Hypertension, atherosclerosis, diabetes mellitus, and many renal diseases are multifactorial disorders in which some continuously and normally varying biologic parameter (BP, plasma lipid concentration, plasma glucose, plasma creatinine, etc.) exceeds the limit compatible with good health. But there are rarely definitive thresholds that are harmful. More than 15% of the adult population in industrialized societies has hypertension, which is one of the principal risk factors for cardiovascular events and end-stage renal disease. However, the pathogenesis of essential hypertension is largely unknown. Although we know that many physiologic systems are involved in determining BP, it has proved difficult to determine which systems are changed in the majority of individual hypertensive patients because of the complex interactions between these systems. As a result, the treatment of hypertensive patients is largely empiric. It is recognized that there is a strong genetic importance in BP variations from studies of twins and the familial aggregation of BP, and substantial efforts have been devoted to the identification of the genes responsible for hypertension in the hope that this will allow less empirical treatments to be developed.
Mendelian Forms of Hypertension
Recently, several genes responsible for severe forms of hypertension showing simple Mendelian patterns of inheritance have been identified.
Glucocorticoid-Remediable Aldosteronism
Glucocorticoid-remediable aldosteronism (GRA) is an autosomal dominant trait, caused by a gene duplication arising from unequal crossover between the genes encoding for aldosterone synthase and 11β-hydroxylase genes. The duplicated gene fuses the 5′ regulatory sequence of the latter gene to the coding sequence of the former resulting in an aldosterone synthase gene whose expression is under the control of adrenocorticotropic hormone (ACTH) rather than angiotensin II (1). This causes ectopic secretion of aldosterone from the adrenal fasciculata, leading to increased salt and water reabsorption in the kidney, plasma volume expansion, hypertension, and suppression of the renin-angiotensin system. Specific antagonists of the targets of aldosterone (the mineralocorticoid receptor and the epithelial type of sodium channel; ENaC) are used to treat the patients with GRA.
Syndrome of Apparent Mineralocorticoid Excess
Syndrome of apparent mineralocorticoid excess (AME) is an autosomal recessive trait caused by 11-β hydroxysteroid dehydrogenase (11-β HSD) deficiency (2,3). In patients with AME, cortisol is not inactivated in the renal collecting duct cells because of the deficit of 11-β HSD. As a result, cortisol is free to activate the mineralocorticoid receptor, leading to stimulated reabsorption of sodium by the ENaC. This salt retention in turn leads to hypertension. Glycyrrhetinic acid present in licorice extracts inhibits 11-β HSD, and overindulgence of licorice presents a syndrome similar to AME (4).
Liddle's Syndrome
Liddle's syndrome is an autosomal dominant disorder characterized by an increased activity of the ENaC, which leads to increased reabsorption of sodium and water in the renal collecting tubules. The ENaC is composed of three subunits, α, β, and γ, and all three subunits are needed for normal activity of the channel (5). Examination of the ENaC genes in patients with Liddle's syndrome revealed mutations in the genes encoding the β or γ subunit of the ENaC, which delete the carboxy terminus of the respective subunits (6,7,8). With these mutations, the ENaC remains inappropriately on the cell surface, and the number of channel proteins expressed at the surface of the apical membrane of the collecting duct is consequently increased. This in turn leads to increased activity of the channel, sodium reabsorption, and hypertension (9).
Although these several simply inherited forms of hypertension are rare, they may provide insight into the pathogenesis of more common forms of hypertension. All forms of hypertension discussed above cause an increase in sodium reabsorption in the renal tubules. Recently, mutations have been found that inactivate the function of electrolyte transporters, such as the thiazide-sensitive NaCl cotransporters (TSC) expressed in the distal convoluted tubules and connecting tubules, and the bumetanide-sensitive NaK2Cl cotransporter (BSC1) expressed in the thick ascending limb of the loop of Henle. These mutations cause a decrease in sodium reabsorption in the renal tubules, and respectively cause Gitelman's syndrome (10) and Bartter's syndrome (11)—both associated with low BP. Loss of function mutations affecting the potassium channel in the thick ascending limb and collecting ducts (ROMK) and the chloride channel in the thick ascending limb (CLCNKB), also cause Bartter's syndrome (12,13). These findings suggest that mutations that increase the activity of these electrolyte transporters are candidates for involvement in essential hypertension.
