B2 Receptor Gene Research Articles

Kininase I Inhibition Prevents Angiotensin II‐Induced Hypertension and Target Organ Damage

Activation of the kinin B1 receptor (B1R), a major component of the kallikrein kinin system (KKS), is associated with the pathogenesis of hypertension. Kininase I (or carboxypeptidase N/M; CPN/CPM), an enzyme primarily responsible for the synthesis of endogenous B1R agonist, has been implicated in the progression of cardiovascular diseases. However, the exact role of kininase I in hypertension has not been investigated. Our preliminary data shows that CPN activity is significantly increased in the brain of angiotensin (Ang) II‐induced hypertensive mice compared to vehicle treated mice (Ang II: 7.1 +0.4 AU/min vs. vehicle: 2.8 +0.3 AU/min, n=5, p<0.05). In this study, our objective was to clarify the role of kininase I in the pathogenesis of hypertension, particularly the effects of this enzyme on blood pressure, cardiac and renal hypertrophy. We hypothesized that inhibition of kininase I will attenuate the development of Ang II‐induced hypertension and target organ damage. To test this hypothesis, 12‐week old male and female C57BL/6NJ wild‐type (WT) and CPN gene deleted (CPNKO) mice, were administered Ang II (600 ng/kg/min; 2 weeks) or saline (vehicle) via osmotic minipumps. Blood pressure was measured using radio telemetry probes and hypertrophy was assessed in the heart and kidney after 14 days of Ang II‐infusion, using weight/tibial length. Baseline blood pressure was similar between WT and CPNKO mice (102 +2 mmHg vs. 104 +2 mmHg). Ang II‐infusion significantly increased the blood pressure in WT mice compared to saline infused mice (147 +5 mmHg, n=5, p<0.05). This Ang II‐increased blood pressure response was attenuated in CPNKO mice (125 +4 mmHg, n=5, p<0.01). Both male and female CPNKO mice did not show any significant differences in blood pressure following Ang II‐infusion. In addition, Ang II‐infusion also induced significant cardiac (heart weight/tibia length: 8.5 +0.9 vs. 6.8 +0.6 mg/mm) and renal (kidney weight/tibia length: 11.8 +1.5 vs. 8.7 +0.9 mg/mm) hypertrophy in WT mice compared to vehicle treated mice. However, CPNKO mice exhibited an attenuation in cardiac and renal hypertrophy following Ang II administration. Furthermore, Ang II‐infusion increased collagen deposition in the heart, measured by picrosirius red staining, which was attenuated in CPNKO mice (percent area 9.8 +1.4 vs. 7.2 +1.4). Ang II‐infusion also significantly increased B1R gene and protein expression in the hypothalamus, heart, kidney, and aorta of WT mice, but this expression was abrogated in CPNKO mice. In conclusion, our data suggest that CPN gene deletion protects from Ang II‐induced hypertension and target organ damage, and indirectly prevents the B1R activation. This is the first evidence to show that inhibition of kininase I prevents the development of Ang II‐induced hypertension. Targeting kininase I might be a novel therapeutic approach for the treatment of hypertension.

Potentiation of B2 receptor signaling by AltB2R, a newly identified alternative protein encoded in the human bradykinin B2 receptor gene

Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3–protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and β-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery.

Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons.

