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Original Article Triptolide attenuates renal interstitial fi brosis in rats with unilateral ureteral obstructionnep_1359200.210 XIAO-PENG YUAN, XIAO-SHUN HE, CHANG-XI WANG, LONG-SHAN LIU and QIAN FU Organ Transplant Centre, First Affi liated Hospital, Sun Yat-sen University, Guangzhou, China KEY WORDS: macrophages, mycophenolate mofetil, myofi broblast, renal fi brosis, triptolide, unilateral ureteral obstruction. Correspondence: Dr Chang-Xi Wang, Organ Transplant Centre, First Affi liated Hospital, Sun Yat-sen University, 58 Zhongshan 2nd Road, Guangzhou 510080, China. Email: wcx6363 Accepted for publication 25 May 2010. Accepted manuscript online 7 June 2010. doi:10.1111/j.1440-1797.2010.01359.x SUMMARY AT A GLANCE In this study, triptolide was reported to reduce infl ammation and fi brosis in an experimental model of unilateral ureteral obstruction. Although the precise mode of action is not clear in the study, the data is interesting because triptolide has been used in China for the treatment of glomerulonephritis. ABSTRACT: Aim:Extracts of Tripterygium wilfordii Hook F. have been used to treat glomerulonephritis for more than 30 years in China. Most of the anti- infl ammatory and immunosuppressive activities of these extracts can be attributed to triptolide (Trip). The present study was to investigate the effect of Trip on renal interstitial fi brosis in a model of unilateral ureteral obstruction (UUO). Methods:UUO or sham-operated rats were randomly assigned to receive mycophenolate mofetil (MMF), Trip or vehicle and were killed on days 7 and 14 after UUO or sham operation. Kidney specimens were fi xed for immu- nohistochemistry for myofi broblasts (a-smooth muscle actin, a-SMA), macrophages (ED-1), monocyte chemoattractant protein-1 (MCP-1) and osteopontin. Interstitial collagen deposition and amounts of transforming growth factor-b1 (TGF-b1) were determined by Sirius red staining and enzyme-linked immunosorbent assay, respectively. The mRNA expression of TGF-b1, connective tissue growth factor (CTGF), MCP-1 and osteopontin were measured by real-time polymerase chain reaction analysis. Results:The scores for the density of a-SMA- and ED-1-positive cells, the staining of MCP-1 and osteopontin, interstitial collagen deposition and amounts of TGF-b1 were signifi cantly reduced by MMF or Trip. MMF or Trip signifi cantly reduced the mRNA expression of TGF-b1, CTGF, MCP-1 and osteopontin. Conclusion: Trip signifi cantly attenuated tubulointerstitial fi brosis in a rat UUO model and the effect of Trip on renal fi brosis was similar to that of MMF. Trip may be useful as a potential candidate in the treatment of renal fi brosis. Renal tubulointerstitial fi brosis is the common pathway in progressive renal disease leading to functional deterioration and eventual loss of renal function, irrespective of the nature of the initial renal injury. It is a multistep process, usually beginning with the release of cytokines from neighbouring infl ammatory cells, which in turn activate fi broblasts and induce their proliferation, contribute to myofi broblast trans- differentiation and excessive synthesis of extracellular matrix components, and ultimately lead to fi brosis.1To date, there is no specifi c and effective treatment to reverse established renal fi brosis. The Chinese herb Tripterygium wilfordii Hook F. (TWHF), known as Thunder God Vine in China, has a long history as an insecticide in traditional Chinese medicine. Extracts of TWHF have been used in the treatment of glomerulonephri- tis and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus for more than 30 years in China.24It has also been investigated as an immunosuppres- sant for kidney transplantation. Study results have shown that it could prevent renal allograft rejection, increase long- term renal allograft survival among adult renal transplant recipients, ameliorate proteinuria dramatically and reduce the therapeutic dose of cyclosporine. Moreover, it could ameliorate the pathological changes of chronic allograft nephropathy such as tubular atrophy, interstitial fi brosis and arteriolar hyalinosis.57Triptolide (Trip), a diterpenoid epoxide sometimes referred to as PG490, is believed to be the major active component of TWHF extracts. Most of the anti- infl ammatory and immunosuppressive activities of extracts can be attributed to Trip.810 Nephrology 16 (2011) 200210 2010 The