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【摘要】 RET, a tyrosine kinase receptor essential for kidney development, has recently been shown to be important for the formation of the urinary tract. When RET is overexpressed in the HoxB7/Ret transgenic mouse, kidneys are small and cystic, and in some of the mice, the ureters are grossly dilated. Here, we report that the observed ureteral dilatation is associated with the urinary tract abnormality vesicoureteric reflux (VUR), in which urine flows retrogradely from the bladder to the ureter. Reflux was determined in vitro by injecting methylene blue into the bladders of HoxB7/Ret and wild-type mice. At postnatal day 1, 30% of HoxB7/Ret mice had VUR compared with 4% of wild-type mice ( P < 0.05). The length of the intravesical ureteral tunnel was shorter in HoxB7/Ret mice compared with wild-type mice, on both the right and the left sides ( P < 0.05), suggesting a basis for the higher incidence of VUR in these mutants. At embryonic day 11, the ureteric bud was found to exit more caudally from the mesonephric duct in HoxB7/Ret mice, and this may predispose them to VUR ( P < 0.05). Wild-type and HoxB7/Ret mice were tested for reflux at embryonic day 17, and both showed a high frequency of VUR (59 and 75%, respectively). These results suggest that VUR may occur transiently during normal urinary tract development before the ureter has completed its insertion into the bladder. In the HoxB7/Ret mouse, overexpression of RET appears to delay the maturation of the distal ureter, resulting in postnatal VUR. The HoxB7/Ret mouse is thus an important model in which to examine how vesicoureteric reflux arises during urinary tract development.
【关键词】 kidney defect urinary tract defect ureteric bud
PRIMARY VESICOURETERIC REFLUX (VUR) is a urinary tract abnormality in which urine flows retrogradely from the bladder to the ureters because of an anatomic defect at the junction of the ureter and the bladder. It affects 1-2% of children and predisposes them to recurrent urinary tract infections and in some cases, end-stage renal disease ( 7, 33 ). The condition is hereditary: it frequently affects multiple family members, and several genetic loci have been identified from linkage studies of affected families ( 5, 6 ).
VUR is normally prevented by a functional valve, the flap-valve mechanism, that forms at the site of the ureteral insertion into the bladder. The flap-valve is dependent on 1 ) the tunnel in which the ureter enters into the bladder, 2 ) the presence of a muscular layer within the bladder and the ureter, and 3 ) the position of the ureteral orifice in the muscular layer within the bladder (the trigone). When the bladder is distended with urine, the tunnel is compressed, and this prevents the retrograde passage of urine from the bladder to the kidneys. The length of the intravesical ureteral tunnel is critical for the flap-valve mechanism: shorter tunnels have been identified in patients with VUR ( 17, 32 ). Other anatomic findings that have been associated with VUR include a lack of musculature within the ureter and its tunnel ( 30 ) and a ureteral orifice that is positioned more laterally relative to the trigone ( 17, 32 ).
All of these anatomic findings may arise because of abnormalities in the development of the urinary tract. The urinary tract arises from the mesonephric ducts, which are paired epithelial tubes extending along the anterior-posterior axis of the developing embryo. The future ureter arises from the ureteric bud, which develops as an outgrowth from the mesonephric duct and invades the future kidney, the metanephric mesenchyme. Several authors have proposed that the final position of the ureteral orifice within the trigone is determined by the site of ureteric budding from the mesonephric duct. A ureteric bud that arises more caudally along the mesonephric duct is more likely to result in a ureteral orifice that is lateral relative to the trigone, casuing a predisposition to VUR ( 18, 28 ).
One approach to understanding how urinary tract abnormalities such as VUR arise is to examine the function of molecules that are known to be expressed by the developing urinary tract. One such molecule is the receptor tyrosine kinase c-RET or RET (for rearranged during transfection) that is expressed by the mesonephric duct, the ureteric bud, and the future bladder ( 1, 13, 24 ). Signaling by RET has been known for some time to be critical for kidney development: targeted deletion of the Ret locus or overexpression of the Ret transcript inhibits formation of the kidneys and drastically reduces their size ( 27, 29 ). More recent evidence suggests that RET also affects urinary tract development. Ret -/- mice have ureteral abnormalities such that the distal ureters are not connected to the bladder but instead remain attached to the sex ducts ( 1 ).
