Milan Normotensive Research Articles

Modulation of Blood Pressure through Microbiome Exchange between Milan Normotensive and Hypertensive Rat Strains

Modulation of Blood Pressure through Microbiome Exchange between Milan Normotensive and Hypertensive Rat Strains

#4127 MODULATION OF BLOOD PRESSURE THROUGH MICROBIOME EXCHANGE BETWEEN MILAN NORMOTENSIVE AND HYPERTENSIVE STRAINS OF RATS

Abstract Background and Aims Hypertension is one of the major health problems contributing to the development of cardiovascular diseases. In contrast to essential hypertension, which often displays complex pathogenesis due to multifactorial aetiology, several monogenic forms of hypertension are caused by mutations in genes associated with renal salt handling. Thus, studies on monogenic forms of hypertension could hold potential for novel therapeutic approaches for essential hypertension. Recently, increasing numbers of studies have demonstrated the alteration of the gut microbiome in pathological states including hypertension. These observations suggest the involvement of the intestinal microbiome in the development and/or maintenance of hypertension. A reciprocal congenic strain of Milan hypertensive rat (NA rat), harbouring a missense mutation in the gene encoding the alpha subunit of the membrane-skeletal protein adducin (Add1), develops hypertension starting from 3 months after birth. Preliminary studies on NA rats and its littermate Milan normotensive (MN) rats showed diverse expression patterns of renal sodium transporters/ion channels in two strains of rats, indicating a putative role of altered renal salt handling on the enhanced blood pressure in NA rats (unpublished data). Based on this background, the aim of this study was to investigate a possible connection between the gut microbiome and blood pressure phenotypes in these two strains. Furthermore, we evaluated whether the exchange of faeces between the two strains could transfer the phenotypes through the gut microbiome. Finally, mechanisms underlying the altered phenotype after microbiome exchange were investigated. Method Hypertensive NA rats and normotensive MN rats were subjected to ‘homogenization (HOM)’ of the microbiome after birth, which consisted of the exchange of bedding containing faeces from either strain, followed by co-housing of MN and NA rats in the same cage (MN-HOM and NA-HOM groups). For the baseline (BSL) condition, rats were homogenized within the same strain (MN-BSL and NA-BSL). At the age of 5 months, blood pressure measurement was performed using a non-invasive tail-cuff BP-2000 Blood Pressure Analysis system. Faeces from each rat were collected for microbiota metataxonomic analysis using next generation sequencing of 16S ribosome encoding DNA. At the end of the treatment, rats were sacrificed for the collections of blood and organs to assess the expressions of genes involved in renal salt reabsorption. Results Systolic blood pressure (SBP) in MN-BSL was averaged at 143.6 mmHg, while NA-BSL showed average SBP 163.3 mmHg, confirming the hypertensive phenotype in NA rats at baseline (P<0.001). Interestingly, MN-HOM showed a significantly enhanced SBP compared to MN-BSL, reaching 153.5 mmHg (P = 0.005). On the other hand, the average SBP of NA-HOM was163.7 mmHg, indicating that homogenization did not significantly impact blood pressure in NA rats. Although the MN-HOM group showed significantly increased SBP compared with MN-BSL group, the average SBP of MN-HOM was still significantly lower than both the NA-BSL and NA-HOM groups. Different gut microbiota compositions were observed in the two strains of rats at baseline, and HOM altered both. Conclusion We have demonstrated that the hypertensive phenotype in NA rats was transferred to normotensive MN rats through the exchange of gut microbiota, indicating that the intestinal microbiota and/or metabolites derived therefrom could eventually modulate blood pressure. Further studies including immunoblotting as well as uremic toxin analyses are required to unveil the molecular mechanisms by which the gut microbiome modulates blood pressure.

