Addition of the Antioxidant Probucol to Angiotensin II Type I Receptor Antagonist Arrests Progressive Mesangioproliferative Glomerulonephritis in the Rat
Shuji Kondo*,
Maki Shimizu*,
Maki Urushihara*,
Koichiro Tsuchiya,
Masanori Yoshizumi,
Toshiaki Tamaki,
Akira Nishiyama,
Hiroshi Kawachi,
Fujio Shimizu,
Mark T. Quinn||,
David J. Lambeth¶ and
Shoji Kagami*
* Pediatrics; Pharmacology, The Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima, Japan; Department of Pharmacology, Kagawa Medical University, Kagawa, Japan; Department of Cell Biology, Institute of Nephrology, Niigata University School of Medicine, Niigata, Japan; || Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana; and ¶ Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia
Address correspondence to: Dr. Shoji Kagami, Department of Pediatrics, The Institute of Health Bioscience, The University of Tokushima Graduate School, Kuramoto-cho-3-chome, Tokushima 770-8503, Japan. Phone: +81-88-633-7135; Fax: +81-886-31-8697; kagami{at}clin.med.tokushima-u.ac.jp
Received for publication May 20, 2005.
Accepted for publication December 29, 2005.
Angiotensin II (Ang II) and reactive oxidative species (ROS)that are produced by NADPH oxidase have been implicated in theprogression of glomerulonephritis (GN). This study examinedthe effect of simultaneously interrupting Ang II and ROS withan Ang II receptor blocker (ARB), candesartan, and a free radicalscavenger, probucol, in a model of progressive mesangioproliferativeGN induced by the injection of anti–Thy-1 antibody intouninephrectomized rats. Nephritic rats were divided into fourgroups and given daily oral doses of the following: Vehicle,1% probucol diet, 70 mg/L candesartan in drinking water, andprobucol plus candesartan. These treatments lasted until day56. Vehicle-treated nephritic rats developed progressively elevatedproteinuria and glomerulosclerosis. Candesartan kept proteinuriasignificantly lower than vehicle or probucol. The addition ofprobucol to candesartan normalized urinary protein excretion.Increases in BP in nephritic rats were lowered by these treatments,except with probucol. It is interesting that both glomerularcell number and glomerulosclerosis were significantly decreasedby candesartan and normalized by the addition of probucol. Immunohistochemicalstudies for TGF-1, collagen type I, and fibronectin revealedthat the combined treatment abolished glomerular fibrotic findingscompared with candesartan. In addition, glomerular expressionof NADPH oxidase components and superoxide production suggestedthat the combined treatment completely eliminated NADPH oxidase–associatedROS production. In conclusion, our study provides the firstevidence that the antioxidant probucol, when added to an AngII receptor blockade, fully arrests proteinuria and diseaseprogression in GN. Furthermore, the data suggest that NADPHoxidase–associated ROS production may play a pivotal rolein the progression of GN. The combination of probucol and candesartanmay represent a novel route of therapy for patients with progressiveGN.
The marked accumulation of extracellular matrix (ECM), includingcollagens and fibronectin, in glomeruli is a hallmark in thedevelopment of human and experimental progressive glomerulonephritis(GN) (1–4). TGF-1, a prime fibrogenic factor, stimulatesthe deposition of ECM by mesangial cells and thereby may induceglomerulosclerosis (2,5).
Recently, the interventions with angiotensin II (Ang II) typeI receptor blocker (ARB) and/or angiotensin-converting enzymeinhibitor (ACEI) have shown their protective potential againsthuman nephropathies, including IgA nephropathy and diabeticnephropathy, and progressive experimental models such as mesangioproliferativeGN and nephrectomized rats (6–10). These clinical andexperimental in vivo studies have strongly suggested that therenoprotective effect of these antihypertensive agents resultsfrom not simply the inhibition of its hemodynamic action butalso blockade of its hemodynamic-independent actions. In fact,many studies, including our previous in vitro studies, havedisclosed the mechanisms and the role of Ang II–inducedECM deposition through mesangial cells (11–13). Ang IIstimulates ECM synthesis through the induction of TGF-1 expressionin cultured rat glomerular mesangial cells (11) and affectsthe plasminogen activator/plasminogen activator inhibitor-1system to result in accelerated deposition of ECM (12), whichconstitutes evidence that Ang II can act as a potent fibroticmolecule independent of its effects on BP. Although the currenttherapy by inhibition of Ang II retards renal fibrosis, it isnot enough to prevent completely the progression of GN. Thissuggests that other fibrogenic factors such as PDGF-BB, TNF-,and IL-1 might also be involved in the progression of GN (3,14).For maximizing the therapeutic potential of antifibrotic treatment,additional therapy that is aimed at suppression of these fibrogenicfactors and their related signaling pathways may be required.