In this study, we examined the HoxB7/Ret mouse in which RET is overexpressed to determine whether the development of the urinary tract is affected. In these transgenic mice, the HoxB7 promoter drives constitutive expression of RET (RET 9 isoform) throughout the ureteric bud, the collecting ducts, and the ureter beginning at embryonic (E) day 9.5 and continuing postnatally ( 15, 29 ). Our studies show that there is a higher incidence of VUR in HoxB7/Ret mice compared with wild-type mice at postnatal (P) day 1. We also demonstrate that reflux occurs during embryogenesis ( E17 ) at a high frequency in both wild-type and HoxB7/Ret mice. These results suggest that VUR may occur transiently during normal urinary tract development before the ureter has completed its insertion into the bladder. In the HoxB7/Ret mouse, overexpression of RET appears to delay the maturation of the distal ureter, resulting in postnatal VUR.
MATERIALS AND METHODS
HoxB7/Ret mouse colony. HoxB7/Ret mice ( 29 ) were established on a CD1 background and used for these studies in accordance with the rules and regulations of the Canadian Council on Animal Care (CCAC). Timed, 2-h early-morning matings were performed to generate the offspring that were used in all of the experiments. The embryos were staged using Theiler's criteria, and crown-rump lengths were measured to confirm the age of the embryos ( 31 ). The results shown represent the progeny from multiple litters.
Genotyping by PCR. Mice were genotyped using tail-clip DNA. Genomic DNA was extracted using a Wizard Extraction Kit (Promega) as per the manufacturer's instructions. Mice with the HoxB7/Ret transgene were identified using a primer in Ret (5'-TTCGGACTCACTGCTGTATGAC-3') and a primer in the -globin sequence in the transgene (5'-ACGATCCTGAGACTTCCACACT-3') ( 29 ). The wild-type Ret allele was amplified as a positive control for each DNA sample using the following primers: P1: 5'-TGGGAGAAGGCGAGTTTGGAAA-3' and P2: 5'-TTCAGGAACACTGGCTACCATG-3' ( 27 ).
Hematoxylin and eosin staining. Kidneys with their ureters and bladder attached were fixed in 4% formaldehyde overnight at 4°C, washed in PBS, dehydrated, and then paraffin embedded. Serial transverse sections were obtained at a thickness of 10 µm and stained with hematoxylin and eosin (Sigma).
VUR experiments. Embryonic and postnatal mice were dissected using an anterior midline incision to expose the kidneys and the urinary tract. Reflux was assessed using one of two methods. In the first method, the bladder was punctured with a 30-gauge needle, and then methylene blue (10 mg/ml) diluted 1:10 with phosphate-buffered saline was manually injected at a rate of 100 µl/min using a 1-ml syringe until dye exited through the urethra or refluxed back up the ureter. In the second method, the 30-gauge needle was attached via tubing to a syringe filled with methylene blue ( 11 ). During injection, the syringe was raised vertically from 30 to 150 cm in relation to the position of the mouse at a rate of 30 cm/min. Therefore, the rate of infusion was determined by the hydrostatic pressure exerted by the weight of the column of methylene blue as it was raised vertically. For each mouse, the experiment was stopped when no reflux was observed at 150 cm for 10 s. The results reported ( Table 1 and see Table 6 ) represent the combined data from both methods. The Cochran-Mantel-Haenszel test and a logistic regression analysis were performed to verify that the occurrence of reflux was not dependent on the method, and therefore the results could be pooled.
Table 1. Incidence of vesicoureteric reflux in HoxB7/Ret and wild-type mice at postnatal day 1
Table 6. Incidence of VUR in HoxB7/Ret and wild-type mice at E17
Kidney surface area and intravesical ureteral tunnel measurements. Postnatal kidneys with the attached ureters and the bladder were carefully dissected. Planar surface area measurements were obtained from each kidney using SPOT RT software (v3.2, Diagnostic Instruments) ( 9 ). Fast green dye (1%) was injected into the pelvis of each kidney using a microinjector (Microinjector IM 300, Narishige) until the dye entered the bladder. The bladder was partially opened to determine the site of exit of methylene blue from the ureteral orifice. The distance between the insertion of the extravesical ureter into the bladder and the exit of dye from the ureteral orifice inside the bladder corresponded to the intravesical ureteral tunnel and was measured using the SPOT RT software. The extravesical ureter was positioned to create a 90° angle relative to the bladder to standardize these measurements.
