Excessive activation of the renin-angiotensin (Ang) system,

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1 Exerts Renoprotective ctions via Peroxisome Proliferator-ctivated Receptor-/Hepatocyte Growth Factor Pathway Independent of ngiotensin II Type 1 Receptor lockade Hiroshi Kusunoki, Yoshiaki Taniyama, Junya zuma, Kazuma Iekushi, Fumihiro Sanada, Rei Otsu, Masaaki Iwabayashi, Keita Okayama, Hiromi Rakugi, Ryuichi Morishita bstract ngiotensin (ng) II type 1 receptor blockers have demonstrated beneficial effects beyond blood pressure control in the treatment of chronic kidney disease. There is clinical evidence that telmisartan is more effective than losartan in reducing proteinuria in hypertensive patients with diabetic nephropathy, because it is a partial agonist of peroxisome-proliferator activated receptor- (PPR), as well as an ng II type 1 receptor blocker (MDEO Study [ comparison of telmisartan versus losrtan in hypertensive type DiabEtic patients with Overt nephropathy]). In this study, we examined the role of PPR activation in the renal protective actions of telmisartan using ng II type 1 receptor deficient mice. Renal injury was induced in ng II type 1 receptor deficient mice by producing unilateral ureteral obstruction, which exhibited severe renal interstitial fibrosis and inflammation. In these mice, telmisartan prevented hydronephrosis induced by unilateral ureteral obstruction more strongly than did losartan. Importantly, the prevention of renal atrophy and fibrosis by telmisartan was significantly attenuated by GW9, a PPR antagonist. Interestingly, the downstream effector of PPR activation by telmisartan is hepatocyte growth factor (HGF), a well-known antifibrotic factor, because renal HGF expression was significantly increased by telmisartan, and a neutralizing antibody against HGF diminished the renal protective action of telmisartan. These beneficial changes by telmisartan were associated with a decrease in the expression of transforming growth factor-1 and other proinflammatory and profibrotic cytokine genes through PPR/HGF activation. Our findings provide evidence of organ protective actions of telmisartan through the PPR/HGF pathway, independent of ng II type 1 receptor blockade. Further development of the next generation of ng II type 1 receptor blockers with added organ protective actions, such as PPR activation, might provide new beneficial drugs to treat renal and cardiovascular diseases. (Hypertension. 1;59:3-31.) Online Data Supplement Key Words: hepatocyte growth factor angiotensin receptors angiotensin antagonists PPR- and - growth factors and cytokines chronic failure (kidney) hypertension (kidney) Excessive activation of the renin-angiotensin (ng) system, specifically ng II, is a key component of the pathogenesis of hypertension, atherosclerosis, coronary artery disease, myocardial infarction, congestive heart failure, and nephropathy. 1 ng II exerts its effects through different receptors, ng II type 1 and type. The binding of ng II to the type 1 receptor (T 1 R) produces vasoconstriction, increases aldosterone release and sympathetic activity, and mediates virtually all of the known adverse cardiovascular effects of ng II. T 1 R blockers (Rs) are selective antagonists of T 1 R and are widely used to treat hypertension and hypertension-related target organ damage. 5 Diabetic nephropathy, the leading cause of end-stage renal disease, is a combination of hemodynamic and metabolic abnormalities that contribute to kidney damage resulting in proteinuria and reduced glomerular filtration rate. mong the available Rs, telmisartan, one of the second-generation Rs known as metabosartans, is reported to be more effective than losartan in reducing proteinuria in hypertensive patients with diabetic nephropathy, despite a similar reduction in blood pressure (MDEO [ comparison of telmisartan versus losrtan in hypertensive type DiabEtic patients with Overt nephropathy]). 7 However, the mechanism explaining why telmisartan has better renal protective effects than losartan is not fully understood, although telmisartan is reported to have greater lipophilicity and a longer half-life than losartan. Received May 15, 11; first decision June, 11; revision accepted November 11, 11. From the Departments of Clinical Gene Therapy (H.K., Y.T., J.., K.I., F.S., R.O., M.I., K.O., R.M.), and Geriatric Medicine and Nephrology (H.K., Y.T., J.., K.I., H.R.), Osaka University, Graduate School of Medicine, Suita, Japan. Correspondence to Ryuichi Morishita and Yoshiaki Taniyama, Department of Clinical Gene Therapy, Osaka University Graduate School of Medicine, - Yamada-oka, Suita 55-71, Japan. morishit@cgt.med.osaka-u.ac.jp and taniyama@cgt.med.osaka-u.ac.jp 1 merican Heart ssociation, Inc. Hypertension is available at DOI: 1.111/HYPERTENSIONH

