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Genes to Cells (2004) 9, 1287-1296. doi:10.1111/j.1365-2443.2004.00805.x
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Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans

Yoshitaka Aoki1,2, Seiichi Mori1, Kazuhito Kitajima1, Osamu Yokoyama2, Hiroshi Kanamaru2, Kenichiro Okada2 and Yoshifumi Yokota1,*

1 Department of Molecular Genetics, School of Medicine, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan
2 Department of Urology, School of Medicine, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Congenital hydronephrosis is one of the most common anomalies found in humans and may cause renal failure in childhood. Half of the cases are due to obstruction at the ureteropelvic junction (UPJ). Here we report that mice lacking Id2, an inhibitor of basic helix-loop-helix (bHLH) transcription factors, exhibit hydronephrosis mimicking the characteristics of human cases such as unilaterality and male preponderance. Hydronephrosis was found even in Id2+/– mice. The penetrance was 67.2% in Id2–/– males, 48.8% in Id2+/– males, 28.0% in Id2–/– females and 20.0% in Id2+/– females. Distortion or high insertion of the ureter at the UPJ was frequently observed and these morphological changes were evident in late embryogenesis. Histologically, the muscle layer, where Id2 is normally expressed, was hypertrophic and/or irregular at the UPJ. Furthermore, gene expression analysis suggested that BMP4 (bone morphogenetic protein 4), which is known to be involved in the development of hydronephrosis, appears to function as an upstream factor of Id2. Our results thus raise the possibility that Id2 is a gene responsible for the pathogenesis of hydronephrosis in man.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Obstructive hydronephrosis is a condition, either congenital or acquired, in which there is progressive dilatation of the renal pelvis and caliceal system. In congenital human cases, it often causes irreversible parenchymal damage or severe bacterial infection of the kidney, leading to significant morbidity (Gulmi et al. 2002). Congenital hydronephrosis may result from a variety of causes including ureteropelvic junction (UPJ) obstruction, primary obstructive megaureter and posterior urethral valves (Churchill & Feng 2001). Among them, UPJ obstruction is the most common cause of congenital hydronephrosis, accounting for approximately 50% of these clinically significant cases, and is recognized in nearly 1 in 1000–1500 live births (Thomas 1990). Little is known about the mechanisms that cause hydronephrosis in humans.

Although spontaneously occurring hydronephrosis in mice and rats has also been described, the lesions are sporadic, unilateral or bilateral, and of moderate severity (Peters 2001). Recently, several gene-deficient mouse lines have been reported to have anomalies that mimic human congenital anomalies of the kidney and urinary tract (CAKUT), although the anomalies are not limited to obstructive hydronephrosis. For example, mice deficient for angiotensin type 2 receptor (Agtr2) have UPJ obstruction, ureterovesical junction stenosis, hypoplastic/aplastic/multicystic/dysplastic kidneys and/or megaureter, and the overall incidence in these mice is 21% in males and 5% in females (Nishimura et al. 1999). Bmp4 heterozygous null mutant mice also display phenotypes of hypoplastic/dysplastic kidneys, megaureter, and/or double collecting system (Miyazaki et al. 2000). On the other hand, deficiency of Adamts1 (a disintegrin and metalloproteinase with thrombospondin motif-1) leads to a more restricted phenotype: 100% of Adamts1 null mutant mice develop bilaterally enlarged renal calicies due to UPJ obstruction associated with fibroblastic changes in the ureter (Shindo et al. 2000).

Id proteins are negative regulators of basic helix-loop-helix (bHLH) transcription factors (such as MyoD) that are crucial for various cell differentiation processes (Benezra et al. 1990; Norton et al. 1998; Yokota 2001; Yokota & Mori 2002). Their mechanism of action is to block the heterodimerization of the bHLH factors at the protein level (Benezra et al. 1990; Norton et al. 1998; Yokota & Mori 2002). They not only inhibit cell differentiation but can stimulate the G1-S transition in the cell cycle (Norton et al. 1998). Four members of the Id protein family, Id1 through Id4, have been identified in mammals (Benezra et al. 1990; Christy et al. 1991; Sun et al. 1991; Riechmann et al. 1994). We previously reported that mice lacking Id2 display defects in secondary lymphoid organ development, natural killer cell differentiation, and lactation (Yokota et al. 1999; Ikawa et al. 2001; Mori et al. 2000; Fukuyama et al. 2002). The variety of phenotypic traits of Id2–/– mice indicate that Id2 is an important player in the control of cell differentiation and proliferation in vivo (Yokota 2001; Yokota & Mori 2002).

