Transfer of a Salt-Resistant Renin Allele Raises Blood Pressure in Dahl Salt-Sensitive Rats
To evaluate the role of the renin gene in the development of hypertension in Dahl salt-sensitive rats (SS/Jr/Hsd), we derived a congenic strain of rats homozygous for the salt-resistant renin allele (S/renrr) and compared them with a control strain homozygous for the salt-sensitive renin allele (S/renss). Mean arterial pressure was significantly higher in 12-week-old S/renrr rats fed a high salt (8.0%) diet for 3 weeks than in S/renss rats or in SS/Jr/Hsd rats rederived from the foundation colony we used to generate the congenic strain (195±3 [n=49] versus 168±3 [n=17] or 161±3 [n=16] mm Hg). Mean arterial pressure was also higher in S/renrr rats than in S/renss rats raised from birth on either a very low salt (0.1%) diet (119±9 [n=6] versus 100±7 [n=4] mm Hg) or a low salt (0.4%) diet (143±1 [n=22] versus 117±3 [n=10] mm Hg). Plasma renin activity of S/renrr rats was significantly higher than that of S/renss rats fed a very low salt diet (5.7±2.0 versus 1.8±0.3 ng angiotensin I/mL per hour), a low salt diet (4.4±1.0 versus 1.1±0.3), or a high salt diet (1.5±0.2 versus 0.9±0.1). Urinary protein excretion was greater in S/renrr rats than in S/renss rats fed a high salt diet (244.2±48.5 versus 43.6±19.5 mg/24 h), and this was associated with significant reductions in renal blood flow (3.3±0.6 versus 4.6±0.5 mL/min per gram kidney weight) and glomerular filtration rate (0.49±0.11 versus 0.82±0.08 mL/min per gram kidney weight). Captopril (20 mg/kg IV) had no effect on blood pressure in S/renss rats fed a low salt diet, but it lowered blood pressure by 20 mm Hg in S/renrr rats to the same level seen in untreated S/renss rats. Chronic administration of captopril (5 mg/100 mL drinking water) reduced blood pressure in S/renrr rats fed a high salt diet (170±5 mm Hg) to the same level seen in untreated S/renss rats, whereas it had no significant effect on blood pressure in S/renss rats. These results indicate that transfer of a salt-resistant renin allele to SS/Jr/Hsd rats raises plasma renin activity and augments the severity of hypertension and renal disease.
The RAS plays a major role in the regulation of body fluid volume and arterial pressure. It is therefore not surprising that inherited abnormalities in the control of renin production and release have long been suspected to be involved in the pathogenesis of hypertension.1 Rapp and colleagues2 were the first to report that a polymorphism in the renin gene cosegregated with BP in an F2 population derived from a cross of SS/Jr rats and SR/Jr rats. Similar results were subsequently reported in F2 populations derived from crosses of spontaneously hypertensive and Lyon hypertensive rats with normotensive strains.3 4 5 However, at the present time, little evidence demonstrates that an abnormality in the RAS contributes to the development of hypertension in SS/Jr rats. Rather, renal, adrenal, and plasma renin activities are lower in SS/Jr rats than SR/Jr rats,6 7 8 9 and SS/Jr rats are relatively resistant to the antihypertensive actions of converting enzyme inhibitors10 or angiotensin II receptor antagonists.11 Moreover, Rapp and colleagues (Alam et al12 ) have reported that there is no difference in the coding region or the first 2000 bp of the 5′ regulatory region of the renin gene in SS/Jr and SR/Jr rats. Thus, it remains to be determined whether the cosegregation of the renin gene with BP in SS/Jr and SR/Jr rats is due to genetically determined strain differences in the expression of this gene or is related to some other closely linked locus on chromosome 13. It also remains to be determined to what extent the suppressed plasma renin levels in SS/Jr rats are genetically determined or are secondary to the development of hypertension.
One approach for addressing these questions would be to construct a congenic strain of salt-sensitive rats homozygous for the salt-resistant renin allele and compare it back to the parental or a coderived control strain. If the congenic and control strains exhibited phenotypic differences in BP and/or PRA, this would strongly support the view that an abnormality in the renin gene contributes to the development of hypertension and/or the strain differences in PRA and renal renin activity. Therefore, in the present study, we derived a congenic strain of SS/Jr rats homozygous for the salt-resistant renin allele (S/renrr) along with a corresponding control strain homozygous for the salt-sensitive renin allele (S/renss). MAP in the congenic S/renrr rats was significantly higher than in the control S/renss rats, regardless of the salt content of the diet. The elevation in BP in S/renrr rats was associated with an increase in PRA and more severe glomerular disease, as evidenced by higher urinary protein excretion, lower RBF, and lower GFR than in the control S/renss strain. Captopril lowered MAP in the congenic S/renrr rats to the same level as in the untreated S/renss rats. These results indicate that the transfer of the salt-resistant renin allele into the SS/Jr/Hsd genetic background elevates PRA and augments the severity of hypertension and glomerular disease.
