Identification of novel loci affecting circulating chromogranins and related peptides.

Chromogranins are pro-hormone secretory proteins released from neuroendocrine cells, with effects on control of blood pressure. We conducted a genome-wide association study for plasma catestatin, the catecholamine release inhibitory peptide derived from chromogranin A (CHGA), and other CHGA- or chromogranin B (CHGB)-related peptides, in 545 US and 1252 Australian subjects. This identified loci on chromosomes 4q35 and 5q34 affecting catestatin concentration (P = 3.40 × 10-30 for rs4253311 and 1.85 × 10-19 for rs2731672, respectively). Genes in these regions include the proteolytic enzymes kallikrein (KLKB1) and Factor XII (F12). In chromaffin cells, CHGA and KLKB1 proteins co-localized in catecholamine storage granules. In vitro, kallikrein cleaved recombinant human CHGA to catestatin, verified by mass spectrometry. The peptide identified from this digestion (CHGA360-373) selectively inhibited nicotinic cholinergic stimulated catecholamine release from chromaffin cells. A proteolytic cascade involving kallikrein and Factor XII cleaves chromogranins to active compounds both in vivo and in vitro.

High intensity focused ultrasound for the treatment of adenomyosis: selection criteria, efficacy, safety and fertility.

Adenomyosis is a disorder of uterus in which endometrial glands and stroma are present within the uterine musculature. The main clinical manifestations are dysmenorrhea and menorrhagia. Adenomyosis has a great impact on both the quality of life and fertility of women. The treatment of adenomyosis remains an immense challenge. Relevant articles were searched through MEDLINE and PubMed between 2000 and March 2017. The search terms of adenomyosis, magnetic resonance imaging (MRI) features of adenomyosis, high intensity focused ultrasound (HIFU), ultrasound-guided HIFU and MRgFUS were used. There were no language restrictions. HIFU is a non-invasive local thermal ablation technique which has been used in the treatment of both focal and diffuse adenomyosis. Several case studies have demonstrated that HIFU presents low rate of minor and/or major complications and, at the same time, a long symptom-relief period. Multiple factors such as the enhancement type of the adenomyotic lesion, volume of the adenomyotic lesions, number of hyperintense foci on T2WI, location of the uterus, location of adenomyotic lesions, thickness of the abdominal wall and distance from the skin to the adenomyotic lesions contribute to the efficacy of HIFU. Consequently, based on these contributing factors, specific and strict selection criteria have been used to achieve higher efficacy. Thus, patients with pelvic endometriosis, adhesions between the bowel and the uterus, or an abdominal surgical scar wider than 10 mm, are not suitable for HIFU treatment. Moreover, HIFU-treated patients with adenomyosis, who wished to conceive, showed high conception and live birth rates. HIFU is a new and promising treatment option for patients with adenomyosis, but its efficacy, safety, cost-effectiveness and fertility outcome must be evaluated by randomized controlled trials.

Polymorphisms at the F12 and KLKB1 loci have significant trait association with activation of the renin-angiotensin system.

BACKGROUND: Plasma coagulation Factor XIIa (Hageman factor; encoded by F12) and kallikrein (KAL or Fletcher factor; encoded by KLKB1) are proteases of the kallikerin-kinin system involved in converting the inactive circulating prorenin to renin. Renin is a key enzyme in the formation of angiotensin II, which regulates blood pressure, fluid and electrolyte balance and is a biomarker for cardiovascular, metabolic and renal function. The renin-angiotensin system is implicated in extinction learning in posttraumatic stress disorder. METHODS & RESULTS: Active plasma renin was measured from two independent cohorts- civilian twins and siblings, as well as U.S. Marines, for a total of 1,180 subjects. Genotyping these subjects revealed that the carriers of the minor alleles at the two loci- F12 and KLKB1 had a significant association with reduced levels of active plasma renin. Meta-analyses confirmed the association across cohorts. In vitro studies verified digestion of human recombinant pro-renin by kallikrein (KAL) to generate active renin. Subsequently, the active renin was able to digest the synthetic substrate angiotensinogen to angiotensin-I. Examination of mouse juxtaglomerular cell line and mouse kidney sections showed co-localization of KAL with renin. Expression of either REN or KLKB1 was regulated in cell line and rodent models of hypertension in response to oxidative stress, interleukin or arterial blood pressure changes. CONCLUSIONS: The functional variants of KLKB1 (rs3733402) and F12 (rs1801020) disrupted the cascade of enzymatic events, resulting in diminished formation of active renin. Using genetic, cellular and molecular approaches we found that conversion of zymogen prorenin to renin was influenced by these polymorphisms. The study suggests that the variant version of protease factor XIIa due to the amino acid substitution had reduced ability to activate prekallikrein to KAL. As a result KAL has reduced efficacy in converting prorenin to renin and this step of the pathway leading to activation of renin affords a potential therapeutic target.