Apoptosis. Kidneys were paraffin embedded and sectioned at 6-µm thickness. Terminal deoxytransferase-mediated dUTP nick end-labeling (TUNEL) staining was performed using a TdT-FragEl DNA Fragmentation Kit (Oncogene, Cambridge, MA) as previously described ( 9 ).
Measurement of the position of the ureteric bud along the length of the mesonephric duct. The embryos were genotyped by extracting DNA from the head, the intra-abdominal organs were removed, and the remaining tissue was fixed overnight in 4% paraformaldehyde. Whole mount in situ hybridization was performed using Ret as a cDNA probe as described by Wilkinson ( 34 ). The embryos were imaged, and the position of the ureteric bud was determined by measuring its distance from the caudal end of the mesonephric duct.
Statistical analysis. Statistical analysis was performed using Microsoft Excel and SAS.
RESULTS
HoxB7/Ret mice have a high incidence of VUR. HoxB7/Ret mice are known to have small cystic kidneys with a decreased number of nephrons and fewer ureteric bud branches ( 29 ), but we also observed gross ureteral dilatation in some of them. Therefore, we hypothesized that the abnormal expression of RET in the urinary tract could affect the insertion of the ureter into the bladder, leading to VUR and ureteral dilatation.
HoxB7/Ret and wild-type mice were tested for VUR by injecting methylene blue into the bladder. Reflux was defined as the passage of methylene blue retrogradely back up the ureter ( Fig. 1, A - C ). At P1, 30% (14/47) of the HoxB7/Ret mice had VUR, whereas virtually none (3/68) of the wild-type mice demonstrated the defect ( Table 1, P < 0.05, Fisher's exact test). Two of the HoxB7/Ret mice demonstrated bilateral reflux. In those cases with unilateral reflux, it appeared to be more right-sided.
Fig. 1. Vesicoureteric reflux (VUR) in mice. A : HoxB7/Ret mouse with unilateral right-sided VUR at postnatal day 1. Magnification = x 1.6. White arrow indicates the passage of dye up to the pelvis of the kidney. B and C : wild-type and HoxB7/Ret mice, respectively, were assessed for reflux at embryonic day 17. Magnification = x 1.6. B : wild-type mouse with unilateral right-sided reflux (white arrow). C : HoxB7/Ret mouse with bilateral reflux and ureteral dilatation (white arrowheads).
HoxB7/Ret mice with VUR have ureteral dilatation. To look for defects in ureteral morphology, histological sections were obtained from paraffin-embedded kidney/bladder/ureteral units of mutant and wild-type mice. At P1, HoxB7/Ret mice with unilateral VUR displayed same-sided ureteral dilation on tissue section ( Fig. 2 A ). This was in contrast to the nonrefluxing ureter that was not dilated. As expected, the ureteral dilatation observed at the level of the kidneys was also seen in sections taken at the level of the bladder ( Fig. 2 B ). Ureteral dilation was not observed in sections from wild-type or HoxB7/Ret mice that did not demonstrate VUR (data not shown).
Fig. 2. Ureteral dilatation in a HoxB7/Ret mouse with VUR. Kidney/bladder/ureteral units from mice at postnatal day 1 were paraffin embedded, sectioned, and stained with hematoxylin and eosin. A : HoxB7/Ret mouse with unilateral right-sided reflux and corresponding right-sided ureteral dilatation from a tissue section (black arrow) taken at the level of the kidneys (black asterix). Magnification = x 40. The nonrefluxing left-sided ureter is also shown (black arrowhead). B : further serial section from the same mouse shown in A showing the refluxing (black arrow) and the nonrefluxing ureter (black arrowhead) at the level of the bladder (red arrow). Magnification = x 40.