Development of Congenic Strains
An inbred male SS/Jr/Hsd rat and female SR/Jr/Hsd rat purchased from Harlan Sprague Dawley, Inc (Indianapolis, Ind) in 1990 were mated to produce an F1 population. Female F1 offspring were backcrossed to male SS/Jr/Hsd rats to produce an N1 backcross generation. The offspring were genotyped by PCR, and females that were heterozygous for the renin allele and that carried the fewest number of SR/Jr alleles at loci tested on chromosomes other than 13 were selected as breeders for the next backcross generation. We did this negative selection for SR/Jr alleles at loci other than the renin locus to reduce the number of generations needed to generate the congenic strain.13 14 Selected females were backcrossed again to male SS/Jr/Hsd rats. This scheme of repetitive genotyping and backcross breeding to SS/Jr/Hsd males was continued for three backcross generations by Dr Theodore Kurtz at the University of California, San Francisco. In June of 1991, female breeders selected from the third backcross generation were transferred to the Medical College of Wisconsin, and the backcross breeding was continued for an additional three generations with male SS/Jr/Hsd rats that were being held in an in-house colony. The rats in this colony retained the hypertensive phenotype and were homozygous for SS/Jr alleles at the six genetic markers that revealed the existence of contamination in the SS/Jr/Hsd rats being supplied by Harlan Sprague Dawley, Inc (Indianapolis, Ind) during 1991-1994.15 16 We did this in an attempt to avoid the introduction of non-SS/Jr/Hsd alleles from the commercially available colony into our congenic line. After the sixth backcross generation, male and female siblings heterozygous for the renin allele were intercrossed to generate the congenic strain, ie, the SS/Jr/Hsd rats homozygous for the salt-resistant renin allele (SS/Jr/Hsd/MCW-renrr; hereafter referred to as S/renrr) and the control SS/Jr/Hsd substrain homozygous for the salt-sensitive renin allele (SS/Jr/Hsd/MCW; hereafter referred to as S/renss). These two strains were subsequently propagated by brother-sister mating for six to eight generations before the phenotyping experiments described herein were performed.
Genomic DNA was isolated from the tail of the rats by a modified guanidinium thiocyanate procedure (DNA-STAT60, TestTEST, Inc). PCR amplification was performed on 100 ng of genomic DNA in a reaction consisting of 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 0.05% Tween 20, 0.05% Nonidet P-40, 1.5 mmol/L MgCl2, 200 μmol/L dNTPs, and 1 μmol/L of each primer in a reaction volume of 100 μL. The reaction tube was heated for 2.5 minutes at 100°C and then cooled to 94°C for 30 seconds when 1 U of Thermus flavus DNA polymerase (Epicentre) was added. The PCR cycle consisted of 30 seconds of denaturation at 94°C, 1 minute of annealing at 50°C, and 1.5 minutes of extension at 72°C for 35 cycles. Fifty microliters of the PCR reaction was separated on a 2% agarose gel (Metaphor, FMC Corp) for 4 hours at 20 V/cm. Gels were then stained with ethidium bromide (0.5 μg/mL) and visualized under ultraviolet light.
We used two polymorphic sites to genotype the rats at the renin locus. First, we designed a pair of primers to amplify across a polymorphic HindIII restriction site in the fifth intron of the renin gene. The PCR product of the SR/Jr renin allele can be cut by HindIII to yield a 122-bp fragment, whereas the PCR product of the SS/Jr renin allele cannot be cut and is 144 bp in length.17 Second, a D13UW1 primer pair purchased from Research Genetics amplifies across a simple sequence length polymorphism in the third intron and yields a 196-bp fragment from the SS/Jr renin allele and a 178-bp fragment from the SR/Jr renin allele.18
We also accomplished genotyping at other loci by PCR analysis of simple sequence length polymorphisms throughout the rat genome to ensure that the strains were inbred and homozygous for SS/Jr alleles. Most of the primer pairs used were obtained from Research Genetics. Most of these markers have been previously described, and the chromosomal locations of these markers have been mapped by Jacob et al.19 A few additional markers used for mapping of the length of the segment on chromosome 13 introgressed from salt-resistant to salt-sensitive rats were described by Remmers et al.20 Table 1⇓ presents a complete list of the primers used in the genotyping experiments.
MAP was measured in several groups of male and female S/renss and S/renrr rats maintained on different salt diets. Group 1 was maintained after weaning at 3 weeks of age on a very low salt (0.1%) diet until they were phenotyped at 12 weeks of age. Group 2 was maintained on a typical low salt (0.4%) diet after weaning. In group 3, S/renss and S/renrr rats were placed on a low salt (0.4%) diet after weaning. When the rats were 9 weeks old, they were switched to a high salt (8.0%) diet for 3 weeks before BP was measured.
Recently, the parental SS/Jr/Hsd strain used to generate our congenic line was rederived from the foundation colony and again became commercially available. We therefore studied rederived SS/Jr/Hsd rats as a second control group. In these experiments, 3-week-old rederived SS/Jr/Hsd rats (nine females, seven males) purchased from Harlan Sprague Dawley, Inc, were placed on the low salt diet until they were 9 weeks old. Then they were switched to the high salt diet for 3 weeks before BP was measured.
