Bradykinin B2 null mice are prone to renal dysplasia: gene-environment interactions in kidney development

SAMIR S. EL-DAHR1,2, LISA M. HARRISON-BERNARD2, SUSANA DIPP1, IGOR V. YOSIPIV1 and SUZANNE MELEG-SMITH3

1 Department of Pediatrics
2 Department of Physiology
3 Department of Pathology, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
El-Dahr, Samir S., Lisa M. Harrison-Bernard, Susana Dipp, Igor V. Yosipiv, and Suzanne Meleg-Smith. Bradykinin B2 null mice are prone to renal dysplasia: gene-environment interactions in kidney development. Physiol Genomics 3: 121–131, 2000.—Congenital abnormalities of the kidney and urinary tract are a common cause of end-stage renal disease in children. Host and environment factors are implicated in the pathogenesis of aberrant renal development. However, direct evidence linking gene-environment interactions with congenital renal disease is lacking. We report an animal model of renal dysgenesis that is dependent on a defined genetic defect and specific embryonic stressor. Specifically, mice that are deficient in the bradykinin type 2 receptor gene (B2) and salt loaded during embryogenesis acquire an aberrant kidney phenotype and die shortly after birth. In contrast, B2 mutant mice maintained on normal sodium intake or salt-loaded wild-type mice do not develop kidney abnormalities. The kidney abnormality is evident histologically on embryonic day 16, shortly after the onset of metanephric B2 gene expression, and consists of distorted renal architecture, foci of tubular dysgenesis, and cyst formation. The dysplastic tubules are of distal nephron origin [Dolichos biflorus agglutinin (DBA)- and aquaporin-2 (AQP2) positive, and angiotensinogen negative]. Neonatal antihypertensive therapy fails to ameliorate the renal abnormalities, arguing against the possibility that the nephropathy is a consequence of early hypertension. Moreover, the nephropathy is intrinsic to the embryo, because B2 homozygous offspring from heterozygous parents exhibit the same renal phenotype as offspring from homozygous null parents. Further characterization of the renal phenotype revealed an important genetic background effect since the penetrance of the congenital nephropathy is increased substantially upon backcrossing of 129/BL6 B2 mutants to a uniform C57BL/6J. We conclude that the type 2 bradykinin receptor is required for the maintenance of metanephric structure and epithelial integrity in the presence of fetal stress. This study provides a "proof-of-principle" that defined gene-environment interactions are a cause of congenital renal disease.

genetic susceptibility; nephrogenesis; G protein-coupled receptors; gene targeting


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NORMAL METANEPHRIC DEVELOPMENT depends on tightly regulated reciprocal interactions between the two nephrogenic compartments: the ureteric bud and metanephric mesenchyme (for a review, see Ref. 33). Results obtained from gene targeting in mice and metanephric organ culture have unraveled essential morphogenetic events that are controlled by growth factor-receptor pairs, transcription factors, cell-matrix interactions, and intracellular signaling molecules (reviewed in Refs. 19 and 39, and in references therein). Some of these factors are required for initial kidney organogenesis (e.g., WT-1, Pax-2, GDNF/c-ret, FGF-2) (18, 24, 26, 27, 32, 34, 36), whereas others are more important for proper mesenchymal-to-epithelial differentiation (e.g., Wnt-4, BMP-7, BF-2) (7, 16, 21, 35) or the maintenance of the tubular epithelial phenotype (e.g., HNF-1 and EGF-R) (29, 37).

Although gene targeting in mice has established a direct role for a number of molecules in normal embryonic development, the vast majority of mutations involving developmentally regulated genes have not resulted in an obvious phenotype. The interpretation of these studies is complex for several reasons. First, most physiological pathways are redundant, and the absence of a factor may be compensated for by one or more systems with overlapping functions. Second, some phenotypic abnormalities are subtle and may not be detected because of lack of appropriate methodologies. Third, some factors perform very specialized functions (e.g., epithelial transport), and the requirement for normal homeostasis may only be revealed by external challenges. This latter concept, i.e., gene-environment interplay, is implicated in the susceptibility of humans to certain diseases affecting the kidney such as essential hypertension and diabetes.