MO068: Paraoxonase 2 Protects the Kidney Tissue from Aldosterone-Promoted Oxidative Stress in Milan Normotensive Rats Strain

Abstract BACKGROUND AND AIMS Kidney plays a pivotal role on the maintenance of blood pressure by the regulation of sodium reabsorption. Aldosterone sensitive distal nephron finely tunes salt reabsorption through several transporters, including the epithelial sodium channel (ENaC). Alteration in ENaC function leads to clinical relevant conditions associated with high or low blood pressure when gain or loss of function mutations occur, respectively. Recently, accumulating evidence suggests the putative role of renal Paraoxonase 2 (PON2) as regulator of ENaC function. PON2 is a membrane-associated enzyme showing hydrolytic activity against 3-oxo-C12 homoserine lactone, a signaling molecule produced by bacteria and mainly Pseudomonas aeruginosa. PON2 also exhibits antioxidant property. In the distal nephron, PON2 is expressed in principal cells, and in vitro it physically interacts with ENaC subunits and inhibits ENaC channel activity in Xenopus oocytes likely by modulating channel assembly in the endoplasmic reticulum. However, the role of PON2 in the kidney is still not known. In the present study, we investigate whether PON2 is regulated by changes in aldosterone state and its role in aldosterone-induced oxidative stress in the kidneys. METHOD We addressed these issues utilizing both in vivo and in vitro approaches, such as RESULTS We first demonstrated that in Milan normotensive rats strain, PON2 is upregulated by low-salt state according with α-ENaC activation. This regulation is aldosterone sensitive since pretreatment with mineralocorticoid receptor antagonist spironolactone prevented the stimulatory effects of sodium restricted diet. Furthermore, low-sodium diet inducing PON2 upregulation was associated with reduced ROS levels, more specifically superoxide levels, but treatment with spironolactone prevented this beneficial effect, by increasing ROS level in kidney cortex. Since antihypertensive drugs spironolactone and canrenone contain a lactone residue in their molecular structure, we tested whether they could directly interact with PON2. We demonstrated in vitro that spironolactone and canrenone were able to inhibit PON2 lactonase activity and so to directly interfere with PON2 function. Finally, Milan hypertensive rats, carrying a mutation of α-adducin as determinant of their hypertensive phenotype, presented with higher level of ROS while PON2 was upregulated. CONCLUSION These results demonstrate PON2 as a novel target of steroid hormone aldosterone. PON2 may serve as a potential ROS scavenger mechanism protecting the kidneys from aldosterone-promoted oxidative stress.

The adducin saga: pleiotropic genomic targets for precision medicine in human hypertension-vascular, renal, and cognitive diseases.

Hypertension is a leading risk factor for stroke, heart disease, chronic kidney disease, vascular cognitive impairment, and Alzheimer's disease. Previous genetic studies have nominated hundreds of genes linked to hypertension, and renal and cognitive diseases. Some have been advanced as candidate genes by showing that they can alter blood pressure or renal and cerebral vascular function in knockout animals; however, final validation of the causal variants and underlying mechanisms has remained elusive. This review chronicles 40 years of work, from the initial identification of adducin (ADD) as an ACTIN-binding protein suggested to increase blood pressure in Milan hypertensive rats, to the discovery of a mutation in ADD1 as a candidate gene for hypertension in rats that were subsequently linked to hypertension in man. More recently, a recessive K572Q mutation in ADD3 was identified in Fawn-Hooded Hypertensive (FHH) and Milan Normotensive (MNS) rats that develop renal disease, which is absent in resistant strains. ADD3 dimerizes with ADD1 to form functional ADD protein. The mutation in ADD3 disrupts a critical ACTIN-binding site necessary for its interactions with actin and spectrin to regulate the cytoskeleton. Studies using Add3 KO and transgenic strains, as well as a genetic complementation study in FHH and MNS rats, confirmed that the K572Q mutation in ADD3 plays a causal role in altering the myogenic response and autoregulation of renal and cerebral blood flow, resulting in increased susceptibility to hypertension-induced renal disease and cerebral vascular and cognitive dysfunction.

