Chronic kidney disease (CKD) leading to end-stage kid-ney failure is associated with interstitial kidney fibrosis re-gardless of the underlying cause.1) As of now, there are nospecific treatments to target fibrosis in the clinic.1,2) Renal in-terstitial fibrosis is characterized by tubular atrophy/dilation,interstitial leukocyte infiltration, and increased interstitialmatrix deposition.3) Although many different cell types andcytokines are involved, a-smooth muscle actin (a-SMA)-positive myofibroblasts are the principal effect cells in fi-brotic kidney and transforming growth factor-b1 (TGF-b1)plays the central role.4,5) For many years a common notionwas that activated myofibroblasts arise primarily from resi-dent fibroblasts, recent evidence has demonstrated that dur-ing fibrosis, activated fibroblasts can also arise from epithe-lial cells via epithelial-to-mesenchymal transition (EMT) andcan be recruited from the bone marrow.6) Therefore, delin-eation of the originality and mechanism of activation of thematrix-producing myofibroblast cells may be indispensablefor designing rational therapeutic strategies for effectivetreatment of renal interstitial fibrosis.

Earlier studies have identified hepatocyte growth factor(HGF) as a potent inhibitor of renal fibrosis. HGF can effi-ciently block myofibroblastic transition from interstitial fi-broblast and tubular epithelial cells triggered by TGF-b .7,8)Administration of HGF protein or its gene prevents renal in-terstitial fibrosis in numerous animal models of renal dis-eases.9—12) But renal fibrosis is a long process, as for theshort half life period and high price, it is impossible to treatrenal fibrosis by HGF in clinic.

Rhubarb, one of popular traditional Chinese herbal medi-cine, has been widely used for the treatment of renal diseasesin traditional Chinese medicine. Although the clinical effi-cacy of rhubarb has been established, the mechanisms againstrenal diseases were still unclear. Rhein is one of the most

bioactive components from rhubarb. Earlier studies haveidentified rhein as a potent inhibitor of hepatic fibrosis in-duced by carbon tetrachloride, which is capable of reducinga-SMA expression, collagen synthesis and deposition inTGF-b1-stimulated hepatic stellate cells.13)

In the present study we explored the therapeutic efficacy ofrhein on renal interstitial fibrosis induced by unilateralureteral obstruction (UUO) and investigated potential mecha-nisms. Our study showed that rhein could ameliorate renalinterstitial fibrosis, reduce a-smooth muscle actin (a-SMA)expression, attenuate deposition of fibronectin and suppresstransforming growth factor-b1 (TGF-b1) and its type I re-ceptor expression in obstructive nephropathy. In vitro, rheinabolished the phenotypic conversion of rat kidney interstitialfibroblasts cells induced by TGF-b1. The results suggest thatrhein may be a potent drug to reduce renal fibrosis duringchronic kidney disease progress, and its therapeutic mecha-nism is, at least in part, blocking interstitial fibroblasts cellsactivation.

MATERIALS AND METHODS

Materials Rhein (purity 99%) was extracted and identi-fied by the Chinese National Institute for the Control of Phar-maceutical and Biological Products (Beijing, China), Recom-binant human TGF-b1 was purchased from Peprotech,U.S.A. Antibodies against a-SMA, fibronectin and TGF-b1type I receptor were from Sigma, U.S.A. Dulbecco’s modi-fied Eagle’s medium/F12 (DMEM/F12), were from Invitro-gen, U.S.A. and fetal calf serum (FCS) was from Sijiqing,China. Horseradish peroxidase-conjugated secondary anti-bodies and cyanine Cy2-conjugated secondary antibodieswere from Sigma, U.S.A. TGF-b1 enzyme-linked immuno-sorbent assay (ELISA) kit was from R&D U.S.A. Bicin-choninic acid protein assay kit was from Sigma, U.S.A.

