Building on the past--challenging the future: will genomics provide the solution to hypertension?

Hypertension is one of the major common polygenic and multifactorial diseases, and continues to constitute a major cause of morbidity and mortality worldwide. The pathophysiological mechanisms underlying hypertension have not been elucidated, which significantly limits our ability to treat and prevent hypertension. A frequently asked question is whether genomics will provide the solution to the many remaining unanswered questions as to the causes of hypertension. Genomics, an art by its own virtue, holds its promises, yet the questions we are asking of genomics must be well defined for genomics to meet at least some of our expectations. The hopes and promises of genomics are high and one of the major hopes is that genomics will improve our understanding of the pathophysiology of hypertension and enable us to classify hypertensive syndromes more succinctly and enhance our ability to provide patients with specific anti-hypertensive therapy as well as predict outcome. There is still much to be done but perseverance in the task and a firm belief in genomics and in the immense potential of the human genome is essential.

The newly inbred cohen diabetic rat: a nonobese normolipidemic genetic model of diet-induced type 2 diabetes expressing sex differences.

The newly inbred Cohen diabetic rat is an exceptional experimental model of diet-induced type 2 diabetes mellitus that is the result of secondary inbreeding nearly 30 years after it originally had been established. Animals from the original colony were selectively inbred by stringent criteria for 10 additional generations, bringing overall inbreeding to >50 generations. The metabolic phenotypes of the resulting contrasting strains, designated as the Cohen diabetic-sensitive (CDs) and -resistant (CDr) rats, were characterized. The phenotype of the CDs strain that was fed a regular diet consisted of fasting normoglycemia, normal glucose tolerance to intraperitoneal glucose loading, normal fasting insulin levels, and a normal insulin response to glucose loading. In contrast, CDs rats that were fed a custom-prepared high-sucrose low-copper diabetogenic diet became overtly diabetic: fasting glucose levels were normal or elevated, and the blood glucose insulin response to glucose loading was markedly abnormal. CDr rats that were fed a regular or diabetogenic diet did not develop diabetes and maintained normal glucose tolerance and insulin secretion. A striking sex difference was observed in CDs rats that were fed a diabetogenic diet: males had a lower growth rate and a more severe glucose intolerance pattern than females. Gonadectomy shortly after weaning did not prevent the development of the diabetic phenotype in its early phase in either sex but markedly attenuated its expression in males at a later phase, abolishing the sex differences. Alternate-day feeding, as opposed to daily feeding, also attenuated the metabolic phenotype in males. The development of the diabetic phenotype in CDs rats that were fed a diabetogenic diet was not accompanied by obesity or hyperlipidemia. The genetic profile of the strains was established using 550 microsatellite markers evenly distributed throughout the rat genome. The rate of homozygosity within strain was > or = 96%. The rate of polymorphism between the contrasting strains was 43%. We conclude that the metabolic phenotypes of the rebred colony of CDs and CDr rats and their genetic makeup render the Cohen diabetic rat a useful experimental model that is highly suitable for studying the interaction between nutritional-metabolic environmental factors and genetic susceptibility (sensitivity and resistance) for the development of type 2 diabetes. The model is also distinctively useful for investigating the effect of sex on the expression of the diabetic phenotype.

Genetic models of hypertension in experimental animals.

Genetic animal models are central to ongoing efforts to elucidate the pathophysiology and genetic basis of hypertension. The rat is the leading species in experimental hypertension. Several rat models of hypertension are available for research, including inbred strains, congenic lines, transgenic animals and recombinant inbred strains. Each of these models has been designed to express different phenotypes, including spontaneous hypertension, salt sensitivity, stress sensitivity and susceptibility to end-organ damage. All these models have been extremely useful in the search for the physiological mechanisms that underlie hypertension, but some of them have been specifically designed for detecting the hypertension genes. This latter task is extremely complex in spontaneous hypertension, but genetic animal models may simplify the task by enabling to focus on specific phenotypes. Despite intensive efforts over nearly 3 decades, the genetic basis of hypertension has not been unveiled so far in the rat or in other species. Recent dense mapping of the rat genome, the development of new strategies and technologies in molecular genetics including differential gene expression, expressed sequence tags and DNA biochips render hope that the formidable task of identification of new candidate genes in hypertension will move another major step forward. Once these genes are identified, their function and role in hypertension will have to be determined, utilizing functional genomic strategies and bioinformatics. Finally, the findings in genetic animal models of hypertension will have to be extrapolated to humans by homology and syntenic mapping strategies.

