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INVITED REVIEW

ERNEST H. STARLING DISTINGUISHED LECTURESHIP OF THE WATER AND ELECTROLYTE HOMEOSTASIS SECTION, 2005
Invited Review:

Genetic variations of tubular sodium reabsorption leading to "primary" hypertension: from gene polymorphism to clinical symptoms

School of Nephrology, University Vita Salute, San Raffaele, Milan, Italy

	ABSTRACT

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The definition of the most appropriate strategy to demonstrate causation of a given genetic-molecular mechanism in a complex multifactorial polygenic disease like hypertension is hampered by the underestimation of the complexity arising from the genetic and environmental interactions. To disentangle this complexity, we developed a strategy based on six steps: 1) isolation of a rodent model of hypertension (Milan hypertensive strain and Milan normotensive strain) that shares some pathophysiological abnormalities with human primary hypertension; 2) definition in the model of the sequence of events linking these abnormalities to a genetic molecular mechanism; 3) determination of the polymorphism of the three adducin genes discovered in the model both in rats and in humans; 4) comparison at biochemical and physiological levels between the rodent models and the hypertensive carriers of the "mutated" gene variants; 5) evaluation of the impact of the adducin genes in hypertension and its organ complications with association and linkage studies in humans, also considering the genetic and environmental interactions; and 6) development of a pharmacogenomic approach aimed at establishing the therapeutic benefit of a drug interfering with the sequence of events triggered by adducin and their effect's size. The bulk of data obtained demonstrates the importance of a multidisciplinary approach considering a variety of genetic and environmental interactions. Adducin functions within the cells as a heterodimer composed of a combination of three subunits. Each of these subunits is coded by genes mapping to different chromosomes. Therefore, the interaction among these genes, taken together with the interactions with other modulatory genes or with the environment, is indispensable to establish the adducin clinical impact. The hypothesis that adducin polymorphism favors the development of hypertension via an increased tubular sodium reabsorption is well supported by a series of consistent experimental and clinical data. Many mechanistic aspects, underlying the link between these genes and clinical symptoms, need to be clarified. The clinical effect size of adducin must be established also with the contribution of pharmacogenomics with a drug that selectively interferes with the sequence of events triggered by the mutated adducin.

genes; blood pressure; Na-K pump; Na channel; adducin

The difficulty lies, not only in the new ideas, but in escaping the old ones.

J. M. Keynes

STARLING'S LONG-LASTING concern was to use physiology as a means to bring basic science to the bedside. Indeed, by integrating the capillary fluid exchange with the cardiac pump function, Starling provided a substantial contribution to bridge the gap between the contemporary basic science of circulation and the clinical symptoms of cardiac failure.

The purpose of my lecture is to honor this Starling's legacy by trying to integrate animal and patient pathophysiology and clinics with genetics to establish some specific molecular causation underlying abnormalities in tubular Na reabsorption leading to hypertension. Thus new diagnostic and therapeutic molecular targets may be used to "classify" and "cure" specific subsets of patients, within the heterogeneous population of patients labeled as "primary hypertensives." The integration of almost all of the biological disciplines (genetics, molecular and cell biology, renal, cardiovascular and nervous pathophysiology, animal models, clinics, pharmacology, epidemiology, and therapeutics) is mandatory to link genetic variations to clinical symptoms, as schematically illustrated in Fig. 1.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Interaction of genetic and environmental backgrounds and biological factors on the sequence of events linking a DNA variation to a clinical symptom. Right: two hypothetical populations carrying the "mutated" and the wild-type gene variant. Because the progressive increase of the influence of the factors (left), the distribution of the values of the two populations tends to overlap as we move from the DNA level to the clinical symptom. Therefore, at this highest level of organization, the separation of the two populations may be very difficult.

Three strategies (45, 47, 131) are available. The "bottom-up" and "top-down" approaches are not based on a priori genetic hypothesis. The former addresses the identification of some DNA regions called BP QTL in the animal model that may be postulated to be relevant for humans when they occur in conserved syntenic regions. These BP QTLs are identified by using the genomic-wide scans in animal genetic crosses or in humans (47). Indeed, some BP QTLs that are involved in these regions have been identified both in rats and humans. However, after the identification of these BP QTLs, the time-consuming procedures aimed at detecting the gene(s) responsible for the BP QTL's effect is hampered by the complexity above mentioned. This strategy may also include the gene expression profiling by the various high-throughput "omics" applied to congenic strains (where, with appropriate backcrosses, a short DNA segment of the affected strain containing the QTL or locus of interest is introgressed into the other control strain), and vice versa, or consomic strain (where a whole chromosome of one strain is introgressed into the other strain) (40, 136). The top-down approach moves from clinical symptoms (or intermediate phenotypes that are alterations in cellular or organ function somehow associated to the clinical symptoms) down to the molecular and DNA abnormalities underlying these phenotypes. The "candidate gene" approach evaluates the possible causation of polymorphisms of gene-encoding proteins known to be involved in those cellular or systemic mechanisms underlying the regulation of blood pressure or organ damage. The hypothetical molecular mechanisms of primary hypertension, based on the previous bulk of knowledge obtained with the "classical" pathophysiological methodology, can be proved (or disproved) by all of the new DNA or genetic tools. Both the bottom-up and the candidate gene approaches have been applied linkage and association studies in humans that try to establish genotype-phenotype relationships (39, 78, 92).