Angiotensin converting enzyme 2 (ACE2) is a critical component of the compensatory axis of the renin angiotensin system. Alterations in ACE2 gene and protein expression, and activity mediated by A Disintegrin And Metalloprotease 17 (ADAM17), a member of the “A Disintegrin And Metalloprotease” (ADAM) family are implicated in several cardiovascular and neurodegenerative diseases. We previously reported that activation of kinin B1 receptor (B1R) in the brain increases neuroinflammation, oxidative stress and sympathoexcitation, leading to the development of neurogenic hypertension. We also showed evidence for ADAM17-mediated ACE2 shedding in neurons. However, whether kinin B1 receptor (B1R) activation has any role in altering ADAM17 activity and its effect on ACE2 shedding in neurons is not known. In this study, we tested the hypothesis that activation of B1R upregulates ADAM17 and results in ACE2 shedding in neurons. To test this hypothesis, we stimulated wild-type and B1R gene-deleted mouse neonatal primary hypothalamic neuronal cultures with a B1R-specific agonist and measured the activities of ADAM17 and ACE2 in neurons. B1R stimulation significantly increased ADAM17 activity and decreased ACE2 activity in wild-type neurons, while pretreatment with a B1R-specific antagonist, R715, reversed these changes. Stimulation with specific B1R agonist Lys-Des-Arg9-Bradykinin (LDABK) did not show any effect on ADAM17 or ACE2 activities in neurons with B1R gene deletion. These data suggest that B1R activation results in ADAM17-mediated ACE2 shedding in primary hypothalamic neurons. In addition, stimulation with high concentration of glutamate significantly increased B1R gene and protein expression, along with increased ADAM17 and decreased ACE2 activities in wild-type neurons. Pretreatment with B1R-specific antagonist R715 reversed these glutamate-induced effects suggesting that indeed B1R is involved in glutamate-mediated upregulation of ADAM17 activity and ACE2 shedding.

The bradykinin system in stress and anxiety in humans and mice

Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p < 0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1a and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.

Expression, distribution and function of kinin B1 receptor in the rat diabetic retina.

The kinin B1 receptor contributes to vascular inflammation and blood-retinal barrier breakdown in diabetic retinopathy (DR). We investigated the changes in expression, cellular localization and vascular inflammatory effect of B1 receptors in retina of streptozotocin diabetic rats. The distribution of B1 receptors on retinal cell types was investigated by immunocytochemistry. Effects of B1 receptor agonist, R-838, and antagonist, R-954, on retinal leukocyte adhesion, gene expression of kinin and VEGF systems, B1 receptor immunoreactivity, microgliosis and capillary leakage were measured. Effect of B1 receptor siRNA on gene expression was also assessed. mRNA levels of the kinin and VEGF systems were significantly enhanced at 2weeks in streptozotocin (STZ)-retina compared to control-retina and were further increased at 6weeks. B1 receptor mRNA levels remained increased at 6months. B1 receptor immunolabelling was detected in vascular layers of the retina, on glial and ganglion cells. Intravitreal R-838 amplified B1 and B2 receptor gene expression, B1 receptor levels (immunodetection), leukostasis and vascular permeability at 2weeks in STZ-retina. Topical application (eye drops) of R-954 reversed these increases in B1 receptors, leukostasis and vascular permeability. Intravitreal B1 receptor siRNA inhibited gene expression of kinin and VEGF systems in STZ-retina. Microgliosis was unaffected by R-838 or R-954 in STZ-retina. Our results support the detrimental role of B1 receptors on endothelial and glial cells in acute and advanced phases of DR. Topical application of the B1 receptor antagonist R-954 seems a feasible therapeutic approach for the treatment of DR.

Effect of AGTR1 and BDKRB2 gene polymorphisms on atorvastatin metabolism in a Mexican population.

Discrepancies in the response to drugs are partially due to polymorphisms in genes involved in drug metabolism and transport. The frequency, pattern and impact of these polymorphisms vary among populations. In the present study, the pharmacokinetics and pharmacogenetics of atorvastatin (ATV) in a Mexican population were investigated. The study cohort exhibited differing ATV metabolizing phenotypes, and in subsequent allelic discrimination assays, single nucleotide polymorphisms in the angiotensinogen, angiotensin II type 1 receptor (AGTR1) and bradykinin B2 receptor (BDKRB2) genes were genotyped and their effects on the pharmacokinetic parameters of ATV were assessed. Additionally, association studies were performed to test for a correlation between metabolizing phenotypes and genetic variants. It was observed that carriers of the genotypes A/C and C/T in AGTR1 and BDKRB2 had higher area under the plasma concentration-time curve values from time 0 to the time of the last measurement and from time 0 extrapolated to infinity, and lower values of clearance of the fraction dose absorbed compared with homozygous carriers (P<0.05). Only the C/C genotype of BDKRB2 was associated with the fast metabolizer phenotype. These data suggest that AGTR1 and BDKRB2 are involved in ATV pharmacokinetics; a novel finding that requires confirmation in further studies.