The lack of a modulating effect of non-genetic factors (age, gonads and maternal environment) on the phenotypic expression of the salt-susceptibility genes in the Sabra rat model of hypertension.

OBJECTIVE: This study was designed to test the hypothesis that non-genetic factors such as age, gonads and maternal environment modulate the expression of the salt-susceptibility genes and affect the blood pressure response to salt-loading (salt-sensitivity and salt-resistance) in the Sabra rat model of hypertension. METHODS: The blood pressure response to salt-loading was studied in Sabra hypertension prone (SBH/y) and Sabra hypertension resistant (SBN/y) rats of both sexes: (1) at 1, 3, 6, 9 and 12 months of age, (2) in adult rats after orchiectomy or oophorectomy, and (3) in animals that had been raised and nourished from birth to weaning by a foster mother from the contrasting strain. In each of the study protocols, systolic blood pressure was measured at baseline by the tail cuff method, animals were salt-loaded with deoxycorticosterone acetate, and blood pressure was measured again after 4 weeks. RESULTS: Basal blood pressure at all the ages studied and in both sexes was on average 10-15 mmHg higher in SBH/y than in SBN/y. Salt-loading in SBN/y of both sexes aged 1-12 months did not induce any significant increment in blood pressure. Salt-loading in SBH/y, in contrast, caused a highly significant rise in systolic blood pressure, of 40 mmHg or more at all the ages studied. There was no age difference or sex dependence in the magnitude of the blood pressure response to salt Oophorectomy or orchiectomy did not affect the levels of basal blood pressure nor prevent the hypertensive response to salt-loading in SBH/y or the lack of a hypertensive response in SBN/y rats. Gonadectomy did not affect blood pressure in salt-loaded hypertensive SBH/y nor in salt-loaded normotensive SBN/y. The basal blood pressure and the blood pressure responses of SBH/y and SBN/y of both sexes raised by foster mothers of the contrasting strains from birth to weaning were not different from those observed when raised by their natural mothers. CONCLUSIONS: This study indicates that salt-sensitivity in SBH/y and salt-resistance in SBN/y are not age-dependent phenomena; that the magnitude of the BP response to salt-loading is not sex-dependent; and that neither gonadectomy nor the maternal environment affect the blood pressure response to salt-loading in the adult animal of either strain. These non-genetic factors thus do not modulate expression of the salt-susceptibility genes in the Sabra genetic model of salt-sensitive hypertension.

Regulation by sodium intake of type 1 angiotensin II receptor mRNAs in the kidney of Sabra rats.

OBJECTIVE: To study the relationship between the sensitivity to sodium content of the diet in terms of development of hypertension and the regulation of the expression of type 1 angiotensin II receptor subtypes by such a diet. METHODS: The expression of angiotensin II receptor subtype (AT1A and AT1B) mRNAs was studied by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the four zones of the kidneys of Sabra rats, sensitive or resistant to DOCA salt-induced hypertension (SBH/y and SBN/y, respectively). Rats were fed a high (8%) or normal (0.4%) NaCl diet. As vasopressin is known to be elevated in SBH/y rats and to be involved in DOCA-salt hypertension, we studied an additional group of SBH/y rats, fed a high sodium diet, enriched in water. RESULTS: With the absence of DOCA, SBH/y rats did not develop hypertension. The high sodium diet induced a greater fall in the plasma renin activity in the SBH/y (-95%) than in the SBN/y (-63%). In the cortex (C) and inner stripe (IS), the high sodium diet decreased AT1A and AT1B mRNAs in SBH/y and SBN/y, with a higher magnitude for SBH/y, than for SBN/y (C, -28 versus -20%; IS, -42 versus -20%). The addition of water to the high sodium diet lessened the effect of sodium in the C and IS, although the plasma renin activity (PRA) was not altered. CONCLUSION: A high sodium diet significantly decreases both AT1A and AT1B gene expression in two specific zones of the rat kidney containing the target cells of angiotensin II (C and IS). This down-regulation is organ-specific since it was observed in the kidney and adrenals, but not in the liver. Finally, SBH/y and SBN/y rats differ in the basal level of AT1 mRNA expression in the IS, and in the ability to modulate AT1 mRNA level under sodium intake.

Farnesol blocks the L-type Ca2+ channel by targeting the alpha 1C subunit.

