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(Hypertension. 1996;27:564-568.)
© 1996 American Heart Association, Inc.

Articles

Cytochrome P4504A Genotype Cosegregates With Hypertension in Dahl S Rats

David E. Stec; Alan Y. Deng; John P. Rapp; Richard J. Roman

From the Department of Physiology, Medical College of Wisconsin, Milwaukee (D.E.S., R.J.R.), and Department of Physiology, Medical College of Ohio, Toledo (A.Y.D., J.P.R.).

	Abstract

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Abstract Recent studies indicate that the production of 20-HETE by a P4504A2 enzyme in the outer medulla of the kidney is reduced in Dahl salt-sensitive (SS/Jr) rats, but the contribution of this abnormality to the elevation in loop Cl^- transport and development of hypertension in this model is unknown. The present study found that alleles at the locus for the P4504A2 gene cosegregate with blood pressure in an F₂ population (n=151) derived from a cross between SS/Jr and Lewis rats (P<.0001). The P4504A2 locus is located in a region on rat chromosome 5 where a blood pressure quantitative trait locus was previously detected. Systolic blood pressure averaged 201±6 mm Hg in rats with the SS genotype (n=36), 192±4 mm Hg in SL genotype rats (n=77), and 169±3 mm Hg in LL genotype rats (n=38). In further studies, we confirmed that there are phenotypic differences in the expression of the P4504A2 gene in the kidneys of SS/Jr and Lewis rats. Although the production of 20-HETE from ¹⁴C-arachidonic acid was similar in microsomes prepared from the renal cortex of SS/Jr and Lewis rats (54±3 versus 55±3 pmol·min^-1·mg protein^-1), the production of 20-HETE in microsomes prepared from the outer medulla (OM) was markedly reduced in SS/Jr rats (2.8±0.8 versus 6.7±1 pmol·min^-1·mg protein^-1). The diminished production of 20-HETE in the OM was due to a threefold reduction in the level of P4504A2 protein. These results suggest that an altered expression of the P4504A2 enzyme in the OM may contribute to the development of hypertension in SS/Jr rats.

Key Words: hydroxyeicosatetraenoic acid • cytochrome P-4504A2 • gene expression • NaCl, dietary • rats, Dahl

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Renal transplantation studies indicate that some form of renal dysfunction underlies the development of hypertension in Dahl SS/Jr rats.^{1 2 3 4} However, the factors responsible for altering kidney function and genes involved remain to be defined. We^{3 5} and others^{6 7 8} have reported an abnormality in renal function that precedes the development of hypertension in genetic rat models of hypertension. In both the SHR and SS/Jr rats, the pressure-natriuresis relation is altered so that the hypertensive strains require a higher renal perfusion pressure to excrete the same amount of water and electrolytes as normotensive strains. In SS/Jr rats, the resetting of the pressure-natriuresis relation is due to an elevation in chloride reabsorption in the TALH.^{5 6 7 8} Previous studies by Carroll et al⁹ and Escalante et al¹⁰ indicate that 20-HETE is the primary metabolite of AA produced in the TALH and that this substance is a potent endogenous inhibitor of the Na⁺, K⁺, 2 Cl^- transporter. In light of these findings, our recent observation that the production of 20-HETE is reduced in the outer medulla of SS/Jr rats¹¹ suggests that a deficiency in the production of 20-HETE may contribute to the elevation of loop Cl^- transport in this model. This hypothesis is further supported by the observation that chronic treatment of SS/Jr rats with clofibrate, which induces the formation of 20-HETE in the kidney, can prevent the development of hypertension.¹² Nevertheless, it is also possible that all of these observations simply reflect strain differences in the renal expression of P450 enzymes and that this system may have nothing to do with the changes in renal function and development of hypertension in SS/Jr rats.

To determine whether alterations in the renal metabolism of AA by P450 enzymes contribute to the development of hypertension, a genetic linkage analysis for the alleles at the P4504A2 enzyme was performed in an F₂ generation of a cross between SS/Jr and Lewis rats. We also evaluated whether there are phenotypic differences in the expression of the P4504A2 protein and enzyme activity in the kidneys of the parental strains used in the cross.