Heritability of Biomarkers of Oxidized Lipoproteins: Twin Pair Study.

OBJECTIVE: To determine whether biomarkers of oxidized lipoproteins are genetically determined. Lipoprotein(a) (Lp[a]) is a heritable risk factor and carrier of oxidized phospholipids (OxPL). APPROACH AND RESULTS: We measured oxidized phospholipids on apolipoprotein B-containing lipoproteins (OxPL-apoB), Lp(a), IgG, and IgM autoantibodies to malondialdehyde-modified low-density lipoprotein, copper oxidized low-density lipoprotein, and apoB-immune complexes in 386 monozygotic and dizygotic twins to estimate trait heritability (h(2)) and determine specific genetic effects among traits. A genome-wide linkage study followed by genetic association was performed. The h(2) (scale: 0-1) for Lp(a) was 0.91±0.01 and for OxPL-apoB 0.87±0.02, which were higher than physiological, inflammatory, or lipid traits. h(2) of IgM malondialdehyde-modified low-density lipoprotein, copper oxidized low-density lipoprotein, and apoB-immune complexes were 0.69±0.04, 0.67±0.05, and 0.80±0.03, respectively, and for IgG malondialdehyde-modified low-density lipoprotein, copper oxidized low-density lipoprotein, and apoB-immune complexes 0.62±0.05, 0.52±0.06, and 0.53±0.06, respectively. There was an inverse correlation between the major apo(a) isoform and OxPL-apoB (R=-0.49; P<0.001) and Lp(a) (R=-0.48; P<0.001) and OxPL-apoB was modestly correlated with Lp(a) (ρ=0.57; P<0.0001). The correlation in major apo(a) isoform size was concordant (R=1.0; P<0.001) among monozygotic twins but not dizygotic twins (R=0.40; P=0.055). Lp(a) and OxPL-apoB shared genetic codetermination (genetic covariance, ρG=0.774±0.032; P=1.09×10(-38)), although not environmental determination (environmental covariance, ρE=0.081±0.15; P=0.15). In contrast, Lp(a) shared environmental but not genetic codetermination with autoantibodies to malondialdehyde-modified low-density lipoprotein and copper oxidized low-density lipoprotein, and apoB-immune complexes. Sib-pair genetic linkage of the Lp(a) trait revealed that single nucleotide polymorphism rs10455872 was significantly associated with OxPL-apoB after adjusting for Lp(a). CONCLUSIONS: OxPL-apoB and other biomarkers of oxidized lipoproteins are highly heritable cardiovascular risk factors that suggest novel genetic origins of atherothrombosis.

Genetic variation at the delta-sarcoglycan (SGCD) locus elevates heritable sympathetic nerve activity in human twin pairs.