We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca2+ channels in vascular smooth muscle cells. To elucidate farnesol's mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca2+ channel alpha 1C subunits. Farnesol dose-dependently and voltage-independently inhibited Ba2+ currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 micromol/L, [corrected] respectively (P=NS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5'-O-(2-thiodiphosphate) (100 micromol/L) [corrected] via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein-independent inhibition of Ca2+ channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift ( approximately 10 mV) of the potential causing 50% channel inactivation in both cell lines (P<0. 001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca2+ channel blockers, farnesol blocked Ca2+ currents in the resting state: initial block was 63+/-8% in A7r5 cells and 50+/-9% in CHOC9 cells at a holding potential of -80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca2+ channel antagonist. Because farnesol is active in cells expressing only the pore-forming alpha1 subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Finally, farnesol has a blood pressure-lowering action that may be relevant in vivo.

Role of chromosome X in the Sabra rat model of salt-sensitive hypertension.

We carried out a total genome screen in the Sabra rat model of hypertension to detect salt-susceptibility genes. We previously reported in male animals the presence of 2 major quantitative trait loci (QTLs) on chromosome 1 that together accounted for most of the difference in the blood pressure (BP) response to salt loading between Sabra hypertension-prone rats (SBH/y) and Sabra hypertension-resistant rats (SBN/y). In females, we reported on 2 major QTLs on chromosomes 1 and 17 that together accounted for only two thirds of the difference in the BP response between the strains. On the basis of phenotypic patterns of inheritance in reciprocal F2 crosses, we proposed a role of the X chromosome. We therefore continued the search for the missing QTL in females that would account for the remaining difference in the BP response between the 2 strains using newly developed microsatellite markers and focusing on chromosome X. We screened an F2 cross, consisting of 371 females and 336 males, using 19 polymorphic chromosome X microsatellite markers. We analyzed the averages of BP by genotype using ANOVA and the individual data using MAPMAKER/QTL. In female F2 progeny, we identified a segment on chromosome X that spans over 33.4 cM and shows significant cosegregation (P<0.001) of 14 microsatellite markers (demarcated by DXRat4 and DXMgh10) with systolic BP after salt loading. This segment has 2 apparent peaks at DXRat4 and DXRat13, with a BP effect of 14 mm Hg for each. Multipoint linkage analysis with a free model detected 3 peaks (logarithm of the odds ratio [LOD] score >4.3) within the same chromosomal segment: One between DXMgh9 and DXMit4 (LOD 4.9; 6.1% of variance), a second between DXMgh12 and DXRat8 (LOD 5.2; 7.2% of variance), and a third between DXRat2 and DXRat4 (LOD 5.8; 7.5% of variance). On the basis of these findings and until congenic strains become available, our working assumption is that within chromosome X, 1 to 3 genetic loci contribute importantly to the BP response of female Sabra rats to salt. In male F2 progeny, we detected no significant cosegregation of any region on chromosome X with the BP response to salt loading. We conclude that in the female rat, salt susceptibility is mediated by 3 to 5 gene loci on chromosomes 1, 17, and X, whereas in the male rat, the X chromosome does not affect the BP response to salt.

[Angiotensin converting enzyme (ACE) gene polymorphism in a diabetic cohort and diabetic nephropathy].

The renin-angiotensin system is thought to play an important role in the pathophysiology of kidney disease in diabetes. Previous studies have shown a possible association between the D allele of the angiotensin converting enzyme (ACE) gene, known to be associated with higher circulating levels of ACE, and increased risk of developing nephropathy in NIDDM. The present study investigated the distribution of ACE gene genotypes in the general population and patients with NIDDM, the association between the D allele and diabetic nephropathy, and the association between the ACE genotype and involvement of other target organs in NIDDM. The ACE genotype (insertion/deletion I/D) was determined in all subjects, subsequently divided into 3 groups based on their polymorphism (DD, DI and II). The presence of nephropathy was defined by an albumin-creatinine ratio of 30 mg/g or greater (mean of 2 first morning urine samples). In the general population most had the D allele (DD or ID) and a minority the II genotype. There was no association between genotype and hypertension, ischemic heart disease, hyperlipidemia, and cerebrovascular or peripheral vascular disease. In diabetics the genotype distribution was not different from that in the general population. Within the diabetic group, there was no association between genotype and hypertension, hyperlipidemia, duration of diabetes, or HbA1C levels. Nephropathy, found in 81 of the 156 with NIDDM, was not associated with genotype. Diabetic nephropathy was not associated with retinopathy, neuropathy, or ischemic heart, cerebrovascular or peripheral vascular disease. We conclude that in the population sampled, there was no association between the D allele of the ACE gene and the risk of developing nephropathy in NIDDM.

Does the Sabra hypertension-prone rat represent a model of salt or mineralocorticoid sensitivity?

