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REGULATION IN GENETICALLY MODIFIED ANIMALS

Renal and blood pressure phenotype in 18-mo-old bradykinin B₂R(-/-)^CRD mice

Departments of ¹Physiology and ²Pediatrics, Section of Pediatric Nephrology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112

Submitted 12 March 2003 ; accepted in final form 12 June 2003

	ABSTRACT

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Aberrant gene-environment interactions are implicated in the pathogenesis of congenital renal dysgenesis (CRD), a leading cause of renal failure in infants and children. We have recently developed an animal model of CRD that is caused by gestational salt stress (5% NaCl diet; HS) of bradykinin B₂R null mice [B₂R(-/-)^CRD; El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Physiol Genomics 3: 121-131, 2000.]. Developing B₂R(-/-)^CRD mice exhibit tubular and glomerular cysts, stromal expansion, and loss of corticomedullary differentiation. In addition, B₂R(-/-)^CRD mice exhibit transient hypertension from 2 to 4 mo of age. The present study was designed to determine the long-term consequences of CRD on renal morphology and salt sensitivity of blood pressure in B₂R(-/-)^CRD mice. One-year- and 18-mo-old B₂R(-/-)^CRD mice exhibited stunted renal growth, glomerular cystic abnormalities, and collecting duct ectasia. Moreover, tumors of mesenchymal cell origin emerged in the dysplastic kidneys of 90% of 1-yr-old and 100% of 18-mo-old B₂R(-/-)^CRD mice but not in age-matched B₂R(-/-) or wild-type mice. When challenged with an HS diet, 18-mo-old B₂R(-/-)^CRD exhibited a significant rise in systolic and diastolic blood pressures and more pronounced natriuresis and diuresis compared with salt-loaded 18-mo-old wild-type mice. Kidney aquaporin-2 expression was decreased by 50%, whereas renin, ANG type 1 receptor, and Na⁺-K⁺-ATPase levels were not different in B₂R(-/-)^CRD mice compared with controls. In conclusion, this study demonstrates that B₂R(-/-)^CRD mice exhibit permanent phenotypic and functional abnormalities in renal growth and differentiation. This novel model of human disease links gene-environment interactions with renal development and blood pressure homeostasis.

kidney development; salt sensitivity; kallikrein-kinin; aquaporin

Several lines of evidence suggest that the aberrant renal phenotype of B₂R(-/-)^CRD mice is the result of impaired terminal epithelial differentiation. First, the renal phenotype appears relatively late in fetal development in concert with morphological and functional tubular differentiation. Second, histomorphometric analysis indicates that the early inductive events of nephrogenesis proceed normally in the B₂R null mice (10). In addition to renal dysplasia, B₂R(-/-)^CRD mice develop early onset hypertension (4). The elevated blood pressure (BP) can be measured as early as 6-8 wk of age and persists until 12-14 wk of age, when it gradually declines toward normal values. The circulating renin-angiotensin system is not activated in B₂R(-/-) mutants on the BL6 genetic background (4), yet these mice are highly sensitive to the chronic hypertensive effect of exogenous ANG II (5). The long-term relation of renal dysplasia with hypertension is not clear because tubular dysgenesis is usually associated with salt wasting (17a). Therefore, the objectives of the current study were to determine the long-term consequences of CRD on renal morphology and salt sensitivity of BP in B₂R(-/-)^CRD mice.

	METHODS

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Animals and experimental protocols. Breeding pairs of B₂R(-/-) mice (C57/BL6) were placed on a high-salt diet of isocaloric chow (HS; 5% NaCl, TD no. 92102; Harlan Teklad, Madison, WI) 1 day before mating and for the duration of gestation to induce CRD in the progeny, as described previously (4, 10). The maternal diet was switched to normal salt (NS; 0.3% NaCl) 1 day postpartum. After weaning (days 21-25), the B₂R(-/-)^CRD progeny were continued on NS. B₂R(-/-) and B₂R(+/+) mice were maintained on life-long NS and served as controls for the effects of age on renal structure and BP. To determine the long-term effects of CRD on BP, systolic BP (SBP) was measured in the same animals at 12 and 17 mo of age in B₂R(-/-)^CRD and B₂R(+/+) mice on a life-long NS diet. To determine the long-term consequences of CRD on salt handling, 17-mo-old B₂R(-/-)^CRD and B₂R(+/+) mice were placed on HS for 4 wk. BP was measured during the 4-wk HS period, and plasma electrolytes and urinary sodium excretion were analyzed at the end of the 4-wk HS diet, as outlined in Fig. 1 and described below.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Experimental design. Congenital renal dysgenesis (CRD) B2 receptor (B2R)-deficient [B2R(-/-)CRD] and B2R wild-type [B2R(+/+)] mice were exposed to a gestational high-salt diet (HS) or a normal salt diet (NS), respectively. All mothers and suckling pups were placed on the NS diet on postnatal day 1. Systolic blood pressures (SBP) were measured by the tail-cuff method in the same animals at 12, 17, and 18 mo of age. Twenty-four-hour urine samples were collected at 17 and 18 mo of age on NS and HS diets, respectively. Acute blood pressures (BP) and blood samples were obtained under pentobarbital sodium anesthesia at the end of the protocol (18 mo only). Kidneys were obtained at postnatal day 2 and 12 and 18 mo of age for histological investigation of renal morphology and protein extraction.

