Effects of L-/N-Type Calcium Channel Blockers on Angiotensin II–Renin Feedback in Hypertensive Patients

Objectives Cilnidipine, an L-/N-type calcium channel blocker (CCB), has unique organ-protective properties due to suppression of hyperactivity in the sympathetic nervous system and renin-angiotensin system (RAS). In this study, we hypothesized that cilnidipine might exert a renoprotective effect by suppressing the RAS. Methods A total of 25 hypertensive patients receiving a RAS inhibitor were randomly assigned to a cilnidipine (n = 12) or amlodipine (n = 13) group. The effects of cilnidipine on proteinuria and angiotensin II–renin feedback were assessed. Results After 6 months of treatment, both systolic and diastolic blood pressures were significantly reduced to a similar extent in both groups. The urine albumin-to-creatinine ratio was significantly lower in the cilnidipine group (p < 0.05) than in the amlodipine group. Amlodipine increased plasma angiotensin I and angiotensin II levels (p < 0.05), whereas cilnidipine did not. Interestingly, the cilnidipine group had a higher ratio of angiotensin-(1–7) (Ang-(1–7)) to angiotensin II in plasma than the amlodipine group (p < 0.05). Conclusions The L-/N-type CCB cilnidipine, but not amlodipine, decreased urinary albumin excretion in hypertensive patients. Cilnidipine also increased the ratio of Ang-(1–7) to angiotensin II in plasma, which might be one factor underlying its beneficial effects.


Introduction
Patients with chronic kidney disease have been shown to have a higher risk of developing cardiovascular diseases and end-stage renal failure as their blood pressure increases [1]. erefore, it is crucial to adequately control their blood pressure, initially either with angiotensin-converting enzyme inhibitors or angiotensin II (Ang II) type 1 receptor blockers (ARBs). However, in clinical practice, these drugs are often used in combination with a calcium channel blocker (CCB) to achieve a sufficient antihypertensive effect.
ere are several types of CCBs that differ according to their duration of action and the calcium channel subtype that they inhibit. e major CCBs exert a strong antihypertensive effect through vasodilation mediated by L-type Ca inhibition, but they can cause reflex stimulation of the sympathetic nervous system and renin-angiotensin system (RAS) by their abrupt hypotensive action. Cilnidipine, a dihydropyridine CCB, is an antihypertensive drug with both L-type and N-type calcium channel antagonist activities [2,3]. Since cilnidipine suppresses hyperactivity of the sympathetic nervous system, it exerts a slow and sustained antihypertensive effect without causing reflex tachycardia during hypotension.
Previous studies showed that cilnidipine demonstrates unique organ-protective properties as a result of its effects on the sympathetic nervous system [4][5][6]. In addition, we reported that cilnidipine exerts a greater renoprotective effect than amlodipine due to its antioxidative properties [7]. Furthermore, previous studies demonstrated that cilnidipine is unique in that it suppresses the RAS. Some previous studies reported that cilnidipine has no effect on plasma renin activity or plasma Ang II levels, while amlodipine, an L-type CCB, significantly increases both parameters [8,9]. Another study showed that cilnidipine but not amlodipine, suppressed the ARB-induced increase in plasma renin activity and plasma Ang II levels [10].

Study Design.
In this 6-month, multicenter, prospective, randomized, open-label clinical trial named the Tokushima Antioxidation Clinical Trial in Hypertensives, Cilnidipine vs. Amlodipine (TACTICAL2) study (UMIN ID: 00006544), 25 hypertensive patients receiving ARB treatment at four hospitals in Japan between August 2013 and June 2016 were enrolled. e study was approved by the Institutional Review Board of e University of Tokushima Clinical Research Center and the review boards of the four hospitals. e study protocol was in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients included in the study. e enrollment criteria were as follows: (1) hypertension defined as a blood pressure (BP) of ≥140/90 mmHg and (2) treatment with ARBs for 3 months or more before the administration of cilnidipine or amlodipine. e exclusion criteria were as follows: (1) age <45 years or >85 years, (2) hypertensive emergency, (3) severe liver failure, (4) pregnancy, and (5) a history of severe adverse effects due to CCBs or ARBs. e overview of the study design is shown in Figure 1. Subjects were randomly assigned to two groups at the time of registration. To achieve the target BP level of 130/85 mmHg, patients received either 10 mg per day of cilnidipine, which was titrated upward to a daily dose of 20 mg, or 5 mg per day of amlodipine, which was titrated upward to a daily dose of 10 mg. e doses of ARBs were not altered during the study period. BP was measured according to the Japanese Society of Hypertension Guidelines for the Measurement of Hypertension.
During the first 3 months of the study, patients received their initial (baseline) assigned drug combination. However, if cilnidipine or amlodipine in combination with the ARB failed to reduce BP to the target level within 3 months, additional antihypertensive medications, such as diuretics, were administered until the target was reached.