OBJECTIVES: Since the Sabra experimental model of hypertension was developed, it has been known as a model of salt-susceptible hypertension. Because the hypertensive response of the Sabra hypertension-prone strain (SBH/y) is classically elicited by salt loading with a combination of deoxycorticosterone acetate (DOCA) and salt, doubt has now been cast on whether the hypertensive response is due to sensitivity to salt or to mineralocorticoids. The present study was designed to resolve this question. MATERIALS AND METHODS: We studied the blood pressure response of SBH/y to various modes of salt loading. Animals were salt-loaded by administration of: 1% NaCl in drinking water and subcutaneous implantation of a 25 mg DOCA pellet (DOCA-salt); DOCA alone; 1% NaCl in drinking water alone; or 8% NaCl in chow alone. Blood pressure was determined by the tail-cuff method in awake and undisturbed animals. RESULTS: Within 4 weeks, the DOCA-salt treatment elicited the full hypertensive response previously reported in the SBH/y strain. Salt loading with 8% NaCl in chow reproduced the full hypertensive response observed with DOCA-salt, except that it occurred only after 7 weeks of treatment. Salt loading with DOCA alone raised blood pressure moderately and to a maximal level within 3 weeks; the magnitude of the blood pressure response was, however, significantly smaller than that observed with DOCA-salt or 8% NaCl in chow. Administration of 1% NaCl in water alone elicited no hypertensive response. CONCLUSIONS: The hypertensive response to salt loading in the Sabra experimental model of hypertension is an expression primarily of salt sensitivity, as it can be fully reproduced with salt alone, but not with DOCA alone. The use of the DOCA-salt mode of salt loading in this model, as opposed to salt loading with 8% salt in chow, is a useful way of accelerating the development of salt-sensitive hypertension in SBH/y, which shortens, and therefore facilitates, phenotyping.

Genetic basis of salt-susceptibility in the Sabra rat model of hypertension.

The Sabra salt-sensitive SBH/y and salt-resistant SBN/y rats constitute a unique experimental model of hypertension in which salt-susceptibility is genetically determined and expressed only after salt-loading, without the development of spontaneous hypertension. To determine the genetic basis of salt-susceptibility in the Sabra rats, the candidate gene and total genome screen approaches were adopted. The likely candidate genes in this model incorporate salt-related physiological mechanisms such as the nitric oxide system, the arginine vasopressin axis and the epithelial sodium channel. In the random genome search scheme for culprit genes, SBH/y and SBN/y were cross-bred. A highly unusual and composite mode of transmission of salt-susceptibility was found in this cross, emphasizing the complexity of the genetic basis of salt-susceptibility. Linkage analysis of the entire rat genome with a large number of widely distributed microsatellite markers identified three putative gene loci on chromosomes 1 and 17 that contribute importantly to salt-sensitivity and/or resistance, and uncovered sex specificity in the role that salt-susceptibility genes fulfill in the development of hypertension.

Salt susceptibility maps to chromosomes 1 and 17 with sex specificity in the Sabra rat model of hypertension.

Random genome screening was initiated in the Sabra rat model of hypertension in search of genes that account for salt sensitivity or salt resistance in terms of the development of hypertension. Female salt-sensitive Sabra hypertension-prone (SBH/y) rats were crossed with male salt-resistant Sabra hypertension-resistant (SBN/y) rats, resulting in an F2 cohort consisting of 100 males and 132 females. Systolic blood pressure (BP) was measured in rats at 6 weeks of age under basal conditions and after 4 weeks of salt loading. Genotypes for 24 polymorphic microsatellite markers localized to chromosome 1 and for 8 markers localized to chromosome 17 were determined in F2 and cosegregation with BP was evaluated by ANOVA and multipoint linkage analysis. Basal BP did not cosegregate with any locus on chromosomes 1 or 17. In contrast, BP after salt loading showed significant cosegregation with three QTLs, two on chromosome 1 and one on chromosome 17, designated SS1a, SS1b, and SS17, respectively; the maximal logarithm of the odds (LOD) scores were 4.71, 4.91, and 3.43, respectively. Further analysis revealed sexual dimorphism. In male F2, BP response to salt loading cosegregated with one QTL (LOD score 4.52) and a second QTL (LOD score 2.98), both on chromosome 1 and coinciding with SS1a and SS1b, respectively. In female rats, BP response cosegregated with one QTL on chromosome 1 (LOD score 3.08) coinciding with SS1b, and with a second QTL on chromosome 17 (LOD score 3.66) coinciding with SS17. In males, the additive effects of the two QTLs on chromosome 1 accounted for most of the BP variance to salt loading, whereas in females the additive effects of the QTLs on chromosomes 1 and 17 accounted for over two thirds of the variance. These results identify three putative gene loci on chromosomes 1 and 17 that contribute importantly to salt sensitivity and/or resistance and uncover sex specificity in the role that salt susceptibility genes fulfill in the development of hypertension.

