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Physiol. Genomics 31: 228-235, 2007. First published June 12, 2007; doi:10.1152/physiolgenomics.00280.2006
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Received 18 December 2006; accepted in final form 11 June 2007.
Physiological Genomics 31:228-235 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Multiple blood pressure loci on rat chromosome 13 attenuate development of hypertension in the Dahl S hypertensive rat

Carol Moreno 1,2, Mary L. Kaldunski 1, Tao Wang 2,3, Richard J. Roman 1,4, Andrew S. Greene 1,5, Jozef Lazar 1,6, Howard J. Jacob 1,2,7 and Allen W. Cowley, Jr. 1

1 Department of Physiology, Milwaukee, Wisconsin
2 Human and Molecular Genetics Center, Milwaukee, Wisconsin
3 Division of Biostatistics, Milwaukee, Wisconsin
4 Kidney Disease Center, Milwaukee, Wisconsin
5 Biotechnology and Bioengineering Center, Milwaukee, Wisconsin
6 Department of Dermatology, Milwaukee, Wisconsin
7 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have indicated that substitution of chromosome 13 of the salt-resistant Brown Norway BN/SsNHsdMcwi (BN) rat into the genomic background of the Dahl salt-sensitive SS/JrHsdMcwi (SS) rat attenuates the development of salt-sensitive hypertension and renal damage. To identify the regions within chromosome 13 that attenuate the development of hypertension during a high-salt diet in the SS rat, we phenotyped a series of overlapping congenic lines covering chromosome 13, generated from an intercross between the consomic SS-13BN rat and the SS rat. Blood pressure was determined in chronically catheterized rats after 2 wk of high-salt diet (8% NaCl) together with microalbuminuria as an index of renal damage. Four discrete regions were identified, ranging in size from 4.5 to 16 Mbp, each of which independently provided significant protection from hypertension during high-salt diet, reducing blood pressure by 20–29 mmHg. Protection was more robust in female than male rats in some of the congenic strains, suggesting a sex interaction with some of the genes determining blood pressure during high-salt diet. Among the 23 congenic strains, several regions overlapped. When three of the "protective" regions were combined onto one broad congenic strain, no summation effect was seen, obtaining the same decrease in blood pressure as with each one independently. We conclude from these studies that there are four regions within chromosome 13 containing genes that interact epistatically and influence arterial pressure.

quantitative trait loci; congenic strain; consomic strain; genetic epistasis; linkage


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ESSENTIAL HYPERTENSION is a multifactorial disease determined by the interaction of genetic and environmental factors (22, 23), making the identification of the causal genes a daunting task. In humans, genome-wide scans have identified many blood pressure quantitative trait loci (QTLs) (19, 24, 29, 37, 38), but confirmation among different populations has been difficult.

The rat is a useful model for the study of complex diseases such as hypertension because multiple hypertensive strains each carry some phenotypic similarities to the human disease (21, 42–44). The Dahl S rat is a commonly used model for the study of salt-sensitive hypertension and has remarkable similarities with phenotypic traits seen in many African Americans with hypertension, including low renin, salt sensitivity, hyperinsulinemia, and early end-stage renal disease (3, 13).

Genetic studies have demonstrated the polygenic nature of salt-sensitive hypertension in the SS rat, and a number of blood pressure QTLs have been identified in different genomic regions, including chromosome 13 (6, 34). Utilization of chromosomal substitution approaches have recently provided even stronger evidence of this, showing that substitution of different chromosomes from the Brown Norway BN/SsNHsdMcwi (BN) strain into the Dahl SS/JrHsdMcwi (SS) strain attenuates the development of salt-induced hypertension (www.pga.mcw.edu). Of particular interest to our laboratory has been consomic strain SS-13BN, which has chromosome 13 from the BN introgressed into the isogenic genetic background of the SS strain. These rats exhibit a marked reduction of salt-induced hypertension and proteinuria (6). However, the specific regions of chromosome 13 that harbor the genes related to salt-sensitive hypertension in the SS rat are unknown (15, 47). Different regions of chromosome 13 have been previously linked to blood pressure. Since Rapp et al. (36) first found linkage between the renin locus and blood pressure, many studies have studied the role of the renin gene in the development of hypertension in the Dahl S rat. Studies by St. Lezin et al. (40) and Jiang et al. (15) found that the renin gene participates in hypertension in the Dahl S rat, but Zhang et al. (47) and DiPaola et al. (8) failed to demonstrate any effect on blood pressure sensitivity when the renin allele from Dahl R rats was transferred to the Dahl S rat. This group found instead a blood pressure QTL in a region above renin. Other groups have found broad QTLs for blood pressure in other regions of chromosome 13 (17, 39, 47).