Here we report that Id2 mutant mice develop hydronephrosis due to UPJ obstruction with a high frequency, showing characteristics that resemble those of congenital UPJ obstruction in humans. The phenotype is detected even in heterozygous Id2 mutant mice, and, thus Id2 mutant mice provide a new UPJ obstruction model useful for delineating the molecular mechanisms underlying the development of congenital hydronephrosis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
By gross morphological inspection, we found that adult Id2–/– mice displayed hydronephrosis, namely an enlarged renal pelvis accompanied by urinary retention, and subsequently noted that Id2 haploinsufficiency also resulted in the same phenotype (Fig. 1A–C). In most cases, hydronephrosis was only observed in the right kidney (Fig. 1). The right kidney is positioned more rostrally than the left kidney in mice and thus the unilaterality of hydronephrosis was biased to the rostral side, as it is in human cases. There were no aberrant blood vessels or ureters directly connected to the lower pole of the kidney, and no obstructive structure surrounding the ureter (data not shown). The ureteropelvic structure of the affected mice displayed stenosis at the level of the UPJ with distortion or with high insertion of the ureter, but without any dilatation of the ureter (Fig. 1D–G). In accordance with these observations, antegrade pyelograms demonstrated an enlarged renal pelvis with no accumulation of contrast medium in the ureter or urinary bladder, while a rapid outflow of contrast medium into the urinary bladder was observed in control mice. The results indicated that there was obstruction of the urinary flow at the UPJ in hydronephrotic kidneys of Id2 mutant mice (Fig. 1H,I). Regarding the lower urinary tract, including the ureterovesical junction, no anatomical anomalies were detected (data not shown).



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  		Figure 1  Macroscopic morphology of hydronephrosis in Id2 mutant mice. A–C, Kidneys and retroperitoneal organs of 8-week-old male mice. Hydronephrosis is apparent in the right kidney of both Id2+/– (B) and Id2–/– mice (C) Their middle-lower ureters and urinary bladders are normal. D–G, Higher magnifications of the right UPJ of adult Id2+/+ (D, E) and Id2–/– (F, G) mice. In adult Id2–/– mice, sharp narrowing at the UPJ (arrow in F) and high insertion of the ureter into the pelvis (arrow in G) are observed H, I, Antegrade pyelogram of the right kidney. Compared to the control, which shows the influx of contrast medium into the urinary bladder (H), the Id2–/– mouse exhibits an enlarged pelvis with sharp narrowing at the UPJ and the urinary bladder is not identifiable (I). Red and yellow arrowheads indicate the positions of the pelvis and bladder, respectively.

 
Since the altered phenotypes in the pelvis and ureter were also detected in Id2 mutant neonates (Fig. 2M,N), we followed the morphological development of the upper urinary tract of Id2 mutant mice during embryogenesis to determine when hydronephrosis becomes apparent. On embryonic day (E) 14, no appreciable difference was observed in renal pelvis and ureteral morphology between Id2+/+ and Id2–/– mice (n = 10–15) (Fig. 2A,H and Table 1). On E16, however, the right renal pelvis was dilated and the ureteropelvic region was twisted in Id2–/– mice, compared with those of Id2+/+ embryos (Fig. 2B–E). The penetrance of hydronephrosis on E16 were 45.5% (5/11) in Id2–/– males, 35.7% (5/14) in Id2+/– males, 25.0% (1/4) in Id2–/– females and 14.3% (2/14) in Id2+/– females, whereas no hydronephrosis was observed in Id2+/+ mice (male = 0/17, female = 0/12) (Table 1). The dilation of the renal pelvis was significantly increased in Id2–/– mice on E18 (Fig. 2I–L). These observations indicated that the hydronephrosis that occurs in Id2 mutant mice develops in the late embryonic period and is a congenital malformation.



