Tomoko Takemitsu

Prorenin Receptor Blockade Inhibits Development of Glomerulosclerosis in Diabetic Angiotensin II Type 1a Receptor–Deficient Mice

Blockade of the renin-angiotensin system slows the progression of diabetic nephropathy but fails to abolish the development of end-stage nephropathy of diabetes. The prorenin-to-active renin ratio significantly increases in diabetes, and prorenin binding to its receptor in diabetic animal kidney induces the nephropathy without its conventional proteolytic activation, suggesting that angiotensin II (AngII) may not be the decisive factor causing the nephropathy. For identification of an AngII-independent mechanism, diabetes was induced in wild-type mice and AngII type 1a receptor gene-deficient mice by streptozotocin treatment, and their development and progression of diabetic nephropathy were assessed. In addition, prolonged inhibition of angiotensin-converting enzyme and prolonged prorenin receptor blockade were compared for their efficacy in preventing the nephropathy that occurred in diabetic AngII type 1a receptor gene-deficient mice. Only the prorenin receptor blockade with a short peptide of prorenin practically abolished the increased mitogen-activated protein kinase (MAPK) activation and nephropathy despite unaltered increase in AngII in diabetic kidney. These results indicate that the MAPK activation signal leads to the diabetic nephropathy but not other renin-angiotensin system-activated mechanisms in the glomeruli. It is not only AngII but also intraglomerular activation of MAPK by the receptor-associated prorenin that plays a pivotal role in diabetic nephropathy.

Nonproteolytic Activation of Prorenin Contributes to Development of Cardiac Fibrosis in Genetic Hypertension

In contrast to proteolytic activation of inactive prorenin by cleavage of the N-terminal 43 residue peptide, we found that prorenin is activated without proteolysis by binding of the prorenin receptor to the pentameric “handle region” I11PLLKK15P. We hypothesized that such activation occurs in hypertensive rats and causes cardiac renin–angiotensin system (RAS) activation and end-organ damage. To test this hypothesis, we devised methods of specifically inhibiting nonproteolytic activation by decapeptide spanning the pentameric handle region peptide as a decoy. In stroke-prone spontaneously hypertensive rats (SHRsp) fed a high-salt diet, arterial pressure started to rise significantly with a marked increase in the cardiac prorenin receptor mRNA level at 8 weeks of age, and cardiac fibrosis had developed by 12 weeks of age. By immunohistochemistry using antibodies to the active site of the renin molecule, we demonstrated increased proteolytic or nonproteolytic activation of prorenin in the heart but not in plasma of SHRsp. Continuous subcutaneous administration of the handle region peptide completely inhibited the increased staining by antibodies to the active site of the renin molecule, indicating the increased nonproteolytic but not proteolytic activation of prorenin in the heart of SHRsp. Administration of the handle region peptide also inactivated tissue RAS without affecting circulating RAS or arterial pressure and significantly attenuated the development and progression of cardiac fibrosis. These results clearly demonstrate the significant role of nonproteolytically activated tissue prorenin in tissue RAS activation leading to cardiac fibrosis and significant inhibition of the cardiac damage produced by chronic infusion of the handle region peptide.

Slowly Progressive, Angiotensin II–Independent Glomerulosclerosis in Human (Pro)renin Receptor–Transgenic Rats