2 Kusunoki et al Protects Kidney via PPR/HGF Pathway 39 a b 1 mm c d e f GW9 a b c 1 mm (-) d e f more reasonable explanation is that telmisartan is a partial agonist of peroxisome proliferator-activated receptor- (PPR),,9 because PPR is a nuclear receptor transcription factor that binds to a putative peroxisome proliferator response element, leading to activation of various genes related to many important physiological processes, including adipose differentiation and lipid and glucose metabolism. 1 Recently, pioglitazone, a synthetic ligand of PPR that is widely used in the treatment of type diabetes mellitus, was demonstrated to reduce cardiovascular events and proteinuria and to delay the progression of diabetic nephropathy, independent of glycemic control. 11,1 There is substantial evidence that activation of PPR leads to suppression of proinflammatory and proatherogenic molecules. Therefore, combined T 1 R antagonism and PPR activation, such as by telmisartan, would be expected to effectively treat the metabolic and inflammatory derangements associated with the metabolic syndrome, which contribute to the development of atherosclerosis, myocardial fibrosis, and glomerulosclerosis. mong numerous downstream effectors of PPR activation, we focused on hepatocyte growth factor (HGF), because peroxisome proliferator response element exists in the promoter region of the HGF gene, which increases local HGF gene expression. 13 Moreover, HGF is well known to prevent the initiation and progression of chronic renal fibrosis and to inhibit the expression and signal of transforming growth factor- (TGF-) in various animal models. 1 1 Recently, our previous study showed that HGF attenuated renal fibrosis through apoptosis of myofibroblasts via TGF-1 suppression. 19 To elucidate the role of PPR activation, HGF, and TGF- in the organ protective actions of telmisartan, we used a hydronephrosis model produced by unilateral ureteral obstruction () in T 1a R-deficient mice. Here, we showed evidence of organ-protective actions of telmisartan through the PPR/HGF pathway, independent of T 1 R blockade. C 7 GW9 Materials and Methods Experimental Model in T 1a R-Deficient Mice was performed on -week old male T 1 ar-deficient mice (Jackson Laboratory, ar Harbor, ME) after 1 week of pretreatment with drugs, according to established procedures. Under general % Renal fibrosis (-) Los Tel GW Figure 1. Renal remodeling and interstitial fibrosis at 1 days after unilateral ureteral obstruction () operation., Typical micrographs of periodic cross-sections of kidney with acid- Schiff staining after ; (a) sham-operated mice, (b) operated mice (control), (c) -operated mice treated with losartan (3 mg/kg per day), (d) -operated mice treated with telmisartan (3 mg/kg per day), (e) -operated mice treated with telmisartan and neutralizing hepatocyte growth factor neutralizing antibody (; g/wk), and (f) -operated mice treated with telmisartan and GW9 (a peroxisomeproliferator activated receptor [PPR]- antagonist,.3 mg/kg per day). ar: 1 mm., Typical micrographs of kidney with Masson trichrome staining. lue color shows fibrotic areas (collagen and fibronectin deposition); (a) sham-operated mice, (b) -operated mice (control), (c) -operated mice treated with losartan, (d) -operated mice treated with telmisartan, (e) -operated mice treated with telmisartan and, and (f) -operated mice treated with telmisartan and GW9. ar: 1 m. C, Quantitative data for percentage of fibrotic area. indicates sham-operated;, -operated mice (control); Los, losartan; Tel, telmisartan; GW, GW9. P5 vs control, P5 vs Tel. Data are shown as meansem (n5).

