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Molecular Physiology Laboratory, Centre for Cardiovascular Science, Queens Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom
ABSTRACT I. INTRODUCTION II. CONTROL OF BLOOD PRESSURE BY THE KIDNEY A. Role of the Kidney in Hypertension B. Impaired Renal Sodium Excretion as a Risk for Hypertension C. Vascular-Dependent Hypertension III. ADDITIONAL HYPERTENSION RISK FACTORS A. Environmental B. Ontogenic C. Prenatal Programming D. Genetic IV. SODIUM BALANCE A. Sodium Transport B. Tubuloglomerular Feedback C. TGF and the Renin-Angiotensin- Aldosterone System V. EVALUATING CANDIDATE GENES A. Transgenesis by Microinjection B. Gene Targeting C. RNA Interference VI. MENDELIAN HYPERTENSION A. Mineralocorticoid Hormone 1. Aldosterone synthase 2. 11beta-Hydroxylase deficiency 3. Syndrome of apparent mineralocorticoid excess B. Mineralocorticoid Receptor Mutations 1. Pseudohypoaldosteronism type I C. Mutations Altering Renal Transport Proteins 1. Proximal convoluted tubule 2. TAL 3. Distal convoluted tubule 4. Collecting duct VII. ESSENTIAL OR MULTIGENIC HYPERTENSION A. Renin-Angiotensin System 1. Renin transgenics 2. Angiotensinogen transgenics 3. ACE 4. ANG II receptor B. Natriuretic Peptides and Receptors 1. Natriuretic peptides 2. Natriuretic peptide receptors 3. Guanylin and uroguanylin C. Endothelin System D. Nitric Oxide Signaling Pathways E. Kallikrein-Kinin System 1. Kallikreins 2. Kinin receptors F. The Dopaminergic System G. Cyclooxygenase Pathway of Arachidonic Acid Metabolism H. Other Potential Contributing Factors VIII. PERSPECTIVE ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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II. CONTROL OF BLOOD PRESSURE BY THE KIDNEY |
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A. Role of the Kidney in Hypertension
If the Guyton hypothesis is valid, hypertension results from either a failure to increase sodium output in response to an increase in intake (i.e., a failure to shift the renal function curve to the left to produce a higher level of excretion at any given pressure; Fig. 1, point B) or a shift in the renal function curve to the right so that a higher equilibrium pressure is required to match sodium output to intake (Fig. 1, point C). All forms of hypertension are predicted to be a consequence of abnormal pressure natriuresis responses (107); blood pressure homeostasis is sacrificed to preserve sodium balance.
In several forms of hypertension the underlying cause of impaired renal natriuretic function is easily identified. Structural changes to the kidney, such as loss of functional renal mass, or a narrowing of the renal arteries (stenosis), can impede salt excretion and increase the risk of hypertension, especially when combined with increased salt intake, as demonstrated by the one-kidney, deoxycorticosterone acetate rat model and by Goldblatt’s two-kidney, one-clip model (18). In some cases, a clear underlying genetic defect can be identified. All of the Mendelian hypertensive disorders arise from mutations in genes encoding proteins directly or indirectly involved with renal sodium handling [see review by Lifton et al. (192) and below].
B. Impaired Renal Sodium Excretion as a Risk for Hypertension
The kidney is usually histologically normal in the early stages of essential hypertension. Nevertheless, a wealth of data, obtained from both humans and experimental models, suggest that an inadequacy in terms of sodium excretion is a risk factor for essential hypertension.
A variety of approaches have found that an inability to excrete sodium leads to increased blood pressure in humans and experimental animals (382). On intravenous infusion of saline, renal sodium excretion is markedly blunted in patients with essential hypertension (209). In a subset of essential hypertensive patients, the "salt retention" is associated with impaired pressure natriuresis response (208). Evidence for the causal link between high salt intake and high blood pressure has been reviewed elsewhere (225).
Cross-transplantation of kidneys between normotensives and hypertensives have provided strong evidence that the kidney plays a key role in primary hypertension (98). Studies in humans show a normalization of blood pressure in six hypertensive patients who, following bilateral nephrectomy, received kidney transplants from normotensive cadaver donors (63). These patients, in whom high blood pressure was resistant to a four-drug antihypertensive treatment, showed a prolonged (
4 yr) lowering of MABP without the need for therapeutic intervention. Conversely, it is noted that the incidence of hypertension in transplant recipients correlated strongly with the familial incidence of hypertension in the donor’s family (99).
Several independent groups performed rodent cross-transplantation studies in the 1970s. Dahl’s original findings (65), confirmed later in a number of studies (119, 227, 279, 313), found that on a 0.3% salt diet, blood pressure was "determined by the genotype of the donor kidney rather than by the genotype of the recipient." Interestingly, the insertion of a control kidney into a DS rat did not prevent blood pressure increases, evoked by a high-salt diet (8%), indicating that extrarenal factors also exert a significant influence on MABP. One possible criticism of these experiments is that they demonstrate the effect of transplanting a kidney already damaged by exposure to sustained hypertension. This issue was addressed in young, Milan hypertensive (MH) rats, studied before the onset of hypertension. Insertion of a normotensive control kidney into a bilaterally nephrectomized MH rat prevented development of hypertension, whereas insertion of an MH kidney into a control rat induced chronically elevated MABP (83). Likewise, cross-transplantation of kidneys from spontaneously hypertensive (SHR) rats, given life-long antihypertensive therapy by angiotensin converting enzyme (ACE) inhibition, and never therefore exposed to high perfusion pressure, conferred hypertension on the genetically normotensive recipient (278).
