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Angiotensin II type I receptor blockade suppresses glomerular renin-angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis
Angiotensin II type I receptor blockade suppresses glomerular renin-angiotensin system activation, oxidative stress, and progressive glomerular injury in rat anti-glomerular basement membrane glomerulonephritis YUKIKO KINOSHITA, SHUJI KONDO, MAKI URUSHIHARA, KENICHI SUGA, SATO MATSUURA, MASANORI TAKAMATSU, MAKI SHIMIZU, AKIRA NISHIYAMA, HIROSHI KAWACHI, and SHOJI KAGAMI TOKUSHIMA, KAGAWA, AND NIIGATA, JAPAN
Excessive renin-angiotensin system (RAS) activation within the kidney induces not only renal oxidative stress but also renal scarring and dysfunction. This study examined the effects of an angiotensin II (Ang II) type I receptor (AT1R) blocker (ARB) on the progression of renal injury in rat anti-glomerular basement membrane glomerulonephritis (GN), with a particular focus on the participation of glomerular RAS activation in glomerular structural alterations, inflammation, and oxidative stress. Nephritic rats were divided into 2 groups and treated with vehicle or ARB until day 28. Treatment with ARB improved proteinuria significantly in nephritic rats. Vehicle-treated nephritic rats developed crescentic GN accompanied by marked macrophage infiltration and the enhanced expression of glomerular a-smooth muscle actin (a-SMA), angiotensinogen (AGT), Ang II, AT1R, and NADPH oxidase (Nox2) on days 7 and 28 of GN. ARB improved pathologic alterations such as crescent formation and glomerulosclerosis, and it had a significant inhibitory effect on the levels of these parameters on day 28 of GN. Enhanced superoxide production in nephritic glomeruli was decreased also by ARB. Moreover, Ang II and transforming growth factor beta (TGF-b) in the supernatant of cultured glomeruli was increased significantly in vehicle-treated nephritic rats whereas ARB inhibited the production of these compounds significantly on day 28. These results indicate that increased glomerular RAS activity and the resulting Ang II play important roles in progressive glomerular injury–the induction of oxidative stress and TGF-b expression, and they suggest that AT1R blockade attenuates proteinuria and progressive glomerular remodeling via the suppression of glomerular RAS activation in GN. (Translational Research 2011;158:235–248)
From the Department of Pediatrics, Institute of Health Bioscience, the University of Tokushima Graduate School, Tokushima, Japan; Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa, Japan; Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Supported by grants-in-aid 18790723, 20591277, and 20591278 for scientific research.
Reprint requests: Shoji Kagami, MD, PhD, Department of Pediatrics, Institute of Health Bioscience, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2011 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2011.05.003
Submitted for publication December 3, 2010; revision submitted May 11, 2011; accepted for publication May 13, 2011.
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Abbreviations: a-SMA ¼ a-smooth muscle actin; AGT ¼ angiotensinogen; Ang II ¼ angiotensin II; ARB ¼ angiotensin II type I receptor blocker; AT1R ¼ angiotensin II type I receptor; DHE ¼ dihydroethidium; ECM ¼ extracellular matrix; ELISA ¼ enzyme-linked immunosorbent assay; EMT ¼ epithelial mesenchymal transition; FITC ¼ fluorescein isothiocyanate; GBM ¼ glomerular basement membrane; GN ¼ glomerulonephritis; IF ¼ immunofluorescent; IgA ¼ immunoglobulin A; MCP-1 ¼ monocytes chemoattractant protein-1; Nox ¼ nicotinamide adenine dinucleotide phosphate-oxidase; O2$- ¼ superoxide; RAS ¼ renin-angiotensin system; ROS ¼ reactive oxygen species; SBP ¼ systolic blood pressure; TGF-b ¼ transforming growth factor beta; WKY ¼ Wistar Kyoto
AT A GLANCE COMMENTARY Kinoshita Y, et al. Background
Recently, the focus of interest on the reninangiotensin system (RAS) has shifted toward the role of the local/tissue RAS in specific tissues. The local RAS in the kidney has several pathophysiologic functions, for regulating not only blood pressure but also renal cell growth and fibrosis. In this study, we focused the molecular mechanisms responsible for glomerular RAS activation, oxidative stress, and inflammation in crescentic glomerulonephritis (GN). Translational Significance
Our data showed that the angiotensin II (Ang II) receptor blocker prevented glomerular RAS activity, suppressing reactive oxygen species and transforming growth factor (TGF)-b expression in a rat model of anti-glomerular basement membrane disease. The targeting and regulation of glomerular RAS activation may provide novel means for preventing glomerular damage and disease progression in GN.
The marked accumulation of extracellular matrix (ECM) and crescent formation in nephritic glomeruli are prominent pathologic features that have been shown to be responsible for progressive human and rat experimental glomerulonephritis (GN).1,2 It has been demonstrated that transforming growth factor beta (TGF-b), oxidative stress, and proinflammatory cytokines play important roles in such progressive GN.1,3,4 Recently, treatment with renin-angiotensin system (RAS) blockers such as angiotensin II (Ang II) type I receptor (AT1R) blocker (ARB) and/or angiotensin converting enzyme inhibitor has been shown to have
protective effects against the progression of several types of human GN, including immunoglobulin A (IgA) nephropathy, and the progression of rat GN models.5-8 These studies indicated that the renoprotective effects of these antihypertensive agents result not only from the inhibition of hemodynamic action but also from the blockade of nonhemodynamic actions.5-8 In fact, many in vivo experimental studies, including ours, have found that Ang II induced proteinuria, renal dysfunction, and glomerular inflammation and ECM accumulation leading to glomerulosclerosis–the increased expression of TGF-b and the production of reactive oxygen species (ROS).9,10 In vitro studies have demonstrated that Ang II stimulates ECM accumulation–the induction of TGF-b expression in rat mesangial cells, which is evidence that Ang II can act as a profibrotic molecule independent of its effects on blood pressure.11 In addition, Ang II induces not only proinflammatory mediators, including monocyte chemoattractant protein-1 and tumor necrosis factor-a but also the production of ROS by all types of glomerular cells, and thereby it contributes to the development of glomerular inflammation and damage.12-14 Recent biochemical, immunohistochemical, and in situ messenger RNA techniques for identifying RAS components have demonstrated that all the RAS components can be found within normal glomeruli and consistent increases in angiotensinogen (AGT) and Ang II can be found in damaged glomeruli in various types of GN.15-20 These studies suggest that glomerular RAS activity and subsequent locally produced Ang II under pathologic conditions may contribute to the development of proteinuria and glomerular injuries observed in progressive GN.15-20 Indeed, isolated glomeruli from streptozotocin-diabetic rats exhibited the increased intraglomerular production of AGT and Ang II compared with control rats, accompanied by a decrease in angiotensin converting enzyme inhibitor 2 activity, which is substantial evidence that glomerular RAS can be activated in the diabetic condition.21 However, little is known about the details of glomerular RAS activation or the deleterious effects of locally produced Ang II on several mediators that influence
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the development and progression of glomerular lesions in progressive GN. To investigate whether local RAS activation occurs in nephritic glomeruli and whether its final effector molecule Ang II contributes to the induction of ROS and inflammation as well as glomerular pathologic alterations, we studied the effects of the ARB candesartan on rat anti-glomerular basement membrane (GBM) antibodyinduced GN by evaluating indexes of glomerular RAS activation, oxidative stress, inflammation, and the expression of TGF-b in GN rats. Additionally, we measured the production levels of AGT, Ang II, and TGF-b in isolated glomeruli obtained from GN rats treated with or without ARB. MATERIALS AND METHODS Animals and experimental protocol. Seven-week-old male Wistar Kyoto (WKY) rats, weighing 160–200 g, were obtained from Charles River Inc, Kanagawa, Japan. They had free access to rat chow and drinking water. All experimental procedures were performed according to the guidelines for the care and use of laboratory animals established by the National Institutes of Health and the Institute for Animal Experimentation, the University of Tokushima Graduate School. Progressive anti-GBM GN was induced in 7-week-old male WKY rats by a single intravenous injection of rabbit anti-rat GBM antiserum (0.2 mg/100 g body weight) via the tail vein.22 Nephritic rats were divided into 2 groups (n 5 36) and given either vehicle or 70 mg/L candesartan (ARB) (Takeda Chemical Industries, Osaka, Japan) daily in drinking water. The dose of ARB (70 mg/L) that was used in this experiment was high enough (10 mg/kg per day) to inhibit Ang II receptor binding to kidney tissue.23,24 Age-matched male WKY rats (n 5 18) without nephritis were used as controls. The rats were sacrificed on days 7 and 28 after disease induction, and the kidneys were removed rapidly for a variety of histologic, biochemical, and molecular analyses. Blood samples were taken from the abdominal aorta before sacrifice. Urine was collected periodically from the rats every week in a metabolic cage (TECNIPLAST, Buguggiate, Italy) during the experiments. Urinary protein excretion was measured by the Bradford method (Bio-Rad, Oakland, Calif). The serum concentration of creatinine was measured using reagents manufactured by WAKO Chemical Industries (Osaka, Japan). Systolic blood pressure (SBP) was measured noninvasively by the tail-cuff method. Histopathology. Kidney tissues were fixed in 10% buffered formalin and embedded in paraffin. The paraffin-embedded tissues were sectioned at a thickness of 3 mm and stained with periodic acid-Schiff reagent.