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  		Figure 2  Id2 mutant mice develop hydronephrosis in late embryogenesis. Kidneys and ureters of Id2+/+ (A–E) and Id2–/– (H–L) embryos. C, E, G, J, L and N are higher magnification of ureteropelvic regions of B, D, F, I, K and M. Arrowheads in J, L and N indicate the UPJ.

 

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  		Table 1  Inceidence of hydron:ephrosis in Id2 mutant embryos
 
Next, the calyx to papilla ratio (C/P ratio) (Tsuchida et al. 1998) was determined by measuring the diameters of the papilla and the maximum calyceal space in Hematoxylin and Eosin-stained coronal sections of kidneys using image analysis software. The C/P ratio is a good indicator of the severity of hydronephrosis (Tsuchida et al. 1998). The C/P ratio at 8 weeks was significantly increased in the right kidney of Id2–/– and Id2+/– mice (P < 0.0001 and P < 0.0005, respectively) as compared to control mice, and the values showed a broad distribution (Fig. 3). Application of our criterion for hydronephrosis (C/P ratio > 4) to all examined samples revealed the penetrance to be 67.2% in Id2–/– males, 48.8% in Id2+/– males, 28.0% in Id2–/– females and 20.0% in Id2+/– females (Table 2), demonstrating male preponderance (the male-to-female ratio was 5 : 2 in both Id2–/– and Id2+/– mice). Bilateral hydronephrosis was also observed (4.7% in Id2–/– males, 7.6% in Id2+/– males, 2.0% in Id2–/– females and 1.5% in Id2+/– females), although the pelvic dilatation was always more prominent in the right kidney (data not shown).



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  		Figure 3  Distribution of C/P ratios in male mice at 8 weeks of age. The genotype of each group is indicated at the bottom. The inset schematizes the method for calculating the C/P ratio. Values are presented as means ± SEM (n = 8, 13 and 20 for Id2+/+, Id2+/– and Id2–/– mice, respectively). *P < 0.05, vs. Id2+/+ mice.

 

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  		Table 2  Incidence of hydronephrosis in adult Id2 mutant mice
 
To gain insight into the cause of the UPJ obstruction, we monitored the intrapelvic pressure by an antegrade perfusion pressure flow test ex vivo (Fig. 4). The hydronephrotic kidneys of Id2–/– and Id2+/– mice had a significantly higher intrapelvic pressure than the control kidneys (26.1 ± 4.0 cmH2O in Id2–/– mice, 31.1 ± 5.2 cmH2O in Id2+/– mice and 11.0 ± 2.9 cmH2O in Id2+/+ mice). These results ruled out hypotony of smooth muscles of the pelvis as a cause of the hydronephrosis and indicated a mechanical obstruction in the hydronephrotic kidney of Id2 mutant mice. In addition, the results excluded the possibility of a neuronal factor as a cause of hydronephrosis, because the examined kidneys were excised from the mice for this assay.



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  		Figure 4  Intrapelvic pressure in hydronephrotic kidney of Id2 mutant mice. Intrapelvic pressure was measured by an ex vivo antegrade perfusion pressure flow test with a pressure transducer. Hydronephrotic kidneys of Id2–/– and Id2+/– mice were examined. Values are presented as means ± SEM (n = 5, 8 and 7 for Id2+/+, Id2+/– and Id2–/– mice, respectively). *P < 0.05, vs. left kidneys.

 
We subsequently examined the histology of the hydronephrotic kidney of adult Id2–/– mice using sectioned specimens stained with Hematoxylin and Eosin. Compared to control kidneys, the kidneys of Id2–/– mice had a hypoplastic papilla and thin medulla, with a highly dilated caliceal space (Fig. 5A). We observed luminal dilation and epithelial flattening of the tubules but no apparent alteration of the cortical structure, including glomeruli. The muscle layer showed irregularity and hypertrophic change at the UPJ, particularly in the internal longitudinal smooth muscles (Fig. 5B). A distorted UPJ was frequently observed (Fig. 5C). There were no neoplasms or urinary stones at the UPJ (data not shown). These findings suggest that the cause of hydronephrosis is smooth muscle hypertrophy at the UPJ. Consistent with this, RNA in situ hybridization showed Id2 expression in the smooth muscles but not in the mesenchymal tissues surrounding the ureter, although a weaker signal was also detected in the epithelial layer of the ureter (Fig. 5D). Using immunohistochemistry, we investigated the expression levels of smooth muscle cell differentiation markers, {alpha}-smooth muscle actin ({alpha}-SMA) (Fig. 5E) and desmin (data not shown), and found no significant difference among the three Id2 genotypes in neonatal and adult mice.