The studies described above suggest that 1) blood pressure can be set by the kidney and 2) the renal defect is genetically determined. Congenic approaches have been used to localize the genomic region responsible for setting of blood pressure by the kidney. For example, congenic SHR rats carrying a segment on chromosome 1 from the normotensive Brown-Norway rat have markedly lower blood pressures than noncongenic SHR rats (55). Elegant cross-transplantation studies between progenitor SHR rats and the congenic strain revealed that the Brown-Norway fragment of chromosome 1 lowered blood pressure. It is important to note that the hypotensive effect was observed whether the fragments were present renally or extrarenally, indicating again that other factors exert powerful influences on MABP (57).
C. Vascular-Dependent Hypertension
It can be difficult to envisage a central role for the kidney in the onset of hypertension, since gross renal abnormalities are mostly absent in the early stages of the disease. Moreover, volume expansion and increased cardiac output would be expected if blunted natriuretic capability plays a primary role in essential hypertension, but neither of these are cardinal features. Guyton’s hypothesis argues that the period during which blood pressure is volume-dependent may only be transitory (104), since elevation of MABP would increase renal salt excretion to restore sodium balance. Failure to return blood pressure to normal is attributed to autoregulatory vasoconstriction in the peripheral vascular beds, triggered locally in response to prolonged exposure to high perfusion pressure. Despite the fact that chronic hypertension, under this model, is maintained by the vasculature, impaired renal sodium excretion remains the initiating event. Data in support of this hypothesis, such as studies showing that prevention of volume expansion following salt loading in DS rats prevents the development of hypertension (95), are reviewed elsewhere (105).
Nevertheless, other studies in salt-sensitive hypertensive models do not find volume expansion to be a key hypertensive event (168, 273). It is known, for example, that an increase in sympathetic nervous system (SNS) activity is often observed in the early stages of hypertension (146). It has been proposed (143) that this increase in sympathetic drive is the initiating hypertensive event. These data suggest that repeated intermittent bouts of sympathetic hyperactivity cause renal vasoconstriction and promote subclinical changes to the renal structure, particularly the afferent arteriole, which in turn leads to altered salt handling (141). Impaired renal sodium excretion persists as a key feature for hypertension but is no longer the initiating event. Instead, the hypertension becomes Guytonian only after the kidney is subjected to repeated ischemic episodes following vasoconstriction and reduced renal plasma flow (142). Moreover, this may be a vicious circle in that small increases in plasma sodium concentration can exert a central pressor effect via activation of both the RAS and SNS (69). For the purposes of the present review, it is unnecessary to reconcile these observations, the germane point being that the kidney remains central to the misregulation of MABP.
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III. ADDITIONAL HYPERTENSION RISK FACTORS |
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Apart from age and gender, environmental factors contributing to persistent or pathological changes in blood pressure include poor diet, lack of exercise, increased body weight, stress, and smoking or alcohol intake. Dietary factors thought to have adverse effects on blood pressure include high sodium, especially in combination with low potassium (383). Also, higher caloric intake, reflected in weight gain and increased adipose tissue, correlates strongly with increase in blood pressure. It has been estimated that 60–70% of hypertension is attributable to adiposity, with 5 mmHg increase in systolic blood pressure (SBP) for every 5 kg weight gain (152).
There are several distinct developmental windows during which certain stimuli affect long-term development of the cardiovascular phenotype. For example, short-term treatment of genetically hypertensive rats with antihypertensive drugs, at 5–9 wk of age, has a long-term beneficial effect (118). If hypertension is deferred, then blood pressure elevation, cardiovascular hypertrophy, and end-organ damage are considerably attenuated, compared with rats developing hypertension during the critical period in development (118, 354). Immature rats are highly susceptible to increased salt intake, whilst dietary calcium has antihypertensive effects. Dietary protein intake also affects blood pressure in juvenile rats (395). An in depth review of dietary salt and development of salt sensitivity has been published elsewhere (225).
It is important to study animals in the transitory phase, when blood pressure is developing, in addition to analyzing the established hypertensive phase. Genetic alterations that occur in the latter phase are likely to reflect mechanisms of target organ damage such as left ventricular hypertrophy, rather than the hypertension per se. In this regard, the inducible hypertensive rat model (153), which places time course and extent of hypertensive damage in the hands of the investigator, should prove invaluable.
The recent description of a mitochondrial tRNA mutation being linked to hypomagnesemia, hypertension, and hypercholesterolemia, each of which showed variable penetrance, poses the interesting possibility that loss of mitochondrial function with age might also contribute to age-related increase in blood pressure (379).
There has been much debate regarding birth weight as a predictor of future hypertension, stemming from observations that lower birth weight was associated with higher death rate from coronary heart disease and stroke (19). There seems to be a small but consistent inverse relationship between birth weight and later blood pressure: a 1-kg increase in birth weight correlates to a 1- to 2-mmHg decrease in SBP (78).
In rats, severe sodium restriction during the last week of gestation causes intrauterine growth retardation and subsequently leads to high blood pressure and renal dysfunction in adulthood (21). Treatment of rats with dexamethasone (a poorly metabolized corticosteroid), during the last week of pregnancy, also causes a reduction in birth weight, glucose intolerance, and increased adult blood pressure. Additionally, carbenoxolone (an inhibitor of 11
-hydroxysteroid dehydrogenase type 2, Hsd11b2) treatment in the last trimester leads to postnatal hyperglycemia, increased blood pressure in adult progeny, and tissue-specific increases in glucocorticoid receptor density leading to increased cortiscosteroid sensitivity (29, 308). Glucocorticoids affect glomerular number and kidney maturation (178) but may also affect blood pressure by altering renal and vasculature catecholamine receptor levels, by inducing growth factors such as insulin-like growth factor (IGF), by indirect effects on carbohydrate and fat metabolism, or through increased vasoconstriction via regulation of catecholamines, nitric oxide, and angiotensinogen synthesis (30).