Recently, reactive oxygen species (ROS) that are produced byNADPH oxidase has been implicated in the development and progressionof GN (15–17). The administration of various natural orsynthetic antioxidants has been shown to be beneficial in theprevention and attenuation of renal scarring in numerous animalmodels of renal disease (18). These compounds include vitamins,N-acetylcysteine, -lipoic acid, lipid-lowering agents, and manyothers. Probucol is a lipid-soluble, cholesterol-lowering drugwith potent antioxidant properties that has been shown to scavengelipid peroxidation (19). This agent seems to play a role ineliminating ROS produced by NADPH oxidase (20).
It is interesting that Ang II is considered a representativestimulator of NADPH oxidase activity that leads to the enhancedproduction of superoxide (O2·–) in several celllines, including mesangial cells and vascular smooth musclecells. These studies have also demonstrated that NADPH oxidasesare composed of gp91phox-homolog and produce ROS including O2·–not only in phagocytes but also in nonphagocytic cells (21–25).In addition, these studies indicated that NADPH oxidase playsan important role as a cell-signaling molecule in addition tobeing a classical mediator of tissue injury (21,22). Whereasgp91phox, which has been called Nox2 (for NADPH oxidase), isgenerally expressed in inflammatory cells such as leukocytes,the Nox family is also composed of various members from Nox1to Nox5, which are expressed in noninflammatory cells, includingmesangial cells and podocytes. In particular, Nox1, Nox2, andNox4 have been investigated in Ang II–induced ROS productionin renal and cardiovascular diseases (21,22,25). Besides, thelevels of these Nox proteins are associated with the activityand O2·– production (21,22). Other fibrogenic factors,such as PDGF-BB, TNF-, and IL-1, all of which are involved inthe progression of GN, also stimulate ROS production in mesangialcells, although the detailed mechanism of their production remainunknown (15,23,24). Therefore, we hypothesized that the blockadeof both the action of Ang II and ROS production associated withNADPH oxidase activation might be a novel strategy for preventingthe progression of glomerulosclerosis. We examined whether simultaneouslyinterrupting Ang II and ROS with the ARB candesartan and probucolcould reduce proteinuria and disease progression in a rat modelof irreversible mesangioproliferative GN.
Antibodies and Reagents
Previously characterized mouse mAb that specifically recognizedNox2 (clone 54.1) and rabbit polyclonal antibody against Nox4were used in this study (26,27). Horseradish peroxidase–conjugatedgoat anti-mouse IgG (H+L; Bio-Rad, Hercules, CA) and horse anti-rabbitIgG (H+L; Cell Signaling Technology Inc., Beverly, MA) wereused for Western blot analysis. For immunohistochemistry, previouslycharacterized rabbit polyclonal anti–TGF-1 antibody wasused, and rabbit antibodies against collagen type I (COL I)and fibronectin (FN) were obtained from Chemicon InternationalInc. (Temecula, CA) (8).
Experimental Design
All experimental procedures were performed according to theguidelines for the care and use of laboratory animals establishedby National Institutes of Health and the Institute for AnimalExperimentation, the University of Tokushima Graduate School.A progressive model of mesangioproliferative GN was inducedas described previously, with some modifications (8). Briefly,rats were uninephrectomized and 1 wk later received an intravenousinjection of 1.0 ml of PBS that contained 2 mg of the nephritogenicanti–Thy-1 mAb 1-22-3. Nephritic rats were divided intofour groups (n = 6) and given daily oral doses as follows: (1)Vehicle, (2) 1% probucol diet, (3) 70 mg/L candesartan in drinkingwater, or (4) probucol plus candesartan. The dose concentrationof probucol in the rat diet and candesartan in drinking waterwere selected on the basis of previous reports (28–31).The therapeutic regimen that was used in our study was pharmacologicallyvalid and efficient according to the manufacturer’s informationfor probucol. For achieving a therapeutically adequate concentrationof probucol, this reagent was given 7 d before the injectionof mAb 1-22-3. The dose of candesartan (70 mg/L) that was usedin this experiment was high enough (10 mg/kg per d) to inhibitAng II receptor binding to kidney tissue (28,29). Candesartanwas started on the same day as disease induction (day 0). Sixrats in each group were killed at 7 d and 56 d after the injectionof mAb 1-22-3. As controls, six rats received injections ofPBS 1 wk after unilateral nephrectomy and were killed on days7 and 56.