The Syrian Cardiomyopathic Hamster (BIO-14.6/53.58 strains) model of cardiac failure, resulting from naturally occurring deletion at the SGCD (delta-sarcoglycan) locus, displays widespread disturbances in catecholamine metabolism. Rare Mendelian myopathy disorders of human SGCD occur, although common naturally occurring SGCD genetic variation has not been evaluated for effects on human norepinephrine (NE) secretion. This study investigated the effect of SGCD genetic variation on control of NE secretion in healthy twin pairs. Genetic associations profiled SNPs across the SGCD locus. Trait heritability (h(2)) and genetic covariance (pleiotropy; shared h(2)) were evaluated. Sympathochromaffin exocytosis in vivo was probed in plasma by both catecholamines and Chromogranin B (CHGB). Plasma NE is substantially heritable (p = 3.19E-16, at 65.2 ± 5.0% of trait variance), sharing significant (p < 0.05) genetic determination with circulating and urinary catecholamines, CHGB, eGFR, and several cardio-metabolic traits. Participants with higher pNE showed significant (p < 0.05) differences in several traits, including increased BP and hypertension risk factors. Peak SGCD variant rs1835919 predicted elevated systemic vascular compliance, without changes in specifically myocardial traits. We used a chimeric-regulated secretory pathway photoprotein (CHGA-EAP) to evaluate the effect of SGCD on the exocytotic pathway in transfected PC12 cells; in transfected cells, expression of SGCD augmented CHGA trafficking into the exocytotic regulated secretory pathway. Thus, our investigation determined human NE secretion to be a highly heritable trait, influenced by common genetic variation within the SGCD locus. Circulating NE aggregates with BP and hypertension risk factors. In addition, coordinate NE and CHGB elevation by rs1835919 implicates exocytosis as the mechanism of release.

Common genetic variants of the human uromodulin gene regulate transcription and predict plasma uric acid levels.

Uromodulin (UMOD) genetic variants cause familial juvenile hyperuricemic nephropathy, characterized by hyperuricemia with decreased renal excretion of UMOD and uric acid, suggesting a role for UMOD in the regulation of plasma uric acid. To determine this, we screened common variants across the UMOD locus in one community-based Chinese population of 1000 individuals and the other population from 642 American twins and siblings of European and Hispanic ancestry. Transcriptional activity of promoter variants was estimated in luciferase reporter plasmids transfected into HEK-293 cells and mIMCD3 cells. In the primary Chinese population, we found that carriers of the GCC haplotype had higher plasma uric acid, and three promoter variants were associated with plasma uric acid. UMOD promoter variants displayed reciprocal effects on urine uric acid excretion and plasma uric acid concentration, suggesting a primary effect on renal tubular handling of urate. These UMOD genetic marker-on-trait associations for uric acid were replicated in the independent American cohort. Site-directed mutagenesis at trait-associated UMOD promoter variants altered promoter activity in transfected luciferase reporter plasmids. Thus, UMOD promoter variants seem to initiate a cascade of transcriptional and biochemical changes influencing UMOD secretion, leading to altered plasma uric acid levels.

Polymorphisms in the myeloperoxidase gene locus are associated with acute kidney injury-related outcomes.

Myeloperoxidase (MPO) is a lysosomal enzyme that may be involved in oxidative stress-mediated kidney injury. Using a two-step approach, we measured the association of four polymorphisms across the length of the MPO gene with systemic markers of oxidative stress: plasma MPO and urinary 15-F(2t)-isoprostane levels. Adverse outcomes were measured in a primary cohort of 262 adults hospitalized with acute kidney injury, and a secondary cohort of 277 adults undergoing cardiac surgery with cardiopulmonary bypass and at risk for postoperative acute kidney injury. Dominant and haplotype multivariable logistic regression analyses found a genotype-phenotype association in the primary cohort between rs2243828, rs7208693, rs2071409, and rs2759 MPO polymorphisms and both markers of oxidative stress. In adjusted analyses, all four polymorphic allele groups had 2-3-fold higher odds for composite outcomes of dialysis or in-hospital death or a composite of dialysis, assisted mechanical ventilation, or in-hospital death. The MPO T-G-A-T haplotype copy-number was associated with lower plasma MPO levels and lower adjusted odds for the composite outcomes. Significant but less consistent associations were found in the secondary cohort. In summary, our two-step genetic association study identified several polymorphisms spanning the entire MPO gene locus and a common haplotype marker for patients at risk for acute kidney injury.

Association of functional kallikrein-1 promoter polymorphisms and acute kidney injury: a case-control and longitudinal cohort study.