The level of protein intake in pregnant rats is inversely proportional to blood pressure in adult offspring (207), although blood pressure can be normalized by treatment with ACE inhibitors at 8 wk of age. Protein restriction causes placental enlargement and is characterized by decreased Hsd11b2 activity, and thus reduced placental protection of the fetus against maternal corticosteroids (29). Exposure of the fetus to high glucocorticoid levels may adversely affect the fetal hypothalamo-pituitary-adrenal (HPA) feedback system that regulates adrenal output. This is likely to have an immediate effect on fetal growth but may also reset the fetal HPA axis, with effects persisting into adulthood. Current evidence suggests that key targets for programming include the HPA axis, the glucocorticoid receptor gene, and Hsd11b2 gene expression (30).
In 1988, Brenner et al. (38) suggested that nephron number was inversely related to MABP. This was based on the observation that inbred hypertensive rats (SHR for example) tended to have smaller kidneys and fewer nephrons than normotensive controls. Similarly, a reduction in nephron number has been found in adults with essential hypertension; whether this is cause or effect remains unknown. Low nephron number does not initially manifest as a reduction in whole kidney glomerular filtration rate, since hyperfiltration of individual glomeruli compensates. Unfortunately, glomerular hyperfiltration is associated with accelerated loss of renal function, and a reduction in nephron number reduces the renal reserve, i.e., the capacity of the kidney to cope with injury, etc. Brenner’s hypothesis is that a congenital reduction in nephron number is prenatally programmed, possibly as a consequence of low birth weight or protein content of the maternal diet (200), and this increases the likelihood of developing hypertension in adulthood, especially in the setting of elevated salt intake. Protein restriction in the latter half of pregnancy, when nephrogenesis normally occurs in the rat, has been found to reduce the number of nephrons in the developing kidney by almost half. Both male and female progeny developed salt-sensitive hypertension, although females were less severely affected (381).
As already mentioned, blood pressure is influenced by a wide variety of physiological systems, that have pleiotropic effects, and interact in complex ways. These include the baroreceptors, which detect acute changes in blood pressure, but are not yet defined at the molecular level; the renin-angiotensin-aldosterone and kinin-kallikrein systems, which influence sodium retention in the kidney and vascular tone; natriuretic peptides produced in response to local increased pressure in the heart and brain; the adrenergic receptor system, which affects cardiac contraction and vascular tone; dopamine receptors, which affect natriuresis; vasodilators such as nitric oxide; and vasoconstrictors such as endothelin. The distinction between primary alteration and secondary adaptive response contributing to long-term high blood pressure may be extremely difficult to discern.
Candidate genes are identified in a number of ways. In some cases, a simple Mendelian form of hypertension (or hypotension) can be identified by pedigree analysis. Detailed linkage analysis may then reveal the chromosome, the subchromosomal region, or even the gene most likely to be involved. Once variants that cosegregate with the hypertensive phenotype have been identified, then detailed sequence analysis may identify the causative genetic mutation. Examples of this include Liddle’s syndrome, caused by a mutation in the - or 
-subunits of epithelial Na+ channel (ENaC) (110, 315).
When hypertension is the result of a complex genetic trait, the power of linkage analysis is more limited. Hypertension may be the result of small increments in blood pressure due to a number of contributing gene variants. Each of these may vary not only in phenotype but may also be affected by polymorphic allele frequencies, low penetrance, and the epistatic effects of other genes. As a consequence, the positions of the quantitative trait loci may fall below detectable LOD (logarhythmic odds) scores, or at best identify very large chromosomal regions.
Genome-wide linkage analyses, which systematically evaluate the human genome for segments containing genes that influence blood pressure, have to be carefully designed (68) and may identify different regions in different populations (172, 385) or even fail to identify any contributing regions (155). These studies suggest that different sets of genes contribute to hypertension in different ethnic groups and that precise analysis is difficult in humans because of their heterogeneous genetic background. Such analyses have been improved by increasing the density of markers, restriction fragment length polymorphisms (RFLPs), single nucleotide polymorphisms (SNPs), and microsatellites, throughout the genome. Recently developed microsatellite databases for the human (58) and the mouse (352) greatly aid whole genome scans.
With the complete analyses of human (221), mouse (248), rat (90), and now chimpanzee (53a) genomes being published, conserved synteny for blood pressure quantitative trait loci (QTLs) will hopefully refine the chromosomal location of blood pressure regulating loci (396). Moreover, comparisons of whole genomes (60) are beginning to reveal highly conserved functional sequences outside the coding sequence of the genes themselves (243). These are short- and long-range cis-regulatory sequences controlling the spatial and temporal expression patterns of the gene or the entire locus (242). Variation in these regions can have as dramatic an effect on gene expression as a mutation within the coding region.
An additional source of candidate genes arises from the study of defects underlying the numerous animal models selected for hypertensive phenotype (276). These include the SHR, the Dahl salt-sensitive rat, and the Milan rat. A wealth of knowledge has been, and continues to be, accumulated for these models, which often prove to be highly complicated. In fact, the selective breeding strategies that generated the models are likely to have "fixed" many allele variants and modifier genes affecting blood pressure homeostasis so that the underlying etiology may not be at all clear. [It should be noted that breeding of strains at different establishments can cause further "fixation" of variants, leading to the development of sublines (6, 92).]
Nevertheless, QTL analyses in animals can be highly informative, because of the large numbers of progeny that can be screened. This phenotype-driven approach (332) capitalizes on natural variation among inbred strains (214, 289). By using crosses between two inbred mouse strains (screening F2 progeny), QTLs associated with blood pressure, heart rate, and heart weight were identified (331).
