Milan Hypertensive Strain Rats Research Articles

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

The role of the kidney in salt-sensitive hypertension

Primary hypertension is one of the leading risk factors for cardiovascular disease. Although the pathogenesis is not completely understood, an imbalance of sodium and chloride homeostasis seems to be relevant both in the induction and in the maintenance of salt-sensitive hypertension. Besides individual renal phenotypes, salt intake is one of the most important environmental determinants of this condition. The Milan hypertensive strain (MHS) of rats is an interesting model to investigate the molecular mechanisms underling the development of salt-sensitive hypertension. In young MHS rats, hypertension is anticipated by a phase of increased salt reabsorption localized along the medullary thick ascending limb associated with the up-regulation of the apical sodium-potassium-chloride cotransporter (NKCC2). Later, the frank hypertensive status of adult MHS rats is accompanied by the activation of the luminal and basal lateral transporters of sodium chloride (NaCl) in the distal convoluted tubule (DCT). Several lines of evidence have proven the key role of DCT in the maintenance of hypertension in MHS rats; more importantly, hypertensive patients carrying a mutation of α-adducin (resembling the MHS model) have a high sensitivity to thiazides, suggesting that the Na(+)-Cl(-) cotransporter also plays a pivotal role in humans.

NKCC2 is activated in Milan hypertensive rats contributing to the maintenance of salt-sensitive hypertension

The Milan hypertensive strain of rats (MHS) develops hypertension as a consequence of the increased tubular Na(+) reabsorption sustained by enhanced expression and activity of the renal tubular Na-K-ATPase. To verify whether the Na-K-2Cl cotransporter (NKCC2) is involved in the maintenance of hypertension in MHS rats, we have analysed the phosphorylation state and the activation of NKCC2 in Milan rats. Western blotting and immunofluorescence experiments were performed using specific antibodies against the regulatory phospho-threonines in the NKCC2 N terminus (R5 antibody). The phosphorylation levels of NKCC2 were significantly increased in the kidney of MHS rats. Moreover, the administration of furosemide in vivo decreased the blood pressure and increased the urine output and natriuresis in MHS rats demonstrating the actual involvement of NKCC2 activity in the pathogenesis of hypertension in this strain of rats. The up-regulation of NKCC2 activity is most probably mediated by a STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylation at serine-325 since it was significantly increased in MHS rats. Interestingly, aldosterone treatment caused an increase in NKCC2 phosphorylation in NKCC2-expressing MDCK cells. In conclusion, we demonstrated an increase in the activity of NKCC2 along the TAL that significantly contributes to the increase in systemic blood pressure in MHS rats. The elevated plasma levels of aldosterone, found in MHS rats, may influence Na(+) balance through a SPAK-dependent regulation of NKCC2 accounting for the maintenance of the hypertensive state in MHS rats.

Altered Expression of Renal Aquaporins and -Adducin Polymorphisms May Contribute to the Establishment of Salt-Sensitive Hypertension

Sodium-sensitive hypertension is caused by renal tubular dysfunction, leading to increased retention of sodium and water. Previous findings have suggested that single-nucleotide polymorphisms of the cytoskeletal protein, α-adducin, are associated with increased membrane expression of the Na/K pump and abnormal renal sodium transport in Milan hypertensive strain (MHS) rats and in humans. However, the possible contribution of renal aquaporins (AQPs) to water retention remains undefined in MHS rats. Kidneys from MHS rats were analyzed and compared with those from age-matched Milan normotensive strain (MNS) animals by quantitative-PCR, immunoblotting, and immunoperoxidase. Endocytosis assay was performed on renal cells stably expressing AQP4 and co-transfected either with wild-type normotensive (NT) or with mutated hypertensive (HT) α-adducin. Semiquantitative immunoblotting revealed that AQP1 abundance was significantly decreased only in HT MHS whereas AQP2 was reduced in both young pre-HT and adult-HT animals. On the other hand, AQP4 was dramatically upregulated in MHS regardless of the age. These results were confirmed by immunoperoxidase microscopy. Endocytosis assays clearly showed that the expression of mutated adducin strongly reduced the rate of constitutive AQP4 endocytosis, thereby increasing its abundance at the plasma membrane. We provide here the first evidence that AQP1, AQP2, and AQP4 are dysregulated in the kidneys of MHS animals. In particular, we provide evidence that α-adducin mutations may be responsible for AQP4 upregulation. The downregulation of AQP1 and AQP2 and the upregulation of AQP4 may be relevant for the onset and maintenance of salt-sensitive hypertension.