Conscious SBP measurements. Serial measurements of conscious SBP were performed on male and female B₂R(-/-)^CRD (n = 20) and male B₂R(+/+) (n = 6) mice at 12, 17, and 18 mo of age using a computer-automated tail-cuff system (Visitech BP-2000 Blood Pressure Analysis System; Visitech Systems; Apex, NC). Animals were placed on a heated platform and underwent 10 preliminary cycles. The average of three 10-cycle measurements, which each have a minimum of 6 out of 10 successful measurement cycles, was used for data analysis.

Anesthetized BP measurements. Before anesthesia, 18-mo-old male and female B₂R(-/-)^CRD (n = 18) and male B₂R(+/+) (n = 4; originally 6 mice, 2 died between 12 and 18 mo of age) mice treated with an HS diet for 4 wk were administered 3 mg/100 g body wt bromodeoxyuridine (BrDU; Zymed Laboratories) intraperitoneally to measure the proliferative index in the kidneys. Animals were anesthetized with 50 mg/kg ip pentobarbital sodium and were placed on a heated surgical table. The right carotid artery was cannulated with a short PE-10 catheter connected to a PE-50 catheter, as previously described (4). Direct arterial BP was measured using a P23XL transducer (Astro-Med) and was converted to a digital signal by using a Pentium 200-MHz computer and an analog-to-digital data-acquisition system (model MP100; Biopac Systems, Santa Barbara, CA) after a 30- to 45-min equilibration period. Average SBP, mean arterial pressure (MAP), diastolic BP (DBP), and heart rate (HR) were obtained over a 10- to 15-min period. Sampling rate was set at 400 samples/s. Blood samples were taken from the carotid artery cannula at the end of the data collection period (5 mM ETDA). Hematocrit and plasma protein concentration (AO Refractometer; American Optical, Buffalo, NY) were measured. Plasma sodium and potassium concentrations were measured as described below. Kidneys were removed, blotted dry, and weighed. One kidney was immersed in liquid nitrogen and stored at -80°C until the time of protein extraction. The other kidney was processed for histology and BrDU staining. SBP and urine excretion were measured in the same mice at 17 mo of age on the NS diet and during the 4-wk period of the HS diet. However, anesthetized BPs, blood samples, and kidney samples were only obtained at the end of the HS diet.

Urine collections. Male B₂R(+/+) (n = 5) and B₂R(-/-)^CRD (n = 7) animals were placed in metabolic cages for a period of 24 h while on the NS diet at 17 mo of age and again at 18 mo of age after 4 wk on the HS diet. Urine was collected and analyzed for volume, sodium concentration, potassium concentration, and osmolality. Water intake was measured on the HS diet. Sodium and potassium were determined with a flame photometer (model 943; Instrumentation Laboratory, Lexington, MA). Osmolality was measured with a vapor pressure osmometer (model 5500; Wescor, Logan, UT).

Western blot analysis of kidney protein. Western blot analysis was performed on kidneys obtained from 18-mo-old B₂R(-/-)^CRD (n = 6) and B₂R(+/+) (n = 4) mice on HS, as previously described (18). The following primary antibodies were used: anti-rat sheep polyclonal angiotensinogen antibody (1:6,000; see Ref. 7), anti-human rabbit polyclonal ANG type 1 receptor (AT₁) antibody (1:200; N-10, sc-1173; Santa Cruz), anti-rat rabbit polyclonal aquaporin-2 (AQP-2) antibody (1:200; AB3066; Chemicon), anti-rabbit mouse monoclonal Na⁺-K⁺-ATPase ₁ (1:1,500; 05-369; Upstate Biotechnology), and anti-human rabbit polyclonal renin antibody (1:2,000; see Ref. 3). Membranes were reprobed with -actin antibody (monoclonal anti--actin antibody, 1:4,000; A5441; Sigma). Signals were detected using enhanced chemiluminescence (Amersham), and protein expression was analyzed densitometrically using the Digital Imaging and Analysis Systems (Apha Innotech).