Animal Model Male CD-1 mice that weighed 18 to 22 gwere obtained from animal center of Nanjing Medical Uni-

August 2011 1219Regular Article

Preventive Effects and Mechanisms of Rhein on Renal Interstitial Fibrosisin Obstructive Nephropathy

Dongyuan HE,a,† Li LEE,a Junwei YANG,b and Xiaoyun WANG*,a

a First Clinical Medical College of Nanjing Medical University; Nanjing 210029, P. R. China: and b Department ofNephrology, 2nd Affiliated Hospital of Nanjing Medical University; Nanjing 210011, P. R. China.Received January 3, 2011; accepted February 28, 2011; published online May 20, 2011

Renal interstitial fibrosis is a common outcome of a variety of chronic renal diseases. Here we evaluated thetherapeutic efficacy of rhein on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) andinvestigated the potential mechanisms. Mice underwent UUO, followed by orally administrated rhein(150 mg/kg/d) or control vehicle. Renal interstitial injury and the degree of fibrosis were evaluated by pathologi-cal staining and Western blot. The possible mechanisms were studied by Western blot, indirect immune-fluores-cence and enzyme-linked immunosorbent assay. Our results showed that rhein therapy markedly amelioratedrenal interstitial fibrotic lesions, reduced aa-smooth muscle actin (aa-SMA) expression, attenuated deposition of fi-bronectin (FN). Rhein also suppressed transforming growth factor-bb1 (TGF-bb1) and its type I receptor expres-sion in obstructed kidneys. In vitro, rhein abolished the aa-SMA and fibronectin expression of rat kidney intersti-tial fibroblasts cells (NRK-49F) induced by TGF-bb1. These observations strongly suggest that rhein is a potentinhibitor of renal interstitial fibrosis, and its therapeutic mechanism is, at least in part, blocking interstitial fibroblasts cells activation.

Key words rhein; renal interstitial fibrosis; interstitial fibroblast; transforming growth factor-b1

Biol. Pharm. Bull. 34(8) 1219—1226 (2011)

© 2011 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: xywang1941@126.com

† Present address: Department of Nephrology, Zhejiang Hospital; Hangzhou310013, P. R. China.

versity. They were housed in the animal facilities of the Nan-jing Medical University, with free access to food and water.Animals were treated humanely by use of the protocols thatwere approved by the Institutional Animal Use and CareCommittee at the Nanjing Medical University. Mice were di-vided randomly into sham group, UUO with rhein therapygroup and UUO with vehicle control group. All the unilateralureteral obstruction (UUO) operations were performed usingan established procedure14) in 3 h. Rhein was dissolved by20% alcohol. Every day after UUO operation the mice weregiven rhein (150 mg/kg) or the same volume of vehicle untilthey were sacrificed. The first administration was done aftermice recovered from anesthesia of operation immediately.The first day after operation, the mice had their second ad-ministration, and so on. Groups of mice (n�6) were sacri-ficed at days 3 and 7 after UUO with or without rhein ther-apy, respectively. One group of sham-operated mice (n�4)was killed at day 7 after surgery. One part of the kidneys wasfixed in 10% phosphate-buffered formalin, followed byparaffin embedding for pathological studies. The remainingkidneys were snap-frozen in liquid nitrogen and stored at�80 °C for ELISA and protein studies.

Histological and Pathological Staining Kidney sectionsfrom paraffin-embedded tissues were prepared at 4-mm thick-ness using a routine procedure. Sections were stained withhematoxylin/eosin for general histology. Another set of sec-tions was stained using the Masson’s trichrome stainingmethod for identifying interstitial collagen by blue color. Amorphological analysis was used for semi-quantitatively de-termining the extent of tubulointerstitial injury. Briefly, 5 vi-sion (upper left, lower left, upper right, lower right and mid-dle) were observed under low magnification per section, eachof 8 parameters of tubulointerstitial injury (tubular epithelialcell degeneration, tubular dilatation, tubular atrophy, red cellcast, protein cast, interstitial edema, interstitial fibrosis andinterstitial infiltration of inflammatory cells) was assigned ascore from 0 to 3 according to severity (0�no abnormality,1�mild, 2�moderate, 3�severe), and these scores added toyield an overall tubulointerstitial score (TIS) from 0 to 24(arbitrary units). The pathologist was unaware of the groupassignment of individual mice.