	Methods

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Rat Strains
Inbred Dahl SS/Jr rats used to breed the F₂ rats for the cosegregation analysis were obtained from the colony maintained at the Medical College of Ohio, Toledo. The Lewis-Wistar (Lew/NCrlBr) rats were purchased from Charles River Laboratories, Wilmington, Mass. The rats were housed in animal care facilities at the Medical College of Ohio and the Medical College of Wisconsin, which are approved by the American Association for Accreditation of Laboratory Animal Care, and had free access to food and water throughout the study. All protocols involving animals were approved by the Animal Care Committees of the Medical College of Ohio and the Medical College of Wisconsin.

The cosegregation analysis was performed on 151 male F₂ rats derived from a cross of SS/Jr rats with Lewis rats and designated as F₂ (SxLew). The rats were weaned 3 weeks after birth and placed on a high salt (8% NaCl) diet (TD82050, Teklad) for the duration of the experiment. Systolic blood pressure was measured at weekly intervals in the population by the tail-cuff microphonic manometer method in conscious, restrained rats. When blood pressures of some of the rats reached 200 mm Hg, the blood pressures of all the rats in the population were measured over a 7- to 10-day period. A daily measurement of pressure was taken as the average of three consistent blood pressure measurements. The blood pressure of each rat was measured on three separate days, and the average pressure recorded over the 3-day period was the blood pressure for that rat.

Genotyping
DNA was extracted from the livers of the rats according to the method of Blin and Stafford.¹³ The rats were genotyped by PCR as previously described¹⁴ at the P4504A2 locus with PCR primers designed around a tandem-repeated element in intron 11 of the P4504A2 gene obtained from a published sequence.¹⁵ The sequences of the primers used were as follows: forward, 5'-TGGCACTGTCCAAATGA-3'; reverse, 5'-GCTTAGGCTCCTGACCT-3'. The linkage analysis for P4504A2 was completed using other markers on rat chromosome 5 as previously described by Deng et al.¹⁶

Renal Metabolism of AA and P4504A Expression Studies
These experiments were performed on 12-week-old male Lewis rats purchased from Charles River Laboratories and Dahl SS/Jr obtained from a colony of rats maintained at the Medical College of Wisconsin. This colony was originally derived from SS/Jr rats purchased from Harlan Industries in 1991, before any evidence of genetic contamination of this strain.¹⁷ The rats have since been maintained by brother-sister mating in a closed breeding colony for 16 additional generations. The rats in this colony all develop severe hypertension when placed on a high salt diet for 3 weeks (with mean arterial pressures >170 mm Hg); the littermates of the rats used in this study (n=8) were extensively genotyped with 40 markers (2 or 3 per chromosome), and no evidence of genetic contamination could be detected (R.S.R., 1995, unpublished observation).

Preparation of Renal Microsomes
Experiments were performed on male SS/Jr and Lewis rats maintained on a normal salt diet (1% NaCl by weight). The rats were anesthetized with pentobarbital (30 mg/kg IP), and the kidneys were rapidly removed, hemisected, sectioned into cortex and outer medulla, and placed into an ice-cold homogenization buffer. The tissue was homogenized in 3 mL potassium phosphate homogenization buffer (10 mmol/L, pH 7.7) containing (in mmol/L) sucrose 250, EDTA 1, and PMSF 0.1. The homogenate was centrifuged at 3000g for 15 minutes to remove large pieces of tissue, and the supernatant was centrifuged at 9000g for 15 minutes, followed by 100 000g for 1 hour. The microsomal pellet was resuspended in a potassium phosphate buffer (100 mmol/L, pH 7.2) that contained 30% glycerol, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, and 0.1 mmol/L PMSF and was stored at -80°C until P450 activity was determined.

