An association study of C9orf3, a novel component of the renin-angiotensin system, and hypertension in diabetes.

The renin-angiotensin system (RAS) is important in the onset and course of cardiovascular, kidney, and metabolic disorders. Previous reports showed that the RAS blockade protects organs and suppress the development of type 2 diabetes mellitus. A novel component of the RAS, namely, chromosome 9 open reading frame 3 (C9orf3), was recently identified, however, its effects are unclear. We evaluated whether the genetic variant of C9orf3 is associated with morbidity of hypertension among subjects with type 2 diabetes. We enrolled 382 subjects with type 2 diabetes, 222 of whom were diagnosed with hypertension. Human leukocyte genomic DNA was isolated and a genetic variant was analyzed for a C/T variant of C9orf3 (rs4385527) via PCR analysis. The relationship between the genotype and hypertension morbidity among subjects with diabetes was examined. The proportion of the respective C9orf3 genetic variants were as follows 247 CC, 119 CT, and 16 TT. The risk of hypertension was determined to be 1.58, with a 95% confidence interval of 1.11-2.27. Moreover, the p value was 0.012 for allelic comparison and for Armitage's trend test, with the C allele identified as the risk factor. Consequently, hypertension was markedly associated with type 2 diabetes in subjects with the C9orf3 variant, exhibiting a nearly 1.6-fold increased risk. The C variant of a new component of the RAS, C9orf3 (rs4385527) might have a considerable impact on the pathogenesis of hypertension in diabetes.

On the top of ARB N/L type Ca channel blocker leads to less elevation of aldosterone.

The activation of the renin-angiotensin system (RAS) is one of the unfavourable characteristics of calcium channel blocker (CCB). N type calcium channel is thought to be involved in renin gene transcription and adrenal aldosterone release. Accordingly, N/L type CCB has a possibility of less elevation of plasma aldosterone concentrations (PAC) among CCBs. In a monotherapy study, we had already demonstrated that N/L type CCB leads to less activation of the RAS compared with L type CCB. The objective of this study is to substantiate the hypothesis that at the condition of additive administration on the top of an angiotensin receptor blocker (ARB), still N/L type CCB leads to less elevation of PAC compared with L type one. Subjects were 60 hypertensives administered with valsartan. As an open label study, amlodipine (L type) or cilnidipine (N/L type) were administered on the top of valsartan (ARB) in a cross-over manner. Results were as follows (valsartan+amlodipine compared with valsartan+cilnidipine): systolic blood pressure (SBP)/diastolic blood pressure (DBP) (mmHg): 132±10/76±10 compared with 131±10/77±9, P=0.95/0.48, plasma renin activity (PRA) (ng/ml·h): 2.41±2.67 compared with 2.00±1.50 P=0.20, PAC (pg/ml): 77.3±31.0 compared with 67.4±24.8, P<0.05, urinary albumin excretion (UAE) (mg/gCr): 105.9±216.1 compared with 73.9±122.2, P<0.05. Thus, PAC at cilnidipine was significantly lower than those at amlodipine in spite of the comparable BP reductions. Besides, UAE was significantly lower at cilnidipine. In conclusion, on the top of the ARB, it is suggested that cilnidipine administration might lead to less elevation of PAC and reduction in UAE compared with amlodipine.

A Genetic Variant in the Distal Enhancer Region of the Human Renin Gene Affects Renin Expression.