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Glomerular injury was evaluated by a person who was unaware of the source of the sections. More than 50 glomeruli were examined in each rat. Glomeruli were considered to exhibit crescent formation when at least 2 layers of cells were observed in Bowman’s space. The ratio of crescent-positive glomeruli to total glomeruli was calculated as the crescent formation ratio. The crescent score in each glomerulus was defined as follows: score 0, glomerulus without crescent; score 1, less than 25% of the circumference; score 2, 25% to 50%; score 3, 50% to 75%; and score 4, more than 75%. The mean score was calculated as the crescent score. Additionally, the characteristics of glomerular crescents on day 28 of GN were analyzed as follows. Glomerular crescents in each glomerulus were categorized into 3 types: cellular, fibrocellular, and fibrous. Their numbers were counted and the ratio of each type of crescent to the total glomeruli with crescents was calculated. Glomerulosclerosis was defined as glomeruli that exhibited adhesion of the capillary tuft to Bowman’s capsule, capillary obliteration, and mesangial expansion. The severity of sclerosis for each glomerulus was graded semiquantitatively as follows: score 0, no lesions; score 1, less than 25% of the glomerular area affected; score 2, 25% to 50% affected; score 3, 50% to 75% affected; and score 4, 75% to 100% affected. The mean score was calculated as the glomerulosclerosis score. Immunohistochemistry. Paraffin-embedded kidneys were sectioned (3 mm), deparaffinized, and incubated with either mouse monoclonal anti-ED1 antibody (MCA341R; Serotech, Oxford, UK) as a macrophage marker, mouse monoclonal anti-a-smooth muscle actin (a-SMA) antibody (1A4; Sigma-Aldrich Co, St. Louis, Mo) as a myofibroblast marker, mouse monoclonal anti-AGT antibody (10R-A131a; Fitzgerald Industries International, Acton, Mass), or mouse monoclonal anti-nicotinamide adenine dinucleotide phosphateoxidase (Nox2) antibody (a kind gift from Dr. Quinn at Montana State University). The tissues were then stained by standard immunoperoxidase procedures according to the manufacturer’s instructions. To evaluate the level of macrophages infiltrating into the glomeruli, the total number of ED-1- positive cells within the glomeruli was counted. The mean number of cells was calculated from at least 50 glomeruli per rat in each group. For the semiquantitative evaluation of glomerular immunostaining with antibody, more than 50 cross-sections of glomeruli were graded based on the extent of the positive area in each glomerulus as follows: score 0, diffuse, very weak, or no staining; score 1, less than 25% of glomerular area exhibits strong staining; score 2, 25% to 50%; score 3, 50% to
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75%; and score 4, more than 75%. The mean score was calculated as the glomerular immunostaining score. To determine the localization of Ang II and AT1R in glomeruli, frozen sections (3 mm) were incubated with either guinea pig anti-Ang II antibody (Peninsula Laboratories, San Carlos, Calif) or goat anti-AT1R antibody (AT1 N-10 sc-1173; Santa Cruz Biotechnology, Santa Cruz, Calif), followed by fluorescein isothiocyanate (FITC)-labeled goat antiguinea pig IgG antibody or FITC-labeled donkey antigoat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa), as appropriate. For the semiquantitative evaluation of glomerular immunofluorescent (IF) staining, more than 50 cross sections of glomeruli were graded based on the extent of the positive area in each glomerulus as follows: score 0, very weak or no staining; score 1, less than 25% of the glomerular area exhibits strong IF staining; score 2, 25% to 50%; score 3, 50% to 75%; score 4, more than 75%. The mean score was calculated as the glomerular IF staining score. Western blot analysis. Rat glomeruli were isolated from the renal cortex by a standard sieving method. Glomerular suspensions consisted of .95% glomeruli as determined by light microscopy. Harvested glomeruli were homogenized and lysed for 30 min on ice in lysis buffer (Cell Signaling Technology, Inc, Beverly, Mass). Supernatants were collected after centrifugation at 15,000 g at 4 C for 20 min. Protein samples (20 mg) were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with either mouse monoclonal anti-Nox2 antibody or mouse monoclonal anti-a-SMA antibody, and then they were incubated with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected with an enhanced chemiluminescence detection system (Amersham Corp, Arlington Heights, Ill). The blots were stripped and probed with mouse monoclonal anti-b-actin antibody (AC-15; Sigma-Aldrich Co, St. Louis, Mo) to confirm equal loading and transfer. In another experiment, protein samples (2 mg) in cell culture supernatants were also analyzed by Western blot using mouse monoclonal anti-AGT antibody. Bands were quantified by ImageJ 1.33u (National Institutes of Health, Bethesda, Md) and the fold expression was indicated as the relative protein level. In situ superoxide (O2$-) production. The in situ production of O2$- was determined as described previously.9 Briefly, 30-mm sections of frozen tissue were incubated with dihydroethidium (DHE; 10 mmol/L) in PBS for 30 min at 37 C in a humidified chamber that was protected from light. DHE is oxidized after
reacting with O2$- to form ethidium bromide, which binds to DNA in the nucleus and fluoresces red. To detect ethidium bromide, we used a 543-nm He-Ne laser combined with a 560-nm long-pass filter. Glomerular O2$- content was estimated by analyzing the mean fluorescence intensity in glomeruli using imaging software from the National Institutes of Health.9 To characterize O2$--producing cells in nephritic glomeruli, we performed IF staining combined with a DHE assay (red) and FITC labeling (green) using either anti-Nox2 antibody or antia-SMA antibody. Measurement of Ang II and TGF-b1 in supernatants from cell cultures of isolated glomeruli. Isolated rat glomeruli
were subjected immediately to 24 h incubation under standard cell culture conditions (1 3 104 glomeruli/mL) in 6-well culture plates. Culture supernatants were then collected after centrifugation at 15,000 g at 4 C for 20 min and stored at –80 C until we measured the production levels of Ang II and TGF-b1. The levels of TGF- b1 protein were measured using a commercial sandwich ELISA kit for TGF- b1 (R&D Systems, Minneapolis, Minn), according to the manufacturer’s instructions. Ang II levels were also determined by a competitive ELISA kit (Peninsula Laboratories). Statistical analyses. Values are expressed as means 6 standard deviation (SD). The differences were evaluated with the StatMate III software package (ATMS Co, Ltd, Tokyo, Japan). The differences between 2 groups were assessed by the unpaired t test, and those among 3 groups were analyzed by a 1-way analysis of variance, followed by the post-hoc Scheffe F test. All experiments were repeated at least 3 times. The values of P , 0.05 were considered statistically significant. RESULTS Clinical characteristics. No difference was found in 24-h urinary protein excretion among any of the groups before the induction of GN (control, 10.9 6 1.2 mg/day; GN, 16.9 6 3.6 mg/day; GN 1 ARB, 21.0 6 15.4 mg/day). Significant proteinuria was observed in vehicle-treated nephritic rats on day 7 of GN (365.2 6 113.8 mg/day) and continued to increase to day 28 (467.9 6 172.9 mg/day). Treatment with ARB reduced the levels of proteinuria (day 7, 164.5 6 46.1 mg/day; day 28, 67.3 6 28.4 mg/day) compared with those in vehicle-treated nephritic rats from day 7 to day 28. SBP was comparable among the groups before disease induction (control, 100.2 6 9.4 mm Hg; GN, 106.6 6 3.6 mm Hg; GN 1 ARB, 105.2 6 4.4 mm Hg), but was increased significantly in vehicle-treated nephritic rats on day 28 (144.0 6 4.6 mm Hg) compared with that in normal control rats (119.0 6 10.8 mm Hg).
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Fig 1. Representative photographs of periodic acid-Schiff-stained sections on days 7 (A–C) and 28 (D–F) in control rats (A and D), vehicle-treated nephritic rats (B and E), and ARB-treated nephritic rats (C and F). Magnification 3400. The nephritic rats showed severe crescent formation and glomerulosclerosis (B and E). Treatment with ARB attenuated those pathologic alterations on day 28 (F) but not on day 7 (C) of nephritic rats.
Treatment with ARB reduced SBP significantly (day 7, 91.9 6 9.6 mm Hg; day 28, 82.0 6 12.3 mm Hg) compared with those in control rats (day 7, 118.6 6 11.1 mm Hg; day 28, 119.0 6 10.8 mm Hg) and vehicle-treated nephritic rats (day 7, 126.9 6 6.5 mm Hg; day 28, 144.0 6 4.6 mm Hg) from day 7 to day 28 of GN. On day 28, the serum level of creatinine in vehicle-treated nephritic rats was significantly higher than that in control rats (0.72 6 0.14 mg/dL vs 0.59 6 0.06 mg/dL). However, no significant difference was found between vehicle-treated nephritic rats and ARBtreated nephritic rats (0.72 6 0.14 mg/dL vs 0.70 6 0.08 mg/dL) on day 28 of GN. Pathology. To examine the effect of ARB treatment on anti-GBM nephritic rats, we performed histologic analyses on days 7 and 28 of GN. Representative light micrographs of glomerular lesions on days 7 and 28 are shown in Fig 1. Nephritic rats showed typical cellular crescent formation in more than 80% of glomeruli on day 7 of GN (Fig 1, B and Fig 2, A). Most of these glomeruli were also accompanied by severe necrotizing lesions, and some were accompanied by glomerulosclerosis. No significant difference was observed in the ratio of crescent formation, the crescent score, and the glomerulosclerosis score between vehicle-treated nephritic rats and ARB-treated nephritic rats on day 7 (Fig 1, B and C and Fig 2, A–C). On day 28, severe glomerular lesions such as fibrocellular crescents and glomerulosclerosis were found in vehicle-treated nephritic rats (Fig 1, E). Compared with vehicle-treated nephritic rats, ARB-treated nephritic rats showed a significant improvement in glomerular crescent formation, the crescent score, and the
glomerulosclerosis score on day 28 of GN (Fig 1, E and F and Fig 2, A–C). In addition, treatment with ARB seemed to inhibit the transition from cellular crescents to advanced fibrous and fibrocellular crescents in GN (Fig 2, D). No apparent effect on renal histology was observed in ARB-treated normal WKY (n 5 12) rats during the course of administration. Histochemical analyses. The effect of ARB on the glomerular accumulation of inflammatory cells in nephritic rats was examined by using anti-ED-1 antibody staining. ED-1-positive macrophages were detected only rarely in the glomeruli of normal rats (Fig 3, A and D). The increased infiltration of ED-1-positive macrophages into nephritic glomeruli was observed on days 7 and 28 in vehicle-treated nephritic rats (22.85 6 2.79 and 20.48 6 4.38/glomerulus, respectively) (Fig 3, B, E, and G). Treatment with ARB decreased these numbers significantly on days 7 and 28 in nephritic rats (12.53 6 1.85 and 10.25 6 1.46/glomerulus, respectively) (Fig 3, C, F, and G). Glomerular cell activation and phenotypic changes were examined by the immunostaining of a-SMA. The expression of a-SMA was increased in vehicletreated nephritic glomeruli with cellular crescents on day 7 and was abundant in fibrocellular crescents of nephritic glomeruli on day 28 (Fig 3, I and L). The treatment with ARB did not change the increased level of a-SMA expression on day 7, but it decreased glomerular a-SMA expression on day 28 (Fig 3, J and M). A semiquantitative evaluation revealed that no significant difference was found in the increased a-SMA score between vehicle- and ARB-treated nephritic rats on day 7 (Fig 3, N). However, ARB-treated
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Fig 2. Effects of ARB on histopathologic parameters of rats. The percentage of crescent formation (A), the crescent score (B), the glomerulosclerosis score (C), and the type of crescent (D) were evaluated. Treatment with ARB was associated with a significant improvement of histopathologic parameters on day 28. *P , 0.01 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN1ARB on day 7. #P , 0.01 versus control on day 28. § P , 0.01 versus GN on day 28.