BACKGROUND: Kallikrein-1 (KLK1) is a highly conserved serine protease that is expressed in the kidney and involved in blood pressure regulation. The activity of this enzyme is diminished in acute kidney injury (AKI). METHODS: We first evaluated the potential role of functional multiallelic KLK1 promoter gene polymorphisms in a case-control study of 481 subjects (214 hospitalized patients with AKI of mixed causes and 267 healthy subjects). The complex, multiallelic G/C-rich repeat region of the proximal KLK1 promoter was determined by direct Sanger/capillary resequencing. RESULTS: 16 alleles were identified in a complex, polymorphic G/C-rich region of the KLK1 proximal promoter; 5 of these alleles (F, G, H, I, and K) were associated with development of AKI. Alleles I and G were classified as risk-alleles (unadjusted OR 1.86; 95% CI 1.23, 2.81; p = 0.003), whereas alleles F, H, and K were classified as protective-alleles (unadjusted OR 0.32; 95% CI 0.22, 0.46; p < 0.001) according to their directional association with development of AKI. After adjustment for sex, race, preexisting chronic kidney disease and APACHE II score, the KLK1 risk-allele (I or G) carrier state was associated with the composite of ≥2-fold increase in serum creatinine, oliguria, or dialysis requirement (adjusted OR 2.71; 95% CI 1.14, 6.44; p = 0.02). The KLK1 risk-allele carrier state was also marginally associated with the composite of ≥2-fold increase in serum creatinine, oliguria, dialysis requirement, or in-hospital death (adjusted OR 2.33; 95% CI 0.98, 5.52; p = 0.06). CONCLUSIONS: KLK1 promoter polymorphisms are associated with development of AKI and adverse outcomes. Further studies are needed to validate these findings.

Genetic covariance between gamma-glutamyl transpeptidase and fatty liver risk factors: role of beta2-adrenergic receptor genetic variation in twins.

BACKGROUND & AIMS: Plasma levels of gamma-glutamyl transpeptidase (GGT) are associated with risk factors for nonalcoholic fatty liver disease (NAFLD), such as dyslipidemia, insulin resistance (IR), and hypertension. Limited data exist on whether there is genetic covariance between plasma levels of GGT and NAFLD risk factors. Variants of beta2-adrenergic receptor gene (ADRB2) have been associated with dyslipidemia, IR, and hypertension, but its effect on GGT secretion is not known. We estimated the heritability of GGT using a twin-study design and examined the genetic covariance between GGT levels, IR, hypertension, levels of low-density lipoproteins and triglycerides, and ADRB2 variants. METHODS: We studied phenotypes of 362 twins; the heritabilities of increased GGT activity and genetic covariance with NAFLD risk factors were estimated by variance-component methodology. ADRB2 genotype associations with plasma GGT activity were examined using generalized estimating equations to account for intra-twinship correlations. RESULTS: GGT activity was heritable at 49% +/- 8% of the twin cohort and had significant covariance with IR; insulin, triglyceride, and uric acid levels; and diastolic blood pressure. In generalized estimating equation models, the most common haplotype of ADRB2 was significantly associated with plasma GGT activity. Five single nucleotide polymorphisms in ADRB2 were associated with levels of GGT; ADRB2 haplotypes displayed pleiotropic effects on GGT and triglyceride levels. CONCLUSIONS: In a twin study, GGT shared genetic codetermination with traits of metabolic syndrome. The ADRB2 gene had pleiotropic effects on plasma levels of GGT and triglycerides, indicating linked pathways (eg, adrenergic) between genetic susceptibility to NAFLD and metabolic syndrome.

Naturally occurring variations in the human cholinesterase genes: heritability and association with cardiovascular and metabolic traits.

Cholinergic neurotransmission in the central and autonomic nervous systems regulates immediate variations in and longer-term maintenance of cardiovascular function with acetylcholinesterase (AChE) activity that is critical to temporal responsiveness. Butyrylcholinesterase (BChE), largely confined to the liver and plasma, subserves metabolic functions. AChE and BChE are found in hematopoietic cells and plasma, enabling one to correlate enzyme levels in whole blood with hereditary traits in twins. Using both twin and unrelated subjects, we found certain single nucleotide polymorphisms (SNPs) in the ACHE gene correlated with catalytic properties and general cardiovascular functions. SNP discovery from ACHE resequencing identified 19 SNPs: 7 coding SNPs (cSNPs), of which 4 are nonsynonymous, and 12 SNPs in untranslated regions, of which 3 are in a conserved sequence of an upstream intron. Both AChE and BChE activity traits in blood were heritable: AChE at 48.8 ± 6.1% and BChE at 81.4 ± 2.8%. Allelic and haplotype variations in the ACHE and BCHE genes were associated with changes in blood AChE and BChE activities. AChE activity was associated with BP status and SBP, whereas BChE activity was associated with features of the metabolic syndrome (especially body weight and BMI). Gene products from cDNAs with nonsynonymous cSNPs were expressed and purified. Protein expression of ACHE nonsynonymous variant D134H (SNP6) is impaired: this variant shows compromised stability and altered rates of organophosphate inhibition and oxime-assisted reactivation. A substantial fraction of the D134H instability could be reversed in the D134H/R136Q mutant. Hence, common genetic variations at ACHE and BCHE loci were associated with changes in corresponding enzymatic activities in blood.

