BACKGROUND:The role of proximal tubule (PT) angiotensinogen (AGT) in modulating blood pressure has previously been examined using mice expressing PT human AGT and human renin, or rat AGT. These animals are hypertensive; however, the question remains whether alterations in mouse PT AGT alone affects arterial pressure.METHODS:Mouse AGT cDNA was knocked-in to the endogenous kidney androgen protein (KAP) gene using an internal ribosomal entry site (IRES)-based strategy.RESULTS:The KAP-mAGT animals showed kidney-specific KAP-AGT mRNA expression; renal in situ hybridization detected KAP-AGT mRNA only in PT. Urinary AGT was markedly increased in KAP-mAGT mice. On a high Na diet, radiotelemetric arterial pressure showed a systolic pressure elevation; no significant difference in arterial pressure was observed on a normal diet. Plasma renin concentration (PRC) was reduced in KAP-mAGT animals given a high Na diet, but was not different between mouse lines during normal Na intake. Plasma AGT concentration was not altered by overexpression of PT mouse AGT.CONCLUSIONS:In summary, PT overexpression of mouse AGT leads to salt-sensitive hypertension without recruitment of the systemic renin-angiotensin system.

BACKGROUND:Much remains unknown about the genetic factors that contribute to essential hypertension. The Utah Genetic Reference Project (UGRP) large pedigree collection provides new opportunities to study quantitative relationships between genetic variation, endophenotypes, and blood pressure. METHODS:We analyzed the relationship between common single-nucleotide polymorphisms (SNPs) and haplotypes spanning the angiotensinogen (AGT) gene and promoter region, plasma AGT levels, and systolic (SBP) and diastolic blood pressure (DBP) in 424 individuals from 41 two-generation UGRP families. RESULTS:Plasma AGT levels are significantly correlated among UGRP family members. Correlations are higher for males than for females. Parent-offspring correlations for plasma AGT (0.30) are higher than those for SBP (0.26) and DBP (0.17) (all P values <0.01). The additive heritability (h(2)) for plasma AGT is high (0.74) and substantially exceeds heritability estimates for SBP (0.26) and DBP (0.16) (all P values <0.01). Significant linkage (logarithm of the odds (LOD) >3) is found between six AGT SNPs and plasma AGT. A model that utilizes three AGT haplotype groups produces the best LOD score (5.1) that exceeds the best single SNP LOD score (3.8). Plasma AGT and blood pressure were not significantly correlated. CONCLUSIONS:Plasma AGT levels demonstrate high heritability in 41 UGRP families. Locus-specific heritability estimates for AGT SNPs and haplotypes approach 67%, indicating that variation at AGT accounts for a large percentage of the heritability of plasma AGT. A three-way haplotype model outperforms single SNPs for quantitative linkage analysis to plasma AGT. In these predominantly normotensive individuals, plasma AGT did not correlate significantly with blood pressure.

The ability to longitudinally monitor portal and splanchnic pressures would greatly enhance the understanding of acute and chronic liver disease by helping to assess the immediate and long-term impact of therapeutic manipulations. However, a technique for measuring portal pressures in the ambulatory setting is not currently available. To overcome this difficulty, we utilized an approach that involved the implantation of a miniature telemetric device, equipped with a specially-designed pressure transmission catheter, into the spleen of an anesthetized mouse. Using this approach, portal pressures were measured continuously over 5 d in conscious, unrestrained animals, the availability of which will help facilitate studies of the portal circulation requiring long-term stability.

Objectives To better understand the relationship between angiotensinogen (AGT) genetic variation and essential hypertension, AGT genotypes and haplotypes were tested for association with hypertensive endophenotypes and essential hypertension.Methods Two hundred and fifty-six Hypertensive Pathotype (HyperPATH)/Specialized Center of Research (SCOR) cases and 126 controls were genotyped for 24 single-nucleotide polymorphisms (SNPs) in the AGT gene. SNPs and AGT haplotypes were tested for association with plasma AGT, renal plasma flow (RPF), and essential hypertension.Results New associations between essential hypertension, plasma AGT, and RPF are reported for alleles -1178G, 6066A, 6152A, 6233C, and 12822C. The maximum odds ratio for association of hypertension and AGT genetic variation was 2.3 [95% confidence interval (CI) 1.5-3.8; P<0.0003] for allele 6233C. Previous associations for -1074T, -532T, -217A, -6A, and 4072C are confirmed (P<0.05). Sodium depletion enhances associations between AGT SNPs and plasma AGT. Most individually associated SNPs, including -6A and 4072C, are found on a common complete AGT haplotype, H4 (frequency = 0.09). Individuals with haplotype H4 have significantly higher plasma AGT and reduced RPF (P<0.003 and P<0.0002, respectively). Other common haplotypes are not associated with increased plasma AGT levels in this data set despite the presence of the -6A and 4072C alleles, suggesting that AGT haplotype H4 is more predictive of elevated plasma AGT than is -6A or 4072C.Conclusion This study demonstrates the importance of analyzing haplotypes in addition to single genotypes in association studies. By demonstrating the dependence of AGT associations on sodium depletion status, it helps to explain previous conflicting association results. J Hypertens 28: 65-75 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the requirement for the PY motif in cAMP-mediated exocytosis of ENaC. We have elucidated how a common genetic variant can underlie significant functional diversity in NEDD4L at the cellular level. We propose a model that describes how that functional variation may influence blood pressure. Moreover, our observations regarding differential function of the NEDD4L isoforms may impact other aspects of physiology that involve this ubiquitin ligase.