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  		Figure 5  Histopathological analysis of hydronephrosis in Id2 mutant mice. (A) Low magnification of renal sections of Id2+/+ and Id2–/– mice at 8 weeks. Horizontal and sagittal sections. (B) Horizontal sections of Id2+/+ and Id2–/– neonatal kidneys and ureters at P7. Upper-UPJ sections are higher magnifications of the areas marked by squares in Kidney-specimens. Two sections, middle- and lower-UPJ taken from more distal parts of the ureter are shown. Much higher magnifications of the areas marked by squares in Middle-UPJ section show distortion and hypertrophy of the smooth muscle in Id2–/– neonatal ureter at P7 (bottom). Bar represented smooth muscle layer. (C) A section at the UPJ of an Id2–/– neonate exhibits a distorted ‘up-and-down’ ureter. (D) In situ hybridization analysis of Id2 mRNA in the ureter on E14. A horizontal section of an embryo was hybridized to the anti-sense riboprobe for Id2 and counter-stained with Hematoxylin and Eosin. Dark field (left) and bright field (right) of the same section. E, Transverse sections of the UPJ and upper ureter of Id2+/+ and Id2–/– mouse at 2 weeks of age were immunostained for {alpha}-smooth muscle actin. Signal was detected by the horse radish peroxidase.

 
Recent studies have identified several genes involved in the pathogenesis of hydronephrosis. Examples include the angiotensin type 1 and 2 receptors (Agtr1 and Agtr2) (Tsuchida et al. 1998; Nishimura et al. 1999), Adamts1 (Shindo et al. 2000) and Bmp4 (Miyazaki et al. 2000). To examine the functional relationship of Id2 to these genes, quantitative real-time RT-PCR analyses were performed using RNA isolated from the renal pelvis and ureter of hydronephrotic kidneys of adult Id2 mutant mice. We found that the level of Agtr1 expression was significantly reduced in Id2 mutant mice, and the reduction was more prominent in Id2–/– mice than in Id2+/– mice (Fig. 6A). In contrast, although there was a tendency for Id2 mutant mice to exhibit a higher level of expression of Bmp4, statistical analysis failed to detect a significant difference among the three Id2 genotypes (Fig. 6A). Regarding other hydronephrosis-related genes, no alteration was observed for angiotensinogen, renin and angiotensin converting enzyme, and expression of Agtr2 was hardly detectable in the adult (data not shown). At the protein level, Agtr1 was reduced in Id2–/– mice but Bmp4 was similarly detected in all three genotypes (Fig. 6B). We failed to detect expression of Agtr2 and ADAMTS-1 proteins in Western blotting (data not shown). These results imply that Agtr1 is a downstream molecule of Id2 in the pathogenesis of congenital hydronephrosis. On E16, however, quantitative real-time RT-PCR analyses failed to detect significant difference in expression of Agtr1, Agtr2 and Bmp4, although there was a tendency for Id2 null mutant mice to exhibit a higher level of expression of Agtr2 and Bmp4 (Fig. 6C). Therefore, reduced expression of Agtr1 seems to be a secondary effect of hydronephrotic changes.



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  		Figure 6  Expression of genes and proteins in the pelvis and ureter. (A) Gene expression of Agtr1, Adamts1 and Bmp4 in the adult pelvis and ureter was analysed by quantitative real-time RT-PCR. RNA isolated from individual adult mice was analysed and representative data are shown (n = 6–8). ß-actin was used as an internal control. Values are presented as means ± SEM. (B) Expression of Agtr1 and BMP4 proteins in the adult pelvis and ureter were analysed by Western blotting. ß-actin was used as an internal control. (C) Gene expression of Agtr1, Agtr2 and Bmp4 in the embryonic pelvis and ureter was analysed by quantitative real-time RT-PCR. RNA isolated from 3 to 6 embryonic tissues was analysed and representative data are shown (n = 3–5). ß-actin was used as an internal control. Values are presented as means ± SEM (D) Gene expression of Id2 in the embryonic pelvis and ureter in response to BMP4 and/or angiotensin II. The pelvis and ureter of five wild-type embryos were dissected out at E14 and cultured for 2 days under the conditions described in the text. RNA was purified and subjected to quantitative real-time RT-PCR (n = 3). ß-actin was used as an internal control. Values are presented as means ± SEM.

