Calcium Channel Blocker Enhances Beneficial Effects of an Angiotensin II AT₁ Receptor Blocker against Cerebrovascular-Renal Injury in type 2 Diabetic Mice

<div><p>Recent clinical trials have demonstrated that combination therapy with renin-angiotensin system inhibitors plus calcium channel blockers (CCBs) elicits beneficial effects on cardiovascular and renal events in hypertensive patients with high cardiovascular risks. In the present study, we hypothesized that CCB enhances the protective effects of an angiotensin II type 1 receptor blocker (ARB) against diabetic cerebrovascular-renal injury. Saline-drinking type 2 diabetic KK-A^y mice developed hypertension and exhibited impaired cognitive function, blood-brain barrier (BBB) disruption, albuminuria, glomerular sclerosis and podocyte injury. These brain and renal injuries were associated with increased gene expression of NADPH oxidase components, NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Treatment with the ARB, olmesartan (10 mg/kg/day) reduced blood pressure in saline-drinking KK-A^y mice and attenuated cognitive decline, BBB disruption, glomerular injury and albuminuria, which were associated with a reduction of NADPH oxidase activity and oxidative stress in brain and kidney tissues as well as systemic oxidative stress. Furthermore, a suppressive dose of azelnidipine (3 mg/kg/day) exaggerated these beneficial effects of olmesartan. These data support the hypothesis that a CCB enhances ARB-associated cerebrovascular-renal protective effects through suppression of NADPH oxidase-dependent oxidative stress in type 2 diabetes.</p> </div

Evaluation of cognitive function, blood-brain barrier (BBB) leakage and mRNA expression of tight junction (TJ) associated proteins by passive avoidance test, Evans blue (EB) dye extravasation and by real-time RT-PCR (reverse transcription-polymerase chain reaction) analyses, respectively.

<p><b>A</b>, Saline-drinking KK-A^y mice showed cognitive impairment, which was markedly improved by olmesartan. However, the combination of olmesartan plus azelnidipine prevented cognitive impairment (n=11 in each group). <b>B</b>, Representative macroscopic image of the pattern of EB dye extravasation (dark blue areas). The background was modified to black without any editing to clarify the picture. <b>C</b>, Quantification of EB dye concentration in whole brain tissue (n=6 in each group). Saline-drinking KK-A^y mice showed widespread EB dye extravasation in brain tissue. Olmesartan treatment showed a marked reduction in the EB dye concentration. However, the combination of olmesartan plus azelnidipine completely inhibited widespread EB dye extravasation in brain tissue. <b>D</b>–<b>F</b>, Saline-drinking KK-A^y mice showed downregulated mRNA expression of TJ associated proteins such as cludin-5, occludin, and zona occludin-1 (ZO-1) (n=8 in each group). Olmesartan markedly prevented the downregulated mRNA expression of TJ associated proteins. However, the combination of olmesartan plus azelnidipine completely prevented this downregulated mRNA expression of TJ associated proteins to levels similar to those in C57BL6 mice. <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl, ^e<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl + olmesartan.</p

NADPH oxidase-dependent superoxide anion production in brain tissues were evaluated by dihydroethidium (DHE) immunofluorescence staining.

<p><b>A</b>, Representative images of DHE immunofluorescence staining (original magnification, ×200). <b>B</b>, DHE fluorescence intensity (n=8 in each group). <b>C</b>, NADPH oxidase activity in homogenized brain tissues evaluated by lucigenin-enhanced chemiluminescence (n=6 in each group). Saline-dinking KK-A^y mice showed superoxide production in brain tissues and were associated with increased NADPH oxidase activity. Treatment with olmesartan markedly attenuated this superoxide production and suppresses NADPH oxidase activity. Furthermore, the combination of olmesartan plus azelnidipine completely ameliorated this NADPH oxidase-dependent superoxide production to a level similar to that in C57BL6 mice. <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl, ^e<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl + olmesartan.</p

Albuminuria and glomerular podocyte injury.

