Atsuhiro Ichihara

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.

The (Pro)renin Receptor/ATP6AP2 is Essential for Vacuolar H+-ATPase Assembly in Murine Cardiomyocytes

Rationale: The (pro)renin receptor [(P)RR], encoded in ATP6AP2, plays a key role in the activation of local renin-angiotensin system (RAS). A truncated form of (P)RR, termed M8.9, was also found to be associated with the vacuolar H+-ATPase (V-ATPase), implicating a non–RAS-related function of ATP6AP2. Objective: We investigated the role of (P)RR/ATP6AP2 in murine cardiomyocytes. Methods and Results: Cardiomyocyte-specific ablation of Atp6ap2 resulted in lethal heart failure; the cardiomyocytes contained RAB7- and lysosomal-associated membrane protein 2 (LAMP2)-positive multivesicular vacuoles, especially in the perinuclear regions. The myofibrils and mitochondria remained at the cell periphery. Cardiomyocyte death was accompanied by numerous autophagic vacuoles that contained undigested cellular constituents, as a result of impaired autophagic degradation. Notably, ablation of Atp6ap2 selectively suppressed expression of the VO subunits of V-ATPase, resulting in deacidification of the intracellular vesicles. Furthermore, the inhibition of intracellular acidification by treatment with bafilomycin A1 or chloroquine reproduced the phenotype observed for the (P)RR/ATP6AP2-deficient cardiomyocytes. Conclusions: Genetic ablation of Atp6ap2 created a loss-of-function model for V-ATPase. The gene product of ATP6AP2 is considered to act as in 2 ways: (1) as (P)RR, exerting a RAS-related function; and (2) as the V-ATPase-associated protein, exerting a non–RAS-related function that is essential for cell survival.

Neuronal nitric oxide synthase modulates rat renal microvascular function

This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 +/- 0.2 microns) and efferent (18.5 +/- 0.2 microns) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-L-thiocitrulline (L-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses to L-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N omega-nitro-L-arginine (L-NNA) were only attenuated by papillectomy. L-SMTC (10 microM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, L-NNA (100 microM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary micro-vasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 ± 0.2 μm) and efferent (18.5 ± 0.2 μm) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-l-thiocitrulline (l-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses tol-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N ω-nitro-l-arginine (l-NNA) were only attenuated by papillectomy.l-SMTC (10 μM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, l-NNA (100 μM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.

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.

Prorenin Receptor Is Essential for Normal Podocyte Structure and Function

The prorenin receptor is an accessory subunit of the vacuolar H(+)-ATPase, suggesting that it has fundamental functions beyond activation of the local renin-angiotensin system. Podocytes express the prorenin receptor, but its function in these cells is unknown. Here, podocyte-specific, conditional, prorenin receptor-knockout mice died of kidney failure and severe proteinuria within 4 weeks of birth. The podocytes of these mice exhibited foot process effacement with reduced and altered localization of the slit-diaphragm proteins nephrin and podocin. Furthermore, the podocytes contained numerous autophagic vacuoles, confirmed by enhanced accumulation of microtubule-associated protein 1 light chain 3-positive intracellular vesicles. Ablation of the prorenin receptor selectively suppressed expression of the V(0) c-subunit of the vacuolar H(+)-ATPase in podocytes, resulting in deacidification of intracellular vesicles. In conclusion, the prorenin receptor is important for the maintenance of normal podocyte structure and function.

(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.

(Pro)renin Receptor–Mediated Signal Transduction and Tissue Renin-Angiotensin System Contribute to Diabetes-Induced Retinal Inflammation

OBJECTIVE The term “receptor-associated prorenin system” (RAPS) refers to the pathogenic mechanisms whereby prorenin binding to its receptor dually activates the tissue renin-angiotensin system (RAS) and RAS-independent intracellular signaling via the receptor. The aim of the present study was to define the association of the RAPS with diabetes-induced retinal inflammation. RESEARCH DESIGN AND METHODS Long-Evans rats, C57BL/6 mice, and angiotensin II type 1 receptor (AT1-R)-deficient mice with streptozotocin-induced diabetes were treated with (pro)renin receptor blocker (PRRB). Retinal mRNA expression of prorenin and the (pro)renin receptor was examined by quantitative RT-PCR. Leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion–labeling technique. Retinal protein levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 were examined by ELISA. Retinal extracellular signal–regulated kinase (ERK) activation was analyzed by Western blotting. RESULTS Induction of diabetes led to significant increase in retinal expression of prorenin but not the (pro)renin receptor. Retinal adherent leukocytes were significantly suppressed with PRRB. Administration of PRRB inhibited diabetes-induced retinal expression of VEGF and ICAM-1. To clarify the role of signal transduction via the (pro)renin receptor in the diabetic retina, we used AT1-R–deficient mice in which the RAS was deactivated. Retinal adherent leukocytes in AT1-R–deficient diabetic mice were significantly suppressed with PRRB. PRRB suppressed the activation of ERK and the production of VEGF, but not ICAM-1, in AT1-R–deficient diabetic mice. CONCLUSIONS These results indicate a significant contribution of the RAPS to the pathogenesis of diabetes-induced retinal inflammation, suggesting the possibility of the (pro)renin receptor as a novel molecular target for the treatment of diabetic retinopathy.

Long-term effects of statins on arterial pressure and stiffness of hypertensives

Although lowering blood pressure (BP) reduces aortic stiffness, achieving the recommended BP goal can be difficult. Recent studies have shown that short-term use of statins can reduce BP significantly. To determine the long-term effects of statins on BP and aortic stiffness, a single-blind randomized prospective study was performed on 85 hyperlipidaemic hypertensive patients whose BP was insufficiently controlled by antihypertensive therapy. Every 3 months, aortic stiffness was assessed by measuring pulse wave velocity (PWV). Patients were randomly allocated to groups treated with pravastatin, simvastatin, fluvastatin, or a nonstatin antihyperlipidaemic drug. No significant differences in patient characteristics, kinds of antihypertensive drugs, BP, ankle brachial index, PWV, or serum lipid, creatinine, or C-reactive protein levels were found between the four groups at the start of the study. During the 12-month treatment period, PWV did not change in the pravastatin group or nonstatin group, but it was transiently reduced in the simvastatin group and significantly decreased in the fluvastatin group, even though the doses of the statins used in this study were lower than the usually prescribed dose. All four antihyperlipidaemic drugs significantly decreased serum cholesterol levels without affecting BP, ankle brachial index, or serum triglyceride levels. The C-reactive protein serum levels decreased significantly in the three statin groups but not in the nonstatin group. These results suggest that long-term use of fluvastatin by hyperlipidaemic hypertensive patients is associated with a significant reduction in aortic stiffness without any effect on BP.