We have reported that the bradykinin B2 receptor gene is developmentally regulated in the kidney and that B2 receptors are expressed in ureteric bud derivatives, suggesting a role for this receptor in distal nephron maturation (9). Progeny of mutant mice lacking the B2 gene have normal kidney development and blood pressure, yet exhibit early salt-sensitive hypertension (1, 4, 22). Thus the requirement for B2 receptors in normal homeostasis was revealed by an environmental stressor such as high-salt intake. The present study was designed to test the hypothesis that the protective effect of the B2 receptor begins in fetal life. An important developmental role for kinins is supported by multiple lines of evidence. First, bradykinin synthesis and B2 gene expression are activated in the developing compared with the adult kidney (8, 9). Second, pharmacological blockade of B2 receptors in neonatal rats retards renal growth (41). Third, activation of the B2 receptor engages intracellular signaling pathways and transcription factors that are coupled to survival pathways, such as the phosphatidylinositol 3-kinase (PI3-kinase)-nuclear factor {kappa}B (NF-{kappa}B) and mitogen-activated protein kinase (MAPK)-AP-1 pathways (10, 25). Accordingly, we examined renal development in wild-type, heterozygous, and homozygous B2-null mice on normal or high gestational salt intake. In addition, the effect of genetic background on the phenotypic penetrance of the renal abnormalities was examined through the generation of a congenic strain of B2-deficient mice.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Transgenic mice.
Bradykinin B2 receptor-deficient transgenic mice were generously provided by Drs. Fred Hess and Howard Chen. The targeting strategy involved the disruption and replacement of exon 4, which contains the entire open reading frame of the murine B2 gene, by a neomycin cassette (2). B2 null mice were originally on a mixed 129/C57BL6J strain but have since been backcrossed to C57BL/6J (10 times) and can therefore be considered a C57BL/6J genetic background. B2 null mice were genotyped by PCR according to the following protocol. The PCR reaction contained 250 ng genomic DNA, 20 pmol each of B2-specific primers, 2.5 U of Taq polymerase, 5 µl of 10x PCR buffer and 0.2 mM dNTPs (94°C, 1 min; 58°C, 30 s; 72°C 1 min). The primers were designed to amplify 572 bp of the B2 coding sequence. The upstream and downstream primers are 5'-AGAACATCTTTGTCCTCAGCG-3' and 5'-CGTCTGGACCTCCTTGAACT-3', respectively. In pilot studies, we determined that 30 cycles of PCR allows the amplification of DNA within the linear range. Using these conditions, we observed no PCR amplicons in the homozygous null mutants, whereas the heterozygous mutants had ~50% the amount of amplified DNA from the wild-type mice. Wild-type C57BL/6J mice were purchased from Charles Rivers Laboratories.

Pairs of male and female B2 +/+ or -/- mice were placed on normal salt (NS, 0.3% NaCl) or high-salt (HS, 5% NaCl) isocaloric chow (Harlan Laboratories, Madison, WI) 1 day before mating. Pregnant +/+ and -/- mice were continued on their diets for the duration of gestation. The progeny consisted of the following: HS/B2 -/- (n = 64), NS/B2 -/- (n = 22), HS/B2 +/+ (n = 17), and NS/B2 +/+ (n = 12). Histological examination of the renal phenotype was performed on embryonic (E) day E15, E16, E17, and E18 and on postnatal (P) day P2, as described below.

To distinguish whether the renal phenotype was due to an intrinsic fetal abnormality or a maternal factor such as placental insufficiency, we produced mutant offspring from B2 heterozygous matings on either NS or HS (n = 15 and 19, respectively). The progeny were evaluated on day P2 and genotyped, and the kidneys were processed for histologic analysis.

Since the combination of B2 deficiency and HS can potentially cause systemic hypertension and consequently renal injury, a subgroup of HS/B2 -/- littermates was treated with either hydralazine (10 mg·kg-1·day-1 sc, n = 5) or sterile water (control, n = 12) from birth until day 20, as previously described (38). The kidneys were then examined histologically at day 20. The thickness of the renal cortex was determined using a micrometer, and the number of glomeruli and cysts per section was determined.

Animal survival was calculated as the percentage of mice surviving the first week of postnatal life. The pups were kept with their mothers until weaning on days 20–30.