Urinary Protein Excretion, Blood Parameters, and BP
Urine was periodically collected from the rats in a metaboliccage during the course of GN. The amount of urinary proteinexcretion was measured by the Bradford method (Bio-Rad, Oakland,CA). The amount of food consumed and water intake were measuredand calculated on day 56. At the end of the study period, serumconcentrations of blood urea nitrogen, creatinine, total cholesterol,and nonesterified fatty acid were measured using the reagentsthat were manufactured by WAKO Chemical Industries (Osaka, Japan).Serum total protein was measured by a BCA protein assay (Pierce,Rockford, IL). The serum level of triglyceride was assessedwith a Lipidos Kit (TOYOBO, Osaka, Japan). Systolic BP (SBP)was measured noninvasively by the tail-cuff method (32). Concentrationsof serum probucol were measured by HPLC at the laboratoriesof Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan).
Histology and Immunohistochemistry
The right kidney of each rat was removed immediately, fixedin 10% buffered formalin and embedded in paraffin, and 4-µmsections were stained with periodic acid-Schiff reagent. Glomerularcell number was assessed on the basis of total glomerular cellcount per glomerular cross-section. It was counted in 30 glomeruliper kidney in a periodic acid-Schiff–stained section,and the mean number of cells per glomerulus was calculated.A pathologist who was blinded to other findings semiquantitativelyanalyzed the glomerulosclerosis score. The percentage of eachglomerulus that was occupied by mesangial matrix was estimatedand assigned a code as follows: 0 = absent, 0.5 = 1 to 5%; 1= 5 to 25%; 2 = 25 to 50%; 3 = 50 to 75%; or 4 = >75%.
Frozen sections (3 µm) were incubated with anti–COLI antibodies, anti-FN antibodies, and anti–TGF-1 Ab for1 h and then with FITC-conjugated secondary antibodies. To evaluatethe level of glomerular staining with each antibody, we performedsemiquantitative analysis as follows: 0 = diffuse, very weak,or absent mesangial staining; 1+ = 1 to 25% of focally increasedmesangial staining; 2+ = 25 to 50% of glomerular tuft demonstratingstrong mesangial staining; 3+ = 50 to 75% of glomerular tuftdemonstrating strong mesangial staining; and 4+ = >75% ofglomerular tuft stained strongly. For each kidney section, 30glomeruli were selected at random and evaluated by the sameblinded pathologist; the mean value per section was calculated(33).
Formalin-fixed tissue sections (3 µm) were deparaffinizedwith xylene and rehydrated with a graded series of ethanol.Endogenous peroxidase was blocked with hydrogen peroxide, andthe samples were heated at 121°C for 15 min in 0.01 mol/Lcitrate buffer (pH 6.0). The sections were incubated with anti–ED-1mAb (Serotec, Kinglington, UK) diluted in PBS that contained1% BSA at 4°C for 24 h. After being washed with PBS, thesections were incubated with biotinylated secondary antibody,avidin-biotin-peroxidase complex (ABC Elite; Vector Laboratories,Burlingame, CA), and then 3,3'-diaminobenzidine (Dojindo, Kumamoto,Japan). Each section was counterstained with Mayer’s hematoxylin(Wako, Tokyo, Japan), dehydrated, and coverslipped. The numberof ED-1–positive cells was counted and calculated in 30full-size glomeruli (34).
Western Blotting
Western blotting was performed as described previously (35).Total protein was extracted from glomeruli that were isolatedby the sieving method (11). Protein samples (30 µg) wereseparated by 12.5% SDS-PAGE and transferred to nitrocellulosemembranes. The membranes were probed with mouse monoclonal anti-Nox2antibodies, rabbit polyclonal anti-Nox4 antibodies, and mousemonoclonal anti–-actin antibodies (Sigma Chemical Co.,St. Louis, MO) as a standard and then incubated with an horseradishperoxidase–conjugated secondary antibodies. Immunoreactiveproteins were detected with an enhanced chemiluminescence detectionsystem (Amersham Corp., Arlington Heights, IL). Densitometricanalysis was performed using an LKB UltroScan XL apparatus (PharmaciaLKB Biotechnology, Uppsala, Sweden).