The various genetic manipulations in mice (homologous gene replacement or knockout) applied to study the role of these genes may also be included in this strategy.

	PERSONAL APPROACH

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Over the past 20 years, our group has combined these approaches both in a rodent model [Milan hypertensive strain (MHS) and Milan normotensive strain (MNS)] and humans. In particular, we have developed a strategy aimed at defining the subset of patients, if any, sharing the largest degree of pathophysiological and genetic similarities with the rodent model. This strategy helped to circumvent the problem of the large genetic, environmental and biological heterogeneity underlying the clinical syndrome of primary hypertension that is responsible for the difficulty of replicating genetic results in different populations and to transfer results in rodent models to humans (134, 154). Such a failure is due to the complexity of the interactions (epistatic, additive, and multiplicative effects) between a given gene and the peculiar genetic network, where this gene works (113, 163). The inbred rodent strains used for these studies have been selected by dozens of brother-sister matings and after having overcome the inbreeding degeneration. The resulting genetic networks differ from those originating from the natural selective pressure driven by the genetic-environmental interactions. Such a difference may influence the comparisons between rat and human genetic data, especially when the associated phenotypes are deeply influenced by the genetic network and the environment (154). For instance, in the presence of an unknown epistasis between two loci, the increase of the sample size would never permit the true estimation of one single locus causation with the available statistic probability models based on randomness (113, 154) because of the difficulty in accounting for the environmental or biological factors (76) (lifestyle factors together with age, gender, BMI, phase of the disease, or previous therapy) that modulate these interactions in humans (113). In spite of the fact that all these sources of variations have been widely recognized also in the so-called monogenic diseases (127, 152), most studies on genetics of hypertension do not measure them or simply try to account for them by mathematical modeling.

On these grounds, we developed a strategy that can be subdivided into the following six steps: 1) look for similarities between the rat model and human at pathophysiological level to support the hypothesis that the model may capture at least a portion of the genetic variation underlying hypertension in humans; 2) use the animal model to identify the pathophysiological mechanism(s) responsible for hypertension and to identify the underlying protein abnormalities that may be an expression of genetic polymorphisms (Use genetic crosses to assess whether the "hypertensive variants" of this polymorphism cosegregate with a significant increase in blood pressure in F2 MHS-MNS populations or in congenic rat strains.); 3) look for polymorphisms on the same genes in humans and, if any, use them to classify patients for more appropriate comparisons between the two species; 4) establish similarities or differences at cell level between the human and rat gene variants using several cellular and molecular biology techniques; 5) apply the classical association or linkage studies to measure the clinical impact of the human gene variants, being aware of the limitations of these methods when not considering the modulatory effect of genetic-environmental and biological factors on the gene of interest; and 6) identify selective inhibitors. The role of a molecular mechanism(s) in a complex disease is almost impossible to establish without selective inhibitors. For instance, after 20 years of fruitless debate, the role of the renin-angiotensin system (RAS) in hypertension and its related disorders became much clearer when angiotensin-converting enzyme (ACE) inhibitors or ANG II antagonists became available. A pharmacogenomic approach could similarly be considered to establish whether a selective interference with the sequence of events triggered by the putative gene variant may offer a therapeutic advantage in carriers of this gene variant. This also may allow an estimation of the clinical effect size of the gene of interest.

The ordered complexity of the biological systems may be better understood through the study of relatively simpler relationships evaluated within the appropriate experimental context and accommodated in a scientific hypothesis, after having accounted for the context-dependent component of the measured effect. This logic underlies the progress of the scientific knowledge toward the truth in any branch of science. However, not always, controls or sham-operated animals account for the influence of a given context or experimental procedure per se on the results obtained. Therefore, consistency among the single clinical or experimental results obtained at various levels of biological organization, together with challenging the result against a variety of contexts, furnishes the two most important arguments to validate the role of a given genetic-molecular mechanism in human diseases. For instance, while evaluating the role of glomerular filtration rate (GFR) in animals prone to develop hypertension, we were presented with contrasting findings (56). Measurement of GFR by infusing inulin at a rate that produced a plasma concentration 1 mg/ml showed lower values in prehypertensive MHS compared with normotensive control MNS (56). A subsequent measurement performed with a plasma inulin 0.1 mg/ml, disclosed higher GFR in the prehypertensive MHS (58). These contrasting results were obtained with the same experimental procedure except plasma inulin. Which is the true GFR in MHS and MNS? When the two results were incorporated within the framework of the other results obtained by studying the cellular response to osmotic pressure (59) and the tubular function measured in isolated kidneys (121, 122) in isolated tubular cells (101) or in cell membranes prepared from these kidneys (52, 72), it is clear that the GFR is elevated in MHS.