To better define the regions of chromosome 13 involved in blood pressure regulation during a high-salt diet, overlapping congenic strains were derived from a cross between SS-13BN consomic and SS rats and phenotyped for arterial pressure and proteinuria. The congenic strains also enabled determination of sexual dimorphism and interaction among the different genomic regions in chromosome 13 in the control of blood pressure.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin (MCW). Experiments were performed on male and female rats of SS, SS-13BN consomic, and congenic strains. The SS colony at MCW was rederived from SS/JrHsd rats originally obtained as a congenic control strain from Dr. T. Kurtz (Univ. of California-San Francisco) in 1991. This colony has been maintained at MCW by strict brother-sister mating for >46 generations. Chromosome 13 from the salt-resistant BN rat, raised at MCW with strict brother-sister mating for >30 generations, was substituted for chromosome 13 of the salt-sensitive SS rat through selective breeding as described previously (6). The SS-13BN consomic strain has since been maintained by brother-sister mating for 20 generations. The rats were raised on a purified AIN76 diet containing 0.4% NaCl (Dyets, Allentown, PA). Experimental rats were switched to an 8.0% NaCl diet at 10 wk of age, 2 wk before surgical implantation of catheters.

Generation of congenic population.
Consomic SS-13BN rats were crossed to SS females, and the first-generation progeny were intercrossed to create the second generation (F2) that captured different regions of chromosome 13 because of recombination events. These F2 rats were backcrossed to SS females for further development of the congenic strains for chromosome 13. Progenitors of each generation were selected by marker-assisted breeding, whereby rats were genotyped with 44 simple sequence length polymorphism (SSLP) genetic markers for chromosome 13 (~1 marker/cM), genetic recombination was evaluated, and the selection of breeders was completed to further generate the congenic strains.

A total of 26 congenic strains were developed covering the entire length of chromosome 13, of which 23 strains were phenotyped. As a positive control, an SS strain was rederived from three overlapping congenic strains, in which all alleles for chromosome 13 were fixed to the SS strain. This strain showed no differences in blood pressure or proteinuria compared with the SS parental inbred strain in response to a high-salt diet, and it was defined and used in the present study as the SS control strain for phenotyping.

Fluorescent genotyping.
A portion of an ear from each animal was collected and incubated overnight in a lysis buffer containing 100 µg/ml of proteinase K. DNA was precipitated with isopropyl alcohol and pelleted by centrifugation at 12,000 g for 10 min. The pellet was washed with 75% ethanol, air dried, and resuspended in Tris-EDTA (TE) buffer, pH 7.5. The concentration of DNA in the samples was determined by measurement of 260-to-280 nm absorption ratios (Beckman DU640). The rats were genotyped with fluorescent M13-labeled primers, as described previously (28). In brief, genomic DNA (25 ng) was amplified by PCR in a 6-µl reaction containing 150 nmol/l of primer-dye conjugate, 10 nmol/l Trizma base, 1.5 mmol/l MgCl2, 50 mmol/l KCl (pH 8.3), 200 µmol/l dNTP, and 1 U/µl Taq DNA polymerase. A "touchdown" PCR reaction was performed, as described previously (9). Samples were run on an ABI 377-96 DNA sequencer for 2 h, and data were analyzed with ABI Genescan and Genotyper. Data were stored in our colony management database.