In Situ Superoxide Production
As described elsewhere, in situ production of O2·–was determined using 30-µm sections of frozen tissue thatwere incubated with dihydroethidium (DHE; 10 µmol/L) inPBS for 30 min at 37°C in a humidified chamber that wasprotected from light. DHE is oxidized upon reacting with O2·–to ethidium bromide, which binds to DNA in the nucleus and fluorescesred. To detect ethidium bromide, we used a 543-nm He-Ne lasercombined with a 560-nm long-pass filter. Glomerular O2·–content was estimated by analyzing the mean fluorescence intensityin glomeruli using National Institutes of Health imaging software(27). To identify O2·–-producing cells in glomeruli,we performed immunofluorescent staining combined with DHE assay(red) and FITC labeling (green) using anti–ED-1 mAb andanti–Thy-1 mAb as markers of macrophages and mesangialcells, respectively.
Statistical Analyses
The values are expressed as mean ± SEM. The differenceswere evaluated with the Stat Mate III software package (ATMSCo., Ltd., Tokyo, Japan). For urinary protein excretion, bloodparameters, and BP, statistical significance was evaluated usinga t test (8,33). The data for histology and immunohistochemistrywere analyzed using the nonparametric Kruskal-Wallis test formultiple comparisons (8). The statistical significance levelwas defined as P < 0.05.
Effect of Probucol and/or ARB on Urinary Protein Excretion, BP, and Blood Parameters
As shown in Table 1, total food intake, total water intake,and body weight were comparable among all groups. The serumlevel of probucol was 11.2 ± 3.8 µg/ml in probucol-treatednephritic rats, 4.5 ± 0.9 µg/ml in probucol plusARB-treated nephritic rats, and below the measurement limitin rats that were not treated with probucol, indicating thatthe serum level of probucol was significantly elevated by a1% probucol diet.
Table 1. Total food intake, total water intake, body weight, and the level of serum probucol in rats that had GN on day 56 and had been treated with probucol and/or ARB (candesartan)a
There was no difference in 24-h urinary protein excretion amongall groups before the induction of GN. As shown in Table 2,proteinuria was significantly increased in all nephritic ratson day 7 after disease induction. In vehicle-treated nephriticrats, proteinuria gradually increased until the end of the experiment.Treatment with ARB or probucol plus ARB reduced proteinuriacompared with vehicle treatment from 2 wk after the inductionof nephritis. Conversely, treatment with probucol did not significantlyreduce proteinuria. Treatment with probucol plus ARB significantlyprevented proteinuria compared with treatment with probucolor ARB on days 7 and 56.
Table 2. Time course of urinary protein excretion (mg/d) in rats that had GN on day 56 and had been treated with probucol and/or ARB (candesartan)a
SBP of vehicle-treated nephritic rats on day 56 was elevated,although not significantly. Treatment with ARB or probucol plusARB significantly reduced SBP compared with vehicle treatment.Conversely, treatment with probucol did not lower SBP (Figure 1).
Figure 1. Systolic BP in rats that had glomerulonephritis (GN) on day 56 and had been treated with probucol and/or angiotensin II receptor blocker (ARB; candesartan). , nephrectomized control rats; , vehicle-treated GN rats; , probucol-treated GN rats; , ARB-treated GN rats;
, probucol + ARB–treated GN rats. Data are mean ± SEM. *P < 0.01 versus control; P < 0.01 versus GN; P < 0.01 versus GN+probucol.
Blood parameters on day 56 are shown in Table 3. Remarkablehypoproteinemia was not detected in any of the groups. A significantelevation of serum total protein was observed in probucol plusARB-treated nephritic rats. The level of blood urea nitrogenin vehicle-treated nephritic rats was elevated, whereas thelevel in probucol plus ARB-treated nephritic rats was significantlylowered to the level in the control. The serum level of creatininein probucol plus ARB-treated nephritic rats was slightly lowerthan that in vehicle-treated nephritic rats, although the differencewas not statistically significant. Hyperlipidemia was observedin vehicle-treated nephritic rats, as shown in Table 3. A significantimprovement of hyperlipidemia was not observed in rats withprobucol or ARB treatment. Combination treatment with probucoland ARB significantly decreased the levels of triglyceride andnonesterified fatty acid, respectively.
Table 3. Serum levels of total protein, BUN, creatinine, T-CHO, TG, and NEFA in rats that had GN on day 56 and had been treated with probucol and/or ARB (candesartan)a
Renal Histology
The effects of treatment with probucol and/or ARB on renal histologywere examined on days 7 and 56 after disease induction. Noneof the treatments significantly affected morphologic changessuch as mesangial cell proliferation accompanied by mild mesangialmatrix accumulation and segmental mesangiolytic lesions seenon day 7 in vehicle-treated nephritic rats (data not shown).Representative light micrographs of glomerular lesions on day56 after each treatment are shown in Figure 2. The most strikingglomerular changes that were observed in vehicle-treated nephriticrats were severe mesangial proliferation and marked ECM accumulation,which were accompanied by adhesion to Bowman’s capsule,glomerulosclerosis, and tubulointerstitial fibrosis (Figure 2B).Treatment with ARB improved these pathologic findings (Figure 2D).Furthermore, glomerular injury could not be detected inprobucol plus ARB-treated nephritic rats (Figure 2E).