A Mutation in γ-Adducin Impairs Autoregulation of Renal Blood Flow and Promotes the Development of Kidney Disease.

The genes and mechanisms involved in the association between diabetes or hypertension and CKD risk are unclear. Previous studies have implicated a role for γ-adducin (ADD3), a cytoskeletal protein encoded by Add3. We investigated renal vascular function in vitro and in vivo and the susceptibility to CKD in rats with wild-type or mutated Add3 and in genetically modified rats with overexpression or knockout of ADD3. We also studied glomeruli and primary renal vascular smooth muscle cells isolated from these rats. This study identified a K572Q mutation in ADD3 in fawn-hooded hypertensive (FHH) rats-a mutation previously reported in Milan normotensive (MNS) rats that also develop kidney disease. Using molecular dynamic simulations, we found that this mutation destabilizes a critical ADD3-ACTIN binding site. A reduction of ADD3 expression in membrane fractions prepared from the kidney and renal vascular smooth muscle cells of FHH rats was associated with the disruption of the F-actin cytoskeleton. Compared with renal vascular smooth muscle cells from Add3 transgenic rats, those from FHH rats had elevated membrane expression of BKα and BK channel current. FHH and Add3 knockout rats exhibited impairments in the myogenic response of afferent arterioles and in renal blood flow autoregulation, which were rescued in Add3 transgenic rats. We confirmed these findings in a genetic complementation study that involved crossing FHH and MNS rats that share the ADD3 mutation. Add3 transgenic rats showed attenuation of proteinuria, glomerular injury, and kidney fibrosis with aging and mineralocorticoid-induced hypertension. This is the first report that a mutation in ADD3 that alters ACTIN binding causes renal vascular dysfunction and promotes the susceptibility to kidney disease.

Impaired myogenic response of the afferent arteriole contributes to the increased susceptibility to renal disease in Milan normotensive rats.

Milan normotensive (MNS) rats are more susceptible to the development of renal disease than Milan hypertensive (MHS) rats, but the genes and pathways involved are unknown. This study compared the myogenic response of isolated perfused afferent arterioles (Af‐Art) and autoregulation of renal blood flow (RBF) and glomerular capillary pressure (Pgc) in 6–9‐week‐old MNS and MHS rats. The diameter of the Af‐Art of MHS rats decreased significantly from 14.3 ± 0.5 to 11.5 ± 0.6 μm when perfusion pressure was elevated from 60 to 120 mmHg. In contrast, the diameter of Af‐Art of MNS rats did not decrease. RBF was well autoregulated in MHS rats, but it increased by 26% in MNS rats. Pgc rose by 11 mmHg when renal perfusion pressure (RPP) was increased from 100 to 140 mmHg in MNS but not in MHS rats. Protein excretion increased from 10 ± 1 to 245 ± 36 mg/day in MNS rats as they aged from 3 to 11 months but it did not increase in MHS rats. We also compared the development of proteinuria in MNS and MHS rats following the induction of diabetes with streptozotocin. Protein excretion rose from 16 ± 3 to 234 ± 43 mg/day in MNS rats, but it remained unaltered in MHS rats. These data indicate that the myogenic response of the Af‐art is impaired in MNS rats and increased transmission of pressure to the glomerulus may contribute to renal injury in MNS rats similar to what is seen in fawn‐hooded hypertensive and Dahl salt‐sensitive rats.