Immunohistochemical analysis of kidney tissue sections. Kidneys from 12- and 18-mo-old mice were fixed in 10% buffered formalin, dehydrated in graded solutions of alcohol, and embedded in paraffin blocks, and 5-µm sections were made and mounted on slides with Vectabond (Vector Laboratories, Burlingame, CA). Immunostaining was performed by the immunoperoxidase technique using the Vectastain Elite kit (Vector Laboratories, as previously described (10). Primary antibodies used include anti-human mouse monoclonal -smooth muscle actin (1:100; NCL-SMA; Nova Castra Laboratories), AQP-2 (1:100; Chemicon International), anti-BrDU mouse monoclonal antibody (1:50; ZBU30; Zymed), anti-human rabbit polyclonal -catenin (1:200; H-102, sc-7199; Santa Cruz Biotechnology), and E-cadherin (1:200; H-108, Santa Cruz Biotechnology). Controls consisted of tissue sections in which the primary antibodies were substituted with PBS or nonimmune serum.

Lectin histochemistry. Tissue sections were incubated with Dolichos biflorus agglutinin (0.0125 mg/ml; Sigma), as previously described (10).

Data analysis. Statistical analyses were performed using SigmaStat Statistical Software on the raw data by two-way ANOVA, followed by Tukey's test or by unpaired t-test, as appropriate. A P value <0.05 was considered statistically significant. All data are presented as means ± SE.

	RESULTS

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Renal morphology. Histological studies were performed on kidney sections of B₂R(+/+) and B₂R(-/-)^CRD mice (Fig. 2). Normal renal morphology is observed on postnatal day 2 of B₂R(+/+) mice (Fig. 2A), 12 mo of age (Fig. 2D), and 18 mo of age (data not shown). Newborn B₂R(-/-)^CRD mice exhibit developmental renal abnormalities similar to what has previously been described (10), consisting of tubular cysts, a disorganized cortex, and poorly developed medullary rays (Fig. 2, B and C). Dysgenic tubules in 18-mo-old B₂R(-/-)^CRD mouse kidneys stain positively for D. biflorus lectin, indicating collecting duct origin (Fig. 2E). The dysplastic tubules stained positively but faintly for AQP-2 (Fig. 2F), consistent with downregulated expression of distal nephron differentiation markers (see results of AQP-2 Western blot below). Hyperplastic islands or "tumorlets" are observed in the dysplastic kidneys of 20/22 1-yr-old and 18/18 18-mo-old B₂R(-/-)^CRD mice. These tumorlets were not observed at birth or at 4 or 6 mo of age (not shown). By immunohistochemistry, proliferating cells (Fig. 2, G-I) are frequently seen in the tumorlets. An average of three to six tumorlets are observed per section and are located in the inner cortex closely associated with renal arteries, veins, and glomeruli (Fig. 2G).

View larger version (166K): [in this window] [in a new window]   Fig. 2. Renal morphology in B2R(+/+) (A and D) and B2R(-/-)CRD (B, C, and E-I) mice at birth and 12 and 18 mo of age. Normal renal morphology is seen in B2R(+/+) mice on postnatal day 2 (A) and at 1 yr(D). Nephrogenic zone (NZ) is demarcated in A, and normal glomerular morphology (arrow) is shown in D. B2R(-/-)CRD mice exposed to gestational HS demonstrate tubular cysts (*) at postnatal day 2 (B and at higher magnification in C) and at 18 mo of age (E). High-power magnification in E shows that the epithelial cells (arrows) lining the renal cysts are positive for the collecting duct marker Dolichos biflorus (DB; E). Aquaporin-2 (AQP-2) expression is downregulated in dysplastic collecting ducts as shown by the presence of a faint immunoreactivity (arrows in F). Renal tumors are found at 18 mo of age in B2R(-/-)CRD mice (G-I). These tumors are positive for bromodeoxyuridine (BrDU; G and H and higher magnification in I; arrows). PT, proximal convoluted tubule; CD, collecting duct; a, artery; v, vein. Original magnification x20 (A, B, and H), x40 (C, D, G, and I), and x100 (E and F).

Figure 3A and higher-magnification Fig. 3B show that smooth muscle -actin positively stained blood vessels are seen at the center of a renal tumorlet, indicating high vascularization. The tumorlets are composed predominantly of mesenchymal, myofibroblastic cells (smooth muscle -actin-positive; Fig. 3B) and, to a lesser extent, vimentin-positive cells (data not shown). Although the periphery of the tumorlet is positive for CD-45, a marker of hematopoietic cells, the tumorlet's core is negative (Fig. 3C). Moreover, the tumorlets are positive for proliferating cell nuclear antigen (Fig. 3D) but negative for tubular epithelial markers, including -catenin (Fig. 3, E and F), AT₁ receptor (Fig. 4, A-C), angiotensinogen (Fig. 4, D and E), AQP-2, Na⁺-K⁺-ATPase, and E-cadherin (data not shown). In the case of epithelial cell markers, no specific staining was observed in the tumorlets in spite of using intentionally high concentrations of antibodies. Importantly, 1-yr-old B₂R(-/-) mice that were maintained on an NS diet during embryogenesis and post-natally (n = 5) have normal renal architecture and no evidence of renal tumorigenesis (Fig. 3, G and H).

View larger version (110K): [in this window] [in a new window]   Fig. 3. Immunohistochemistry of tumorlets in 12-mo-old B2R(-/-)CRD dysplastic kidneys. Control section without primary antibody (A) and consecutive section immunostained for smooth muscle -actin (B) showing the localization of a blood vessel at the center of the tumor (arrow). Tumorlets are negative for CD45, a marker of hematopoietic cell lineage (C). The dotted line outlines the core of the tumorlet, which is CD45 negative. The tumorlet is positive for proliferating cell nuclear antigen (PCNA; arrow), a proliferation marker (D). Anti--catenin antibody staining (E and F) shows positive immunoreactivity in the tubules (arrow), whereas the tumorlets are -catenin negative (arrowhead). Kidney sections from 12-mo-old NS/B2R(-/-) mice (maintained on an NS diet since the time of conception) show normal morphology and an absence of dysplastic tubules and tumors (G and higher magnification in H). Original magnification x20 (A, C, D, and G) and x40 (B, E, F, and H).

View larger version (48K): [in this window] [in a new window]   Fig. 4. Immunohistochemistry of tumorlets in 12-mo-old B2R(-/-)CRD dysplastic kidneys. A-C: ANG II type 1 (AT1) receptor immunoreactivity is present in renal tubules (arrowhead in B) and blood vessels (C) but not in the tumorlet or the dysplastic primitive duct (arrow in B). D and E: angiotensinogen (AGT) is expressed in tubules (arrows) but not in the tumorlet. The background nonspecific staining is the result of the use of intentionally high concentrations of AGT antibody. A glomerular cyst (glom cyst) is seen in D. Original magnification x20 (A) and x40 (B-E).

Excretory function of B₂R(-/-)^CRD and B₂R(+/+) mice on NS and HS diets. A 24-h urine collection was obtained from 17-mo-old male B₂R(+/+) (n = 5) and B₂R(-/-)^CRD (n = 7) mice while on the NS diet. Body weights were significantly higher in NS/B₂R(-/-)^CRD than NS/B₂R(+/+) mice (Table 1). No significant weight change occurred in either group after 4 wk of HS diets (Table 1). Twenty-four-hour urine volume, urinary Na⁺ and K⁺ excretion, factored for body weight, and urine osmolality, are not different between 17-mo-old NS/B₂R(-/-)^CRD and NS/B₂R(+/+) mice (Table 1). A second 24-h urine collection and 24-h water intake were measured after placing these two groups of mice on an HS diet for 4 wk. HS/B₂R(+/+) mice showed a fivefold increase in urinary sodium excretion (P = 0.08) and a significant increase in urine volume compared with NS (P < 0.05; Table 1). The degree of natriuresis in HS/B₂R(+/+) mice is similar to that reported for wild-type mice on a 1-wk HS diet by Oliverio et al. (17). HS/B₂R(-/-)^CRD mice had statistically significant increases in urine volume (3-fold), sodium excretion (10-fold), and potassium excretion (40%) compared with values on the NS diet (Table 1). Importantly, urine volume (2-fold), sodium excretion (3-fold), and potassium excretion (3-fold) of HS/B₂R(-/-)^CRD mice, factored for body weight, were significantly greater than HS/B₂R(+/+) (Table 1). Twenty-four-hour water intake, factored for body weight, was similar in HS/B₂R(-/-)^CRD and HS/B₂R(+/+) mice (Table 1).