Figure 2. Light micrographs showing the effects of probucol and/or ARB (candesartan) on glomerular structural changes in rats with GN on day 56. (A) Control. (B) Vehicle-treated GN. (C) Probucol-treated GN. (D) ARB-treated GN. (E) Probucol + ARB–treated GN. Periodic acid-Schiff–stained sections. Magnification, x200.
To examine the effects of each treatment on nephritic glomeruli,we performed histologic and histochemical analyses on days 7and 56 of the study period (Figure 3). On day 7, the glomerularcell number and the glomerulosclerosis score were significantlyincreased to similar levels in all nephritic rats that receivedeach treatment (Figure 3, A and B). Conversely, the increasednumber of ED-1–positive macrophages seen in day 7 vehicle-treatednephritic rats was significantly reduced in rats that were givenprobucol and/or ARB (Figure 3C). On day 56, a striking increasein both glomerular cell number and the glomerulosclerosis scorewas observed in vehicle-treated nephritic rats (Figure 3, Dand E). Treatment with ARB but not probucol led to a considerablereduction in both parameters. Furthermore, a remarkable reductionof these parameters to the control level was achieved in probucolplus ARB-treated nephritic rats. There were no significant changesin the number of ED-1–positive macrophages in any of thegroups (Figure 3F).
Figure 3. The glomerular cell number (A and D), the glomerulosclerosis score (B and E), and the number of ED-1–positive macrophages (C and F) in rats that had GN on days 7 (A through C) and 56 (D through F) and had been treated with probucol and/or ARB (candesartan). , nephrectomized control rats; , vehicle-treated GN rats; , probucol-treated GN rats; , ARB-treated GN rats;
, probucol + ARB–treated GN rats. Data are mean ± SEM. *P < 0.01 versus control; P < 0.01 versus GN; P < 0.01 versus GN+ARB.
Renal Expression of TGF-1 and ECM Proteins
Representative immunostaining micrographs on day 56 after diseaseinduction showed that a marked increases in the expression ofTGF-1, COL I, and FN were observed in the mesangial area in>90% of the glomeruli from vehicle-treated nephritic rats(Figure 4). Treatment with ARB significantly reduced glomerularimmunofluorescence staining for TGF-1, COL I, and FN, whereastreatment with probucol did not. The administration of probucolplus ARB improved staining to the control level. Glomerularstaining scores for TGF-1, COL I, and FN are shown in Figure 5.A semiquantitative analysis on day 7 revealed that the glomerularexpression of COL I and FN but not TGF-1 was upregulated inall nephritic rats regardless of treatment (Figure 5A). Conversely,on day 56, the levels of immunofluorescence for these factorsindicated that combined treatment with probucol plus ARB normalizedimmunofluorescence intensity for the expression of TGF-1 andECM proteins to the control level, in parallel with histologicimprovement (Figure 5B).
Figure 4. Representative immunohistochemical micrographs showing the effects of probucol and/or ARB (candesartan) on the expression of TGF-1, collagen type I (COL I), and fibronectin (FN) in rats with GN on day 56. Magnification, x200.
Figure 5. Semiquantitative assessment of the expression of TGF-1, COL I, and FN in rats that had GN on days 7 (A) and 56 (B) and had been treated with probucol and/or ARB. , nephrectomized control rats; , vehicle-treated GN rats; , probucol-treated GN rats; , ARB-treated GN rats;
, probucol + ARB–treated GN rats. Data are mean ± SEM. *P < 0.01 versus control; #P < 0.05 versus control; P < 0.01 versus GN; P < 0.01 versus GN+ARB; P < 0.05 versus GN+ARB.