Several reasons supported these conclusions: a plasma inulin concentration of 1 mg/ml exerts an osmotic force that may produce different cellular reactions; according to the genetic network (58), only a higher GFR is consistent with 1) the findings in isolated MHS and MNS kidneys (121, 122); 2) a constitutive increase in renal Na reabsorption (as it occurs in other contexts, i.e., mineralcorticoid administration), either measured in the whole kidneys (121, 122) or inferred from the Na transport measurements across the cell membranes removed from MHS and MNS kidneys (52, 53, 72); 3) a mild and transient renal Na retention during development of hypertension (15). Also in humans, GFR may be higher or lower in offspring of hypertensive parents compared with offspring of normotensive parents (17, 46, 145). These differences may be due to either genetic or environmental factor heterogeneity or to the experimental procedure per se. For instance, an experimental procedure that does not minimize the sympathetic drive to the kidney (by reducing stress or postural effects) may detect the kidney effects of a possible increase in sympathetic drive to the kidney in offspring of hypertensive parents (43) and not the intrinsic renal function characteristics of these subjects. There are other data supporting the notion that the control group does not capture all the noise due to the experimental procedure per se (16).

	COMPARISON BETWEEN THE RODENT MODELS AND HUMANS

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The comparison shown in Table 1 between the data on the MHS and MNS rodent model and those on humans (22, 56) must be examined by keeping in mind the above considerations, and particularly, the content of Fig. 1. In fact, the context's influence on the genetic mechanisms of interest progressively increases moving from the lower levels of biological organization (molecular and cellular biochemistry) to the higher ones (organs or intact organisms). Therefore, some inconsistencies between human and rat data may be anticipated when higher levels are examined. Collectively, these data show some pathophysiological similarities between the two species, thus suggesting that the model may capture at least a portion of the genetic and/or environmental heterogeneity of human primary hypertension. Of course, this tentative conclusion requires further corroboration with appropriate experimental data.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Gene structure of ADD1, ADD2, and ADD3 and single-nucleotide polymorphisms (SNPs) identified in human and rat hypertension. Exon-intron structure of ADD genes is indicated by boxes and lines; gray and open boxes represent the exons for coding and noncoding regions of mRNA, respectively. Solid boxes indicate the alternatively spliced exons. Exons that contain alternative donor or acceptor sites within the coding sequence are presented as a/b (exon X for ADD1, exons VII, XII, and XIII for ADD2). The position of human and rat (see underline) SNPs is indicated by the arrow. The SNP coordinate is based on the amino acid change (one letter code, italics) for missense mutation, and nucleotide change for silent or intronic mutation. dbSNP ID for human SNP is reported in brackets. The human and rat gene location is reported on the right (23).

The availability of naturally occurring polymorphisms within the same genes in the rodent model and human in spite of differences at the mutation sites (see Fig. 2) allows comparisons at the various levels of biological organization (protein-protein interactions, cellular biochemistry, organ function, etc.) and the development of specific inhibitors of the sequence triggered by the gene variant of interest.

Rat and human lineages separated 40 millions years ago. The persistence of similarities (if any) of the adducin effects on biological systems involved in blood pressure regulation, despite the large number of the genetic and environmental variations, can provide an important argument to support the inclusion of the newcomer protein adducin within the list of "pressure regulatory" molecules. In fact, from the previously accumulated knowledge on the adducin cellular effects, a pressor regulatory mechanism could not be easily anticipated.

	RELATIONSHIP BETWEEN ADD POLYMORPHISM AND RELEVANT INTERMEDIATE PHENOTYPES IN RATS AND HUMANS

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Cellular effect of adducin variant transfected in tubular cell culture. Tubular cells transfected with the cDNA MHS Add1 variant displayed the following differences compared with cells transfected with the MNS variant. Na-K-ATPase at V_max increased because of an increased number of Na-K pump sites on the basolateral membrane (142), as already observed in isolated tubuli cells from MHS rats (49, 101) (measured as Na-K pump activity at V_max); this was associated with a reduction in the protein endocytosis (49). The actin bundles were thicker, and surface expression of integrin and other membrane proteins was increased (142).

The human hypertensive adducin variant produces the same effects on the Na-K pump when transfected in CV1 Origin SV40 (49) or human renal tubular cells (Torielli L., personal communication). These findings provided strong support to the hypothesis that adducin polymorphism may affect the constitutive tubular capacity for Na reabsorption by acting on the driving mechanism (that is, the Na-K pump on basolateral membrane).