Relationship of Bradykinin B2 Receptor Gene C-58T Variation with Total-Cholesterol and Glucose in Essential Hypertension

Relationship of Bradykinin B2 Receptor Gene C-58T Variation with Total-Cholesterol and Glucose in Essential Hypertension

The relationship of Bradykinin B₂ receptor gene variation with obesity, hypertension and lipid variables in obese patients.

This study examined the association of C-58T genotypes with obesity/hypertension related parameters and serum lipids in obese (n=108) and non-obese (n=80) patients. Bradykinin receptor (B₂R) C-58T genotypes were determined by PCR-RFLP. B₂R gene C-58T frequencies for T/T (homozygous wild type), T/C (heterozygous) and C/C (homozygous polymorphic) genotypes for obese and non-obese patients were respectively: 36.1%, 37.5%; 45.4%, 52.5% and 18.5%, 10%. Obese patients using diuretic medication had lower C/C genotype frequency compared to T/T and T/C genotypes. Total cholesterol (T-Chol) (p=0.035) levels were found to be associated with B₂R C-58T polymorphism, where the T/T genotype had higher total cholesterol levels compared to the T/C genotype in obese patients. Non-obese patients using oral antidiabetic medication had higher C/C genotype frequency than that of T/T and T/C genotypes. Waist circumference (p=0.016) and diastolic blood pressure (p=0.01) levels were elevated in the non-obese subjects with the C/C genotype compared to T/C and T/T. Although B₂R C-58T gene polymorphism was not found to be effective on obesity with logistic regression analysis in the whole study population in obese subjects, the T-Chol decreasing effect of the B₂R gene C allele and the higher waist circumference measurements in the non-obese subjects may indicate there may be a link between B₂R gene C-58T polymorphism and obesity in study populations of higher numbers.

Length polymorphism of the B2-VNTR minisatellite repeat of the bradykinin B2 receptor gene in healthy Russians and patients with coronary heart disease

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, and pain. This receptor attracts special attention as one of the factors that have cardioprotective and infarct-limiting effects. Certain genetic variants of the coding and noncoding regions of the bradykinin B2 receptor gene (BDKRB2) may play a role in modulating its expression. The 3′-untranslated region of BDKRB2 exon 3 harbors a minisatellite repeat (B2-VNTR), which affects the mRNA stability. Hence, it is of interest to study a possible association of B2-VNRT alleles with various forms of coronary heart disease (CHD). In our work the allele and genotype frequency distributions of B2-VNTR were compared between healthy individuals and patients with CHD (angina pectoris or myocardial infarction (MI)) of the Russian ethnic group. Based on its length polymorphism, B2-VNTR was classed with low-polymorphic non-hypervariable minisatellites. Three B2-VNTR alleles, which consisted of 43, 38, and 33 repeats, were observed in all investigated cohorts. The alleles with 43 and 33 repeats were the most prevalent. The allele and genotype frequencies of B2-VNTR did not significantly differ between males and females in control group, and also between healthy males and males with angina pectoris or MI. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of CHD in males. However, we do not exclude the possibility of an association of the short B2-VNTR alleles (38 and 33 repeats) with a cardioprotective effect in females with CHD. This hypothesis requires further investigation.

The bradykinin B1 receptor antagonist BI113823 reverses inflammatory hyperalgesia by desensitization of peripheral and spinal neurons.