 
We also investigated if Id2 is positioned downstream of BMP4 and/or angiotensin II in the respective signalling pathways. To this end, we dissected out the ureteropelvic region of E14.5 wild-type embryos, cultured them ex vivo for 2 days with BMP4 or angiotensin II, and then examined their level of Id2 expression by quantitative real-time RT-PCR. With BMP4 stimulation, Id2 mRNA expression was elevated twofold compared to the control (Fig. 6D), and the extent of the induction was comparable to that of Msx2, which is known to be a downstream target of BMP4 (Hollnagel et al. 1999). In contrast, angiotensin II, a ligand of Agtr1, did not induce Id2 gene expression, while expression of p21, a CDK inhibitor, was suppressed by the application of angiotensin II, as reported (Mueller et al. 2002). These results, together with data shown in Fig. 6A, suggest that Id2 plays a role in the normal development of the kidney by participating in the BMP4 signalling pathways.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The present study demonstrated that both heterozygous and homozygous Id2 mutant mice developed congenital hydronephrosis. Macroscopic inspection and antegrade pyelogram identified obstruction at the UPJ. The observed hydronephrosis was congenital, and exhibited male preponderance, unilaterality and incomplete penetrance, features that parallel those of human congenital hydronephrosis with UPJ obstruction. Among a variety of phenotypes that are found in Id2 null mutant mice (Yokota et al. 1999; Mori et al. 2000; Ikawa et al. 2001; Yokota 2001; Fukuyama et al. 2002), hydronephrosis is the only phenotype to which Id2 haploinsufficiency leads. An ex vivo antegrade perfusion pressure flow test indicated that the cause of UPJ obstruction was intrinsic to the urinary tract. Analysis of the expression of Id2 in developing embryos suggested that the UPJ obstruction appears to be derived from a developmental defect in smooth muscles around the UPJ. Quantitative RT-PCR analysis, furthermore, revealed that expression of Agtr1 is perturbed in the ureteropelvic region of Id2–/– mice. On the other hand, BMP4 induced Id2 mRNA expression in ex vivo cultures of the ureteropelvic structure of wild-type embryos. Our present study thus revealed a novel role of Id2 in normal kidney development and a potential molecular basis for the pathogenesis of congenital hydronephrosis.

Although congenital renal anomalies include a variety of conditions and many factors seem to be involved in their development, studies using gene-deficient mice have identified two secreted molecules, BMP4 and angiotensin, that play important roles in the morphogenesis of the urinary system. BMPs are pleiotropic soluble factors that play indispensable roles in many aspects of pattern formation during development by regulating cell proliferation and differentiation (Hogan 1996; Miyazawa et al. 2002). Accumulating evidence has indicated that BMPs induce expression of Id genes in various cell types (Ogata et al. 1993; Hollnagel et al. 1999; Miyazawa et al. 2002; Ying et al. 2003). Consistent with this notion, we observed that Id2 expression is induced in the embryonic ureteropelvic region in the presence of BMP4. Our work has thus identified the BMP4-Id2 axis as an important regulator of normal kidney development. The fact that haploinsufficiency of either Bmp4 or Id2 leads to congenital renal anomalies may suggest the requirement for critical regulation of this axis for the normal development of the kidney. With respect to the phenotypes displayed by these mutant mice, Bmp4 heterozygous null mutant mice show a wide spectrum of CAKUT phenotypes including not only hydronephrosis but also megaureter and renal hypoplasia/dysplasia, whereas the defect in the urinary system of Id2 mutant mice is confined to hydronephrosis due to UPJ obstruction. It is highly probable that BMP4 regulates diverse genes besides Id2, and that dysregulation of these genes results in a variety of CAKUT phenotypes. Conversely, there may be genes that are not dependent of the BMP4 signalling cascade but are under the control of Id2. Elucidation of gene activation/suppression networks governed by BMP4 and Id2 will help to clarify the molecular basis of the development of congenital renal anomalies.