Measurement of the Urinary Parameters.
Morning spot urine samples were obtained. e urinary levels of albumin and creatinine were measured using a turbidimetric immunoassay and enzyme assay, respectively (LSI Medience Corporation, Tokyo, Japan).

Measurement of RAS.
e levels of Ang I, Ang II, and Ang-(1-7) concentrations in human plasma were measured by liquid chromatography (LC)/mass spectrometry (MS)/ MS (SRL, Tokyo, Japan).

Statistical Analysis.
e data in this study are expressed as the mean ± SD. Differences between the two groups were analyzed by Student's unpaired t-test for continuous variables and the chi-square test for categorical variables. Changes before and after treatment administration were assessed by Wilcoxon's signed-rank test.

Baseline Characteristics.
e baseline characteristics of the subjects are shown in Table 1. ere were no significant differences between the two groups in terms of age, sex, systolic and diastolic BP, heart rate, HbA1c, or total cholesterol level. ere was no additional use of diuretics in either group during the study period.

Safety.
No adverse effects related to the antihypertensive treatments occurred during the study period.

Changes in BP and Heart Rate.
e systolic and diastolic BP values were significantly lower in both the cilnidipine and amlodipine groups at 6 months after the initiation of therapy than at baseline (p < 0.01). However, there were no significant differences in BP values between the two groups at either time point (shown in Figure 2). Moreover, the heart rate showed no changes over time in either group (shown in Figure 3).

Changes in Urinary Albumin.
While the urine albuminto-creatinine ratio did not change over time in the amlodipine group, it decreased significantly after 6 months of treatment in the cilnidipine group (p < 0.05; shown in Figure 4).

Changes in Plasma
Ang I and Ang II Levels. After 6 months of treatment, the plasma Ang I and Ang II levels were significantly increased in the amlodipine group (p < 0.05) but not in the cilnidipine group (shown in Figure 5). (PRA). Although neither plasma aldosterone levels nor PRA differed significantly between the two groups, there was a nonsignificant increase in both measurements from pre-to post-treatment in the amlodipine group (shown in Figure 6). (1)(2)(3)(4)(5)(6)(7) to Ang II in Plasma. Compared to the amlodipine group, the cilnidipine group had a significantly higher ratio of Ang-(1-7) to Ang II in plasma at the end of treatment (p < 0.05; shown in Figure 7).