BACKGROUND: The high heritability of plasma renin activity was confirmed in recent investigations. A variation located near the strong enhancer of the human renin gene (REN), C-5312T, has been shown to have different transcription activity levels depending on its allele: the 5312T allele shows transcription levels that are 45% greater than those of the 5312C allele. The purpose of this study was to confirm the hypothesis that variations in the enhancer region of the REN gene are involved in regulating renal expression of renin. METHODS: Sixty-four subjects with biopsy-proven renal diseases were included in this study (male/female: 35/29, age 41.9 ± 20.9 years, SBP/DBP 123.1 ± 23.7/73.4 ± 14.8 mmHg, s-Cr 0.93 ± 0.63 mg/dl). A genetic variant of REN, C-5312T, was assayed by PCR-RFLP and the TaqMan method. Total RNAs from a small part of the renal cortex were reverse-transcribed and amplified for REN and GAPDH with a real-time PCR system. RESULTS: Logarithmically transformed expression values of the relative ratio of REN to GAPDH (10-3) were as follows (mean ± SE): CC (26 cases), 0.016 ± 0.005; CT (33 cases), 0.047 ± 0.021 (p = 0.41 vs. CC); TT (5 cases), 0.198 ± 0.194 (p = 0.011 vs. CC, p < 0.031 vs. CT). Thus, significant differences in REN expression were observed among the genetic variants. CONCLUSION: The results suggest that variants in the enhancer region of the human renin gene have an effect on the expression levels of renin in renal tissue; this observation is in good accordance with the results of the transcriptional assay.

Determinants of plasma renin activity: role of a human renin gene variant as a genetic factor.

The plasma renin activity (PRA) is affected by a number of environmental factors. However, significant heritability has been shown for the activity. A hypothesis that a candidate regulatory single-nucleotide polymorphism, C-5312T, of human renin gene should have a significant effect on PRA was elucidated and updating of independent determinants of PRA was attempted. Cross sectional study. Outpatient study. We enrolled consecutive 810 subjects who had consulted our hospitals for lifestyle-related diseases. Genotypes were assayed with genomic DNA for C-5312T. Among the genetic variants, the difference of PRA was evaluated. Monovariate linear regression analysis was performed to test the correlation between PRA and clinical variables. Finally, stepwise multiple regression analysis was performed to evaluate the independent determinants. On comparing 2 genotype groups, CC/CT and T allele homozygote, the geometric means of PRA were 0.778 and 0.941 ng/ml/h, respectively (F = 5.992, P = 0.015). Monovariate linear regression analysis revealed that a number of variables have a significant correlation with the activity, including urinary salt excretion. A stepwise multivariate regression analysis revealed that renin C-5312T variant (TT) is one of the independent determinants of PRA. Thus, for the first time, a human renin gene variant was associated with a significant increase in PRA as a genetic factor and the independent determinants for the activity were updated including genetic factor.

Genetic variant of the renin-angiotensin system and prevalence of type 2 diabetes mellitus: a modest but significant effect of aldosterone synthase.

Recent genome-wide association studies have identified multiple variants that confer risk of type 2 diabetes mellitus (DM). However, established associations explain only a part of the heritability. Thus, even at the genome-wide association studies era, candidate gene approach should be still useful. Recent interventional studies against the renin-angiotensin system (RAS) showed reduction in new onset of DM, implying the system is involved in the onset. We substantiated the hypothesis that genetic variants of RAS have significant association with prevalence of DM. We enrolled to the study consecutive 782 subjects who had consulted our hospitals for mainly lifestyle related diseases. They consisted of 282 (36.1 %) diabetes cases. Genotypes were assayed with genomic DNA for conventional four genes of the RAS, i.e., angiotensin converting enzyme (ACE) insertion/deletion variant, angiotensinogen (AGT) M235T variant, angiotensin II type I receptor (AT1) A1166C variant, and aldosterone synthase (CYP11B2) C-344T variant. Association between the genetic variants of the RAS and prevalence of type 2 DM was tested. A significant association of DM and CYP11B2 genotype was obtained. There was no significant association between DM and ACE, AGT and AT1 variants. A multivariate logistic regression showed that age, gender, and CYP11B2 genotype were independent factors for association to diabetes, the DM risk of CC/CT to TT being 1.40 (95 % CI 1.04-1.90, p = 0.029). Thus, it is concluded that a genetic variant of the RAS should have a modest but significant impact on the onset of type 2 diabetes mellitus.

A crossover comparison of urinary albumin excretion as a new surrogate marker for cardiovascular disease among 4 types of calcium channel blockers.

