Title:  Method of assaying modulators of hypertension

United States Patent:  6,844,483

Issued:  January 18, 2005

Inventors:  Ruiz-Opazo; Nelson (Westwood, MA); Herrera; Victoria L. M. (Westwood, MA)

Assignee:  NoMI LLC (Wellesley, MA)

Appl. No.:  040722

Filed:  January 7, 2002

Abstract

The invention features methods for assaying compounds that affect hypertension by using an animal model with a functionally variant hypertension susceptibility gene.

Description of the Invention

FIELD OF THE INVENTION

The invention relates to methods useful for delaying or ameliorating diseases associated with hypertension.

BACKGROUND OF THE INVENTION

Essential hypertension (EHT; 1) is a paradigmatic, complex; and multifactorial condition. Genes that mediate EHT have therefore been difficult to isolate and characterize, requiring multiple lines of evidence to establish their roles in EHT pathogenesis.

In view of the wide range of disorders that are associated with hypertension, it would be desirable to identify compounds for the treatment or prevention of hypertension.

SUMMARY OF THE INVENTION

Here we present evidence of the identification and characterization of an EHT susceptibility gene. The invention provides methods for identifying compounds which affect hypertension.

In one aspect, the invention features a method of assaying a test compound, by providing a non-human mammal with a functionally variant hypertension susceptibility gene, administering said test compound to said non-human mammal, and determining whether the test compound affects hypertension parameters in the non-human mammal relative to a non-human mammal containing a wild type hypertension susceptibility gene.

In preferred embodiments of the invention, the hypertension susceptibility gene is the .alpha.1 Na,K ATPase gene and the non-human mammal is a rat, preferably the Dahl S rat.

By "test compound" is meant any chemical compound, be it naturally-occurring or artificially-derived. Test compounds may include, for example, peptides, polypeptides, synthesized organic molecules, naturally occurring organic molecules, and nucleic acid molecules.

By "affects" is meant changes, either by increase or decrease.

By "determining" is meant analyzing the effect of a test compound on the test system. The readout of the analysis may be measurement of life span, blood pressure, renal pathology, and other hypertension parameters known to those skilled in the art.

The invention provides a means for assaying compounds that affect hypertension by means of an animal model in which a hypertension susceptibility gene has been identified.

DETAILED DESCRIPTION OF THE INVENTION

Given the difficulty of isolation and characterization of genes mediating EHT, delineation of a putative EHT susceptibility gene should meet the following criteria: 1, identification of a functionally significant structural mutation in the relevant gene; 2, concordance of the observed genetic dysfunction with a pathophysiologic mechanism logical to the hypertension pathogenesis; 3, association of the putative hypertension susceptibility gene with hypertension in validated genetic animal models or human hypertensive patients; and 4, delineation of the mechanistic role in an in vivo model (1, 2). To date, no EHT susceptibility gene has been identified that meets all these criteria.

To simplify the molecular genetic characterization of an EHT susceptibility gene, one subtype of EHT, salt-sensitive hypertension (SS-EHT), was studied. The hypothesis that variants of the .alpha.1 Na,K-ATPase gene mediate SS-EHT in a genetic rat model of hypertension, was tested using the Dahl S hypertensive rat strain (3, 4). Because .alpha.1 Na,K-ATPase is the sole active Na+ transporter in the renal basolateral epithelia throughout the nephron (5, 6), it is a logical candidate gene to be considered in the assessment of the abnormal renal sodium handling in the Dahl S rat (7).

Two of the four criteria stated above (1, 2) and required to define the .alpha.1 Na,K-ATPase gene as an EHT susceptibility gene have been met. For criterion 1, a Q276L substitution in the .alpha.1 Na,K-ATPase gene in inbred Dahl S rats from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) (Dahl SHSD) has been characterized (8, 9). In contrast to the non-detection by PCR sequencing reported by Simonet et al. (10), the Q276L .alpha.1 Na,K-ATPase variant was confirmed in Dahl S genomic DNA by using PCR error-independent assays (polymerase allele specific amplification, PASA, and 3' mismatch correction assay) and ligase chain reaction assay; in kidney RNA by RT^th -PCR; and in cDNA clones by resequencing (9). Likewise, detection of the wild-type (wt) Q276 sequence (11) was confirmed in Dahl R genomic DNA and in resequenced cDNA clones (9).

The Q276L mutation results in decreased K+(⁸⁶ R.beta.+) influx detected in Xenopus oocyte expression experiments using both Dahl S kidney polyA+ RNA, as well as in vitro transcribed variant Q276L-specific cRNA transcript, in contrast to control Dahl R rat kidney polyA+ RNA and in vitro transcribed wt Q276 cRNA transcript, respectively (8). Kinetic studies of .alpha.1 Na,K-ATPases in red blood cell flux experiments, comparing Dahl S and Dahl R .alpha.1 Na,K-ATPases corroborated decreased K+(⁸⁶ R.beta.+) influx and revealed normal Na+ transport resulting in an increased Na:K coupling ratio in the Dahl S Q276L .alpha.1 Na,K-ATPase variant (12). For criterion 2, simulated modeling studies have revealed that consequences of an increased Na:K coupling ratio (from 3:2 to 3:1) observed in the Q276L .alpha.1 Na,K-ATPase variant results in an altered set point for cellular Na+ metabolism, with higher sodium reabsorption at unchanged Na,K-ATPase levels in the proximal convoluted tubule, as well as in the thick ascending limb of the loop of Henle (13), thus providing a mechanistic hypothesis for increased Na+ reabsorption in Dahl S rats.