Upregulation of Na+/Ca2+exchanger and TRPC6 contributes to abnormal Ca2+homeostasis in arterial smooth muscle cells from Milan hypertensive rats

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca(2+) signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca(2+) signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 +/- 2 vs. 113 +/- 1 mmHg, P < 0.05). Resting cytosolic free Ca(2+) concentration (measured with fura-2) and ATP-induced Ca(2+) transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na(+)/Ca(2+) exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by approximately 13-fold), whereas the expression of plasma membrane Ca(2+)-ATPase was not altered. Ca(2+) entry via the reverse mode of NCX1 evoked by the removal of extracellular Na(+) induced a rapid increase in cytosolic free Ca(2+) concentration that was significantly larger in MHS arterial myocytes. The expression of alpha(1)/alpha(2) Na(+) pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca(2+) signaling. We suggest that the increased Ca(2+) signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats.

Functional alterations of mesenteric small resistance arteries in Milan hypertensive and normotensive rats

The Milan hypertensive rat strain (MHS) is a genetic strain in which cardiovascular phenotypes seem to be dependent, at least in part, on adducin gene polymorphisms. The aim of our study was to evaluate the structure, contractile responses and endothelium-dependent vasodilation in mesenteric small resistance arteries in 12-week-old MHS, (n=7), age-matched Milan normotensive rats (MNS, n=7) and congenic strains in which the DNA segments carrying the alpha-adducin locus from the MHS have been introgressed into the MNS (MNA, n=7). Systolic blood pressure (tail cuff) and left ventricular weight to body weight were measured. Mesenteric small arteries were dissected and mounted on a micromyograph; the media:lumen ratio was then calculated. Concentration-response curves to acetylcholine and to norepinephrine (NE) were created. Systolic blood pressure was significantly increased in the MHS and MNA strains compared with the MNS. No significant difference in mesenteric small resistance artery structure was observed among the groups; however, a slightly more elevated media:lumen ratio was observed in MNA compared with the MNS. In contrast, left ventricular weight to body weight was significantly increased and ACH-induced dilatation was significantly impaired in the MHS and in MNA compared with MNS. The concentration-response curve to NE in the MHS showed significantly reduced sensitivity to NE; however, maximum contraction was increased in the MHS vs. the other groups. The MHS presents cardiac (but not vascular) remodeling, endothelial dysfunction and a peculiar contractile response to NE, compared with the other groups. The systolic blood pressure increase and trend to vascular remodeling in MNA support the pathogenic role of alpha-adducin.

Aquaporin 2 and Adducin Polymorphism Impact on Essential Hypertension

Aim: In this study we tested the hypothesis that alteration in Aquaporin 2 (AQP2) expression/trafficking may contribute to the extracellular volume expansion observed in hypertension.Methods: To test this hypothesis we monitored the urinary AQP2 excretion in patients with essential hypertension carrying polymorphisms of the cytoskeletal protein adducin (ADD) known for increasing tubular sodium reabsorption. AQP2 urinary excretion was also measured in Milan Hypertensive Strain (MHS) rats carrying single nucleotide polymorphisms (SNPs) of ADD genes. AQP2 trafficking to the apical plasma membrane was also evaluated in MCD4 cells upon overexpression of human SNPs of ADD1 gene.Results: AQP2 excretion was significantly higher in hypertensive patients than in controls. In addition, after two hours of acute sodium load, AQP2 excretion was significantly higher in patients carrying ADD mutation. MHS rats showed a significant increase in total AQP2 protein and an increased expression at the apical plasma membrane compared to normotensive rats. In MCD4 cells, AQP2 trafficking to the apical plasma membrane was significantly increased upon co‐expression of mutated ADD.Conclusions: Taken together, these observations suggest that increase in AQP2 expression/trafficking possibly related to alteration of cytoskeleton structure, may contribute to the extracellular volume expansion observed in hypertension.