One of the early decisions in what became the Human Genome Project was to recruit families that would serve as a reference set, thereby focusing efforts to create human genetic maps on the same sets of DNA samples. The families recruited from Utah provided the most widely used samples in the Centre d'Etudes du Polymorphisme Humain (CEPH) set, were instrumental in generating human linkage maps, and often serve as the benchmark for establishing allele frequency when a new variant is identified. In addition, the immortalized cell lines created from the peripheral blood cells of these subjects are a broadly used resource and have yielded insights in many areas, from the genetics of gene expression to the regulation of telomeres. More recently, these families were recontacted and underwent extensive, protocol-based evaluation to create a phenotypic database, which will aid in the study of the genetic basis of quantitative traits. As with the earlier efforts, this project involved collaborations among many investigators and has yielded insights into multiple traits.

The ubiquitin ligase, NEDD4L, participates in plasma volume and blood pressure regulation by controlling cell surface expression of kidney epithelial sodium channel (ENaC). Impairment of ENaC internalization with disrupting Nedd4L binding PY motif in the C‐terminal of the protein showed rare Mendelian human hypertension disorder, Liddle syndrome. Therefore, possible involvements of ENaC‐Nedd4L system abnormalities were suggested in development of human hypertension. Previously, we discovered a common variant, named v13(G/A), that affects the formation of an evolutionally new C2‐encoding isoform of the Nedd4L gene in humans. The aim of this study was to test a hypothesis whether this variant is involved in essential hypertension; we performed the association study in a sample of the Japanese population. We found a gender‐specific genotypic and allelic association with statistical significance achieved only in males. Our data suggest that this genetic variation may contribute to essential hypertension in Japanese male patients through the mechanism of disrupting the C2 encoding isoform in vivo. Furthermore, molecular and functional studies will be required to elucidate the mechanism by which genetic variation in NEDD4L contributes to the development of essential hypertension.

Although progress in the genetics of essential hypertension may seem disappointing, it has considerable potential in defining research directions that will ultimately translate into clinical practice. The hypothesis that genetic variation at the angiotensinogen locus impacts on individual susceptibility to develop essential hypertension has motivated a substantial body of research by us and many others. We examine how analyses of the mechanisms by which variation in angiotensinogen expression may contribute to disease susceptibility and may have arisen in human populations have progressed in recent years. Although the objective of personalized medicine is still in the future, a genetic hypothesis based on human variation can uniquely empower functional genomics approaches to reach such an ultimate goal.

Although progress in the genetics of essential hypertension may seem disappointing, it has considerable potential in defining research directions that will ultimately translate into clinical practice. The hypothesis that genetic variation at the angiotensinogen locus impacts on individual susceptibility to develop essential hypertension has motivated a substantial body of research by us and many others. We examine how analyses of the mechanisms by which variation in angiotensinogen expression may contribute to disease susceptibility and may have arisen in human populations have progressed in recent years. Although the objective of personalized medicine is still in the future, a genetic hypothesis based on human variation can uniquely empower functional genomics approaches to reach such an ultimate goal.

Objective: Women who develop pre-eclampsia, show significantly less hypervolemia of pregnancy, compared with controls. We have shown that chronically elevated angiotensinogen expression increases a woman's risk of developing pre-eclampsia. Our objective was to determine whether increased angiotensinogen expression is sufficient to cause failed hypervolemia.Study design: To isolate the effects of elevated angiotensinogen expression, we studied transgenic mice with either 2 or 3 copies of the in urine angiotensinogen gene. Plasma volume was measured by Evans blue dye dilution, and kidney sections were immunostained for angiotensinogen and renin.Results: Three-copy mice failed to maintain hypervolemia after midgestation (P < .01) and failed to up-regulate renin expression in the distal nephron, compared with 2-copy controls. Intrarenal angiotensinogen was up-regulated during pregnancy in both genotypes.Conclusion: Chronically elevated angiotensinogen expression is sufficient to cause failed hypervolemia of pregnancy. Whether this observation is related to failed up-regulation of distal tubule renin expression requires further study. (c) 2006 Mosby, Inc. All rights reserved.