Detection of B2 mRNA by RT-PCR.
RNA extraction, reverse transcription, and PCR were performed from kidneys as previously described (9). One-half of the PCR mixture was subjected to Southern blot analysis followed by hybridization with a full-length random-primed 32P-labeled rat B2R cDNA (23).

Analysis of the renal phenotype.
Kidneys were fixed by immersion in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin blocks. Two-micrometer frontal corticomedullary sections taken through the hilus were stained with periodic acid-Schiff (PAS) to delineate the proximal tubular brush border and glomerular mesangial matrix. Additional 5-µm sections were used for immunohistochemical studies as described below. Upon examination of the stained sections, particular attention was focused on the integrity of the nephrogenic zone, the organization of the medullary rays, the presence of tubular cysts/dysplasia and glomerular cysts, and the thickness and integrity of the renal microvessels. Renal hypoplasia was defined as a reduction in the number of mature glomeruli and generations of nephrons. Four microscopic fields (800 µm x 530 µm) were counted in each kidney. "Dysplasia" referred to abnormal spatial organization of nephron segments within the cortex or medulla associated with cystic dilatation or "ectasia" of the tubules and primitive glomeruli. Histological evaluations of kidney tissue sections were performed in a blinded fashion by three investigators (S. S. El- Dahr, L. Harrison-Bernard, and S. Meleg-Smith).

Immunohistochemistry.
Kidney tissue sections were immunostained by the immunoperoxidase technique using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) as described previously (15). The type, source, and working dilutions of the primary antibodies used in this study are shown in Table 1. The sections were counterstained with hematoxylin and lithium carbonate, washed with tap water, dehydrated, and coverslipped. Consecutive control sections were incubated in nonimmune serum or in the absence of the primary antibody. Slides were photographed using an Olympus model SC35 camera mounted to an Olympus model BH-2 microscope, and digital images were captured using the Adobe Photoshop 5.0 software.


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Table 1. Antibodies used in the immunohistochemical studies

 
Lectin histochemistry.
Tissue sections were deparaffinized in xylene, rehydrated in ethanol, and rinsed in PBS. Endogenous peroxidase activity was abolished by treatment in 1% H2O2 for 10 min. The slides were incubated with a solution of Dolichos biflorus agglutinin (DBA; 0.0125 mg/ml, Sigma Chemical, St. Louis, MO) in PBS for 30 min at room temperature and rinsed in PBS, and the peroxidase reaction was developed using diaminobenzidine as the chromogen.

Western blot analysis.
Kidney extracts (50 µg) were electrophoretically separated by 3–10% stacking Tris-glycine gel at 100 V for 2 h (10% SDS, 24 mM Tris base, 192 mM glycine) and transferred (20% methanol, 12 mM Tris base, 96 mM glycine) to nitrocellulose membrane (0.45; Bio-Rad) for 90 min at 25 V according to the manufacturer’s specifications (Xcell II Mini-Cell; Novex) as previously described (8). Molecular weight markers (10- to 250-kDa rainbow, Amersham) were used to approximate molecular mass. Blots were incubated with the primary antibody for 3 h, washed, incubated with the secondary antibody conjugated to horseradish peroxidase (1:1,500) for 1 h, and washed. Detection was accomplished using enhanced chemiluminescence Western blotting (ECL; Amersham), and the blots were exposed to X-ray film (Hyperfilm-ECL; Amersham). Equal protein loading and transfer were documented by Ponceau-S stain of the nitrocellulose membrane.

Data analysis and statistics.
Results are expressed as means ± SE. Data were analyzed using unpaired t-test or ANOVA followed by Tukey’s test using SigmaStat v.2.03 software. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Spatiotemporal expression of B2R.
RT-PCR/Southern blotting using B2-specific primers documented the expression of B2 mRNA during murine nephrogenesis (days E16, E18, and P5; Fig. 1A). The distribution of B2 receptors within the normal embryonic kidney (day E15) was assessed immunohistochemically and is shown in Fig. 1, B and C. B2 receptors are expressed on the apical membrane of ureteric bud branches (Fig. 1, B and C) and on apical and basolateral membranes of mature collecting ducts (day P12; Fig. 1D). As expected, the B2 receptor protein was undetectable in kidneys of homozygous null mice (Fig. 1E).