Essential Hypertension
Essential hypertension is high BP without any obvious cause. Although there is a strong genetic component to the condition, in only a small percentage of individuals is high BP a consequence of changes in single genes inherited in a simple Mendelian manner as we have seen above. Since families in which hypertension is inherited simply account for only a very small portion of individuals with hypertension, one may conclude that the condition is usually not due to single dominant or recessive mutations. In the majority of cases, the pattern of inheritance is not clear, suggesting that BP is determined by actions of many genes. A likely hypothesis is that essential hypertension results from combinations of small genetic variations, many of which can be considered to be within the range of normal variations and may not be significantly harmful individually. The particular combination of variations in different hypertensive individuals may not be the same.
Tools to Identify a Candidate Gene
The experimental tools to identify genes that contribute to BP can be categorized as analytic or synthetic. A typical analytic experiment may aim to identify a candidate gene by looking at the segregation patterns of genetic markers vis-a-vis BP. Jacob et al., using this method, found cosegregation between genetic markers close to and including the angiotensin-converting enzyme gene locus and BP in rats (14). Another type of analytic approach is to look directly for gene variations that correlate with the phenotype. Good examples include a renin gene variation in rats (15) and a polymorphism in the human angiotensinogen (methionine versus threonine at position 235). Individuals who inherit the threonine variety are more likely to have higher BP and higher plasma angiotensinogen than those who inherit the methionine variety (16).
In general, analytic experiments demonstrate a correlation (and often genetic linkage) between specific genetic variants and BP. However, it is often difficult to prove that the detected change in a candidate gene is the cause of the hypertension. In particular, these experiments usually do not exclude the possibility that the changes in the phenotype are the consequence of a difference in another gene linked to the scored variant. To prove causation in a complex system requires experiments designed to detect the effect of changing only one variable at a time. Properly designed synthetic experiments in the mouse with a targeted gene meet this requirement and allow proof of genetic causation. In carrying out a synthetic experiment of this type, it is first necessary to pick a test gene based on previously acquired data from physiologic or analytic experiments. For example, in the case of angiotensinogen, the previous analytic experiments suggested that it would be a valuable test to generate mice with threonine at position 235 instead of methionine. However, in practice, a simple experiment of this type is not possible in the mouse because there is no methionine at position 235 in mouse angiotensinogen.
Gene Titration
For this reason, and because a difference in the plasma concentration of angiotensinogen was associated with the methionine/threonine difference, a “gene titration” method was devised to alter the plasma level of angiotensinogen by a genetic manipulation (17). Gene titration enables one to vary the expression of a gene by generating animals having different numbers of copies of the gene. Thus, the heterozygotes for gene disruption allow one to study what happens when the expression of a gene is decreased (but not abolished). Conversely, by adding a copy of the candidate gene at its normal chromosomal location by duplication of the gene, one can observe what happens when the amount of the gene product is increased. The level of decrease or increase achieved by this type of experiment is typically -50% to +100% of normal, which is of the order of magnitude of normal physiologic variations. At first sight, one might think that by changing gene copy number, some compensation will occur so that no change in the phenotype will be seen. However, in the earlier literature there are data predicting that a gene titration experiment is really likely to work. Thus, back in the 1980s, Charles Epstein looked at data for more than 40 genes and reported that for every gene he examined, the amount of gene product in individuals who had three copies is 1.5 times normal, while individuals who had one copy of the gene had half the product. These data suggest the existence of quantitatively precise gene dosage effects (18), and these gene dosage effects suggest that control mechanisms to regulate the final amount of a gene product at a fixed level do not exist. This is the theoretical basis of gene titration experiments.