BACKGROUND: At the intervention for cardiovascular disease (CVD), albuminuria is a new pivotal target. Calcium channel blocker (CCB) is one of the most expected agents. Currently CCBs have been classified by delivery system, half-life and channel types. We tested anti-albuminuric effect among 4 types of CCBs. METHODS: Subjects were 50 hypertensives (SBP/DBP 164.7±17.1/92.3±12.2mmHg, s-Cr 0.81±0.37mg/dl, urinary albumin excretion (UAE) 69.4 (33.5-142.6) mg/gCr). Four CCBs were administered in a crossover setting: nifedipine CR, a long biological half-life L type by controlled release; cilnidipine, an N/L type; efonidipine, a T/L type; and amlodipine, a long biological half-life L type. RESULTS: Comparable BP reductions were obtained. UAE at endpoints ware as follows (mg/gCr, *P<0.01): nifedipine CR 30.8 (17.3-81.1),* cilnidipine 33.9 (18.0-67.7),* efonidipine 51.0 (21.2-129.8), amlodipine 40.6 (18.7-94.7). By all agents, significant augmentations were observed in PRA, angiotensin I and angiotensin II (AngII). AngII at cilnidipine was significantly lower than that at amlodipine. PAC at cilnidipine and efonidipine was significantly lower than that at amlodipine. Nifedipine CR significantly reduced ANP concentration. CONCLUSIONS: It is revealed that only nifedipine CR and cilnidipine could reduce albuminuria statistically. Thus, it is suggested that the 2 CCBs might be favorable for organ protection in hypertensives.

A new-generation N/L-type calcium channel blocker leads to less activation of the renin-angiotensin system compared with conventional L type calcium channel blocker.

OBJECTIVE: Calcium channel blocker (CCB) is one of the most useful antihypertensive agents. However, the activation of the renin-angiotensin system (RAS) is an unfavorable characteristic. N-type calcium channel is thought to be involved in catecholamine's release. Accordingly, N/L-type CCB has a probability of less activation of the RAS. We substantiated the hypothesis that N/L-type CCB, cilnidipine, leads to less activation of the RAS compared with conventional L-type CCB, amlodipine. DESIGN: Randomized, cross-over study. SETTING: Outpatient study. PARTICIPANTS: Participants were 110 hypertensive patients [male/female 46/64, age 66.3 ± 10.8 years, systolic blood pressure (SBP)/diastolic blood pressure (DBP) 161.8 ± 16.9/92.9 ± 12.4 mmHg, s-Cr 0.77 ± 0.32 mg/dl, plasma renin activity (PRA) 0.65 ± 0.63 ng/ml per h, angiotensin I (AngI) 70.5 ± 77.3 pg/ml, angiotensin II (AngII) 5.2 ± 3.9 pg/ml, plasma aldosterone concentration (PAC) 76.3 ± 35.9 pg/ml, urinary albumin excretion (UAE) 108.1 ± 284.2 mg/gCr]. Amlodipine besilate or cilnidipine was administered for 12 weeks in a cross-over manner as a monotherapy with an intention-to-treat fashion by titrating doses. Final doses of amlodipine besilate and cilnidipine were 6.6 ± 2.7 and 13.7 ± 5.1 mg/day, respectively. MAIN OUTCOME MEASURES: Changes in blood pressure, PRA, AngI, AngII, PAC, UAE of baseline and each end of amlodipine besilate and cilnidipine administration. RESULTS: Results were as follows (amlodipine vs. cilnidipine): SBP/DBP (mmHg): 135.2 ± 11.7/79.8 ± 9.6 vs. 136.7 ± 13.2/79.5 ± 10.9, P = 0.22/0.74; PRA (ng/ml per h): 1.16 ± 1.03 vs. 0.95 ± 0.78, P < 0.01; AngI (pg/ml): 155.0 ± 306.4 vs. 101.8 ± 92.0, P < 0.05; AngII (pg/ml): 12.0 ± 12.3 vs. 7.1 ± 4.5, P < 0.001; PAC (pg/ml): 81.6 ± 37.9 vs. 74.3 ± 36.2, P < 0.05; UAE (mg/gCr): 145.4 ± 424.5 vs. 58.8 ± 125.1, P < 0.05. Thus, in spite of the comparable blood pressure reductions, each level of components of the RAS at cilnidipine administration was significantly lower than those at amlodipine. Apart from this, UAE at cilnidipine administration was also significantly lower than that at amlodipine. CONCLUSION: It is suggested that cilnidipine leads to less activation of the RAS compared with amlodipine for the first time in human clinical patients and therefore cilnidipine might be expected to be superior in organ protection in addition to the antialbuminuric effect.