SBP profile in B₂R(-/-)^CRD and B₂R(+/+) mice on the NS diet. We have previously reported that B₂R(-/-)^CRD mice exposed to gestational HS and switched to an NS diet postnatally exhibit significantly higher SBP at 2 and 3 mo of age with a gradual return of SBP to normal values by 4 mo of age (4). SBP of B₂R(-/-)^CRD mice measured at 12 and 17 mo of age averaged 116 ± 2(n = 12) and 114 ± 2(n = 20) mmHg, respectively. SBP of B₂R(+/+) mice measured at 12 and 17 mo of age averaged 112 ± 2 (n = 8) and 115 ± 5 (n = 5) mmHg, respectively. There were no significant differences in SBP at 12 and 17 mo of age in B₂R(-/-)^CRD mice compared with B₂R(+/+) mice maintained on the NS diet throughout postnatal life (Fig. 5).

View larger version (30K): [in this window] [in a new window]   Fig. 5. SBP at 2, 3, 4, 12, and 17 mo of age in NS/B2R(-/-)CRD (solid bars; n = 10-20) and NS/B2R(+/+) (hatched bars; n = 5-8) mice. Data at 2, 3, and 4 mo adapted from Ref. 4. *P < 0.05 vs. B2R(+/+) mice at a given time point.

Effect of HS diet on SBP profile in B₂R(-/-)^CRD and B₂R(+/+) mice. In response to 4 wk of elevated dietary salt, 18-mo-old HS/B₂R(-/-)^CRD exhibited a significant rise in SBP, whereas SBP in HS/B₂R(+/+) was not altered (Fig. 6). SBP of HS/B₂R(-/-)^CRD increased significantly from 114 ± 2 to 127 ± 3 mmHg after 18 days compared with the NS diet. SBP continued to increase over the following week in HS/B₂R(-/-)^CRD and was significantly higher on days 24 and 28 compared with HS/B₂R(+/+) mice. SBP averaged 133 ± 3 in HS/B₂R(-/-)^CRD and 118 ± 8 mmHg in HS/B₂R(+/+) (P < 0.05) at the end of 4 wk of the HS diet. The impact of salt loading on SBP is evidenced by the increase of 18 vs. 4 mmHg in HS/B₂R(-/-)^CRD and HS/B₂R(+/+) mice, respectively (Fig. 6, inset).

View larger version (26K): [in this window] [in a new window]   Fig. 6. SBP profile of B2R(-/-)CRD (; n = 13-20) and B2R(+/+) (; n = 4-5) mice beginning at 17 mo of age. Bars indicate time points for NS and HS diets. SBP was not different between the groups on NS, remained constant in B2R(+/+) on the HS diet, but increased significantly in B2R(-/-)CRD mice on the HS diet. The change in SBP from the last day of NS to the last day of HS is shown in inset [solid bar, B2R(-/-)CRD; hatched bar, B2R(+/+)]. *P < 0.05 vs. NS. P < 0.05 vs. B2R(+/+) mice.

BP profile in anesthetized B₂R(-/-)^CRD and B₂R(+/+) mice on the HS diet. Direct measurements of arterial BP were collected in anesthetized mice to confirm the observation of salt sensitivity in B₂R(-/-)^CRD mice made by tail-cuff SBP measurement. HS/B₂R(-/-)^CRD mice exhibited higher DBP compared with HS/B₂R(+/+) mice when studied under pentobarbital sodium anesthesia [66 ± 3 and 51 ± 3 mmHg, respectively (P < 0.05); Fig. 7C]. MAP (Fig. 7A) and SBP (Fig. 7B) tended to be higher in HS/B₂R(-/-)^CRD than HS/B₂R(+/+) mice, averaging 79 ± 4 and 62 ± 3 mmHg, respectively (P = 0.06). HR was not different between HS/B₂R(-/-)^CRD and HS/B₂R(+/+) mice (449 ± 24 vs. 444 ± 61 beats/min; Fig. 7D).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Hemodynamic profile of anesthetized B2R(-/-)CRD (solid bars; n = 14) and B2R(+/+) (hatched bars; n = 4) mice on the HS diet. Mean arterial (A) and systolic blood (B) pressures of B2R(-/-)CRD mice tended to be higher than B2R(+/+) mice. Diastolic BP (C) was significantly elevated in B2R(-/-)CRD compared with B2R(+/+) mice. Heart rate (D) was not different between the two groups. *P < 0.05 vs. B2R(+/+) mice. bpm, beats/min.