Cell Culture and Treatment Normal rat kidney intersti-tial fibroblast cells (NRK-49F) were obtained from AmericanType Culture Collection. The immortalized rat renal intersti-tial fibroblasts (NRK-49F) was cultured in DMEM/F12 con-taining 100 U/ml penicillin, 100 mg/ml streptomycin and10% FCS at 37 °C in 5% CO2. The NRK-49F cells wereseeded on six-well culture plates to 60 to 70% confluence inthe complete medium containing 10% fetal bovine serum for16 h, and then changed to serum-free medium after washingtwice with medium. Recombinant human TGF-b1 was addedto the culture at a final concentration of 2 ng per ml. Rheinwas dissolved by dimethyl sulfoxide (DMSO). For dose-dependent studies, rhein was added to the culture at a finalconcentration of 0.01, 0.1, 1 ng per ml, respectively. The cells were typically incubated for 48 h after addition of cyto-kines before harvesting and subjecting to Western blot or immunofluorescence staining.

Western Blot Kidney samples were homogenized byusing a homogenizer in radioimmune precipitation assaylysis buffer (1% Nonidet P-40, 0.1% sodium dodecyl sulfate

(SDS), 100 mg/ml phenylmethylsulfonyl fluoride, 0.5%sodium deoxycholate, 1 mmol/l sodium orthovanadate,2 mg/ml aprotinin, 2 mg/ml antipain, and 2 mg/ml leupeptininphosphate-buffered saline) on ice. The supernatants werecollected after centrifugation at 13000�g at 4 °C for 20 min.Protein concentration was determined using a bicinchoninicacid protein assay kit, and whole tissue lysates were mixedwith an equal amount 2�SDS loading buffer (125 mmol/lTris–HCl, 4% SDS, 20% glycerol, 100 mmol/l dithiothreitol,and 0.2% bromphenol blue). Cells were lysed with SDS sam-ple buffer. Samples were heated at 100 °C for 5 to 10 min be-fore loading and were separated on 10% SDS-polyacryl-amide gels. The proteins were electrotransferred to a nitro-cellulose membrane in transfer buffer containing 48 mmol/lTris–HCl, 39 mmol/l glycine, 0.037% SDS, and 20%methanol at 4 °C for 1 h. Nonspecific binding to the mem-brane was blocked for 1 h at room temperature with 5% non-fat milk in TBS buffer (20 mmol/l Tris–HCl, 150 mmol/lNaCl, and 0.1% Tween 20). The membranes were then incu-bated for 16 h at 4 °C with various primary antibodies inblocking buffer containing 5% milk at the dilutions specifiedby the manufacturers, followed by incubation with horserad-ish peroxidase-conjugated secondary anti-body for 1 h in 5%nonfat milk dissolved in Tris-buffered saline. Membraneswere then washed with Tris-buffered saline buffer, and thesignals were visualized using the enhanced chemilumines-cence system.

Indirect Immunofluorescence Indirect immunofluores-cence staining was performed using an esablishedprocedure.15) Briefly, cells that were cultured on coverslipswere washed with cold phosphate buffered saline (PBS)twice and fixed with cold methanol : acetone (1 : 1) for10 min at �20 °C. After extensive washing three times withPBS that contained 0.5% BSA, the cells were blocked with20% normal donkey serum in PBS buffer and then incubatedwith the specific primary antibodies against a-SMA and fi-bronectin. For visualizing primary antibodies, cells werestained with cyanine Cy2-conjugated secondary antibodies.Cells were double stained with DAPI (4�,6-diamidino-2-phenylindole, HCl) to visualize the nuclei. Stained cellsviewed under Nikon Eclipse E600 Epi-fluorescence micro-scope equipped with a digital camera.

