offer as intensity targets (V2R bypasses) for a diagnosis for XNDI. To establish which GPCRs have been voiced by rodent middle medullary pciking up channel (IMCD) cells, you initial rebuilt sum RNA from IMCD tubule suspensions regulating C57BL/6 WT mice (10-week-old males). We afterwards totalled receptor countenance levels around real-time quantitative RT-PCR (qRT-PCR) regulating rodent GPCR form plates containing specific primers as good as TaqMan probes for all rodent GPCRs, except for a odorant, taste, as good as pheromone receptors. These studies showed which rodent IMCD cells denote many GPCRs together with a V2R, which valid to be a many rarely voiced GPCR (Supplemental Table 2). Besides a V2R, this research identified 1 a singular some-more Gs-coupled receptor which was voiced in rodent IMCD cells during poignant levels, a EP4 subtype of a PGE2 receptor (or marked down EP4 receptor; Supplemental Table 2). Real-time qRT-PCR research showed which a TMX-induced erasure of a V2R gene had no poignant goods upon EP4 receptor transcript levels in rodent IMCD cells (data not shown).
A singular injection of ONO, a resourceful EP4 receptor agonist, ameliorates a pass manifestations of XNDI. To exam a probability which kick of renal EP4 receptors is profitable for a diagnosis of XNDI, V2R-KO mice (TMX-treated V2Rfl/yEsr1-Cre mice; age, 7 months) were given a singular sip of a resourceful EP4 receptor agonist ONO (0.05 or 0.5 mg/kg s.c.; ref. 13). Figure 4, A as good as B, shows which ONO diagnosis led to dose-dependent increases in urine osmolality in V2R-KO mice (urine osmolalities were totalled rught away prior to as good as 1.52 hours after ONO injection). The higher ONO sip (0.5 mg/kg) augmenting urine osmolalities in V2R-KO mice by you estimate 2.5-fold (Figure 4B). Treatment of carry out littermates (TMX-treated V2Rfl/y mice) with 0.5 mg/kg of ONO led to a many not as big (~1.2-fold) enlarge in urine osmolalities (Figure 4C).
Acute goods of ONO upon urine osmolality, urine output, as good as H2O consumption.(A as good as B) Acute urine-concentrating outcome of ONO. V2R-KO mice (TMX-treated V2Rfl/yEsr1-Cre mice; age, 6.5 months) perceived a singular sip of ONO (0.05 or 0.5 mg/kg s.c.) or car (n = 4 or 5). Urine was picked up rught away prior to as good as 1.52 hours after ONO or car injection. (A) Urine osmolality interpretation achieved with particular mice have been shown. (B) Summary of interpretation shown in A. (C) Summary of urine osmolality interpretation achieved with ONO-treated carry out mice. Control mice (TMX-treated V2Rfl/y mice; age, 6.5 months) perceived a singular sip of ONO (0.5 mg/kg s.c.; n = 6) or car (n = 9). (D) Time march of changes in urine osmolality totalled in ONO-treated V2R-KO mice. V2R-KO mice (age, 7 months) were treated with colour with colour with colour with possibly car (n = 5) or ONO (0.5 mg/kg s.c., n = 6). (E) Cumulative urine prolongation totalled in ONO-treated V2R-KO mice. V2R-KO mice (age, 7 months) were injected with ONO (0.5 mg/kg s.c., n = 6) or car (n = 5). (F) Water money coming in of ONO-treated V2R-KO mice. V2R-KO mice (age, 7 months) were injected with ONO (0.5 mg/kg s.c., n = 6) or car (n = 5). Water money coming in was totalled 6 hours after injection. Data have been shown as meant SEM. *P < 0.05; ***P < 0.001.
To acquire report about a duration of a urinary concentrating goods of ONO, you injected V2R-KO mice with 0.5 mg/kg (s.c.) of ONO as good as monitored urine osmolalities as good as accumulative urine outlay over a 6-hour period. V2R-KO mice showed an you estimate 3-fold enlarge in urine osmolality 1 hour after ONO injection as good as an you estimate 2-fold enlarge in urine osmolality during a 2-hour time indicate (Figure 4D). At later time points, urine osmolalities returned to baseline values (~160 mOsmol/kg; Figure 4D), indicating which ONO has a comparatively marked down biological half-life in mice (<3 hours). However, during present, no pharmacokinetic interpretation have been accessible for ONO (Takayuki Maruyama, personal communication).