Body weight was not different in male and female 18-mo-old HS/B₂R(-/-)^CRD and male HS/B₂R(+/+) mice (Table 2). Plasma sodium and potassium concentrations were not different between the groups. Plasma protein concentration was significantly higher (P < 0.05); however, hematocrit values were not different in HS/B₂R(-/-)^CRD compared with HS/B₂R(+/+) mice. Consistent with the postnatal phenotype of renal dysgenesis, total kidney weight and kidney-to-body weight ratios were reduced significantly in adult HS/B₂R(-/-)^CRD compared with HS/B₂R(+/+) mice (Table 2).

Kidney protein expression in B₂R(-/-)^CRD and B₂R(+/+) mice on the HS diet. To assess whether the urinary excretory abnormalities in HS/B₂R(-/-)^CRD are associated with changes in tubular sodium and water transport mechanisms, the abundance of the renin-angiotensin system components, Na⁺-K⁺-ATPase, and AQP-2 protein expressions were determined in kidneys obtained from 18-mo-old HS/B₂R(-/-)^CRD and HS/B₂R(+/+) mice by Western blot analysis. AQP-2 protein expression was significantly lower by 52% in kidneys of HS/B₂R(-/-)^CRD compared with HS/B₂R(+/+) mice (P < 0.05; Fig. 8). Na⁺-K⁺-ATPase, renin, and AT₁ receptor protein expressions were not different between the two groups, but there was a 50% elevation in angiotensinogen protein in HS/B₂R(-/-)^CRD (P < 0.05; data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 8. Kidney AQP-2 protein expression in B2R(-/-)CRD (solid bar; n = 6) and B2R(+/+) (hatched bar; n = 4) mice, analyzed by Western blot analysis. Protein extracts were prepared from kidneys of 18-mo-old mice after 4 wk of the HS diet (20 µg total protein/lane). The membrane was reprobed with -actin antibody. Data are expressed as the ratio of densitometric units to -actin. AQP-2 protein expression was 50% lower in HS/B2R(-/-)CRD mouse kidneys compared with HS/B2R(+/+). *P < 0.05 vs. B2R(+/+) mice. MW, mol wt.

	DISCUSSION

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Genetic and environmental factors are implicated in the pathogenesis of CRD. The environmental component includes the fetal-maternal milieu. As such, we have developed a model of CRD resulting from the combination of an environmental stressor (gestational HS) combined with genetic deletion of the bradykinin B₂ receptor. This animal model exhibits congenital collecting duct dysgenesis (Ref. 10 and this study). Long-term follow-up in the present study demonstrates that the collecting duct dysgenesis is permanent and irreversible. In addition, microscopic tumors of mesenchymal cells emerge in the dysplastic kidneys. Functional studies performed at 17-18 mo of age revealed that CRD mice show abnormalities in salt and water handling and a propensity to develop salt-induced hypertension. Importantly, 1-yr-old B₂R(-/-) maintained on an NS diet since the time of conception revealed no abnormalities in renal development and had no renal tumors. Collectively, these findings present direct genetic evidence linking aberrant intrauterine gene-environment interactions with abnormal renal development and salt-sensitive hypertension.

A surprising finding of this study is the emergence of tumor growths in kidneys of 1-yr- and 18-mo-old B₂R(-/-)^CRD mice. Interestingly, studies in humans have described the emergence of nodular blastema tumors within dysplastic kidneys (16). Immunohistochemical staining of 1-yr-old and 18-mo-old B₂R(-/-)^CRD kidneys identified the cellular masses as proliferative (BrDU positive) and of mesenchymal cell origin (smooth muscle -actin and vimentin positive). In comparison, the tumorlets were negative for tubular epithelial cell markers such as angiotensinogen, Na⁺-K⁺-ATPase, AQP-2, E-cadherin, and -catenin. Given the proximity of the tumorlets to glomeruli, the possibility that these cells originated from the extraglomerular mesangium was considered. Unfortunately, there are no specific markers for mesangial cells in the mouse. Finally, the core of the tumorlet was CD45 negative, indicating that the cells are not of hematopoeitic origin. We hypothesize that the tumorlets originated from a population of renal stromal stem cells that maintained uncontrolled proliferation in the aberrant microenvironment of the dysplastic kidney. Additional studies are necessary to elucidate the cell biology of abnormal growth regulation in this model, particularly in light of the fact that hypertensive patients are pre-disposed to renal cancer (6, 13).