Enzyme-Linked Immunosorbent Assay To measurerenal TGF-b1 levels, kidneys from mice were homogenizedin the extraction buffer containing 20 mM Tris–HCl, pH 7.5,2 M NaCl, 0.1% Tween 80, 1 mM ethylenediamine tetra-acetate, and 1 mM phenylmethylsulfonyl fluoride, and the supernatant was recovered after centrifugation at 19000 gfor 20 min at 4 °C. Renal tissue TGF-b1 level was determinedby using the commercial Quantikine TGF-b1 ELISA kit inaccordance with the protocol specified by the manufacturer.This kit measures the abundance of active TGF-b1 proteinthat binds to its soluble type II receptor precoated onto a mi-croplate. Total protein levels were determined by using abicinconinic acid protein assay kit as described above. Theconcentration of TGF-b1 in kidneys was expressed aspicograms per milligram of total protein.

Statistical Analysis All data examined were expressedas mean�S.E. Statistical analysis was performed using Sig-maStat software. Comparison between groups was madeusing one-way analysis of variance followed by Student–

1220 Vol. 34, No. 8

Newman–Keuls test. A p value of less than 0.05 was consid-ered to be statistically significant.

RESULTS

Rhein Reduces Renal Fibrosis in Obstructive Nephro-pathy To assess the potential effect of rhein on renal fibro-sis, mice were given rhein (150 mg/kg/d) or control vehicleafter common UUO. Figure 1 shows representative micro-graphs of the Hematoxylin–Eosin (HE) and Masson’strichrome staining of renal tissue sections at 3 or 7 d afterUUO. In mice treated with control vehicle, extensive peribil-iary and interstitial collagen deposition was evident, asshown by positive Masson’s trichrome staining. However, de-livery of rhein largely inhibited renal interstitial collagen ac-cumulation after UUO. Collagen staining was much weakerin the enlarged periductal area.

To quantitatively evaluate the therapeutic efficacy of rheinon renal interstitial injury induced by UUO, we determinedthe tubulointerstitial score from 8 parameters as mentionedpreviously. As shown in Fig. 1M, the tubulointerstitial scorewas progressively increased at 3 and 7 d after UUO. How-ever, rhein treatment substantially reduced the tubulointersti-tial score in the renal tissue sections at different time points.The main difference between rhein treatment group andUUO group is mainly due to the interstitial fibrosis grading.The extent of tubular epithelial cell degeneration, tubular atrophy and interstitial infiltration of inflammatory cells arealso reduced in the rhein treatment group. These results sug-gest that rhein is capable of protecting the kidney from thedevelopment of tubulointerstitial lesions after UUO.

Rhein Inhibits Myofibroblasts Activation and Extracel-lular Matrix Accumulation in Obstructive NephropathyWe further examined activation of myofibroblasts and the ac-cumulation and deposition of the specific extracellular matrixcomponents by Western blot. Because a-SMA-positive myo-fibroblasts are the effector cells that are primarily responsiblefor the overproduction of extracellular matrix at pathogenicconditions and there is almost no myofibroblasts in the normal renal tissues, we choose a-SMA as the mark of myofibroblasts activation. Extracellular matrix accumulationand deposition is the other character of interstitial fibrosis, fi-bronectin (FN) was selected as representative of extracellularmatrix. As demonstrated in Fig. 2, renal tissues at 3 and 7 dafter UUO displayed stronger protein expression of a-SMAand FN, suggesting activation of the renal myofibroblasts andextracellular matrix accumulation following UUO-inducedinjury. Consistent with the pathological staining, Westernblot results showed that delivery of rhein largely blocked therenal a-SMA and FN expression, which means rhein treat-ment could suppress the activation of myofibroblasts and ac-cumulation of the extracellular matrix.