Consistent with a urine osmolality measurements, urine outlay was dramatically marked down in V2R-KO mice during a initial 2 hours after ONO injection (Figure 4E). At a finish of a 6-hour regard period, accumulative urine outlay was you estimate 50% reduce in ONO-treated than in vehicle-treated V2R-KO mice (Figure 4E). At a same time point, accumulative H2O expenditure was additionally decreased by you estimate 50% in ONO-treated V2R-KO mice, as compared with vehicle-treated V2R-KO mice (Figure 4F). Taken together, these commentary indicate which ONO is rarely influential in augmenting urine osmolality as good as reducing urine outlay as good as H2O expenditure in a deficiency of V2Rs.
Beneficial goods of enlarged ONO diagnosis in XNDI mutant mice. Since a singular s.c. injection of ONO had usually a comparatively short-lasting outcome (Figure 4D), you infused V2R-KO mice (TMX-treated V2Rfl/yEsr1-Cre mice; age, 3 months) with ONO by regulating osmotic minipumps for up to 3 weeks (55 g ONO/d/mouse s.c.). Prolonged ONO distillate led to a distinguished rebate in urine outlay as good as H2O money coming in of V2R-KO mice as good as a consequent enlarge in urine osmolality, as compared with preinfusion values (Figure 5, AD). The enlarge in urine osmolality was many conspicuous after a initial day of ONO distillate (Figure 5D). Since ongoing kick of GPCRs is good good known to trigger multiple cellular events which moderate hankie responses in a the singular after an one more presence of an activating ligand (receptor desensitization), a singular probability is which receptor desensitization events have been obliged for a regard which a ONO goods upon urine osmolality were marked down during later time points (Figure 5D).
To establish possibly enlarged ONO diagnosis had any outcome upon a pathological changes in kidney morphology customarily compared with XNDI, you in use MRI which relies upon a capability of a chelated gadolinium contrariety representative to amass in a urinary space (21). V2Rfl/yEsr1-Cre mice perceived possibly car or ONO (55 g/d/mouse) around s.c. distillate for 2 weeks regulating osmotic minipumps. Mice were serially imaged, initial prior to a 6-day TMX injection period, afterwards 3 days after a final TMX injection, as good as eventually 2 weeks after a begin of car or ONO distillate (infusions were instituted 5 days after a final TMX injection). In vehicle-treated V2R-KO mice, a distension of a renal pelvis caused by TMX-mediated intrusion of a V2R gene became some-more conspicuous during a march of a examination (Figure 5, E as good as F). Figure 5F shows which a volume of pelvic liquid augmenting by you estimate 2-fold during a 2-week car distillate period. In contrast, ONO diagnosis of V2R-KO mice utterly prevented a offer expansion of renal pelvic space (Figure 5, E as good as F).
Immunoblotting studies showed which s.c. ONO diagnosis (55 g/d/mouse) of V2R-KO mice for 2 weeks augmenting renal AQP2 levels by you estimate 70% (P < 0.01; Figure 6, A as good as B). Mean AQP3 levels were additionally augmenting by you estimate 40% after a 2-week ONO distillate duration (this effect, however, unsuccessful to reach statistical significance; P = 0.12; Figure 6, A as good as B).
Analysis of a resource underlying a profitable goods of ONO in a diagnosis of XNDI mice. As already mentioned above, a antidiuretic wake up of a V2R depends upon a capability to turn upon a stimulatory G protein, Gs, leading to augmenting cAMP levels, which, around a cascade of biochemical events, in a future trigger an enlarge in H2O permeability of a kidney pciking up channel cells. Like a V2R, a EP4 prostanoid receptor is a Gs-coupled receptor (22, 23) which is voiced in rodent pciking up channel cells (Supplemental Table 2). Consistent with this observation, diagnosis of WT rodent IMCD tubule suspensions with possibly ONO or dDAVP led to concentration-dependent increases in intracellular cAMP levels (Figure 7, A as good as B). The magnitude of a dDAVP-mediated cAMP reply was greater than which of a corresponding ONO response, substantially as a consequence of a really tall V2R countenance levels in IMCD cells (Supplemental Table 2).
To investigate a outcome of ONO upon a H2O permeability of kidney pciking up channel cells, cortical pciking up channel segments were microdissected from rodent kidneys as good as perfused in vitro regulating an removed tubule microperfusion technique (24). Osmotic H2O permeability (Pf in m/s) was totalled fluorometrically regulating a fluorescent luminal pen (fluorescein sulfonate). Consistent with a in vitro cAMP measurements (Figure 7A), incubation of removed tubules with ONO (100 nM) led to an you estimate 2.5-fold enlarge in H2O permeability (Pf in m/s: control, 35 33; ONO, 83 31; n = 5 per group [preparations were achieved from 5 opposite animals]; P < 0.05).
In vivo studies showed which ongoing s.c. distillate of V2R-KO mice with ONO (55 g every day per mouse) had no poignant outcome upon diastolic as good as systolic red red red blood vigour as good as heart rate (Supplemental Figure 2), incompatible a probability which altered cardiovascular parameters have been obliged for a capability of ONO to reduce urine outlay in a deficiency of organic V2Rs. Moreover, enlarged ONO diagnosis of V2R-KO mice had no poignant outcome upon food intake, strongly suggesting which ONO does not cause ubiquitous sadness (Supplemental Figure 3).
Finally, to bar a probability which a ONO-induced diminution in urinary outlay in V2R-KO mice was due to a rebate in glomerular filtration rate (GFR), you carried out GFR measurements regulating unwavering V2R-KO mice as good as carry out littermates (TMX-treated V2Rfl/y mice) chronically treated with colour with colour with colour (s.c.) with possibly car or ONO (55 g/d/mouse) around osmotic minipumps. Vehicle-treated carry out mice showed a GFR of 395 26 l/min (Figure 6C). The GFR of ONO-treated carry out mice was not significantly opposite from this worth (344 fourteen l/min; Figure 6C). In contrast, vehicle-treated V2R-KO mice showed a poignant rebate (by ~40%) of GFR (246 eighteen l/min), as compared with vehicle-treated carry out mice (P < 0.05; Figure 6C). Remarkably, distillate of V2R-KO mice with ONO easy normal GFR (428 26 l/min; Figure 6C).
Taken together, these observations denote which a profitable goods of ONO in ameliorating a symptoms of XNDI in V2R-KO mice have been due to activation of EP4 prostanoid receptors voiced by renal pciking up channel cells.
In this study, you describe what you hold is a initial viable rodent indication of XNDI, a many usual form of inborn NDI. In these newly grown mutant mice, a V2R gene can be deleted in a redeeming (TMX-dependent) conform in a kidneys of adult mice. The ensuing V2R-KO mice showed all pass symptoms of XNDI, together with a prolongation of vast amounts of dilute urine (polyuria) as good as polydipsia. Following dDAVP administration, a V2R mutant mice were incompetent to enlarge their urine osmolality, unchanging with a finish deficiency of renal V2R-binding sites celebrated in radioligand-binding assays.
Kidneys from V2R-KO mice additionally showed a distension of a renal pelvis, a characteristic morphological necessity which is in all celebrated with NDI mutant mice (12, 16, 2529). Moreover, immunoblotting studies showed which renal AQP2 as good as AQP3 countenance levels were drastically marked down (by ~70%80%) in V2R-KO mice. The kidney pciking up channel AQP2 as good as AQP3 H2O channels have been good good known to fool around a pass purpose in mediating AVP-dependent H2O reabsorption (4, 3032). These observations strongly indicate which tellurian XNDI is additionally compared with conspicuous decreases in AQP2 as good as AQP3 countenance levels.
In addition, unwavering V2R-KO mice showed an you estimate 40% rebate in GFR (as compared with carry out littermates), despite carrying normal red red red blood pressure. This regard is obviously of intensity clinical interest, given no systematic GFR measurements have been carried out in XNDI patients (Daniel Bichet, unpublished observations). The precise resource underlying this phenotypic change stays unclear during present, though this outcome competence be delegate to ongoing polyuria as good as dilatation of a renal pelvis with urinary reflux.
Current drug diagnosis strategies for XNDI have been problematic as good as nonspecific in nature. They include a administration department department department of thiazide diuretics (e.g., hydrochlorothiazide [HCT]) along with a rebate in salt intake, mostly in multiple with prostaglandin synthesis inhibitors (e.g., indomethacin) or potassium-sparing diuretics (e.g., amiloride) (13). However, these drug usually lead to a moderate rebate in urine production, as good as their use is mostly compared with serious side effects, together with disturbances in electrolyte change as good as renal as good as gastrointestinal complications (1, 2). Moreover, in XNDI, thiazides as good as indomethacin have been expected to reduce GFR (3), thereby augmenting a risk of nephropathy. Thus, there is a transparent need for brand brand brand new classes of XNDI drug endowed with augmenting efficacy as good as marked down side effects.
We speculated which renal pciking up channel cells competence denote Gs-coupled receptors (besides a V2R) which could offer as some-more specific targets for a pharmacotherapy of XNDI. Real-time qRT-PCR studies regulating rodent GPCR form plates demonstrated which rodent IMCD cells denote an one more Gs-coupled receptor (besides a V2R), a EP4 prostanoid receptor, during poignant levels. This receptor subtype has additionally been shown to be voiced by rodent IMCD cells (33). The EP4 receptor is a singular of a 4 receptor subtypes by which PGE2 exerts a pharmacological actions (22, 23).
To exam a intensity utility of EP4 receptor agonists for a care of XNDI, you treated with colour with colour with colour V2R-KO mice with ONO, a drug which is ordinarily used to investigate EP4 receptor function. Importantly, ONO is endowed with a really tall grade of selectivity for a EP4 receptor subtype (13, 34). Remarkably, both strident as good as ongoing diagnosis of V2R mutant mice with ONO severely marked down all vital manifestations of XNDI, leading to distinguished reductions in urine outlay as good as H2O money coming in as good as conspicuous increases in urine osmolality.
Long-term complications of XNDI include vast dilatations of a renal pelvic space as good as kidney disaster delegate to shared hydronephrosis (1, 2, 5). Interestingly, MRI studies showed which enlarged ONO diagnosis of V2R-KO mice could prevent a offer progression of renal pelvic distension compared with a polyuric state caused by a intrusion of a V2R gene.
As mentioned above, V2R-KO mice displayed an you estimate 40% rebate in GFR. Interestingly, enlarged diagnosis of V2R-KO mice with ONO easy normal GFR, incompatible a probability which a ONO-mediated diminution in urine outlay in V2R-KO mice is simply a consequence of marked down GFR. The ONO-mediated normalization of GFR in V2R-KO mice competence produce an a singular some-more benefit for a intensity diagnosis of XNDI, given it reduces a risk of nephropathy compared with a use of current XNDI drug (see above). The ONO-mediated enlarge in GFR celebrated in a deficiency of organic V2Rs competence be due to EP4 receptormediated vasodilator goods upon afferent renal arterioles (35, 36).
Chronic ONO diagnosis had no outcome upon red red red blood pressure, heart rate, as good as food intake, incompatible a probability which changes in cardiovascular parameters or ubiquitous sadness contributed to a ONO-mediated diminution in urine output.
Immunoblotting studies demonstrated which enlarged diagnosis of V2R-KO mice with ONO augmenting renal AQP2 levels by you estimate 70%, substantially due to EP4 receptormediated elevations of cAMP levels in kidney pciking up channel cells (20, 3739). Since AQP2 plays a pass purpose in renal H2O reabsorption (4, 3032), this regard is of substantial healing relevance.
ONO diagnosis of pciking up channel tubule preparations led to a conspicuous enlarge in cAMP levels as good as enhanced H2O permeability. Taken together, these interpretation await a judgment which a profitable goods of ONO in a diagnosis of XNDI mice have been due to a approach movement upon EP4 receptors voiced by kidney pciking up channel cells.
ONO is comparatively unstable in aqueous resolution (Takayuki Maruyama, unpublished observations). Moreover, due to a limited solubility of ONO, you could not broach some-more than 55 g of ONO per day (per mouse) in a ongoing distillate experiments. These factors competence explain because ONO usually partially topsy-turvy a phenotypic changes displayed by a XNDI mutant mice. The expansion of some-more stable EP4 receptor agonists with improved pharmacokinetic properties is thus approaching to produce agents which vaunt augmenting efficacy in a diagnosis of XNDI. However, it should be remarkable which even a drug which shows usually moderate wake up in reducing urine flow is approaching to significantly urge a quality of hold up of people pang from XNDI.
Whereas ONO-mediated activation of renal EP4 receptors leads to marked down urine output, intrarenal PGE2 distillate is good good known to foster diuresis (40), substantially around activation of alternative PGE2 receptor subtypes mediating predicament of salt as good as H2O fullness along a nephron (22). Inhibition of these latter PGE2 goods by indomethacin, which reduces hankie PGE2 levels around nonselective predicament of cyclooxygenase 1 as good as 2, is thought to minister to a capability of this drug to reduce urine prolongation in XNDI patients.
It should be remarkable which a EP4 receptor is not usually voiced in a kidney, though additionally in multiform alternative viscera as good as dungeon sorts (23). Studies with EP4 receptordeficient mice as good as EP4 receptorselective agonists have shown which EP4 receptors fool around a purpose in facilitating a closure of a ductus arteriosus in newborns, compelling bone growth, protecting opposite inflammatory bowel disease, as good as facilitating a emigration as good as maturation of Langerhans cells in a skin as good as in multiform alternative physiological functions (23).
Interestingly, GPCR countenance research demonstrated which rodent IMCD cells additionally denote many orphan GPCR transcripts (Supplemental Table 2). The identification of activating ligands for these receptors competence vaunt novel Gs-coupled receptors as intensity targets for a diagnosis of XNDI. Moreover, in a recent review, Bouley et al. (39) due a series of brand brand brand new strategies which competence infer utilitarian for a diagnosis of NDI by bypassing V2R signaling. The intensity clinical utility of these assorted strategies can right away be tested in a newly generated XNDI mutant mice.
Whereas many patients with inborn NDI bay inactivating mutations in a V2R gene, a tiny subpopulation of these patients (~10%) vaunt mutations in a AQP2 gene, causing NDI with possibly autosomal dominant or recessive inheritance (2, 4, 31). As has been celebrated with V2R-KO mice, inactivation of a AQP2 gene in mice around expansion results in neonatal death (25, 27). However, multiform viable rodent models of a autosomal form of inborn NDI have been generated recently (16, 2629, 32, 41).
In conclusion, you have generated what you hold is a initial viable rodent indication of tellurian XNDI. Our interpretation await a judgment which resourceful EP4 receptor agonists competence represent a brand brand brand new class of drug utilitarian for a diagnosis of XNDI due to their approach movement upon kidney pciking up channel cells. The newly generated V2R mutant mice should infer in all utilitarian for contrariety brand brand brand new strategies for a diagnosis of XNDI.
Mouse maintenance as good as diet. Mice were housed in a specific pathogenfree separator facility (4 to 5 mice per cage) as good as maintained upon a 12-hour light/12-hour dim cycle. Mice were fed ad libitum with a customary rodent food (4% [w/w] fat content; Zeigler). Bedding as good as H2O bottles were transposed every day in cages housing V2R-KO mice. All studies were carried out with masculine littermates maintained upon a C57BL/6 background. All experiments were authorized by a Animal Care as good as Use Committee of a National Institute of Diabetes as good as Digestive as good as Kidney Diseases.
Generation of redeeming V2R-KO mice. The V2R-targeting erect (Figure 1A) was derived from a pPN2T vector (42) containing a PGK-neomycin insurgency gene (neo cassette) as good as 2 copies of a herpes simplex virus thymidine kinase (HSV-TK) gene. The rodent V2R coding sequence, which codes for 371 amino acids, is interrupted by 2 marked down introns, referred to as intron 1 as good as 2 (Figure 1A). The neo cassette, flanked by 2 loxP sequences, was extrinsic in to intron 2, as described by Yun et al. (12) (Figure 1A). In addition, a third loxP site was extrinsic in to intron 1 (185 bp downstream of a ATG begin codon). The final targeting erect contained you estimate 6 kb of genomic process upstream of a initial loxP site (5 arm) as good as 1.4 kb of genomic process downstream of a neo gene (3 arm), respectively.
The newly generated targeting erect (Figure 1A) was linearized with NotI as good as electroporated in to 129 SvEv ES cells (43). G418- as good as FIAU-resistant clones were removed as good as screened for proper integration of a V2R targeting erect around Southern blotting, regulating BamHI-digested DNA as good as an external 3 examine (Figure 1, A as good as B). Properly targeted ES dungeon clones were microinjected in to C57BL/6 rodent blastocysts to generate chimeric mice. Female chimeric mice were afterwards corresponding with masculine WT C57BL/6 mice (Taconic) to acquire F1 offspring. F1 mice containing a floxed V2R allele were identified around Southern blotting (Figure 1, A as good as B). To eliminate a neo cassette, womanlike F1 mice carrying a floxed V2R allele were corresponding with EIIa-Cre transgenic mice (official strain name: B6.FVB-Tg(EIIa-cre)C5379Lmgd/J; genetic background: C57BL/6; The Jackson Laboratory), which denote Cre recombinase in a early bud (14, 44). The successful dismissal of a neo gene was reliable by PCR research of tail genomic DNA regulating a plan shown in Figure 1C. Female mice heterozygous for a floxed V2R allele not in a neo cassette were backcrossed to WT masculine C57BL/6 mice (Taconic) for 2 generations to safeguard germline delivery of a mutant V2R gene. In a subsequent step, womanlike mice heterozygous for a floxed V2R allele not in both a neo cassette as good as a EIIa-Cre transgene were crossed to Esr1-Cre transgenic mice (official strain name: Tg(cre/Esr1)5Amc/J; genetic background: C57BL/6; The Jackson Laboratory). Esr1-Cre transgenic mice denote a tamoxifen-inducible Cre recombinase/mouse mutant estrogen receptor alloy protein underneath a transcriptional carry out of a duck -actin promoter/enhancer joined with a CMV immediate-early enhancer (15, 16). This mating schedule yielded mice of opposite genotypes, together with masculine hemizygous V2R mutant pups harboring a Esr1-Cre transgene (V2Rfl/yEsr1-Cre mice; redeeming V2R-KO mice). Male V2Rfl/y mice which did not contain a Esr1-Cre transgene served as carry out animals in many experiments.
Induction of V2R gene deletion. A stock resolution of TMX (Sigma-Aldrich) in ethanol (250 mg/ml) was widely separated in corn oil to 5 mg/ml. Male V2Rfl/yEsr1-Cre as good as V2Rfl/y mice (controls) which were 7 to 8 weeks aged perceived singular every day i.p. injections of TMX (0.1 ml of a 5 mg/ml suspension) over 6 consecutive days, following a protocol identical to which described by Yang et al. (16).
PCR genotyping analysis. Mouse tail biopsies were used for PCR genotyping studies by regulating a rapid genotyping kit (Extract-N-Amp Tissue PCR Kit; Sigma-Aldrich). The sequences of a PCR primers used have been listed in Supplemental Table 3. The presence of a Esr1-Cre transgene was rescued by regulating PCR primers Cre1 as good as Cre2. The presence of a loxP site in intron 1 was verified by regulating primers p1 as good as p2. The successfully dismissal of a neo cassette was reliable by regulating primers p3 as good as p4. The surgery of coding exon 2 was verified by regulating primers p1 as good as p4. All PCR reactions were run underneath a following conditions: 94C for 5 minutes, followed by 31 cycles during 94C for 45 seconds, 55C for 30 seconds, as good as 72C for 30 seconds. The approaching sizes of a opposite PCR products have been indicated in Figure 1C.
Southern blot analysis. A 414-bp PCR product amplified with primers 3.1-F (5-AATTCATGTTCTGTGCCACAG) as good as 3.1-R (5-CACAGGAGCTAAAGCCTGGTA) was used as a examine (3 probe; Figure 1A). The assorted recombination events were reliable by Southern hybridization (Figure 1A). In brief, 10 g of tail genomic DNA was digested with BamHI, electrophoresed, transferred to a Nylon+ surface (Schleicher & Schuell BioScience), as good as hybridized with a 32P-labeled 3 probe.