Association between VUR and kidney malformation in the HoxB7/Ret mouse. To determine whether the presence of reflux in HoxB7/Ret mice correlated with the severity of the kidney phenotype, the planar surface area of kidneys from HoxB7/Ret mice with reflux was measured and compared with those without reflux. The size of kidneys from HoxB7/Ret mice with reflux was significantly smaller than those without reflux ( Table 2, P < 0.05, Student's t -test). The discrepancy in renal size in a comparisom of HoxB7/Ret mice with and without reflux could have been due to a defect in apoptosis. To explore this possibility, kidneys were sectioned and the TUNEL assay was performed. As shown in Table 3, HoxB7/Ret mice with reflux had a significantly greater number of apoptotic cells per square millimeter section surface area than mutant mice without reflux ( P < 0.05, Student's t -test). Although there were more apoptotic cells in HoxB7/Ret mice with reflux than those without reflux, the regions of the kidney and the cell types affected were not different between these two groups ( Fig. 3 ). Most apoptotic cells were noted within cystic structures and mesenchymal elements in the nephrogenic zone and within the medullary papilla.
Table 2. Comparison of kidney size in HoxB7/Ret mice with and without VUR
Table 3. Renal apoptosis in HoxB7/Ret mice with and without reflux
Fig. 3. Renal apoptosis in HoxB7/Ret mice with and without VUR and in wild-type mice. All mice were tested for reflux at postnatal day 1, and then the kidneys were paraffin embedded and sectioned for terminal deoxytransferase-mediated dUTP nick end-labeling assay. Apoptotic cells were identified using a streptavidin-horseradish peroxidase conjugate. Counterstaining with methyl green was used to enhance images. Representative sections from the papilla were imaged. Magnification = x 400. An increased number of apoptotic bodies (black arrowheads) are noted in HoxB7/Ret mice with reflux compared with those without reflux and to wild-type mice.
Assessment of the flap-valve mechanism in HoxB7/Ret and wild-type mice. Some humans with VUR are reported to have a shortened intravesical ureteral tunnel, leading to a defective flap-valve mechanism ( 17, 32 ). To determine the length of the intravesical ureteral tunnel in mutant and wild-type mice, fast green dye was injected into the pelvis of each kidney at P1. Dye passed along the length of the ureter and exited through the ureteral orifice within the trigone of the bladder ( Fig. 4 ). The intravesical ureteral tunnel was measured as the distance between the extravesical insertion of the ureter into the bladder and the intavesical ureteral orifice ( Fig. 4 ). The length for both the right and the left intravesical ureteral tunnel was significantly shorter in HoxB7/Ret mice than in wild-type mice ( Table 4, P < 0.05, Student's t -test).
Fig. 4. Intravesical ureteral tunnel length. Kidney/bladder/ureteral units were dissected en bloc. Methylene blue was injected into the pelvis of each kidney using a microinjector. The distance between the insertion of the extravesical ureter into the bladder and the exit of dye from the ureteral orifice inside the bladder was defined as the intravesical ureteral tunnel (demarcated by red line). Right and left sides for wild-type and HoxB7/Ret newborn pups are shown in the left (magnification = x 1.6) column. The right column shows higher power images of the tunnel (magnification = x 3.2).