Rhein Suppresses the Expression of TGF-bb1 and ItsType I Receptor in the Obstructed Kidneys BecauseTGF-b1 is a fibrogenic cytokine that is believed to play acentral role in regulating tissue fibrosis, we next investigatedthe effect of rhein on renal TGF-b1 and its type I receptorexpression after UUO operation. As presented in Fig. 3A,UUO induced a dramatic increase in renal TGF-b1 proteinexpression, as determined by ELISA, The steady-state levelsof TGF-b1 in the kidney at 7 d after UUO were increased

about more than 10-fold, compared to the sham controls.However, rhein markedly suppressed renal TGF-b1 proteinabundance. The expression of TGF-b1 type I receptor(Tb1R-I) in the kidney was also examined at different timepoints after UUO by Western blot. As shown in Figs. 3B andC, rhein significantly altered renal Tb1R-I expression at 3and 7 d after UUO.

Rhein Abrogates TGF-bb1-Induced Myofibroblastic Ac-tivation Because a-SMA expression is the hallmark formyofibroblasts, we investigated the myofibroblastic activationfrom renal interstitial fibroblast cells by examining the a-SMA protein expression after incubation with various agents.As shown in Fig. 4, TGF-b1 markedly induced de novo a-SMA expression in renal interstitial fibroblast NRK-49Fcells. Simultaneous incubation with rhein dramatically re-pressed TGF-b1-initiated a-SMA expression of NRK-49Fcells in a time and dose-dependent manner. This result wasindependently confirmed by an indirect immunofluorescencestaining for a-SMA in NRK-49F cells. As shown in Fig. 4F,TGF-b1 induced de novo expression of a-SMA that was as-sembled into abundant a-SMA-positive microfilament fibersin the cytoplasm of NRK-49F cells. Simultaneous incubationwith rhein primarily abolished the a-SMA staining inducedby TGF-b1 (Fig. 4G). These results indicate that rhein elicitsits anti-fibrotic actions by suppressing myofibroblastic activa-tion of renal interstitial fibroblasts initiated by profibrotic cy-tokine TGF-b1.

Rhein Inhibits FN Expression in NRK-49F Cells In-duced by TGF-bb1 Western blot and indirect immune-fluo-rescence staining were used to examine the effect of rhein onthe expression of interstitial matrix components in NRK-49Fcells activated by TGF-b1. As shown in Fig. 5, NRK-49Fcells at basal conditions expressed trivial amount of FN.However, after myofibroblastic activation by TGF-b1, theNRK-49F cells became activated, principal matrix producingcells that expressed a striking amount of interstitial FN. Si-multaneous incubation with rhein blocked the expression ofFN induced by TGF-b1. Indirect immune-fluorescence stain-ing for FN in NRK-49F cells confirmed this result. Thus, onactivation by TGF-b1, renal interstitial fibroblast cells aretransformed into activated myofibroblasts that produce largeamounts of interstitial matrix; and rhein completely blocksthis process of myofibroblastic activation and its subsequentmatrix accumulation and deposition.

Rhein’s Doesn’t Inhibit the Expression of TGF-bb1 TypeI Receptor in NRK-49F Cells Activated by TGF-bb1Western blot was used to examine the effect of rhein on theexpression of TGF-b1 type I receptor (Tb1R-I) in NRK-49Fcells activated by TGF-b1. As shown in Fig. 6, after incu-bated with TGF-b1 (2 ng/ml) or/and rhein (1 ng/ml) for 24 or48 h, the protein expression of Tb1R-I in NRK-49F cells didhave a little change, but the difference had no statistical sig-nificance.

DISCUSSION

Under most circumstances when patients are diagnosedwith chronic kidney disease, their kidneys may already dis-play different degrees of renal fibrosis. Hence, a key to an ef-fective therapy for CKD is to develop a strategy that blocksthe progression of an established renal fibrosis. The purpose