For defining the pathogenic effects of the (pro)renin receptor-transgenic rat, strains that overexpressed the human receptor were generated. Although transgenic rats were normotensive and euglycemic and had a renal angiotensin II (AngII) level that was comparable to that of wild-type rats, transgenic rats developed proteinuria with aging and significant glomerulosclerosis at 28 wk of age. In kidneys of 28-wk-old transgenic rats, mitogen-activated protein kinases (MAPK) were activated without recognizable tyrosine phosphorylation of the EGF receptor, and expression of TGF-beta1 was enhanced. In vivo infusion of the (pro)renin receptor blocker peptide (formerly handle region decoy peptide) significantly inhibited the development of glomerulosclerosis, proteinuria, MAPK activation, and TGF-beta1 expression in the kidneys, but the angiotensin-converting enzyme inhibitor did not attenuate these changes despite a significant decrease in the renal AngII level. In addition, recombinant rat prorenin stimulated MAPK activation in the human receptor-expressed cultured cells, but human receptor was unable to evoke the enzyme activity of rat prorenin. Thus, human (pro)renin receptor elicits slowly progressive nephropathy by AngII-independent MAPK activation in rats. This study clearly provided in vivo evidence for the AngII-independent MAPK activation by human (pro)renin receptor and induction of glomerulosclerosis with increased TGF-beta1 expression.

Regression of Nephropathy Developed in Diabetes by (Pro)renin Receptor Blockade

Activation of prorenin by (pro)renin receptor stimulates the tissue renin-angiotensin system and plays a significant role in the development of nephropathy in diabetic animals. This study examined whether (pro)renin receptor blockade inhibits the progression of nephropathy that has already developed in diabetic rats. Seventeen-week-old heminephrectomized streptozotocin-induced diabetic rats with an increased urinary protein excretion and a significant glomerulosclerosis had been treated for 12 wk with the (pro)renin receptor blocker (PRRB), angiotensin-converting enzyme inhibitor (ACEi), or vehicle peptide by using subcutaneously implanted osmotic minipumps. At the end of observation, in diabetic rats that were treated with vehicle, urinary protein excretion was progressively increased and a significant progression of glomerulosclerosis was observed. In diabetic rats that were treated with PRRB, however, no further increase in urinary protein excretion or glomerulosclerosis was observed, but 12-wk treatment with ACEi only attenuated further increases in urinary protein excretion and glomerulosclerosis. The enhanced expression of activated prorenin was observed in the kidneys of diabetic rats that were treated with vehicle, whereas it was markedly suppressed in the kidneys of diabetic rats that were treated with PRRB but not ACEi. These results suggest that (pro)renin receptor blockade does not only inhibit the progression of nephropathy but also reverses the glomerulosclerosis that has already developed in diabetic rats.

(Pro)Renin Receptor–Mediated Activation of Mitogen-Activated Protein Kinases in Human Vascular Smooth Muscle Cells

Blockade of (pro)renin receptor has benefits in diabetic angiotensin II type-1a-receptor–deficient mice, suggesting the importance of (pro)renin receptor–mediated intracellular signals. To determine the mechanism whereby the human (pro)renin receptor activates mitogen-activated protein kinases in human vascular smooth muscle cells (hVSMC), we treated the cells with recombinant human prorenin. Prorenin enhanced hVSMC proliferation and activated extracellular-signal–related protein kinase (ERK) in a dose- and time-dependent manner but did not influence activation of p38 or c-Jun NH2-terminal kinase. The activated ERK level was reduced to the control level by the tyrosine kinase inhibitor genistein, and the MEK inhibitor U0126 markedly reduced the activated ERK level to the control level, whereas the level of activated ERK was unaffected by the angiotensin-converting enzyme inhibitor imidaprilat or the angiotensin II receptor blocker candesartan. A human (pro)renin receptor was present in hVSMCs, and its knockdown with small interfering RNA (siRNA) significantly inhibited the prorenin-induced ERK activation. These results suggest that prorenin stimulates ERK phosphorylation in hVSMCs through the receptor-mediated activation of tyrosine kinase and subsequently MEK, independently of the generation of angiotensin II or the activation of its receptor. The (pro)renin receptor–mediated ERK signal transduction is thus a possible new therapeutic target for preventing vascular complications.