Renal Metabolism of AA
Renal P4504A enzyme activity was measured by incubating microsomes prepared from the renal cortex and outer medulla of the rats (0.5 mg protein) with [¹⁴C]AA (0.1 µCi/mL, 20 µmol/L) in 1 mL of a potassium phosphate buffer (pH 7.4) containing (in mmol/L) MgCl₂ 5, EDTA 1, and NADPH 1 and an NADPH-regenerating system (10 mmol/L isocitric acid and isocitrate dehydrogenase 0.4 U/mL, Sigma). Samples were incubated at 37°C for 30 minutes after the addition of NADPH. The reactions were terminated by acidification to pH 4.0 with 0.1 mol/L formic acid, the samples were extracted twice with ethyl acetate and dried under N₂ gas, and the residue was resuspended in 500 µL of 100% ethanol. The metabolites were separated by use of a Hitachi 655A-1 high-performance liquid chromatography gradient system equipped with a C18 reverse-phase column (2.1x250 mm, 5 µm; Supleco) and a 2-cm guard column (Supleco). A linear elution gradient ranging from acetonitrile/water/acetic acid (50:50:2 vol/vol/vol) to acetonitrile/acetic acid (100:0.2, vol/vol) was used. The rate of change was 1.25% per minute at a flow rate of 0.5 mL/min. Metabolites were monitored by a radioactive flow detector (Flo-oneBeta, series A-120, Radiomatic Instruments) specially modified with lead shielding for a low background. The mean production rate for each metabolite was calculated and expressed as picomoles formed per minute per micrograms of protein.

Immunoblots
Microsomal protein prepared from the renal cortex (15 µg) and outer medulla (50 µg) of the Dahl S and Lewis-Wistar rats was separated by electrophoresis on a 7.5% sodium dodecyl sulfate gel (15x15 cm) for 14 hours at 80 V. Proteins were transferred electrophoretically to a nitrocellulose membrane (Trans-Blot, Bio-Rad) at 40 V in a buffer consisting of 25 mmol/L Tris-HCl, 192 mmol/L glycine, and 20% methanol for 4 hours at 4°C. The membrane was blocked for 2 hours by immersion in a buffer (TBST-20) containing 10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.8% Tween-20, and 5% nonfat dry milk and incubated with a 1:2000 dilution of a polyclonal antibody raised against a 20-mer synthetic peptide in the rat P4504A1 sequence that recognizes all three of the 4A isoforms.¹⁸ The membrane was washed in TBST-20 buffer and incubated for 1 hour with a 1:1000 dilution of an alkaline phosphatase–coupled goat anti-rabbit second antibody (Zymed). The membrane was then placed in a color development solution (Bio-Rad), and the relative intensities of the bands in the 50- to 52-kD range were quantified with a densitometer (Personal Densitometer SI, Molecular Dynamics).

Statistics
Mean±SEM values are presented. The significance of differences in mean values was evaluated with either a paired or an unpaired t test. A value of P<.05 was considered to be statistically significant. QTLs were identified with the MAPMAKER program.¹⁹

	Results

Top Abstract Introduction Methods Results Discussion References

 
Renal Metabolism of AA
Experiments were performed to determine whether there are phenotypic differences in the expression of P4504A enzymes in the kidney of parental strains used to derive the F₂ population studied in the genetic linkage analysis. A comparison of P4504A enzyme activity in microsomes prepared from the cortex and outer medulla of male Dahl SS/Jr and Lewis-Wistar rats maintained on a normal salt diet (1% NaCl) is presented in Fig 1. The production of 20-HETE by microsomes prepared from the renal cortex was not significantly different in SS/Jr and Lewis rats (Fig 1A). In contrast, the production of 20-HETE by microsomes prepared from the outer medulla was threefold lower in SS/Jr than in Lewis rats (Fig 1B).