3 31 Hypertension February 1 anesthesia, complete ureteral obstruction was performed by double ligation of the left ureter with 5- silk after midline abdominal incision. -operated mice had their ureters exposed and manipulated but not ligated. 17 Mice were divided into 11 groups: (1) sham, () -operated mice with no drug treatment (control), (3) operated mice treated with losartan ( mg/kg per day), () -operated mice treated with telmisartan ( mg/kg per day), (5) -operated mice treated with telmisartan ( mg/kg per day) and neutralizing HGF antibody (; mg/wk), () -operated mice treated with telmisartan (3 mg/kg per day) and GW9 (a PPR antagonist,.3 mg/kg per day), (7) operated mice treated with losartan ( mg/kg per day) and neutralizing ( g/wk), () -operated mice treated with losartan ( mg/kg per day) and GW9 (.3 mg/kg per day), (9) -operated mice treated with telmisartan (1 mg/kg per day), (1) -operated mice treated with losartan (. mg/kg per day), and (11) -operated mice treated with losartan (1 mg/kg per day; 5 7 mice for each group). Drugs were dissolved in water and administered ad libitum. Mice were housed in the animal facilities of Osaka University with free access to food and water. Mice were euthanized at weeks after operation. Cell Isolation and Culture In in vitro experiments, dermal fibroblasts obtained from T 1a R- deficient mice were used. Dermal fibroblasts were obtained using a dermapunch (Maruho, Osaka, Japan) as instructed by the manufacturer, followed by culture in DMEM (Nacalai, Kyoto, Japan) plus 1% FS (Sanko Junyaku, Tokyo, Japan) and antibiotics and incubation at 37 C in a humidified 5% CO and 95% air atmosphere. Studies were performed by growing the cells to % confluence and rendering them quiescent by replacing the complete medium with serum-free medium for hours and then incubating them in incremental concentrations of TGF-1 (1 ng/ml). Cells were pretreated with drugs (telmisartan: 1 mol/l, losartan: 1 mol/l, : 1 nmol/l, and GW9: 1 mol/l) for 1 hours before TGF-1 stimulation. Western blotting was used to examine the cells for expression of -smooth muscle actin (SM) protein induced by TGF-1. Lysates of the cells were obtained after hours of TGF-1 stimulation. Western blotting was performed using primary antibodies, mouse monoclonal anti -SM, and anti -actin (Sigma-ldrich, St Louis, MO). There is more information about materials and methods in the Materials and Methods section of the online Data Supplement, available at Statistical nalysis Data are expressed as meansem. Comparisons were made using NOV followed by Turkey simultaneous multiple comparisons. Values with P5 were considered significant. Results Prevention of Hydronephrosis by Is Stronger Than That by, in T 1a R-Deficient Mice In this study, to elucidate the role of PPR activation by telmisartan, we used a renal injury model using T 1a R- deficient mice, because traditional Rs theoretically fail to demonstrate organ protective actions in the absence of T 1 R. To induce renal injury, we generated a model, because this model exhibits severe renal interstitial fibrosis and inflammation in the kidney on the obstructed side at 1 days after operation. Indeed, our present study clearly demonstrated the typical appearance of renal atrophy in control mice after operation (Figure 1). s expected, losartan-treated kidneys showed more marked hydronephrosis than that in telmisartan-treated kidneys but seemed to HGF protein in kidney (ng/mg tissue) Serum HGF protein (ng/ml) (-) Los Tel Tel GW (-) show less marked hydronephrosis than that in untreated s. also seemed to exert renoprotective effects to prevent hydronephrosis, but such effects were weaker than those of telmisartan (Figure 1 and Figure S1, available in the online Data Supplement). Prevention of hydronephrosis by telmisartan was also confirmed by measurement of the fibrotic area in obstructed kidneys (Figure 1 and 1C). In the obstructed kidneys of T 1a R-deficient mice treated with losartan, Masson trichrome staining showed areas of severe renal fibrosis similar to control (Figure 1 and 1C). In contrast, quantification of renal fibrosis demonstrated a significant reduction in fibrotic area in the telmisartan-treated group, as compared with the losartan-treated group and control group (Figure 1C; P5). There were no significant differences in blood pressure, heart rate (Table S), serum creatinine level, and serum urea nitrogen among the groups (Table S3). These data clearly demonstrated the potential contribution of PPR activation, because telmisartan stimulated PPR activity separately from T 1 R blockade. This hypothesis is further supported by the observation that the prevention of renal atrophy and fibrosis by telmisartan in the model of T 1a R-deficient mice was significantly attenuated by treatment with GW9 (a PPR antagonist; Figure 1C and Figure S1; P5). To further explore the role of PPR activation, we focused on the downstream effectors of PPR activation. Los Tel Tel GW Figure. Concentration of hepatocyte growth factor (HGF) in kidney and serum at 1 days after unilateral ureteral obstruction () operation., HGF protein in kidney on side (nanograms per milligram of tissue)., Serum HGF concentration (nanograms per milliliter). indicates sham-operated;, -operated mice (control); Los, losartan; Tel, telmisartan; GW, GW9. P1 vs control, P1 vs Tel. Data are shown as meansem (n5).

