Genetic Characterization of the ‘New’ Harlan Sprague Dawley Dahl Salt-Sensitive Rats
Abstract In 1994, it was reported that Dahl salt-sensitive SS/Jr rats supplied by Harlan Sprague Dawley were genetically contaminated and resistant to the pressor effects of a high salt diet. Harlan Sprague Dawley subsequently developed new pedigree expansion and production colonies from their foundation colony to supply new, purportedly inbred, Harlan Sprague Dawley SS/Jr (SHSD). To evaluate the genetic integrity and salt sensitivity of these new SHSD, we performed genotyping (microsatellite DNA markers) and phenotyping (radiotelemetric arterial pressure) of 12 SHSD, 16 “authentic” SS/Jr from the inbred colony of John Rapp (SRapp), 9 Harlan Sprague Dawley salt-resistant SR/Jr (RHSD), and (genotyping only) 6 known “contaminated” Harlan Sprague Dawley Dahl SS/Jr (S*). In the genotyping studies, 20 of 22 markers revealed polymorphisms between SRapp and S* and 18 were polymorphic between SRapp and RRapp, but none of the 22 markers revealed polymorphisms between SRapp and the new SHSD. The phenotyping studies showed that during an ultra–low salt diet, mean arterial pressure was higher (P<.05) in both authentic SRapp (129±2 mm Hg; mean±SE) and new SHSD (120±2 mm Hg) than in RHSD (93±1 mm Hg). A high salt diet increased mean arterial pressure in every SHSD and SRapp. Increases in mean arterial pressure after 4 weeks of a high salt diet were significantly (P<.05) greater in authentic SRapp (+51±3 mm Hg) than in new SHSD (+39±3 mm Hg). In addition, salt-induced mortality was significantly greater in SRapp (62.5%) than SHSD (8.3%) after 8 weeks (P<.01). SHSD were genotypically indistinguishable from SRapp, had an elevated arterial pressure on a low salt diet, and had a pressor response to salt. Thus, the new SHSD supplied to us had several characteristics of inbred Dahl SS/Jr and did not have evidence of the previously detected genetic contamination. However, phenotypic characteristics such as body weight, salt-induced hypertension, and mortality were significantly different in SHSD compared with SRapp. This may reflect genetic differences between these two strains or differences in environmental factors and suggests that the SHSD and SRapp may now constitute distinct substrains of Dahl SS/Jr.
In 1985, Rapp and Dene1 2 reported the development and characteristics of inbred strains of Dahl salt-sensitive and salt-resistant rats. The inbred SS/Jr is exquisitely and consistently sensitive to the pressor effects of a high salt diet, whereas the SR/Jr is insensitive. These inbred rat strains are valuable animal models for the study of salt-induced hypertension. To ensure the ready availability of these strains, Rapp provided inbred SS/Jr and SR/Jr to Harlan Sprague Dawley, Inc (Indianapolis, Ind) in 1986, which then began to provide investigators with purportedly inbred SS/Jr and SR/Jr. Concurrently, Rapp and colleagues perpetuated their own colonies of each strain.
In 1994, St. Lezin et al3 reported that SS/Jr supplied to them by HSD between October 1992 and July 1993 were genetically contaminated and not consistently responsive to the pressor effects of a high salt diet. Lewis et al4 subsequently confirmed this genetic contamination.
HSD uses a breeding plan that includes three geographically separate colonies: a foundation colony, a pedigree expansion colony, and a production colony. The foundation colony is self-propagating. Offspring of the foundation colony become breeders for the pedigree expansion colony. Offspring of the pedigree expansion colony become breeders for the production colony, whose offspring are distributed to investigators.4 The analysis by Lewis et al4 indicated that both the pedigree expansion and production colonies of HSD SS/Jr were genetically contaminated. However, screening 10 breeder pairs from the foundation colony with a small number of markers failed to reveal any genetic contamination. They concluded that the HSD SS/Jr foundation colony was not genetically contaminated.4 Accordingly, HSD reported in 1994 that they had eliminated the pedigree expansion and production colonies and were reestablishing new such colonies from the purportedly inbred foundation colony.5
We report here a genotypic and phenotypic analysis of SS/Jr from these HSD colonies (SHSD). For comparison, we also performed genotyping and phenotyping on SS/Jr from Rapp’s colony (SRapp) and SR/Jr from HSD (RHSD). For additional comparison, we performed genotyping on DNA from genetically contaminated SS/Jr from the “old” HSD pedigree expansion and production colonies (S*).