Effects of amlodipine and valsartan on vascular damage and ambulatory blood pressure in untreated hypertensive patients

The present study was performed to compare the long-term effects of 24-h ambulatory blood pressure (BP) control with amlodipine versus valsartan on vascular damage in untreated hypertensive patients. Amlodipine and valsartan have benefits on cardiovascular mortality and morbidity in hypertensive patients. Although ambulatory BP is associated with severity of target-organ damage in hypertensive patients, beneficial effects of ambulatory BP control with amlodipine versus valsartan on vascular damage have not been compared. Pulse wave velocity (PWV), intima–media thickness (IMT) of the carotid arteries, urinary albumin excretion (UAE) and 24-h ambulatory BP were determined in 100 untreated hypertensive patients before and 12 months after the start of antihypertensive therapy with amlodipine or valsartan. Amlodipine and valsartan decreased ambulatory BP similarly, but the variability of 24-h and daytime ambulatory systolic BP was significantly reduced by amlodipine but not by valsartan. The reduced variability of ambulatory systolic BP caused by amlodipine significantly contributed to the improvement of PWV, although both drugs decreased PWV similarly. Carotid IMT was unaffected by treatment with either drug. Valsartan significantly decreased UAE independently of its depressor effect, but amlodipine had no effect on UAE. These results suggest that the 24-h control of ambulatory BP with amlodipine had functionally improved the stiffened arteries of hypertensive patients by the end of 12 months of treatment, in part through reducing BP variability, whereas ambulatory BP control with valsartan decreased the arterial stiffness to the same degree as amlodipine without affecting BP variability maybe through some pleiotropic effects.

Cardio-Ankle Vascular Index and Ankle Pulse Wave Velocity as a Marker of Arterial Fibrosis in Kidney Failure Treated by Hemodialysis

BACKGROUND Patients with kidney failure treated with hemodialysis have a high incidence of cardiovascular diseases caused by accelerated arteriosclerosis. However, accurate evaluation of the extent of arteriosclerosis is difficult. This study sought to compare the strength of predictions of arterial fibrosis by using a new parameter, the cardio-ankle vascular index (CAVI), versus pulse wave velocity (PWV) in patients with kidney failure treated with hemodialysis. STUDY DESIGN Diagnostic test study. SETTING & PARTICIPANTS 103 patients with kidney failure undergoing surgical construction of an arteriovenous access for hemodialysis therapy. INDEX TEST CAVI and PWV. REFERENCE TEST Arterial fibrosis, evaluated by using Masson trichrome stain on part of the brachial artery obtained during surgery, expressed as percentage of fibrosis of the layer of vascular smooth muscle cells. RESULTS Median percentage of arterial stiffness was 52.85%. Mean PWV and CAVI were 18.3 +/- 5.6 (SD) m/s and 9.9 +/- 2.6, respectively. Multivariate regression analysis showed that arterial fibrosis was significantly associated with older age (0.247%/y; 95% confidence interval, 0.013 to 0.482) and CAVI (6.117%/unit; 95% confidence interval, 4.764 to 4.740), but not with systolic blood pressure (0.039%/mm Hg; 95% confidence interval, -0.076 to 0.153) or PWV (-0.044%/m/s; 95% confidence interval, -0.646 to 0.558). The area under the receiver operating characteristic curve to predict greater than median percentage of arterial stiffness was 0.892 for CAVI and 0.779 for PWV (P = 0.006). LIMITATION It is unclear whether arterial fibrosis of the brachial artery evaluated by using CAVI is applicable for arteriosclerosis of other arterial districts, and clinical outcomes were not evaluated in this study. CONCLUSION CAVI reflects the histological arterial fibrosis of hemodialysis patients and is a useful clinical marker for evaluating arterial stiffness in these patients.

Add-on amlodipine improves arterial function and structure in hypertensive patients treated with an angiotensin receptor blocker.