<p><b>A</b>, KK-A^y mice showed albuminuria, which was exacerbated by further increased saline intake (n=11 in each group). <b>B</b>, Glomerular podocyte injury was detected by desmin immunostaining. Representative desmin-stained images (scale bar shows the values), and <b>C</b>, the desmin-positive area as a percentage of the total glomerular area. KK-A^y mice showed larger desmin-positive areas (brown staining) in the glomeruli, which were further increased by saline intake. Olmesartan markedly prevented these changes. However, the combination of olmesartan plus azelnidipine almost completely abrogated albuminuria and completely prevented glomerular podocyte injury. <b>D</b>, Glomerular sclerosis was evaluated by examining periodic acid-Schiff (PAS) staining. Representative micrographs of PAS-stained renal sections (scale bar shows the values), and <b>E</b>, the PAS-positive area within the total glomerular area. KK-Ay mice exhibit severe glomerular sclerosis, which was further exacerbated by saline intake. Olmesartan markedly prevented glomerular sclerosis. However, the combination of olmesartan plus azelnidipine exhibited greater protective efficacy against glomerular sclerosis (n=7 in each group). <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl, ^e<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl + olmesartan.</p

NADPH oxidase-dependent superoxide anion production in kidney tissues.

<p><b>A</b>, Representative images of dihydroethidium (DHE) staining (original magnification, ×100). <b>B</b>, DHE fluorescence intensity (n=8 in each group). <b>C</b>, NADPH oxidase activity in homogenized renal cortical tissues (n=6 in each group). KK-A^y mice showed increased superoxide production in kidney tissues and were associated with increased NADPH oxidase activity, which were further increased by saline intake. Olmesartan markedly prevented superoxide production and NADPH oxidase activity in kidney tissues. Interestingly, the combination of olmesartan plus azelnidipine completely prevented NADPH oxidase-dependent superoxide production in kidney tissues. <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl, ^e<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl + olmesartan.</p

Systemic oxidative stress was evaluated by measuring plasma and urine 8-hydroxy-2’-deoxyguanosine (8-OHdG).

<p><b>A</b>, KK-A^y mice showed elevated plasma 8-OHdG, and <b>B</b>, increased urinary excretion of 8-OHdG, which were exacerbated by further increased saline intake (n=6 in each group). Treatment with olmesartan suppresses the elevated plasma and urinary excretion of 8-OHdG. Furthermore, the combination of olmesartan plus azelnidipine exhibited greater suppressive efficacy (n=8 in each group). <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl, ^e<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl + olmesartan.</p

Systolic blood pressure (SBP) measured by tail-cuff plethysmography and postprandial blood glucose (PPBG) profiles.

<p><b>A</b>, Saline-drinking KK-A^y mice showed hypertension, which was attenuated to similar levels by olmesartan, and the combination of olmesartan plus azelnidipine. <b>B</b>, None of the treatments significantly altered PPBG levels (n=11 in each group). <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl.</p

Renal tubulointerstitial fibrosis was detected by Azan staining.

<p><b>A</b>, Representative micrographs of Azan-stained renal sections (scale bar shows the values), and <b>B</b>, the Azan-positive area. <b>C</b>, Gene expression of α-smooth muscle actin (α-SMA), and <b>D</b>, type 1 collagen. KK-A^y mice exhibited increased Azan-positive area (blue staining) in the tubulointerstitium and profibrotic gene expression in renal cortical tissues, which were further exacerbated by saline intake. Olmesartan markedly prevented these changes. However, the combination of olmesartan plus azelnidipine exhibited greater protective efficacy (n=7 in each group). <b><i>^a</i></b><i>P</i> < 0.05 vs. C57BL/6, ^b<i>P</i> < 0.05 vs. C57BL/6 + 0.9% NaCl, ^c<i>P</i> < 0.05 vs. KK-A^y, ^d<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl, ^e<i>P</i> < 0.05 vs. KK-A^y + 0.9% NaCl + olmesartan.</p