Glomerular barrier dysfunction in glomerulosclerosis‐ resistant Milan rats with experimental diabetes: the role of renal haemodynamics

Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease. Genetically determined hypertrophy of intrarenal arteries has been suggested as the putative mechanism preventing transmission of systemic hypertension to the glomerular microcirculation or diabetes-induced loss of autoregulation, which lead to glomerular hypertension and consequent podocyte injury and proteinuria. This study aimed to investigate glomerular barrier function and structure in ageing and diabetic MHS rats under basal conditions and after injection of 2.5 g of bovine serum albumin (BSA) causing increased workload and possibly removing haemodynamic protection by inducing renal cortical vasodilatation. Genetically related rats of the Milan normotensive strain (MNS) served as a proteinuric counterpart. No change in renal function or structure was detected in diabetic MHS rats, whereas MNS rats developed diabetic nephropathy superimposed on that occurring spontaneously in this strain. Diabetic, but not non-diabetic, MHS rats showed significantly reduced synaptopodin and nephrin expression, though to a lesser extent than non-diabetic and diabetic MNS rats, together with unchanged podocyte number, density and structure and no proteinuria. Agrin expression was significantly altered in diabetic versus non-diabetic MHS animals, whereas collagen I was expressed only in diabetic MHS rats and collagen IV content did not change significantly between the two groups. Upon BSA injection, proteinuria increased markedly and abundant BSA was detected only in kidneys from diabetic MHS rats. BSA injection was associated with changes in intrarenal arteries suggesting vasodilatation, without any influx of inflammatory cells. These data indicate that while MNS rats show marked changes in the glomerular filtration barrier with either age or diabetes, glomerulosclerosis-resistant MHS rats develop only minor diabetes-induced podocyte (and extracellular matrix) alterations, which are not associated with proteinuria unless they are unmasked by an increased workload or removal of the haemodynamic protection.

Adducin Polymorphisms and the Treatment of Hypertension

Hypertension is an important public health problem affecting more than 50 million individuals in the USA alone. The most common form, essential hypertension, results from the complex interplay between genetic predisposition and environmental influences. Epidemiological, migration, intervention and genetic studies in humans and animals provide very strong evidence of a causal link between high salt intake and high blood pressure. One of the candidate genes for salt-sensitive hypertension is adducin. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2 and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through enhanced constitutive tubular sodium reabsorption. An example of a prospective efficacy of pharmacogenetics and pharmacogenomics is the detection and impact of adducin polymorphisms on hypertension. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar blood pressure reduction in carriers of the mutated adducin may support new strategies aimed at optimizing the use of new antihypertensive agents for the prevention of hypertension-associated organ damage.

Pharmacogenomics and Pharmacogenetics of Hypertension

There is a growing literature on the potential prospective use of genome information to enhance success in finding new medicines. An example of a prospective efficacy of pharmacogenetic and pharmacogenomics is the detection and impact of adducin polymorphism on hypertension. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2, and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through an enhanced constitutive tubular sodium reabsorption. In particular, six linkage studies, 18 of 20 association studies, and four of five follow-up studies that measured organ damage in hypertensive patients support the clinical impact of adducing polymorphism. As many modulatory genes and environment affect the adducin activity, the context must be taken into account to measure the clinical effect size of adducins. Pharmacogenomics is giving an important contribution to this end. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar BP reduction in carriers of the mutated adducin may support new strategies that aim to optimize the use of antihypertensive agents for the prevention of hypertension-associated organ damage.

Role of TGF-β/GLUT1 axis in susceptibility vs resistance to diabetic glomerulopathy in the Milan rat model