At 12 weeks of age, all rats were anesthetized with injection of ketamine (30 mg/kg IM) and xylazine (2 mg/kg IM), and polyvinyl catheters were implanted in the femoral artery, with the use of sterile technique, for direct measurement of arterial pressure and collection of arterial blood. The catheter was tunneled to the back of the neck and brought out through a stainless steel spring and swivel device. After a 3-day recovery period, arterial pressure was directly measured with a pressure transducer and computerized recording system for 3 hours per day between noon and 3 pm on 3 consecutive days while the rat was conscious and unrestrained in its home cage. No one was allowed in the recording room while pressure was being measured so that environmental stress was minimized. The amplified analog pressure signal was sampled at 100 Hz, and the digitized signals were stored on an Apollo computer system. We then processed the digitized signals using the SignifiCat Software package to compute daily averages of systolic, diastolic, and mean BPs.
Measurement of PRA
After 3 days of arterial pressure measurements, a blood sample (1 mL) was collected into an ice-cold tube containing potassium EDTA via the femoral artery catheter while the rats were resting quietly in their cages. The sample was immediately centrifuged at 4°C, and the plasma was frozen and stored at −20°C. PRA was determined by measurement of angiotensin I generated in plasma incubated at 37°C for 3 hours with the use of a radioimmunoassay as previously described.21 PRA was expressed as nanograms angiotensin I formed per milliliter of sample per hour. At the end of each experiment, the rats were placed in metabolic cages, and a 24-hour urine sample was collected for measurement of urine volume and protein excretion. Urine protein concentration was measured by the Bradford colorimetric method with bovine serum albumin as a standard (Bio-Rad).
Effects of Captopril on Arterial Pressure in S/renss and S/renrr Rats
To determine whether the observed differences in PRA contributed to the differences in arterial pressure observed between the strains, we performed acute and chronic experiments using the converting enzyme inhibitor captopril. In the chronic experiments, 9-week-old rats were treated with captopril (5 mg/100 mL in drinking water) for 3 days before and throughout the 3 weeks while the rats were challenged with a high salt (8.0%) diet. Arterial pressure was then measured as described above. In the acute experiments, baseline BP was measured in 12-week-old S/renss (n=6) and S/renrr (n=8) rats maintained on a low salt (0.4%) diet. Then, a bolus dose of captopril (20 mg/kg) was injected via the arterial catheter, and BP was monitored for an additional 60 minutes.
Determination of Renal Function in S/renss and S/renrr Rats
Renal function was assessed in 10 SR/Jr/Hsd, 5 S/renrr, and 7 S/renss rats maintained from birth on a low salt (0.4%) diet. The rats were 12 weeks old at the time of the study. The rats were anesthetized with ketamine (30 mg/kg IM) and thiobutabarbital (Inactin, 30 mg/kg IM) and placed on a thermostatically controlled warming table for maintenance of body temperature at 36.5°C. Cannulas were placed in the right external jugular vein for intravenous infusions, in the right femoral artery for measurement of arterial pressure, and in the ureter for collection of urine. A flow probe (2 mm ID) was placed around the left renal artery for measurement of RBF with an electromagnetic flowmeter (model 501, Carolina Medical Instruments). During surgery, the rats received a 2-mL intravenous infusion of 0.9% NaCl solution containing 6.0% albumin to replace fluid losses. Thereafter, the rats received an intravenous infusion of 3% bovine serum albumin in 0.9% NaCl solution at a rate of 50 μL/min throughout the experiment. [3H]Inulin (1 μCi/mL) was included in the infusion solution for measurement of GFR.
After surgery and a 1-hour equilibration period, urine flow, hematocrit, RBF, GFR, MAP, and urinary excretions of sodium, potassium, and chloride were measured during two 30-minute baseline clearance periods. The rats then received 0.9% NaCl solution intravenously in an amount equal to 5% of their body weight over a 30-minute period, and urine and plasma samples were again collected during an additional 30-minute clearance period.
Urine flow was measured gravimetrically. Chloride concentrations of urine and plasma samples were measured by electrometric titration (Chloride Analyzer 926, Corning Instruments). Sodium and potassium concentrations of urine and plasma samples were measured with a flame photometer (model 143, Instrumentation Laboratories). [3H]Inulin concentration was determined with a liquid scintillation spectrophotometer (model 2450, Packard Instruments). GFR was calculated as the ratio of urine to plasma inulin concentration times urine flow. Urine flow, sodium and potassium excretions, GFR, and RBF were all factored per gram of kidney weight.
Data are presented as mean±SE. The significance of differences in mean values within and between groups was determined with ANOVA for repeated measures followed by Duncan's multiple range test. A value of P<.05 (two-tailed test) was considered significant.