nephritic rats showed a significant decrease in the a-SMA score compared with that in nephritic rats without treatment on day 28 (P , 0.01; Fig 3, N). Glomerular expression of a-SMA. In parallel with the degree of a-SMA immunostaining, Western blot analyses using isolated glomeruli showed a prominent increase in the a-SMA protein level in both vehicle- and ARB-treated nephritic rats, and these enhanced levels were not significantly different on day 7 of GN (Fig 4, A and C). Treatment with ARB significantly decreased the level of a-SMA protein in nephritic rats compared with that in vehicle-treated nephritic rats on day 28 (Fig 4, B and C). Glomerular expression of RAS components. The expression of glomerular RAS components such as AGT, Ang II, and AT1R was studied by immunohistochemistry (Fig 5). AGT was detected mainly in proximal tubular cells and was weakly positive in glomerular endothelial cells of control rats on days 7 and 28 (Fig 5, A and D). Enhanced staining of AGT was observed in mesangial cells and crescentic cells on days 7 and 28 in vehicletreated nephritic rats (Fig 5, B and E). Treatment with
ARB did not affect glomerular AGT staining on day 7 but significantly decreased such staining on day 28 in nephritic rats (P , 0.01) (Fig 5, C, F, and G). Ang II, which is an effector molecule of RAS, was detected weakly in the mesangial area in normal control rats on days 7 and 28 (Fig 5, H and K). Consistent with the level of AGT immunostaining, strong staining of Ang II was observed in glomeruli with crescents in vehicletreated nephritic rats on days 7 and 28 (Fig 5, I and L). ARB did not influence the staining intensity of glomerular Ang II on day 7, but it decreased such staining on day 28 in nephritic rats (P , 0.05) (Fig 5, J, M, and N). The glomerular expression of AT1R protein was found weakly in all of the glomerular cells in normal rats, as described previously15 (Fig 5, O and R). Notably, AT1R was expressed strongly in crescentic cells of vehicle-treated nephritic rats on days 7 and 28 (Fig 5, P and S), whereas such expression was reduced in ARB-treated nephritic rats on day 28 of GN (P , 0.01; Fig 5, T and U). Glomerular production of AGT, Ang II, and TGF-b1. To demonstrate the glomerular production of AGT, Ang
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Fig 3. Infiltrating ED-1-positive macrophages in glomeruli on days 7 (A–C) and 28 (D–F) in control rats (A and D), vehicle-treated nephritic rats (B and E), and ARB-treated nephritic rats (C and F). Magnification 3400. The number of ED-1-positive cells was increased in vehicle-treated nephritic rats on days 7 and 28. In contrast, treatment with ARB significantly decreased the number of these cells on days 7 and 28 (G). Immunohistochemical analysis of the expression of a-SMA on days 7 (H–J) and 28 (K–M) in control rats (H and K), vehicletreated nephritic rats (I and L), and ARB-treated nephritic rats (J and M). Magnification 3400. Treatment with ARB was associated with a significant decrease in the a-SMA score compared with vehicle-treated rats on day 28 (N). *P , 0.01 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN 1 ARB on day 7. #P , 0.01 versus control on day 28. §P , 0.01 versus GN on day 28. {P , 0.05 versus GN on day 28.
II, and TGF-b1 in nephritic glomeruli with or without ARB treatment, several experiments using culture supernatants of isolated nephritic glomeruli on day 28 after disease induction were performed (Fig 6). In accordance with immunohistochemical data, Western blot analysis showed a significant increase in the production of glomerular AGT protein in vehicletreated nephritic rats, whereas treatment with ARB attenuated the production of AGT protein significantly on day 28 of GN (Fig 6, A and B). Similarly, an ELISA examination showed that the Ang II concentration in culture supernatant was increased significantly in vehicle-treated nephritic rats (P , 0.05). Treatment with ARB reduced the concentration of Ang II significantly to almost the control level (P , 0.05; Fig 6, C). TGF-b1 levels were measured using a sandwich ELISA. In parallel with the Ang II concentration in culture supernatant, TGF- b1 levels
were increased significantly in vehicle-treated nephritic rats (307.92 6 14.31 pg/mL) compared with those in normal rats (60.77 6 0.74 pg/mL). Nephritic rats treated with ARB showed a decrease in the production of TGF-b1 (86.22 6 12.21 pg/mL; Fig 6, D). Glomerular expression of Nox2 and in situ production of O2$-. To investigate whether ROS plays a role in the
development and progression of this model of GN, the levels of Nox2 expression, a major component of NADPH oxidase, and O2$- production were studied (Fig 7). Strong Nox2 expression was detected in nephritic glomeruli, particularly in crescentic cells on days 7 and 28 in nephritic rats (Fig 7, B and E). Several Nox2-positive cells were also observed in the interstitial area in vehicle-treated nephritic rats on day 28. Treatment with ARB did not influence the level of glomerular Nox2 staining on day 7 but significantly decreased this level on day 28 in nephritic rats
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Fig 4. a-SMA protein expression in isolated glomeruli. Representative results of Western blot are shown (A and B). The samples were adjusted to a standard content (20 mg) and analyzed by Western blot using the mouse monoclonal anti-a-SMA antibody. Data for a-SMA protein levels were normalized by b-actin protein levels. Each band was scanned and subjected to densitometry and expressed as a fold increase relative to the results in control rats (C). The a-SMA protein level was increased in both vehicle- and ARB-treated nephritic rats on day 7, but it was significantly decreased in ARB treated nephritic rats on day 28. *P , 0.01 versus control on day 7. iP , 0.05 versus control on day 7. #P , 0.01 versus control on day 28. §P , 0.01 versus GN on day 28.
(P , 0.01) (Fig 7, C, F, and G). Furthermore, an examination of in situ O2$- production in nephritic glomeruli by DHE dye revealed that glomerular O2$production was increased significantly in vehicletreated day 7 nephritic rats and was further enhanced in day 28 nephritic rats (Fig 7, I, L, and N). Treatment of nephritic rats with ARB did not affect the level of glomerular O2$- production significantly in day 7 nephritic rats, whereas this treatment reduced O2$production to the level in control rats on day 28 (Fig 7, J, M, and N). Western blot analysis of isolated glomeruli confirmed that the level of Nox2 protein was increased significantly in both vehicle- and ARBtreated day 7 nephritic rats compared with that in control rats (Fig 8, A–C). A significant increase in Nox2 protein was still observed in vehicle-treated
nephritic rats on day 28, whereas this increased level of Nox2 protein was attenuated by approximately 53% in day 28 ARB-treated nephritic rats. Finally, double-IF staining in combination with antibody and DHE dye showed clearly that Nox2-positive or a-SMA-positive crescentic cells were similarly double stained with DHE dye, indicating that glomerular crescentic cells strongly produced O2$-, possibly via Nox activation in this rat model of GN (Fig 9). DISCUSSION
Intrarenal RAS activation has been implicated in the development and progression of GN and diabetic nephropathy.5-8 The current study suggested that
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Fig 5. Immunohistochemical study of AGT (A–F), Ang II (H–M) and AT1R (O–T) on days 7 (A–C, H–J, and O–Q) and day 28 (D–F, K–M and R–T). Arrows show crescents. Magnification 3400. Glomerular AGT expression was weak in normal control rats (A and D), and it was enhanced in mesangial cells and crescents in vehicletreated nephritic rats (B and E). Treatment with ARB decreased its expression significantly compared with that in vehicle-treated nephritic rats on day 28 (F and G). Enhanced staining of Ang II was observed mostly in the crescent area in nephritic rats on day 7 (I and J). ARB-treated nephritic rats showed significantly weak staining compared with that in vehicle-treated nephritic rats on day 28 (M and N). AT1 receptor was only weakly expressed in the glomeruli of normal control rats (O and R). In contrast, strong expression was observed in the crescent area (P and S). In vehicle-treated nephritic rats, the expression of AT1 receptor was significantly stronger on day 28 (S and U), but treatment with ARB reduced this expression significantly on day 28 (T and U). *P , 0.01 versus control on day 7. iP , 0.05 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN 1 ARB on day 7. §P , 0.01 versus GN on day 28. {P , 0.05 versus GN on day 28.
glomerular RAS activity leading to Ang II production play important roles in glomerular structural remodeling characteristic of progressive GN. Based on the results from biochemical analyses using isolated glomeruli, it seems likely that the beneficial molecular effect of ARB is caused by inhibition of the enhanced glomerular production of AGT and Ang II as well as the glomerular expression of Nox2, which is a major component of Nox (ROS-generating system) and TGF-b in nephritic glomeruli.