Glomerular Expression of NADPH Oxidase Components and In Situ Production of Superoxide
To examine whether probucol and/or ARB affected the levels ofNox2 and Nox4 protein, the major component of NADPH oxidase,we performed Western blotting on days 7 and 56 after diseaseinduction (Figure 6). On day 7, a significant increase in Nox2protein was observed in only vehicle-treated nephritic rats,whereas Nox4 protein was significantly increased in all nephriticrats regardless of treatment (Figure 6, A through C). The increasedlevel of Nox2 protein in vehicle-treated nephritic rats seemedto be slightly suppressed by probucol and/or ARB administrationeven though this effect was NS (Figure 6, A and B). On day 56,the levels of Nox2 and Nox4 protein in vehicle-treated nephriticrats were upregulated 4.6- and 1.6-fold, respectively (Figure 6,D through F). This increased level of Nox2 protein was significantlydecreased in probucol-treated nephritic rats and was attenuatedby approximately 50% in ARB- or probucol plus ARB-treated nephriticrats (Figure 6, D and E). In addition, ARB or probucol plusARB treatment tended to decrease the level of Nox4 in nephriticrats, although these changes were not statistically significant(Figure 6, D and F).
Figure 6. Expression of Nox2 and Nox4 in rats that had GN on days 7 (A through C) and 56 (D through F) and had been treated with probucol and/or ARB (candesartan). Total protein was extracted from glomeruli that were isolated by the sieving method. The samples were adjusted to a standard content (30 µg) and analyzed by Western blotting using the mouse monoclonal anti-Nox2 antibody and rabbit polyclonal anti-Nox4 antibody. (A and D) Representative Western blotting is shown. (B, C, E, and F) Results of densitometric analysis. The results represent the mean ± SEM. , nephrectomized control rats; , vehicle-treated GN rats; , probucol-treated GN rats; , ARB-treated GN rats;
, probucol + ARB–treated GN rats. *P < 0.01 versus control; P < 0.05 versus GN; P < 0.01 versus GN.
For evaluation of the effect of probucol and/or ARB on glomerularO2·– content, in situ superoxide production ofdays 7 and 56 was detected by DHE (Figure 7). A significantincrease in glomerular O2·– production was observedin only vehicle-treated nephritic rats on day 7 (Figure 7F).Treatment of nephritic rats with probucol or ARB seemed to decreaseits production, although it was numerically NS. Treatment withprobucol plus ARB significantly attenuated glomerular O2·–production to near the control level. On day 56, O2·–production was strikingly enhanced in vehicle-treated nephriticrats, and this was significantly reduced to the control levelin probucol- or ARB-treated nephritic rats (Figure 7, B throughD and G). Of note, combined treatment completely eliminatedthe glomerular O2·– content from day 56 nephriticglomeruli (Figure 7, E and G).
Figure 7.In situ detection of superoxide (O2·–) in glomeruli of rats that had GN and had been treated with probucol and/or ARB (candesartan). Representative fluorescence micrographs of glomeruli that were stained with the O2·–-sensitive dye dihydroethidium (DHE; red fluorescence) were from nephrectomized control rats (A), vehicle-treated GN rats (B), probucol-treated GN rats (C), ARB-treated GN rats (D), and probucol plus ARB-treated GN rats (E) on day 56 after disease induction. Semiquantitative assessment of mean fluorescence intensity for glomerular O2·– content detected on days 7 (F) and 56 (G) in all groups that received each treatment. , nephrectomized control rats; , vehicle-treated GN rats; , probucol-treated GN rats; , ARB-treated GN rats;
, probucol + ARB–treated GN rats. Data are mean ± SEM. *P < 0.01 versus control; P < 0.01 versus GN; P < 0.01 versus GN+probucol or ARB; P < 0.05 versus control.
Next, double-immunofluorescent staining of mesangial cells ormacrophages combined with the detection of O2·–by DHE on frozen sections was performed to identify the O2·–-producingcells in day 56 nephritic glomeruli. Figure 8, A through C,shows that immunostaining of Thy-1–positive mesangialcells was co-localized with most of the nuclear staining ofO2·–-producing cells. Staining of O2·–was also detected on a few ED-1–positive macrophages thathad infiltrated nephritic glomeruli (Figure 8, D through F).These results suggest that mesangial cell O2·–production, possibly via NADPH oxidase activation, is involvedmainly in the level of glomerular injury, including morphologicparameters in this rat model of GN.
Figure 8. Representative double-fluorescence study with immunofluorescence staining of Thy-1–positive mesangial cells or ED-1–positive macrophages and dye DHE fluorescence in vehicle-treated nephritic rats on day 56. In A and D, green indicates Thy-1–positive mesangial cells and ED-1–positive macrophages, respectively. In B and E, red indicates nuclear staining of superoxide (O2·–)-producing cells. In C and F, merged images show O2·–-producing mesangial cells and macrophages, respectively. Magnification, x400.