Molecular mechanisms underlying the cellular adducin effects. Incubation of the different human and rat -adducin variants with Na-K pump in a cell-free medium showed that the hypertensive variants from both species activate Na-K pump at a lower concentration than the normotensive ones (55). Moreover, the rat MHS variant accelerated actin polymerization and bundling to a greater extent (142). Adducin may be immunoprecipitated from transfected cells associated with a protein phosphatase A₂ (PPA₂) (49). However, the amount of PPA₂ is lower in cells transfected with the MHS adducin that also have higher levels of adaptin2 (AP_2-µ₂) phosphorylated protein (49). Therefore, the most likely explanations for the reduced endocytosis in MHS adducin-transfected cells are (see also Fig. 3) 1) an impaired phospho-dephospho cycle of AP_2-µ_2, which promotes the clathrin-dependent Na-K pump endocytosis; and 2) a less permissive and stiffer cortical actin cytoskeleton that obstacles the Na-K pump endocytosis.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Influence of adducin polymorphism of Na-K pump endocytosis and renal Na reabsorption. A: the process of insertion and removal (endocytosis) of the Na-K pump on basolateral renal cell membranes of a tubular cell. Reduced endocytosis increases the Na-K pump molecules on basolateral membrane, thus leading to increased sodium reabsorption. B: the interaction among some proteins involved in these processes (49). In the basal condition, the PPA2 -adducin association is reduced in cell transfected with mutated adducin (please note the smaller dimension of the phosphatase PPA2 protein symbol associated to mutated adducing). Because the phosphorylation state of a protein results from the balance between protein kinase and protein phosphatase (PPA2), the reduced PPA2-adducin association favors the AP2-µ2 phosphorylated state, thus impairing the phosphor-dephospho cycle of AP2-µ2. This cycle promotes the association of AP2 to Na-K pump that is a key event for the recruitment of clathrin and the formation of clathrin-coated vesicles (CCV). Therefore, the impairment of this cycle may represent the molecular mechanism underlying the reduced constitutive and dopamine-induced Na-K pump endocytosis observed in the presence of mutated adducin. This schematric does not include another possible mechanism of deficient endocytosis represented by the less permissive, stiffer, cortical actin cytoskeleton of cells transfected with the mutated adducin (23).

Comparison between MHS rats and humans carrying the mutated ADD1. Table 3 shows the direction of changes in rat and in W (or mutated Trp) ADD1 allele hypertensive humans with respect to their appropriate controls, either MNS rats or wild-type ADD1 homozygous hypertensive subjects. Erythrocytes from MHS rats or W ADD1 patients display lower Na content (59, 69, 71) and plasma renin levels (11, 15, 42, 70, 80, 155), higher affinity for Na of the Na transport systems (69, 71), and renal tubular reabsorption (25, 57, 94), whereas the rate of various Na transport systems in erythrocytes tends to be higher (19, 57, 69) with some contrasting results in human subjects (71). The difference in Na content and transport systems between MHS and MNS mimics the differences existing in proximal tubular cells between the two strains (57). This parallelism justifies the use of erythrocytes to carry out comparison between rats and humans. Taken together, these findings support the notion that mutated adducin, either in rats or in humans, tends to maintain a lower intracellular Na because of an increase affinity of the Na transport systems that, together with the increased number of Na-K pump on the basolateral membrane, may drive the increased renal tubular reabsorption with a consequent decrease in plasma renin. When the relationship between renal Na excretion and blood pressure is examined during acute saline infusion in never-treated, mildly hypertensive patients, carriers of the mutated W adducin allele, compared with homozygotes for wild adducin, need a higher perfusion pressure to excrete the same Na load (11, 95).

	RELATIONSHIP BETWEEN ADDUCIN AND HORMONES AFFECTING RENAL Na HANDLING

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In never-treated early hypertensive subjects, the plasma levels of renin, aldosterone, and ouabain are lower in carriers of the mutated adducin compared with the other subjects (87). The downregulation of the hormones promoting hypertension and renal Na retention at the early stages of hypertension suggests a counterregulatory mechanism that tends to limit the rise of blood pressure sustained by other mechanisms, including a primary increase in tubular Na reabsorption. Studies in a predominantly normotensive general population showed an interesting interrelationship among plasma ouabain, blood pressure, and 24-h renal Na excretion taken as an index of Na intake (149). At lower Na excretion, blood pressure is higher in subjects with plasma ouabain above the median value of the population than in subjects with plasma ouabain below this median value. Conversely, at higher level of Na excretion, blood pressure is lower in subjects with higher plasma ouabain (149). A dose-dependent effect of the mutated W ADD1 allele on plasma ouabain is found in the general population, being that the plasma levels of homozygous W allele carriers were 20% higher than those of wild-type homozygous carriers (149).

Taken together, all these findings suggest a relationship between ADD1 polymorphism and plasma ouabain based on an interaction with the Na-K pump. We may tentatively propose that ouabain and adducin polymorphism play a key role in the homeostatic regulation of blood pressure in response to variations in Na intake and that a ouabain increase may limit either the rise of blood pressure in the case of a high-salt diet or the fall in blood pressure in the case of a low-salt diet.

The molecular mechanisms underlying the regulation of this set point, as well as the difference in the mutated adducin-plasma ouabain relationship between the general population and the never-treated early hypertensive subjects, remain to be clarified.