Abstract 063: Impaired Myogenic Response of the Af-art Contributes to the Increased Susceptibility to Hypertension and Diabetic Induced Renal Disease in Milan Normotensive Rats

Previous studies have indicated that Milan normotensive (MNS) rats are more susceptible to the development of hypertension and diabetic induced renal injury than Milan hypertensive (MHS) rats, but the genes and pathways involved are unknown. MNS also develop proteinuria and chronic kidney disease (CKD) as they age, whereas hypertensive MHS do not. We compared the myogenic response of isolated perfused Af-Art and autoregulation of RBF and glomerular capillary pressure in 6-9 week old MNS and MHS rats. The diameter of Af-Art of MNS rats increased from 14.0 ± 0.5 to 14.2 ± 0.6 μm (n=6) when elevation in perfusion pressure from 60 to 120 mmHg. In contrast, the diameter of the Af-Art decreased significantly from 14.3 ± 0.5 to 11.5 ± 0.6 μm (n=6) in MHS rats. In vivo, RBF increased by 26% when RPP was increased from 100 to 140 mmHg in MNS rats but it remained unchanged in MHS rats. Glomerular capillary pressure rose by 11 mmHg in MNS following the elevation in RPP from 100 to 140 mm Hg but not in MHS rats. Protein excretion increased from 8.9 ± 0.7 to 158.2 ± 23.1 mg/day in MNS rats as the increased in age from 3 to 9 months of age but it did not increase in MHS rats. In com-parison to other strains susceptible and resistant to CKD, we noticed that both MNS and Fawn Hooded hypertensive (FHH) rats that do not autoregulate RBF also share the same sequence variant in the Adducin 3 gene. We performed a genetic complementation study to test whether this mutation might be responsible for the impaired myogenic response in MNS. The diameter of the Af-Art isolated from an F1 cross of MNS &FHH rats increased from 17.2 ± 0.9 to 18.5 ± 0.9 μM (n=5) in response to increase in perfusion pressure and RBF was not efficiently autoregulated in these animals. These data indicate a mutation in Adducin 3 which impairs myogenic response of the Af-Art and increased transmission of pressure to the glomerular capillaries may contribute to the development of CKD in MNS rats similar to what is seen in FHH rats.

Impaired myogenic responses of the Af‐Art contributes to chronic kidney disease in Milan Normotensive rats

We have recently reported that the development of glomerular injury, proteinuria and chronic kidney disease (CKD) in Dahl Salt Sensitive and Fawn Hooded Hypertensive rats are associated with an impaired myogenic response of the afferent arteriole (Af-Art) and autoregulation of renal blood flow (RBF), whereas the myogenic response is enhanced in Spontaneously Hypertensive rats that are resistant to the development of renal disease. Milan normotensive rats (MNS) are more susceptible to development of CKD than the Milan Hypertensive rat (MHS) even though blood pressure is much lower. This study compared the myogenic response in the Af-Art and autoregulation of RBF in 6-9 week old MNS and MHS rats. Autoregulation of RBF was measured following an increase in renal perfusion pressure (RPP) from 100 to 140 mmHg. The diameter of Af-Art of MNS rats increased from 14.0 ± 0.5 to 14.1 ± 0.6 µm (n=6, p>0.05) in response to an increase in perfusion pressure from 60 to 120 mmHg. In contrast, the diameter of the Af-Art decreased significantly from 14.0 ± 0.8 to 11.7 ± 1.1 µm (n=6, p<0.05) in MHS rats. In vivo, MNS rats exhibited poor autoregulation of RBF, it increased by 27% when RPP was increased from 100 to 140 mmHg. In contrast, MHS rats exhibited nearly perfect autoregulation and RBF was not significantly altered. Glomerular capillary pressure estimated from measurement of proximal tubular stop flow pressure increased by 10 mmHg in MNS following the elevation in RPP, but not in MHS rats. These data indicate that impaired myogenic response of the Af-Art contributes to the progression of proteinuria and renal injury in MNS rats. This studies were supported by NIH Grants HL36279 and DK104184-01.