View larger version (31K): [in this window] [in a new window]   Figure 1. Metabolism of AA by P450 in the cortex and outer medulla of male Dahl S and Lewis rats on a normal salt diet (1% NaCl wt/wt). Microsomes were prepared from the cortex and outer medulla of SS/Jr and Lewis rats, and the production of 20-HETE was measured by incubating the microsomes with [14C]AA for 30 minutes at 37°C in the presence of 1 mmol/L NADPH. Metabolites were separated by reverse-phase high-performance liquid chromatography, and the enzyme activity is expressed as picomoles of product formed per minute per milligram protein. A, Results from cortical microsomes prepared from SS/Jr (n=10) and Lewis (n=11) rats. B, Outer medullary microsomes prepared from SS/Jr (n=9) and Lewis (n=8) rats. *Significant difference (P<.05) from corresponding value in Lewis rats.

Expression of P4504A Protein in the Kidney
Western blot experiments were performed with an antibody raised against a synthetic peptide sequence in rat P4504A1 protein that recognizes all three 4A isoforms in the liver and kidney of rats.¹⁸ The purpose of these experiments was to determine whether the differences observed in enzyme activity in SS/Jr and Lewis rats were associated with changes in the levels of P4504A protein in the kidneys of these rats. The results of these experiments are presented in Fig 2. The P4504A2 enzyme was the only isoform detected in microsomes prepared from either the renal cortex or outer medulla of male SS/Jr and Lewis rats. The levels of 4A2 protein were threefold higher in the renal cortex of Lewis than in SS/Jr rats. In the outer medulla, the levels of 4A2 protein were also threefold greater in Lewis rats compared with the levels in SS/Jr rats (Fig 2B).

View larger version (19K): [in this window] [in a new window]   Figure 2. Immunoblot of P4504A isoforms expressed in the cortex and outer medulla of male SS/Jr and Lewis rats maintained on a normal salt diet (1% NaCl wt/wt). A, Lanes 1 through 6 were loaded with microsomes (15 µg) prepared from the cortex of male SS/Jr rats, lanes 8 through 13 were loaded with microsomes (15 µg) prepared from the cortex of male Lewis rats, and lane 7 was loaded with microsomes (25 µg) prepared from rat liver that expresses all three P4504A isoforms. Values are mean±SEM from 6 rats. B, Lanes 1 through 4 were loaded with microsomes (50 µg) prepared from the outer medulla of male SS/Jr rats, lanes 6 through 9 were loaded with microsomes (50 µg) prepared from the outer medulla of male Lewis rats, and lane 5 was loaded with microsomes (15 µg) prepared from rat liver that expresses all three P4504A isoforms. Values are mean±SEM from 4 rats. *Significant difference (P<.05) from corresponding value in Lewis rats.

Cosegregation Analysis
The F₂ (SxLew) population was genotyped for the P4504A2 locus. The P4504A2 locus mapped to a region on rat chromosome 5, 3.3 cM (centiMorgans) from a glucose transporter locus and 2.6 cM away from the endothelin 2 locus. Both of these loci have previously been reported to cosegregate with blood pressure in this cross.¹⁶ The results of the present cosegregation analysis are presented in Fig 3. The P4504A genotype cosegregated with systolic blood pressure (P<.0001), and the effect of the P450 genotype on systolic pressure was quite large, with average pressures of 201±6 mm Hg in the rats with an SS genotype (n=36), 192±4 mm Hg in heterozygote rats with an SL genotype (n=77), and 169±3 mm Hg in rats with the LL genotype (n=38).