In this study, we demonstrated that the combined administrationof probucol and ARB fully arrests proteinuria and the progressionof severe mesangioproliferative GN in the rat. It seems likelythat the beneficial effect of such combined treatment is dueto the synergistic action of Ang II inhibition with a receptorantagonist and the elimination of ROS with a radical scavenger.
At present, blocking of Ang II action is one of the most usefulstrategies for treating chronic progressive mesangioproliferativeGN, including IgA nephropathy. ARB and/or ACEI limits proteinuriaand reduces the decline in GFR and the risk for ESRD more effectivelythan other antihypertensive treatments (6,36–38). Severalin vivo animal experiments have indicated that the effect ofARB and/or ACEI in preventing the progression to end-stage renalfailure is due to the ability of ARB and ACEI to modulate theeffects of Ang II on the production of TGF-1 and ECM as wellas on glomerular hypertension (8,10,39). In vitro studies havedemonstrated that Ang II stimulates TGF-1 expression, whichpromotes the synthesis of ECM proteins and inhibits ECM turnoverby decreasing protease activity and increasing the productionof protease inhibitors (11–13). Our findings that an ARB,candesartan, considerably reduced proteinuria, the level ofTGF-1, and ECM accumulation at the late stage in this rat modelof GN and finally inhibited the progression of glomerulosclerosisare consistent with the above concept.
ROS has been implicated in the development and progression ofGN (15–17,40,41). NADPH oxidase is a major source of ROSin phagocytes as well as non-phagocytic cells, including fibroblasts,vascular smooth muscle cells, and glomerular cells (21–25).NADPH oxidases are composed of gp91phox-homolog, called Noxprotein and several subunits that contain p22phox, p47phox,p67phox, and small GTPase rac. Nox2 indicates that gp91phox,the main subunit of the phagocyte NADPH oxidase, and Nox4, whichseems to share the same overall structure as Nox2, are abundantin the vascular system, kidney cortex, and mesangial cells (21,22,25).In fact, an increase in glomerular ROS and NADPH-dependent oxidaseactivity has been reported in an acute model of Thy-1 GN thatwas induced by a single injection of anti–Thy-1 antibody(15). In addition, our data clearly showed that an increasein glomerular O2·– content and Nox2 and 4 proteinswas detected in the early and advanced stages of chronic progressiveGN in rats (17). Recently, it was shown that ROS that is producedby NADPH oxidase activates intracellular signal molecules, suchas mitogen-activated protein kinases (MAPK) and activator protein-1,and thereby might contribute to cell proliferation, ECM production,and ECM remodeling involved in the progression of GN (42). Indeed,Budisavljevic et al. (16) suggested that ROS might mediate glomerularinjury by activating extracellular signal–regulated kinasethat could induce mesangial cell proliferation and increaseof glomerular TGF-1 expression in acute Thy-1 GN. We have alsoreported in renal injury in Dahl salt-sensitive hypertensiverats that the renal expression of p22phox and Nox1 and phosphorylationof MAPK are associated with the level of progressive scleroticand proliferative glomerular changes (43). Tempol, a superoxidedismutase, ameliorated not only those glomerular changes butalso the expression of p22phox, Nox1, and MAPK activities, suggestingthat ROS, generated by NADPH oxidase, contributes to the progressionof renal injury through MAPK activation in Dahl salt-sensitivehypertensive rats (43).
Of note, Ang II as well as other fibrogenic factors, such asPDGF-BB, TNF-, and IL-1, are involved in ROS production (3,14,44).We showed in this study that ARB significantly decreased thelevel of glomerular O2·– production on day 56.Western blot experiments indicated that this reduction of glomerularO2·– content is probably due to the decreased expressionof glomerular Nox2 protein by ARB, because several reports basedon cell culture studies have demonstrated that Ang II stimulatedthe protein and mRNA expression of Nox2 (45,46). Therefore,the reduction of glomerular O2·– content in GNby ARB might be linked to some of the renoprotective effectsthat are induced by Ang II blockade.
Probucol, a cholesterol-lowering drug with potent antioxidantproperties, reduced glomerular O2·– productionin GN to almost the normal level, although a significant ameliorationof proteinuria, glomerular pathology, and expression of TGF-1and ECM was not seen in probucol-treated nephritic rats. Probucolseems to act mainly as an antioxidant in nephritic rats becausethe level of serum cholesterol was not significantly reduced.The level of Nox2 protein was slightly decreased on day 7 andsignificantly downregulated on day 56 by probucol. This suppressiveeffect on Nox2 might lead to significant O2·– reductionin long-term probucol-treated nephritic rats via coordinationwith the action as a radical scavenger.