Hypertension-Linked Mutation of α-Adducin Increases CFTR Surface Expression and Activity in HEK and Cultured Rat Distal Convoluted Tubule Cells

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

Mitochondrial polymorphisms in rat genetic models of hypertension

Hypertension is a complex trait that has been studied extensively for genetic contributions of the nuclear genome. We examined mitochondrial genomes of the hypertensive strains: the Dahl Salt-Sensitive (S) rat, the Spontaneously Hypertensive Rat (SHR), and the Albino Surgery (AS) rat, and the relatively normotensive strains: the Dahl Salt-Resistant (R) rat, the Milan Normotensive Strain (MNS), and the Lewis rat (LEW). These strains were used previously for linkage analysis for blood pressure (BP) in our laboratory. The results provide evidence to suggest that variations in the mitochondrial genome do not account for observed differences in blood pressure between the S and R rats. However, variants were detected among the mitochondrial genomes of the various hypertensive strains, S, SHR, and AS, and also among the normotensive strains R, MNS, and LEW. A total of 115, 114, 106, 106, and 16 variations in mtDNA were observed between the comparisons S versus LEW, S versus MNS, S versus SHR, S versus AS, and SHR versus AS, respectively. Among the 13 genes coding for proteins of the electron transport chain, 8 genes had nonsynonymous variations between S, LEW, MNS, SHR, and AS. The lack of any sequence variants between the mitochondrial genomes of S and R rats provides conclusive evidence that divergence in blood pressure between these two inbred strains is exclusively programmed through their nuclear genomes. The variations detected among the various hypertensive strains provides the basis to construct conplastic strains and further evaluate the effects of these variants on hypertension and associated phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-010-9259-5) contains supplementary material, which is available to authorized users.

α- and β-Adducin polymorphisms affect podocyte proteins and proteinuria in rodents and decline of renal function in human IgA nephropathy

Adducins are cytoskeletal actin-binding proteins (α, β, γ) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates α- and β-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of α- and β-adducin on glomerular function and disease using β-adducin null mice, congenic substrains for α- and β-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of β-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, α-actinin, ZO-1, Fyn). The introgression of polymorphic MHS β-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, α-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS α-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic β-adducin (ADD2, 1797T, rs4984) with a significant interaction with α-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-009-0549-x) contains supplementary material, which is available to authorized users.

Opportunities and limitations of genetic analysis of hypertensive rat strains

Genetic influences contribute upto 30–50% to blood pressure variability in human essential hypertension [1]. Intense efforts have been dedicated to unraveling the role of genes in this disease, following two parallel and interacting tracks. Studies on human populations have mostly focused on the candidate gene linkage approach, associating single nucleotide polymorphic (SNP) markers with the hypertensive phenotype. In spite of some promising observations [2], most attempts to replicate association studies have not been encouraging [3]. There are calls for application of stricter quality criteria for genetic studies, including better definition of phenotype characteristics, increased attention to environmental factors, sharing and combining data resources and more emphasis on whole genome-linkage scans in the context of collaborative multidisciplinary approaches [4–17]. Recently, however, there has been impressive progress in designing studies, understanding the mechanisms of disease and genotyping technology [17]. The second parallel track is the development and study of rodent experimental models of disease. Genetically hypertensive rat strains have been developed over the past 60 years. The models were obtained from initial random rat populations by brother—sister mating selecting for a high blood pressure phenotype. The better characterized genetically hypertensive strains are the spontaneously hypertensive rat (SHR) [18], stroke-prone SHR (SPSHR) [19], Dahl saltsensitive [20], Milan hypertensive (MHS) [21], New Zealand genetically hypertensive [22], Sabra hypertensive (SBH) [23] and the Lyon hypertensive rats [24]. Comparisons are generally made with inbred normotensive strains derived from the original rat population and sharing most of the genetic background with the corresponding hypertensive strain. The most common normotensive strains are the Dahl salt-resistant, Wistar—Kyoto (WKY), Milan normotensive (MNS), genetically normotensive, Sabra normotensive (SBN) and Lyon normotensive and Lyon low blood pressure rats [18–24]. Physiological and pharmacological approaches to the study of these models have enhanced our understanding of many systems for blood pressure control. During the 1990s, technological advances allowed the use of quantitative trait locus (QTL) mapping to identify chromosomal regions with possible genetic variants affecting blood pressure. Many candidate genes for a role in human hypertension were identified as a consequence of findings in rodent animal models [25–30].