Authors Nephrology 2010 Asian Pacifi c Society of Nephrology200 The experimental model of unilateral ureteral obstruction (UUO) is a widely used model for progressive renal fi bro- sis.11,12 The obstructed kidney after UUO exhibits signifi cant interstitial infl ammatory cell infi ltration and tubulointersti- tial fi brosis. Immunosuppressive agents such as rapamycin and mycophenolate mofetil (MMF) have been testifi ed to be able to attenuate renal interstitial fi brosis following UUO in rats.12,13In this present study, we hypothesized that the administration of Trip in vivo might attenuate renal tubu- lointerstitial fi brosis after UUO. METHODS Animals The studies were performed on adult male SpragueDawley rats, approximately 3 months of age and weighing 200230 g, obtained from Sun Yat-sen University Animal Centre (Guangzhou, China). Animals were allowed free access to a standard laboratory chow and tap water and were housed in an environment of constant tempera- ture (21 1 2C) and humidity with regular light cycles (12 : 12 h) during the experiments. All experimental procedures were per- formed according to the animal care and ethics legislation and were approved by the Animal Care Committee of Sun Yat-sen University. Preparation of Trip Trip was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Trip was reconstituted in dimethylsulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA), stored at -20C and diluted to the appropriate concentrations with water before use in experiments. Experimental protocols Unilateral ureteral obstruction was performed as described previ- ously.12,14In brief, under i.p. pentobarbital anaesthesia, the left kidney and ureter were exposed through a fl ank incision. The left ureter was ligated with 5-0 silk at two points and cut between the ligatures in order to prevent retrograde urinary tract infection. Finally, the wound was closed in layers. Sham animals underwent identical surgical procedures, but the left ureter was simply manipu- lated. Rats were given Trip (0.6 mg/kg per day in 1 mL vehicle), MMF (20 mg/kg per day in 1 mL vehicle; Roche, Shanghai, China), or vehicle (0.5% DMSO) by daily gastric gavage. The treatments began within 2 h after the surgical procedure and continued until the rats were killed at day 7 and day 14. In both experimental sets, the rats were divided into the same four groups: (i) Sham group, animals underwent sham operations and were treated with vehicle (n = 6); (ii) UUO group, the rats underwent UUO but were treated with vehicle (n = 6); (iii) MMF group, the rats underwent UUO and were treated with MMF (n = 6); and (iv) Trip group, the rats under- went UUO and were treated with Trip (n = 6). In experiments of delayed administration of Trip, the rats were divided into six groups: (i) Sham group (n = 6); (ii) UUO group (n = 6); (iii) Trip day 0 group (n = 6); (iv) Trip day 3 group (n = 6); (v) Trip day 7 group (n = 6); and Trip day 10 group (n = 6). In groups (iiivi), the rats underwent UUO and were treated with Trip (0.6 mg/kg per day) initiated at 0, 3, 7 or 10 days after UUO, respec- tively. All rats were killed at day 14. Tissue preparation At the end of the study (day 7 and day 14), all rats were killed by pentobarbital anaesthesia (50 mg/kg bodyweight, i.p.). The abdomi- nal wall was sectioned and the left kidney was harvested and then prepared for analysis. Half of the left kidney, obtained through a mid-coronal section, was immersed in a 4% buffered paraformalde- hyde solution for histopathological and immunohistochemical evaluation. The other half of the left kidneys were snap-frozen in liquid nitrogen, and stored at -80C for further mRNA analysis. Sirius red staining Collagen was identifi ed using Sirius red staining by a method modi- fi ed from a previous report.15 Briefl y, 4 mm, formalin-fi xed, paraffi n- embedded tissue sections were deparaffi nized using xylene and absolute ethanol, rehydrated with tap water, stained with 0.1% Sirius Red F3BA solution (Sigma-Aldrich) in saturated aqueous picric acid (Sigma-Aldrich) overnight (12 h) at room temperature, washed in 0.01 N hydrochloric acid for 2 min, dehydrated, cleared and mounted on coverslips. Immunohistochemistry Sections were subjected to microwave irradiation in Tris-ethylene diamine tetraacetic acid (EDTA; pH 9.0) buffer to enhance antigen retrieval for a-smooth muscle actin (a-SMA) staining, or treated with trypsin for ED-1 staining, or microwave