Experimental Animals and Diets
We performed phenotyping and genotyping on several rat groups. First, we studied a total of 12 SHSD (8 females and 4 males). These rats were sent to us in two shipments. Shipment 1 consisted of 7 SHSD (4 females and 3 males) born on July 22, 1994, from the new HSD production colony and was received by us on August 24, 1994, at 4 weeks of age. Shipment 2 consisted of 5 SHSD (4 females and 1 male) from the foundation colony, born on September 23, 1994, and was received by us on October 24, 1994, at 4 weeks of age. Second, we studied 9 RHSD (5 females and 4 males). These rats were born and received at the same times as the first and second shipments of SHSD. Third, we studied 16 female SRapp from the Medical College of Ohio colony (Toledo). Only female SRapp were available to us. These rats were sent to us in two shipments. The first group of 8 rats was born on December 4, 1994, and was received on January 20, 1995, at 6 weeks of age. The second group of 8 SRapp was born on March 24, 1995, and was received on May 3, 1995, at 6 weeks of age. We also performed genotyping on DNA supplied to us by James L. Lewis (University of Alabama at Birmingham) from 6 S* known to contain contaminated alleles4 and on DNA from 3 SR/Jr from Rapp’s colony (RRapp).
On arrival at the University of Iowa Animal Care Facility, rats were fed an ultra–low salt diet (0.13% NaCl and 1.06% KCl; ICN Nutritional Biochemicals) until starting a high salt diet (8.0% NaCl and 1.06% KCl; ICN Nutritional Biochemicals). Rats were allowed tap water ad libitum and housed in an automatic temperature-controlled room (22°C) with a 12-hour light (6 am to 6 pm) and dark (6 pm to 6 am) cycle. Animal care and all procedures were approved by the University of Iowa and Iowa City Veterans Affairs Animal Research Committees.
Measurements of Arterial Pressure
At 73 to 81 days of age (approximately 11 weeks old), when the rats were sufficiently large for surgery, each rat was weighed and anesthetized with methohexital, sodium salt (Brevital, 50 mg/kg IP; Eli Lilly). The caudal artery was cannulated and a 200 μL blood sample collected in a heparinized syringe for DNA analysis. After the abdominal aorta was exposed, a radiotelemetry transducer (model TA11PA-C40, Data Sciences, Inc) was inserted and glued into the vessel. After implantation, the rat was given postoperative antibiotics (LA-200, 10 U IM; Pfizer) and allowed to recover for 3 weeks in an individual cage with ample access to tap water and the ultra–low salt diet. Measurements of systolic pressure, diastolic pressure, and MAP were made for 10 seconds every 5 minutes in each rat. These radiotelemetry measurements were stored for later analysis with Dataquest IV software (Version 2.2, Data Sciences, Inc). When the rats reached an age of 100 days (approximately 14 weeks), they were switched to the high salt diet, thus allowing adequate time to ensure recovery from surgery and obtain sufficient arterial pressure data during the ultra–low salt diet. Radiotelemetry measurements were continued until the rats were killed or died. Body weight, total kidney weight, and dry ventricular heart weight were measured postmortem.
Radiotelemetry data were averaged over 3.5-day intervals and given as absolute values. In addition, arterial pressure was averaged over the last 2 weeks of the ultra–low salt diet, and change from this baseline was calculated after 4 and 8 weeks of the high salt diet. For continuous variables (arterial pressure and weights), statistical analysis was performed with ANOVA and Duncan’s multiple range tests. Mortality differences were assessed statistically by Fisher’s exact test. A comparison value of P<.05 was considered statistically significant.