	STATISTICAL AND OTHER GENETIC APPROACHES AT THE WHOLE ORGANISM LEVEL

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Studies in rats. Studies involving rat genetic crosses demonstrate that only the MHS Add1 variant cosegregated with a significant increase in blood pressure in MNS-MHS F2 generation (21), whereas MHS Add2 and Add3 variants interact with Add1 in determining the level of blood pressure (21, 160). Also, a whole genome scanning in the F2 population revealed a QTL containing the Add1 locus together with other BP QTLs (160). Moreover, an interaction is present between the Add1 locus and markers on Add2 and Add3 loci (160).

The development of congenic rats by introgressing in MHS or MNS a short (143) DNA segment containing the Add1 locus of the other strain, confirmed the effect of this locus. From the results of all these studies, it is not possible to distinguish between the effect of the Add1 locus from that of a gene mapping in the close vicinity. However, two observations support the involvement of this locus: 1) the interaction between the adducin's loci, as discussed above and 2) in congenic rats, besides the blood pressure level, the increase of renal Na-K-ATPase activity is also transmitted with the Add1 locus. This observation is important because this activity is also increased by the transfection of the cDNA MHS Add1 variant in tubular cells, as discussed in Cellular effect of adducin variant transfected in tubular cell culture (142).

Studies in humans. According to a PubMed search, 67 linkage or association or population studies evaluated the role of adducin in hypertension, the related phenotype, and organ damage. Seven studies were from our group (11, 29, 42, 87, 94, 95, 126). Sixteen referred to clinical data collected and analyzed by others but were blindly genotyped by us (8–10, 32, 44, 69, 70, 129, 133, 138, 146–150, 157). Forty-five described results obtained by others on African, American, Chinese, and Japanese populations (2, 3, 13, 14, 26–28, 30, 36–38, 48, 64, 71, 73, 74, 79–81, 83, 84, 88, 100, 103, 106–108, 110–112, 115–119, 124, 128, 132, 140, 144, 151, 153, 155, 156–162). Six human linkage studies (28, 42, 44, 81, 138–147) showed positive results when a DNA marker mapping within 30 kb from the ADD1 locus or single-nucleotide polymorphisms (SNPs) on 1 or 3 adducin genes were considered either alone or in combination with each other or with ACE D allele or salt intake. When DNA markers mapping at a much larger distance from the ADD1 locus were used, negative results were found in four studies (26, 36, 73, 112). Within this large distance, many haplotype blocks were included (International HapMap Project, www.hapmap.org); thus the clinical significance of these findings is poor. In 18 (11, 14, 30, 42, 44, 64, 69, 70, 71, 94, 95, 108, 118, 126, 132, 138, 150, 153) out of 20 (37, 144) studies, positive associations were found between adducin polymorphism and blood pressure or one of the variables involved in blood pressure and renal Na handling regulation, such as RAS, renal function, nitric oxide, and response to diuretics. There were mixed results in case control studies. In 10 populations, a positive association was found (10, 13, 32, 42, 80, 116, 119, 124, 156), whereas in two populations, a positive association was found only in women (128) or young subjects (132). In 13 populations, no association was present (2, 3, 38, 70, 74, 79, 84, 88, 100, 116, 119, 124, 151). In three studies (116, 119, 124), both positive and negative associations are observed in different populations. It is noteworthy that the hypertensive carriers of the mutated W (Trp) ADD1 allele show a lower renin and a larger response to thiazide diuretics than the carriers of the wild allele, even in a population (70) where no difference in the W ADD1 allele frequency is detected between hypertensive and normotensive patients.

Also, in predominantly normotensive populations or in normotensive subjects, the results may be either negative (26–28, 83) or positive (129, 138, 148). Positive findings are obtained only when interactions between ADD1 and ACE or ADD2 polymorphisms are considered in subsets of patients.

Four (42, 70, 118, 126) of five (144) studies showed a selective beneficial effect of diuretics in carriers of the mutated ADD1. Twelve (8, 9, 14, 48, 106, 110, 111, 117, 118, 148, 150, 155) of 16 (103, 107, 115, 162) studies found that ADD1 polymorphism alone or in combination with that of ACE positively associates with stroke or coronary heart disease or renal or vascular dysfunctions. In conclusion, when context is taken into account, the results obtained both in rats and in humans are consistent with the notion that the interaction among the adducin loci or between ADD1 and ACE are involved in human and rat hypertension and related disorders.

Clinically relevant genetic interactions. Beside the interactions between ADD1 and ADD2 (21) and between ADD1 and ADD3 on blood pressure or pulse pressure (44) of both rat and humans, another interaction was found in never-treated early hypertensive patients and in a predominantly normotensive population between adducin and ACE. The effect of the W ADD1 allele on the fall in plasma renin (11) and on the increase in blood pressure after a sodium load (11), on the incidence of hypertension in a general population (129), on the intima-media thickness (9) and stiffness (8) of femoral artery, on the decrease of GFR (150), and on the increase in urine protein excretion (150) is augmented in the deleted (DD) ACE genotype and is diminished in the ACE II genotype, compared with the average values of the general population. Cell surface ACE activity in fibroblasts isolated from patients with various ADD1 genotypes was higher in W ADD1 carriers than in wild-type ADD1 homozygotes (161). This may suggest that the W allele increases the number of ACE molecules on the plasma membrane similarly to the effect on Na-K pump.