Bradykinin is a neuropeptide released after tissue damage which plays an important role in inflammatory pain. The up-regulation of the bradykinin B1 receptor in response to inflammation makes it an attractive target for drug development. Aim was to investigate if the selective B1 receptor antagonist BI113823 reduces inflammation-induced mechanical hyperalgesia and if the effect is mediated via peripheral and/or spinal B1 receptor antagonism. Electrophysiological recordings of peripheral afferents and spinal neurons were combined with behavioural experiments to better understand the underlying mechanisms of B1 receptor antagonism. Experiments were performed 24 h after injection of complete Freund's adjuvant (CFA) or saline into the paw of Wistar rats. A gene expression analysis for the B1 receptor was performed in different tissues. BI113823 was administered orally or intrathecally to assess effects on CFA-induced hyperalgesia. Peripheral afferents of the saphenous nerve as well as spinal wide dynamic range (WDR) and nociceptive-specific (NS) neurons were recorded, and mechanosensitivity was measured before and after BI113823 administration. BI113823 reduced CFA-induced mechanical hyperalgesia when administered orally or intrathecally. An increased B1 receptor gene expression was found in peripheral and spinal neural tissue. BI113823 significantly reduced mechanosensitivity of peripheral afferents and spinal NS neurons, but had no effect on WDR neurons. The selective bradykinin B1 receptor antagonist BI113823 reduces CFA-induced mechanical hyperalgesia which is mediated via antagonism of peripheral as well as spinal bradykinin B1 receptors. The selective modulation of CFA-sensitized spinal NS neurons by BI113823 could be a promising property for the treatment of inflammatory pain.

Novel Kinin B1 Receptor Splice Variant and 5′UTR Regulatory Elements Are Responsible for Cell Specific B1 Receptor Expression

The kinin B1 receptor (B1R) is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood.We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV) of human B1R gene with a shorter 5′untranslated region. This novel SV is ≈35% less stable than the wild-type (WT) transcript in lung adenocarcinoma cells (H2126), but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE) −1020 to −766 bp in H2126; positive response element (PRE) −766 to −410 bp in 16HBE; −410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells) were found.These findings reveal complex regulation of B1R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer.

Genetic variation and gender determine bradykinin type 1 receptor responses in human tissue: implications for the ACE-inhibitor-induced effects in patients with coronary artery disease

The efficacy of the ACE (angiotensin-converting enzyme) inhibitor perindopril in coronary artery disease [EUROPA (European trial on reduction of cardiac events with perindopril in stable coronary artery disease) study] is associated with the rs12050217 A/G single nucleotide polymorphism in the B1 receptor (bradykinin type 1 receptor) gene. To investigate the underlying mechanism, we examined the effect of this polymorphism on B1-receptor-mediated coronary artery dilation and peripheral blood mononuclear cell activation. Vasorelaxant responses of human coronary microarteries from subjects without coronary disease to des-Arg(9)-bradykinin and to bradykinin were studied in organ bath experiments. Des-Arg9-bradykinin responses were endothelium-dependent and exclusively mediated by B1 receptors, whereas responses to bradykinin were induced through B2 receptors (bradykinin type 2 receptors). The presence of the G allele reduced the response to 3 × 10(-8) mol/l des-Arg(9)-bradykinin by 29% [AA (n=13) compared with AG/GG (n=8); P<0.03], and tended to lower concentration-related responses (P=0.065) to this agonist, whereas the responses to bradykinin were unaffected by the rs12050217 genotype. In freshly obtained human mononuclear cells 1 μmol/l des-Arg(9)-bradykinin increased expression of the pro-inflammatory factors CXCL5 (CXC chemokine ligand 5) and IL6 (interleukin-6). These responses were not affected by genotype and exclusively occurred in blood cells from women, correlating (in the case of CXCL5) with their plasma 17β-oestradiol levels (r(2)=0.32, P=0.02; n=17). IL-1β (interleukin-1β) increased CXCL5 and IL6 expression in both genders, and this response was not associated with 17β-oestradiol levels. The gender difference in responses to B1 receptor stimulation in blood mononuclear cells implies possible gender differences in the response to ACE inhibitor therapy, which needs to be studied more comprehensively. The observed decrease in coronary vasodilator response might contribute to the impaired treatment response to perindopril of G allele carriers found in the EUROPA study.