The recombinant inbred strain platform, generated between the SHR and the Brown-Norway rat (270, 272), has been useful for identifying QTLs for numerous cardiovascular phenotypes including arterial pressure. The development of a new framework marker-based linkage map, which clearly defines the strain distribution patterns (137), will greatly enhance the utility of these recombinant inbred (RI) strains. An equally useful resource is provided by the panel of consomic rat strains (61), in which an entire chromosome from one inbred strain is introgressed onto the background of a second inbred strain. These can be used to assign traits and QTLs to any given chromosome.
Congenic strains can be rapidly developed using the marker-assisted speed congenic approach (135) to locate the QTL more accurately over narrow regions of the chromosome. Arguably this may disrupt the very gene-trait relationships being sought, but consomics with multiple chromosome substitutions, and double congenic strains, can be used to investigate such gene-gene interactions in the future (217). It must be stressed that the identification of any candidate gene in an animal model does not automatically imply its involvement in hypertension in humans. This may be due to species specificity, a topic that will be revisited later in the review, or simply to a lack of genetic variation in some species.
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IV. SODIUM BALANCE |
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Sodium is freely filtered at the glomerulus, with 99% of the filtered load being reabsorbed along the nephron, by an integrated system of ion channels, ion exchangers. and ion transporters (Fig. 2A). In the proximal convoluted tubule, 50% of filtered sodium is reabsorbed. Although there are 20 different sodium transporters in the apical membrane, most of these couple to "substrates" (such as amino acids and carbohydrates), and collectively they mediate only 10% of the proximal tubule sodium reabsorption. The sodium-hydrogen exchanger, NHE3, mediates the majority of Na+ reabsorption (Fig. 2B).
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20% of the filtered load) but water is not, thereby creating a steep osmotic gradient in the medullary interstitium, which permits vasopressin-dependent water reabsorption in the collecting duct. In the TAL, almost all sodium transport results directly or indirectly from Na+-K+-2Cl– cotransport (96). Efficient operating of this transporter (NKCC2) requires K+ to recycle across the apical membrane through a K+ channel (ROMK) and chloride to exit basolaterally through a chloride channel (CLCNKB; Fig. 2C). Potassium recycling creates a potential difference, which drives the reabsorption of cations through the paracellular pathway. This is especially important for divalent ions as shown by the hypercalciuria of Bartter’s disease. Sodium reabsorption in the early distal tubule (DCT1 and DCT2) is mediated by the thiazide-sensitive NaCl cotransporter (NCC) (Fig. 2D) and also, to a lesser extent, by sodium-hydrogen exchange (NHE2). The remaining reabsorption is achieved in the connecting tubule and cortical collecting duct via ENaC, and it is this segment in which the fine-tuning of sodium reabsorption occurs, under the control of aldosterone (Fig. 2E).
The early distal tubule is the site of the macula densa (MD), a collection of specialized epithelial cells able both to "sense" the NaCl concentration of the tubular fluid and to influence filtration at the glomerulus of origin (Fig. 3). This mechanism, called tubuloglomerular feedback (TGF), rapidly stabilizes acute increases in Na+ delivery by vasoconstriction of the afferent arteriole and reduction in single-nephron glomerular filtration rate (SNGFR): delivery of sodium to the MD is thus held within narrow boundaries, ensuring that the distal tubule and cortical collecting duct (CCD) are not overloaded. Equally important is the increase in SNGFR following a drop in distal Na+ delivery, which serves to restore Na+ concentration to levels compatible with the maintenance of K+ and H+ secretion. TGF responses are rapid, and the sensitivity of response is dictated by afferent arteriolar tone. Thus TGF is modulated by agents such as angiotensin II (ANG II), nitric oxide (NO), and the eicosanoids and can be reset during chronic perturbations in MD sodium delivery and altered volume status (299). Moreover, studies in inbred hypertensive rat strains show that TGF is inappropriately sensitized during the development of hypertension, i.e., GFR, and thus filtered sodium load, is lower for a given delivery of sodium to the MD than normal, reducing the ability of the kidney to shed sodium (265).
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C. TGF and the Renin-Angiotensin- Aldosterone System
The intimate connection between TGF and the RAS is shown by cessation of TGF response in mice lacking angiotensin AT1A receptors (300). The RAS can certainly modulate TGF but represents a distinct mechanism for regulating sodium excretion depending on the requirements of systemic sodium balance. Thus, if enhanced sodium delivery to the MD is maintained, then renin secretion is reduced, local ANG II levels fall, and SNGFR rises to promote sodium excretion. Likewise, reduced salt levels in the TAL lead to release of renin. Increased renin levels lead to increased production of ANG II, which acts on the adrenal glomerulosa cells, via a G protein-coupled receptor, to increase aldosterone synthase activity, and thus aldosterone secretion. Aldosterone targets the mineralocorticoid receptors in the distal nephron, and ultimately causes an increase in sodium reabsorption and potassium secretion. Any mutation that causes sustained salt retention concomitantly increases water retention and will thus raise blood pressure. Likewise, any mutation that causes salt wasting will lead to hypotension.
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V. EVALUATING CANDIDATE GENES |
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Ultimately it is desirable to alter the expression of a candidate gene, to ascertain or confirm its mode of action, and the pleitropic effects, including hemodynamic parameters, which might result from altered expression. Detailed analysis of transgenic models may reveal the presence of previously unidentified modifier loci, which attenuate or exacerbate the phenotype, on different genetic backgrounds.
Genetic modification can be achieved in a number of ways, including microinjection and homologous recombination. Each strategy is detailed in the following sections.