Alfie et al. (1) reported that SBP and MAP were higher in adult B₂R(-/-) mice maintained on the HS diet for 8 wk compared with B₂R(-/-) on the NS diet. Madeddu et al. (15) have shown that SBP and MAP were higher in B₂R(-/-) than B₂R(+/+) mice on the NS diet. It is possible that the difference between the two studies is related to the genetic background of the mice under study (129Sv vs. mixed 129/BL6). We have previously shown that B₂R null mice exposed to a "postnatal" HS diet for 4 mo develop hypertension yet do not show signs of renal dysplasia (4). Only the combination of gestational HS diet and lack of B₂R receptors leads to renal dysplasia. We have capitalized on this unique model of renal dysplasia to investigate the long-term effects of salt and water handling on BP, and renal morphology. The results of the present study show that, under conditions of normal salt intake, B₂R(-/-)^CRD mice remain normotensive up to 17 mo of age. However, B₂R(-/-)^CRD mice have a propensity to develop hypertension when challenged with a chronic dietary salt load. Loss of bradykinin's natriuretic actions cannot be the sole reason for salt sensitivity in HS/B₂R(-/-)^CRD, since bradykinin's actions via the B₂R are natriuresis and diuresis, and these mice exhibited substantial increases in both salt and water excretion in response to chronic salt loading. Therefore, additional studies are warranted to elucidate the mechanisms of hypertension in 18-mo-old HS/B₂R(-/-)^CRD mice.

In the present study, we observed decreased kidney/collecting duct AQP-2 expression in HS/B₂R(-/-)^CRD compared with age- and sex-matched wild-type mice, which may contribute, at least partly, to the diuresis. Interpretation of the exaggerated natriuresis in HS/B₂R(-/-)^CRD mice is more complex, because estimates of dietary salt intake are not available. It is important to note that higher body weights were observed in B₂R(-/-)^CRD compared with B₂R(+/+) mice in both the mice on the NS and HS diets (Table 1), yet exaggerated natriuresis was only observed in the HS/B₂R(-/-)^CRD group. Also, differences in appetite cannot account for the natriuresis because a primary increase in salt appetite, resulting in a threefold increase in steady-state urinary sodium excretion, would be expected to cause a significant weight gain as a result of higher caloric intake. However, this did not occur because NS/B₂R(-/-)^CRD and HS/B₂R(-/-)^CRD had similar body weights on NS and HS diets (Table 1). The precise mechanisms of hypertension in HS/B₂R(-/-)^CRD remain to be defined. From a "clinical" standpoint, patients born with CRD initially present with complex electrolyte disturbances secondary to renal tubular salt wasting and/or concentrating defects, renal tubular acidosis, and varying degrees of renal functional impairment. Treatment involves careful replacement of fluid and electrolyte losses to maintain euvolemia (12). It is unknown whether euvolemic CRD patients are susceptible to salt-induced hypertension, similar to the B₂R(-/-)^CRD mice.

In summary, 1-yr-old and 18-mo-old B₂R(-/-)^CRD mice with CRD exhibit persistent structural abnormalities such as renal tubular ectasia and glomerular cysts. An unexpected new finding was the development of mesenchymal-type tumor growth in the kidneys of B₂R(-/-)^CRD mice. BP, urine flow, and sodium excretion are normal at 17 mo of age in B₂R(-/-)^CRD mice maintained on an NS diet throughout postnatal life. However, when challenged with the HS diet for 4 wk, 18-mo-old B₂R(-/-)^CRD mice exhibit a rise in SBP and greater natriuretic and diuretic responses than salt-loaded B₂R(+/+) mice. The diuresis is consistent with the downregulation of kidney AQP-2 expression. The development of salt-induced hypertension in the face of enhanced urinary salt/water excretion in 18-mo-old B₂R(-/-)^CRD is puzzling. To our knowledge, there are no published studies that have addressed renal handling of a salt load in adult humans born with renal dysgenesis and whether these individuals have increased susceptibility to salt-induced hypertension. Our study suggests such possibilities. In conclusion, the B₂R(-/-)^CRD mouse is a novel model of human disease that links gene-environment interactions with renal development and BP homeostasis.

	DISCLOSURES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-56264 (to S. S. El-Dahr). L. M. Harrison-Bernard was supported by American Heart Association Scientist Development Grant 9930120N and NIDDK Grant DK-62003-01. Digital images were obtained at the Tulane Hypertension and Renal Center of Excellence Imaging Core Facility made possible by the HEF2001-06-07 Louisiana Board of Regents through the Millennium Trust Health Excellence Fund.