Kinin B1 receptor gene ablation affects hypothalamic CART productionb

A role for the kinin B1 receptor in energy-homeostatic processes was implicated in previous studies; notably, the studies where kinin B1 receptor knockout mice (B1-/-) were shown to have impaired adiposity, impaired leptin and insulin production, lower feed efficiency, protection from liver steatosis and diet-induced obesity when fed a high fat diet (HFD). In particular, in a model where the B1 receptor is expressed exclusively in the adipose tissue, it rescues the plasma insulin concentration and the weight gain seen in wild type mice. Taking into consideration that leptin participates in the formation of hypothalamic nuclei, which modulate energy expenditure, and feeding behavior, we hypothesized that these brain regions could also be altered in B1-/- mice. We observed for the first time a difference in the gene expression pattern of cocaine and amphetamine related transcript (CART) in the (lateral hypothalamic area (LHA) resulting from the deletion of the kinin B1 receptor gene. The correlation between CART expression in the LHA and the thwarting of diet-induced obesity corroborates independent correlations between CART and obesity. Furthermore, it seems to indicate that the mechanism underlying the 'lean' phenotype of B1-/- mice does not stem solely from changes in peripheral tissues but may also receive contributions from changes in the hypothalamic machinery involved in energy homeostasis processes.

Clinical impact of an angiotensin I-converting enzyme insertion/deletion and kinin B2 receptor +9/-9 polymorphisms in the prognosis of renal transplantation

There is a consensus in the scientific literature that supports the importance of the kallikrein kinin and renin angiotensin systems in renal physiology, but few studies have investigated their importance after renal transplantation. The aim of this study was to investigate the clinical effects of the insertion/deletion polymorphism in the angiotensin I-converting enzyme (ACE) gene and the +9/-9 polymorphism in the kinin B2 receptor (B2R) gene in kidney-transplanted patients (n=215 ACE, n=203 B2R) compared with 443 healthy individuals. Demographic results showed that there is a higher frequency of the D allele (high plasma ACE activity) and +9 allele (lower B2R expression) in transplant patients compared with control individuals. We also observed a higher frequency of these alleles in patients who had an elevated level of plasma creatinine. At day 7 post-transplantation, we found a higher prevalence of individuals with the DD genotype with elevated plasma creatinine level. Furthermore, individuals with the DD genotype had a higher chronic allograft dysfunction and graft loss compared with the II patient genotype, which showed no loss of graft. Taken together, our data suggest that the DD genotype is an indicator of an unfavorable prognosis following renal transplantation and could be related to kinin modulation.

Contributions of Renin-Angiotensin System-Related Gene Interactions to Obesity in a Chinese Population

BackgroundGene-gene interactions may be partly responsible for complex traits such as obesity. Increasing evidence suggests that the renin-angiotensin system (RAS) contributes to the etiology of obesity. How the epistasis of genes in the RAS contributes to obesity is still under research. We aim to evaluate the contribution of RAS-related gene interactions to a predisposition of obesity in a Chinese population.Methodology and Principal FindingsWe selected six single nucleotide polymorphisms (SNPs) located in angiotensin (AGT), angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AGTR1), MAS1, nitric oxide synthase 3 (NOS3) and the bradykinin B2 receptor gene (BDKRB2), and genotyped them in 324 unrelated individuals with obesity (BMI ≥28 kg/m2) and 373 non-obese controls (BMI 18.5 to <24 kg/m2) from a large scale population-based cohort. We analyzed gene-gene interactions among 6 polymorphic loci using the Generalized Multifactor Dimensionality Reduction (GMDR) method, which has been shown to be effective for detecting gene-gene interactions in case-control studies with relatively small samples. Then we used logistic regression models to confirm the best combination of loci identified in the GMDR. It showed a significant gene-gene interaction between the rs220721 polymorphism in the MAS1 gene and the rs1799722 polymorphism in the gene BDKB2R. The best two-locus combination scored 9 for cross-validation consistency and 9 for sign test (p = 0.0107). This interaction showed the maximum consistency and minimum prediction error among all gene-gene interaction models evaluated. Moreover, the combination of the MAS1 rs220721 and the BDKRB2 rs1799722 was associated with a significantly increased risk of obesity (OR 1.82, CI 95%: 1.15–2.88, p = 0.0103).Conclusions and SignificanceThese results suggest that the SNPs from the RAS-related genes may contribute to the risk of obesity in an interactive manner in a Chinese population. The gene-gene interaction may serve as a novel area for obesity research.