Measurement of short-term goods of drug upon urinary osmolality. Urine was picked up rught away prior to as good as 1.5 to 2 hours after injection of dDAVP (0.4 g/kg i.p.; Sigma-Aldrich) or ONO (0.05 or 0.5 mg/kg s.c.; present from Takayuki Maruyama, Ono Pharmaceutical Co. Ltd., Osaka, Japan). Urine samples were picked up by fixation mice upon a wire filigree height in a purify potion beaker.
Metabolic cage studies as good as urine osmolality measurements. Urine outlay as good as H2O expenditure were totalled in metabolic cages (Hatteras Instruments) adapted for mice (for details, see Supplemental Methods). Urine osmolalities were totalled around customary procedures regulating a vapor vigour osmometer (model 5520; Wescor).
Urine as good as serum chemistry. Blood samples were picked up in heparinized potion tubes by puncture of a periorbital venous sinus. Urine as good as serum chemistry measurements were carried out by a Laboratory of Animal Medicine as good as Surgery of a National Heart, Lung, as good as Blood Institute.
[3H]-AVP saturation contracting studies. Whole kidneys were picked up from 8-month-old masculine mice, as good as surface preparations were achieved radically as described (12). Membrane preparations (200 g protein/tube) were incubated with augmenting concentrations (0.1254.0 nM) of [3H]-AVP (61.9 Ci/mmol; PerkinElmer). Incubations were carried out for 2 hours during room temperature. To block renal V1a receptors, 2 nM of HO-LVA (a present from Maurice Manning, The University of Toledo, Toledo, Ohio, USA), a rarely resourceful V1a receptor antagonist (18, 19), was combined to all tubes. Reactions were terminated by rapid filtration over GF/C filters (Brandel). Nonspecific contracting was defined as contracting in a presence of 5 M dDAVP. Binding interpretation were analyzed by a nonlinear 1-binding-site curve-fitting procedure regulating Prism (version 4.0; GraphPad Software Inc.).
Immunoblotting studies. Whole-kidney homogenates were rebuilt from rodent kidneys as good as solubilized in Laemmlis reagent. Immunoblotting studies were achieved regulating rabbit affinity-purified polyclonal antibodies, as described formerly (45). Thirty g of protein were installed per lane. The blots were grown regulating an Alexa Fluor 680conjugated delegate antibody (Molecular Probes). Bands were visualized as good as analyzed regulating LI-CORs Odyssey infrared imaging complement (LI-COR) (45). To facilitate comparisons, you normalized band intensities such which a meant for a carry out mice was defined as 1 (AU).
Preparation of IMCD tubule suspensions. Mouse IMCD tubule suspensions were rebuilt as described formerly with slight modifications (46). Inner medullas were dissected out from kidneys of C57BL/6 WT mice (10-week-old males), minced, as good as enzymatically digested in sucrose resolution (250 mM sucrose, 10 mM triethanolamine, pH 7.6) containing 3 mg/ml collagenase B as good as 2000 U hyaluronidase during 37C for 90 minutes. The ensuing cessation was centrifuged during 80 g for 30 seconds. IMCD-enriched pellets were harvested after 3 washes with sucrose solution.
cAMP assays. cAMP assays were achieved regulating rodent IMCD tubule suspensions rebuilt as described above. For any particular experiment, IMCD suspensions were rebuilt as good as pooled from a kidneys of 7 C57BL/6 WT mice (10-week-old males). Aliquots of IMCD tubule suspensions were preincubated with a cAMP phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) as good as Ro-20 (0.1 mM each; Sigma-Aldrich) for 10 mins during 37C. Subsequently, opposite concentrations of ONO (0.1100 M) or dDAVP (0.1100 nM) were added, as good as reactions were carried out for 30 mins during 37C. Total intracellular cAMP was dynamic around ELISA according to a manufacturers instructions (cAMP Biotrak; GE Healthcare). Protein concentrations were dynamic by regulating a Bradford process (Bio-Rad). cAMP interpretation were analyzed regulating Prism program (version 4.0; GraphPad Software Inc.).