Regarding angiotensin, deficiency of any one of the components that are involved in the renin-angiotensin system, i.e. angiotensinogen, renin, angiotensin-converting enzyme, Agtr1 and Agtr2, causes renal anomalies in mice, although the phenotypes depend on the impaired component and are not all the same (Krege et al. 1995; Miyazaki et al. 1998; Okubo et al. 1998; Tsuchida et al. 1998; Nishimura et al. 1999; Yanai et al. 2000). By quantitative RT-PCR analysis, we found that the gene expression of Agtr1 is substantially reduced in the region around the UPJ of hydronephrotic kidneys of adult Id2 mutant mice in a manner dependent on the gene dosage of Id2. However, this reduction was not observed at the stage of E16. Since hydronephrosis is apparent in Id2 mutant mice on E16, reduced expression of Agtr1 in adult Id2 mutant mice seems to be a secondary effect of hydronephrotic changes of the kidney. In accordance with this notion, renal anomalies found in mice deficient for Agtr1 are different from those of Id2 mutant mice, showing functional obstruction of the urinary tract due to impaired peristalsis of the ureter (Miyazaki et al. 1998).

As demonstrated by in situ hybridization, Id2 is expressed mainly in the smooth muscle layer of the ureter, suggesting that Id2 deficiency in smooth muscles of the ureter is the main cause of hydronephrosis. In fact, the smooth muscles of the ureter of hydronephrotic kidneys show irregularity and hypertrophy, although the pathological findings may in part be secondary effects of hydronephrosis. The fact that Id proteins are negative regulators of bHLH transcription factors raises the intriguing possibility that some bHLH factor may be involved in ureteropelvic morphogenesis, and that its functional dysregulation resulting from Id2-deficiency causes hydronephrosis. We, however, have failed to detect significant differences between Id2 mutant mice and wild-type mice in expression of several bHLH factors, such as Capsulin, that are known to be expressed in smooth muscles (Y. Aoki and Y. Yokota, unpublished observation). Further investigations will be required to elucidate the role of Id2 in ureteropelvic development.

Compared to other gene-deficient mice with hydronephrosis, Id2 mutant mice exhibit a more similar battery of features to that of human cases of UPJ obstruction. Furthermore, it is intriguing that haploinsufficiency of Id2 can cause hydronephrosis. These observations raise the possibility that ID2 is a gene responsible, at least in part, for the pathogenesis of congenital hydronephrosis in humans. To determine whether ID2 is a susceptibility gene for congenital hydronephrosis, we are currently investigating the ID2 gene locus of familial cases of hydronephrosis.

Hydronephrosis is found in Id2 mutant mice of a mixed NMRI and 129/Sv genetic background, but not in those of the 129/Sv genetic background (data not shown), suggesting the involvement of other genes in the pathogenesis, as suggested in both humans and mice (Tsuchida et al. 1998; Miyazaki et al. 2000). In accord with this, Id2 mutant mice with the mixed genetic background show incomplete penetrance of hydronephrosis, and the severity of hydronephrosis varies among Id2 mutant mice. Genetic analyses may identify the modifiers responsible for the variable penetrance and/or severity of hydronephrosis.

In conclusion, we have shown that Id2 mutant mice develop hydronephrosis with congenital obstruction at the UPJ, the characteristics of which show a close resemblance to those of human congenital hydronephrosis. Elucidation of the role of Id2 in the morphogenesis of the ureteropelvic structure will facilitate our understanding of how hydronephrosis develops. Furthermore, mutational analysis of the Id2 gene locus in patients with familial hydronephrosis will tell us whether Id2 is indeed a gene responsible for hydronephrosis.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Mice

Id2-deficient mice of mixed 129/Sv and NMRI genetic background were analysed (Yokota et al. 1999). For in situ hybridization analysis and in vitro culture of the renal pelvis and ureter, ICR or mixed genetic background (129/Sv and NMRI) mouse embryos were used. All animal procedures were performed in accordance with the guidelines of the University of Fukui for animal experiments.

Antegrade pyelogram

After abdominal opening, iopamidol (Iopamiron, Nihon Schering), a contrast medium, was infused into the kidney with a 22-G needle and subsequently X-rays were taken.