Contemporary approaches to genetic influences on hypertension.

PURPOSE OF REVIEW: Essential hypertension has long been considered to be primarily 'genetic,' though recent studies have only revealed minor contributions to blood pressure. Technology has advanced tremendously in the recent years, with much focus on DNA studies utilizing both candidate gene and genome-wide association studies. However, many new areas that need continued investigation have arisen. RECENT FINDINGS: In addition to DNA studies, genetic studies are actively pursuing previously unexplored areas of potential variation, such as that which occurs posttranscriptionally in RNA and posttranslationally in protein structure. Advances have also been made in animal models and systems biology for large-scale integrative approaches. However, many other areas need continued investigation in the genetics of hypertension, including improved phenotyping and trait definition, gene-by-gene interactions (epistasis), and gene-by-environment interactions. 'Next generation' sequencing will assist researchers in performing more extensive genetic studies even more quickly, especially on unusual (rare) genetic variants. SUMMARY: Hypertension appears to have many genetic contributions from each regulatory area ranging from DNA to RNA to protein to postprotein to interactive influences of the environment on genes. New technologies have enabled such research to advance in the recent years. However, for this complex trait of hypertension, continued efforts must progress in all of these areas as well as in increased modeling and sequencing, so that the knowledge may be united for a comprehensive understanding of this common disease, such that diagnosis and treatment options in hypertensive patients and those at risk are facilitated.

Catecholamine storage vesicles: role of core protein genetic polymorphisms in hypertension.

Hypertension is a complex trait with deranged autonomic control of the circulation. The sympathoadrenal system exerts minute-to-minute control over cardiac output and vascular tone. Catecholamine storage vesicles (or chromaffin granules) of the adrenal medulla contain remarkably high concentrations of chromogranins/secretogranins (or "granins"), catecholamines, neuropeptide Y, adenosine triphosphate (ATP), and Ca(2+). Within secretory granules, granins are co-stored with catecholamine neurotransmitters and co-released upon stimulation of the regulated secretory pathway. The principal granin family members, chromogranin A (CHGA), chromogranin B (CHGB), and secretogranin II (SCG2), may have evolved from shared ancestral exons by gene duplication. This article reviews human genetic variation at loci encoding the major granins and probes the effects of such polymorphisms on blood pressure, using twin pairs to probe heritability and individuals with the most extreme blood pressure values in the population to study hypertension.

Renal kallikrein excretion and epigenetics in human acute kidney injury: expression, mechanisms and consequences.