It is well known that glomeruli can generate Ang II at levels that are several orders of magnitude higher than that detected in circulation, which supports the existence of a glomerular RAS that can act independently of the systemic RAS.19 Recently, Singh et al16 reported using diabetic rat glomeruli that glomerular Ang II levels are increased because of an increase in AGT substrate and an increase in the formation of Ang II, suggesting that the development of diabetic nephropathy is associated with glomerular RAS activation and a resultant
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Fig 6. Effects of treatment with ARB on AGT (A and B), Ang II (C), and TGF-b1 (D) levels in supernatant from cell culture of isolated glomeruli on day 28. #P , 0.01 versus control. iP , 0.05 versus control. *P , 0.01 versus GN. §P , 0.05 versus GN. These parameters were increased in vehicle-treated nephritic rats. ARB treatment significantly attenuated those levels.
production of Ang II. Furthermore, a large body of evidence indicates that intrarenal RAS activity (Ang II activity) depends on the amount of renal AGT production and is associated with renal damage.25-27 Similarly, our previous immunohistochemical study found that the level of glomerular AGT staining paralleled those of glomerular Ang II staining and glomerular injury in patients with IgA nephropathy, indicating a close relationship between glomerular RAS activity and glomerular injury even in human GN.15 Collectively, our current data constitute substantial evidence of glomerular RAS activation in anti-GBM GN rats and that AGT expression plays an important role as a substrate for RAS activation in nephritic glomeruli. Ang II participates in not only systemic and glomerular hypertension but also proteinuria, inflammation, and abnormal pathologic remodeling in damaged kidney via AT1R signaling.7,28 Ang II enhances the infiltration of macrophages, which are key inflammatory cells, into damaged glomeruli, via the upregulation of adhesion molecules and monocyte chemoattractant protein1(MCP-1) by AT1R-expressing glomerular cells.29-31
Hypertension per se promotes the progression of renal disease by worsening glomerular injury and proteinuria.32 In the current study, treatment with ARB decreased the levels of SBP and proteinuria, and it prevented macrophage accumulation in inflamed glomeruli on days 7 and 28 in nephritic rats by inhibiting the action of Ang II, suggesting that changes in these parameters are at least in part involved in pathologic improvement in day 28 nephritic rats treated with ARB. Therefore, subsequent studies with Ang II-independent antihypertensive drugs that could induce a compatible reduction in BP with candesartan or MCP-1 null mice are required to explore the more direct role of glomerular RAS activity in glomerular injury in anti-GBM GN rats. The current study also suggested that locally produced Ang II plays a critical role in glomerular structural remodeling–the induction of TGF-b and ROS, which are representative mediators in progressive GN.1,3 Accumulating evidence suggests that both mediators contribute to activated mesangial cell (a-SMA-positive)-mediated ECM accumulation, leading to glomerulosclerosis, a hallmark for
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Fig 7. NADPH oxidase isoform Nox2 staining and in situ detection of superoxide (O2$-) in glomeruli of rats. Magnification 3400. Representative photographs of Nox2 staining on days 7 (A–C) and 28 (D–F) in control rats (A and D), vehicle-treated nephritic rats (B and E), and ARB-treated nephritic rats (C and F). Nox2 was markedly expressed in glomeruli and was particularly increased in crescentic cells on day 7 (B and C). It had spread to the interstitial area in vehicle-treated nephritic rats on day 28 (E). In contrast, treatment with ARB suppressed Nox2 staining significantly on day 28 (F and G). Representative fluorescence micrographs of glomeruli that were stained with the O2$--sensitive dye DHE (red fluorescence) on days 7(H–J) and 28 (K–M) in control rats (H and K), vehicle-treated nephritic rats (I and L), and ARB-treated nephritic rats (J and M) are shown. Semiquantitative assessment of the mean fluorescence intensity for glomerular O2$-content was detected on days 7 and 28 (N). No significant difference was found between vehicle-treated nephritic rats and ARB-treated nephritic rats on day 7 (I and J), but ARB treatment reduced O2$- production to the level in control rats on day 28 (M and N). * P , 0.01 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN 1 ARB on day 7. § P , 0.01 versus GN on day 28.
progressive GN. RAS blockers significantly attenuate sclerotic lesions via the suppression of these mediators.1,3 We stress that ARB significantly inhibits crescent formation in nephritic rats, which is another pathologic feature of progressive GN. Recent studies regarding the molecular mechanisms of crescent formation suggested that increased TGF-b expression in nephritic glomeruli induces the transition of glomerular parietal epithelial cells to myofibroblasts (a-SMA-positive cells), the so-called epithelial mesenchymal transition (EMT), and thereby contribute to crescent formation in rat anti-GBM GN.22,33,34 Notably, in this study, the strong
expression of RAS components such as AGT, Ang II, and AT1R as well as Nox2, O2$-, and a-SMA in cellular and fibrocellular crescents was detected in nephritic rats. Double IF focusing on crescentic cells indicated that O2$--producing cells are Nox2-positive and a-SMA-positive crescentic cells. Nox2 is a main component of Nox, which is a major source of renal ROS.3 The long-term inhibition of Ang II action with ARB decreased the glomerular expression of these compounds as well as TGF-b production, and it induced histologic improvements on day 28. Because Ang II has been shown to induce ROS production via Nox activation in glomerular cells, and ROS is now
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Fig 8. Expression of Nox2 protein in isolated glomeruli. The representative results of Western blot are shown (A and B). The samples were adjusted to a standard content (20 mg) and analyzed by Western blot using the mouse monoclonal anti-Nox2 antibody. The data for Nox2 protein levels were normalized by b-actin protein levels. Each band was scanned and subjected to densitometry and expressed as a fold increase relative to the results in control rats (C). Along with the results of an immunohistochemical analysis, these findings show that Nox2 protein in isolated glomeruli was increased in both vehicle-treated nephritic rats and ARB-treated nephritic rats on day 7 (A and C). Treatment with ARB decreased Nox2 protein to the control level (B and C) on day 28. *P , 0.01 versus control on day 7. #P , 0.01 versus control on day 28. {P , 0.05 versus GN on day 28.
recognized as a potential inducer of EMT phenomena in various pathologic settings,35-37 it is conceivable that the enhanced production of Ang II in nephritic glomeruli induces ROS production and TGF-b expression and drives the EMT phenomena for crescent formation in GN. Overall, treatment with ARB may block the action of Ang II on changes in the cell phenotype responsible for glomerular structural remodeling and prevent glomerulosclerosis and crescent formation by inhibiting the expression of ROS and TGF-b in GN. In contrast with this beneficial effect of ARB on pathologic findings, no significant efficacy on renal function as evaluated with serum creatinine was observed in ARB-treated nephritic rats. The explanation for discrepancy between pathologic improvement and persistence of
impaired renal function under ARB treatment is uncertain but might be related to the well-known facts that extensive RAS blockade slightly lowers the GFR in rat and human with renal disease functionally via decreased systemic BP, and therefore it induces a slight increase of serum creatinine in treated subjects.38,39 Subsequent experiments to clarify this discrepancy are needed when one considers translating our findings into treatment strategies of patients with crescentic GN. Speculations. In conclusion, the current study provides evidence that glomerular RAS activation occurs after injury, and the level of Ang II produced is associated closely with glomerular pathologic alterations such as sclerosis and crescent formation, ROS production, TGF-b expression, and proteinuria in rat anti-GBM
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Fig 9. Representative double-fluorescence study with immunofluorescence staining of Nox2-positive cells (A and C) or a-SMA-positive cells (B and D) and DHE fluorescence in vehicle-treated nephritic rats on day 7 (A and B) and day 28 (C and D). Magnification 3400. The red area indicates nuclear staining of superoxide (O2$-)-producing cells. Merged images show that glomerular crescentic cells produced O2$- strongly, possibly via Nox activation.
GN. ARB may inhibit local RAS activity and Ang II signaling in nephritic glomeruli, and finally it attenuates the progression of GN, possibly by decreasing deleterious mediators such as ROS and TGF-b in GN. We are grateful to Dr. Quinn at Montana State University for kindly providing mouse anti-Nox2 antibody. We also thank Ms. Naomi Okamoto for her excellent technical assistance. Candesartan was kindly provided by Takeda Chemical Industries (Osaka, Japan).
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1. Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Engl J Med 1994;331:1286–92. 2. Atkins RC, Nikolic-Paterson DJ, Song Q, Lan HY. Modulators of crescentic glomerulonephritis. J Am Soc Nephrol 1996;7:2271–8. 3. Shah SV, Baliga R, Rajapurkar M, Fonseca VA. Oxidants in chronic kidney disease. J Am Soc Nephrol 2007;18:16–28. 4. Tipping PG, Holdsworth SR. Cytokines in glomerulonephritis. Semin Nephrol 2007;27:275–85. 5. Coppo R, Peruzzi L, Amore A, et al. IgACE: a placebo-controlled, randomized trial of angiotensin-converting enzyme inhibitors in
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children and young people with IgA nephropathy and moderate proteinuria. J Am Soc Nephrol 2007;18:1880–8. Lewis EJ, Hunsicker LG, Clarke WR, et al. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med 2001;345: 851–60. Taal MW, Brenner BM. Renoprotective benefits of RAS inhibition: from ACEI to angiotensin II antagonists. Kidney Int 2000; 57:1803–17. Suzuki Y, Shirato I, Okumura K, et al. Distinct contribution of Fc receptors and angiotensin II-dependent pathways in anti-GBM glomerulonephritis. Kidney Int 1998;54:1166–74. Kondo S, Shimizu M, Urushihara M, et al. Addition of the antioxidant probucol to angiotensin II type I receptor antagonist arrests progressive mesangioproliferative glomerulonephritis in the rat. J Am Soc Nephrol 2006;17:783–94. Whaley-Connell AT, Chowdhury NA, Hayden MR, et al. Oxidative stress and glomerular filtration barrier injury: role of the renin-angiotensin system in the Ren2 transgenic rat. Am J Physiol Renal Physiol 2006;291:F1308–14. Kagami S, Border WA, Miller DE, Noble NA. Angiotensin II stimulates extracellular matrix protein synthesis–induction of transforming growth factor-beta expression in rat glomerular mesangial cells. J Clin Invest 1994;93:2431–7.
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12. Sachse A, Wolf G. Angiotensin II-induced reactive oxygen species and the kidney. J Am Soc Nephrol 2007;18:2439–46. 13. Ruiz-Ortega M, Ruperez M, Lorenzo O, et al. Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney. Kidney Int 2002;(suppl):S12–22. 14. Hsu HH, Hoffmann S, Endlich N, et al. Mechanisms of angiotensin II signaling on cytoskeleton of podocytes. J Mol Med 2008;86: 1379–94. 15. Takamatsu M, Urushihara M, Kondo S, et al. Glomerular angiotensinogen protein is enhanced in pediatric IgA nephropathy. Pediatr Nephrol 2008;23:1257–67. 16. Singh R, Singh AK, Leehey DJ. A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat glomeruli. Am J Physiol Renal Physiol 2005;288:F1183–90. 17. Del Prete D, Gambaro G, Lupo A, et al. Precocious activation of genes of the renin-angiotensin system and the fibrogenic cascade in IgA glomerulonephritis. Kidney Int 2003;64:149–59. 18. Ingelfinger JR, Zuo WM, Fon EA, Ellison KE, Dzau VJ. In situ hybridization evidence for angiotensinogen messenger RNA in the rat proximal tubule. An hypothesis for the intrarenal renin angiotensin system. J Clin Invest 1990;85:417–23. 19. Seikaly MG, Arant BS Jr, Seney FD Jr. Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat. J Clin Invest 1990;86:1352–7. 20. Obata J, Nakamura T, Takano H, et al. Increased gene expression of components of the renin-angiotensin system in glomeruli of genetically hypertensive rats. J Hypertens 2000;18:1247–55. 21. Leehey DJ, Singh AK, Bast JP, Sethupathi P, Singh R. Glomerular renin angiotensin system in streptozotocin diabetic and Zucker diabetic fatty rats. Transl Res 2008;151:208–16. 22. Shimizu M, Kondo S, Urushihara M, et al. Role of integrin-linked kinase in epithelial-mesenchymal transition in crescent formation of experimental glomerulonephritis. Nephrol Dial Transplant 2006;21:2380–90. 23. Taal MW, Zandi-Nejad K, Weening B, et al. Proinflammatory gene expression and macrophage recruitment in the rat remnant kidney. Kidney Int 2000;58:1664–76. 24. Fabiani ME, Dinh DT, Nassis L, Casley DJ, Johnston CI. In vivo inhibition of angiotensin receptors in the rat kidney by candesartan cilexetil: a comparison with losartan. Am J Hypertens 2000; 13:1005–13. 25. Kobori H, Ozawa Y, Suzaki Y, Nishiyama A. Enhanced intrarenal angiotensinogen contributes to early renal injury in spontaneously hypertensive rats. J Am Soc Nephrol 2005;16:2073–80.