View larger version (31K): [in this window] [in a new window]   Figure 3. Systolic blood pressures of male rats of the F2 (SxLew) generation sorted by P4504A2 genotype. Pressures were measured by the tail-cuff method as described in the "Methods" section. Numbers at bottom indicate number of animals with the corresponding genotype. *Significant difference from the corresponding value in F2 rats with an LL genotype (P<.0001).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous studies have established that an elevation in Cl^- transport in the TALH underlies the resetting of the pressure-natriuresis relation toward higher pressures in SS/Jr rats.^{5 6 7 8} More recently, we reported that this is associated with an abnormality in the renal metabolism of AA via the P450 pathway. Specifically, the production of 20-HETE in the outer medulla of SS/Jr rats is reduced in comparison with SR/Jr rats.¹¹ 20-HETE is the primary metabolite of AA produced by TALH cells,⁹ in which it serves as an endogenous inhibitor of Na⁺, K⁺, 2 Cl^- transporter¹⁰ ; therefore, a deficiency in the renal production of this substance may play a role in the resetting of the pressure-natriuresis relation and the development of hypertension in this strain. In the present study, a cosegregation analysis was performed on an F₂ population derived from a cross of SS/Jr and Lewis rats. The results indicate that the P4504A2 alleles cosegregate with blood pressure in this population. The P4504A2 locus maps to a region of chromosome 5 between the endothelin-2 and glucose transporter loci. This region of chromosome 5 has previously been identified by Deng et al¹⁶ to contain a major QTL for high blood pressure. Since abnormalities in the endothelin-2 or glucose transporter could potentially influence vascular tone and/or renal sodium transport, it is obvious that this region contains potential candidate genes other than P4504A2 that may be responsible for the cosegregation with hypertension in this cross. One useful way to begin to sort out which of the potential candidate genes within the chromosomal region may actually be the QTL is to determine whether there are any differences in the coding or regulatory regions in the gene or phenotypic differences in the expression or function of the system between the parental strains used in generating the F₂ population.

In the present study, we compared the renal metabolism of AA by P450 and the expression of the P4504A proteins in the kidneys of the parental strains. Despite significantly higher levels of P4504A2 protein in the cortex of Lewis rats compared with SS/Jr rats, no significant differences were observed in the cortical production of 20-HETE between SS/Jr and Lewis rats. The reason why 20-HETE is not elevated in the renal cortex despite an elevation in P4504A2 protein is unknown. One possibility is that there may be differences in levels of other cofactors such as cytochrome P450 reductase and B₅ in the renal cortex in Lewis rats that compensate for the difference in 4A2 protein. It is also possible that some of the immunoreactive P4504A2 protein detected in the cortex of Lewis rats may be inactive. Clearly, further investigation will be required to examine the reasons behind this observation.

The production of 20-HETE was reduced in the outer medulla of the kidney of SS/Jr rats in comparison with the levels seen in Lewis rats, and this was associated with threefold lower levels of the P4504A2 protein in the outer medulla of SS/Jr rats compared with Lewis rats. These results establish that there is a marked difference in the production of 20-HETE in the appropriate region of the kidney between the parental strains of the rats used to generate the F₂ population for the cosegregation studies. Moreover, we have confirmed in micropuncture studies that the P4504A system directly influences Cl^- transport in the TALH²⁰ and that an abnormality in this system appears to contribute to the elevation in Cl^- transport seen in SS/Jr rats. In these experiments, addition of an inhibitor of the production of 20-HETE to fluid perfusing the loop of Henle of SR/Jr rats increased Cl^- transport to the same level as that observed in SS/Jr rats, and it had no effect on loop Cl^- transport in SS/Jr rats. Similarly, addition of the putative mediator, 20-HETE, to the perfusate normalized loop Cl^- transport in SS/Jr rats, but it had much less effect in Dahl R rats, in which the endogenous levels of this compound are higher. These biochemical and functional results are also consistent with our previous observation that induction of the renal production of 20-HETE can prevent the development of hypertension in SS/Jr rats fed a high salt diet.¹²

In summary, the present findings indicate that the renal metabolism of AA by P450 and the steady state levels of the P4504A2 protein in the outer medulla of the kidney are lower in SS/Jr than in Lewis rats and that the P4504A genotype cosegregates with blood pressure in an F₂ cross derived from these strains. The product of this enzyme, 20-HETE, serves as an endogenously formed inhibitor of Cl^- transport in the TALH of SS/Jr rats.^{9 10} This system appears to be a viable candidate gene that may contribute to the elevation in loop Cl^- transport,^{5 6 7 8} the resetting of the pressure-natriuresis relation,³ and the development of hypertension in SS/Jr rats.