BACKGROUND: Renal kallikrein (KLK1) synthesis and urinary excretion are reportedly diminished during AKI (acute kidney injury) in animal models, and provision of kallikrein abrogates renal injury in this setting, but data in human AKI is limited. Therefore we first examined KLK1 renal excretion in human AKI, and then probed potential endocrine and epigenetic mechanisms for its alterations. METHODS: KLK1 enzymatic activity excretion was evaluated in urine from patients with established or incipient AKI, versus healthy/non-hospital as well as ICU controls. Endocrine control of KLK1 excretion was then probed by catecholamine and aldosterone measurements in established AKI versus healthy controls. To examine epigenetic control of KLK1 synthesis, we tested blood and urine DNA for changes in promoter CpG methylation of the KLK1 gene, as well as LINE-1 elements, by bisulfite sequencing. RESULTS: Patients with early/incipient AKI displayed a modest reduction of KLK1 excretion, but unexpectedly, established AKI displayed substantially elevated urine KLK1 excretion, ~11-fold higher than healthy controls, and ~3-fold greater than ICU controls. We then probed potential mechanisms of the change. Established AKI patients had lower SBP, higher heart rate, and higher epinephrine excretion than healthy controls, though aldosterone excretion was not different. Promoter KLK1 CpG methylation was higher in blood than urine DNA, while KLK1 methylation in blood DNA was significantly higher in established AKI than healthy controls, though KLK1 methylation in urine tended to be higher in AKI, directionally consistent with earlier/incipient but not later/established changes in KLK1 excretion in AKI. On multivariate ANOVA, AKI displayed coordinate changes in KLK1 excretion and promoter methylation, though directionally opposite to expectation. Control (LINE-1 repetitive element) methylation in blood and urine DNA was similar between AKI and controls. CONCLUSIONS: Unexpectedly, increased KLK1 excretion in AKI patients was found; this increase is likely to be due in part to increments in adrenergic tone during BP depression. Epigenetic changes at KLK1 may also play a role in early changes of KLK1 expression and thus AKI susceptibility or recovery.

Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk.

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.

Progression of chronic kidney disease: Adrenergic genetic influence on glomerular filtration rate decline in hypertensive nephrosclerosis.

BACKGROUND: African-Americans are likely to develop hypertension and hypertensive nephrosclerosis. This grave prognosis, coupled with familial aggregation of end-stage renal disease (ESRD) in Blacks, prompts a search for genetic risk factors for ESRD. Recent evidence implicates a crucial role for the sympathetic nervous system in progressive renal disease. METHODS: We used the African-American Study of Kidney Disease to probe whether beta2-adrenergic receptor (ADRB2) predicts glomerular filtration rate (GFR) decline rate. A total of 580 participants were included. Baseline GFR was 51.2 +/- 0.5 ml/min/1.73 m2. Subjects were randomized in a 2 x 3 block design: to intensively lowered (MAP < or = 92 mm Hg) versus 'usual' (MAP = 102-107 mm Hg) blood pressure goal groups, and also divided by three randomized antihypertensive drugs (ramipril, metoprolol, or amlodipine). We scored 4 SNPs at the ADRB2 locus. RESULTS: Haplotypes at ADRB2 predicted chronic GFR decline rate, GFR declined more slowly in individuals with haplotype-1 (-804G-->173T-->16Gly-->27GIn), and faster in those who carried haplotype-3 (-804G-->173T-->16Arg-->27Gln). ADRB2 genotype interacted with antihypertensive drug class to influence GFR slope (p = 0.001-0.037). We extended our findings to an independent case/control sample of Black hypertensive ESRD, in which we found that variant Gly16Arg that tagged the GFR slope-determining ADRB2 haplotype also conferred risk for the ESRD trait in Blacks. CONCLUSIONS: The GFR decline/progression rate in hypertensive renal disease is controlled in part by genetic variation within the adrenergic pathway.

Common charge-shift mutation Glu65Lys in K+ channel ß1-Subunit KCNMB1: pleiotropic consequences for glomerular filtration rate and progressive renal disease.

BACKGROUND: Glomerular filtration rate (GFR) is a heritable trait, and hyperfiltration (GFR increment in remnant nephrons) may accelerate renal functional decline in chronic kidney disease (CKD). Mesangial and vascular smooth myocytes control GFR by contraction, dependent on voltage-gated Ca(2+) influx, which is controlled by the regulatory ß1-subunit (KCNMB1) of large-conductance heteromeric K+ ('BK') channels. KCNMB1 gain-of-function variant Glu65Lys results in generalized vasorelaxation and thus protection against systemic hypertension. Here we asked whether the Glu65Lys variant influences GFR, in the basal state or during progressive renal decline. METHODS: We explored Glu65Lys effects on GFR in three populations spanning two ethnicities and two diseases (hypertension and nephrosclerosis). GFR was either estimated (eGFR from serum creatinine) or directly measured (iothalamate clearance). RESULTS: The 65Lys variant was relatively common, occurring on ∼5-10% of chromosomes in different biogeographic ancestry groups, and 65Lys carriers exhibited higher eGFR in two primary care populations: extreme BP values in Kaiser clinics (p = 0.029, accounting for ∼0.2% of trait variance), or treated hypertensives in VA clinics (p = 0.017, accounting for ∼0.9% of trait variance). In blacks with progressive renal disease (NIDDK AASK), 65Lys carriers displayed a steeper slope in GFR chronic decline (p = 0.030, accounting for ∼0.4% of trait variance), and Glu65Lys genotype also predicted time of onset of renal failure (log rank p = 0.019). CONCLUSIONS: Common KCNMB1 gain-of-function variant Glu65Lys influences GFR, and 65Lys carriers exhibit not only elevated baseline GFR, but also more rapid GFR decline (and consequent development of renal failure) in CKD. The results suggest that profiling patients at Glu65Lys can assist in gauging renal prognosis as well as selection of rational therapy in hypertension with progressive renal disease.