To generate animals having different gene copy numbers for a gene titration experiment, both gene-disrupted and gene-duplicated animals are needed. Figure 1 shows how this is possible from the point of view of gene targeting. Homologous recombination between the targeting construct and the gene locus happens during a gene disruption in the manner shown in the left side of the figure. In a gene duplication experiment, however, the DNA fragments used to make the targeting construct are assembled in a different arrangement, and the result is different. Thus, during homologous recombination with this type of construct, the gap between the 5′ and 3′ ends of the gene fragments that were used for making the targeting construct is first repaired, and the crossover then continues in a manner that generates two copies of the gene on one chromosome. Animals with one gene copy are the heterozygotes for gene disruption. Animals with two copies are wild type. Animals with three and four copies are heterozygotes and homozygotes for gene duplication.
;)
Figure 1: Two models of gene targeting. (A) Gene disruption. (B) Gene duplication. The top lines represent the target gene in its natural chromosomal position; the middle lines, the targeting constructs; the bottom lines, the modified genes after homologous recombination. Neo designates neomycin-resistant gene for positive selection. The orientation of Neo transcription is shown in the open arrows. TK indicates thymidine kinase gene for negative selection.
The advantages of gene duplication compared to classic transgenesis for studies of quantitative variation are twofold. First, with a duplicated gene, the gene product and the regulation of its expression are very well conserved, because the duplicated gene is located at the normal chromosomal position and the cis-acting elements are the same. Second, the changes in expression caused by the changes in copy number from one to four are generally of the same order of magnitude as is observed physiologically. This is an important contrast to classic transgenic animals, in which the number of transgene copies often reaches hundreds, and the position where classic transgenes integrate varies. Therefore, the levels and regulation of their expression are often nonphysiologic, and sometimes integration of the transgenes disrupts another gene. For these reasons, gene titration experiments match naturally occurring quantitative variations more closely than do classic transgenesis experiments.
Gene Disruption (Zero Copy)
The value of generating animals homozygous for gene disruption (gene “knockout” animals) to determine the effects of complete absence of a gene product needs little emphasis here. A great deal of information on gene function has been obtained from zero-copy animals. Generating them in their own right is of course worthwhile. They can also be obtained as a byproduct from one-copy animals made in the course of a gene titration experiment.
Natriuretic Peptide System
Gene Titration of the Natriuretic Peptide Receptor 1
The natriuretic peptide receptor 1 (Npr1; also referred to as Npra or GC-A), mediates the biologic functions of both the atrial natriuretic peptide (ANP) and the type B natriuretic peptide (BNP). Ligand binding activates the guanylate cyclase (GC) activity of the receptor. As the natriuretic peptide receptor 1 (Npr1) gene copy number is increased, a linear progressive increase in GC activity is observed (Figure 2) (19). The BP of mice having one copy of the Npr1 gene averaged 9 mmHg above normal and mice having three copies of the Npr1 gene had BP 5 mmHg below normal (19). These results establish a clear causation and prove that a decrease in the expression of the Npr1 gene causes BP to rise while increasing expression causes a decrease in BP. It is important to note that these changes are observed in animals that have all homeostatic mechanisms intact, and so represent a net effect despite the existence of various means of normalizing BP.
Figure 2: Renal guanylate cyclase activity as a function of copy number of the natriuretic peptide receptor 1 (
Npr1) gene. Rates of atrial natriuretic peptide (ANP)-stimulated cGMP synthesis in kidney membrane preparations from mice with zero, one, two, three, and four copies of the
Npr1 gene are shown as a percentage of that of wild type (two-copy). Error bars represent the SEM. Modified from reference (
21) and unpublished data.