Table 4. Measurement of ureteral tunnel lengths in HoxB7/Ret and wild-type mice at postnatal day 1
Site of the exit of the ureteric bud from the mesonephric duct in HoxB7/Ret and wild-type mice. In 1975, Mackie and Stephens ( 18 ) proposed that defects in urinary tract development could explain the origin of ureteral anomalies such as VUR. They hypothesized that if ureteral budding occurred from an abnormal position along the mesonephric duct, then the final site of the ureteral orifice would be modified. To determine whether the higher incidence of VUR in the HoxB7/Ret mouse was correlated with an abnormality in the site of ureteral budding from the mesonephric duct, whole mount in situ hybridizations were performed using a probe for Ret to label the mesonephric duct and the ureteric bud in embryos at E11. Because of the curvilinear form of the embryo, a method was developed to standardize the position of exit of the ureteric bud using the caudal edge of the mesonephric duct as a reference point ( Fig. 5 ). A line was drawn from the caudal edge of the mesonephric duct to the start point of the ureteric bud, and the distance was measured. The ureteric bud exited from a more caudal location along the mesonephric duct on both the right and the left sides in HoxB7/Ret mice compared with wild-type mice, as shown by the shorter distance from the end of the mesonephric duct to the start point of the ureteric bud ( P < 0.05, Student's t -test) ( Table 5 ). An alternative, independent method was also used to evaluate the relative position of the ureteric bud. In this case, the position of exit of the ureteric bud from the mesonephric duct was measured relative to the caudal edge of the hindlimb as described ( 8 ). A line was drawn along the length of the mesonephric duct, and two lines were then drawn perpendicular to it: one transected the ureteric bud at its point of exit from the mesonephric duct, and the other crossed the caudal edge of the hindlimb. Because the ureteric bud was observed to be either proximal or distal to the hindlimb, negative and positive distances were measured. The results of these analyses also showed that the ureteric bud exited from a more caudal location along the mesonephric duct on both the right and the left sides in the mutant mice, although this wasn't statistically significant on the left [right ureteric bud distance (reported as mean distance in mm in relation to position 0 at hindlimb ± SE): wild-type -0.12 ± 0.027, n = 24 vs. HoxB7/Ret 0.004 ± 0.047, n = 12, P < 0.05; left ureteric bud distance: wild-type -0.065 ± 0.029, n = 26 vs. HoxB7/Ret -0.043 ± 0.048, n = 13, P 0.05].
Fig. 5. Position of exit of the ureteric bud from the mesonephric duct. A : in situ hybridizations were performed on embryonic day 11 wild-type and HoxB7/Ret embryos using a Ret cDNA probe that labeled the mesonephric duct and the ureteric bud. The embryos were imaged, and the distance of the ureteric bud from the caudal edge of the mesonephric duct was measured as shown (demarcated by red line). Top : right and left mesonephric duct and ureteric bud for a wild-type embryo. Bottom : the same structures for a HoxB7/Ret embryo. Magnification = x 2.5.
Table 5. Position of ureteric bud along the mesonephric duct as measured in relation to the caudal end of the mesonephric duct in wild-type and HoxB7/Ret mice at E11
HoxB7/Ret and wild-type mice demonstrate VUR during embryogenesis. Urinary tract dilatation discovered on antenatal ultrasounds is not always detected on postnatal imaging ( 14, 25 ). This raises many questions regarding its natural history, including the possibility that dilatation can occur as a transient finding during normal human embryogenesis ( 20 ). We speculated that urinary tract dilatation from VUR could occur normally during embryogenesis before the formation of a competent flap-valve mechanism. To test this hypothesis, we looked for the presence of VUR in HoxB7/Ret and wild-type mice at E17.
We found that 59% (30/51) of wild-type mice had reflux and that an even higher proportion of HoxB7/Ret mice, 75% (41/55), had reflux, although this did not reach statistical significance ( Fig. 1, B and C ) ( Table 6, P 0.05, Fishers exact test). Bilateral reflux was more frequently seen in the HoxB7/Ret mice. Interestingly, in both wild-type and HoxB7/Ret mice, there was a much higher incidence of right-sided compared with left-sided unilateral reflux.
DISCUSSION
Our progress in understanding the underlying basis for VUR has been limited by a lack of suitable models and candidate molecules. We have described and characterized a new genetic model of VUR: the HoxB7/Ret mouse. In this model, the HoxB7 promoter drives constitutive expression of RET throughout the ureteric bud, the collecting ducts, and the ureter beginning at E9.5 and continuing postnatally. Affected transgenic mice have small cystic kidneys with fewer nephrons and VUR, a urinary tract defect. Notably, those mice with VUR had smaller kidneys and more apoptosis than those without VUR. To understand how VUR occurs, the flap-valve mechanism was examined, and the intravesical ureteral tunnel was found to be shorter in HoxB7/Ret mice compared with wild-type mice. During formation of the urinary tract, the future ureter arises from the ureteric bud, an outgrowth of the mesonephric duct. The proximal part of the ureteric bud induces the metanephric mesenchyme to form the kidney, whereas the distal portion grows vertically toward the future bladder and becomes the ureter. The ureter then becomes laterally displaced from the mesonephric duct and grows into the bladder, forming the ureteral orifice. This morphogenetic step is accompanied by the formation of an epithelial wedge at the base of the mesonephric duct that is believed to contribute to the muscular layer of the bladder known as the trigone. The site of ureteric bud exit from the mesonephric duct is a critical step during urinary tract development: it determines where the final ureteral orifice will be situated within the bladder trigone. In the HoxB7/Ret mouse, the ureteric bud was found to exit from a more caudal position along the mesonephric duct, and we suggest that this may predispose the mice to VUR by leading to an abnormally positioned ureteral orifice. To determine when the flap-valve mechanism becomes competent during development, wild-type and HoxB7/Ret mice were tested at E17. Interestingly, both showed a high incidence of VUR at this stage.