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Fig. 1. A: reverse-transcription PCR/Southern blot of bradykinin B2 receptor cDNA during murine kidney development. Total kidney RNA was pooled from one litter per age group. E, embryonic day; P, postnatal day. B–E: B2 receptor immunohistochemistry. Tissue sections are counterstained with hematoxylin. B: a low-power magnification (x4) of E15 mouse kidney. UB, ureteric bud branch; CD, collecting duct. C: high-power magnification (x90) of the nephrogenic cortex in E15 kidney. Amp, ampullar region of ureteric bud branch; V, vesicle; C, condensate. B2 immunostaining is present in the collecting ducts of wild-type (+/+) (arrows) (D) but is absent in homozygous null (-/-) (E) mice (postnatal day 12). Bar in D = 33 µm.

 
General characteristics of animal groups.
Average litter size at birth was not different among the groups. Similarly, body and kidney weights at birth were not different (Table 2). Examination of the progeny from heterozygous matings indicated that intrauterine survival to term was not affected by the combination of B2 deficiency and HS since the proportion of +/+ (n = 11), +/- (n = 25), and -/- (n = 9) pups followed approximately the expected Mendelian inheritance pattern (1:2:1). However, the percentage of mice surviving beyond the first week of life was significantly lower in HS/B2 -/- (38%) compared with NS/B2 -/- and HS/B2 +/+ mice (>=84%).


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Table 2. Characteristics of the experimental groups

 
Embryonic salt stress impairs renal development in B2-deficient and not wild-type mice.
Histological examination of PAS-stained tissue sections revealed that newborn NS/B2 -/- mice (day 2) have normal tubular and glomerular development (Fig. 2, AC). In contrast, HS/B2 -/- pups (day 2) manifest aberrations in epithelial nephrogenesis involving both kidneys (Fig. 2, DF). The structures in the renal cortex of HS/B2 -/- mice are disorganized, with areas of tubular microcysts and primitive glomeruli. The cortical medullary rays, consisting of collecting tubules and proximal and distal straight tubules are scarce or absent. Furthermore, the number of mature glomeruli and glomerular generations were not different from control animals (Table 2), suggesting that the renal lesion is dysplastic not hypoplastic. Examination of embryos at different stages of development revealed that the earliest histological signs of aberrant nephrogenesis were detectable on day E16 and consists of focal areas of tubular ectasia (Fig. 3, CF). Embryonic HS had no effect on kidney development in the progeny of wild-type C57BL/6J mice (Figs. 3A and 4, A and B). Thus the induction of the abnormal renal phenotype requires both B2 deficiency and embryonic HS.



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Fig. 2. Renal morphology in NS/B2 -/- (A–C) and HS/B2 -/- (D–F) on postnatal day 2 (day P2): Periodic acid-Schiff (PAS) stain. Renal development is normal in NS/B2 -/-. In contrast, kidneys of HS/B2 -/- pups exhibit disorganized cortex, poorly developed medullary rays, tubular ectasia/cysts (*), and primitive glomeruli. Bars: in A = 25 µm, in B = 33 µm, and in C = 50 µm.

 


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Fig. 3. Onset of the renal abnormalities in salt-stressed B2-deficient mice during embryonic development: PAS stain. A: day E15, HS/B2 -/-. B: day E16, NS/B2 -/-. C and D: day E16, HS/B2 -/-. E and F: day E18, HS/B2 -/-. Magnification in A and B is x3, in C is x7, in D and E is x15, and in F is x75 (D is the box from C, and F is the box from E). G, glomerulus; CD, collecting duct; PT, proximal tubule. In F, note the normal PAS-positive proximal tubules and the hyperproliferative epithelium lining the collecting duct cyst.

 


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Fig. 4. Renal morphology in HS/B2 +/+ mice on day 5 of postnatal life (day P5): PAS stain. A: low-power view of the renal cortex showing normal development of the nephrogenic cortex and intact corticomedullary differentiation. B: higher power view of the renal cortex in the same animal showing normal glomerular and tubular structure. Bars: in A = 25 µm; and in B = 33 µm.