Nitric oxide synthase and renin-angiotensin system gene expression in salt-sensitive and salt-resistant Sabra rats.

The molecular mechanisms of salt sensitivity and the contribution of the kidney to salt-induced hypertension in Sabra rats are imperfectly defined. We investigated the expression of the nitric oxide (NO) system (endothelial, inducible, and neural NO synthases) and renin-angiotensin system (renin, angiotensinogen, and angiotensin II type 1A receptor) gene components in the kidneys of SBN/y (salt-resistant) and SBH/y (salt-sensitive) Sabra rat substrains, with and without deoxycorticosterone acetate (DOCA)-salt treatment. We also looked for immunocytochemical evidence of angiotensin II, the effector peptide of the renin-angiotensin system. Inducible and neural NO synthase gene expression values were lower in SBH/y than in SBN/y before and after DOCA-salt treatment. The gene expression level of endothelial NO synthase was not different in SBH/y and SBN/y, either with or without DOCA salt. Renin gene expression was significantly higher in kidneys of SBN/y than in kidneys of SBH/y rats, whereas angiotensinogen gene expression was significantly lower in SBN/y. After DOCA-salt treatment, renin gene expression was strongly suppressed in both strains but more so in SBH/y. Angiotensinogen gene expression, on the other hand, was increased by DOCA salt in SBN/y rats so that the two strains were no longer different. Angiotensin II immunoreactivity was significantly higher in SBN/y than in SBH/y; however, after DOCA salt, immunoreactivity in both strains was no longer detectable. Angiotensin II type 1A receptor gene expression was not different between the two strains, either before or after DOCA-salt administration. We conclude that DOCA salt induced a decrease in the activity of the renin-angiotensin system but did not change NO synthase gene expression in SBH/y and SBN/y. Inducible and neural NO synthase gene expression values were less in SBH/y than in SBN/y, independent of DOCA-salt administration. Thus, the NO system could explain, at least in part, the salt resistance of SBN/y.

Pressure natriuresis in salt-sensitive and salt-resistant Sabra rats.

Salt-resistant (SBN/y) and salt-sensitive (SBH/y) Sabra rats are a useful model of salt-sensitive hypertension with incompletely explored renal mechanisms. We investigated their pressure-natriuresis curves, with and without deoxycorticosterone acetate (DOCA)-salt treatment. To differentiate between extrinsic neural and hormonal mechanisms and intrinsic renal influences, we performed experiments with neural denervation, adrenalectomy, and infusions of vasopressin, norepinephrine, 17-hydroxycorticosterone, and aldosterone as well as without these maneuvers. In untreated SBN/y without controlled neural and circulating hormonal factors, urine flow and sodium excretion increased from 32 to 95 microL/min per gram kidney weight (gkwt) and from 4 to 17 mumol/min per gkwt, respectively, as renal perfusion pressure was increased from 85 to 146 mm Hg. Renal blood flow and glomerular filtration rate were autoregulated and averaged 7.5 and 1.2 mL/min per gkwt. In untreated SBN/y with controlled neural and circulating factors, pressure-diuresis and -natriuresis curves were shifted toward the right, and renal blood flow and glomerular filtration rate ranged between 4.2 and 9.1 or 1 and 1.3 mL/min per gkwt as perfusion pressure was increased from 99 to 164 mm Hg. In both protocols, values in SBH/y did not differ. DOCA-salt increased blood pressure in SBH/y. In SBH/y without controlled neural and hormonal factors, pressure-diuresis and -natriuresis curves were shifted approximately 20 mm Hg toward the right. Fractional sodium and water excretion curves, renal blood flow, and glomerular filtration rate were shifted rightward in parallel. On the other hand, SBH/y with DOCA-salt and controlled neural and hormonal factors had lower sodium and water excretion rates only at the renal perfusion pressure of 150 mm Hg as well as decreased renal blood flow and glomerular filtration rate compared with DOCA-salt SBN/y. These data suggest that both extrinsic and intrinsic factors are responsible for reduced sodium and water excretory capacity in DOCA-salt SBH/y; however, the extrinsic factors may be more important.

Polymorphisms in the carboxy-terminus of the epithelial sodium channel in rat models for hypertension.