Multiple genetic loci have been associated with VUR from linkage studies of affected families ( 5, 6 ), but to date only a few genes have been identified. Humans with mutations in Pax-2, a transcription factor important for kidney and urinary tract development, have a syndrome including malformed kidneys, VUR, and ocular abnormalities ( 26 ). Mutations in the angiotensin type 2 receptor (AGTR2), which mediates the effects of renin and angiotensin, result in kidney and urinary tract abnormalities in humans and mice including cystic kidneys and VUR ( 10, 21, 22 ). In mice, when uroplakin III (UPIII), a cytokeratin that forms plaques on the apical surfaces of uroepithelial cells, is inactivated, VUR occurs ( 11 ). So far, no humans have been reported that have mutations in UPIII and reflux ( 12 ). Taken together, only a few gene mutations have been implicated in human disease, and only a few mouse models are known to have VUR. Therefore, the HoxB7/Ret transgenic model is an important addition to our understanding of this condition. Although no humans have VUR resulting from this genetic defect, there is good evidence that genes upstream of Ret, such as Pax-2, are directly responsible for human VUR ( 3, 26 ). Characterization of this signaling pathway and its regulation during urinary tract development are therefore important for an understanding of VUR in humans.
The development of the kidney and urinary tract is tightly linked such that urinary tract abnormalities are frequently observed in patients with kidney malformations ( 33 ). Indeed, VUR is frequently seen in combination with small, malformed kidneys, and this may partly explain how this condition predisposes patients to end-stage renal disease ( 4, 5 ). The HoxB7/Ret mouse has both a kidney and a urinary tract defect: it has small, malformed kidneys as well as VUR. Furthermore, those HoxB7/Ret mice with reflux had smaller kidneys than those without reflux, and we demonstrate that this may have arisen from a greater amount of apoptosis. Our data support the growing body of evidence that mutations in genes expressed in the developing mesonephric duct are directly responsible for combined urinary tract and kidney malformations and suggest that these defects could arise through similar cellular mechanisms.
From studies in humans, several components of the flap-valve mechanism are important to prevent VUR ( 17, 30, 32 ). We measured the length of the intravesical ureteral tunnel, which has been shown to be shorter in some humans with VUR. At P1, HoxB7/Ret mice had shorter intravesical ureteral tunnels compared with their wild-type littermates. Due to technical limitations, we were unable to rigorously evaluate other components of the flap-valve mechanism. However, there did not appear to be any gross deficiency in the muscular layer encompassing the distal ureter and bladder in sections from HoxB7/Ret vs. wild-type mice (data not shown).
Mackie and Stephens ( 18 ) proposed a model whereby abnormal budding of the ureteral bud from the mesonephric duct would lead to an abnormally situated ureteral orifice, predisposing patients to VUR. While there is clinical evidence that children with VUR do have ectopically situated ureteral orifices, the theory of Mackie and Stephens does not explain how other anatomic findings associated with VUR might arise. It has been frequently noted that children with VUR have ectopic ureteral orifices that are situated lateral to the trigone and short intravesical ureteral tunnels. Because we were not able to visualize the trigone together with the ureteral orifices within the bladder mucosa, we could not determine whether HoxB7/Ret mice have both abnormalities.