Development of diabetic nephropathy in the Milan normotensive strain, but not in the Milan hypertensive strain: Possible permissive role of hemodynamics

Rats of the Milan normotensive strain develop spontaneous glomerulosclerosis, whereas those of the Milan hypertensive strain are resistant to renal disease, possibly due to intrarenal artery hypertrophy protecting from systemic hypertension. To assess the role of hemodynamic versus metabolic factors in diabetic nephropathy, we investigated whether streptozotocin-induced diabetes accelerates glomerulosclerosis in Milan normotensive and/or removes (the hemodynamic) protection in Milan hypertensive rats by reducing preglomerular vascular resistance. Diabetic and nondiabetic Milan normotensive, hypertensive, and progenitor Wistar rats were followed for 6 months for the assessment of renal function and structure. Proteinuria increased in nondiabetic and diabetic normotensive and, to a lesser extent, in diabetic Wistar, but not hypertensive rats. Serum creatinine increased and creatinine clearance decreased in nondiabetic and diabetic normotensive rats at 6 months. At 1.5 months, diabetic normotensive, but not hypertensive rats showed increased glomerular filtration rate and filtration fraction, suggesting glomerular hypertension. Diabetic nephropathy was detected in diabetic normotensive and Wistar, but not hypertensive rats. Glomerular extracellular matrix and TGF-beta mRNA levels increased with diabetes (and age) in normotensive, but not hypertensive rats. Arterioles and interlobular arteries showed increased media thickness in hypertensive versus normotensive rats, with diabetes reducing it only in the normotensive. These data show that Milan hypertensive rats are not susceptible to diabetic nephropathy, at variance with glomerulosclerosis-prone Milan normotensive rats, thus indicating the importance of genetic background. Our study suggests that the nature of this (genetic) protection might be hemodynamic, with intrarenal artery hypertrophy preventing diabetes-induced loss of autoregulation.

Altered expression of renal apical plasma membrane Na+transporters in the early phase of genetic hypertension

The present study explores whether the development of hypertension in the Milan strain of rats (MHS) rats is preceded or paralleled by alterations of mRNA and/or protein levels of the major luminal Na+ transporters. MHS rats were studied at 23-25 days after birth; age-matched Milan normotensive (MNS) rats were used as controls. The glomerular filtration rate (GFR), measured by inulin clearance, was higher in MHS than in MNS rats, while the mean blood pressure was not different in the two strains of animals indicating that the MHS rats were still in the prehypertensive state. Type 3 sodium/hydrogen exchanger (NHE3), bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC) and alpha-ENaC mRNA abundances were quantified by competitive PCR. In MHS compared with MNS, mRNA abundance was unchanged for NHE3 in proximal tubules, higher for NKCC2 in medullary thick ascending limbs of Henle's loops (TAL) and lower for NCC in distal convoluted tubules (DCT) and for alpha-ENaC along collecting ducts (CD). Western blot experiments revealed 1) unchanged NHE3; 2) a significant increase in NKCC2 in the outer medulla; 3) a significant decrease in NCC in the renal cortex and of alpha-ENaC in both the renal cortex and outer medulla, whereas beta- and gamma-ENaC remained unchanged. These data indicate that, in MHS rats, there is a strong upregulation of NKCC2 along the TAL associated with increased GFR, robust inhibition of NCC cotransporter along the DCT and modest downregulation of alpha-ENaC along the CD. The interplay of the various Na+ transporters may well explain why, at this age, the rats are still in the prehypertensive state.