Microperfusion of removed renal tubules. Cortical pciking up channel segments were creatively microdissected from medullary rays of a kidney of masculine Sprague-Dawley rats (100120 g; Taconic Farms), mounted upon potion pipettes, as good as perfused in vitro during 37C regulating a technique of Burg (24). Osmotic H2O permeability (Pf in m/s) was totalled fluorometrically regulating a fluorescent luminal pen (1 mM of fluorescein sulfonate; Molecular Probes, Invitrogen), as described formerly (47). Basal Pf values were achieved after a 30-minute equilibration period. Subsequently, prewarmed bath resolution containing a ONO devalue was added, as good as Pf values were dynamic after an a singular some-more 30-minute equilibration period. Three liquid samples were picked up for Pf determinations during a finish of any of a 2 equilibration periods.
Photography of rodent kidneys. Mouse right kidneys were bisected slightly off a center. Photographic images were taken with a Sony CCD camera (model DKC-5000) mounted upon a Wild M-8 dissection microscope.
GFR measurements in unwavering mice. GFR was dynamic in unwavering mice by measuring a rate of disappearance of plasma FITC-inulin as formerly described (48, 49). In brief, a bolus of you estimate 2.5% FITC-inulin (3.7 l/g physique weight; Sigma-Aldrich) was injected retroorbitally underneath marked down isoflurane anesthesia, as good as red red red blood was picked up in to heparinized 5 l microcaps (Drummond Scientific) during 3, 7, 10, 15, 35, 55, as good as 75 mins after a injection. The picked up plasma was widely separated 1:10 in 500 mM HEPES (Sigma-Aldrich) of neutral pH, as good as fluorescence was totalled in a Nanodrop-ND-3300 spectrometer (Nanodrop Technologies). GFR was dynamic by analyzing FITC-inulin plasma disappearance regulating a 2-compartment indication of 2-phase exponential decay.
MRI imaging of rodent kidneys. To acknowledge changes in kidney morphology, MRI experiments were carried out regulating a 7.0 T, 16-cm plane Bruker MR imaging complement (Bruker) with Bruker ParaVision 4.0 software. Mice were anesthetized with 1.5%2.5% isoflurane as good as imaged regulating a 38-mm Bruker birdcage volume coil. Respiration as good as physique heat were monitored around a computerized monitoring complement (SA Instruments) around a experiment. Gadolinium-DTPA (Gd-DTPA; Magnevist, Berlex) was used as a contrariety representative to daydream renal morphology. Magnevist was widely separated 1:20 in 0.9% salty as good as afterwards administered s.c. during a sip of 0.2 mmol/d/kg physique weight. T1 weighted spin relate images (TR/TE = 700/10.8 ms, 6 averages, 0.5-mm cut thickness, eleven slices, 2.8 cm 5.4 cm field of view, 512 256 matrix, with fat saturation) were acquired in a coronal plane by a kidneys commencement 10 mins after Magnevist injection. Mice were imaged serially, initial prior to a 6-day TMX injection period, afterwards 3 days after a final TMX injection, as good as eventually 2 weeks after a begin of s.c. delivery of possibly car or ONO around osmotic minipumps (infusions were instituted 5 days after a final TMX injection). The liquid volume in a kidney showing tall signal intensity was calculated regulating CAAS-MRV_FARM program (Pie Medical Imaging).
Statistics. Data have been voiced as meant SEM for a indicated series of observations. For comparisons in between 2 groups, a interconnected or unpaired Students t exam (2-tailed) was used, as appropriate. For multiple comparisons, 1-way ANOVA was applied. P < 0.05 was considered statistically significant.
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This research was upheld by a Intramural Research Program budgets of a National Institute of Diabetes as good as Digestive as good as Kidney Disorders (NIDDK) as good as a National Heart, Lung, as good as Blood Institute (NHLBI) (project series ZO1-HL-001285). We thank Brenda Klaunberg as good as Vivian Diaz (In Vivo NMR Center, National Institute of Neurological Disorders as good as Stroke) for their help with a red red red blood vigour measurements, Shawn Kozlov (NHLBI) for analyzing urine as good as serum samples, as good as Cuiling Li as good as Yinghong Cui (NIDDK) for consultant technical assistance.
Conflict of interest: The authors have spoken which no conflict of seductiveness exists.
Citation for this article:J. Clin. Invest. doi:10.1172/JCI39680.
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