The present study was designed to determine whether adding amlodipine further improved functional and structural cardiovascular damage in hypertensive patients whose blood pressure was already well controlled with an angiotensin II type 1 receptor blocker (ARB). The cardiothoracic ratio on chest radiographs, level of urinary albumin excretion, pulse wave velocity (PWV), intima-media thickness (IMT) of the carotid arteries, and 24 hour ambulatory blood pressure (BP) were evaluated before and 12 months after the start of add-on of amlodipine or placebo in 50 hypertensive patients being treated with an ARB. The add-on amlodipine therapy significantly improved the PWV from 1689 ± 61 to 1447 ± 47 cm/s and the IMT from 0.88 ± 0.08 to 0.75 ± 0.06 mm in the hypertensive patients treated with an ARB without altering their mean 24 hour ambulatory BP values, but did not alter the cardiothoracic ratio or urinary albumin excretion. Amlodipine also significantly decreased the variability of ambulatory BP, but the decrease did not significantly contribute to the changes in PWV or IMT. Thus, the add-on low-dose amlodipine therapy had benefits in terms of the vascular function and vascular structure of hypertensive patients treated with an ARB that were independent of its depressor effects. The antiatherogenic pleiotropic properties of amlodipine have a preventive effect on the progression of arterial stiffness in hypertensive patients treated with an ARB.

Ambulatory blood pressure variability and brachial-ankle pulse wave velocity in untreated hypertensive patients.

Blood pressure (BP) variability is estimated as the standard deviation of 24-h ambulatory BP. The present study was performed to determine the effect of the mean 24-h ambulatory BP values and standard deviations on arterial wall stiffness assessed by brachial–ankle pulse wave velocity (baPWV). Brachial–ankle pulse wave velocity, carotid intima–media thickness (IMT), urinary albumin excretion (UAE) and 24-h ambulatory BP were measured before the start of antihypertensive therapy in 203 newly diagnosed hypertensive patients (53.3±0.7 years old; clinic systolic/diastolic BP: 154±1/98±1 mm Hg), and univariate and multivariate regression analyses of these clinical and biological parameters were performed. Univariate regression analyses revealed a significant association between mean baPWV values and the standard deviations of ambulatory systolic/diastolic BP. Mean ambulatory systolic/diastolic BP values were also associated with UAE, and the standard deviations of ambulatory systolic BP were associated with maximum carotid IMT. Quintile analyses showed that patients with a mean 24-h ambulatory mean BP value and standard deviation below 110 and 20 mm Hg, respectively, had the lowest baPWV. Moreover, the multivariate regression analyses confirmed a significant correlation between baPWV and the standard deviation of 24-h ambulatory systolic BP. In conclusion, untreated hypertensive patients with a higher 24-h ambulatory systolic BP variability had stiffer arterial walls. Ambulatory systolic BP variability may be involved in stiffening of the arteries of hypertensive patients.

Benefits of candesartan on arterial and renal damage of non-diabetic hypertensive patients treated with calcium channel blockers.

Background/Aim: Although long-term, intensive blood pressure (BP) control with calcium channel blockers (CCBs) reduced arterial stiffness and renal damage of hypertensive patients, combination therapy with antihypertensive drugs is frequently needed to maintain the intensive BP control. The present study was conducted to examine add-on benefits of candesartan therapy on hypertensive patients treated with CCBs for at least 12 months. Methods: Pulse wave velocity (PWV), urinary albumin excretion (UAE), intima-media thickness (IMT) of the carotid arteries, and 24-hour ambulatory BP were determined in 50 non-diabetic hypertensive patients treated with CCBs before and 12 months after the start of therapy with candesartan or placebo. Results: Candesartan significantly decreased clinic BP and tended to decrease ambulatory BP, but the decreases were similar to those in the placebo group except nocturnal BP decrease, which was significantly enhanced by candesartan. Add-on candesartan significantly decreased PWV and UAE compared to placebo, but IMT was unchanged with candesartan or placebo. The decrease in clinic BP or nocturnal BP decrease did not contribute to the improvement of PWV or UAE. Conclusion: Add-on candesartan functionally improved the stiffened arteries of hypertensive patients treated with CCBs by the end of 12 months of treatment independently of its effects on BPs.