Further chromosomal mapping of a blood pressure QTL in Dahl rats on chromosome 2 using congenic strains

Both linkage and use of congenic strains have shown that a region on rat chromosome 2 (Chr 2) of Dahl salt-sensitive rats (S) contained a quantitative trait locus (QTL) for blood pressure (BP). A congenic strain was made by replacing a segment of the S rat by the homologous region of the Milan normotensive (MNS) rat. Since the region was roughly 80 cM in size, a further reduction is required toward the final identification of the QTL. Currently, three congenic substrains were made by replacing smaller sections within the 80 cM. Each strain contains a specific region of MNS in the S genetic background. Two of the three congenic strains shared a segment in common, and both showed a BP-lowering effect. One of the three congenic strains carried a unique segment and had the same BP as S. Deducing the fragment shared in the two substrains having an effect, the BP QTL has to be present in a region of roughly 15 cM. In contrast to BP, heart rates of all the congenic rats were the same as that of the S rat. Thus BP and the heart rate are under the control of independent genetic determinants.

Mapping a blood pressure quantitative trait locus to a 5.7-cM region in Dahl salt-sensitive rats

A region on rat Chromosome (Chr) 2 of the Dahl salt-sensitive rat (S) was shown previously to contain a quantitative trait locus (QTL) for blood pressure (BP). This was achieved first by linkage, followed by the use of congenic strains. A congenic strain, designated S.MNS-D2Mit6/Adh, contained a segment of Chr 2 from the Milan Normotensive (MNS) rat in the S genetic background. Since the region containing the QTL was roughly 80 cM in size, a further reduction was needed towards the positional or candidate gene cloning. Currently, two congenic substrains were made from the original strain S.MNS-D2Mit6/Adh. One of these two substrains showed a BP-lowering effect, whereas the other substrain did not. Deducing the segment not shared in the two substrains, the BP QTL has to be present in a chromosome region of roughly 5.7 cM between the marker D2Rat303 and the locus for the neutroendopeptidase gene (Nep). Nep is not included within the segment. This region does not seem to contain any candidate genes well known for the BP control. Thus, the final identification of the QTL will most likely lead to the discovery of a brand new gene for the BP regulation.

Mapping of Two Quantitative Trait Loci (QTL) for Blood Pressure (BP) on Rat Chromosome (CHR.) 2 Using Congenic Strains by Telemetry

P141 We previously mapped a BP QTL on rat Chr. 2 near the Na,K-ATPase ( Atp1a1) locus in two congenic strains. In the strain, S.MNS- Adh / D2Mit5, the Dahl salt sensitive (S) Chr. region, which was replaced by that of the Milan normotensive (MNS) strain, was quite large (roughly 80 cM). Now, we made 6 substrains from the original S.MNS- Adh / D2Mit5 strain, and then measured BP by telemetry for two (and will continue to measure for the rest) substrains. These two substrains do not contain overlapping chromosome regions. One, S.MNS-D2Mit6 / D2Rat166 (S.MNS1) , encompasses the angiotensin receptor AT1B ( Agtr1b ) and the other substrain, S.MNS-D2Mit9/ Camk2d (S.MNS2) includes Atp1a 1. Both substrains showed a significant (p Agtr1b. The BP effects of these QTL from the two separate substrains appeared to exceed those of the same number of QTL combined in one strain. This implies that these individual QTL on the same chromosome interact epistatically with one another.