4 Kusunoki et al Protects Kidney via PPR/HGF Pathway 311 a b c a C b c 5 mm 1 mm (-) (-) d e f d e f Glomerular injury index GW9 D (-) Los Tel GW (-) Los Tel GW Tubulointerstitial injury score. GW9 Figure 3. Glomerular injury index and tubulointerstitial injury score. Data show histological analyses of glomerular injury and tubulointerstitial injury by semiquantitative morphometric evaluation., Typical micrographs of renal glomerular sections with acid-schiff staining to evaluate mesangial proliferation: (a) sham, sham-operated mice; (b) unilateral ureteral obstruction ()-operated mice (control); (c) -operated mice treated with losartan (3 mg/kg per day); (d) -operated mice treated with telmisartan (3 mg/kg per day); (e) -operated mice treated with telmisartan and neutralizing hepatocyte growth factor neutralizing antibody (; g/wk), and (f) -operated mice treated with telmisartan and GW9 (a peroxisome-proliferator activated receptor [PPR]- antagonist,.3 mg/kg per day). ar: 5 m., Summary of glomerular injury index. indicates sham-operated;, -operated mice (control); Los, losartan; Tel, telmisartan; GW, GW9. P5, P1 vs control, P5 vs Tel. Data are shown as meansem (n5). C, Typical micrographs of renal tubulointerstitial sections with acid-schiff staining. Dat show tubulointerstitial injury including macrophage infiltration and increased interstitial space as a result of extracellular matrix (ECM) deposition: (a) sham, sham-operated mice; (b) operated mice (control); (c) -operated mice treated with losartan (3 mg/kg per day); (d) -operated mice treated with telmisartan (3 mg/kg per day); (e) -operated mice treated with telmisartan and neutralizing ( g/wk); (f) -operated mice treated with telmisartan and GW9 (a PPR- antagonist,.3 mg/kg per day). ar: 1 m. D, Summary of tubulointerstitial injury score. P1 vs control, P5 vs Tel. Data are shown as meansem (n5). Thus, we measured renal HGF concentration, because the promoter region of the HGF gene contains peroxisome proliferator response element. Importantly, protein expression of HGF was significantly increased in the obstructed kidneys of T 1a R-deficient mice treated with telmisartan, whereas losartan had no significant effect (Figure ; P1). Similarly, serum HGF concentration was also significantly increased in mice treated with telmisartan (Figure ; P1). The increase in HGF expression by telmisartan was attributed to PPR activation, because treatment with GW9 significantly decreased renal and serum HGF concentrations (Figure ; P1). ecause HGF is well known to inhibit and reduce fibrosis, we administered a neutralizing antibody against HGF (). s shown in Figure 1 through 1C, significantly diminished the renal protective action of telmisartan (P5). These data suggest that telmisartan exerts additional renal protective actions through PPR/HGF activation, separate from T 1 R blockade, more strongly than does losartan. Molecular Mechanisms of Organ-Protective ctions of -PPR/HGF ctivation in T 1a R Deficient Mice Next, we histologically evaluated renal injury by acid-schiff staining. Glomerular changes were observed in the model, characterized by lipid deposits in the mesangium and capillaries and glomerular hyperplasia with deposition of extracellular matrix in the mesangium. Tubulointerstitial changes including macrophage infiltration and a modest increase in the interstitial space as a result of deposition of extracellular matrix were also detected in the control group. These changes also included glomerular capillary dilation, increased capillary and mesangial lipid deposits, mesangial expansion with increased infiltration by inflammatory cells, and glomerulosclerosis with glomerular tuft occlusion (Fig-