Gene “Knockout” of Npr1
Hearts from Npr1 zero-copy animals show severe cardiac hypertrophy, which are nearly twice as large as those of wild-type animals (20). Although hypertension is a known risk factor for ventricular hypertrophy, the degree of hypertrophy in the Npr1 zero-copy animals is greater than that expected from their high BP because other mouse models with a similar degree of hypertension do not show this degree of cardiac hypertrophy; comparable models include endothelial-type nitric oxide synthase zero-copy mice and angiotensinogen four-copy mice. The mechanism leading to the cardiac hypertrophy of the Npr1 zero-copy animals is now under investigation. Its extent was not expected before execution of the experiment. Overall, these observations suggest that it will be useful to look for any unique variations of the natriuretic peptide receptor gene in human patients with cardiac hypertrophy. Lopez et al. have also studied animals with the Npr1 gene disrupted. These investigators reported that the animals had hypertension that was not affected by salt intake (21).
Atrial Natriuretic Peptide
Changes in the amount of ANP, one of the ligands of Npr1, had also been studied by gene disruption. The BP levels of heterozygote and wild-type animals were not different when they were fed regular chow (0.5% NaCl) (22). However, when the animals were fed a high salt diet (8% NaCl), the BP of heterozygote animals was increased by 28 mmHg, while the BP of wild-type animals was not changed (22). These results demonstrate that a genetic reduction in ANP expression can cause salt-sensitive hypertension. In the opposite sense, transgenic mice overexpressing ANP or BNP show lower BP (23,24).
Renin-Angiotensin System
Gene Titration of Angiotensinogen
Angiotensinogen (AGT) is produced in the liver and secreted into the blood where it is cleaved by renin. This reaction yields the inactive peptide angiotensin I, from which angiotensin II is formed by the action of angiotensin-converting enzyme. Angiotensin II, the active peptide, is a potent vasoconstrictor that is also able to stimulate the synthesis and secretion of aldosterone in the adrenal glomerulosa. The levels of AGT in the plasma progressively increase with the increase in the gene copy number in a gene titration experiment (17). In this case, BP rises linearly as the number of copies of the Agt gene increases (17). Again, except for the targeted gene, nothing else is altered and the animals have all of the homeostatic mechanism intact. And again they do not get their BP levels back to normal. So this experiment definitely demonstrates that different levels of the functions of the Agt gene will directly cause significant changes in BP. Subsequent to this work, a base pair difference was found between the promoter of the human 235 threonine type of AGT gene and the 235 methionine type, which changes the effectiveness of the promoter in the expected direction (25). The data suggest that the methionine/threonine substitution is in fact an irrelevant difference that is linked to the real causative difference; however, without an experimental test of causation, it is difficult to be certain. Tanimoto and coworkers have also disrupted the Agt gene, but they did not observe any differences in BP between the wild-type and the heterozygous animals (26). They used embryonic stem cells derived from a C56BL/6/CBA F1 embryo and mated their chimeras to outbred ICR mice. The genetic heterogeneity inherent to this scheme might have made the detection of the subtle difference between the wild-type mice and the heterozygotes difficult. Indeed, the design of matings to allow the detection of subtle changes in a phenotype is extremely important in this type of quantitative experiment, as has been further discussed in a previous review (27).
Kidney from Angiotensinogen Zero-Copy Mice
Homozygotes for disruption of the Agt gene not only have reduced BP, but also have thickened arterial walls in their kidneys accompanied by an increased number of smooth muscle cells, atrophy of the cortex with interstitial fibrosis, and an infiltration of chronic inflammatory cells (17,28). Similar observations have been reported in the kidneys from angiotensin-converting enzyme zero-copy mice (29) and double knockout mice for type 1A and 1B receptors for angiotensin II (30), indicating that the renin-angiotensin system is indispensable for preserving normal kidney form including postnatal development and function. An increase in transforming growth factor and in platelet-derived growth factor expression may be involved in these changes (28). However, the detailed mechanism behind these changes remains to be elucidated. The kidneys of Agt one-copy animals are indistinguishable from wild type.
Renin
Some strains of mice (including C56BL/6) have one copy of renin gene (Ren-1c) per haploid genome, and other strains (including 129) naturally have two closely linked renin genes (Ren-1d and Ren-2). Clark and coworkers have disrupted the Ren-1d gene and found that this decreased BP in heterozygote females and homozygote females, respectively, by 8 and 13 mmHg compared to wild type (31). However, they did not find any difference in BP of the males. Disruption of the Ren-2 gene did not lead to any changes in BP (32).
Angiotensin-Converting Enzyme
The angiotensin-converting enzyme (Ace) gene consists of two homologous regions and codes for two isozymes: somatic ACE and testis ACE (33,34,35). The testis-specific form of ACE has its own promoter in intron 12 and is encoded by the 3′ half of the gene; it is found only in postmeiotic spermatogenic cells and sperm (36). When the Ace gene was disrupted so as to prevent the synthesis of both isozymes, the BP levels of heterozygotes were not different from wild type, despite the reduction of plasma ACE levels to 50% normal (37). Similarly, when the Ace gene was duplicated, BP levels of three-copy heterozygotes and four-copy homozygotes were normal despite their increased plasma ACE (150, 200% normal) (37). Thus, the gene titration experiment shows that changes in the copy number of the Ace gene do not affect BP. This result suggests that the genetically determined quantitative changes in ACE known to be prevalent in the general human population are unlikely to affect BP.
This result raises an obvious question: Why do ACE inhibitors work? When ACE decreases by 50%, as in heterozygotes for the Ace gene disruption, angiotensin I levels increase, and this increase compensates for the decreased level of ACE. The net result is that angiotensin II levels change only slightly and BP levels do not change. However, this compensation cannot continue indefinitely when ACE levels are more substantially decreased, as can occur using ACE inhibitors. As the inhibition gets more profound, eventually angiotensin I levels reach a plateau. Further decreases in ACE activity beyond this plateau point now cause decreases in angiotensin II levels, so at this point the system becomes sensitive to further ACE inhibition. These effects of ACE inhibitors have been simulated using a computer program based on the STELLA system (High Performance Systems, Inc., Hanover, NH) (38). The data obtained by simulation are supported by previous experimental data by Campbell et al. (39).
Angiotensin II Receptors
Three different receptors for angiotensin II have been identified: type 1A, type 1B, and type 2. Each of these receptors is encoded by a different gene on a different chromosome. The majority of the known physiologic effects of angiotensin II are mediated by the type 1A receptor. The gene coding for the angiotensin II type 1A receptor (Agtr 1a) has been disrupted by two groups, and both male and female heterozygotes show about a 12-mmHg decrease in BP compared with wild-type animals (40,41). Disruption of the type 1B receptor gene alone did not show any change in BP (42). The type 2 receptor of angiotensin II is encoded on the X chromosome. The gene (Agtr 2) has been disrupted by two groups: Ichiki and coworkers found a 13-mmHg increase in BP in their zero-copy animals, rather than the expected decrease (43), but Hein et al. did not find any change in BP (44). The reasons behind these differences are not known, but may relate to differences in the genetic backgrounds of the experimental animals used by the two groups of investigators.
Nitric Oxide Synthase
There are three types of nitric oxide synthase (NOS), 1, 2, and 3, or neuronal, inducible, and endothelial, respectively. The endothelial type of NOS expressed in endothelial cells produces nitric oxide, which reduces vascular tone. Heterozygotes for the Nos3 gene disruption show a tendency toward higher BP but the data did not reach significance (45). Homozygotes for inactivation of the Nos3 gene have 18 mmHg higher BP levels compared with wild-type animals (45,46). In contrast, disruption of either the neuronal (47) or the inducible type of NOS did not cause significant changes in BP.