Mapping of Two Quantitative Trait Loci (QTL) for Blood Pressure (BP) on Rat Chromosome (CHR.) 2 Using Congenic Strains by Telemetry

P141 We previously mapped a BP QTL on rat Chr. 2 near the Na,K-ATPase ( Atp1a1) locus in two congenic strains. In the strain, S.MNS- Adh / D2Mit5, the Dahl salt sensitive (S) Chr. region, which was replaced by that of the Milan normotensive (MNS) strain, was quite large (roughly 80 cM). Now, we made 6 substrains from the original S.MNS- Adh / D2Mit5 strain, and then measured BP by telemetry for two (and will continue to measure for the rest) substrains. These two substrains do not contain overlapping chromosome regions. One, S.MNS-D2Mit6 / D2Rat166 (S.MNS1) , encompasses the angiotensin receptor AT1B ( Agtr1b ) and the other substrain, S.MNS-D2Mit9/ Camk2d (S.MNS2) includes Atp1a 1. Both substrains showed a significant (p&lt;0.0001) effect in reducing the mean arterial pressure (MAP) in mmHg (see graph). This proves the presence of a QTL around Agtr1b. The BP effects of these QTL from the two separate substrains appeared to exceed those of the same number of QTL combined in one strain. This implies that these individual QTL on the same chromosome interact epistatically with one another.

Short– and Long–Term Effects of Fish Oil on Proteinuria, Morphology and Renal Hemodynamics in the Milan Normotensive Rat Model of Spontaneous Glomerulosclerosis

Background/Aims: A diet rich in polyunsaturated Ω3 fatty acids has been shown to modulate the course of several experimental models of renal disease. The short– and long–term effects of an 8% fish oil (FO) chow on proteinuria, renal blood flow and glomerular morphology were evaluated in Milan normotensive rats that spontaneously develop progressive glomerulosclerosis. Methods: Eight rats each were pairfed FO– versus cholesterol–enriched or control diets for either 2 or 32 weeks. 4/48 animals died (2–week trial: 1 rat on the FO and 1 rat on the control diet; 32–week trial: 1 rat on the cholesterol and 1 rat on the control diet) and were excluded from all statistic analyses. Results: After 2 weeks the renal blood flows were higher in the FO animals versus controls (8.75±2.19 vs. 6.87±1.91 ml/min/g, p<0.05), and the prostaglandin E₂/thromboxane B₂ ratio shifted towards the vasodilatative prostaglandin E₂ (1.76±0.18 vs. 0.91±0.19, p<0.05). During the long–term trial proteinuria in the FO animals progressed faster and to a higher level (176.5±32.2 vs. 82.7±36.7 mg/24 h at week 32, p<0.01). After 32 weeks the renal blood flow was significantly lower in th FO group 2.8±1.1 vs. 4.6±1.9 ml/min/g, (p<0.05), and the rats had an accelerated development of nephrosclerosis, with sclerotic lesions in 60.3±6.6% of the glomeruli as compared with 46.5±9.8% in the cholesterol and 39.8±5.9 in the control group (p<0.05). Conclusion: The short–time effects of FO on renal hemodynamics did not alleviate the progress of renal damage in Milan normotensive rats, but the morphologic and functional signs of injury were rather pronounced with FO feeding.

Is the nitric oxide system involved in genetic hypertension in Dahl rats?

To address this issue, a series of genetic tests were carried out. Linkage studies showed that the inducible nitric oxide synthase (Nos2) locus cosegregated with blood pressure in three F2 populations originated from crosses of Dahl salt-sensitive (S) rats with rats of various normotensive strains. However, the brain nitric oxidase synthase (Nos1) and endothelial nitric oxide synthase (Nos3) loci did not cosegregate with blood pressure in five F2 populations. Thus, only Nos2, but not Nos1 and Nos3, was considered as a candidate gene for being a quantitative trait locus (QTL) for blood pressure in the S rat. To further test this hypothesis, congenic strains were constructed by substituting regions on Chromosome 10 of the S rat with the homologous regions of the Milan normotensive (MNS) rat. Results showed that the chromosome region including Nos2 did not contain a blood pressure QTL. In consequence, Nos2 per se is not supported as a candidate QTL capable of causing a blood pressure difference between the S and MNS rats. Nevertheless, the nitric oxide system appears to be involved secondarily in the regulation of blood pressure in the S rat, as evidenced by physiological data.