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26. Kobori H, Nangaku M, Navar LG, Nishiyama A. The intrarenal renin-angiotensin system: from physiology to the pathobiology of hypertension and kidney disease. Pharmacol Rev 2007;59:251–87. 27. Ingelfinger JR, Jung F, Diamant D, et al. Rat proximal tubule cell line transformed with origin-defective SV40 DNA: autocrine ANG II feedback. Am J Physiol 1999;276:F218–27. 28. Schmieder RE. Mechanisms for the clinical benefits of angiotensin II receptor blockers. Am J Hypertens 2005;18:720–30. 29. Nikolic-Paterson DJ, Atkins RC. The role of macrophages in glomerulonephritis. Nephrol Dial Transplant 2001;16:3–7. 30. Hisada Y, Sugaya T, Yamanouchi M, et al. Angiotensin II plays a pathogenic role in immune-mediated renal injury in mice. J Clin Invest 1999;103:627–35. 31. Ruiz-Ortega M, Bustos C, Hernandez-Presa MA, et al. Angiotensin II participates in mononuclear cell recruitment in experimental immune complex nephritis–nuclear factor-kappa B activation and monocyte chemoattractant protein-1 synthesis. J Immunol 1998; 161:430–9. 32. Ravera M, Re M, Deferrari L, Vettoretti S, Deferrari G. Importance of blood pressure control in chronic kidney disease. J Am Soc Nephrol 2006;17:S98–103. 33. Huang XR, Chung AC, Zhou L, Wang XJ, Lan HY. Latent TGFbeta1 protects against crescentic glomerulonephritis. J Am Soc Nephrol 2008;19:233–42. 34. Zhou A, Ueno H, Shimomura M, et al. Blockade of TGF-beta action ameliorates renal dysfunction and histologic progression in anti-GBM nephritis. Kidney Int 2003;64:92–101. 35. Rhyu DY, Yang Y, Ha H, et al. Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol 2005;16:667–75. 36. Djamali A, Vidyasagar A, Adulla M, Hullett D, Reese S. Nox-2 is a modulator of fibrogenesis in kidney allografts. Am J Transplant 2009;9:74–82. 37. Bondi CD, Manickam N, Lee DY, et al. NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts. J Am Soc Nephrol 2010;21:93–102. 38. Sebekova K, Lill M, Boor P, Heidland A, Amann K. Functional and partial morphological regression of established renal injury in the obese zucker rat by blockade of the renin-angiotensin system. Am J Nephrol 2009;29:164–70. 39. Bakris GL, Weir MR. Angiotensin-converting enzyme inhibitorassociated elevations in serum creatinine: is this a cause for concern? Arch Intern Med 2000;160:685–93.
Excessive renin-angiotensin system (RAS) activation within the kidney induces not only renal oxidative stress but also renal scarring and dysfunction. This study examined the effects of an angiotensin II (Ang II) type I receptor (AT1R) blocker (ARB) on the progression of renal injury in rat anti-glomerular basement membrane glomerulonephritis (GN), with a particular focus on the participation of glomerular RAS activation in glomerular structural alterations, inflammation, and oxidative stress. Nephritic rats were divided into 2 groups and treated with vehicle or ARB until day 28. Treatment with ARB improved proteinuria significantly in nephritic rats. Vehicle-treated nephritic rats developed crescentic GN accompanied by marked macrophage infiltration and the enhanced expression of glomerular a-smooth muscle actin (a-SMA), angiotensinogen (AGT), Ang II, AT1R, and NADPH oxidase (Nox2) on days 7 and 28 of GN. ARB improved pathologic alterations such as crescent formation and glomerulosclerosis, and it had a significant inhibitory effect on the levels of these parameters on day 28 of GN. Enhanced superoxide production in nephritic glomeruli was decreased also by ARB. Moreover, Ang II and transforming growth factor beta (TGF-b) in the supernatant of cultured glomeruli was increased significantly in vehicle-treated nephritic rats whereas ARB inhibited the production of these compounds significantly on day 28. These results indicate that increased glomerular RAS activity and the resulting Ang II play important roles in progressive glomerular injury–the induction of oxidative stress and TGF-b expression, and they suggest that AT1R blockade attenuates proteinuria and progressive glomerular remodeling via the suppression of glomerular RAS activation in GN. (Translational Research 2011;158:235–248)
From the Department of Pediatrics, Institute of Health Bioscience, the University of Tokushima Graduate School, Tokushima, Japan; Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa, Japan; Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan. Supported by grants-in-aid 18790723, 20591277, and 20591278 for scientific research.
Reprint requests: Shoji Kagami, MD, PhD, Department of Pediatrics, Institute of Health Bioscience, The University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2011 Mosby, Inc. All rights reserved. doi:10.1016/j.trsl.2011.05.003
Submitted for publication December 3, 2010; revision submitted May 11, 2011; accepted for publication May 13, 2011.
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Abbreviations: a-SMA ¼ a-smooth muscle actin; AGT ¼ angiotensinogen; Ang II ¼ angiotensin II; ARB ¼ angiotensin II type I receptor blocker; AT1R ¼ angiotensin II type I receptor; DHE ¼ dihydroethidium; ECM ¼ extracellular matrix; ELISA ¼ enzyme-linked immunosorbent assay; EMT ¼ epithelial mesenchymal transition; FITC ¼ fluorescein isothiocyanate; GBM ¼ glomerular basement membrane; GN ¼ glomerulonephritis; IF ¼ immunofluorescent; IgA ¼ immunoglobulin A; MCP-1 ¼ monocytes chemoattractant protein-1; Nox ¼ nicotinamide adenine dinucleotide phosphate-oxidase; O2$- ¼ superoxide; RAS ¼ renin-angiotensin system; ROS ¼ reactive oxygen species; SBP ¼ systolic blood pressure; TGF-b ¼ transforming growth factor beta; WKY ¼ Wistar Kyoto
AT A GLANCE COMMENTARY Kinoshita Y, et al. Background
Recently, the focus of interest on the reninangiotensin system (RAS) has shifted toward the role of the local/tissue RAS in specific tissues. The local RAS in the kidney has several pathophysiologic functions, for regulating not only blood pressure but also renal cell growth and fibrosis. In this study, we focused the molecular mechanisms responsible for glomerular RAS activation, oxidative stress, and inflammation in crescentic glomerulonephritis (GN). Translational Significance
Our data showed that the angiotensin II (Ang II) receptor blocker prevented glomerular RAS activity, suppressing reactive oxygen species and transforming growth factor (TGF)-b expression in a rat model of anti-glomerular basement membrane disease. The targeting and regulation of glomerular RAS activation may provide novel means for preventing glomerular damage and disease progression in GN.
The marked accumulation of extracellular matrix (ECM) and crescent formation in nephritic glomeruli are prominent pathologic features that have been shown to be responsible for progressive human and rat experimental glomerulonephritis (GN).1,2 It has been demonstrated that transforming growth factor beta (TGF-b), oxidative stress, and proinflammatory cytokines play important roles in such progressive GN.1,3,4 Recently, treatment with renin-angiotensin system (RAS) blockers such as angiotensin II (Ang II) type I receptor (AT1R) blocker (ARB) and/or angiotensin converting enzyme inhibitor has been shown to have
protective effects against the progression of several types of human GN, including immunoglobulin A (IgA) nephropathy, and the progression of rat GN models.5-8 These studies indicated that the renoprotective effects of these antihypertensive agents result not only from the inhibition of hemodynamic action but also from the blockade of nonhemodynamic actions.5-8 In fact, many in vivo experimental studies, including ours, have found that Ang II induced proteinuria, renal dysfunction, and glomerular inflammation and ECM accumulation leading to glomerulosclerosis–the increased expression of TGF-b and the production of reactive oxygen species (ROS).9,10 In vitro studies have demonstrated that Ang II stimulates ECM accumulation–the induction of TGF-b expression in rat mesangial cells, which is evidence that Ang II can act as a profibrotic molecule independent of its effects on blood pressure.11 In addition, Ang II induces not only proinflammatory mediators, including monocyte chemoattractant protein-1 and tumor necrosis factor-a but also the production of ROS by all types of glomerular cells, and thereby it contributes to the development of glomerular inflammation and damage.12-14 Recent biochemical, immunohistochemical, and in situ messenger RNA techniques for identifying RAS components have demonstrated that all the RAS components can be found within normal glomeruli and consistent increases in angiotensinogen (AGT) and Ang II can be found in damaged glomeruli in various types of GN.15-20 These studies suggest that glomerular RAS activity and subsequent locally produced Ang II under pathologic conditions may contribute to the development of proteinuria and glomerular injuries observed in progressive GN.15-20 Indeed, isolated glomeruli from streptozotocin-diabetic rats exhibited the increased intraglomerular production of AGT and Ang II compared with control rats, accompanied by a decrease in angiotensin converting enzyme inhibitor 2 activity, which is substantial evidence that glomerular RAS can be activated in the diabetic condition.21 However, little is known about the details of glomerular RAS activation or the deleterious effects of locally produced Ang II on several mediators that influence
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the development and progression of glomerular lesions in progressive GN. To investigate whether local RAS activation occurs in nephritic glomeruli and whether its final effector molecule Ang II contributes to the induction of ROS and inflammation as well as glomerular pathologic alterations, we studied the effects of the ARB candesartan on rat anti-glomerular basement membrane (GBM) antibodyinduced GN by evaluating indexes of glomerular RAS activation, oxidative stress, inflammation, and the expression of TGF-b in GN rats. Additionally, we measured the production levels of AGT, Ang II, and TGF-b in isolated glomeruli obtained from GN rats treated with or without ARB. MATERIALS AND METHODS Animals and experimental protocol. Seven-week-old male Wistar Kyoto (WKY) rats, weighing 160–200 g, were obtained from Charles River Inc, Kanagawa, Japan. They had free access to rat chow and drinking water. All experimental procedures were performed according to the guidelines for the care and use of laboratory animals established by the National Institutes of Health and the Institute for Animal Experimentation, the University of Tokushima Graduate School. Progressive anti-GBM GN was induced in 7-week-old male WKY rats by a single intravenous injection of rabbit anti-rat GBM antiserum (0.2 mg/100 g body weight) via the tail vein.22 Nephritic rats were divided into 2 groups (n 5 36) and given either vehicle or 70 mg/L candesartan (ARB) (Takeda Chemical Industries, Osaka, Japan) daily in drinking water. The dose of ARB (70 mg/L) that was used in this experiment was high enough (10 mg/kg per day) to inhibit Ang II receptor binding to kidney tissue.23,24 Age-matched male WKY rats (n 5 18) without nephritis were used as controls. The rats were sacrificed on days 7 and 28 after disease induction, and the kidneys were removed rapidly for a variety of histologic, biochemical, and molecular analyses. Blood samples were taken from the abdominal aorta before sacrifice. Urine was collected periodically from the rats every week in a metabolic cage (TECNIPLAST, Buguggiate, Italy) during the experiments. Urinary protein excretion was measured by the Bradford method (Bio-Rad, Oakland, Calif). The serum concentration of creatinine was measured using reagents manufactured by WAKO Chemical Industries (Osaka, Japan). Systolic blood pressure (SBP) was measured noninvasively by the tail-cuff method. Histopathology. Kidney tissues were fixed in 10% buffered formalin and embedded in paraffin. The paraffin-embedded tissues were sectioned at a thickness of 3 mm and stained with periodic acid-Schiff reagent.