A. Transgenesis by Microinjection
Transgenesis by microinjection generally leads to overexpression of the transgene. A transgene construct is microinjected into the pronucleus of a fertilized oocyte (see Fig. 4), which is then placed in the oviduct of a pseudopregnant host and allowed to develop to term. Resultant pups are screened for potential founders, from which transgenic lines can be derived. The transgene integrates at random in the genome; therefore, every transgenic line is unique. The researcher has no control over the number of copies inserted, which are present in addition to the endogenous copies of the gene. The site of insertion can have a profound effect on expression of the transgene (114). If it inserts in a silent region of the chromosome, then expression may be completely suppressed. On the other hand, insertion near a strong enhancer may result in aberrant patterns of expression. Integration within a gene may insertionally inactive it, causing a completely unrelated and unexpected phenotype.
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With the development of homologous recombination systems in Escherichia coli (184, 236), BACs and PACs can be easily engineered to carry reporter genes, or to place the transgene under the control of inducible or conditional promoters. There are sophisticated systems for conditionally turning genes on or off by dietary changes (153), or antibiotic induction or suppression [e.g., using tetracycline/doxycycline (394)]. Strategies using the Cre-lox recombination system (237) or FRT-flp technology (281) have proven particularly useful. A detailed discussion is beyond the scope of this review, but it is worth pointing out that many of these strategies require the generation of two independent transgenic lines. For example, one line might carry the transgene of interest, flanked by loxP sites (floxed) to allow conditional knockout, or have an upstream floxed stop cassette to allow conditional knock-in. The second line would carry tissue-specifically or developmentally expressed Cre recombinase. Crossing the two lines would result in conditional Cre-mediated recombination yielding the desired alteration in gene expression.
Reporter strains, which express -galactosidase (325) or enhanced green fluorescent protein (156, 293) following Cre recombinase activation, indicate the tissue specificity and extent of recombination for any given Cre strain. This should be tested, since Cre expression may be leaky, ectopic, or mosaic. The latter observation has been used to advantage, recently, for generating Cre-mediated germline mosaicism (124, 187). It is also worth noting that the loxP footprint left behind after Cre recombination may affect gene expression. (See detailed reviews of conditional transgenic technologies in Refs. 35 and 290.)
Gene targeting and gene knockout is achieved when a modified copy of the target gene is introduced into embryonic stem (ES) cells (Fig. 5). Under appropriate selective pressure, the transgene replaces the endogenous gene by homologous recombination. The targeted ES cells are introduced into blastocysts, where they have the potential to contribute to all tissues of the developing embryo. The resultant chimeric pups are bred to screen for germline transmission of the transgene. This technique can be used not only for loss-of-function analysis, but also for replacement of the targeted gene with a reporter, such that the developmental and tissue-specific expression patterns of the endogenous gene can be visualized. An obvious potential drawback to loss-of-function targeting is the possibility of embryonic lethality due to a requirement for gene expression early in development. To overcome this, sophisticated strategies for making transgene expression conditional are available. With the advent of BAC homologous technology, much larger targeting constructs can be engineered, and multiple alterations can be achieved in one round of ES cell recombination (344). High-throughput engineering technology is now established, which can replace precisely any given gene of interest with a reporter, or generate point mutations or deletions (355). Coupled with high-resolution expression analysis, this will be a powerful tool in functional genomics.
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RNA interference, using small interfering RNAs (siRNAs), is a powerful new tool for analyzing gene knockdown phenotypes. RNA interference is an evolutionarily conserved mechanism mediated by double-stranded microRNA (miRNA), which targets mRNA for degradation by cellular enzymes, effectively limiting translation by binding to sites within introns, or in the 3'-untranslated region (139). The dsRNA is processed into small duplex RNA molecules (20–25 nucleotides) by an RNase III enzyme called dicer. The siRNAs interact with an RNA-induced silencing complex, leading to sequence-specific binding and cleavage of the target mRNA. Introduction of siRNA molecules directly into somatic mammalian cells circumvents the nonspecific response against larger dsRNA molecules (53).
At present, knockout technology is not available for the rat, since rat ES cells have proven technically difficult to produce. Chemically synthesized siRNAs and short hairpin RNAs (shRNAs) have recently been shown to transiently or stably knock-down gene expression in transgenic mice and, significantly, in rats (113, 189, 218, 257). This technology, together with others discussed in future perspectives, promises to widen the application of transgenic technology to the rat and other species.
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VI. MENDELIAN HYPERTENSION |
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Aldosterone synthase deficiency, arising through either point mutation or microdeletion (261), results in severe impairment of salt reabsorption, leading to reduced plasma volume and severe hypotension, and impaired secretion of K+ and H+ leading to hyperkalemia and acidosis. Mice completely lacking aldosterone synthase (Cyp11b2) have been reported to have a broadly similar phenotype (185): hypotension, abnormal electrolyte homeostasis, and increased urine production, together with high levels of circulating renin and ANG II. They also showed increased cyclooxygenase-2 (COX-2) expression in the macula densa, which was somewhat enlarged (206). Interestingly, all the abnormalities, except for low blood pressure, were substantially corrected on administration of a high-salt diet.
Glucocorticoid remedial aldosteronism (GRA; Ref.261) occurs when unequal crossing over between aldosterone synthase and the closely related gene 11-hydroxylase puts aldosterone synthesis under the control of adrenocorticotropic hormone (ACTH), which normally controls adrenal 11-hydroxylase expression and cortisol secretion. Constitutive aldosterone secretion causes sodium retention, increased plasma volume and blood pressure, and increased secretion of K+ and H+, leading to hypokalemia and alkalosis. As the name suggests, exogenous glucocorticoids completely suppress the aldosterone secretion, through negative feedback of ACTH production.
The complementary hybrid gene resulting from unequal crossover between 11-hydroxylase and aldosterone synthase places the chimeric gene under the aldosterone synthase promoter (108, 267). The gene is induced by ANG II and K+, and its expression is restricted to the zona glomerulosa, leading to 11-hydroxylase deficiency, hypertension, and congenital adrenal hyperplasia (CAH).