Discussion
In this study, we evaluated whether cilnidipine might exert a renoprotective effect by suppressing the RAS. e results suggested that cilnidipine, unlike amlodipine, had a greater suppressive effect on the RAS when used in combination with a RAS inhibitor to treat hypertensive patients. In addition, the finding that cilnidipine had greater antiproteinuric effects than amlodipine was consistent with our previous study.
In this study, amlodipine increased both plasma Ang I and Ang II levels (p < 0.05), whereas cilnidipine did not. Furthermore, cilnidipine raised the ratio of Ang-(1-7) to Ang II in plasma more than amlodipine did. ese results suggest that cilnidipine might shift the balance of the 2 arms of the RAS from the ACE-Ang II-AT1R receptor to the ACE2-Ang-(1-7)-Mas receptor. According to previous studies, the predominant effects of the RAS might be related to the balance between these 2 arms [13][14][15]. is effect of cilnidipine may be partly caused by N-type calcium channel blockade via the close conjunction between the ACE2-Ang-(1-7) pathway and sympathetic nerve activity [16]. It is still unknown if Ang-(1-7) is predominantly formed from Ang II via ACE2 or from Ang-(1-9) via ACE. However, despite the similar hypotensive effects of cilnidipine and amlodipine when combined with RAS inhibitor therapy, the greater increase in the ratio of Ang-(1-7) to Ang II by cilnidipine might contribute to its superior renal and cardiovascular protective effects. is hypothesis is supported by previous reports showing that Ang-(1-7) administration to streptozotocin (STZ)-induced diabetic rats reduced proteinuria and restored vascular reactivity in isolated renal artery segments [17] and that treatment with Ang-(1-7) attenuated NAPDH oxidase activation and reduced proteinuria in spontaneously hypertensive, STZ-induced diabetic rats [18].
We previously showed that in hypertensive patients, cilnidipine decreased urinary levels of both 8-hydroxy-20deoxyguanosine (OHdG), a biomarker of oxidative stress, and liver-type fatty-acid-binding protein (L-FABP), a useful biomarker for renal tubulointerstitial injury [7]. Cilnidipine may suppress oxidative stress by modulating the renal RAS system. In practice, the increased ratio of Ang-(1-7) to Ang II might have attenuated the significant oxidative stress that contributed to renal injury in this study, because previous reports have shown that Ang-(1-7) has an antioxidative effect in vascular tissues whereas Ang II has an oxidative effect [19,20]. In agreement with these data, other studies have shown that Ang-(1-7) mediates the effects of ACE2 in suppressing Ang II-induced oxidative stress and ERK1/2 signaling in cultured cardiomyocytes and fibroblasts [21] and that ACE2 attenuates cardiac, kidney, and vascular oxidative stress in SHR [22].
We previously reported that cilnidipine, but not amlodipine, reduced urinary albumin excretion in hypertensive patients receiving RAS inhibitor treatment [7]. In addition, the Cilnidipine vs. Amlodipine Randomized Trial for Evaluation in Renal Disease (CARTER) study showed that cilnidipine was more beneficial than amlodipine in hypertensive patients [23]. Supporting these previous reports, this study showed that only cilnidipine reduced urinary albumin excretion in hypertensive patients receiving ARB treatment. A recent study reported that L/N-type CCBs exert the antialbuminuric and renoprotective effects at least in part by reducing glomerular hypertension through both efferent and afferent arteriolar vasodilation [24]. Furthermore, another study reported that in patients with diabetes, the combination of cilnidipine with a RAS inhibitor had an additive effect in suppressing urinary albumin excretion [25].
Although the present study revealed the association between the renoprotective effects of cilnidipine and the systemic RAS, it remains unknown whether cilnidipine also have an effect on the renal ACE2-Ang-(1-7)-Mas receptors which might contribute to the renoprotection. Fan Figure 1: Study design. Patients received either 10 mg per day of cilnidipine, which was increased to a daily dose of 20 mg, or 5 mg per day of amlodipine, which was increased to a daily dose of 10 mg. Doses of RAS inhibitors were not altered when the combination with amlodipine failed to reduce BP to the target level within 3 months. Instead, additional antihypertensive medications, such as diuretics, were administered to achieve the target BP. glomerular epithelial cells [26]. In addition, they showed that treatment with cilnidipine, but not amlodipine, significantly suppressed the renal Ang II levels and podocyte injury in spontaneously hypertensive rat/ND mcr-cp, suggesting that the renoprotective mechanism of cilnidipine may involve the suppression of RAS. Furthermore, Toba et al. showed that cilnidipine inhibited the renal renin-angiotensinaldosterone system, including measurements of renal ACE activity and aldosterone content in the DOCA-salt hypertensive rats [27]. Further studies are needed to clarify the

Limitations.
Our study had several limitations. First, serial measurements of urinary and RAS parameters could only be performed in a limited number of patients at 2 time points. Second, measurements related to the RAS might have fluctuated over the time and position of the patient during the blood sampling. ird, the failure to consider the severity of chronic kidney disease may have influenced the results of the study. Fourth, we did not clarify the precise mechanism whereby cilnidipine suppresses the RAS and oxidative stress. Finally, since the sample size was small in this study, we need to increase the number of samples in future evaluations.

Conclusion
In conclusion, cilnidipine and ARB combination therapy reduced urinary albumin excretion and increased the ratio of Ang-(1-7) to Ang II in plasma in hypertensive patients. Better renoprotective effects may be achieved by combining a RAS inhibitor with an L-/N-type CCB rather than an L-type CCB.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval e study was approved by the Institutional Review Board of e University of Tokushima Clinical Research Center and the review boards of the four hospitals. e study protocol was in accordance with the Declaration of Helsinki.

Consent
Written informed consent was obtained from all patients included in the study. to Ang II in plasma before and after treatment. After 6 months of treatment, the cilnidipine group had a significantly higher ratio Ang-(1-7) to Ang II in the plasma than the amlodipine group. Ang-(1-7), angiotensin; Ang II, angiotensin II. 6 International Journal of Hypertension