The bulk of data on isolated fibroblasts and on the various intermediate phenotypes measured in patients supports the hypothesis that the combination of these two genotypes may favor the development of a specific clinical entity characterized by consistent alterations at DNA, cellular, renal, and cardiovascular phenotypes, favoring the development of hypertension and organ damage. The proposal of this new clinical entity is also reinforced by the findings that combination of these genotypes also modulates the magnitude of blood pressure fall after diuretics (126). The strength of this proposal relies on the consistency among data collected from different contexts. The weakness is mainly due to the lack of a proper follow-up on a large group of patients in whom all of these characteristics are simultaneously measured.

	RELATIONSHIP BETWEEN ADDUCIN AND CARDIOVASCULAR ABNORMALITIES, MORBIDITY, AND MORTALITY IN RELATION TO BLOOD PRESSURE AND OTHER FACTORS

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Two (106, 117) out of three studies (118) demonstrate an association between W ADD1 allele and coronary disease in hypertensive patients. A fourth study (140) shows a lower frequency of this allele in myocardial infarction (MI) survivors aged 75 years compared with controls of the same age. Therefore, it is unclear whether these findings suggest a protective effect of the W ADD1 allele in controls or a high risk of premature death in W ADD1 allele carriers with MI. Ventricular hypertrophy has been shown to occur in WW ADD1 genotype (155). The intima-media thickness (9) and femoral artery stiffness (8) are increased in W ADD1 and D ACE carriers. A recent prospective study on 2,235 Belgian residents followed for several years (89) shows that the W ADD1 allele may be a risk factor for total and cardiovascular morbidity and mortality when systolic blood pressure at baseline is included in the analysis as a continuous variable. The hazard ratio for cardiovascular complications associated with the W allele relative to wild homozygotes after adjustment for other risk factors is 2.94 P = 0.01 in patients with stage 2 systolic hypertension (160 mmHg) and 0.83 P = 0.32 in the other subjects. For each 10-year increment in age, the relative hazard ratio associated to the Trp allele increased by 39.7% (89).

W ADD1 allele frequency is around 8% in African populations (10), 12% in African Americans (124), 22–25% in Caucasians (42, 129), and above 50% in Asian populations (81, 147). How can this trend implying some positive effect on biological fitness with the pathological influence on cardiovascular and overall mortality be reconciled? Two possible mechanisms may be proposed. The W ADD1 allele may exert a protective effect at a lower level of blood pressure (89) or at younger ages (32). Second, several studies on epistatic interaction show that the pathological effect of W ADD1 allele occurs in the presence of some other genotypes (for instance ACE DD) (8, 9, 11, 129, 150). Conversely, while in the presence of other genotypes (for instance ACE II), the W allele may reduce the value of "pathological" intermediate phenotypes below the level of the general population. Therefore, the increase in the frequency of the W allele in Caucasian or Asian populations may be due to the "positive" effect on biological fitness of this allele in specific subsets of population. According to the trade-off hypothesis (114), natural selection will increase the frequency of mutations that produce beneficial effects early in life, even though these mutations may be deleterious later in life. This may also account for the high frequency of alleles, such as the D ACE allele, which certainly accelerates cardiovascular and renal damage at older ages (130).

Pharmacogenomics. For many decades, the possibility of selectively blocking a given molecular mechanism has provided a very important tool to disentangle the role of that mechanism within the complex regulatory network of feed-back loops underlying biological homeostasis. By analogy, the demonstration of a selective therapeutic benefit of a drug interfering with the sequence of events triggered by a gene variant (or a combination of gene variants) may provide an additional important argument in favor of their role (23). Although many genes involved in drug metabolism may affect the therapeutic effect (125), such interference should be equally distributed between carriers and noncarriers of the genotypes of interest, provided that a large enough number of patients is used. Among the action mechanisms of the available drugs, that of diuretics is the one that more selectively interferes with that of adducin on tubular Na transport (126). It may therefore be anticipated that diuretics may normalize the faster constitutive tubular Na reabsorption in carriers of the W allele without triggering counterregulatory mechanisms (75). Five studies examined this issue. Two from our group (42, 126), one study from another group that we blindly genotyped (70), and two studies that were carried out by others (118, 144). Measurement of blood pressure falls in newly discovered, never-treated, relatively mildly hypertensive patients after a 2 mo diuretic treatment disclosed a greater effect in carriers of the W ADD1 allele. This selective effect cannot be found in patients with various types and duration of antihypertensive treatment discontinued 4 wk before the diuretic administration lasting 1 mo (144). The early rise of blood pressure after discontinuation of therapy may occur via mechanisms different from those responsible for the slow development of hypertension over the years (133). Indeed, long-term administration of various renin-angiotensin-aldosterone system blockers or diuretics produce an increase of ANG I or II that, per se, may contribute to the increase of blood pressure when therapy is discontinued (41, 133). The most interesting finding is described by Psaty et al. (118) on 1,038 hypertensive patients followed for several years. The incidence of MI and stroke in W ADD1 carriers regularly treated with diuretics is halved compared with that occurring in W ADD1 carriers receiving a nondiuretic antihypertensive treatment producing a similar fall in blood pressure. This selective diuretic effect is not present in homozygotes for the wild adducin allele (118). These results bring us to the heart of the issue regarding the application of pharmacogenomics to improve our ability to prevent organ damage in hypertension in the subset of patients carrying a specific genotype or combination of genotypes.