5 31 Hypertension February 1 % Positive area for macrophages (F/) % Positive area for myofibroblasts (α-sm) C % Positive area for TGF- β (-) Los Tel GW ure 3 through 3C). In contrast, glomerular injury index and tubulointerstitial injury score were significantly reduced in the telmisartan group as compared with the losartan group and control group (Figure 3; P5). Treatment with HGF- b, as well as GW9, significantly diminished the decrease in glomerular injury index and tubulointerstitial injury score by telmisartan (P5). n early event in the development of tubulointerstitial fibrosis is the recruitment of inflammatory cells like macrophages. In the obstructed kidney at 1 days after surgery, macrophage infiltration was observed in the renal (-) Los Tel GW (-) Los Tel GW Figure. Quantitative data of immunohistochemical staining for macrophages (F/), myofibroblasts (-smooth muscle actin [SM]), and transforming growth factor (TGF)-1., Quantitative data of macrophage infiltration area (F/)., Percentage of positive area for myofibroblasts (-SM). C, Percentage of positive area for TGF-1. indicates sham-operated;, unilateral ureteral obstruction () operated mice (control); Los, losartan; Tel, telmisartan;, hepatocyte growth factor neutralizing antibody; GW, GW9. P1 vs control, P1 vs Tel. Data are shown as meansem (n5). tubulointerstitial area, as assessed by the F/-positive area. s shown in Figure and Figure S, prevention of macrophage infiltration by telmisartan is stronger than that by losartan (P1), although losartan also seemed to prevent macrophage infiltration, but such effects were weaker than those of telmisartan. Consistently, treatment with, as well as GW9, significantly diminished the decrease in the area of macrophage infiltration by telmisartan (P1). More exciting findings of the present study were the expression of -SM, which is the molecular hallmark of myofibroblasts, with localization and quantification of myofibroblasts, denoting the intensity of the fibrogenic response. Renal interstitial myofibroblast cells are -SM positive activated matrix-producing cells responsible for relentless accumulation and deposition of extracellular matrix in the interstitial compartment of diseased kidneys. s shown in Figure and Figure S, telmisartan treatment resulted in a significant decrease in interstitial -SM expression as compared with losartan treatment or control (P1), whereas treatment with, as well as GW9, significantly diminished the decrease in interstitial -SM expression induced by telmisartan (P1). These beneficial changes by telmisartan were associated with a decrease in renal expression of TGF-1, a fibrogenic cytokine in the obstructed kidney. n increase in TGF-1 protein was detected mainly in the tubular epithelial cells of the obstructed kidney in control mice. However, telmisartan treatment significantly reduced interstitial TGF-1 expression as compared with the losartan-treated group and control group (Figure C and Figure SC; P1). The decrease in TGF-1 expression by telmisartan was attributed to PPR/ HGF activation, because, as well as GW9, attenuated the decrease in TGF-1 expression induced by telmisartan (P1). Renoprotective Effects of Were lso Inhibited by and GW9 In this model of kidney injury, losartan might exert beneficial effects on some parameters, but such effects would be weaker than those of telmisartan. The weak PPR agonistic effects of losartan may affect the antifibrotic and anti-inflammatory actions of losartan. When and GW9 were administered together with losartan, the degrees of glomerular injury and of macrophage infiltration observed were similar to those in mice receiving no drugs (Figure S3). Therefore, it is supposed that the weak PPR agonistic effects of losartan may induce the beneficial changes seen in these models. Renoprotective Effects of Each Drug re Not Dependent on Dose If both losartan and telmisartan have similar effects on renal protection via the same pathway, a higher dose of losartan may test just as effective as telmisartan. Therefore, experiments were performed to test that telmisartan is more effective than losartan even when higher doses of losartan are used. dministration of 1 mg/kg per day of losartan showed no significant improvement in renal fibrosis, macrophage infiltration, glomerular injury index, and tubulointerstitial