Chronic phenotyping protocol.
Experiments were performed in control SS and SS-13BN and congenic rats (n = 10–15 for each sex of each strain). Animals were anesthetized with an intramuscular injection of ketamine (40 mg/kg), xylazine (2.5 mg/kg), and acepromazine (0.6 mg/kg), and a microrenathane catheter was implanted in the left femoral artery for measurement of arterial blood pressure. The catheter was tunneled to the back of the neck and brought out through a stainless steel spring and attached to a swivel device. After surgery, the rats were given 10 mg/kg Baytril to prevent infections and 1 mg/kg Buprenex to prevent pain. The rats were placed in stainless steel metabolic cages, and food and water were provided ad libitum. After a 2-day recovery period, heart rate and systolic, diastolic, and mean arterial pressure (MAP) were recorded between 9 AM and 1 PM on 3 consecutive days and averaged. A urine sample was collected for 24 h during the second day of the blood pressure recording for measurement of urine volume, sodium, and potassium and total protein and albumin excretion.

Statistics.
Mean ± SE values are presented, and significance of differences in corresponding values between SS, SS-13BN, and congenic rats was determined by analysis of variance followed by a Dunnett's post hoc test (SigmaStat version 2.03, SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 1 shows a schematic representation of the 23 overlapping congenic strains from the SS-13BN consomic rat phenotyped in the present study. The size of the congenic regions ranged from 4.6 Mbp (congenic strain 9) to 50 Mbp (congenic strain 4). Some of the congenic strains covered large regions of the chromosome, enabling evaluation of possible gene-gene interactions. Other strains covered small regions of chromosome 13, representing a possible step toward positional cloning of the genes involved in the development of hypertension.


Figure 1
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  		Fig. 1. Schematic representation of the overlapping congenic strains developed, in which segments of various lengths of chromosome 13 from the Brown Norway rat (black bars) were introgressed in the genetic background of the SS rat, by marker-assisted breeding. Delimitation of the congenic regions is defined by the first outward SS marker allele. Thin bars represent chromosome crossover regions.

 
As expected, the SS strain developed hypertension after 2 wk of high-salt diet (181 ± 2.9 mmHg in male and 177 ± 5 mmHg in female rats). Substitution of the entire chromosome 13 from the BN rat in the SS-13BN consomic rat greatly attenuated the development of hypertension after high-salt diet in both male and female rats (142 ± 1.3 and 137 ± 1.9 mmHg in male and female consomic rats, respectively; P < 0.05 from SS), validating our previous observations (6).

The average MAP values for control and 23 congenic strains are detailed in Table 1. In male rats there were six congenic strains (lines 1, 2, 5, 10, 17, and 18) in which MAP was significantly lower than the SS strain after 2 wk on high-salt diet (Fig. 2A). In contrast, in the female congenics a total of 11 strains exhibited lower MAP than the SS strain (Fig. 2B). Although there were no sex differences in blood pressure in the control SS and SS-13BN strains, there were some congenic strains that exhibited significant differences between male and female rats. In some congenic strains the values of blood pressure were higher in males than in females, suggesting an interaction between sex and genetic determinants of blood pressure salt sensitivity in the congenic regions. The specific congenic strains that showed sexual dimorphism in the development of hypertension were strains 1, 4, 7–11, 13, 16, 19, 22, and 26 (see Table 1).


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  		Table 1. Blood pressure in male and female congenic and parental strains after 2 wk of high-salt diet

 

Figure 2
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  		Fig. 2. A: mean arterial pressure values after 2 wk of high salt for male SS, SS-13BN consomic, and overlapping congenic strains of chromosome 13. *P < 0.05 vs. SS strain. B: mean arterial pressure values after 2 wk of high salt for female SS, SS-13BN consomic, and overlapping congenic strains of chromosome 13. *P < 0.05 vs. SS strain.

 
Four discrete nonoverlapping regions were identified in female congenic rats that had an effect reducing MAP. These are represented by congenic strains 1 (12.4 Mbp), 5 (14 Mbp), 9 (4.5 Mbp), and 26 (16 Mbp). Each of these congenic strains independently provided protection from salt-induced hypertension and reduced blood pressure by 20–29 mmHg compared with the control SS female rats. Figure 3 represents a schematic of the different congenic strains showing protection from hypertension. Two of the four congenics/regions identified in the female congenic rats were also evident in the male congenic population (congenic lines 1 and 5). Nevertheless, the congenic strains harboring the renin gene (congenic strains 7, 8, and 9), and congenic line 26 showed no differences in blood pressure from SS in the male rats, although they did exhibit protection from hypertension in the female rats.