Diuretics also have other activities (on K, glucose metabolism, RAS, and sympathetic system) that, in the long run, may limit their beneficial effect (91). Therefore, a more selective blockade of the adducin and ouabain effects may offer therapeutic advantages on this subset of patients. In a previous paragraph, the peculiarity of the interactions between these two mechanisms in the regulation of blood pressure at various levels of Na intake is discussed. A compound has been developed that is able to block the effect of mutated adducin (60) or ouabain (63) on the Na-K pump in isolated renal tubular cells. This effect occurs at concentrations that are five order of magnitude lower than those producing other biochemical or pharmacological effects (60, 63). Therefore, this compound may be the appropriate pharmacological tool to explore the impact of these two molecular mechanisms on cardiovascular diseases. Preliminary clinical data (61) seem to suggest that the magnitude of the antihypertensive activity of this compound is associated with 1) the polymorphism of genes encoding adducin and some enzymes involved in endogenous ouabain synthesis and 2) the level of Na intake (61).

	HYPOTHESIS

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The following previous findings are appropriate for a full understanding of the biological and clinical meaning of the adducin data described here.

First, in anesthetized rats the increase in kidney perfusion pressure is associated with the removal of Na transport systems from the luminal and basolateral membranes (93). This phenomenon, which occurs after a few minutes from the pressure increase, is reversible (93, 159).

Second, aldosterone and dopamine affect tubular reabsorption, Na excretion, and blood pressure by mechanisms involving the regulation of the residential time of the Na channel or other transport systems on the luminal and basolateral membranes (35, 50, 135).

Third, most of the genetic mechanisms affect blood pressure via a modulation of the residential time of Na channel or other transport systems on membrane of tubular cells (67, 90). Also adducin and ouabain affect Na transport and blood pressure by regulating the Na-K pump residential time on the basolateral membrane (49, 63). In both the erythrocytes (59, 62) and tubular cells (12) of rats and in the erythrocytes of humans carrying the W ADD1 allele (69, 71), intracellular Na is lower. This strongly supports the primacy of the Na-K pump activity alteration over that of luminal transport system in determining the increase in the tubular cell constitutive capacity of Na reabsorption. All together, these findings support the hypothesis that the molecular mechanisms regulating the residential time on the tubular cell membranes of the Na transport systems may be the biochemical component of the complex transducer system involved in the resetting of the relationship between kidney perfusion pressure and Na excretion.

	LIMITS AND FUTURE DIRECTIONS

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The tubular effect of adducin or ouabain is certainly modulated by a variety of factors (physical, hormonal, luminal) either intrinsic or extrinsic to the kidney together with the genetic network underlying Na tubular transport, blood pressure regulation, and the dietary sodium intake. These interactions hamper the determination of the clinical effect size of adducin. Failure to recognize this complexity (that stands from the results of more than 50 years of pathophysiological research) in the evaluation of the role of adducin (as the role of any other candidate gene) is the main source of confusion and mixed results characterizing the present status of knowledge on genetics of hypertension. After almost 15 years of conflicting data on this issue, the most orthodox geneticists may recognize that the current genetic strategies, generated from monogenic diseases, are unable to demonstrate genetic causation in complex multifactorial diseases like hypertension and the related organ damage (105, 113, 137). The dream to discover a major gene effect in these disorders, using the most sophisticated "homic" high-throughput methodologies, shall be replaced by a more realistic goal to detect major specific gene causation in subsets of patients. These genes may or may not be relevant to the whole heterozygous population that we label as "essential" hypertensive patients (up to 40% of adult population of industrialized countries).

However, the following mechanistic aspects linking the various molecular, cellular, and whole body adducin effects require clarification.

First, the adducin effects on organ damage may be due to renal Na retention that may promote organ damage, per se, as it occurs in a high-salt diet (5, 7). A slight increase in body sodium may favor the production of reactive oxygen species (4, 33, 102, 123), a well-known mediator of organ damage. Also an alteration of actin cytoskeleton associated with an increase of cell-surface expression of integrin or focal adhesion sites may, per se, favor cell-to-cell contact and organ damage (34, 85). The respective role of these mechanisms must be addressed by future studies.

Second, adducin is composed of three subunits with many isoforms that may be tissue or cell specific (99). Therefore, the function of the various heterodimers resulting from these subunits must also be assessed.