	Selected Abbreviations and Acronyms

 

20-HETE = 20-hydroxyeicosatetraenoic acid AA = arachidonic acid PCR = polymerase chain reaction PMSF = phenylmethylsulfonyl fluoride QTL = quantitative trait locus SHR = spontaneously hypertensive rats SR/Jr = salt-resistant John Rapp rats SS/Jr = salt-sensitive John Rapp rats TALH = thick ascending loop of Henle TBST = Tris-buffered saline with Tween-20

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grant HL-36279 (Dr Roman). D.E. Stec is the recipient of a Predoctoral Fellowship (94-pre-20) from the American Heart Association, Wisconsin Affiliate. This work was also supported in part by grants from the National Institutes of Health and the Helen and Harold McMaster fund to Dr Rapp. The authors wish to thank Lisa Henderson for her excellent technical assistance.

	Footnotes

 
Reprint requests to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.

	References

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2. Sterzel RB, Luft FC, Gao Y, Schnermann J, Briggs JP, Ganten D, Waldherr R, Schnabel E, Kriz W. Renal disease and the development of hypertension in salt-sensitive Dahl rats. Kidney Int. 1988;33:1119-1129. [Medline] [Order article via Infotrieve]

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5. Roman RJ, Kaldunski ML. Enhanced chloride reabsorption in the loop of Henle of Dahl S rats. Hypertension. 1991;17:1018-1024. [Abstract/Free Full Text]

6. Kirchner KA. Greater loop chloride reabsorption contributes to blunted pressure-natriuresis in Dahl salt-sensitive rats. J Am Soc Nephrol. 1990;1:180-186. [Abstract]

7. Kirchner KA. Increased loop chloride uptake precedes hypertension in Dahl salt-sensitive rats. Am J Physiol. 1992;262:R263-R268. [Abstract/Free Full Text]

8. Kirchner KA. Segmental chloride transport in the Dahl S rat kidney during L-arginine administration. J Am Soc Nephrol. 1995;5:1567-1572. [Abstract]

9. Carroll MA, Sala A, Dunn CE, McGiff JC, Murphy RC. Structural identification of cytochrome P450-dependent arachidonic acid metabolites formed by rabbit renal medullary thick ascending limb cells. J Biol Chem. 1991;266:12306-12312. [Abstract/Free Full Text]

10. Escalante B, Erlij D, Falck JR, McGiff JC. Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science. 1991;251:799-801. [Abstract/Free Full Text]

11. Ma Y-H, Schwartzman ML, Roman RJ. Altered renal P-450 metabolism of arachidonic acid in Dahl salt-sensitive rats. Am J Physiol. 1994;267:R579-R589. [Abstract/Free Full Text]

12. Roman RJ, Ma Y-H, Frohlich B, Markham B. Clofibrate prevents the development of hypertension in Dahl salt-sensitive rats. Hypertension. 1993;21:985-988. [Abstract/Free Full Text]

13. Blin N, Stafford DW. Isolation of high-molecular weight DNA. Nucleic Acids Res. 1976;3:2303-2308.

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15. Kimura S, Hanioka N, Matsunaga E, Gonzalez FJ. The rat clofibrate-inducible CYP4A gene subfamily, I: complete intron and exon sequence of the CYP4A1 and CYP4A2 genes, unique exon organization, and identification of a conserved 19-bp upstream element. DNA. 1989;8:503-516. [Medline] [Order article via Infotrieve]

16. Deng AY, Dene H, Pravenec M, Rapp JP. Genetic mapping of two new blood pressure quantitative trait loci in the rat by genotyping endothelin system genes. J Clin Invest. 1994;93:2701-2709.

17. St Lezin E, Pravenec M, Wong A, Merriouns JM, Newton S, Stec DE, Roman RJ, Morris RC, Kurtz TW. Genetic contamination of Dahl SS/Jr rats: impact on studies of salt-sensitive hypertension. Hypertension. 1994;23:786-790. [Abstract/Free Full Text]

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20. Zou A-P, Drummond HA, Roman RJ. Role of 20-HETE in elevating loop chloride reabsorption in Dahl SS/Jr rats. Hypertension. 1996;27(part 2):631-635.

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