Figure 3
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  		Fig. 3. Schematic representation of the overlapping congenic strains for chromosome 13, with bars representing the BN allele regions. Four regions for blood pressure were identified in females, represented by congenic strains 1, 5, 9, and 26. Black bars indicate congenic strains that showed no reduction in blood pressure; gray bars represent congenic strains in which protection from salt-induced hypertension was observed in both males and females (striped bars) or was sex specific (solid bars).

 
When two or three of the "protective" regions were combined on one broad congenic strain of female rats (Fig. 3), no summation effect was observed in congenic strains 4, 7, and 11, which obtained a similar decrease in blood pressure as with each smaller congenic strain independently (Table 1). This suggests that there is an epistatic interaction among these regions in the control of blood pressure.

Epistasis was more dramatically illustrated by the fact that the decrease in blood pressure achieved by the some of the smaller congenic strains was comparable to that observed in the consomic strain, which carries the entire chromosome (and therefore all 4 regions) from the BN rat. The sum of effects of each individual strain was much greater than that obtained when the four regions were combined, and even greater than the consomic rat (Fig. 4). These observations indicate the existence of epistatic interactions between the different genetic regions of chromosome 13 that affect blood pressure. There are a number of possibilities that can explain this observation. Blood pressure regulation is a complex, multifactorial trait that is regulated by a variety of physiological systems. Many pathways have been identified that can alter blood pressure regulation, in particular those impacting the kidney. In the SS rat, alterations in renal, vascular, nervous, and cardiac function have been described, each of which could independently impact blood pressure. Connections between each of the congenic regions and these independent intermediate effectors of blood pressure would provide a mechanism by which apparent epistatic interactions could occur. Epistasis occurring through a direct gene-gene interaction at two or more loci has been observed in a number of studies of complex phenotypes and has been proposed to be a common feature in human disease (26). Given the extent of the congenic regions in this study and the number of genes in each of the regions, such interactions are likely. However, it is unlikely that these direct gene-gene interactions are responsible for the observed phenomenon since there is certainly not one final common biochemical pathway for blood pressure reduction. Instead, it is most likely that, given the central role of the kidney in blood pressure regulation, the observed epistatic effect is determined by genes that commonly alter intrinsic or extrinsic structure-function relationships in the kidney's ability to excrete sodium and water. Appendix 1 in the supplemental material for this article lists the genes located within the four regions identified in our study.1 We used GeneInfo, a literature data mining tool that identifies genes associated with specific disease, to search PubMed for genes related to blood pressure regulation and/or hypertension (48), and we found that of the 361 genes in the region, only a handful of genes have been related to blood pressure or hypertension (none in congenic line 1; Cxcr4 and Ctse in congenic line 5; Ren1, Adora1, Adipor1, and Rnpep in congenic line 9; and Pl2g4a, Ptgs2, Fmo3, and Sele in congenic line 26). It is notable that within the strain 1 congenic region there are no genes that are known to influence arterial blood pressure. This is consistent with our understanding that the complex trait of arterial blood pressure is indeed importantly influenced by a variety of biochemical and cellular signaling pathways that can ultimately influence vascular, cardiac, neural, endocrine, and renal functions. The absence of known connections of any of the genes within this congenic to arterial pressure emphasizes that there is considerably more to learn about the complex biochemical and cellular regulation of cardiovascular function.


Figure 4
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  		Fig. 4. Impact on salt-induced hypertension of the 4 congenic regions on chromosome 13 of the BN rat introgressed into the background of the SS. *P < 0.05 vs. SS.

 
Albumin excretion was also measured in parental and congenic strains after 2 wk of high-salt diet (Table 2). Higher albumin excretion was observed in the SS compared with the SS-13BN consomic in both male and female rats. None of the congenic strains showed a significant reduction in albumin excretion compared with the SS rat, despite the fact that blood pressure was reduced in many of the congenic strains. A clear sex effect in this parameter was observed, however, because albumin excretion was much higher in males than in females in the parental SS and in 17 of the 23 congenic strains (Table 2), but not in the SS-13BN consomic strain, where albumin excretion in both males and females was low.