 
The renal dysgenesis involves the distal nephron.
To determine the segmental origin of the dysplastic tubules, we performed immunohistochemical staining for distal nephron [DBA and aquaporin-2 (AQP2)] and proximal tubule (angiotensinogen) markers. The cystic-dysplastic tubules stained positively for DBA (Fig. 5A) and AQP2 (Fig. 5B). In contrast, angiotensinogen-positive proximal tubules appeared relatively spared of dysgenic abnormalities (Fig. 5C). Thus the dysplastic tubules are of ureteric bud origin, which is consistent with the expression of B2 in the differentiating distal nephron.



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Fig. 5. Tubular dysplasia in HS/B2 -/- mice (day 2) is restricted to the distal nephron. A: histochemical staining for Dolichos biflorus agglutinin (DBA, arrows) (x90). B: immunostaining for aquaporin-2 (AQP2, arrows) (x90). C: immunostaining for angiotensinogen (x18); hematoxylin counterstain. Note that the cystic-dysplastic tubular segments are positive for the collecting duct lectin, DBA, and the water channel, AQP2, but are negative for the proximal tubule protein, angiotensinogen. *Angiotensinogen-negative dysplastic tubules.

 
Gestational salt loading suppresses metanephric renin expression.
In all species examined to date, renin is widely distributed along the developing renal microvasculature (14). Along with these findings, we observed renin immunostaining along the afferent arterioles of NS/B2 -/- (Fig. 6A). In contrast, salt-stressed B2 -/- pups exhibit suppressed immunoreactive renin in the juxtaglomerular cells (Fig. 6B) and significantly lower percentage of juxtaglomerular apparatuses expressing renin (39 ± 7 vs. 6.0 ± 1.0%, P < 0.001) (Fig. 6C), indicating that embryonic salt loading was achieved. The suppression of immunoreactive renin in the kidney by HS was confirmed by Western blotting (Fig. 6D).



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Fig. 6. Kidney renin localization and expression on day 2. Renin immunohistochemistry in kidneys of NS/B2 -/- (A) and HS/B2 -/- (B); aa, afferent arteriole. Note suppression of juxtaglomerular renin by high salt. C: quantitative analysis of the percentage of juxtaglomerular afferent arterioles expressing immunostainable renin as assessed by immunohistochemistry. D: quantitative analysis of immunoreactive renin by Western blotting.

 
The renal phenotype is intrinsic to the fetus.
To determine the role of fetal and maternal factors in the renal dysgenesis, we examined the offspring of heterozygous matings on gestational HS. The results showed that kidney development proceeds normally in HS/B2 +/+ and +/- pups (Fig. 7A). In contrast, their HS/B2 -/- littermates (Fig. 7B) manifested the renal abnormalities observed in the homozygous offspring from homozygous null parents. Therefore, the aberrant renal phenotype is a consequence of B2 receptor deficiency in the embryo and not the mother.



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Fig. 7. Renal morphology in progeny of heterozygous matings on day 2. PAS stain. A: kidney development is normal in HS/B2 +/- pups. B: a littermate with homozygous deficiency of B2 showing aberrant tubular morphology and cystic changes (arrowheads) (x3).

 
Antihypertensive therapy does not modify the renal phenotype.
To assess the contribution of hypertension to the renal abnormalities observed in salt-stressed B2-deficient mice, we treated HS/B2 -/- mice with hydralazine or sterile water from birth until day 20. Histological examination of the kidneys of control HS/B2 -/- mice showed expanded interstitial spaces, cortical microcysts, and dilated Bowman’s space (Fig. 8A). Antihypertensive therapy with hydralazine did not modify the severity of the renal abnormalities as assessed by measurement of cortical thickness, number glomeruli or cystic changes (Fig. 8B and Table 3).



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Fig. 8. Effect of chronic antihypertensive therapy (birth to day 20) on the renal phenotype of HS/B2 -/- mice: PAS stain. A: saline-treated control. B: hydralazine treated. Arrows point to cystic structures. Hydralazine therapy had no effect on the severity of renal dysgenesis in HS/B2 -/- mice (x9).