Histology

After systemic perfusion of mice with PBS containing 4% paraformaldehyde, the kidneys and ureters were dissected out and postfixed with the same fixative on ice overnight and then embedded in paraffin according to standard procedures. Sections of 4–10-µm thickness were stained with Hematoxylin and Eosin and examined. To determine the calyx to papilla ratio (C/P ratio) (Tsuchida et al. 1998), the diameters of the papilla and the maximum calyceal space were measured in coronal sections of kidneys using NIH Image 1.61 image analysis software (URL: http://rsbweb.nih.gov/nih-image/index.html). For Immunohistochemistry, the sections were reacted with an antibody against {alpha}-SMA (1 : 200, DAKO) followed by a goat anti-mouse immunogloblin conjugated with a horseradish peroxidase-labelled dextran polymer (Envision+, DAKO). Immunoreactivity was visualized with diaminobenzidine.

In situ hybridization

In situ hybridization was performed with paraffin-embedded sections of kidneys or embryos essentially as previously described (Mori et al. 1999). An 35S-labelled anti-sense riboprobe spanning nt 61–759 of the mouse Id2 cDNA was used. Specimens were hybridized and washed at high stringency and autoradiographed with NTB2 emulsion (Eastman Kodak). A sense probe of cytokeratin 18 was used as a negative control (data not shown).

Ex vivo antegrade perfusion pressure flow test

After the excision of the kidneys together with the ureters, the renal pelvis was punctured with a 22-G needle to allow measurement of the intrapelvic pressure in PBS. A catheter was connected to a pump (TE-311; Terumo) for the continuous infusion of saline, and to a pressure transducer (TP-400T; Nihon-Kohden). The intrapelvic pressure was measured ex vivo under infusion of physiological saline into the renal pelvis at a rate of 0.05 mL/min at room temperature.

Organ culture

The region around UPJ was dissected out from 14.5 d.p.c. (days post coitus) mouse embryos and cultured in DMEM supplemented with 20% foetal calf serum in the presence of 50 ng/mL BMP4 (R&D Systems) and/or 1 µM angiotensin II (Peptide Institute) under a 5% CO2 atmosphere at 37 °C. After the ureters were cultured for 48 h, RNA was purified from the organs and subjected to RT-PCR.

Quantitative real-time RT-PCR

Total RNA was prepared from the pelvis and ureter with the acid guanidium thiocyanate/phenol/chloroform extraction procedure (Chomczynski & Sacchi 1987). For RT-PCR, cDNAs were synthesized by using the SuperScript II‘ First Strand Synthesis System (Life Technologies). Specific primer pairs were designed with ABI PRISM Primer Express software (PE Applied Biosystems). Quantitative real-time RT-PCR was performed with each primer set using SYBR Green PCR Master Mix (PE Applied Biosystems), and run on an ABI PRISM® 7000 sequence detection system (PE Applied Biosystems). Data were normalized relative to ß-actin amplification. Primer pairs and reaction conditions are available upon request.

Western blotting

Tissue samples were excised from the region around UPJ and lysed in 2x RIPA buffer (300 mM NaCl, 1% Triton X-100, 0.1% SDS and 0.4% sodium deoxycholate). Protein samples (10 µg each) were separated on a SDS-PAGE gel and blotted with a standard method. Each blot was treated with primary antibodies. Anti-Agtr1, BMP4 and ß-actin antibodies were purchased from Santa Cruz Inc., Vector laboratories Inc., and Sigma, respectively. A horseradish peroxidase-conjugated secondary antibody was used to detect primary antibodies (1 : 5000). Signals were detected by the enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech).

Statistical analysis

All numeral data were expressed as mean ± s.e.m. The Student's t-test or ANOVA was used to determine the significance of differences. A P-value of less than 0.05 was considered statistically significant.


   Acknowledgements
 
We are grateful to H. Itoh and Y. Nakamura for kindly sharing equipment, Y. Matsui for secretarial assistance, and T. Tsubota and E. Kawai for technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Y.Y. #13470034 and #15390102), by The Naito Foundation (Y.Y.) and by 21st Century COE program (medical Sciences) (Y.Y.).


   Footnotes
 
Communicated by: Shuh Narumiya

*Correspondence: E-mail: yyokota{at}fmsrsa.fukui-med.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 1 June 2004
Accepted: 17 September 2004




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