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  		Table 2. Microalbuminuria in male and female congenic and parental strains after 2 wk of high-salt diet

 
We also measured sodium excretion to assure ourselves that all rats had similar food intake, and therefore similar salt loading. There were no differences among the strains, within sex, in this parameter.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Previous studies indicate that transfer of chromosome 13 from BN to SS rats greatly attenuates the development of hypertension and renal disease (6), but the genomic regions involved remain to be determined. In the present study, we developed and phenotyped a series of 23 overlapping congenic strains across the entire length of chromosome 13, to narrow the region containing the genes that protect against the development of hypertension during a high-salt diet.

The four narrow regions on chromosome 13 identified in our study, represented by congenic strains 1, 5, 9, and 26, each show an effect on blood pressure salt sensitivity and represent genomic regions that range in physical size from 4.6 to 16 Mbp. The renin region, represented by strain 9 (4.6 Mbp), has been identified in previous studies as being related to blood pressure (20, 33, 36). Although renin is an obvious candidate for this region because of the important role that the renin-angiotensin system plays in the regulation of blood pressure, the role of the renin gene in the development of hypertension in the Dahl S rat has been an issue of controversy. Since Rapp et al. (36) initially reported linkage of the renin gene to blood pressure in the Dahl S rat, the renin region has been linked to the development of hypertension in other hypertensive strains (10, 33) and some human populations (1, 12), although there is no evidence of functional sequence variants within the renin gene itself (8, 15, 40, 47). The present study implicates a role for the region around the renin gene in the development of hypertension in female rats, but this region was not protective in males.

The genomic region identified in the p end of the chromosome (congenic strain 1) and the chromosomal region above renin (congenic strain 5) have not previously been reported to be related to blood pressure in any rat strain. The region of chromosome 13 above renin corresponds, by comparative mapping, to a region in human chromosome 2 that has been linked to systolic blood pressure in the Quebec Family Study (38) and in a sib-pair study as part of the Family Blood Pressure Program (19). Also, a homologous region corresponding to congenic strain 1 has been found to be associated with blood pressure in two different human study populations (18, 32).

With respect to the region found toward the q end of the chromosome, represented by congenic strain 26, there have been previous studies in which QTLs for blood pressure spanned this genomic area. For instance, in the SS/Rapp rat Zhang et al. (47) found that when this region of chromosome 13 from the Dahl R rat was introgressed onto the genomic background of the SS/Rapp rat, there was a 24-mmHg attenuation in blood pressure sensitivity to a high-salt diet. Also, a QTL for blood pressure was found in this region in a cross between the Lyon normotensive and the Lyon hypertensive strains (10), although the peak of this QTL was located at the renin gene. QTLs covering the region have also been found in crosses from SHR and WKY rats (39) and SHR and diabetic BB rats (17). It is interesting and relevant that the region of congenic strain 26 on chromosome 13 corresponds to a genomic region in human chromosome 1 that has been found to harbor several blood pressure-related QTLs in French Canadian (14) and African American populations (16).

Linkage analysis has very little power to detect QTLs adjacent to each other within a genomic region. One of the most important findings of the present study is that the congenic strains have enabled the clear detection of four separate genomic regions involved in blood pressure during a high-salt diet on chromosome 13 of the SS rat. While the phenotypic effects of the four genomic regions defined by congenic rat strains 1, 5, 9, and 26 were similar (in the female population), the genes responsible are obviously different. Moreover, the sum of the protective effects of each of the discrete regions of chromosome 13 (–116 mmHg) was greater than the protective effects of the whole consomic SS-13BN strain (–40 mmHg), as seen in the female rats (Fig. 4). This suggests the existence of epistatic interactions between these genomic regions in the regulation of blood pressure. Gene interactions in the regulation of blood pressure were initially suggested by Deng and Rapp (7) and later confirmed in several other studies, mainly by the same group (4, 11, 25, 31, 35).