Human tyrosine hydroxylase natural allelic variation: influence on autonomic function and hypertension.

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)(6) and (TCAT)(10i), predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk.

Naturally occurring genetic variants in human chromogranin A (CHGA) associated with hypertension as well as hypertensive renal disease.

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.

Phenylethanolamine N-methyltransferase gene polymorphisms and adverse outcomes in acute kidney injury.

BACKGROUND/AIMS: The catecholaminergic pathway is important in the physical stress response; however, its role is not well understood in acute kidney injury (AKI). We studied single nucleotide polymorphisms (SNPs) of phenylethanolamine N-methyltransferase (PNMT), the terminal enzyme of the catecholaminergic pathway, and their association with adverse outcomes in AKI. METHODS: We performed a case-control study of 961 Caucasian subjects (194 with AKI and 767 controls). The PNMT promoter G-161A (rs876493) and coding A+1543G (rs5638) SNPs were genotyped and haplotypes generated. The outcomes of interest were the development of AKI, in-hospital mortality, dialysis requirement, oliguria, and hemodynamic shock. Urine catecholamines were measured in cases to explore genotype-phenotype correlations. RESULTS: The PNMT +1543 G allele was associated with AKI [odds ratio (OR) 2.19, 95% confidence interval (CI): 1.04-4.60]. For AKI cases, each PNMT -161 A allele was associated with lower mortality (OR 0.58, 95% CI: 0.35-0.99) and hemodynamic shock (OR 0.63, 95% CI: 0.40-1.00). The PNMT +1543 G allele was associated with oliguria (OR 3.35, 95% CI: 1.13-9.95). Urine adrenaline was associated with increased hemodynamic shock and mortality, but was lowest in PNMT -161 A/A carriers. CONCLUSION: In Caucasians, PNMT SNPs are associated with the development of AKI, disease severity, and in-hospital mortality. The adrenergic pathway provides another area of focus in the study of AKI.

Direct vasoactive effects of the chromogranin A (CHGA) peptide catestatin in humans in vivo.

Catestatin is a bioactive peptide of chromogranin A (CHGA) that is co-released with catecholamines from secretory vesicles. Catestatin may function as a vasodilator and is diminished in hypertension. To evaluate this potential vasodilator in vivo without systemic counterregulation, we infused catestatin to target concentrations of approximately 50, approximately 500, approximately 5000 nM into dorsal hand veins of 18 normotensive men and women, after pharmacologic venoconstriction with phenylephrine. Pancreastatin, another CHGA peptide, was infused as a negative control. After preconstriction to approximately 69%, increasing concentrations of catestatin resulted in dose-dependent vasodilation (P = 0.019), in female subjects (to approximately 44%) predominantly. The EC(50) (approximately 30 nM) for vasodilation induced by catestatin was the same order of magnitude to circulating endogenous catestatin (4.4 nM). No vasodilation occurred during the control infusion with pancreastatin. Plasma CHGA, catestatin, and CHGA-to-catestatin processing were then determined in 622 healthy subjects without hypertension. Female subjects had higher plasma catestatin levels than males (P = 0.001), yet lower CHGA precursor concentrations (P = 0.006), reflecting increased processing of CHGA-to-catestatin (P < 0.001). Our results demonstrate that catestatin dilates human blood vessels in vivo, especially in females. Catestatin may contribute to sex differences in endogenous vascular tone, thereby influencing the complex predisposition to hypertension.