To date, there is no gold standard for measuring the position of the ureteric bud along the mesonephric duct ( 16, 23 ). Miyazaki et al. ( 19 ) looked at Bmp4+/ - mice and found a more cranially placed ureteric bud that they scored in relation to the nearest somite. This method is limited by the fact that embryos at this stage are curvilinear; therefore, picking the nearest somite is not straightforward. In this paper, we measured the position of the ureteric bud in relation to the caudal end of the mesonephric duct and found that this distance was shorter in the HoxB7/Ret mutants than in wild-type mice. We also measured the position of the ureteric bud using the hindlimb as the reference point and found similar results ( 8 ). This evidence suggests that the VUR seen in the mutant mice arises from ectopic ureteral budding from the mesonephric duct and that this is associated with a shorter intravesical ureteral tunnel.
Little is known as to when the flap-valve mechanism becomes competent during urinary tract development. To explore this further, we tested mice at E17 and noted a high rate of VUR in both wild-type and HoxB7/Ret mice. This finding is highly significant because it suggests that VUR may occur during normal urinary tract development. Urinary tract dilatation is frequently discovered on antenatal ultrasounds, but it often disappears on postnatal imaging ( 20 ). Certainly, this might arise if the defect were subtle and not picked up by routine imaging. Alternatively, urinary tract dilatation might occur normally during urinary tract development, perhaps due to the occurrence of VUR before the formation of a competent flap-valve mechanism. In reviewing the long-term follow-up of antenatal urinary tract dilatation, there are always some cases where previously observed abnormalities are no longer detected in the postnatal period ( 14, 25 ). Our data suggest that in wild-type mice, VUR resolves in the newborn period because the flap-valve mechanism has now become competent. In the HoxB7/Ret mouse, the flap-valve mechanism fails to mature and VUR persists.
Our studies show that when RET is overexpressed, ureteral insertion into the bladder is affected. Batourina et al. ( 1 ) have also shown that RET is important for urinary tract development. They showed that mRNA for Ret was expressed in the trigonal wedge where the ureter inserts into the bladder at E13. In addition, they noted that in Ret knockout mice, the ureters either terminated proximally or failed to join the bladder and remained connected to the mesonephric duct. Therefore, the absence of RET also affects the insertion of the ureter into the bladder. Although we have not explored the roles of the ligand, glial-derived neurotrophic factor (GDNF), and the coreceptor, GDNF family receptor (GFR) -1, in RET signaling, they are likely to be equally important because they are expressed in the mesenchyme adjacent to the developing mesonephric duct and the trigonal wedge ( 1, 13 ).
The specific cellular events that are disrupted during kidney and urinary tract development in the HoxB7/Ret mouse remain poorly characterized. Based on the higher rate of renal apoptosis, our results suggest that there may also be more apoptosis within the developing urinary tract of these mice. It is somewhat puzzling though if RET is a survival factor why its overexpression would lead to an increased rate of apoptosis. We speculate as have others ( 29 ) that this may be due to the fact that within the HoxB7/Ret mouse there is a relative lack of ligand and coreceptor compared with the amount of receptor, and therefore the signaling complex can no longer mediate cell survival. This may create a milieu where the balance is tipped in favor of more apoptosis ( 2 ). Even so, many questions remain as to how more apoptosis would lead to VUR.
The results reported here show clearly the importance of RET in the formation of the urinary tract. Overexpression of RET causes the ureteric bud to come off more caudally from the mesonephric duct and leads to the formation of a shorter intravesical ureteral tunnel. These two developmental abnormalities are likely responsible for the higher incidence of VUR in the HoxB7/Ret mouse. VUR occurs transiently during normal urinary tract development, but it persists in the HoxB7/Ret transgenic mouse. In future studies, it will be important to establish the significance of these findings for human kidney and urinary tract development.
ACKNOWLEDGMENTS
We thank Dr. F. Costantini who generously provided the HoxB7/Ret mice and the Ret probe, Dr. H. Tenenhouse and Dr. R. Jednak for discussions and feedback on the manuscript, and Pavle Vrljicak for technical support and helpful insights.
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作者单位：1 Departments of Pediatrics and 2 Human Genetics, Montreal Children‘s Hospital, McGill University, Montreal, Quebec, Canada H3H 1P3