Third, as already discussed, we have described two possible molecular mechanisms underlying the increased Na-K pump number on the basolateral membrane of tubular cell produced by the mutated W ADD1 allele. However, most of the proteins involved in these processes are still unknown.

Fourth, the interaction between adducin and ouabain is of particular interest because of the peculiar common effect on the Na-K pump. The data so far available suggest their involvement in the homeostatic control of blood pressure at various levels of Na intake. However, the distinction between a "beneficial" effect on biological fitness and a "detrimental" one on cardiovascular morbidity and mortality must be clarified.

Fifth, a derangement in the regulation of the residential time of Na transport system on the renal tubular cell membrane seems to be one of the mechanisms underlying the alteration of the blood pressure-sodium excretion relationship promoting a "primary" form of hypertension. Therefore, the molecular mechanisms responsible for this phenomenon may constitute a new target for selective pharmacological intervention aimed at normalizing blood pressure without counterregulatory reactions (or side effects) in the subsets of patients in which this mechanism operates. Along this line, a novel compound has been developed (51, 60, 61, 63). However, high-throughput technologies addressed to exploit the therapeutic potential of this pressure regulatory mechanism can be considered.

Sixth, the involvement of vascular resistance also needs to be explored because of the key role of Na-K pump on the regulation of vascular tone (104). Preliminary data seem to exclude any interaction of the mutated adducin with vascular Na-K pump activity (Torielli unpublished). This observation is relevant to explain why an increased Na-K pump activity on the basolateral renal membrane may raise blood pressure. In fact, a similar increase in the vascular smooth muscle cell may decrease the vascular tone, thus buffering the adducin pressor effect at the kidney level. However, the molecular mechanism underlying this differential adducin effect on these two types of cells must be elucidated.

Seventh, this review does not mention the work so far done on Add3. Briefly, studies on spontaneously hypertensive rats, which carries the same Add3 mutation as MHS (141), have shown a lower level of Add3 mRNA and protein in the brain stem (157). Intracellular delivery of Add3 antibodies in the neuronal cell culture, increases their firing rate to a similar extent as the incubation with ANG II (158). MHS also show an Add3 reduction in the same brain areas. These findings open a new area of research regarding the genetic molecular mechanisms underlying the kidney-brain crosstalk in the regulation of blood pressure and body sodium.

Eighth, finally, the estimation of the clinical impact size of adducin polymorphism is of paramount importance for planning health interventions and costs. The new chemical entity mentioned above is the first of a series of compounds that in the future may help in finding this type of answer regarding either adducin or other candidate genes. This new compound interferes with the effect of ouabain at concentrations or doses that are 1–2 orders of magnitude lower than those affecting the adducin effects (60, 63). This may help in assessing the respective clinical impact of these two mechanisms on the various populations.

	DISCLOSURES

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The author worked as a consultant for Prassis Research Institute, Sigma Tau, Milan, Italy.

	GRANTS

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This work was, in part, supported by grants from the Ministero Università e Ricerca Scientifica of Italy (Fondo per gli Investimenti della Ricerca di Base Grant RBNE01724C_001 to G. Bianchi and Programma di Ricerca di Interesse Nazionale Grant 2004069314_01 to G. Bianchi), from Ministero della Salute (Instituto di Cura Scientifico 110.4/RF02353), and from European Network to Develop Genetic Markers of Essential Hypertension, European Community, funded research (Grant QLG1–2000-01137).

	ACKNOWLEDGMENTS

 
The work described in this Starling Lecture has been carried out by a large team of highly dedicated scientists trained in clinical science, physiology, pharmacology, biochemistry, molecular biology, and genetics, in both animal and humans, working at San Raffaele Hospital and at Prassis Sigma Tau (Milan) or in other institutions either in Italy or abroad. I would have never achieved the present results without the long-lasting fundamental contribution and thoughts of Patrizia Ferrari, Daniele Cusi, Paolo Manunta, Grazia Tripodi, Mara Ferrandi, Lucia Torielli, Cristina Barlassina, and Sergio Salardi. The honor of presenting the Starling Lecture must be shared with all these people and many others, particularly, Jan Staessen, who, in the most recent years, has provided substantial insights regarding the clinical and epidemiological impact of the genetic mechanisms described in this lecture.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. Bianchi, School of Nephrology, Univ. Vita Salute San Raffaele, Division of Nephrology, Dialysis and Hypertension, San Raffaele Hospital, Via Olgettina 60, 20132 Milan, Italy (e-mail: bianchi.giuseppe{at}hsr.it)

Distinguished Lectureship Awards are named after outstanding contributors to the disciplinary areas of physiology represented by 12 APS Sections. The recipient is chosen by a Section as a representative of the best within the discipline. Lecturers present and are active participants at the Experimental Biology meeting. Each year, 4 of the 12 lecturers give plenary lectures that incorporate the main meeting topic. In the years that sections do not have plenary lectures, the lecturer presents 1 h of a featured topic programmed by the section.

	REFERENCES

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