 

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Table 3. Effect of chronic antihypertensive treatment on the renal phenotype of HS/B2 -/- mice

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrates that embryonic salt stress of B2 null mice causes aberrant renal development and postnatal lethality. In contrast, nephrogenesis and survival are normal in unstressed B2 null homozygotes, and salt-stressed wild-type and heterozygote mice. Thus the B2-deficient mouse exhibits a genetic susceptibility to renal dysgenesis in the presence of embryonic stress. To our knowledge, this is the first animal model of congenital renal disease that is induced by the cooperation of a specific gestational stressor and a defined genetic defect.

The kinin B2 receptor is a developmentally regulated G protein-coupled receptor that is implicated in renal and cardiovascular maturation (reviewed in Ref. 11). Kininogen, bradykinin, and B2 gene expression are upregulated during distal nephron differentiation (8, 9). Pharmacological antagonism of B2 receptors in newborn rats impairs renal growth (41). In addition, studies in cell culture systems have shown that B2 signaling is coupled to activator protein-1 (10), a transcription factor believed to mediate growth and differentiation during early development (30). With the development of B2 knockout mice, it is now possible to investigate the developmental role of this receptor.

The present study demonstrates that B2-deficient mice challenged with high salt during embryogenesis exhibit renal dysgenesis that shares many (but not all) features with human renal cystic dysplasia, including disorganization of the nephrogenic cortex, loss of corticomedullary differentiation, and collecting duct ectasia/cysts (40). Although this study was not designed to elucidate the mechanisms mediating the effects of embryonic salt stress and B2 deficiency on kidney development, the results do provide important clues concerning potential mechanisms. First, the renal phenotype is not apparent histologically until embryonic day E16. In the mouse, metanephric development begins on day E11.5 and nephrogenesis continues until the second week of postnatal life. Thus the renal phenotype appears relatively late during renal development. Indeed, the histomorphometric analysis indicated that the early inductive events of nephrogenesis proceed normally in the salt-stressed B2 -/- mice since the number of nephrons (glomeruli) is comparable to control mice. Thus the aberrant renal phenotype is probably the result of a disturbance in terminal epithelial differentiation leading to dysplasia. Further studies are clearly required to test this hypothesis.

To gain some insights into the possible role of genetic background on the renal phenotype, we compared the prevalence of renal abnormalities in HS/B2 -/- mice on a mixed 129/BL6 genetic background (F4) to mice backcrossed onto C57BL/6J for 10 generations (F10). The results showed an increase in the percentage of mice exhibiting the renal abnormalities from 57% (8/14) in F4 to 84% (38/45) in F10 (P < 0.05), demonstrating an important genetic background effect on the penetrance of the renal phenotype.

Another important observation in this study is that aberrant nephron development is predominantly localized to the distal nephron. Dysplastic tubules and cysts are positive for two collecting duct-specific markers, DBA and the water channel, AQP2 (17), but negative for a proximal tubule-specific protein, angiotensinogen. This restricted distribution corresponds well with the predominant expression of B2 receptors in the distal nephron during kidney development. Therefore, the renal dysgenesis cannot simply result from a generalized toxic effect of high salt on the renal parenchyma and is consistent with the hypothesis that kinins specifically promote or protect the process of distal nephron differentiation.

The present study also demonstrates that the susceptibility to salt-induced nephropathy is intrinsic to the fetus and results from B2 receptor deficiency in the embryo, since B2 homozygous offspring from heterozygous parents exhibit the same renal phenotype as offspring from homozygous null parents. This makes the contribution of maternal factors, such as placental insufficiency, to the fetal renal phenotype less likely. It should be emphasized here that heterozygous B2 (+/-) mice develop mild hypertension on a high-salt diet (22). Therefore, maternal hypertension may be a contributing factor. Renal abnormalities intrinsic to B2 null animals may occur due to the receptor’s role in maintaining normal vascular tone or its role in regulating cell growth and differentiation. At the present time, it is difficult to discriminate between these factors. However, restriction of differentiation impairment to epithelial cell types that normally express the B2 receptor lends support to the hypothesis that growth/differentiation promoting functions of the receptor are crucial to normal development. We have recently shown that pharmacological blockade of B2 receptors in concert with elevated gestational salt intake in rats results in a fetal kidney abnormality (12) that is strikingly similar to the one seen here following genetic ablation. This provides independent validation of the role of B2 in this kidney phenotype.