To fulfill criteria 3 and 4, the following questions were addressed: does the Q276L .alpha.1 Na,K-ATPase variant contribute to the salt-sensitive hypertension phenotype? and does the functionally aberrant Q276L .alpha.1 Na,K-ATPase allele cosegregate with salt-sensitive hypertension? These questions were addressed using transgenic experiments and a standard intercross linkage analysis strategy.

Because of the inadvertent genetic contamination of the Dahl S^HSD strain (19, 24), transgenic studies were performed using only Dahl S^HSD rats from the foundation colonies at Harlan Sprague, Inc. confirmed as to genotype and salt-sensitive phenotype (19). The F2 intercross was also done using Dahl S^HSD and Dahl R^HSD rats confirmed for both genotype and phenotype. Unequivocal observations are thus ascertained (19).

The results obtained from the transgenic and cosegregation studies fulfill the requirements of the criteria set out to definitively assign the .alpha.1 Na,K-ATPase gene as a susceptibility gene for hypertension using the Dahl SHSD genetic hypertension rat model. The concordance of improvement not just in all measures of blood pressure, but also in renal disease and life span, provides holistic support strengthening the ascertainment of the mechanistic role of .alpha.1 Na,K-ATPase in salt-sensitive hypertension as modeled in the Dahl S^HSD rat. The results indicate that phenotypic differences observed in the transgenic Tg[wt.alpha.1] rats are most likely due to the functional heterozygosity of wt and Q276L variant .alpha.1 Na,K-ATPases rather than an additive overabundance of .alpha.1 Na,K-ATPases. This is consistent with the observation made from blood pressure data of F1(SxR) rats, indicating that hypertension is a recessive trait and that normotension is a dominant trait.

Other genetic studies have documented previous linkage of the .alpha.1 Na,K-ATPase locus. The .alpha.1 Na,K-ATPase locus was found to be the closest candidate gene in a total chromosome 2 scan analyzing two F2 cohorts, one involving the Dahl SxMilan normotensive strain, and one involving Dahl SxWistar Kyoto normotensive strain (22). Recent studies on chromosome 2 analyzing F2 crosses derived from the stroke-prone spontaneously hypertensive rat and the normotensive Wistar-Kyoto rat (25) and derived from the spontaneously hypertensive rat and the Wistar-Kyoto rat (26) have also detected a QTL for high blood pressure close to the .alpha.1 Na,K-ATPase locus. The cosegregation study presented herein independently confirms these previous results and with P<0.003, meets the required nominal P<0.01 criterion for confirmed linkage (27).

The pronounced improvement in blood pressure (.about.40%) by the transgenic manipulation of a single gene suggests that hypertension, being polygenic, does not, most likely, follow a simple additive model of genetic inheritance, but rather involves a di- or multigenic interaction within a polygenic context. With normotension being dominant, transgenic experiments designed to correct hypertension in the inbred hypertensive strain would be more robust in investigating the effects of interacting hypertension susceptibility genes rather than F2 intercross studies with polymorphic markers as shown herein.

The improvement of multiple pathogenic events in transgenic Tg[wt.alpha.1] Dahl S rats is consistent with observations in human hypertensive patients, wherein lowering of blood pressure has been shown to decrease mortality and target organ complications (28). The greater reduction in the degree of renal pathology (50%) and greater improvement in life span (75.6%) compared with blood pressure parameters (.about.40%) seen in the transgenic rats could be attributed to an "early" intervention, as the transgenic rats have the corrective transgene from one-cell embryo stage--a finding which promotes the value of early preventive interventions for some complex diseases. Additionally, the .alpha.1 Na,K-ATPase gene might play a role in hypertensive renal complication pathogenesis that is distinct from its role in hypertension pathogenesis, and/or a threshold phenomenon might be involved in the pathogenesis of hypertensive target organ complications.

The inability to detect the A¹⁰⁷⁹ -T¹⁰⁷⁹ transversion in Dahl S rat genomic DNA via amplification-based methods and sequencing of genomic clones underscores the importance of a multifaceted analysis of such refractory mutations encompassing structural and functional approaches. The demonstration, therefore, of functionally significant differences between Dahl S and Dahl R .alpha.1 Na,K-ATPases and, more significantly, the partial correction of salt-sensitive hypertension in the Dahl S rat via transgenesis support the contention that the Q276L mutation exists in Dahl S rats as shown by error-independent assays and that it plays a role in salt-sensitive hypertension. The observation of an amplification error-prone genomic DNA region raises the question that other mutations might be similarly refractory to detection by conventional amplification-based methods. Amplification-independent assays provide an alternative and suitable approach to structurally assess these "refractory" mutations.

Altogether, the results presented herein demonstrate that the Na,K-ATPase locus is a SS-EHT susceptibility gene and showcases the strength of a "forward genetics approach" testing functionally significant variant alleles at biologically relevant loci (1, 29)--as was done recently in the study of the variant the value of a multifaceted molecular genetic approach (1, 31), wherein transgenic rat experiments in an inbred model organism might allow one to deduce the role of a gene in complex disease pathogenesis. The success in the significant alleviation of salt-sensitive hypertension by the manipulation of a single gene validates the potential for gene therapy for complex cardiovascular diseases and other multifactorial disorders. Moreover, the proposed criteria and approach in animal models provide evidence that make analogous studies of homologous human genes in hypertension compelling.

Claim 1 of 2 Claims

What is claimed is:

1. A method of assaying a test compound for an effect on hypertension parameters, said method comprising:

(a) providing a Dahl Salt-sensitive^HSD rat whose genome comprises a functional variant .alpha.1 Na,K-ATPase hypertension susceptibility gene;

(b) administering said test compound to said rat; and

(c) determining whether the test compound affects hypertension parameters in said rat relative to hypertension parameters in a rat whose genome comprises a wild type .alpha.1 Na,K-ATPase gene.

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