6 Kusunoki et al Protects Kidney via PPR/HGF Pathway 313 TGF- β1 TGF- β1 α-sm α-sm β-actin β-actin α-sm/β-actin TGF- β1 α-sm/β-actin TGF- β1 Figure 5. Western blotting analysis of -smooth muscle actin (-SM). Effects of transforming growth factor (TGF)-1 (1 ng/ml), telmisartan (1 mol/l), losartan (1 mol/l), and hepatocyte growth factor neutralizing antibody (; 1 nmol/l) on -SM protein in dermal fibroblasts from angiotensin II type 1 receptor deficient mice., Top, Typical example of Western blotting for comparing telmisartan and losartan. ottom, Quantitative analysis of Western blotting. ar graphs show -SM/-actin ratio. P1 vs control, P5 vs telmisartan. Data are shown as meansem (n)., Top, Typical example of Western blotting for evaluating the effects of. ottom, Quantitative analysis of Western blotting. ar graphs show -SM/-actin ratio. P1 vs control, P5 vs telmisartan. Data are shown as meansem (n). injury score as compared with a dosage of 3 mg/kg per day of losartan (Figures S and S5). Unexpectedly, even high doses (1 mg/kg per day) of telmisartan did not improve these parameters as compared with 3 mg/kg per day of telmisartan. Therefore, the renal protective effects of these drugs were shown to be independent of their administration dosages. Inhibited TGF-1 Mediated Phenotype Change in T 1a R-Deficient Fibroblasts More Significantly Than Finally, to identify the relationship between HGF and TGF-1 in phenotype modulation, we performed in vitro experiments using dermal fibroblasts from T 1a R-deficient mice, because TGF-1 initiates a phenotypic response in tubular epithelial cells to undergo epithelial-mesenchymal transition (EMT), becoming fibroblasts that migrate to the interstitium. Consistent with previous reports, 1 TGF-1 significantly induced a myofibroblast phenotype, as indicated by increased -SM protein expression (Figure 5; P1)., but not losartan, significantly inhibited the increase in expression of -SM induced by TGF-1 (Figure 5; P5). We examined the role of HGF in myofibroblast phenotype change. s expected, significantly reduced the inhibition of -SM expression by telmisartan (Figure 5; P5). s shown in Figure, telmisartan significantly increased HGF expression in fibroblasts from T 1a R-deficient mice (P1), whereas losartan did not increase HGF. The increased expression of HGF by telmisartan was significantly diminished by GW9, consistent with in vivo data (Figure ; P5). In addition, although TGF-1 significantly increased proinflammatory and profibrotic cytokine genes, such as monocyte chemoattractant protein 1, interleukin, collagen I, collagen IV, and plasminogen activator inhibitor 1 (P1), telmisartan significantly inhibited the enhanced expression of these cytokines induced by TGF-1 (Figure through F; P5). However, as shown in in vivo studies, losartan also inhibited the enhanced expression of some of these parameters (collagen I, collagen IV, and plasminogen activator inhibitor 1; Figure D through F; P5), but such effects are weaker than those of telmisartan. The contribution of PPR/HGF activation was also confirmed by the observation that both and GW9 significantly attenuated the decrease in expression of these cytokines by telmisartan (Figure through F; P5). Discussion Using a renal injury model in T 1a R-deficient mice, the present study provided direct evidence of renal protective actions of telmisartan through the PPR/HGF pathway, independent of T 1 R blockade. In this study, telmisartan, but not losartan, prevented tubulointerstitial fibrosis and activation of myofibroblasts (expressing -SM) and macrophages, which play a pivotal role in the progression of renal obstructive injury after. Especially because tubulointerstitial fibrosis is an integral part of the structural changes in the kidney in chronic progressive renal failure, 3 the present study is important for clinical practice. In general, Rs have beneficial effects on proteinuria and renal dysfunction that appear to be mediated by factors additional to their effects on blood pressure. The most intriguing question is whether there are some differences in organ protection among various Rs, because telmisartan and irbesartan are multifunctional Rs or metabosartans that activate PPR.