Genotyping of S/renss and S/renrr Strains
Fig 1⇓ presents a representative gel illustrating genotyping of six S/renrr rats and six S/renss rats with a primer pair that amplifies across a polymorphic HindIII restriction site in the fifth intron of the renin gene. DNA extracted from the livers of authentic SS/Jr and SR/Jr rats obtained from Dr Rapp's colony at the Medical College of Ohio (Toledo) were used as controls. The PCR product from the SS/Jr renin allele could not be cut by HindIII and had an apparent molecular weight of 160 bp when run under nondenaturing conditions in a 2% agarose gel. HindIII cut the PCR product from the SR/Jr renin allele to a fragment with an apparent molecular weight of 130 bp. Compared with the authentic SS/Jr and SR/Jr DNA standards, all of the S/renss rats were homozygous for the SS/Jr renin allele, and all of the S/renrr rats were homozygous for the SR/Jr renin allele. Similar results were obtained with the D13 UW1 primer pair, which amplifies across a simple sequence length polymorphism in the third intron of the renin gene.
To further define the length of chromosome 13 transferred from the SR/Jr/Hsd rat into the S/renrr strain, we genotyped these rats using six additional markers (D13Mit1, D13N1, Syt2, D13NaF, D13Mit3, and D13Mit5) on chromosome 13 that are polymorphic in SS/Jr and SR/Jr rats. Fig 2⇓ summarizes the locations of these markers relative to the renin locus. The results indicate that all of the S/renrr and S/renss rats studied were homozygous for the SS/Jr alleles at all of the six markers tested on chromosome 13. Thus, the maximum size of the segment of chromosome 13 that could have been transferred from SR/Jr/Hsd rats to the S/renrr strain lies between the D13N1 and Syt2 markers, a distance estimated to be 10 cm in length on the basis of previously published maps of rat chromosome 13.19 20
To determine the extent to which these strains were inbred and genetically alike, we genotyped the rats at 49 additional loci scattered throughout the genome. The markers chosen were all polymorphic in SS/Jr and SR/Jr rats and are summarized in Table 1⇑. All of the S/renss and S/renrr rats studied were homozygous for SS/Jr alleles at all of the autosomal markers on chromosome 1 through chromosome 20, as determined with DNA standards extracted from the livers of authentic SS/Jr and SR/Jr rats. We also tested the rats with nine additional markers on the X chromosome. Only one marker (DXMgh12) was found to be informative. The results with this marker indicate that the S/renrr rats were fixed with the SS/Jr allele. However, the control S/renss rats were not fixed at this locus. Approximately 30% of the male rats exhibited an SR/Jr allele, and 50% of the female rats were heterozygous. To determine whether this strain difference at this locus on the X chromosome could contribute to the differences in BP seen in the S/renss and S/renrr rats, we genotyped the entire S/renss population at the DXMgh12 locus. MAP and PRA did not differ significantly in the S/renss rats homozygous for the SS/Jr DXMgh12 allele versus those that carried one SR/Jr allele. In addition, MAP during the high salt diet was not significantly different in the S/renss rats and the newly rederived SS/Jr/Hsd rats that are homozygous for the SS/Jr allele at this locus.
Phenotyping of S/renss and S/renrr Strains
No significant difference in MAP was observed between male and female rats in any of the groups studied; therefore, the data from both groups were pooled and are presented together. As summarized in Fig 3⇓, MAP was higher in S/renrr rats than in the control S/renss rats, regardless of the salt content of the diet. During a very low salt (0.1%) diet, MAP was 19 mm Hg higher in S/renrr than control S/renss rats. During a typical low salt (0.4%) diet, MAP was 26 mm Hg higher in S/renrr than S/renss rats. After exposure of rats to a high salt (8.0%) diet for 3 weeks, MAP was 30 mm Hg higher in S/renrr rats than either S/renss rats or SS/Jr/Hsd rats rederived from the foundation colony we originally used to generate our congenic strain. MAP values in S/renss rats or SS/Jr/Hsd rats fed a high salt diet for 3 weeks did not differ significantly.
As shown in Fig 4⇓, the congenic S/renrr rats exhibited higher PRA than the control S/renss rats, regardless of the salt content of the diet. It is also of interest that PRA increased significantly as expected in the S/renrr rats when the salt content of the diet was reduced, but it did not differ significantly in the groups of S/renss rats fed different salt diets.
To determine the extent to which the higher arterial pressure measured in the S/renrr rats might depend on an elevated PRA, we compared the antihypertensive effects of captopril in the S/renrr and S/renss strains. Fig 5⇓ presents the results of the acute experiments. A bolus injection of captopril (20 mg/kg) markedly lowered MAP in S/renrr rats maintained on a low salt diet to the same level seen in the untreated S/renss rats (P<.05), whereas captopril had no effect on MAP in S/renss rats fed the same diet.
In the chronic experiments, captopril (5 mg/100 mL) was added to the drinking water for 3 days before and throughout 3 weeks while the rats were fed a high salt diet. As shown in Fig 6⇓, captopril lowered MAP in the congenic S/renrr rats fed a high salt diet to 170±5 mm Hg (P<.05), which is comparable to the BP seen in untreated S/renss rats. Chronic captopril treatment had no significant effect on BP in control S/renss rats fed a high salt diet. Moreover, the antihypertensive effect of captopril in the S/renrr rats was reversible. Three days after withdrawal of captopril treatment, the BP of S/renrr rats rose by 20 mm Hg to a level comparable to that in untreated S/renrr rats fed a high salt diet (P<.05). In contrast, removal of captopril from the drinking water did not significantly alter the BP of the S/renss rats.
Comparison of Renal Function in S/renss and S/renrr Strains
Table 2⇓ presents the results of experiments comparing renal function in the S/renrr and S/renss strains. Also presented for comparison are data recently reported by our group for SR/Jr/Hsd rats using an identical protocol.22 MAP was not significantly different in SR/Jr/Hsd rats versus values in control S/renss rats maintained from birth on a low salt (0.4%) diet. In contrast, MAP was significantly higher in S/renrr rats than either S/renss or SR/Jr/Hsd rats. RBF and GFR were approximately 25% lower in S/renss than SR/Jr/Hsd rats, and these values were even lower in the S/renrr rats relative to the values in control S/renss rats. Urine flow and urinary sodium, potassium, and chloride excretions were all significantly reduced in S/renss rats relative to the values seen in SR/Jr/Hsd rats, and all of these values were even lower in the congenic S/renrr rats relative to values in control S/renss rats. Fractional excretions of urinary sodium and chloride were similar in S/renss and S/renrr rats, and both were significantly lower than the corresponding values in SR/Jr/Hsd rats.
Previous studies have indicated that outbred Dahl salt-sensitive rats (Brookhaven; Dahl S) and SS/Jr/Hsd rats maintained on a low salt diet exhibit an impaired ability to excrete an isotonic saline load relative to outbred Dahl salt-resistant (Dahl R) or SR/Jr/Hsd rats.21 23 Therefore, we compared the natriuretic response to an intravenous infusion of 0.9% NaCl solution in S/renss and S/renrr strains. These results are presented in Fig 7⇓. Consistent with previous results in outbred Dahl S and SS/Jr/Hsd rats, the natriuretic response to infusion of isotonic saline was less in S/renss than SR/Jr/Hsd rats. This response was even more severely impaired in S/renrr rats. Over the 1-hour course of these experiments, S/renrr rats excreted only 31±4% of the saline load compared with 102±10% in SR/Jr/Hsd and 76±11% in S/renss rats.
It has also been reported that the excretion of total protein24 and/or albumin25 is elevated in SS/Jr/Hsd rats fed a high salt diet. Therefore, we compared the 24-hour excretion of urinary protein in the S/renss and S/renrr strains. The results of these experiments are presented in Fig 8⇓. The 24-hour excretion of protein was significantly greater in S/renrr than S/renss rats maintained for 3 weeks on the high salt diet (P<.05).
Previous studies have indicated that a polymorphism in the renin gene cosegregates with BP in an F2 population derived from a cross of SS/Jr and SR/Jr rats.2 However, at the present time, there is little evidence indicating that an abnormality in the RAS contributes to the development of hypertension in this model of hypertension. In fact, renal, adrenal, and plasma renin activities were reported to be lower in Dahl S than Dahl R rats even before the development of hypertension,6 7 8 9 and SS/Jr/Hsd rats have been reported to be relatively resistant to the antihypertensive effects of converting enzyme inhibitors or angiotensin II receptor antagonists.10 11 Therefore, it remains uncertain whether the cosegregation of the renin gene with BP is due to genetically determined strain differences in the expression of the renin gene or is related to another gene physically linked to the renin locus on chromosome 13.
The present study describes the development and phenotypic characterization of a congenic strain derived from SS/Jr/Hsd rats homozygous for the SR/Jr renin allele along with a control strain homozygous for the SS/Jr renin allele. PCR analysis of 49 genetic markers polymorphic in SS/Jr and SR/Jr rats indicates that except for a segment of chromosome 13 flanking the renin allele, both S/renss and S/renrr strains were homozygous for the SS/Jr alleles at all the autosomal markers tested on chromosomes 1 through 20. This finding, however, does not mean that the S/renss and S/renrr strains are genetically identical or that the S/renss strain can be considered to be genetically equivalent to the SS/Jr/Hsd parental strain or genetically similar to the original SS/Jr strain. Indeed, we did find that the S/renss and S/renrr rats differed at least at the DXMgh12 locus on the X chromosome. This finding was not unexpected in that the percentage of the genome that would be fixed following six generations of backcross breeding plus six generations of brother-sister mating would at best be about 99%.26
Genotyping of other markers on chromosome 13 revealed that the segment of chromosome 13 transferred from SR/Jr/Hsd rats to the S/renrr strain is located between the D13N1 and D13Mit5 markers, a region that is approximately 10 cm in length. Moreover, 10 of the 49 markers used have been reported to be polymorphic in the contaminated SS/Jr/Hsd rats sold by Harlan Sprague Dawley, Inc, between 1991 and 1994 and the rats from the original or rederived SS/Jr/Hsd colonies.15 16 The fact that none of these markers revealed the presence of non-SS/Jr alleles in the S/renss or S/renrr rats indicates that we managed to avoid contaminating the congenic strain over the 5 years it took to develop the strains.
In the present study, we observed several phenotypic differences in BP and PRA in S/renrr rats compared with S/renss rats. MAP was about 20 mm Hg higher in the S/renrr than the S/renss rats, regardless of the salt content of the diet. After rats were exposed to a high salt diet, BP was also higher in S/renrr rats than SS/Jr/Hsd rats rederived from the foundation colony we used to create the congenic strain. The higher BPs in the S/renrr rats were also associated with significantly higher PRA than in S/renss rats. The results of our additional studies indicating that the difference in BP between these two strains was eliminated after captopril administration suggest that the increment in BP in S/renrr rats depends on the elevated PRA in this strain.
Overall, our results suggest that transfer of the renin gene from SR/Jr/Hsd rats into the SS/Jr/Hsd genetic background elevates PRA and BP. These findings are consistent with previous observations that PRA and renal renin content are lower in SS/Jr than SR/Jr rats.6 7 8 9 They also are complementary to the results of a parallel study recently published by St Lezin et al27 indicating that the transfer of the SS/Jr renin allele into the SR/Jr genetic background lowered systolic arterial pressure, PRA, and renal renin mRNA levels in the congenic strain compared with the levels seen in the progenitor SR/Jr rats.
We cannot exclude the possibility that the differences in PRA and BP observed in the present study in the S/renrr and S/renss strains might be due to another gene in the transferred region on chromosome 13 or some other residual genetic differences in S/renrr and S/renss strains such as the one we identified at the DXMgh12 locus. However, it does not appear that differences at the DXMgh12 locus can account for the elevated BP in the congenic S/renrr rats, because there was no significant difference in arterial pressure in S/renss rats homozygous for the SS/Jr allele versus those that carried one SR/Jr allele at this locus. Moreover, BP was also higher in the congenic S/renrr rats than in the rederived SS/Jr/Hsd rats that are homozygous for the SS/Jr DXMgh12 allele.
Overall, the most direct interpretation of the present results is that there is some molecular variation in the renin gene that contributes to the differences in PRA and BP observed in the S/renss and S/renrr strains. However, previous studies by Alam et al12 failed to reveal any sequence difference in the coding region of the renin gene in SS/Jr and SR/Jr rats, nor has any difference been found in the first 2000 bp of the 5′ regulatory region of this gene.12 These findings indicate that if structural differences in the renin gene exist that are responsible for the differential expression of this gene in the kidneys of SS/Jr/Hsd and SR/Jr/Hsd rats, they must be proximal to the first 2000 bp of the transcription start site. This remains a distinct possibility, especially in light of the recent discovery that there are transcriptional regulatory elements, more than 2.5 to 5 kb proximal from the transcription start site in the human renin gene, that are essential for the determination of tissue-specific and high expression levels of this gene.28 29 Alternatively, it remains possible that some of the strain differences identified in noncoding regions of the renin gene in SS/Jr and SR/Jr rats might affect the expression of this gene.17
The results of the present study, and of that by St Lezin et al,27 in which the renin allele from SS/Jr/Hsd rats was introgressed into an SR/Jr/Hsd genetic background, indicate that the SS/Jr renin allele is associated with lower PRA and BP compared with the SR/Jr renin allele. This observation is consistent with previous findings of lower PRA and renal renin activity in SS/Jr than SR/Jr rats6 7 8 9 and the fact that this model is considered a non–renin-dependent model of hypertension and is relatively resistant to the antihypertensive actions of converting enzyme inhibitors and angiotensin II type 1 receptor antagonists.10 11 However, these observations are opposite to what we originally expected to find on the basis of the previous report that the SS/Jr renin allele cosegregates with elevated BP in an F2 population derived from a cross of SS/Jr and SR/Jr rats. The reason for this inconsistency remains to be determined, but one possibility is that the SS/Jr rat may carry a renin allele that promotes lower PRA and BP than the renin allele that SR/Jr rats carry and that the linkage of renin with BP in the previously reported cross of SS/Jr and SR/Jr rats was due to the presence of another major hypertensive gene on chromosome 13 linked to but distinct from the SS/Jr renin gene. Since the resolution of mapping studies with a limited number of rats and markers can only resolve quantitative trait loci to broad regions of a chromosome, it is possible that this putative gene for high BP lies outside of the limited region of chromosome 13 that we transferred from SR/Jr/Hsd to SS/Jr/Hsd rats. If this is the case, comparison of BP data in congenic strains carrying different portions of chromosome 13 might be useful in tracking down the location of this major putative quantitative trait locus for hypertension on chromosome 13. Another likely possibility that cannot be excluded is that the original inbred SS/Jr rats and the SS/Jr/Hsd colony that we used to derive our congenic strains have diverged over the many years that they have been separately maintained so that now they are two related but genetically distinct substrains of SS/Jr rats. Detailed genetic mapping of the different colonies of SS/Jr rats is needed to resolve this question.