irradiation in citrate buffer (pH 6.0) for monocyte chemoattractant protein-1 (MCP-1) and osteopontin (OPN) staining. The renal tissue was then incubated with 1:100 monoclonal anti-ED-1 antibody (MCA341R; Serotec, Oxford, UK), 1:200 monoclonal anti-a-SMA antibody (M0851; Dako, Glostrup, Denmark), 1:400 rabbit polyclonal to MCP-1 (ab7202; Abcam, Hong Kong, China) and 1:800 rabbit polyclonal to OPN (ab8448, Abcam). The incubation of primary antibodies was carried out overnight at 4C in a humidifi ed chamber, followed by a second reaction with envision polymer anti-rabbit/mouse antibody for 30 min (EnVision Detection Kit; Dako). Finally, a diaminobenzi- dine reaction was performed on the section, using a kit (Dako), and haematoxylin was used as the counterstain. Interstitial staining of a-SMA and collagen, staining of MCP-1 and OPN were measured using computerized morphometry by Image Pro Plus ver. 6.0.0 (Media Cybernetics, Silver Spring, MD, USA).14 Stained areas of 20 randomly selected fi elds in cortex were quanti- fi ed at a magnifi cation of 400 and expressed as percentage of total measured area. Because it was diffi cult to count ED-1-positive cells, the positive areas stained with anti-ED-1 antibody were also evalu- ated as a percentage of the total examined area.16 Enzyme-linked immunosorbent assay (ELISA) To measure the level of transforming growth factor-b1 (TGF-b1), tissue homogenates were prepared using a homogenizer IKA Ultra- Turnax (2 45 s, 0C) in the extraction buffer containing 20 mmol/L Triptolide attenuates renal fi brosis 2010 The Authors Nephrology 2010 Asian Pacifi c Society of Nephrology201 Tris-HCl (pH 7.5), 2 mol/L NaCl, 0.1% (w/v) Tween-80, 1 mmol/L EDTA and 1 mmol/L phenylmethylsulfonyl fl uoride. Homogenates were then centrifuged at 15 000 g for 20 min at 4C, and the super- natants were used for ELISA. Rat TGF-b1 level was determined using a Biosource Immunoassay Kit in accordance with the protocol sup- plied by the manufacturer (KAC1688; Invitrogen, Camarillo, CA, USA). The resultant optical density was determined using a micro- plate reader at 450 nm. Protein was determined by the BCA Protein Assay kit (Pierce, Rockford, IL, USA) according to the manufactur- ers protocol using bovine serum albumin as a standard. Results were expressed as ng/g protein. Real-time polymerase chain reaction (PCR) analysis Quantitative real-time PCR following reverse transcription was used to assess transcript levels of TGF-b1, connective tissue growth factor (CTGF), MCP-1 and OPN. RNA was extracted from frozen tissue by homogenization in Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA), and after DNAse I (Promega, Madison, WI, USA) treat- ment and inhibition, 2 mg aliquots of RNA were used in reverse transcription reaction with M-MLV reverse transcriptase (Promega). The resulting cDNA was used as template for qPCR analysis. Primers were obtained from Sangon Biological Engineering Technology and Services (Shanghai, China), specifi c primers were designed as follows: 18S rRNA 5-CCT GGA TAC CGC AGC TAG GA-3 (sense) and 5-GCG GCG CAA TAC GAA TGC CCC-3 (antisense); TGF-b1 5-TGC TTC AGC TCC ACA GAG AA-3 (sense) and 5-TGG TTG TAG AGG GCA AGG AC-3 (antisense); CTGF 5-TGT TCA CTA GCG CAC AGT-3 (sense) and 5-ACA CAC CCA GCT CTT GCT A-3 (anti- sense); MCP-1 5-GTC ACC AAG CTC AAG AGA-3 (sense) and 5-GAA CCA GGA TTC ACA GAG-3 (antisense); and OPN 5-ACA TCA GAG CCA CGA GTT-3 (sense) and 5-TAC AGT GGT GTC TGC ATG-3 (antisense). Primer specifi city in real-time PCR reactions was confi rmed using reverse transcription PCR. A 20 mL of real-time PCR reaction solution included SYBR Green PCR Master Mix (TOYOBO, Shanghai, China) was amplifi ed according to the manufacturers instructions. Real-time quantifi cations were performed in duplicate on the ABI PRISM 7300 Sequence Detection System (Applied Bio- systems, Foster City, CA, USA). The calculation of relative change in Fig. 1 Representative micrographs of a-smooth muscle actin staining in kidney tissue of (A) vehicle-treated rats with sham operation on day 7, unilateral ureteral obstruction rats treated with (B) vehicle, (C) mycophenolate mofetil and (D) triptolide on day 7. (Original magnifi cation 400.) X-P Yuan et al. 2010 The Authors Nephrology 2010 Asian Pacifi c Society of Nephrology202 mRNA was performed using the deltadelta method,17with normal- ization for the housekeeping gene 18S rRNA. Collagen and hydroxyproline assays The quantitative