Endothelin-1
Interesting unexpected results following disruption of the gene coding for endothelin-1 have been reported by Kurihara and coworkers (48). Homozygotes for disruption of the endothelin-1 gene died soon after birth consequent to craniofacial abnormalities. Heterozygotes had 11 mmHg higher BP levels compared with wild-type animals instead of the lower BP anticipated. This observation that reduction of endothelin leads to an increase in BP was unexpected because of the known potent pressor effects of endothelin-1.
B2 Receptor for Bradykinin
Bradykinin modulates water and electrolyte metabolism by increasing intracellular calcium activity via the bradykinin B2 receptor, which leads to activation of the endothelial type of NOS and also to prostaglandin synthesis. These changes lower BP. The results of inactivating the Bdkrb2 gene have been reported by two groups. Madeddu and coworkers reported that heterozygotes for disruption of this gene did not show significantly higher BP compared with wild-type animals, whereas the homozygotes had 15 to 19 mmHg higher BP than wild-type mice or heterozygotes (49). If the animals were fed a high salt diet, they showed much higher BP, and then even the heterozygotes had higher BP than wild type. Alfie et al. observed higher BP in homozygotes and only when they were fed a high salt diet (50).
Classification of Genes Affecting BP
Many genes related to BP control have now been disrupted, and some of them have also been duplicated. The genes can be classified according to the changes in BP, which accompany quantitative changes in their expression (not including complete absence of gene function). In the first group of genes, BP changes are observed when expression of the gene or the number of gene copies is changed. Npr1 is an example: BP increases as its expression decreases. In the second group of genes, changes in gene copy number do not change BP unless another environmental factor is changed. The ANP gene is an example: BP increases in the heterozygotes for disruption but only when the animals are fed a high salt diet. In the third group of genes, no differences in BP have been detected as expression is changed genetically with or without additional environmental factors. Clearly, many genes will be in the third group because their function is completely unrelated to cardiovascular/renal function. But modest changes in the expression of genes that code for a product that is essential for normal cardiovascular function may still have no effect on BP. The Ace gene is an example.
Combining Mutations Together through Breeding
The magnitude of the changes in BP observed when the level of expression of a candidate gene is changed may suggest the importance of the gene in influencing basal BP. Little is known, however, of the interaction between different genes, which can best be studied by combining deliberately engineered variants through breeding. One might guess that an animal that has high BP could be made by combining pressure-raising mutations in two different systems. It will be interesting, for example, to see the BP in an animal having one copy of the Npr1 gene and so a decreased amount of the natriuretic peptide receptor, and four copies of the Agt gene and so an increased amount of angiotensinogen.
Conclusion and Future Direction
We have discussed the use of gene targeting for studying hypertension with special emphasis on gene titration experiments. Gene titration is a powerful tool for analyzing the effects of genetically controlled quantitative variations such as are likely to be observed in many common diseases with a complicated pattern of inheritance. The method has particular value in allowing proofs of causation between a specific gene variation and the phenotype of interest. Many common kidney diseases as well as hypertension are likely to prove to be quantitative genetic traits arising from different combinations of genetic variants and gene—environment interactions. Animal models have the advantage that controlled matings can be used that allow the detection of synergistic or antagonistic interactions between mutant genes free from all other genetic variations and unintentional variations in environmental factors. Yet the effects of intentional variations in environmental factors such as dietary salt can easily be determined, thus providing opportunities to evaluate the interaction between gene variation and environment. More complex, but still approachable, are the interactions between systems, such as the interactions between hypertension and atherosclerosis, or between hypertension and end-stage renal diseases. These kinds of studies will help us understand the pathophysiology of common diseases with complicated patterns of inheritance, develop useful genetic screening tests, and identify potential targets for therapies, so that we can improve the efficacy of treatment and reduce the mortality and morbidity from these serious common diseases.
Acknowledgments
We thank Nobuyo Maeda, Hyung-Suk Kim, and Thomas M. Coffman for their help and discussion.
American Society of Nephrology
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