The pathogenesis of embryonic salt stress-induced renal dysgenesis in B2-deficient mice is likely multifactorial. We have considered several possibilities. As shown in this study, salt-loaded B2-deficient fetuses exhibited suppressed renal renin. Consequently, downregulation of the fetal renin-angiotensin system (combined with B2 ablation) may contribute to the renal dysgenesis. Another possibility is altered expression of growth factor/receptor pairs that are required for full and proper differentiation of the distal nephron such as the EGF-R (37). Alterations in transcription factors are also a possibility. For example, we have recently identified the B2 receptor gene as a direct transcriptional target for the p53 tumor suppressor protein (31). Ongoing studies in our laboratory are exploring the potential role of p53 activation in the pathogenesis of the renal dysgenesis in salt-stressed B2-deficient embryos. It is also conceivable that altered fluid handling by the fetal kidney could contribute to the cystic distension of the collecting ducts.

We also considered the hypothesis that the renal phenotype may result, at least partly, from fetal hypertension and consequently renal vasoconstriction and ischemia. We have shown in this study that gestational high salt suppresses juxtaglomerular renin expression in the progeny of B2 null mice, an indicator of fetal salt loading. The latter (high salt) could potentially cause fetal hypertension if kinins are inactive as a result of B2 ablation. However, we do not favor this hypothesis for several reasons. First, the observed renal phenotype does not include typical pathological changes of chronic hypertension such as vascular thickening. Second, the hypertension does not explain the predominant localization of the renal abnormalities in the distal nephron. Finally, neonatal hydralazine treatment was ineffective in preventing or attenuating the renal abnormalities observed in weanling HS/B2R -/- mice, suggesting that neonatal hypertension, if it existed, was not responsible for the persistent renal dysgenesis. We recognize, however, the limitations of our approach. First, we are limited by the technical inability to verify that blood pressure was adequately controlled in the preweaning pups due to their small size. Second, administration of antihypertensive drugs to the mother (to treat fetal hypertension) is hazardous and may impair placental blood flow. Thus whether the nephroprotective role of bradykinin depends on its ability to promote epithelial cell differentiation and survival or its vasoactive properties or both remains a formidable challenge for future investigations.


Significance and relevance to human disease.
The present study demonstrates that mice that are deficient in the B2 bradykinin receptor and salt loaded during embryonic development acquire an abnormal kidney phenotype. This finding is highly significant, since it represents the creation of an animal model of renal disease that is reproducibly dependent on both a defined genetic abnormality (mutation of B2) and a specific environmental stress (salt loading). Many diseases affecting the kidney, such as essential hypertension and diabetes, appear to involve interactions between genetic and environmental factors. Thus understanding how salt loading worsens the phenotype of B2 mutant mice could be relevant to many other genetic diseases affecting kidney and cardiovascular development. Congenital abnormalities of kidney and urogenital tract development are a major cause of renal failure in early childhood, accounting for 40% of end-stage renal disease in children under 4 years of age (20, 28). Thus insights gained from this animal model of abnormal kidney development are relevant to important human diseases.


    ACKNOWLEDGMENTS
 
We are grateful to F. Hess and H. Chen (Merck Research Laboratories, Rahway, NJ) for providing the bradykinin B2 receptor null mice, Werner Müller-Esterl (Frankfurt, Germany) for the bradykinin B2 receptor antibody, Mark Knepper (National Institutes of Health, Bethesda, MD) for the AQP2 antibody, Conrad Sernia (University of Queensland, Australia) for the angiotensinogen antibody, and D. Catanzaro and J. Sealy (Cornell School of Medicine) for the renin antibody. We also thank Novartis Pharmaceuticals (Summit, NJ) for providing us with hydralazine (Apresoline).

The present study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R21-DK-53595 and RO1-DK-56264 (to S. S. El-Dahr).

Portions of this work were presented at Annual Meeting of the American Society of Nephrology, in San Antonio, TX, 1998, and have been published in abstract form (J Am Soc Nephrol 9: 361, 1998).


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S. S. El-Dahr, Section of Pediatric Nephrology, Dept. of Pediatrics, SL-37, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: seldahr{at}tmcpop.tmc.tulane.edu).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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