measurement of collagen and total protein content in formalin-fi xed, paraffi n-embedded tissue sections was performed as described previously.12 Briefl y, the method uses the selective binding of Sirius red to collagen protein and Fast Green FCF to non-collagen protein when both are dissolved in aqueous saturated picric acid. When the dye was eluted from tissue sections with sodium hydroxide-methanol, the absorbances of 540 and 605 nm were determined for Sirius red and Fast Green FCF-binding proteins. The absorbances provided a relative measurement of collagen/total protein (mg/mg) quantity. Hydroxyproline, a constituent of collagen, was measured using a colorimetric assay as previously described.14 Briefl y, the samples were immersed in acetone for 24 h at 4C and dried in an oven at 60C; 20 mg of dry samples were completely hydrolyzed in 6 mol/L HCl. A fraction of the samples were derivatized using chloramine T solution and Ehrlichs reagent, and optical density was measured at 560 nm. The concentration was estimated by a standard curve using a pure solution of hydroxyproline (Sigma-Aldrich). Results were expressed as mg/mg dry tissue. Statistical analysis All data were expressed as the mean 1 standard error of the mean. Statistical calculations were performed using the SPSS ver. 16.0 software (SPSS, Chicago, IL, USA). Intergroup comparisons were made by a one-wayANOVA, with Bonferronis t-test when variances were homogeneous, or Tamhanes t-test when variances were not homogeneous. The results were considered signifi cant at P 0.05. RESULTS Effect of Trip on expression of a-SMA after UUO We examined the effect of Trip on interstitial myofi broblasts characterized by the expression of a-SMA. The a-SMA was Fig. 2 Representativemicrographsofanti-ED-1cellstaininginkidneytissueof(A)vehicle-treatedratswithshamoperationonday7,unilateralureteralobstruction rats treated with (B) vehicle, (C) mycophenolate mofetil and (D) triptolide on day 7. (Original magnifi cation 400.) Triptolide attenuates renal fi brosis 2010 The Authors Nephrology 2010 Asian Pacifi c Society of Nephrology203 essentially expressed in vascular smooth muscle cells in SpragueDawley rats. As shown in Figure 1, compared to rats with sham operation (Fig. 1A), rats with UUO developed severerenaltubulointerstitial fi brosiswithnumerous a-SMA-positive cell accumulation (Fig. 1B), which was attenuated by MMF and Trip (Fig. 1C,D). The 0.6 mg/kg per day Trip signifi cantly reduced the score of a-SMA expression in cortical interstitium of UUO rats by 70.8% on day 7 and by 67.9% on day 14 (from 19.5% to 5.7% and from 25.2% to 8.1%, respectively, both P 0.01, Fig. 6A). MMF reduced the score by 75.9% on day 7 and by 73.0% on day 14. No statistically signifi cant differences were observed between MMF and Trip treatments (Fig. 6A). Effect of Trip on macrophage infi ltration after UUO The infi ltration of ED-1-positive macrophage was present as reported previously (Fig. 2B).16Trip treatment resulted in an effective reduction in the percentage of ED-1-positive stain- ing areas by 81.0% on day 7 and by 87.5% on day 14 (from 2.1% to 0.4% and from 6.4% to 0.8%, respectively, both P 0.01, Fig. 6B). MMF treatment decreased the percentage by 76.2% on day 7 and by 82.8% on day 14. No statistically signifi cant differences were observed between MMF and Trip treatments (Fig. 6B). Effect of Trip on interstitial collagen deposition after UUO Collagen was diffusely increased in the interstitium of obstructed kidneys. Figure 3 were representative pictures showing that the interstitial collagen deposition of the obstructed kidney was signifi cantly increased after UUO as compared to that of control kidneys. Trip administration signifi cantly blunted the increase in the score of interstitial collagen deposition of the obstructed kidney by 55.7% on day 7 and by 48.9% on day 14 (from 16.7% to 7.4% and Fig. 3 Representative micrographs of Sirius red staining in kidney tissue of (A) vehicle-treated rats with sham operation on day 7, unilateral ureteral obstruction rats treated with (B) vehicle, (C) mycophenolate mofetil and (D) triptolide on day 7. (Original magnifi cation 400.) X-P Yuan et al. 2010 The Authors Nephrology 2010 Asian Pacifi c Society of Nephrology204 from 21.9% to 11.2%, respectively, both P 0.01, Fig. 6C). MMF administration signifi cantly blunted the increase by 50.3% on day 7 and by 39.3% on day 14. No statistically signifi cant differenc