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Glomerular injury was evaluated by a person who was unaware of the source of the sections. More than 50 glomeruli were examined in each rat. Glomeruli were considered to exhibit crescent formation when at least 2 layers of cells were observed in Bowman’s space. The ratio of crescent-positive glomeruli to total glomeruli was calculated as the crescent formation ratio. The crescent score in each glomerulus was defined as follows: score 0, glomerulus without crescent; score 1, less than 25% of the circumference; score 2, 25% to 50%; score 3, 50% to 75%; and score 4, more than 75%. The mean score was calculated as the crescent score. Additionally, the characteristics of glomerular crescents on day 28 of GN were analyzed as follows. Glomerular crescents in each glomerulus were categorized into 3 types: cellular, fibrocellular, and fibrous. Their numbers were counted and the ratio of each type of crescent to the total glomeruli with crescents was calculated. Glomerulosclerosis was defined as glomeruli that exhibited adhesion of the capillary tuft to Bowman’s capsule, capillary obliteration, and mesangial expansion. The severity of sclerosis for each glomerulus was graded semiquantitatively as follows: score 0, no lesions; score 1, less than 25% of the glomerular area affected; score 2, 25% to 50% affected; score 3, 50% to 75% affected; and score 4, 75% to 100% affected. The mean score was calculated as the glomerulosclerosis score. Immunohistochemistry. Paraffin-embedded kidneys were sectioned (3 mm), deparaffinized, and incubated with either mouse monoclonal anti-ED1 antibody (MCA341R; Serotech, Oxford, UK) as a macrophage marker, mouse monoclonal anti-a-smooth muscle actin (a-SMA) antibody (1A4; Sigma-Aldrich Co, St. Louis, Mo) as a myofibroblast marker, mouse monoclonal anti-AGT antibody (10R-A131a; Fitzgerald Industries International, Acton, Mass), or mouse monoclonal anti-nicotinamide adenine dinucleotide phosphateoxidase (Nox2) antibody (a kind gift from Dr. Quinn at Montana State University). The tissues were then stained by standard immunoperoxidase procedures according to the manufacturer’s instructions. To evaluate the level of macrophages infiltrating into the glomeruli, the total number of ED-1- positive cells within the glomeruli was counted. The mean number of cells was calculated from at least 50 glomeruli per rat in each group. For the semiquantitative evaluation of glomerular immunostaining with antibody, more than 50 cross-sections of glomeruli were graded based on the extent of the positive area in each glomerulus as follows: score 0, diffuse, very weak, or no staining; score 1, less than 25% of glomerular area exhibits strong staining; score 2, 25% to 50%; score 3, 50% to
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75%; and score 4, more than 75%. The mean score was calculated as the glomerular immunostaining score. To determine the localization of Ang II and AT1R in glomeruli, frozen sections (3 mm) were incubated with either guinea pig anti-Ang II antibody (Peninsula Laboratories, San Carlos, Calif) or goat anti-AT1R antibody (AT1 N-10 sc-1173; Santa Cruz Biotechnology, Santa Cruz, Calif), followed by fluorescein isothiocyanate (FITC)-labeled goat antiguinea pig IgG antibody or FITC-labeled donkey antigoat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa), as appropriate. For the semiquantitative evaluation of glomerular immunofluorescent (IF) staining, more than 50 cross sections of glomeruli were graded based on the extent of the positive area in each glomerulus as follows: score 0, very weak or no staining; score 1, less than 25% of the glomerular area exhibits strong IF staining; score 2, 25% to 50%; score 3, 50% to 75%; score 4, more than 75%. The mean score was calculated as the glomerular IF staining score. Western blot analysis. Rat glomeruli were isolated from the renal cortex by a standard sieving method. Glomerular suspensions consisted of .95% glomeruli as determined by light microscopy. Harvested glomeruli were homogenized and lysed for 30 min on ice in lysis buffer (Cell Signaling Technology, Inc, Beverly, Mass). Supernatants were collected after centrifugation at 15,000 g at 4 C for 20 min. Protein samples (20 mg) were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with either mouse monoclonal anti-Nox2 antibody or mouse monoclonal anti-a-SMA antibody, and then they were incubated with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected with an enhanced chemiluminescence detection system (Amersham Corp, Arlington Heights, Ill). The blots were stripped and probed with mouse monoclonal anti-b-actin antibody (AC-15; Sigma-Aldrich Co, St. Louis, Mo) to confirm equal loading and transfer. In another experiment, protein samples (2 mg) in cell culture supernatants were also analyzed by Western blot using mouse monoclonal anti-AGT antibody. Bands were quantified by ImageJ 1.33u (National Institutes of Health, Bethesda, Md) and the fold expression was indicated as the relative protein level. In situ superoxide (O2$-) production. The in situ production of O2$- was determined as described previously.9 Briefly, 30-mm sections of frozen tissue were incubated with dihydroethidium (DHE; 10 mmol/L) in PBS for 30 min at 37 C in a humidified chamber that was protected from light. DHE is oxidized after
reacting with O2$- to form ethidium bromide, which binds to DNA in the nucleus and fluoresces red. To detect ethidium bromide, we used a 543-nm He-Ne laser combined with a 560-nm long-pass filter. Glomerular O2$- content was estimated by analyzing the mean fluorescence intensity in glomeruli using imaging software from the National Institutes of Health.9 To characterize O2$--producing cells in nephritic glomeruli, we performed IF staining combined with a DHE assay (red) and FITC labeling (green) using either anti-Nox2 antibody or antia-SMA antibody. Measurement of Ang II and TGF-b1 in supernatants from cell cultures of isolated glomeruli. Isolated rat glomeruli
were subjected immediately to 24 h incubation under standard cell culture conditions (1 3 104 glomeruli/mL) in 6-well culture plates. Culture supernatants were then collected after centrifugation at 15,000 g at 4 C for 20 min and stored at –80 C until we measured the production levels of Ang II and TGF-b1. The levels of TGF- b1 protein were measured using a commercial sandwich ELISA kit for TGF- b1 (R&D Systems, Minneapolis, Minn), according to the manufacturer’s instructions. Ang II levels were also determined by a competitive ELISA kit (Peninsula Laboratories). Statistical analyses. Values are expressed as means 6 standard deviation (SD). The differences were evaluated with the StatMate III software package (ATMS Co, Ltd, Tokyo, Japan). The differences between 2 groups were assessed by the unpaired t test, and those among 3 groups were analyzed by a 1-way analysis of variance, followed by the post-hoc Scheffe F test. All experiments were repeated at least 3 times. The values of P , 0.05 were considered statistically significant. RESULTS Clinical characteristics. No difference was found in 24-h urinary protein excretion among any of the groups before the induction of GN (control, 10.9 6 1.2 mg/day; GN, 16.9 6 3.6 mg/day; GN 1 ARB, 21.0 6 15.4 mg/day). Significant proteinuria was observed in vehicle-treated nephritic rats on day 7 of GN (365.2 6 113.8 mg/day) and continued to increase to day 28 (467.9 6 172.9 mg/day). Treatment with ARB reduced the levels of proteinuria (day 7, 164.5 6 46.1 mg/day; day 28, 67.3 6 28.4 mg/day) compared with those in vehicle-treated nephritic rats from day 7 to day 28. SBP was comparable among the groups before disease induction (control, 100.2 6 9.4 mm Hg; GN, 106.6 6 3.6 mm Hg; GN 1 ARB, 105.2 6 4.4 mm Hg), but was increased significantly in vehicle-treated nephritic rats on day 28 (144.0 6 4.6 mm Hg) compared with that in normal control rats (119.0 6 10.8 mm Hg).
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Fig 1. Representative photographs of periodic acid-Schiff-stained sections on days 7 (A–C) and 28 (D–F) in control rats (A and D), vehicle-treated nephritic rats (B and E), and ARB-treated nephritic rats (C and F). Magnification 3400. The nephritic rats showed severe crescent formation and glomerulosclerosis (B and E). Treatment with ARB attenuated those pathologic alterations on day 28 (F) but not on day 7 (C) of nephritic rats.