7 31 Hypertension February 1 HGF/GPDH mrn C IL-/GPDH mrn E TGF-β1 Tel Los GW Collagen /GPDH mrn.5. TGF-β1 Tel Los GW TGF-β1 Tel Los GW # MCP-1/GPDH mrn D Collagen /GPDH mrn F PI-1/GPDH mrn # # Figure. Real-time PCR analysis. Effects of transforming growth factor (TGF)-1 (1 ng/ml), telmisartan (1 mol/l), losartan (1 mol/ L), GW9 (1 mol/l), and HGF neutralizing antibody (; 1 nmol/l) on, hepatocyte growth factor (HGF)/GPDH mrn relative expression;, monocyte chemoattractant protein (MCP)-1/GPDH mrn ; C, interleukin (IL)-/GPDH mrn ; D, collagen I/GPDH mrn ; E, collagen IV/GPDH mrn ; and F, plasminogen activator inhibitor (PI)-1/GPDH mrn in dermal fibroblasts from angiotensin II type 1a receptor deficient mice. P1 vs no treatment, P5 vs TGF-1telmisartan, #P5 vs TGF-1. Data are shown as meansem (n). ased on the present study, the prevention of renal fibrosis and injury, at least in hydronephrosis, by telmisartan is mediated by PPR activation, leading to a local increase in HGF and a decrease in TGF-, associated with the inhibition of the transdifferentiation of tubular epithelial cells into myofibroblasts, an event underlying progressive chronic kidney disease. HGF has been shown to exhibit a marked ability to block this phenotypic transition both in vitro and in vivo. 1 HGF was also shown to ameliorate renal fibrosis and preserve kidney function in some chronic kidney disease models. 15,1 In contrast, among many fibrogenic factors that regulate fibrotic processes, TGF- is a strong inducer of transdifferentiation in renal tubular cells. Under the stimulus of TGF-1 produced by macrophages or other cells, fibroblasts synthesize stress fibers and differentiate further into myofibroblasts. Myofibroblasts are contractile and augment the deposition of the extracellular matrix, leading to progressive interstitial fibrosis and the structural changes within the kidney in chronic progressive renal failure. 5 previous study demonstrated that HGF prevented the initiation and progression of chronic renal fibrosis and inhibited TGF-1 expression in a wide variety of animal models. 1 On the other hand, PPR agonists also significantly reduced TGF- and attenuated renal interstitial fibrosis and inflammation in models of. Thiazolidines have been proven to mediate anti-inflammatory effects and antifibrotic effects in animal models of diabetic nephropathy, 7 consistent with several clinical studies in which thiazolidines reduced urine albumin excretion and prevented the development of renal injury. 11,1, The present in vivo study demonstrated that the

8 Kusunoki et al Protects Kidney via PPR/HGF Pathway 315 increase in HGF by PPR activation mediated by telmisartan prevented renal injury and fibrosis, associated with a decrease in TGF-1. These effects of PPR activation by telmisartan were also confirmed by in vitro experiments using fibroblasts from T 1a R-deficient mice. Similarly, in fibroblasts derived from T 1a R-deficient mice, mrn of inflammatory cytokines, such as monocyte chemoattractant protein 1 and interleukin, and profibrotic cytokines, such as plasminogen activator inhibitor 1, collagen I, and collagen IV, were upregulated by TGF-1, whereas telmisartan reduced the increase in expression of these genes mediated by TGF-1. Importantly, these effects of telmisartan were inhibited by a neutralizing HGF antibody and GW9, a PPR antagonist. There are some limitations of this study. The mice used in this study were T 1a R-deficient mice. In the losartan group, the area positively immunostained for F/ and TGF-1 was attenuated compared with that in the control group. lso, it has already been proven that losartan has weak PPR agonistic activity as well. 9 We chose T 1a R-deficient mice, so the T 1b R-blocking action of losartan may have antiinflammatory effects. Crowley et al 3 showed previously that T 1b receptor activation increased podocyte injury and expression of inflammatory mediators using T 1a R-deficient lpr mice. lso, administration of losartan reduced markers of kidney disease, such as proteinuria, glomerular pathology, and inflammatory cytokine expression. lso in this model, losartan might have beneficial effects on kidney injury (glomerular injury and macrophage infiltration, etc), but such effects are weaker than those of telmisartan. These beneficial effects induced by losartan were attenuated by cotreatment with or GW9, and it is supposed that the weak PPR agonistic effects of losartan may induce the beneficial changes seen in these models. T 1b R-blocking action of losartan may not play the crucial role, even if it exists. In the kidney, it has been reported that T 1b receptor expression is largely limited to the glomerulus. 