The present congenic rat study indicates that there are multiple regions that can attenuate the development of hypertension in SS rats. These observations differ markedly from previous QTL analysis studies. Indeed, there has been considerable controversy about the localization of blood pressure loci on chromosome 13 in the Dahl S rat. The initial linkage analysis that launched the search for genes of hypertension was a study by Rapp et al. (36) in 1989 indicating that the renin gene cosegregated with blood pressure in a cross of Dahl S and R rats. A previous linkage study performed by our group (27, 41) in an F2 population derived from SS and BN rats failed to find significant linkage for blood pressure on chromosome 13 in either male or female rats. However, in a subsequent study (6), we found that transfer of chromosome 13 from BN to SS had a very large protective effect on salt-induced hypertension. In the present study, at least four loci located in neighboring regions of chromosome 13 were found (see Figs. 1 and 4). In the initial F2 linkage study, the presence of several QTLs segregating at other chromosomes could have reduced the power for QTL detection of interval mapping (2).

We hypothesize that multiple loci within a single chromosome could reduce the ability of a linkage analysis to predict their exact location and would require very large numbers of rats to achieve adequate recombination. This was tested during the process of developing the overlapping congenic strains for this study. We performed a linkage analysis for blood pressure in an F2 population (n = 187 rats phenotyped) derived from backcrossing the SS-13BN consomic rats with SS rats (unpublished observations). In this F2 population, the whole genome was SS and segregation of alleles could only occur on chromosome 13. This linkage analysis resulted in only a single, albeit broad but highly significant, QTL (LOD score of 9.41), with its peak at 45 Mbp. This broad QTL with 95% confidence limits of nearly 60 Mbp fell over the margins of congenic strain 26 but failed to detect the other three loci on chromosome 13. These data suggest that QTL mapping, although useful in the initial detection of genetic regions linked to a phenotype/disease, can lack precision when multiple loci are located nearby on a chromosome.

Regarding the influence of sex on development of hypertension and renal injury, there were sex differences in the effect of some genomic regions on blood pressure. The most noticeable was the lack of effect of the renin region in male rats, while significant attenuation of blood pressure during a high-salt diet was found in female rats. Although a mechanistic study of this phenomenon is beyond the scope of the present study, it suggests that some genes affecting blood pressure are sex specific, and that sex should be taken into account in linkage and association studies. These observations are consistent with sex-specific gene effects that have been described in linkage studies with the SS rat (27, 41) and in other rat models of hypertension like the Sabra (46) or the Genetically Hypertensive rat (5). In humans, association and genetic linkage performed for different complex diseases, including hypertension, have also been found to be sex specific (30, 45).

Protein excretion follows blood pressure in susceptible strains, but it is known also to be determined by different genetic loci (27, 41). The SS-13BN consomic strain showed both reduced blood pressure and protein excretion; however, to our surprise, none of the congenic strains evidenced a significant reduction of albumin excretion, including those that had decreased blood pressure with respect to the SS strain. The reason for this is not apparent, although one can speculate that a combination of genes at opposite locations in chromosome 13 may be needed to protect the kidney from renal damage. It is possible that the necessary gene combination was not achieved in the present study with these overlapping congenic strains.

In summary, we have confirmed that development of overlapping congenic strains is a very powerful tool for narrowing genomic regions containing blood pressure-related genes. We have identified four distinct regions in chromosome 13 containing genes that participate in the regulation of blood pressure during high-salt diet. These regions act epistatically in the regulation of blood pressure, with two of them being sex specific. This could provide an explanation of why it has been so difficult in the past to map blood pressure QTLs to this chromosome. Finally, it should be emphasized that although these studies were completed in strains derived from SS and BN rats, these QTLs may translate to humans, providing a guide for candidate regions to look for disease mutations.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-54998 and HL-82798.


   ACKNOWLEDGMENTS
 
The authors thank Gina Tadisch-Rhodes and Kristin Bastian for phenotyping assistance and Barbara Meinecke, Holly Kinservik, and Michael Tschannen for genotyping.


   FOOTNOTES
 
Address for reprint requests and other correspondence: A. W. Cowley Jr., Dept. of Physiology, Medical College of Wisconsin, Milwaukee, WI 53266 (e-mail: cowley{at}mcw.edu).

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

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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