Treatment with ARB reduced SBP significantly (day 7, 91.9 6 9.6 mm Hg; day 28, 82.0 6 12.3 mm Hg) compared with those in control rats (day 7, 118.6 6 11.1 mm Hg; day 28, 119.0 6 10.8 mm Hg) and vehicle-treated nephritic rats (day 7, 126.9 6 6.5 mm Hg; day 28, 144.0 6 4.6 mm Hg) from day 7 to day 28 of GN. On day 28, the serum level of creatinine in vehicle-treated nephritic rats was significantly higher than that in control rats (0.72 6 0.14 mg/dL vs 0.59 6 0.06 mg/dL). However, no significant difference was found between vehicle-treated nephritic rats and ARBtreated nephritic rats (0.72 6 0.14 mg/dL vs 0.70 6 0.08 mg/dL) on day 28 of GN. Pathology. To examine the effect of ARB treatment on anti-GBM nephritic rats, we performed histologic analyses on days 7 and 28 of GN. Representative light micrographs of glomerular lesions on days 7 and 28 are shown in Fig 1. Nephritic rats showed typical cellular crescent formation in more than 80% of glomeruli on day 7 of GN (Fig 1, B and Fig 2, A). Most of these glomeruli were also accompanied by severe necrotizing lesions, and some were accompanied by glomerulosclerosis. No significant difference was observed in the ratio of crescent formation, the crescent score, and the glomerulosclerosis score between vehicle-treated nephritic rats and ARB-treated nephritic rats on day 7 (Fig 1, B and C and Fig 2, A–C). On day 28, severe glomerular lesions such as fibrocellular crescents and glomerulosclerosis were found in vehicle-treated nephritic rats (Fig 1, E). Compared with vehicle-treated nephritic rats, ARB-treated nephritic rats showed a significant improvement in glomerular crescent formation, the crescent score, and the
glomerulosclerosis score on day 28 of GN (Fig 1, E and F and Fig 2, A–C). In addition, treatment with ARB seemed to inhibit the transition from cellular crescents to advanced fibrous and fibrocellular crescents in GN (Fig 2, D). No apparent effect on renal histology was observed in ARB-treated normal WKY (n 5 12) rats during the course of administration. Histochemical analyses. The effect of ARB on the glomerular accumulation of inflammatory cells in nephritic rats was examined by using anti-ED-1 antibody staining. ED-1-positive macrophages were detected only rarely in the glomeruli of normal rats (Fig 3, A and D). The increased infiltration of ED-1-positive macrophages into nephritic glomeruli was observed on days 7 and 28 in vehicle-treated nephritic rats (22.85 6 2.79 and 20.48 6 4.38/glomerulus, respectively) (Fig 3, B, E, and G). Treatment with ARB decreased these numbers significantly on days 7 and 28 in nephritic rats (12.53 6 1.85 and 10.25 6 1.46/glomerulus, respectively) (Fig 3, C, F, and G). Glomerular cell activation and phenotypic changes were examined by the immunostaining of a-SMA. The expression of a-SMA was increased in vehicletreated nephritic glomeruli with cellular crescents on day 7 and was abundant in fibrocellular crescents of nephritic glomeruli on day 28 (Fig 3, I and L). The treatment with ARB did not change the increased level of a-SMA expression on day 7, but it decreased glomerular a-SMA expression on day 28 (Fig 3, J and M). A semiquantitative evaluation revealed that no significant difference was found in the increased a-SMA score between vehicle- and ARB-treated nephritic rats on day 7 (Fig 3, N). However, ARB-treated
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Fig 2. Effects of ARB on histopathologic parameters of rats. The percentage of crescent formation (A), the crescent score (B), the glomerulosclerosis score (C), and the type of crescent (D) were evaluated. Treatment with ARB was associated with a significant improvement of histopathologic parameters on day 28. *P , 0.01 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN1ARB on day 7. #P , 0.01 versus control on day 28. § P , 0.01 versus GN on day 28.
nephritic rats showed a significant decrease in the a-SMA score compared with that in nephritic rats without treatment on day 28 (P , 0.01; Fig 3, N). Glomerular expression of a-SMA. In parallel with the degree of a-SMA immunostaining, Western blot analyses using isolated glomeruli showed a prominent increase in the a-SMA protein level in both vehicle- and ARB-treated nephritic rats, and these enhanced levels were not significantly different on day 7 of GN (Fig 4, A and C). Treatment with ARB significantly decreased the level of a-SMA protein in nephritic rats compared with that in vehicle-treated nephritic rats on day 28 (Fig 4, B and C). Glomerular expression of RAS components. The expression of glomerular RAS components such as AGT, Ang II, and AT1R was studied by immunohistochemistry (Fig 5). AGT was detected mainly in proximal tubular cells and was weakly positive in glomerular endothelial cells of control rats on days 7 and 28 (Fig 5, A and D). Enhanced staining of AGT was observed in mesangial cells and crescentic cells on days 7 and 28 in vehicletreated nephritic rats (Fig 5, B and E). Treatment with
ARB did not affect glomerular AGT staining on day 7 but significantly decreased such staining on day 28 in nephritic rats (P , 0.01) (Fig 5, C, F, and G). Ang II, which is an effector molecule of RAS, was detected weakly in the mesangial area in normal control rats on days 7 and 28 (Fig 5, H and K). Consistent with the level of AGT immunostaining, strong staining of Ang II was observed in glomeruli with crescents in vehicletreated nephritic rats on days 7 and 28 (Fig 5, I and L). ARB did not influence the staining intensity of glomerular Ang II on day 7, but it decreased such staining on day 28 in nephritic rats (P , 0.05) (Fig 5, J, M, and N). The glomerular expression of AT1R protein was found weakly in all of the glomerular cells in normal rats, as described previously15 (Fig 5, O and R). Notably, AT1R was expressed strongly in crescentic cells of vehicle-treated nephritic rats on days 7 and 28 (Fig 5, P and S), whereas such expression was reduced in ARB-treated nephritic rats on day 28 of GN (P , 0.01; Fig 5, T and U). Glomerular production of AGT, Ang II, and TGF-b1. To demonstrate the glomerular production of AGT, Ang
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Fig 3. Infiltrating ED-1-positive macrophages in glomeruli on days 7 (A–C) and 28 (D–F) in control rats (A and D), vehicle-treated nephritic rats (B and E), and ARB-treated nephritic rats (C and F). Magnification 3400. The number of ED-1-positive cells was increased in vehicle-treated nephritic rats on days 7 and 28. In contrast, treatment with ARB significantly decreased the number of these cells on days 7 and 28 (G). Immunohistochemical analysis of the expression of a-SMA on days 7 (H–J) and 28 (K–M) in control rats (H and K), vehicletreated nephritic rats (I and L), and ARB-treated nephritic rats (J and M). Magnification 3400. Treatment with ARB was associated with a significant decrease in the a-SMA score compared with vehicle-treated rats on day 28 (N). *P , 0.01 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN 1 ARB on day 7. #P , 0.01 versus control on day 28. §P , 0.01 versus GN on day 28. {P , 0.05 versus GN on day 28.
II, and TGF-b1 in nephritic glomeruli with or without ARB treatment, several experiments using culture supernatants of isolated nephritic glomeruli on day 28 after disease induction were performed (Fig 6). In accordance with immunohistochemical data, Western blot analysis showed a significant increase in the production of glomerular AGT protein in vehicletreated nephritic rats, whereas treatment with ARB attenuated the production of AGT protein significantly on day 28 of GN (Fig 6, A and B). Similarly, an ELISA examination showed that the Ang II concentration in culture supernatant was increased significantly in vehicle-treated nephritic rats (P , 0.05). Treatment with ARB reduced the concentration of Ang II significantly to almost the control level (P , 0.05; Fig 6, C). TGF-b1 levels were measured using a sandwich ELISA. In parallel with the Ang II concentration in culture supernatant, TGF- b1 levels
were increased significantly in vehicle-treated nephritic rats (307.92 6 14.31 pg/mL) compared with those in normal rats (60.77 6 0.74 pg/mL). Nephritic rats treated with ARB showed a decrease in the production of TGF-b1 (86.22 6 12.21 pg/mL; Fig 6, D). Glomerular expression of Nox2 and in situ production of O2$-. To investigate whether ROS plays a role in the
development and progression of this model of GN, the levels of Nox2 expression, a major component of NADPH oxidase, and O2$- production were studied (Fig 7). Strong Nox2 expression was detected in nephritic glomeruli, particularly in crescentic cells on days 7 and 28 in nephritic rats (Fig 7, B and E). Several Nox2-positive cells were also observed in the interstitial area in vehicle-treated nephritic rats on day 28. Treatment with ARB did not influence the level of glomerular Nox2 staining on day 7 but significantly decreased this level on day 28 in nephritic rats
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Fig 4. a-SMA protein expression in isolated glomeruli. Representative results of Western blot are shown (A and B). The samples were adjusted to a standard content (20 mg) and analyzed by Western blot using the mouse monoclonal anti-a-SMA antibody. Data for a-SMA protein levels were normalized by b-actin protein levels. Each band was scanned and subjected to densitometry and expressed as a fold increase relative to the results in control rats (C). The a-SMA protein level was increased in both vehicle- and ARB-treated nephritic rats on day 7, but it was significantly decreased in ARB treated nephritic rats on day 28. *P , 0.01 versus control on day 7. iP , 0.05 versus control on day 7. #P , 0.01 versus control on day 28. §P , 0.01 versus GN on day 28.