31 Moreover, Zhou et al 3 reported that, in mouse kidney, the T 1a receptor is the predominant subtype of T 1 R. These previous reports support our results. might also have anti-inflammatory effects in cultured fibroblasts derived from T 1a R-deficient mice, but such effects would be weaker than those of telmisartan as shown in in vivo studies. The same mechanism may operate in in vitro studies as seen in in vivo studies. Moreover, even when the administration dosage of losartan was increased, the renal protective effects were not changed significantly. Therefore, the renal protective effects of these drugs appear to be independent of their administration dosage. nother potential limitation of this study is the use of the tail-cuff technique to measure blood pressure. The tail-cuff technique does not give -hour blood pressure, blood pressure variability, and so forth. Thus, it is possible that some of the greater renoprotective effects of telmisartan versus losartan might have been because of greater effects of telmisartan on -hour blood pressure or other components of blood pressure not measured by the tail-cuff technique. In conclusion, the present study provides direct evidence of organ-protective actions of telmisartan through the PPR/ HGF pathway, independent of T 1 R blockade. also provided the similar effects, but those effects are weaker than those of telmisartan. ecause inhibition of the renin-ng system does not control all of the pathophysiological mechanisms of hypertension or cardiovascular risk, the development of the next generation of Rs with added organ protective actions that can target additional mechanisms of hypertension, cardiovascular disease, and diabetes mellitus is clearly important. Indeed, next-generation Rs that are intended to not only antagonize T 1 R but also to block endothelin receptors, function as NO donors, inhibit neprilysin activity, and increase natriuretic peptide level or stimulate PPR are now becoming available. Perspectives In T 1a R-deficient mice with unilateral ureteral obstruction, we have found that the renoprotective actions of telmisartan may not simply depend on T 1 receptor blockade and may be partially mediated by its ability to activate PPR and increase the expression of HGF. HGF appears to be an important downstream effector molecule of the organprotective effects of PPR agonists. To the extent that the renal protective effects of telmisartan extend beyond the model, it is possible that Rs that activate PPR and stimulate HGF may someday prove useful in limiting the progression of a variety of forms of chronic kidney disease. Further drug design to add PPR activation to Rs might provide new beneficial drugs to treat renal and cardiovascular disease in diabetes mellitus, obesity, and the metabolic syndrome. cknowledgments We thank the members of the Department of Clinical Gene Therapy, Osaka University Graduate School of Medicine, for much helpful discussion and technical support. Sources of Funding This work was partially supported by a grant from the Osaka Kidney Foundation, a Grant-in-id from the Organization for Pharmaceutical Safety and Research, a Grant-in-id from the Ministry of Public Health and Welfare, a Grant-in-id from Japan Promotion of Science, SENSHIN Medical Research Foundation, and through special (coordinated) funds of the Ministry of Education, Culture, Sports, Science and Technology, of the Japanese Government. None. Disclosures References 1. Imai Y, Kuba K, Ohto-Nakanishi T, Penninger JM. ngiotensinconverting enzyme (CE) in disease pathogenesis. Circ J. 1;7: Parving HH, Lehnert H, röchner-mortensen J, Gomis R, ndersen S, rner P, Group IiPwTDaMS. The effect of irbesartan on the development of diabetic nephropathy in patients with type diabetes. N Engl J Med. 1;35: Lewis EJ, Hunsicker LG, Clarke WR, erl T, Pohl M, Lewis J, Ritz E, tkins RC, Rohde R, Raz I, Group CS. 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