	ACKNOWLEDGMENTS

 
We thank Drs. Conrad Sernia and Dan Catanzaro for generously providing the angiotensinogen and renin antibodies and Dr. Kenneth D. Mitchell for critical review of the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. S. El-Dahr, Dept. of Pediatrics, SL37, Tulane Univ. Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail: seldahr{at}tulane.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Alfie ME, Sigmon DH, Pomposiello SI, and Carretero OA. Effect of high salt intake in mutant mice lacking bradykinin-B₂ receptors. Hypertension 29: 483-487, 1997.[Abstract/Free Full Text]
3. Campbell Jr WG, Gahnem F, Catanzaro DF, James GD, Camargo MJF, Laragh JH, and Sealey JE. Plasma and renal prorenin/renin, renin mRNA, and blood pressure in Dahl salt-sensitive and salt-resistant rats. Hypertension 27: 1121-1133, 1996.[Abstract/Free Full Text]
4. Cervenka L, Harrison-Bernard LM, Dipp S, Primrose G, Imig JD, and El-Dahr SS. Early onset salt-sensitive hypertension in bradykinin B₂ receptor null mice. Hypertension 34: 176-180, 1999.[Abstract/Free Full Text]
5. Cervenka L, Maly J, Karasova L, Simova M, Vitko S, Hellerova S, Heller J, and El-Dahr SS. Angiotensin II-induced hypertension in bradykinin B₂ receptor knockout mice. Hypertension 37: 967-973, 2001.[Abstract/Free Full Text]
6. Chow WH, Gridley G, Fraumeni Jr JF, and Jarvholm B. Obesity, hypertension, and the risk of kidney cancer in men. N Engl J Med 343: 1305-1311, 2000.[Abstract/Free Full Text]
7. Darby IA and Sernia C. In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys. Cell Tissue Res 281: 197-206, 1995.[Web of Science][Medline]
8. El-Dahr SS. Development biology of the renal kallikrein-kinin system. Pediatr Nephrol 8: 624-631, 1994.[Web of Science][Medline]
9. El-Dahr SS, Figueroa CD, Gonzalez CB, and Müller-Esterl W. Ontogeny of bradykinin B₂ receptors in the rat kidney: implications for segmental nephron maturation. Kidney Int 51: 739-749, 1997.[Web of Science][Medline]
10. El-Dahr SS, Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Bradykinin B2 null mice are prone to renal dysplasia: gene-environment interactions in kidney development. Physiol Genomics 3: 121-131, 2000.[Abstract/Free Full Text]
11. Guyton AC, Coleman TG, Cowley AW, Schell KW, Manning RD, Manning RA, and Norman RA. Arterial pressure regulation: overriding dominance of the kidneys in long-term regulation and in hypertension (Abstract). Am J Med 52: 584, 1972.[Web of Science][Medline]
12. Limwongse C, Clarren S, and Cassidy S. Syndromes and malformations of the urinary tract. In: Pediatric Nephology, edited by Barratt T, Avner E, and Harmon MF. Philadelphia, PA: Lippincott, 1999, p. 427-452.
13. Lindgren A, Pukkala E, Nissinen A, Kataja V, Notkola IL, and Tuomilehto J. Cancer incidence in hypertensive patients in North Karelia, Finland. Hypertension 37: 1251-1255, 2001.[Abstract/Free Full Text]
14. Mackenzie HS, Lawler E, and Brenner B. Congenital oligonephropathy: the fetal flaw in essential hypertension. Kidney Int 49: S30-S34, 1996.
15. Madeddu P, Varoni MV, Palomba D, Emanueli C, Demontis MP, and Dessi-Fulgheri P. Cardiovascular phenotype of a mouse strain with disruption of bradykinin B₂ receptor gene. Circulation 96: 3570-3578, 1997.[Abstract/Free Full Text]
16. Noe HN, Marshall JH, and Edwards OP. Nodular renal blastema in the multicystic kidney. J Urol 142: 486-488, 1989.[Medline]
17. Oliverio MI, Best CF, Smithies O, and Coffman TM. Regulation of sodium balance and blood pressure by the AT(1A) receptor for angiotensin II. Hypertension 35: 550-554, 2000.[Abstract/Free Full Text]
17. Watkins SL, McDonald RA, and Avner ED. Renal dysplasia, hypoplasia, and miscellaneous cystic disorders. In: Pediatric Nephrology, edited by Barratt T, Avner E, and Harmon WE. Philadelphia, PA: Lippincott, 1999. p. 415-425.

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