Yasukazu Makino

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 catecholamines 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.

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.

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. J. Cell. Biochem. 102: 1043–1050, 2007.

A CROSS-OVER COMPARISON OF ANTI-ALBUMINURIC EFFECTS AMONG 4 TYPES CALCIUM CHANNEL BLOCKERS ON CHRONIC KIDNEY DISEASE: PP.33.310

Background and Objectives: At the intervention for hypertension with chronic kidney disease (CKD), albuminuria is one of the pivotal targets as well as strict blood pressure (BP) control for organ protection. Calcium channel blocker (CCB) is one of the most expected agents for CKD. Currently CCBs have been classified by half-life, drug delivery system and channel types. We tested anti-albuminuric effect and humoral factors level of 4 types of CCBs in CKD. Methods: Subjects were 50 hypertensives with CKD (male/female 22/28, age 69.8±10.8yrs, SBP/DBP 164.7±17.1/92.3±12.2 mmHg, s-Cr 0.81±0.37 mg/dl, urinary albumin excretion (UAE) 69.4 (33.5–142.6) mg/gCr). CCBs were administered for 12 weeks in a crossover manner. Tested agents were nifedipine CR, a long biological half-life L type CCB with controlled release system, cilnidipine, an N/L type CCB, efonidipine, a T/L type CCB and amlodipine, a long biological half-life L type CCB with trans-membrane approach. Results: Comparable BP reductions were obtained. UAE at endpoints ware as follow (mg/gCr, *P < 0.05): 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 for the first time that, although all tested drugs reduced UAE, only nifedipine CR and cilnidipine could reach statistical difference. Thus, it is suggested that, in respect of albuminuria reduction, the 2 CCBs might be favorable for organ protection in CKD.

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.

AbstractThe 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.

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.

ASSOCIATION STUDY OF URIC ACID TRANSPORTER GLUT9 GENOTYPE WITH THE RENIN-ANGIOTENSIN SYSTEM

Objective: Uric acid is thought to be one of risk factors for atherosclerotic disorders. The mechanisms have been thought to include endothelial dysfunction, inflammation, oxidative stress and the renin angiotensin system (RAS) activation. It is known that the RAS plays a pivotal role in the atherosclerotic disorders. However, the evidence that uric acid is involved in the activation is not sufficient. We therefore tested the hypothesis that a genetic variant of a uric acid transporter, glucose transporter 9 (GLUT9) could show significant association with prevalence of hyper-reninemic state. Design and method: We enrolled consecutive 804 subjects who had consulted our hospitals for life style related diseases (statistic power 80%, significance level 0.05). We defined the subjects with plasma renin activity (PRA) equal with or more than 0.70 mg/ml/hr. as hyper-reninemic group as cases of the study and the subjects with PRA less than 0.70 mg/ml/hr as normo-reninemic group as controls. Genomic DNA was isolated from human leukocytes. Genotypes were assayed with genomic DNA for a C/T variant of GLUT9 (rs1014290) using real-time PCR system by TaqMan method. Association between the genetic variant and the prevalence of hyper-reninemic state was tested. Results: They consisted of cases (51.0%) and controls (49.0%). The serum uric acid (mg/dl) with each genotype of GLUT9 were as follows: CC (127 cases) 5.04 ± 1.45, CT (392 cases) 5.08 ± 1.49, TT (286 cases) 5.44 ± 1.45 (CC vs CT, p = 0.77; CC vs TT, p = 0.011; CT vs TT, p = 0.002). The numbers of individuals with each genotype were as follows (CC, CT and TT): 52, 204 and 154 for cases and 75, 187 and 132 for controls. Accordingly, the risk for hyper-reninemic state was 1.24 (95% confidence interval; 1.02–1.52), p = 0.033 for allelic comparison and also p = 0.032 for Armitages trend test, the T allele being the risk allele. Conclusions: The uric acid concentration is associated with a genetic variant of transporter GLUT9 and the subjects with genetic variant of high uric acid have hyper-reninemic constitution. Thus, from a view point of Mendelian randomization theory, it is found that high uric acid state may have a significant impact on the RAS activation.

GENETIC VARIATION IN THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM AND RENAL SURVIVAL IN JAPANESE PATIENTS WITH DIABETIC NEPHROPATHY

Objective: Genetic polymorphisms of the renin-angiotensin-aldosterone system (RAAS) may play a pivotal role in the development and progression of diabetic nephropathy. Therefore, we aimed to investigate whether genetic variation in the RAAS is related to renal survival in patients with diabetic nephropathy. Design and method: We enrolled 246 Japanese patients with diabetic nephropathy who consulted nephrologists in our hospital between January 1995 and December 2010. One hundred and fifty-three (62.2%) patients progressed to end stage renal disease (ESRD) at an average age of 63.8 years. We estimated the association between 5 polymorphisms of RAAS and cumulative renal survival. We investigated the following genetic polymorphisms: renin enhancer region (REN) C-5312T, angiotensinogen M235T, angiotensin converting enzyme (ACE) insertion/deletion, angiotensin II type I receptor A1166C, and aldosterone synthase CYP 11B2 C-344T. We used cumulative survival analysis using the Kaplan–Meier method with the log-rank test for statistical analysis of the time course of progression to ESRD. Results: Cumulative renal survival in diabetic nephropathy was significantly lower in those with the DD/DI genotype of the ACE I/D polymorphism (log-rank, P = 0.038; X2 = 4.298). There was no association between cumulative survival and the REN C-5312T, M235T, A1166C, and C-344T polymorphisms. Conclusions: The ACE I/D polymorphism may play a role in diabetic nephropathy progression in that it may directly affect prognosis in these patients.