Rat genomic DNA was purified from whole blood with a commercially available kit, according to the manufacturer’s protocol (QIAamp Blood Kit, QIAGEN). Approximately 20 μg DNA was usually isolated from 200 μL whole blood. DNA was analyzed by PCR with oligonucleotide primers (MapPairs) rat markers purchased from Research Genetics. These markers have been described by Jacob et al6 and Serikawa et al7 and amplify regions within the rat genome that contain short tandem repeat sequences.
The rat DNA was initially genotyped with eight microsatellite rat markers previously reported by St. Lezin et al3 and Lewis et al4 to show genetic contamination in S*. We subsequently extended the genotyping studies by including 14 more markers for a total of 22 markers found on at least 14 different chromosomes. Amplification of short tandem repeat markers was done in final reaction volumes of 8.35 μL, containing 1.25 μL 10× PCR buffer (100 mmol/L Tris-HCl [pH 8.8], 500 mmol/L KCl, 15 mmol/L MgCl2, 0.01% gelatin [wt/vol]), 200 μmol/L of each dNTP, 2.5 pmol of each primer, 0.25 U Taq DNA polymerase (Boehringer Mannheim), and 40 ng DNA. Samples were denatured at 94°C for 5 minutes and subjected to 35 cycles of amplification at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. The amplified PCR products were mixed with formamide loading buffer (90% formamide, 10 mmol/L Tris-HCl [pH 8.0], 1 mmol/L EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol), denatured for 5 minutes at 94°C, and run on 6% denaturing polyacrylamide gels containing 7.7 mol/L urea (30×40×0.02 cm) for 2 to 4 hours at 60 W. The PCR products were visualized by silver staining.8
Fig 1⇓ shows an example of the genetic analyses performed on DNA from SHSD, SRapp, S*, and RHSD using two short tandem repeat markers. Table 1⇓ summarizes the genotyping results for all 22 markers in these rats. There are three important points. First, 20 of the 22 markers demonstrated polymorphisms between SRapp and S*. Thus, we identify another 12 loci that demonstrate polymorphisms between SRapp and S*. Second, we could detect no polymorphisms using the 22 markers between any of the SRapp and SHSD tested. Third, 18 of the 22 markers were polymorphic between DNA from SRapp and RRapp. We detected no polymorphisms between DNA from RHSD and RRapp (data not shown).
During the ultra–low salt diet, arterial pressure was higher (P<.05) in both groups of salt-sensitive rats than in the RHSD (Table 2⇓, Fig 2⇓). In addition, SRapp had a significantly higher (P<.05) arterial pressure than SHSD. Since the SRapp (n=16) were all female, the arterial pressures for only female SHSD and RHSD were also analyzed to determine whether there were any sex effects on the observed results. The top panel of Fig 2⇓ gives MAP versus weeks on diet for all rats studied, and the bottom panel shows the data for female rats only. Although the numbers of rats are small, it was observed that the male rats (n=4) shifted the RHSD curve to a higher MAP, and the male rats (n=4) moved the SHSD curve to a lower MAP (Fig 2⇓, bottom).
Table 2⇑ lists systolic and diastolic pressures and MAP for SRapp, SHSD, and RHSD on the ultra–low salt diet and after 4 weeks of the high salt diet. Arterial pressure did not significantly increase in RHSD, even after 8 weeks of the high salt diet. In contrast, every SRapp and SHSD had an increase in arterial pressure. Specifically, after 4 weeks of high salt, MAP rose by at least 22 mm Hg in each SHSD and by at least 29 mm Hg in each SRapp. Interestingly, the salt-induced increases in arterial pressure were significantly greater (P<.05) in SRapp than in new SHSD (+51±3 and +39±3 mm Hg, respectively; P<.05) after 4 weeks of the high salt diet. After 8 weeks of the high salt diet, the difference in blood pressure between the surviving SRapp (n=6) and SHSD (n=7) was not significant (+64±4 and +59±4 mm Hg, respectively). Arterial pressure during the ultra–low salt diet and the response to the high salt diet did not differ significantly between SHSD from the HSD foundation colony and the new production colony (data not shown). Likewise, arterial pressure and the response to salt did not differ significantly between the two shipments of SRapp (data not shown).
Body Characteristics and Mortality
Body weights were significantly less in the RHSD compared with female SHSD and SRapp at the time of surgical implantation of the transducer (Fig 3⇓). The average body weights of both male and female rats did not differ significantly in the three groups during low salt (Table 3⇓). During high salt feeding, the SRapp failed to gain further weight, and at death, these rats weighed significantly less than RHSD or new SHSD. Both RHSD and SHSD gained weight after 8 weeks on the high salt diet. The SRapp showed an increased susceptibility to salt-induced mortality (Fig 4⇓). After 8 weeks of high salt, 10 of 16 SRapp died, whereas only 1 female rat of 12 SHSD had died. Postmortem kidney and ventricular heart weights were significantly greater (P<.05) in SRapp and SHSD than in RHSD (Table 3⇓). However, kidney and ventricular heart weights did not differ between SRapp and SHSD.
In this study, SHSD from the HSD foundation and new production colonies were (1) genotypically indistinguishable from SRapp using 22 microsatellite DNA markers, (2) had elevated arterial pressure on an ultra–low salt diet compared with RHSD, and (3) consistently displayed a salt-induced increase in arterial pressure. However, there were phenotypic differences between SRapp and SHSD. SHSD had a lower arterial pressure than SRapp during the ultra–low salt diet; they were less sensitive than SRapp to the pressor effects of a high salt diet; they weighed more after the high salt diet; and they survived longer than the SRapp. This suggests that SHSD and SRapp may now constitute distinct substrains of SS/Jr and should not be compared within or between studies.
Are SHSD Genetically Identical to SRapp?
We found no evidence to indicate a genetic difference between SHSD and SRapp. Given the limited number of markers used (n=22), it is still possible that there are allelic differences between SRapp and new SHSD. We are not certain that existing statistical methods permit us to make a valid calculation of the probability of genetic differences between these two purportedly inbred groups of rats using our findings of no allelic differences with 22 markers. Therefore, we have not attempted to calculate such odds and cannot be absolutely certain that the SHSD and SRapp are genetically identical. In contrast, we can conclude that the SHSD sent to us are very different from the previously reported genetically contaminated S*. Our results underscore the extent of this previously reported contamination. Of the 14 new markers we selected to represent a variety of loci in many chromosomes, 12 exhibited polymorphisms between S* and SRapp. These findings provide further evidence that the contamination was not subtle. We did not find evidence of this previous contamination in the SHSD supplied to us.
What Is the Basis for the Phenotypic Differences Between SHSD and SRapp?
The new SHSD displayed three phenotypic characteristics of inbred SS/Jr: (1) arterial pressure was elevated during a low salt diet; (2) a high salt diet induced a consistent increase in arterial pressure; and (3) cardiac hypertrophy was present. Thus, these new SHSD exhibited several features of inbred SS/Jr. However, as described above, there were demonstrable phenotypic differences between SRapp and SHSD. We considered several possible explanations for these differences.
The first potential explanation is a sex effect. Male SS/Jr are known to develop more severe hypertension on a high salt diet than female SS/Jr.9 All our SRapp were female, whereas 4 of the 12 SHSD were male. However, when we compared only female SHSD and SRapp, the differences in arterial pressure were still present.
A second possible explanation for the phenotypic differences between SHSD and SRapp is an environmental effect. We made every effort to ensure an identical environment for these rats at our institution. The diets, the age at transducer implantation, the ambient temperature, and the age at which the high salt diet was started were similar for all SHSD, RHSD, and SRapp. However, the SHSD and SRapp were born and raised for the first 4 to 6 weeks of life at different institutions. Both Rapp rats and HSD rats were maintained on the same diet before being sent to us. Differences in maternal influences may have contributed to the phenotypic differences we observed. In addition, the SHSD and SRapp were studied at different times of the year at our institution. However, arterial pressure results were similar in the two shipments of SHSD and also in the two shipments of SRapp, despite the fact that the shipments were studied at different times. Thus, a seasonal effect appears unlikely.
A third possibility is that the phenotypic differences between SHSD and SRapp reflect genetic differences not detected by the use of only 22 markers. If unidentified genetic differences explain the phenotypic differences, there are two possible sources of genetic variation between SHSD and SRapp: genetic contamination and genetic drift. We can exclude the presence of the previously reported genetic contamination in the SHSD, but we cannot completely exclude a new type or source of contamination. However, it is also theoretically possible that a genetic difference between the SHSD and SRapp might reflect genetic drift or spontaneous mutations in two inbred strains derived from the same colony, outbred separately for almost 10 years.
Our results prompt two observations regarding SS/Jr. First, even though there are no detectable genotypic differences between SHSD and SRapp using a modest number of markers, the rats from these two sources are phenotypically dissimilar. Thus, it is important for investigators to clearly state the origin of the SS/Jr used in their work because the SHSD and SRapp may now constitute separate substrains of the SS/Jr strain. Second, in assessing the magnitude of salt-induced increases in arterial pressure in SS/Jr, one cannot rely only on a single measurement of arterial pressure in salt-sensitive and salt-resistant rats during a high salt diet because some of the difference in pressure may occur even in the absence of a high salt diet. To accurately evaluate the magnitude of salt-sensitive hypertension, one must either compare pressures in age-matched salt-sensitive rats fed either a low or high salt diet or (as in our study) measure arterial pressure before and during a high salt diet. Indeed, we submit that only by measuring arterial pressure before and during a high salt diet in each rat could one detect an occasional rat that fails to exhibit the characteristic salt-sensitive arterial pressure phenotype. Such monitoring may be important in detecting early signs of genetic contamination and could prompt a genotyping survey. Serial measurement of arterial pressure was a key element in the initial detection of the genetic contamination in SS/Jr from HSD.3
In summary, SHSD supplied to us were genotypically indistinguishable from SRapp using 22 markers and displayed arterial pressure responses to a high salt diet that are characteristic of inbred SS/Jr. However, despite the apparent lack of genotypic differences, SHSD when compared with SRapp displayed lower arterial pressures during a low salt diet, had less salt-induced hypertension, and had lower mortality on a high salt diet. This suggests that SHSD and SRapp may now constitute distinct substrains of SS/Jr. Further studies are required to elucidate the mechanism or mechanisms underlying the phenotypic differences between these colonies of SS/Jr.
Selected Abbreviations and Acronyms
|HSD||=||Harlan Sprague Dawley|
|MAP||=||mean arterial pressure|
|PCR||=||polymerase chain reaction|
|RHSD||=||Dahl SR/Jr rats from Harlan Sprague Dawley colonies|
|RRapp||=||Dahl SR/Jr rats from Rapp’s colony|
|S*||=||SS/Jr rats from genetically contaminated Harlan Sprague Dawley colonies|
|SHSD||=||Dahl SS/Jr rats from Harlan Sprague Dawley colonies|
|SR/Jr||=||Dahl salt-resistant rat(s)|
|SRapp||=||Dahl SS/Jr rats from Rapp’s colony|
|SS/Jr||=||Dahl salt-sensitive rat(s)|
This research was supported by grants HL-44546 and HL-43514 from the National Heart, Lung, and Blood Institute and by funds from the Department of Veterans Affairs. William G. Haynes is the recipient of a Wellcome Trust Advanced Training Fellowship (No. 04215/114). The authors wish to acknowledge John S. Beck for his technical assistance in DNA analysis and Nancy Davin for her secretarial assistance. The authors also wish to thank (1) Dr John Rapp of Toledo, Ohio, for the generous supply of 16 inbred SS/Jr from his colony, (2) Dr James Lewis of Birmingham, Ala, for providing DNA from genetically contaminated Dahl SS/Jr (S*) for genetic analysis, and (3) Harlan Sprague Dawley in Indianapolis, Ind, for the use of SS/Jr from their foundation and new production colonies. Finally, our sincere gratitude to Dr Val Sheffield for the use of his laboratory and for his scientific advice.
Reprint requests to Dr Allyn L. Mark, 220 MAB, College of Medicine, University of Iowa, Iowa City, IA 52242-1101. E-mail firstname.lastname@example.org.
↵1 These three authors contributed equally to this work.
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