GLUT1 upregulation and increased glucose transport activity may contribute to extracellullar matrix (ECM) accumulation characterizing diabetic nephropathy (DN). Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease, due to a haemodynamic protection. On the contrary, those of the Milan normotensive strain (MNS) develop spontaneous glomerulosclerosis, and when rendered diabetic, show typical morphological and haemodynamic changes. To assess whether susceptibility to diabetic glomerulopathy in MNS rats is associated with higher glucose transporter 1 (GLUT1) expression (and glucose transport activity) vs MHS rats, diabetic and nondiabetic MNS and MHS rats were followed for 6 months and mesangial cells derived from these animals were exposed to high glucose (HG) vs normal glucose (NG) conditions. Glomerular expression of GLUT1 protein and ECM and transforming growth factor-beta (TGF-beta) mRNA was significantly upregulated in diabetic vs nondiabetic MNS, but not MHS rats. Upon exposure to HG and/or TGF-beta, mesangial cells from 1- and 8-month-old MNS rats showed higher glucose transport activity and GLUT1 membrane expression than those from age-matched MHS rats. Likewise, ECM and TGF-beta production increased more markedly in response to HG and/or TGF-beta in MNS vs MHS mesangial cells. These data indicate that susceptibility to diabetic glomerulopathy in MNS rats is associated with increased GLUT1-dependent glucose transport activity in response to hyperglycaemia and/or TGF-beta, which may amplify ECM overproduction. Conversely, the haemodynamic protection from glomerulosclerosis in MHS rats is associated with lack of upregulation of TGF-beta/GLUT1 axis, thus supporting the concept that this axis may represent the link between haemodynamic and metabolic mechanisms of injury.

Carotid arteriotomy induces different temporal gene expression profiles in normotensive and hypertensive rat strains

Analysis of gene expression profiles in patients or in animal models affected by cardiovascular diseases may provide insight into therapeutic strategies. In this study, 3 rat strains, Wistar Kyoto (WKY), spontaneously hypertensive rats (SHR) and the Milan hypertensive rat strain (MHS), have been investigated to assess the influence of genetic background and/or of hypertension on gene expression in arteriotomy-injured carotid arteries (CAs). Expression profiles of genes, c-myc, AT1, AT2, ETA, ETB, Bcl-2, Bax and Bcl-X, were determined by reverse transcription-polymerase chain reaction (RT-PCR) in the acute phase, from 1 to 48 h, following CA arteriotomy. WKY, SHR and MHS show significant differences in gene expression profiles after CA arteriotomy. c-Myc mRNA is activated earlier and/or to a greater extent in hypertensive strains than in WKY (p<0.05). AT1 mRNA increases in WKY after injury, while it decreases in both SHR and MHS (p<0.05). AT2 shows the opposite behaviour, decreasing in WKY and increasing in hypertensive strains (p<0.05). ETA mRNA decreases in all strains although with different timing and levels, associated with a replacement by ETB mRNA (p<0.05). Bcl-2/Bax ratio gradually decreases in WKY, while it decreases only transiently in SHR and MHS 4 h after injury (p<0.05). Overall data indicate that therapeutic strategies for stenosis prevention should carefully consider the gene expression profile after injury, the genetic background, the kind of vascular trauma and the diseases affecting the animal model or the patient.

Adducin Polymorphism

Adducin is a heterodymeric cytoskeleton protein, the 3 subunits of which are encoded by genes (ADD1, ADD2, ADD3) mapping to 3 different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function may cause hypertension through an enhanced constitutive tubular sodium reabsorption. Six human linkage studies showed positive results when a DNA marker mapping to 30 kb from the ADD1 locus or single-nucleotide polymorphisms (SNPs) of 1 of the 3 adducin genes were considered either alone or in combination with each other or angiotensin-converting enzyme (ACE) D allele or salt intake. When DNA markers mapping at much larger distance from the ADD1 locus were used, negative results were found by 4 studies. Positive results were also obtained in 18 of 20 association studies that, in addition to blood pressure, investigated variables reflecting body sodium or the renin-angiotensin system. Mixed results regarded case-control studies or studies in predominantly normotensive populations that did not consider the above-mentioned variables. Four of 5 studies showed a selective beneficial effect of diuretics in carriers of the mutated ADD1. Twelve of 16 studies found that ADD1 polymorphism alone or in combination with that of ACE positively associates with stroke or coronary heart disease or renal or vascular dysfunctions. In conclusion, when context is taken into account, the impact of adducin in hypertension and its related disorders is clear.

Glomerular number and size in Milan hypertensive and normotensive rats: their relationship to susceptibility and resistance to hypertension and renal disease.