Previous studies have indicated that activation of the RAS promotes the progression of glomerular disease in diabetes, following reductions in renal mass, and in several models of hypertension.30 31 32 Therefore, we compared renal function in the S/renss and S/renrr strains in which PRA differs. The data we obtained from the S/renss rats were nearly identical to those we have previously reported for SS/Jr/Hsd rats.22 33 34 RBF and GFR in S/renss rats were reduced by about 25% compared with values seen in SR/Jr/Hsd rats, even though the rats were maintained on a low salt diet to prevent the development of hypertension. S/renss rats also excreted less sodium, chloride, and water than SR/Jr/Hsd rats when studied during a mild saline diuresis. In addition, S/renss rats exhibited an impaired natriuretic response to an intravenous infusion of isotonic saline relative to SR/Jr/Hsd rats, and they developed severe proteinuria and glomerular disease after exposure to a high salt diet.25 The transfer of the SR/Jr renin allele into the SS/Jr/Hsd strain resulted in more severe impairment of renal function. RBF and GFR were significantly lower in the S/renrr than S/renss strain, and the degree of proteinuria after exposure to a high salt diet was significantly greater. S/renrr rats also retained more sodium, chloride, and water than S/renss rats during a mild saline diuresis, and they exhibited only a small natriuretic response to an acute 5% body weight load of isotonic saline. It remains to be determined in future studies to what extent the impairment of renal function and more severe renal disease in S/renrr than in S/renss rats can be attributed to activation of the RAS or the greater degree of hypertension seen in this strain.
Although the Dahl SS/Jr/Hsd rat is the most widely used animal model of salt-sensitive hypertension, some characteristics have limited the applicability of this model to studies of salt-sensitive hypertension in humans. For example, hypertension in SS/Jr/Hsd rats is generally induced by feeding the rats a high salt diet containing 4% or 8% NaCl for several weeks. Many investigators have questioned the utility of studying salt-sensitive hypertension with a strain of rats in which sodium intake is typically elevated by more than 20-fold to induce the disease. Salt-sensitive patients generally develop hypertension on a normal salt intake and exhibit changes in arterial pressure when salt intake is changed even modestly.35 In this regard, it is interesting to note that MAP of the S/renrr rats in the present study was in the normotensive range (<120 mm Hg) when the rats were fed a very low salt (0.1%) diet, and they became overtly hypertensive when raised on a typical low salt (0.4%) diet. In contrast, the SS/Jr/Hsd and S/renss rats remained relatively normotensive (BP <120 mm Hg) when raised on a 0.4% NaCl diet. These findings indicate that the S/renrr strain appears to be more salt sensitive than the S/renss and the original SS/Jr/Hsd strains. In this regard, the S/renrr strain might be useful as a model of salt-sensitive hypertension that does not require a high salt intake to develop hypertension.
Another limitation of the SS/Jr model is that it is a low-renin model of hypertension that is relatively resistant to antihypertensive treatment with drugs that target the RAS, but SS/Jr rats readily respond to diuretic treatment.10 36 37 This has limited the ability of researchers to use Dahl SS/Jr rats as a model to study the important interactions between the RAS and end-organ damage in the heart, kidney, and peripheral vasculature that occur after the development of hypertension. In the present study, we found that the S/renrr rats exhibited appropriate changes in PRA in response to changes in salt intake, in contrast to the lack of change in PRA seen in S/renss rats or as previously reported in SS/Jr rats8 9 when they are switched from a high to a low salt diet. Moreover, acute and chronic blockade of the RAS with captopril significantly lowered arterial pressure in the S/renrr rats, whereas S/renss and SS/Jr10 rats typically exhibit little response to the same maneuvers. Thus, the S/renrr strain may be an interesting model for investigation of the role of the RAS in the pathogenesis of glomerular disease and cardiac and vascular hypertrophy during the development of salt-sensitive hypertension as opposed to the original SS/Jr/Hsd model.
In summary, we transferred a segment of chromosome 13 flanking the renin locus, approximately 10 cm in length, from SR/Jr/Hsd to SS/Jr/Hsd rats using backcross breeding. MAP of the S/renrr rats was significantly higher than that measured in control S/renss rats, regardless of the salt content of the diet. The BP elevation in S/renrr rats was associated with increased PRA and the development of more severe glomerular injury, as evidenced by a higher 24-hour urinary protein excretion and lower RBF and GFR than values observed in control S/renss rats. Captopril lowered MAP of the congenic S/renrr rats to the same level seen in untreated S/renss rats. These results suggest that the transfer of a segment of chromosome 13 flanking the renin gene from SR/Jr/Hsd to SS/Jr/Hsd rats raises PRA and results in more severe hypertension and renal damage.