The combination of probucol and ARB not only completely eliminatedO2·– production but also normalized urinary proteinexcretion and the expression of TGF-1 and ECM seen in GN, resultingin full prevention of the progression of GN. Long-term combinationtherapy with ARB and probucol may confer a strong, synergisticantioxidative action on nephritic glomeruli by inhibiting Nox2protein expression associated with NADPH oxidase activity andby scavenging glomerular O2·– content and therebyenhance the renoprotective effect of ARB against the progressivenature of the disease in this rat model of GN. It is interestingthat HMG-CoA reductase inhibitors or statins, which are cholesterol-loweringagents, also show strong antioxidant ability involved in theinhibition of Rac1 geranylgeranylation and enhance the beneficialeffects of RAS inhibition on several types of organ damage (47,48).Nie et al. (49) reported in a model of CCl4-induced liver fibrosisthat pitavastatin enhances the antifibrogenesis effects of candesartan.Pitavastatin significantly enhanced the suppressive effectsof ARB on TGF-1 expression and fibrogenesis, whereas it alonedid not affect liver fibrogenesis or TGF-1 expression. Regardingthe effect of statins on glomerular injury, Zoja et al. (39)reported that therapy with either ACEI and ARB or cerivastatinhad limited effects, but combination therapy with these agentsfurther reduced glomerulosclerosis and urinary protein excretionin a rat model of Heymann nephritis. The expression of TGF-1was significantly decreased by triple combined therapy, whereasACEI, ARB, and cerivastatin did not significantly affect TGF-1expression in GN. Although neither study mentioned substantialchanges in O2·– production in damaged tissues bystatins, several studies have demonstrated that statins reducedoxidative stress in damaged tissues (47,48). Therefore, thecombination of an antioxidant and Ang II blockade might be auseful strategy for preventing the progression of chronic GN.Further investigations, including in vitro studies, are neededto explore the molecular mechanisms that are responsible forthe inhibitory effects of combined therapy on the progressionof GN by especially focusing on the signal pathways that leadto the suppression of the expression of TGF-1.
Probucol is a well-established pharmacologic agent that hasbeen shown to reduce restenosis after coronary balloon angioplastyby improving vascular remodeling in humans (50). Furthermore,Haas et al. (51) reported that probucol also had favorable renaleffects in membranous nephropathy in a clinical study. Therefore,further studies on rat models of progressive GN and patientswith chronic GN, such as IgA nephropathy and diabetic nephropathy,will be necessary to address whether combined therapy with probucoland ARB could have clinical significance.
Our study provides important evidence that the antioxidant probucolwhen added to ARB fully arrests proteinuria and the progressionof GN. The data suggest that the beneficial effects of thiscombined therapy might be mediated through the elimination ofROS that is produced mainly by NADPH oxidase in GN. The combinationof probucol and ARB, both of which are frequently used in dailyclinical practice, may represent a novel route of therapy forpatients with progressive GN.
Acknowledgments
This work was supported by grants from the Japanese Ministryof Welfare (16591035).
Parts of this work were published in abstract form at the 37thAnnual Meeting of the American Society of Nephrology; October28 to November 1, 2004; St. Louis, MO.
We are grateful to Dr. Masuko Ushio-Fukai and Dr. Tohru Fukai(Emory University) and Dr. Katsuyuki Matsui (Teikyo University)for helpful discussions. We also thank Dr. Masanori Takamatsuand Ms. Naomi Okamoto for excellent technical assistance. Probucoland candesartan were kindly provided by Otsuka PharmaceuticalCo. Ltd. and Takeda Chemical Industries (Osaka, Japan), respectively.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
1, collagen type I, and fibronectin revealed that the combined treatment abolished glomerular fibrotic findings compared with candesartan. In addition, glomerular expression of NADPH oxidase components and superoxide production suggested that the combined treatment completely eliminated NADPH oxidase–associated ROS production. In conclusion, our study provides the first evidence that the antioxidant probucol, when added to an Ang II receptor blockade, fully arrests proteinuria and disease progression in GN. Furthermore, the data suggest that NADPH oxidase–associated ROS production may play a pivotal role in the progression of GN. The combination of probucol and candesartan may represent a novel route of therapy for patients with progressive GN. 
, and IL-1
, nephrectomized control rats; 
, vehicle-treated GN rats; 
, probucol-treated GN rats; 
, probucol + ARB–treated GN rats. Data are mean ± SEM. *P < 0.01 versus control; 
