Tsutomu Nakagawa

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.

(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.

Nine polymorphisms of angiotensinogen gene in the susceptibility to essential hypertension

Even if the importance of angiotensinogen (AGT) gene has been known in gene targeting animals and humans genetic studies, its precise mechanism and the interaction among AGT gene variants, plasma AGT concentration and risk for hypertension remain uncertain. We examined whether AGT gene variants predispose to hypertension via an increase of plasma AGT concentration. Plasma AGT concentration was estimated from plasma angiotensin I which was cleaved by an excess amount of human renin and measured by RIA. Using 9 AGT gene variants which included new polymorphisms (G-152A and T+31C), we examined the association with hypertension and with plasma concentration by a case-control study. Haplotype analysis revealed that G-6A, T+31C and M235T polymorphisms were in absolute linkage disequilibrium and were associated with hypertension but not with plasma AGT level. On the other hand, -1074t;T235 haplotype was associated with an increase of AGT level but not with hypertension. In the haplotype analysis, only H3 haplotype frequency, which contained G-6, T+31 and M235 alleles, was significantly increased in normotensive subjects, suggesting that this haplotype is associated with a hypotensive effect. According to combined haplotype analysis of diallele and microsatellite markers, it remains a possibility that M235T, T+31C, G-6A, A-20C and G-1074T polymorphisms may play an important role in increased risk for essential hypertension. Our results suggest that the positive association between AGT polymorphism and hypertension is not simply explained by an increase of plasma AGT concentration.

Role of "handle" region of prorenin prosegment in the non-proteolytic activation of prorenin by binding to membrane anchored (pro)renin receptor.

A role of the handle region in the prorenin prosegment sequence was investigated to demonstrate the crucial non-proteolytic activation of prorenin by binding to the recombinant (pro)renin receptor on the COS-7 cell membrane. The plasmid DNA containing either rat or human (pro)renin receptor was transfected into the COS-7 cells. The highest amount of receptor was observed on the COS-7 cell membrane after 18 h transfection. Of the total rat and human prorenin, 90% and 50% were bound to each of the respective receptors, respectively. The Kd values were 0.89 and 1.8 nM, respectively. Rat prorenin was activated non-proteolytically by the receptor. The Km was determined 1.0 microM when sheep angiotensinogen was used as the substrate. Human prorenin was also activated by the receptor. The Km was 0.71 microM. Additionally, decapeptides (10P-19P) known as decoy peptide and pentapeptides (11P-15P) named handle region peptide, were observed to inhibit the binding of both prorenins to receptors, respectively. The Ki were similar around 7 nM for both the peptides. Other two region peptides in the prosegment did not interfere the binding. These results show that the handle region probably plays a crucial role in prorenin binding to the receptor and in its enzymic activity by non-proteolytic activation.

Prorenin has high affinity multiple binding sites for (pro)renin receptor.

An important role of the decoy peptide sequence has recently been suggested in vitro for the binding of prorenin to the (pro)renin receptor [PRR]. In this study, other prospective crucial regions in renin and prorenin responsible for their interaction with PRR were investigated using various kinds of peptides, e.g., the hinge S149QGVLKEDVF158 designed from the structure of renin also common to prorenin, L1PPTDTTTF8P, L1PPTDTTTFKRIFLKR15P and the decoy (R10PIFLKRMPSI19P) designed from the predicted structure of prorenin. For the kinetic analysis, the recombinant hPRR was immobilized on the biosensor surface through a specific anti-PRR antibody. In case of the equilibrium state analysis, the PRR was directly adsorbed on plastic wells for observing the bindings of renin/prorenin. The dissociation constants (KD) for the bindings of renin and prorenin to the pre-adsorbed receptors were 4.5 and 1.0 nM, respectively, similar to those stated in the kinetic study by BIAcore assay. The hinge region peptide bound to PRR in a dose-dependent manner with a KD estimated 17.0 nM which was five times higher than that of the decoy. The KD values for L1PPTDTTTF8P and L1PPTDTTTFKRIFLKR15P were 52 and 7.6 nM, respectively. The hinge peptide, as the decoy, inhibited the bindings of renin and prorenin to PRR. The inhibition constant (Ki) for the binding of renin and prorenin by the decoy and hinge were 16.7 and 15.1, and 37.1 and 30.7 nM, respectively. These in vitro studies suggest that renin has a single and prorenin has at least two high affinity binding sites for the PRR.

Efficient Production of Recombinant Human (Pro)renin Utilizing a Decahistidine Tag Attached at the C-Terminus

Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin.

Non-proteolytic activation of prorenin: activation by (pro)renin receptor and its inhibition by a prorenin prosegment, "decoy peptide".

Prorenin is the enzymatically inactive precursor of renin. Recent interest has focused on the nonproteolytic activation of prorenin by antibodies and renin/prorenin receptors since markedly increased levels of circulating prorenin have been associated with both physiological and pathological changes. Prorenin has been considered to be activated in vivo proteolytically and/or non-proteolytically. It has been demonstrated in vitro the gate and handle regions in the prorenin molecule is crucial for its non-proteolytic activation by a protein-protein interaction. Prorenin was also activated by the renin/prorenin receptors. Decapeptides (10P-19P) known as decoy peptide and pentapeptides (11P-15P) named as handle region peptide, were observed to inhibit the binding of both prorenins to receptors. The handle region plays an important role in prorenin binding to the receptor and its enzymatic activity by non-proteolytic activation. Prorenin receptors so far revealed by animal experiments have indicated that the decoy peptide prevented diabetes nephropathy and retinopathy. It was postulated the existence of novel regulative system that stimulated signal transduction as well as that of renin-angiotensin system. These findings help to find out the clue to design useful drug with greater benefit on the end-organ damage in diabetes and hypertension than those of conventional renin-angiotensin system inhibitors.

Site-1 protease is required for the generation of soluble (pro)renin receptor.

The extracellular domain of the (pro)renin receptor [(P)RR] is cleaved to generate the soluble form of (P)RR [s(P)RR]. Multiple clinical studies have revealed the association between serum/plasma s(P)RR levels and certain diseases, thereby suggesting a potential role for s(P)RR as a disease biomarker. Here, we investigated whether site-1 protease (S1P) is responsible for cleaving (P)RR to generate s(P)RR. Reduction of endogenous S1P with siRNA attenuated s(P)RR generation in Chinese hamster ovary (CHO) cells exogenously expressing human (P)RR with a C-terminal decahistidine tag [CHO/h(P)RR-10His cells]; conversely, overexpression of S1P by transient transfection increased s(P)RR generation. The S1P inhibitor PF429242 suppressed s(P)RR generation in CHO/h(P)RR-10His and human cervical carcinoma HeLa cells; however, the ADAM inhibitor GM6001 had no effect. The furin inhibitor Dec-RVKR-CMK had no effect on the amount of s(P)RR, but caused a slight increase in the size of the s(P)RR. Moreover, the reversible vesicle-trafficking inhibitor brefeldin A (BFA) enhanced the generation of large-sized s(P)RR; PF429242, but not Dec-RVKR-CMK, suppressed this BFA-induced s(P)RR formation. The size of s(P)RR generated during BFA treatment was reduced after removal of BFA; Dec-RVKR-CMK, but not PF429242, suppressed this conversion. Together, these results suggest that s(P)RR is generated by sequential processing by S1P and furin.