Structural analysis, including morphometric computation of glomerular size and number, was applied to analyse the divergence between propensity to hypertension and renal damage, expressed by rats of the Milan hypertensive strain (MHS) and Milan normotensive strain (MNS), respectively. MHS, MNS rats and progenitor Wistar rats were investigated at age 9 weeks and 9 months. Classical morphometric methods were complemented by the dissector/fractionator technique to count glomeruli. At 9 weeks, when nephrogenesis was completed and hypertension established, MHS rats exhibited significantly lower kidney weight, cortical volume, glomerular number and volume compared to coeval MNS rats. In Wistar rats, these parameters were similar to those of MNS rats, except for lower glomerular volume. At 9 months, MHS rats showed significantly lower expansion of glomerular volume compared to MNS and Wistar rats. MNS rats had 10% sclerotic glomeruli, which was associated with reduced renal function and heavy proteinuria; conversely, sclerosis was rare in coeval MHS and Wistar rats. Media thickness was higher, whereas lumen diameter was lower, in intrarenal arteries of MHS versus MNS rats at both time points. These data indicate that structural changes other than a tubular defect may play a role in the development of hypertension in MHS rats. The lack of significant glomerular hypertrophy and damage in this strain, despite reduced glomerular number, could be related to their (haemodynamic) protection from hypertensive renal disease, possibly due to the hypertrophy of intrarenal arteries. The larger size of glomeruli of MNS rats may be linked to their susceptibility to glomerulosclerosis.

Interaction of the SH2 domain of Fyn with a cytoskeletal protein, beta-adducin.

Fyn is a Src family tyrosine kinase expressed abundantly in neurons and believed to have specific functions in the brain. To understand the function of Fyn tyrosine kinase, we attempted to identify Fyn Src homology 2 (SH2) domain-binding proteins from a Nonidet P-40-insoluble fraction of the mouse brain. beta-Adducin, an actin filament-associated cytoskeletal protein, was isolated by two-dimensional gel electrophoresis and identified by tandem mass spectrometry. beta-Adducin was tyrosine phosphorylated by coexpression with wild type but not with a kinase-negative form of Fyn in COS-7 cells. Cell staining analysis showed that coexpression of beta-adducin with Fyn induced translocation of beta-adducin from the cytoplasm to the periphery of the cells where it was colocalized with actin filaments and Fyn. These findings suggest that tyrosine-phosphorylated beta-adducin associates with the SH2 domain of Fyn and colocalizes under plasma membranes.

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats.

Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na+,K+-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA+,K+-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting.

Genetics of renal mechanisms of primary hypertension: the role of adducin.

To summarize data concerning the identification of adducin as a 'candidate' gene in the Milan hypertensive strain of rats (MHS), a genetic model of essential hypertension, and in human essential hypertension. The sequence of events from renal function to cell membrane ion transports and finally to the molecular defect has been established in MHS rats. This led to the identification of polymorphisms in the cytoskeletal protein adducin. These polymorphisms are involved in blood pressure regulation in these rats. A linkage and an association study on Caucasian populations support the involvement of adducin in human hypertension also. A polymorphism of alpha-adducin gene is significantly associated with human hypertension. In particular, both in humans and in rats, adducin polymorphisms affect kidney function by modulating the overall capacity of tubular epithelial cells to transport ions. Adducin polymorphisms account for only a portion of hypertension both in humans and rats. Therefore additive or epistatic interactions with other genes involved in renal sodium handling need to be studied.

High glucose level unmasks a genetic predisposition to enhanced extracellular matrix production in mesangial cells from the Milan normotensive strain.