The most common cause of CAH (accounting for 90% of cases) is 21-hydroxylase deficiency, while up to 8% of classic CAH is caused by 11-hydroxylase deficiency (375). Decreased or absent cortisol secretion stimulates ACTH secretion, which results in accumulation of steroid precursors and increased androgen levels. Patients suffer from masculinization and short adult stature due to precocious pseudopuberty, and increased levels of deoxycorticosteroid may cause hypertension because of its mineralocorticoid activity. The nonclassic form of 11-hydroxylase deficiency has milder symptoms and is characterized by missense mutations, which have been shown to affect enzyme activity in vitro (138). Severity of symptoms appears to be determined by the nature of the point mutation or deletion in the gene (171). No transgenic models of 11-hydroxylase (Cyp11b1) deficiency have been reported to date.
3. Syndrome of apparent mineralocorticoid excess
The syndrome of apparent mineralocorticoid excess (SAME) is autosomal recessive and occurs when the enzyme HSD11B2 is defective. This enzyme normally converts active cortisol (or corticosterone in rodents), which can act at the mineralocorticoid receptor (MR) to inactive cortisone (or 11-dehydrocorticosterone), effectively protecting the receptor from the high circulating levels of cortisol. In SAME, physiological levels of glucocorticoids illicitly activate the MR in the distal tubule. This leads to sodium retention, early-onset hypertension, hypokalemia, and alkalosis, but additionally, suppressed plasma renin and absence of circulating aldosterone are observed due to feedback inhibition. Interestingly, glycyrrhetinic acid, the active ingredient of licorice, inhibits HSD11B2 and induces hypertension, presenting as pseudohyperaldosteronism (358).
A mouse transgenic model, in which Hsd11b2 was knocked out (169), developed early-onset hypertension and demonstrated major features of SAME, including polyuria and hypokalemia. Homozygous mutants appear normal at birth, but 50% show motor weakness and reduced suckling, and die within 48 h, presumably related to the severity of hypokalemia. Surviving null animals exhibit enlarged kidneys, associated with distal tubule enlargement due to hyperplasia and hypertrophy of epithelia. Administration of dexamethasone (the synthetic glucocorticoid, which suppresses the HPA axis and hence corticosterone production), which is not a ligand for MR, increases the urinary Na+/K+ ratios to wild-type levels (see Fig. 7), while electrolyte abnormality is recreated following corticosterone administration (123).
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1. Pseudohypoaldosteronism type I
Pseudohypoaldosteronism type I (PHA-1) is an autosomal dominant condition caused by loss-of-function mutations in the mineralocorticoid receptor (89). Despite elevated levels of aldosterone, affected patients have severe neonatal salt wasting and hypotension, with hyperkalemia and metabolic acidosis. The MR deficiency is potentially lethal, suggesting that two copies of functional MR are required by the neonate. All cases improve with age, and by adulthood, a normal-salt diet is asymptomatic.
Mineralocorticoid receptor (Nr3c2)-deficient mice die around day 9 or 10 after birth. By day 8, the animals show symptoms of pseudohypoaldosteronism, with high levels of plasma renin, ANG II, and aldosterone, high renal salt wasting, hyponatremia, and hyperkalemia (27, 127). They also exhibit histological changes in the kidney, with enlargement of the macula densa segment of the distal tubule and recruitment of renin-producing cells along the afferent arteriole. Daily subcutaneous injections of isotonic NaCl solution until weaning, followed by continued oral NaCl, rescue the pups (34). Under these conditions, plasma Na+ concentration is normal, but the animals remain hyperkalemic, their fractional renal excretion of Na+ (FENa) is still enhanced and plasma renin and aldosterone levels remain high. Interestingly, the mRNA abundance of 
-ENaC is reduced by 30% in the kidney (though that of the - and -ENaC subunits is unaltered) and ENaC activity is almost absent. Recently, it has been shown that glucocorticoid treatment restores plasma K+ and almost normalizes FENa (304). This is achieved by increasing the mRNA abundance of -ENaC in the kidney.
Interestingly, cardiac-specific, conditional expression of an antisense mRNA of murine MR caused cardiac fibrosis and heart failure in the absence of chronic hypertension or chronic aldosteronism (22). Antisense mRNA could be up- or downregulated by Tet or Dox, respectively, the latter leading to rapid improvement. This model demonstrates the potential to interrogate gene function in specific cell types, rather than by constitutive alteration, which has both systemic and local effects.
As further evidence for the involvement of MR in hypertension, a missense mutation in the mineralocorticoid receptor ligand-binding domain has been shown to cause an autosomal dominant form of hypertension accelerated by pregnancy. The missense mutation renders the receptor activatable by steroids that would not normally bind to MR, including progesterone. Because progesterone levels increase 100-fold during pregnancy, a rapid acceleration of hypertension associated with complete suppression of the RAS is observed (88).
Overexpression of the human mineralocorticoid receptor in mice (directed by its P1 promoter, which is transcriptionally active in all MR-expressing tissues and importantly directs appropriate transgene expression in the distal nephron) leads to alterations in cardiac and renal functions. These mice have a decreased urinary sodium/potassium concentration ratio, consistent with an increased aldosterone action in the connecting tubule and collecting duct, and this is exacerbated on sodium depletion (186).
C. Mutations Altering Renal Transport Proteins
Animals with null mutations in renal NaCl and water transporter genes provide a powerful tool for studying the physiological balance between glomerular filtration rate and tubular reabsorption (296). In general, all Na+ transport deficiencies cause a permanent reduction in extracellular fluid volume, with symptoms such as increased renin expression and aldosterone synthesis, and a tendency to hypotension. This leads to compensatory activation of Na+ conserving mechanisms, such as the TGF. If Na+ balance can be reinstated, then the volume depletion is self-limiting and a steady state is achieved. When sodium balance cannot be achieved, salt wasting and volume depletion would lead to death.