OBJECTIVE: To investigate whether mutations in the C-terminus of the three subunits of the rat epithelial sodium channel (alphabetagamma-rENaC) contribute to the hypertensive phenotype in five rat models for essential hypertension. DESIGN: We sequenced the C-terminal regions of alpha-, beta- and gamma-rENaC genes in five different hypertensive rat strains [spontaneously hypertensive rats (SHR), Dahl salt-sensitive (SS/Jr) rats, Milan hypertensive (MHS) rats, Sabra hypertensive (SBH) rats and Lyon hypertensive rats (LHR)] and their normotensive controls [Wistar-Kyoto (WKY) rats, Dahl salt-resistant (SR/Jr) rats, Milan normotensive (MNS) rats, Sabra normotensive (SBN) rats and Lyon normotensive rats (LNR)]. Identified polymorphisms were tested for cosegregation with blood pressure as well as for increased epithelial sodium channel (ENaC) activity. METHODS: Genomic DNA extracted from hypertensive and normotensive rat strains was amplified by the polymerase chain reaction and polymerase chain reaction fragments were sequenced. Cosegregation analysis was performed to test for correlations between blood pressure and different genotypes. The effects of a polymorphism on ENaC activity were assessed by functional expression in Xenopus laevis oocytes. The chromosomal location of the gene for gamma-ENaC was determined by linkage analysis in an F2 (MHS x MNS) population. RESULTS: We found no polymorphisms at the C-terminus of alpha- and beta-rENaC in the five rat models tested. We identified two polymorphisms at the C-terminus of the gamma-subunit, one leading to an amino acid change. Milan strains (MNS and MHS) were polymorphic for this mutation. By cosegregation analysis we could exclude the possibility that there was a correlation between blood pressure and this polymorphism. Functional expression of the polymorphism caused no increase in ENaC activity assessed by measurement of the amiloride-sensitive sodium current in Xenopus oocytes. The gene for the gamma-ENAC was located on rat chromosome 1. CONCLUSIONS: No polymorphisms at the C-terminus of the three subunits of the epithelial sodium channel cosegregating with blood pressure were detected in five different genetic rat models for hypertension. If an altered ENaC activity contributes to the pathogenesis of hypertension in these rats, it must thus arise from mutations in other parts of the protein, from mutations outside the coding region impairing the proper regulation of one of the subunits or from mutations in an ENaC-associated protein.

Glomerular basement membrane polyanionic sites and nitric oxide in genetically salt-sensitive and resistant hypertensive rats.

Cationic colloid gold, a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, including heparan sulfate protoglycan in genetic salt-sensitive (SBH/Y) and resistant (SBN/Y) hypertensive rats, with or without high dietary salt intake. GBM morphology, renal function and nitric oxide, as measured by plasma and urine nitrite (NO2) and nitrate (NO3) were also determined. In the salt-sensitive rats the high-salt dietary intake resulted in severe hypertension, proteinuria and decreased glomerular filtration rate. After 1 month of high-salt intake, the average width of the GBM of salt-sensitive rats was higher by 27% than that of salt-resistant rats. The number of GBM anionic sites (lamina rata externa and interna) was much lower in both salt-sensitive and salt-resistant groups after 1 month of salt loading, 8.04+/-0.36 and 7.8+/-0.25 counts/cm, respectively, compared to the respective values of non-salt-loaded animals, 20.58+/-1.08 counts/cm in the SBH/Y (p < 0.001) and 21+/-1.86 counts/cm in the SBN/Y (p < 0.001). A decreased nitric oxide production was found in the salt-sensitive rats before and after salt loading compared with the salt-resistant group. No correlation was found between the nitric oxide changes and the GBM modifications. It is concluded that high-salt intake may be deleterious to the permselectivity of the GBM. It is suggested that salt restriction in hypertension may have a beneficial effect in preventing GBM permselectivity changes and proteinuria.

Development, genotype and phenotype of a new colony of the Sabra hypertension prone (SBH/y) and resistant (SBN/y) rat model of slat sensitivity and resistance.