Selected Abbreviations and Acronyms
|GFR||=||glomerular filtration rate|
|Hsd||=||rats from Harlan Sprague Dawley, Inc|
|MAP||=||mean arterial pressure|
|PCR||=||polymerase chain reaction|
|PRA||=||plasma renin activity|
|RBF||=||renal blood flow|
|S/renrr||=||SS/Jr/Hsd rat homozygous for salt-resistant renin allele|
|S/renss||=||SS/Jr/Hsd rat homozygous for salt-sensitive renin allele|
|SR/Jr||=||Dahl salt-resistant (rat)|
|SS/Jr||=||Dahl salt-sensitive (rat)|
This work was supported in part by a grant from the National Heart, Lung, and Blood Institute (HL-36279, R.J.R.) and grants from the National Institute of Diabetes and Digestive and Kidney Diseases (H.J.J.). D.E.S. and J.J. were recipients of Predoctoral Fellowships from the American Heart Association, Wisconsin Affiliate. The authors wish to thank Dr John Rapp (Medical College of Ohio, Toledo) for providing livers from SS/Jr and SR/Jr rats for preparation of DNA standards, and Dr Theodore Kurtz (University of California, San Francisco) for providing us with the founder breeding pairs from which the strains described in this article were derived. The authors also wish to thank Mary Kaldunski and Bruce Frohlich for providing excellent technical assistance in these experiments.
- Received May 3, 1996.
- Revision received June 20, 1996.
- Revision received September 4, 1996.
Rapp JP, Wang SM, Dene H. A genetic polymorphism in the renin gene of Dahl rats cosegregates with blood pressure. Science. 1989;243:542-544.
Kurtz TW, Simonet L, Kabra PM, Wolfe S, Chan F, Hjelle BL. Cosegregation of the renin allele of the spontaneously hypertensive rat with an increase in blood pressure. J Clin Invest. 1990;85:1328-1332.
Hilbert P, Lindpaintner K, Beckmann JS, Serikawa T, Soubrier F, Dubay C, Cartwright P, Degouyon B, Julier C, Takahashi S, Vincent M, Ganten D, Georges M, Lathrop GM. Chromosomal mapping of two genetic loci associated with blood pressure regulation in hereditary hypertensive rats. Nature. 1991;353:521-529.
Iwai J, Dahl LK, Knudsen KD. Genetic influence on the renin angiotensin system: low renin activities in hypertension-prone rats. Circ Res. 1973;32:678-684.
Baba K, Mulrow PJ, Franco-Saenz R, Rapp JP. Suppression of adrenal renin in Dahl salt-sensitive rats. Hypertension. 1986;8:1149-1153.
Campbell WG Jr, Gahnem F, Catanzaro DF, James GD, Camargo MJF, Laragh JH, Sealey JE. Plasma and renal prorenin/renin, renin mRNA and blood pressure in Dahl salt-sensitive and salt-resistant rats. Hypertension. 1996;27:1121-1133.
Alam KY, Wang Y, Dene H, Rapp JP. Renin gene nucleotide sequence of coding and regulatory regions in Dahl rats. Clin Exp Hypertens. 1993;15:599-614.
Lande R, Thompson R. Efficiency of marker-assisted selection in the improvement of quantitative traits. Genetics. 1990;124:743-746.
Hospital F, Chevalet C, Mulsant D. Using markers in gene introgression breeding programs. Genetics. 1992;132:1199-1210.
Lewis JL, Russell RS, Warnock DG. Analysis of the genetic contamination of salt-sensitive Dahl/Rapp rats. Hypertension. 1994;24:255-259.
Sealy JE, Laragh JH. How to do a plasma renin assay. Cardiovasc Med. 1977;2:1079-1092.
Zou AP, Drummond HA, Roman RJ. Role of 20-HETE in elevating loop chloride transport in Dahl SS/Jr rats. Hypertension. 1996;27:631-635.
Roman RJ, Osborn JL. Renal function and sodium balance in conscious Dahl S and R rats. Am J Physiol. 1987;252:R833-R841.
Falconer DS. Introduction to Quantitative Genetics. 3rd ed. Essex, UK: Longman Scientific and Technical Corp; 1989.
Anderson S, Jung FF, Ingelfinger JR. Renal renin-angiotensin system in diabetes: function, immunohistochemical, and molecular biological relations. Am J Physiol. 1993;265:F477-F486.
Mackenzie HS, Troy JL, Rennke HG, Brenner BM. TCV116 prevents progressive renal injury in rats with extensive renal mass ablation. J Hypertens. 1994;12:S11-S16.
Roman RJ, Kaldunski ML. Pressure natriuresis and cortical and papillary blood flow in inbred Dahl S rats. Am J Physiol. 1991;261:R595-R602.
Campese VM. Salt sensitivity in hypertension: renal and cardiovascular implications. Hypertension. 1994;23:531-550.
Tobian L, Langer J, Iwai J, Hiller K, Johnson MA, Goossens P. Prevention with thiazide of NaCl-induced hypertension in Dahl S rats. Hypertension. 1979;1:316-323.