Association and Interaction of −58C>T and ±9 bp Polymorphisms of BDKRB2 Gene Causing Susceptibility to Essential Hypertension

Introduction. Bradykinin, a vasodilator by nature has been documented to have a protective role against hypertension and cardiovascular complications. Polymorphisms of bradykinin B2 receptor (BDKRB2) gene are reported to be predisposing factors for hypertension. Evaluation of the association between −58C>T and ±9 bp polymorphisms of BDKRB2 with essential hypertension (EHT) was attempted. Methods. Two hundred and fourteen primary hypertensives and 249 controls were genotyped for the selected markers by polymerase chain reaction, gel electrophoresis (±9 bp), and SSCP (−58C>T). Results. While −58C>T polymorphism did not reveal any association with EHT, ±9 bp polymorphism showed a significant association with high risk for heterozygotes (+9/−9) when tested against the pooled frequencies of homozygotes (OR [odds ratio] = 1.63, 95% confidence interval [CI] = 1.12–2.38, P = .02), and this risk was 1.7 folds high in males (OR = 1.74, 95% CI = 1.05–2.86, P = .06) and 1.9 folds high in familial cases (OR = 1.96, 95% CI = 1.09–3.53, P = .04). In contrast, significant protective effect was observed for −9/–9 genotype against EHT when tested under dominant model in general (OR = 0.59, 95% CI = 0.41–0.86, P = .01), in males (OR = 0.49, 95% CI = 0.30–0.82, P = .01), and in familial cases (OR = 0.50, 95% CI = 0.28–0.89, P = .04). Significant risk for +9 bp allele was observed in general (OR = 1.39, 95% CI = 1.05–1.86, P = .04) and in males (OR = 1.65, 95% CI = 1.13–2.41, P = .02). The interaction information analysis revealed a synergistic effect between the two polymorphisms contributing to EHT. +9/+9 genotype of ±9 bp polymorphism when present in combination with CC genotype of −58C>T polymorphism showed 2.2-fold higher risk for developing EHT. Conclusions. The results suggest that allele +9 bp might be a risk factor for EHT in general and specially in males. Markers −58C>T and ±9 bp may act synergistically causing susceptibility to EHT.

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism

Kinin B1 and B2 receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B1 (B1−/−) and B2 receptor (B2−/−) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B1−/− and B2−/− in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B1−/− and B2−/−. In contrast, in the presence of l-arginine, Ca2+ and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B1−/− and B2−/−. The present study demonstrated that targeted deletion of B1 or B2 receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B1−/− and B2−/− vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.

A Proposal----The Renal Kallikrein-Kinin System as a Compensating System for Salt Accumulation after Excess Salt Intake