A growing body of evidence indicates that the individual genetic background plays a role in the pathogenesis of diabetic glomerular disease by either favoring or protecting against injury produced by hyperglycemia. Two genetically related rat strains, the Milan normotensive strain (MNS) and the Milan hypertensive strain (MHS) display different susceptibilities to develop glomerulosclerosis with age. Glomerular sclerosing lesions occur in the MNS rats, which remain normotensive throughout their entire life-span, but not in the MHS rats, despite the presence of arterial hypertension. Previous studies have reported that extracellular matrix production and cell proliferation increased with donor-aging in mesangial cells isolated from MNS rats, but not in those from MHS rats, thus suggesting the existence of an inherited defect in the regulation of cell and matrix turnover, which translates into an abnormal response to growth-promoting stimuli favoring the development of glomerulosclerosis. In the study presented here, it was hypothesized that, in addition to donor-aging, other independent risk factors for the development of glomerular disease, such as metabolic injury by hyperglycemia, would be able to trigger and/or precipitate the occurrence of these changes in mesangial cells from the susceptible normotensive strain, but not in those from the protected hypertensive strain. To test this hypothesis, mesangial cells obtained from these rat strains (before the onset of either glomerulosclerosis or hypertension) were used to assess the effects of prolonged (4 wk) exposure to high (30 mmol/L) versus normal (5.5 mmol/L) glucose concentrations on extracellular matrix and cytokine production and cell proliferation. The accumulation and/or gene expression of the matrix components fibronectin, laminin, and collagen IV, and of the cytokines insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) did not change under normal glucose and increased progressively in response to high glucose in both MNS and MHS cells. These increases, with the exception of the increment in TGF-beta gene expression, were significantly more pronounced in MNS cells than in MHS cells. In contrast, the proliferative response to serum was not affected by high glucose, but increased in MNS cells, and decreased, although not significantly, in MHS cells during the 4-wk period, thus mimicking the changes previously observed in these rat strains as a function of age. These results indicate that high glucose unmasks a genetic tendency to produce increasing amounts of extracellular matrix, not yet evident under normal glucose conditions, and suggest that a genetically determined propensity of mesangial cells to hyperrespond to chronic hyperglycemia may be implicated in the pathogenesis of diabetic glomerular disease.

Association between Plasma Membrane (Ca+Mg) ATPase and Calpain/Calpastatin System in Rat Erythrocytes

We studied the activity of plasma membrane (Ca+Mg)ATPase from erythrocytes of Milan hypertensive rat strain (MHS) and Milan low calpastatin rat strain (MLCS), that show an activity level of the specific calpain inhibitor, calpastatin, about five fold reduced in comparison with the Milan normotensive rat strain (MNS), while the protease activity level is similar. This imbalance of calpain:calpastatin ratio leads to a decrease of the erythrocyte plasma membrane (Ca+Mg)ATPase activity and to the appearance of 124 kDa fragments, which are the typical products of proteolytic calpain action on the 136 kDa (Ca+Mg)ATPase native form.

Age-dependency and dietary influence on the hypothalamic ouabain-like factor in Milan hypertensive rats

Previous studies have demonstrated that the hypothalamus of the adult Milan hypertensive rat strain (MHS) contains a higher proportion of ouabain-like factor than Milan normotensive (MNS) controls. The present study was designed to demonstrate that the rat standard diet contains a ouabain-like factor similar to that extracted from rat tissue and to investigate the influence of low or high dietary ouabain-like factor content on tissue ouabain-like factor levels at different ages in MHS and MNS rats. MHS and MNS rats were reared on two controlled batches of standard rat diet containing low (batch A 0.09 mu g/kg) and high (batch B 0.7 mu g/kg) concentrations of ouabain-like factor. The mothers of these rats had also been fed with the diet throughout pregnancy and lactation. The hypothalamic content of ouabain-like factor was measured in both strains at 21, 30 and 90 days of age by high performance liquid chromatography fractionation. (1) The dietary ouabain-like factor content did not influence either the hypothalamic ouabain-like factor yield or systolic blood pressure, either in MHS or MNS rats. (2) As a function of age, the hypothalamic ouabain-like factor content was constant between 21 and 30 days of age in MHS rats, and then decreased by 60% at 90 days. In MNS rats, ouabain-like factor was decreased by 80 and 90%, respectively, at 30 and 90 days, compared to the age of 21 days. (3) At the age of 21 days, MHS rats had 30% lower levels of ouabain-like factor than MNS rats, but 60% higher levels at 30 and 90 days. Hypothalamic ouabain-like factor and systolic blood pressure are not influenced by dietary ouabain-like factor, thus excluding a process of passive tissue accumulation. Different mechanisms regulate the age-dependent endogenous ouabain-like factor production and accumulation in MHS and MNS rats, suggesting that the maintenance of higher ouabain-like factor levels in MHS than in MNS at the age of 30 and 90 days contributes to the development and maintenance of hypertension in this strain.