(P , 0.01) (Fig 7, C, F, and G). Furthermore, an examination of in situ O2$- production in nephritic glomeruli by DHE dye revealed that glomerular O2$production was increased significantly in vehicletreated day 7 nephritic rats and was further enhanced in day 28 nephritic rats (Fig 7, I, L, and N). Treatment of nephritic rats with ARB did not affect the level of glomerular O2$- production significantly in day 7 nephritic rats, whereas this treatment reduced O2$production to the level in control rats on day 28 (Fig 7, J, M, and N). Western blot analysis of isolated glomeruli confirmed that the level of Nox2 protein was increased significantly in both vehicle- and ARBtreated day 7 nephritic rats compared with that in control rats (Fig 8, A–C). A significant increase in Nox2 protein was still observed in vehicle-treated
nephritic rats on day 28, whereas this increased level of Nox2 protein was attenuated by approximately 53% in day 28 ARB-treated nephritic rats. Finally, double-IF staining in combination with antibody and DHE dye showed clearly that Nox2-positive or a-SMA-positive crescentic cells were similarly double stained with DHE dye, indicating that glomerular crescentic cells strongly produced O2$-, possibly via Nox activation in this rat model of GN (Fig 9). DISCUSSION
Intrarenal RAS activation has been implicated in the development and progression of GN and diabetic nephropathy.5-8 The current study suggested that
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Fig 5. Immunohistochemical study of AGT (A–F), Ang II (H–M) and AT1R (O–T) on days 7 (A–C, H–J, and O–Q) and day 28 (D–F, K–M and R–T). Arrows show crescents. Magnification 3400. Glomerular AGT expression was weak in normal control rats (A and D), and it was enhanced in mesangial cells and crescents in vehicletreated nephritic rats (B and E). Treatment with ARB decreased its expression significantly compared with that in vehicle-treated nephritic rats on day 28 (F and G). Enhanced staining of Ang II was observed mostly in the crescent area in nephritic rats on day 7 (I and J). ARB-treated nephritic rats showed significantly weak staining compared with that in vehicle-treated nephritic rats on day 28 (M and N). AT1 receptor was only weakly expressed in the glomeruli of normal control rats (O and R). In contrast, strong expression was observed in the crescent area (P and S). In vehicle-treated nephritic rats, the expression of AT1 receptor was significantly stronger on day 28 (S and U), but treatment with ARB reduced this expression significantly on day 28 (T and U). *P , 0.01 versus control on day 7. iP , 0.05 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN 1 ARB on day 7. §P , 0.01 versus GN on day 28. {P , 0.05 versus GN on day 28.
glomerular RAS activity leading to Ang II production play important roles in glomerular structural remodeling characteristic of progressive GN. Based on the results from biochemical analyses using isolated glomeruli, it seems likely that the beneficial molecular effect of ARB is caused by inhibition of the enhanced glomerular production of AGT and Ang II as well as the glomerular expression of Nox2, which is a major component of Nox (ROS-generating system) and TGF-b in nephritic glomeruli.
It is well known that glomeruli can generate Ang II at levels that are several orders of magnitude higher than that detected in circulation, which supports the existence of a glomerular RAS that can act independently of the systemic RAS.19 Recently, Singh et al16 reported using diabetic rat glomeruli that glomerular Ang II levels are increased because of an increase in AGT substrate and an increase in the formation of Ang II, suggesting that the development of diabetic nephropathy is associated with glomerular RAS activation and a resultant
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Fig 6. Effects of treatment with ARB on AGT (A and B), Ang II (C), and TGF-b1 (D) levels in supernatant from cell culture of isolated glomeruli on day 28. #P , 0.01 versus control. iP , 0.05 versus control. *P , 0.01 versus GN. §P , 0.05 versus GN. These parameters were increased in vehicle-treated nephritic rats. ARB treatment significantly attenuated those levels.
production of Ang II. Furthermore, a large body of evidence indicates that intrarenal RAS activity (Ang II activity) depends on the amount of renal AGT production and is associated with renal damage.25-27 Similarly, our previous immunohistochemical study found that the level of glomerular AGT staining paralleled those of glomerular Ang II staining and glomerular injury in patients with IgA nephropathy, indicating a close relationship between glomerular RAS activity and glomerular injury even in human GN.15 Collectively, our current data constitute substantial evidence of glomerular RAS activation in anti-GBM GN rats and that AGT expression plays an important role as a substrate for RAS activation in nephritic glomeruli. Ang II participates in not only systemic and glomerular hypertension but also proteinuria, inflammation, and abnormal pathologic remodeling in damaged kidney via AT1R signaling.7,28 Ang II enhances the infiltration of macrophages, which are key inflammatory cells, into damaged glomeruli, via the upregulation of adhesion molecules and monocyte chemoattractant protein1(MCP-1) by AT1R-expressing glomerular cells.29-31
Hypertension per se promotes the progression of renal disease by worsening glomerular injury and proteinuria.32 In the current study, treatment with ARB decreased the levels of SBP and proteinuria, and it prevented macrophage accumulation in inflamed glomeruli on days 7 and 28 in nephritic rats by inhibiting the action of Ang II, suggesting that changes in these parameters are at least in part involved in pathologic improvement in day 28 nephritic rats treated with ARB. Therefore, subsequent studies with Ang II-independent antihypertensive drugs that could induce a compatible reduction in BP with candesartan or MCP-1 null mice are required to explore the more direct role of glomerular RAS activity in glomerular injury in anti-GBM GN rats. The current study also suggested that locally produced Ang II plays a critical role in glomerular structural remodeling–the induction of TGF-b and ROS, which are representative mediators in progressive GN.1,3 Accumulating evidence suggests that both mediators contribute to activated mesangial cell (a-SMA-positive)-mediated ECM accumulation, leading to glomerulosclerosis, a hallmark for
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Fig 7. NADPH oxidase isoform Nox2 staining and in situ detection of superoxide (O2$-) in glomeruli of rats. Magnification 3400. Representative photographs of Nox2 staining on days 7 (A–C) and 28 (D–F) in control rats (A and D), vehicle-treated nephritic rats (B and E), and ARB-treated nephritic rats (C and F). Nox2 was markedly expressed in glomeruli and was particularly increased in crescentic cells on day 7 (B and C). It had spread to the interstitial area in vehicle-treated nephritic rats on day 28 (E). In contrast, treatment with ARB suppressed Nox2 staining significantly on day 28 (F and G). Representative fluorescence micrographs of glomeruli that were stained with the O2$--sensitive dye DHE (red fluorescence) on days 7(H–J) and 28 (K–M) in control rats (H and K), vehicle-treated nephritic rats (I and L), and ARB-treated nephritic rats (J and M) are shown. Semiquantitative assessment of the mean fluorescence intensity for glomerular O2$-content was detected on days 7 and 28 (N). No significant difference was found between vehicle-treated nephritic rats and ARB-treated nephritic rats on day 7 (I and J), but ARB treatment reduced O2$- production to the level in control rats on day 28 (M and N). * P , 0.01 versus control on day 7. †P , 0.01 versus GN on day 7. ‡P , 0.01 versus GN 1 ARB on day 7. § P , 0.01 versus GN on day 28.
progressive GN. RAS blockers significantly attenuate sclerotic lesions via the suppression of these mediators.1,3 We stress that ARB significantly inhibits crescent formation in nephritic rats, which is another pathologic feature of progressive GN. Recent studies regarding the molecular mechanisms of crescent formation suggested that increased TGF-b expression in nephritic glomeruli induces the transition of glomerular parietal epithelial cells to myofibroblasts (a-SMA-positive cells), the so-called epithelial mesenchymal transition (EMT), and thereby contribute to crescent formation in rat anti-GBM GN.22,33,34 Notably, in this study, the strong
expression of RAS components such as AGT, Ang II, and AT1R as well as Nox2, O2$-, and a-SMA in cellular and fibrocellular crescents was detected in nephritic rats. Double IF focusing on crescentic cells indicated that O2$--producing cells are Nox2-positive and a-SMA-positive crescentic cells. Nox2 is a main component of Nox, which is a major source of renal ROS.3 The long-term inhibition of Ang II action with ARB decreased the glomerular expression of these compounds as well as TGF-b production, and it induced histologic improvements on day 28. Because Ang II has been shown to induce ROS production via Nox activation in glomerular cells, and ROS is now
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Fig 8. Expression of Nox2 protein in isolated glomeruli. The representative results of Western blot are shown (A and B). The samples were adjusted to a standard content (20 mg) and analyzed by Western blot using the mouse monoclonal anti-Nox2 antibody. The data for Nox2 protein levels were normalized by b-actin protein levels. Each band was scanned and subjected to densitometry and expressed as a fold increase relative to the results in control rats (C). Along with the results of an immunohistochemical analysis, these findings show that Nox2 protein in isolated glomeruli was increased in both vehicle-treated nephritic rats and ARB-treated nephritic rats on day 7 (A and C). Treatment with ARB decreased Nox2 protein to the control level (B and C) on day 28. *P , 0.01 versus control on day 7. #P , 0.01 versus control on day 28. {P , 0.05 versus GN on day 28.
recognized as a potential inducer of EMT phenomena in various pathologic settings,35-37 it is conceivable that the enhanced production of Ang II in nephritic glomeruli induces ROS production and TGF-b expression and drives the EMT phenomena for crescent formation in GN. Overall, treatment with ARB may block the action of Ang II on changes in the cell phenotype responsible for glomerular structural remodeling and prevent glomerulosclerosis and crescent formation by inhibiting the expression of ROS and TGF-b in GN. In contrast with this beneficial effect of ARB on pathologic findings, no significant efficacy on renal function as evaluated with serum creatinine was observed in ARB-treated nephritic rats. The explanation for discrepancy between pathologic improvement and persistence of
impaired renal function under ARB treatment is uncertain but might be related to the well-known facts that extensive RAS blockade slightly lowers the GFR in rat and human with renal disease functionally via decreased systemic BP, and therefore it induces a slight increase of serum creatinine in treated subjects.38,39 Subsequent experiments to clarify this discrepancy are needed when one considers translating our findings into treatment strategies of patients with crescentic GN. Speculations. In conclusion, the current study provides evidence that glomerular RAS activation occurs after injury, and the level of Ang II produced is associated closely with glomerular pathologic alterations such as sclerosis and crescent formation, ROS production, TGF-b expression, and proteinuria in rat anti-GBM
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Fig 9. Representative double-fluorescence study with immunofluorescence staining of Nox2-positive cells (A and C) or a-SMA-positive cells (B and D) and DHE fluorescence in vehicle-treated nephritic rats on day 7 (A and B) and day 28 (C and D). Magnification 3400. The red area indicates nuclear staining of superoxide (O2$-)-producing cells. Merged images show that glomerular crescentic cells produced O2$- strongly, possibly via Nox activation.
GN. ARB may inhibit local RAS activity and Ang II signaling in nephritic glomeruli, and finally it attenuates the progression of GN, possibly by decreasing deleterious mediators such as ROS and TGF-b in GN. We are grateful to Dr. Quinn at Montana State University for kindly providing mouse anti-Nox2 antibody. We also thank Ms. Naomi Okamoto for her excellent technical assistance. Candesartan was kindly provided by Takeda Chemical Industries (Osaka, Japan).
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