OBJECTIVES: Variations in the blood pressure response to salt-loading, the lack of quality control measures, and the need to prepare the strains for genetic studies led to renewed secondary inbreeding of the original colony of Sabra hypertension prone (SBH) and resistant (SBN) rats in order to regain genotypic and phenotypic homogeneity of the substrains. METHODS: Animals from the original breeding colony were selectively inbred for basal normotension and for susceptibility or resistance to the development of hypertension following salt-loading with deoxycorticosterone acetate (DOCA)-salt. Efficacy of inbreeding was tested by genome screening with 416 microsatellite primer sets. Phenotyping was based on measurements of systolic blood pressure by the tail-cuff methodology in awake, undisturbed animals maintained on standard diet and after salt-loading with DOCA-salt. Telemetric measurements of blood pressure were performed in a small number of animals to validate tail-cuff measurements. RESULTS: Animals from the new colony were designated SBH/y and SBN/y to differentiate from the original colony. Fourteen generations have been inbred over the past 4 years. Of the 402 microsatellites that amplified, 183 (45.5%) were polymorphic between the two substrains, and not a single locus was found to be heterozygous in either substrain. Phenotypic characteristics are provided for SBH/ y and SBN/y rats with respect to tail-cuff systolic blood pressure. The values obtained, which were validated by telemetry, demonstrate classical features of salt sensitivity or resistance, respectively. CONCLUSIONS: The genetic homogeneity found in SBH/y and SBN/y, the phenotype demonstrating salt-sensitivity or salt-resistance in terms of development of hypertension, and the relatively high frequency of informative genetic markers identify this Sabra rat model as highly suited for studies concerning the molecular genetics of gene-environment interactions affecting blood pressure regulation.

Disparate expression of the AVP gene in Sabra hypertension-prone and hypertension-resistant rats.

We recently re-inbred the original colony of SBH-SBN rats, a model of salt-induced hypertension. In the course of phenotyping the new colony, SBH/y were found to excrete a lower urine flow with a higher urine osmolality than SBN/y. Thus disparate water handling between the substrains, a phenotype characteristic of the original colony, was retained throughout the selection procedure and transmitted down the generations to the new colony. As water handling is directly linked to arginine vasopressin (AVP) and in view of potential linkage of this phenotype to salt sensitivity or resistance in terms of the development of hypertension, the AVP axis was further investigated in the new substrains. Basal plasma AVP levels were higher in SBH/y (2.86 +/- 0.22 pg/ml; n = 10) than in SBN/y (1.98 +/- 0.11 pg/ml; n = 10, P < 0.05). Water deprivation for 48 h increased plasma AVP levels severalfold in both substrains to similar levels. Niravoline, a kappa receptor agonist that inhibits central release of AVP, produced at 0.6 and 0.9 mg/kg a more profound diuretic effect in SBN/y than in SBH/y, suggesting greater pituitary release of AVP into the circulation of SBH/y. AVP mRNA contents were compared in SBH/y and SBN/y rats in whole hypothalamic extracts and in the supraoptic (SON) and paraventricular (PVN) nuclei by RNA protection assay. Under basal conditions, AVP mRNA content (in ng) in the hypothalamus of SBH/y was 4.48 +/- 0.52 (n = 29) and of SBN/y was 3.13 +/- 0.35 (n = 30), P < 0.05; in the SON of SBH/y, AVP mRNA content was 3.62 +/- 0.44 (n = 11) and of SBN/y was 2.21 +/- 0.54 (n = 10), P < 0.05; in the PVN of SBH/y, AVP mRNA content was 0.78 +/- 0.16 (n = 9) and of SBN/y was 0.77 +/- 0.13 (n = 11, not significant). Thus the differences in hypothalamic AVP mRNA were primarily in the SON. Water deprivation as well as salt loading (8% NaCl) induced a significant elevation in AVP mRNA content in SBN/y but a blunted response in SBH/y. These data suggest that there is genetically transmitted enhanced hypothalamic expression of the AVP gene in SBH/y compared with SBN/y which results, under basal conditions, in greater pituitary release of AVP, in higher plasma AVP levels, and in increased renal concentrating activity. As AVP has been implicated in various forms of hypertension, these findings render AVP a candidate gene for salt sensitivity or resistance in the Sabra rat model of hypertension.

Processing of 125I-insulin by polarized cultured kidney cells.

Renal clearance of insulin is achieved by glomerular filtration and by passage from the postglomerular peritubular circulation into the renal interstitium. In the proximal tubule, filtered insulin binds to the apical membrane and is internalized and degraded while insulin in the interstitium is taken up by receptor-mediated endocytosis and degraded. To study these processes we have utilized cultured opossum kidney cells. These cells have proximal-like features and process insulin in a manner consistent with that described in vivo. To study apical and basolateral uptake and metabolism of insulin independently, cells were grown on filters suspended in culture wells. insulin was degraded to large insulin-size intermediates and low-molecular-weight products. This occurred whether the protein was internalized from the apical or basolateral pole of the cells. Analysis of the intermediate products by reverse-phase high-performance liquid chromatography revealed that products formed after apical or basolateral internalization were similar. Since products were preferentially released from the side of uptake, it is likely that apically and basolaterally internalized insulin is degraded in comparable organelles located in different regions of the cell. Most of the internalized insulin traversed the degradative pathway but some insulin followed a retroendocytic or minor transcytotic pathway. Degradation was inhibited by chloroquine, which also selectively increased the release of internalized insulin from the apical pole irrespective of the side of uptake. Thus while the polar degradative processes appear to be similar in nature, the polar exocytotic processes appear to be different.