The Na+/H+ exchanger, NHE3, is the major pathway for sodium reabsorption in the proximal tubule (see Fig. 2B), and as such, knockout of the exchanger would be expected to markedly impair tubule function and result in renal salt loss. However, despite a significant (50–60%) reduction in proximal tubule fluid reabsorption, NHE3 (Slc9a3) knockouts have only mild volume depletion and hypotension (301, 374). A major compensatory mechanism is the reduction in SNGFR that reduces filtered sodium load to match impaired proximal reabsorption capability or capacity, to the extent that delivery to the end of the tubule segment is comparable to that in controls (197). SNGFR is reduced through the action of TGF. Because sodium delivery to the macula densa is not increased in Slc9a3 –/– mice, chronic volume depletion must cause resetting of the TGF response.
A "targeted proteomics" approach (semiquantitative immunoblotting), profiling sodium transporter expression along the entire renal tubule (40) demonstrated upregulation of both the proximal tubule Na+-phosphate cotransporter, and the collecting duct -ENaC subunit. Upregulation of NHE2 in the early distal tubule has also been demonstrated (16). Together with the reduced GFR, changes in transporter expression (which may relate to the increased aldosterone and renin levels, Ref. 301) compensate for the loss of NHE3 so efficiently that absolute urinary sodium excretion is significantly lower in knockouts than controls (16, 40). The volume depletion and hypotension may relate to salt/fluid loss attributed to an intestinal sodium reabsorption defect, since the knockouts have pronounced diarrhea. In support of this, partial transgenic rescue through restoration of NHE3 expression in the small intestine improves the volume-pressure defects (244).
Aquaporin-1 (AQP1) is a water channel highly expressed in the proximal tubule, thin limbs of Henle, and the vasa recta (363). Aqp1 knockout mice are polydipsic and have moderate hypotension. Like the Slc9a3 knockout mouse, Aqp1 deficiency results in a marked deficit in proximal tubule reabsorption which is compensated for by activation of TGF (298). Loss of Aqp1 in the thin limb of Henle and in the vasa recta disrupts the countercurrent multiplication system and thus knockout mice are unable to concentrate urine in response to water deprivation, even during administration of vasopressin (363).
From these two examples, it is apparent that impaired proximal tubule function results in modest hypotension, despite the quantitative importance of the proximal tubule in renal sodium reabsorption. This is because the TGF loop provides a powerful compensatory mechanism, which limits the severity of the genetic defect. One would predict that an inoperative TGF, coupled with a proximal tubule disorder, would result in severe salt wasting. To investigate this hypothesis, the Aqp1 –/– mouse was crossed with the adenosine A1a receptor knockout mouse, which has a defective TGF. Surprisingly, the double knockouts were able to maintain salt balance, despite having a higher GFR and lower proximal reabsorption than Aqp1 –/– mice (112). This demonstrates the remarkable redundancy of renal sodium transport and illustrates the compensatory power of distal sodium reabsorption.
Mutations in the Na+-K+-2Cl– cotransporter, NKCC2, or any of the genes encoding ion channels required for its operation (ROMK, CLCNKB) cause Bartter’s syndrome (see Fig. 8), characterized by severe polyuria, low plasma potassium, high blood pH, hypercalciuria, proteinuria, and low blood pressure (116). The importance of Nkcc2 (Slc12a1) is demonstrated by the fact that homozygous knockout mice die within 2 wk of birth from severe volume depletion (335). [Animals heterozygous for the mutation show no phenotype (334).] Indomethacin (a potent nonselective COX inhibitor), administered from birth, rescues the phenotype, implicating prostaglandins in the regulation of renal salt excretion. Surviving adults exhibit all the features of Bartter’s syndrome and develop severe hydronephrosis.
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Loss-of-function mutations in CLCNKb or its associated protein Barttin produce Bartter’s syndrome types III and IV, respectively (116). Both proteins (Clcnkb being the mouse ortholog) are localized to the mouse TAL, but transgenic models have not yet been reported.
Sodium reabsorption in the DCT occurs via the apical thiazide-sensitive NaCl cotransporter (NCC), mutations of which cause Gitleman’s disease (see Fig. 8). Patients with Gitelman’s syndrome often present at adolescence with hypokalemia, metabolic alkalosis, and mild hypotension (320). In contrast to Bartter’s syndrome, hypocalciuria is observed as a consequence of an increased driving force for Ca2+ reabsorption in the DCT. Mice lacking NCC (Slc12a3) have no overt salt wasting phenotype unless sodium restricted (302). A targeted proteomics approach noted an increase in -ENaC in Slc12a3 knockout mice (40), relating perhaps to stimulation of the RAS.
Patients with Gordon’s syndrome (pseudohypoaldosteronism type II, a rare autosomal dominant condition) exhibit low-renin low-aldosterone hypertension, hyperkalemia, and metabolic acidosis. The syndrome can be corrected with thiazide diuretics, suggesting increased NCC activity as the underlying cause. There is, however, no significant linkage between Gordon’s syndrome and the NCC gene (SLC12A3) locus (85). The clinical features, in a subset of these patients, arise from independent mutations in two members of a serine-threonine kinase family, WNK4 (378), and WNK1 (239) which regulate sodium and potassium transport proteins in the distal nephron. The hypertension stems from both impaired retrieval of NCC from the apical membrane of the DCT cell (380) and increase in paracellular chloride flux (147). The hyperkalemia arises from a gain-of-function mutation in WNK4 (independent of its kinase activity), which increases the inhibition of K+ secretion via endocytotic retrieval of ROMK (148). Thus WNK4 has a dual role in controlling renal sodium and potassium excretion (33). The association between an SNP near the WNK1 promoter and severity of hypertension suggests that increased WNK1 expression might also contribute to increased blood pressure (239).
The autosomal recessive form of human pseudohypoaldosteronism type I is caused by loss-of-function mutations in any of the three ENaC subunits (, , and ; see Fig. 8). It is characterized by salt wasting, hyperkalemia, and high mortality immediately after birth (49, 330). Unlike patients with the autosomal dominant form of pseudohypoaldosteronism type 1, patients fail to improve with age and require massive salt supplementation. Mice with knockout mutations in the Scnn1b or Scnn1g genes encoding the - or -subunits of the sodium channel die shortly after birth from dehydration and hyperkalemia (20, 219). Mice with targeted deletion of the -subunit (Scnn1a) die from failure to clear their lungs (128). Mice expressing an -ENaC transgene on an Scnn1a knockout background have 50% perinatal mortality, and salt wasting, but can compensate for the deficiency as adults (129). Animals surviving to adulthood develop compensated pseudohypoaldosteronism, though aldosterone levels are sixfold higher than normal (371). These observations suggest that Na+ malabsorption in the terminal nephron causes severe salt wasting and that compensatory mechanisms for severe volume depletion in the newborn are relatively inefficient under these conditions. Interestingly, when -ENaC was specifically inactivated in the collecting duct only, knockout animals were completely viable. This points to the connecting tubule as being critical for achieving sodium and potassium balance (288).
When the Scnn1b gene is "knocked down" and mRNA expression is reduced to 1% of wild-type levels in mouse kidney and lung (268), homozygous mutants develop normally on a normal-salt diet. Adults exhibit significantly reduced ENaC activity in the colon and increased levels of plasma aldosterone, but they have normal plasma Na+ and K+ concentrations, normal blood pressure, and no weight loss. On a low-salt diet, however, the mice develop acute pseudohypoaldosteronism type 1, indicating that -ENaC is important for Na+ conservation during salt deprivation.
Liddle’s syndrome is characterized by early-onset hypertension, hypokalemic alkalosis, suppressed plasma renin activity, and low plasma aldosterone levels. The autosomal dominant syndrome is caused by mutations at the conserved PY motif in either the - or the -subunit of ENaC, which delete or modify their cytoplasmic COOH termini, resulting in increased ENaC activity (294), and increased water and salt reabsorption in the renal collecting tubules. The number of channels in the membrane is effectively increased due to their reduced clearance from the cell surface. Normally, a ubiquitin-protein ligase, Nedd4, binds to the PY motif of ENaC subunits leading to ubiquitination and degradation. In cells derived from the mouse collecting duct, it has been shown that Nedd4–2 is the isoform responsible for binding to the ENaC complex and negatively regulating it (150, 151; see Fig. 6). No knockout models of Nedd4l have as yet been published, but in vitro analysis has shown that siRNA against Nedd4–2 specifically increases amiloride-sensitive Na+ current (324), whilst the mutation associated with Liddle’s syndrome (R566X) abolishes the effect of the siRNA.
A mouse model for Liddle’s syndrome has been generated by Cre/loxP-mediated recombination (269). Animals heterozygous for the -ENaC-mutated allele containing the floxed neo gene were crossed with hemizygous mice ubiquitously expressing the EIIa-Cre transgene. Mice showing complete excision of the neo gene were then used to generate new mouse lines. Under normal-salt diet, mice heterozygous (L/+) and homozygous (L/L) for the Liddle mutation (L) develop normally during the first 3 mo of life. Blood pressure is normal, despite increased sodium reabsorption in the distal colon and low plasma aldosterone, suggesting chronic hypervolemia. Under high salt intake, the Liddle mice develop high blood pressure, metabolic alkalosis, and hypokalemia accompanied by cardiac and renal hypertrophy. This animal model reproduces to a large extent the human form of salt-sensitive hypertension and establishes a causal relationship between dietary salt, ENaC expression, and hypertension.
The earliest effect of aldosterone is to increase ENaC activity without increasing its mRNA or protein levels, suggesting the presence of an ENaC regulator. To identify aldosterone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from a renal distal nephron-derived cell line (50). With the use of a coexpression assay, a serum and glucocorticoid-regulated kinase, sgk, was found to stimulate ENaC activity sevenfold in Xenopus laevis oocytes. Induction of sgk mRNA by aldosterone was detected in kidney cortex and medulla as early as 30 min after hormonal application, and independent of de novo protein synthesis, suggesting that the response is mediated by occupancy of mineralocorticoid receptor (314). Sequence comparison has revealed a homologous serine-threonine kinase in rats and frogs, suggesting an ancient kinase adapted to control epithelial Na+ transport (264).
The sgk knockout mouse was found to have impaired ability to retain renal Na+ (384) when placed under dietary NaCl restriction, despite increases in plasma aldosterone. Unlike most serine-threonine kinases, SGK1 is under dual control: protein levels are controlled by aldosterone, through effects on gene transcription, while its activity is dependent on phosphatidylinositol 3-kinase (PI3K) activity. Thus insulin and other activators of PI3K are key regulators of SGK1. SGK1 exerts its positive affect on ENaC activity through phosphorylation-dependent inhibition of Nedd4–2 (263, 364; see Fig. 6).
In summary, loss of function of sodium transporters tends to have more severe consequences, the more distally the loss occurs, despite the fact that, in absolute terms, the distal transporters account for a small proportion of overall sodium reabsorption. This may reflect the loss of compensatory capacity; there are no more lines of defense for the body to fall back on (295).