Genetic variant of the Renin-Angiotensin system and diabetes influences blood pressure response to Angiotensin receptor blockers.

OBJECTIVE Recent studies have proven the favorable effects of angiotensin receptor blockers (ARBs) on cardiovascular and renal disorders. However, determinants of the response to ARBs remain unclear. We substantiated the hypothesis that genetic variants of the renin-angiotensin system (RAS) have significant impacts on the response to ARBs. RESEARCH DESIGN AND METHODS Subjects comprised 231 consecutively enrolled hypertensive individuals including 45 type 2 diabetic subjects. Five genetic variants of the RAS, i.e., renin (REN) C-5312T, ACE insertion/deletion, angiotensinogen M235T, angiotensin II type 1 receptor A1166C, and angiotensin II type 2 receptor C3123A were assayed by PCR and restriction fragment-length polymorphism. A dose of 40-160 mg/day of valsartan was administered for 3 months as a monotherapy. RESULTS Changes in diastolic blood pressure significantly differed between genotypes of REN C-5312T: 10.7-mmHg reduction (from 95.9 +/- 12.9 to 85.2 +/- 11.4) in CC versus 7.0-mmHg reduction (from 94.7 +/- 14.0 to 87.7 +/- 12.6) in CT/TT (P = 0.02 for interactive effects of valsartan and genotype). Responder rates also differed between the genotypes: 72.8% in CC versus 58.0% in CT/TT (P = 0.03). Univariate analysis indicated a significant association of response to valsartan with blood pressure, diabetes, plasma aldosterone concentration, and CC homozygotes of REN C-5312T. Finally, multiple logistic regression analysis revealed that systolic blood pressure, CC homozygotes of REN C-5312T, and diabetes were independent predictors for responders with odds ratios (95% CI) of 2.49 (1.41-4.42), 2.03 (1.10-3.74), and 0.48 (0.24-0.96), respectively. CONCLUSIONS This study provides strong support that a genetic variant of REN C-5312T and diabetes contribute to the effects of ARBs and are independent predictors for responder. Thus, in treatment of hypertension with ARBs, a new possibility for personalized medicine has been shown.

A proximal direct repeat motif characterized as a negative regulatory element in the human renin gene.

The regulation of renin gene expression is thought to be fundamental to regulation of the total renin-angiotensin system. The human renin gene contains a direct repeat (DR) motif AGGGGTCAC-AGGGCCA in the proximal region (-259/-245 bp), which contains similar sequence for nuclear receptor superfamily binding core motif, AGGTCA, and is the most similar to COUP-TFII consensus. The DR motif was evaluated as a functional cis-element with renal cortex and chorio-decidual cells by footprint assay, electromobility shift assay (EMSA) and reporter assay. The DR motif site was protected by footprint analysis with a clear hypersensitive and a minor hypersensitive region in good accordance with the DR of the consensus. One of the binding proteins was strongly suspected to be COUP-TFII-consensus-specific by EMSA. The DNA/protein complexes obtained with nuclear extract of renin producing cells could be completely blocked by homologous competitor and strongly blocked by the second-half mutant oligonucleotide of the DR motif but not by the first-half mutant oligonucleotide. Finally, the transcriptional activity of second-half mutant construct is slightly elevated and that first-half mutant construct is significantly stronger by twofold compared with wild type construct in reporter assay. These findings suggest that the DR motif site of the human renin gene functions as a negative regulatory element involved in a twofold repression of transcription and that member(s) of nucleic receptor superfamily bind the site and play important roles in the human renin gene expression with a possibility that one of the binding protein is COUP-TFII.