The effects of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct.

It has been previously demonstrated that adenosine induces natriuresis when administered directly into the renal circulation of the rat. It was postulated that the mechanism was inhibition of tubule Na+ reabsorption. In the current study, the hypothesis was tested that adenosine inhibits ion reabsorption across the inner medullary collecting duct (IMCD), a tubule segment which is rich in adenosine receptors. IMCD epithelium from rat kidney was grown in primary culture as a confluent monolayer on Costar filters, allowing selective access to the basolateral and apical surfaces of the cells. Transepithelial resistance was taken as a measure of epithelial permeability and ion conductance. Na+ uptake was studied using 22Na+ and used to determine the permeability of the epithelial monolayer specifically to Na+. Exposure of the basolateral aspect of the monolayer to adenosine (10(-8)-10(-7) M) increased transepithelial resistance in a dose- and time-dependent manner; in parallel, adenosine (10(-7)-10(-6) M) reduced apical Na+ uptake from 20 +/- 5 to 10 +/- 2 nmol/cm2. 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX, 5 x 10(-9) M), an adenosine antagonist with selectivity for the A1 receptor, inhibited the rise in transepithelial resistance and the decrease in Na+ uptake following the addition of adenosine. The effects of adenosine on transepithelial resistance were reproduced with the A1 receptor selective adenosine analogue N6-cyclohexyladenosine (CHA, 10(-8)-10(-7) M) but not with the A2 selective analogues, 5'-N-ethylcarboxamidoadenosine (NECA) or CGS 21680. CHA (10(-7) M) inhibited apical Na+ uptake by 50%, an effect abolished by PACPX. (ABSTRACT TRUNCATED AT 250 WORDS)

Effect of extended exposure to hypertonicity on vasopressin messenger ribonucleic acid content in hypothalamo-neurohypophyseal explants.

The feasibility of using organ-cultured explants of the rat hypothalamo-neurohypophyseal system (HNS) to study the mechanisms regulating the vasopressin (VP) mRNA content of the HNS was examined by evaluating the effect of exposure to hypertonicity on the VP mRNA content of these explants. Different effects were observed after a step increase in osmolality and a gradual increase in the same amount over 24 h. The VP mRNA content of control HNS explants determined from a RNA protection assay was 22 +/- 6 pg. It gradually decreased to 23% and 9% of the control value during 24 and 48 h in culture, respectively. Northern blot analysis revealed a single band of VP mRNA approximately 700 bases long in explants cultured for 36 h. Explants exposed to the step increase in osmolality were maintained in static culture. The control explants were placed directly into isotonic medium (299 mosmol/kg H2O). The explants exposed to the step increase were placed directly into hypertonic medium (greater than 304 mosmol/kg H2O). After 24 h in culture, basal VP release was measured, and all explants were then exposed to a further acute 15 mosm/kg H2O increase in osmolality. The highest basal release of VP was observed in the explants maintained under isotonic conditions (299 mosm/kg H2O). These explants significantly increased VP release in response to the acute increase in osmolality. Basal VP release was lower in explants maintained in hypertonic medium (greater than 304 mosmol/kg H2O), and these explants did not respond to the acute hypertonic pulse. VP mRNA content was significantly decreased in explants maintained for 24 or 48 h in hypertonic medium compared to that in explants maintained in isotonic medium (47 +/- 10% and 57 +/- 6%, respectively; P less than 0.01). No significant difference existed in the VP content of the posterior pituitary between the groups. To achieve a slow increase in osmolality, explants were perifused in individual chambers with medium at 2.1 ml/h. A gradual increase in osmolality (16 mosmol/kg H2O medium) was achieved by increasing the NaCl concentration in the perifusion medium. In response to this stimulus there was a significant increase in VP release, which was sustained for 9 h. VP mRNA content in the hypertonic group was 165 +/- 19% of that in control explants (P less than 0.001), but no difference existed in VP content in the posterior pituitary compared to that in time control explants.(ABSTRACT TRUNCATED AT 400 WORDS)