Terrestrial mammals, like human, must reserve water and NaCl to counter the effects of dry-environments. Their kidneys need to reabsorb as much water and sodium as possible along the proximal tubules and collecting duct. Thus, excess sodium intake tends to sodium accumulation in the body. The renal (tissue) kallikrein-kinin system (KKS) exists to prevent excessive sodium reabsorption. Renal kallikrein is secreted from the distal connecting tubule cells, which are located just distal to the major reabsorbing system. Kallikrein releases kinins, which inhibit sodium reabsorption along the collecting ducts. The kinins in the tubules are quickly destroyed by kidney-specific destroying enzymes (kininases), carboxypeptidase Y-like exopeptidase and neutral endopeptidase, which differ completely from those in plasma. Ebelactone B and poststatin were discovered as selective inhibitors of urinary kininases. Excess sodium intake causes hypertension, since sodium accumulation in the blood vessel walls increases sensitivity to hypertensive agents. Potassium, and ATP-sensitive potassium channel blockers cause renal kallikrein secretion. In bradykinin B2 receptor gene knockout mice, tissue kallikrein gene knockout mice, and kininogen-deficient rats, the knockout or deficiency does not itself induce hypertension, but these animals are salt-sensitive. Mutant kininogen-deficient (kinin-free) rats on 2% sodium intake quickly develop hypertension. Urinary kininase inhibitors reduce high blood pressure due to sodium accumulation on high sodium intake. Our ample supporting data suggest that the renal KKS prevents sodium accumulation, and that reduced levels of renal kallikrein may cause salt-sensitive hypertension. Keywords: Excess salt intake, The renal kallikrein-kinin system, Potassium, ATP-sensitive potassium channel blockers, Ebelactone B, Poststatin, Neutral endopeptidase, Renal Kallikrein-Kinin System, Renal kallikrein, Y-like exopeptidase, urinary kininases, Mutant kininogen-deficient, kallikrein-kinin system, renin-angiotensin system, systemic blood pressure, angiotensin-converting enzymes, lysyl-bradykinin, bradykinin, kallikrein-secreting cells, Henle's loop, BK-B2 antagonist, carboxypeptidase Y-like exopeptidase, human urinary kininases, epithelial Na channels, Rattus norvegicus, LMW kininogens, cerebrospinal fluid, deoxycorticosterone acetate, Weinberger's group

Kinin B1 Receptor Enhances the Oxidative Stress in a Rat Model of Insulin Resistance: Outcome in Hypertension, Allodynia and Metabolic Complications

BackgroundKinin B1 receptor (B1R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B1R activation could perpetuate the oxidative stress which leads to diabetic complications.Methods and FindingsYoung Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8–12 weeks. A selective B1R antagonist (SSR240612) was administered acutely (3–30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B1R expression, aortic superoxide anion (O2 •−) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3–30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O2 •−, NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B1R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O2 •− in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10–100 µM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe8]des-Arg9-BK (20 µM; B1R agonist). Data show that the greater aortic O2 •− production induced by the B1R agonist was blocked only by apocynin.ConclusionsActivation of kinin B1R increased O2 •− through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B1R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B1R gene expression in this model.

Mechanisms of p53-mediated repression of the human polycystic kidney disease-1 promoter

We previously reported that the tumor suppressor protein p53 participates in a negative feedback loop to fine-tune PKD1 gene expression. This physiological pathway is believed to prevent polycystin-1 overexpression and thus renal cysts. The present study examined the mechanisms of p53-mediated repression of PKD1. The 5′-upstream region of the human PKD1 gene is TATA-less, GC-rich, and contains four consensus p53 binding sites at positions −2.7 kb (BS4), −1.2 kb (BS3), −0.8 kb (BS2), and −0.2 kb (BS1), respectively. PKD1 BS1 – 4 are bound to endogenous p53 in vivo and in vitro. Transient transfection assays in inner medullary collecting duct cells revealed that disruption of PKD1 BS1 enhances baseline PKD1 promoter activity; in contrast, disruption of PKD1 BS4 suppressed PKD1 transcription. PKD1 BS1 confers p53-mediated repression when substituted for the p53 enhancer element in the bradykinin B2 receptor gene, indicating that PKD1 BS1 is a bona fide p53 repressor element. Moreover, PKD1 BS1 requires intact BS2-4 and cellular histone deacetylase activity for full functional activity. Indeed, the PKD1 BS1/4 regions are occupied by a complex containing HDAC1/2 and mSin3. These findings suggest a model whereby p53 exerts a biphasic control on PKD1 gene transcription, depending on cellular context and the cognate cis-acting element.