Synergistic expression of angiotensin-converting enzyme (ACE) and ACE2 in human renal tissue and confounding effects of hypertension on the ACE to ACE2 ratio.

Angiotensin-converting enzyme (ACE) 2, a newly emerging component of the renin-angiotensin system, is presumed to be a counterregulator against ACE in generating and degrading angiotensin II. It remains to be elucidated how mRNA levels of these two genes are quantitatively regulated in the kidney and also what kind of clinicopathological characteristics could influence the gene expressions in humans. Seventy-eight cases of biopsy-proven renal conditions were examined in detail. Total RNA from a small part of each renal cortical biopsy specimen was reverse transcribed, and the resultant cDNA was amplified for ACE, ACE2, and glyceraldehyde-3-phosphate dehydrogenase with a real-time PCR system. Then we investigated the relationship between clinicopathological variables and mRNA levels adjusted for glyceraldehyde-3-phosphate dehydrogenase. Statistically significant correlation was not observed between any clinicopathological variables and either of the gene expressions by pairwise comparison. However, a strong correlation was observed between the gene expressions of ACE and those of ACE2. Moreover, the ACE to ACE2 ratio was significantly higher in subjects with hypertension (HT) than that in subjects without HT. Whereas parameters of renal function, e.g. urinary protein excretion (UPE) and creatinine clearance (Ccr), are not significantly related to the ACE to ACE2 ratio as a whole, the HT status may reflect disease-induced deterioration of renal function. That is, UPE and Ccr of subjects with HT are significantly different from those without HT, in which a significant correlation is also observed between UPE and Ccr. Finally, stepwise regression analysis further revealed that only the HT status is an independent confounding determinant of the ACE to ACE2 ratio among the variables tested. Our data suggest that ACE2 might play an important role in maintaining a balanced status of local renin-angiotensin system synergistically with ACE by counterregulatory effects confounded by the presence of hypertension. Thus, ACE2 may exert pivotal effects on cardiovascular and renal conditions.

Tissue gene expression of renin-angiotensin system in human type 2 diabetic nephropathy.

OBJECTIVE: Recent studies have proved that blockade of the renin-angiotensin system (RAS) retards the progression of diabetic nephropathy, whereas hyporeninemia is known as a typical state in diabetic subjects. The purpose of this study is to determine whether expression levels of RAS differ between nondiabetic and diabetic renal tissues with accurate quantitative method. RESEARCH DESIGN AND METHODS: Subjects were 66 nondiabetic and 8 diabetic patients with biopsy-proven renal diseases. The eight diabetic subjects suffered from type 2 diabetes with overt proteinuria. Renal histology revealed typical diffuse or nodular lesions with linear IgG deposit on immunofluorescent staining and thickened basement membrane on electronic microscopy. Total RNA from a small part of the renal cortical biopsy specimens was reverse-transcribed, and the resultant cDNA was amplified for new major components of RAS (i.e., renin, renin receptor, angiotensinogen, ACE, ACE2, angiotensin II type 1 receptor, and angiotensin II type 2 receptor) and measured. RESULTS: Among these components, a significant upregulation was observed in the ACE gene in diabetic renal tissue. CONCLUSIONS: The results suggest that renal tissue RAS might be activated in the respect that ACE gene expression is upregulated in spite of a tendency to low renin expression in type 2 diabetic nephropathy.

Candidate cis-elements for human renin gene expression in the promoter region.

The regulation of renin gene expression, the rate-limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis-elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A-F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP-TFII (ARP-1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis-elements were conducted to a consensus-specific binding assay to compare renin-producing and non-renin-producing cells by EMSA and electromobility super-shift assay. Different sequence-specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP-TFII (ARP-1) motif like site and possibly with footprint F site. The results implicate these putative cis-elements and each corresponding trans-factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression.