Yuko Izuhara

Renoprotective Properties of Angiotensin Receptor Blockers beyond Blood Pressure Lowering

Clinical studies have demonstrated that some antihypertensive agents provide renoprotection independent of BP lowering. Recent in vitro and in vivo studies evaluated the mechanisms involved in this protection. First, the in vitro effects of several angiotensin II type 1 receptor blockers (ARB), calcium channel blockers (CCB), and beta blockers (BB) on various mediators were compared: Formation of pentosidine (an advanced glycation end product), hydroxyl radical-induced formation of o-tyrosine, and transition metals-induced oxidation of ascorbic acid (the Fenton reaction). All of the six tested ARB but neither the six CCB nor the nine BB inhibited pentosidine formation. ARB, as well as BB but not CCB, inhibited hydroxyl radicals-mediated o-tyrosine formation. ARB but neither BB nor CCB inhibited efficiently transition metals-catalyzed oxidation of ascorbic acid. Second, the in vivo consequences for the kidney of these various in vitro effects were evaluated. Hypertensive, type 2 diabetic rats with nephropathy, SHR/NDmcr-cp, were given for 20 wk either olmesartan (ARB) or nifedipine (CCB), or atenolol (BB). Despite similar BP reduction, only ARB significantly reduced proteinuria and prevented glomerular and tubulointerstitial damage (mesangial activation, podocyte injury, tubulointerstitial injury, and inflammatory cell infiltration). It is interesting that only ARB prevented abnormal iron deposition in the interstitium, corrected chronic hypoxia, reduced expressions of heme oxygenase and p47phox (a subunit of NADPHoxidase), and inhibited pentosidine formation (which correlates well with proteinuria). These observations confirm unique renoprotective properties of ARB, independent of BP lowering but related to decreased oxidative stress (hydroxyl radicals scavenging and inhibition of the Fenton reaction), correction of chronic hypoxia, and inhibition of advanced glycation end product formation and of abnormal iron deposition. These benefits of ARB may contribute to the renoprotection observed beyond BP lowering.

A Novel Class of Prolyl Hydroxylase Inhibitors Induces Angiogenesis and Exerts Organ Protection Against Ischemia

Objective—Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to ischemic conditions. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD). Methods and Results—We discovered 2 unique compounds (TM6008 and TM6089), which inhibited PHD and stabilized HIF activity in vitro. Our docking simulation studies based on the 3-dimensional structure of human PHD2 disclosed that they preferentially bind to the active site of PHD. Whereas PHD inhibitors previously reported inhibit PHD activity via iron chelation, TM6089 does not share an iron chelating motif and is devoid of iron chelating activity. In vitro Matrigel assays and in vivo sponge assays demonstrated enhancement of angiogenesis by local administration of TM6008 and TM6089. Their oral administration stimulated HIF activity in various organs of transgenic rats expressing a hypoxia-responsive reporter vector. No acute toxicity was observed up to 2 weeks after a single oral dose of 2000 mg/kg for TM6008. Oral administration of TM6008 protected neurons in a model of cerebrovascular disease. The protection was associated with amelioration of apoptosis but independent of enhanced angiogenesis. Conclusions—The present study uncovered beneficial effects of novel PHD inhibitors preferentially binding to the active site of PHD.

Inhibition of Plasminogen Activator Inhibitor-1: Its Mechanism and Effectiveness on Coagulation and Fibrosis

Objective—Serine protease inhibitors (serpin) play a central role in various pathological processes including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serpins may prove therapeutic. As yet, however, only very few small molecule serpin inhibitors have been reported. For the first time, we apply a new approach of virtual screening to discover novel, orally active, small molecule serpin inhibitors and report their effectiveness. Methods and Results—We focused on a clinically important serpin, plasminogen activator inhibitor-1 (PAI-1), whose crystal structure has been described. We identify novel, orally active molecules able to enter into the strand 4 position (s4A) of the A β-sheet of PAI-I as a mock compound. In vitro they specifically inhibit the PAI-1 activity and enhance fibrinolysis activity. In vivo the most effective molecule (TM5007) inhibits coagulation in 2 models: a rat arteriovenous (AV) shunt model and a mouse model of ferric chloride–induced testicular artery thrombosis. It also prevents the fibrotic process initiated by bleomycin in mouse lung. Conclusions—The present study demonstrates beneficial in vitro and in vivo effects of novel PAI-1 inhibitors. Our methodology proves to be a useful tool to obtain effective inhibitors of serpin activity.

A novel inhibitor of plasminogen activator inhibitor-1 provides antithrombotic benefits devoid of bleeding effect in nonhuman primates.

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterialvenous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latters therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time.

Overexpression of the serpin megsin induces progressive mesangial cell proliferation and expansion.

Mesangial cells maintain normal glomerular function by mediating ECM remodeling and immune complex disposal. We have recently identified megsin, a novel member of the serine protease inhibitor (serpin) superfamily predominantly expressed in the mesangium. While our previous studies suggested a role for megsin in the pathogenesis of human glomerular diseases, its exact biological significance remained unknown. Here we produced two lines of megsin transgenic mice. Overexpression of megsin led to progressive mesangial matrix expansion and an increase in the number of mesangial cells. These glomerular lesions were accompanied by an augmented immune complex deposition, together with Igs and complement. Binding and functional assays in vitro identified plasmin as one biological substrate of megsin and confirmed its activity as a proteinase inhibitor. Transgenic animals exhibiting nephritis as a result of treatment with anti--glomerular basement membrane antiserum showed significantly more persistent expansion of the mesangial ECM than was seen in parental mice. Megsin therefore exerts a biologically relevant influence on mesangial function, and on the mesangial microenvironment, such that simple overexpression of this endogenous serpin engenders elementary mesangial lesions.

A novel inhibitor of advanced glycation and endoplasmic reticulum stress reduces infarct volume in rat focal cerebral ischemia

We have developed a novel, non-toxic inhibitor of advanced glycation and oxidative stress, TM2002, devoid of effect on blood pressure. In transient focal ischemia, TM2002 significantly decreased infarct volume compared with vehicle (79.5+/-18.7 vs. 183.3+/-22.9 mm3, p<0.01). In permanent focal ischemia, TM2002 (2.79, 5.58, and 11.16 mg/kg twice a day) dose-dependently reduced infarct volume (242.1+/-32.3, 201.3+/-15.1, and 171.3+/-15.2 mm3, respectively), and improved neurological deficits. Reduction of infarct volume is demonstrable, provided that TM2002 was administered within 1.5 h after the occlusion. To unravel TM2002s mechanism of action, we examined its in vitro effect on endoplasmic reticulum (ER) stress, using aortic smooth muscle cells isolated from ORP 150(+/-) mice and F9 Herp null mutated cells. Cell death induced by ER stress (tunicamycin or hypoxia) was dose-dependently prevented by TM2002. In vivo immunohistochemical study demonstrated a significant reduction of ORP- and TUNEL-positive apoptotic cells, especially in the penumbra. Inhibition of advanced glycation and oxidative stress was confirmed by a significantly reduced number of cells positive for advanced glycation end products and heme oxygenase-1. TM2002 reduced the levels of protein carbonyl formation in ischemic caudate. The efficacy of TM2002 is equivalent to that of a known neuroprotective agent, NXY-059. In conclusion, TM2002 significantly ameliorates ischemic cerebral damage through reduction of ER stress, advanced glycation, and oxidative stress, independently of blood pressure lowering.

A Novel Sartan Derivative With Very Low Angiotensin II Type 1 Receptor Affinity Protects the Kidney in Type 2 Diabetic Rats.

Background—Antihypertensive angiotensin II receptor blockers (ARBs) protect the kidney, at least in part, independently of blood pressure lowering. Still, the extent to which blood pressure lowering is related to renoprotection remains unclear. Methods and Results—139 newly synthesized ARB-derivatives were assayed for inhibition of advanced glycation (AGEs). The 9 most powerful compounds were then tested for transition metal chelation, angiotensin II type 1 receptor (AT1R) affinity, and pharmacokinetic parameters. R-147176 was eventually selected as it strongly inhibits advanced glycation but is 6700 times less effective than olmesartan in AT1R binding. It is orally bioavailable and toxicologically safe. Despite a minimal blood pressure lowering effect, it provides significant renoprotection in 3 experimental rat models with renal injury, ie, obese, hypertensive, type 2 diabetic rats (SHR/NDmcr-cp), normotensive type 2 diabetic rats (Zucker diabetic fatty), and remnant kidney rats. Conclusion—R-147176 retains renal protective properties despite a minimal blood pressure–lowering effect. Clearly, the renal benefits of ARBs do not necessarily depend on blood pressure lowering and AT1R affinity, but rather on the inhibition of AGEs and oxidative stress inherent to their chemical structure. R-147176 opens new avenues in the treatment of cardiovascular and kidney diseases.

Inhibition of Advanced Glycation End Products

Several factors have been incriminated in the genesis of diabetic nephropathy. To elucidate their interplay, we have used a hypertensive, obese, diabetic rat model with nephropathy (SHR/NDmcr‐cp) that mimics human type 2 diabetes. This model is characterized by hypertension, obesity with the metabolic syndrome, diabetes with insulin resistance, and intrarenal advanced glycation end product (AGE) accumulation. In order to achieve renoprotection, which was evaluated by histology and albuminuria, various therapeutic approaches were used: caloric restriction, antihypertensive agents (angiotensin II receptor blocker [ARB] and calcium channel blocker), lipid‐ (bezafibrate) or glucose‐lowering (insulin and pioglitazone) agents, and cobalt chloride (a hypoxia‐inducible factor activator). Altogether, renoprotection is not necessarily associated with blood pressure or glycemic control. By contrast, it is almost always associated with decreased AGE formation, with the exception of insulin, which induces hyperinsulinemia, eventually leading to an overproduction of transforming growth factor‐β. AGE formation is reduced directly by in vitro active compounds (e.g., ARBs) or indirectly by in vitro inactive compounds (e.g., pioglitazone and cobalt). In the latter cases, AGE reduction may reflect a decreased oxidative stress as it is concomitant with a marked reduction of oxidative stress markers. It remains to be seen whether the renoprotection offered by these various approaches may be additive.

The role of megsin, a serine protease inhibitor, in diabetic mesangial matrix accumulation

In diabetic nephropathy decreased activities of matrix metalloproteinase (MMP)-2, MMP-9 and plasmin contribute to mesangial matrix accumulation. Megsin, a novel member of the serine protease inhibitor superfamily, is predominantly expressed in mesangial cells and is up-regulated in diabetic nephropathy and its overexpression spontaneously induces progressive mesangial expansion in mice. High-glucose stimulated megsin mRNA expression in an in vivo model of type II diabetic nephropathy as well as in vitro in cultured mesangial cells. Megsin potentially inhibits total enzymatic activities of MMP-2 and -9 and plasmin, indicating decreased degradation of mesangial matrix. A specific monoclonal anti-megsin neutralizing antibody restored MMP activity in a transforming growth factor-beta independent manner. Our study suggests that the mesangial matrix accumulation caused by hyperglycemia in diabetes might be due at least in part to up-regulation of megsin which can inhibit plasmin and MMP activities.

Very high doses of valsartan provide renoprotection independently of blood pressure in a type 2 diabetic nephropathy rat model.

Aim:  Angiotensin II type 1 receptor blockers (ARB) retard the progression of hypertensive diabetic kidney disease. Clinical evidence suggests that the dose of ARB required to correct hypertension is suboptimal for renoprotection evaluated by proteinuria. No systematic, prospective study has yet evaluated separately the effect of increasing doses of ARB on blood pressure and proteinuria.