August 2011 1221

of this study was to test whether rhein, which has beenshown to be preventive in retarding the onset of hepatic fibro-sis induced by carbon tetrachloride,13) also has therapeutic ef-fects to ameliorate renal fibrosis. Although the pathologicalmechanism underlying chronic obstructive nephropathy isnot completely elucidated, the fibrogenic process clearlyplays a critical role in ultimately leading to permanent loss ofthe normal structural and functional integrity of the kid-

ney.5,16,17) Our results demonstrate that rhein markedly ame-liorate renal tubulointerstitial fibrosis in UUO mice (see inFig. 1). The character of renal tubulointerstitial fibrosis is thedestruction of normal tissue and excess production and depo-sition of extracellular matrix components. And a-SMA-posi-tive interstitial myofibroblast cells are responsible for relent-less accumulation and deposition of extracellular matrix inthe interstitial compartments of diseased kidneys. Suppress

1222 Vol. 34, No. 8

Fig. 1. HE/Masson Staining (A—L) and Tubulointerstitial Score (M) of Sham, UUO 3 or 7 d with or without Rhein Treatment

Rhein treatment can significantly ameliorate the degree of obstructive nephropathy and reduce the tubulointerstitial score. ∗ p and # p�0.01 versus sham, ∗∗ p and ## p�0.05 ver-sus UUO 3 d and UUO 7 d, respectively.

renal myofibroblast activation can attenuates renal interstitialmatrix deposition in the obstructed kidneys. Because myofi-broblast cells are a-SMA-positive and not present in normalkidneys, we can evaluate the activation degree of myofibro-blast by observing the a-SMA expression in the kidneys. Asseen in Fig. 2, the protein expression levels of a-SMA inUUO mice renal tissues were significantly higher than thenormal control group, rhein significantly reduced protein ex-pressin of a-smooth muscle actin in kidneys with unilateralureteral obstruction. That means rhein can inhibit myofibro-blast activation in obstructed kidneys. And consistent withreduction of a-SMA expression, the extracellular matrix protein expressions (FN) are also suppressed by rhein.

The origin of myofibroblast cells under pathological condi-tions is uncertain. They are often presumed to derive fromlocal resident interstitial fibroblasts. Recently, emerging evi-dence suggests that these cells may also come from tubularepithelial cells via a process known as epithelial-to-mesen-chymal transition.6) In response to tissue injury, renal intersti-tial fibroblasts undergo an activation process to become a-

SMA-positive myofibroblast cells. Profibrotic TGF-b1 is theprime stimulator of this phenotypic activation, as shown inprevious studies.18) We use rat kidney interstitial fibroblastscells (NRK-49F) as an in vitro system, compare the expres-sion of protein levels of a-smooth muscle actin and fi-bronectin between the normal control group, transforminggrowth factor-b1 activated group and rhein interventiongroup by Western blots and indirect immunofluorescence.Western blot detection showed that rhein significantly inhib-ited a-SMA protein expression of NRK-49F cells induced byTGF-b1 in a time and dose-dependent manner. As the sameway, rhein reduced fibronectin expression of NRK-49F cellsinduced by TGF-b1. Indirect immunofluorescence resultswere consistent with Western blot. So rhein may reduce theextent of renal interstitial fibrosis of unilateral ureteral ob-struction mice, at least in part, by inhibit renal interstitial fi-broblasts activation.

Rhein blockade of myofibroblastic activation from intersti-tial fibroblast cells is likely mediated by antagonizing TGF-b1 signaling. TGF-b1, on binding to its type II and type I re-ceptors that contains a cytoplasmic serine/threonine kinasedomain, initiates a cascade of signaling transduction eventsinvolving intermediate mediator Smad proteins.19,20) Thereare three classes of Smads: the receptor-regulated Smads (R-Smads), the common Smad (Co-Smad), and the inhibitorySmads (I-Smads), each of which have distinct functions.21,22)

Earlier studies reveal that TGF-b1 specifically initiatesSmad-2 and -3 phosphorylation, which in turn bind to Co-Smad-4 and translocate into the nuclei, where they controlthe transcription of TGF-b1-responsive genes.23,24) Thus, dis-ruption of any steps in this cascade of signal transductionprocesses may potentially lead to interception of TGF-b1signaling that results in blockage of myofibroblastic activa-

August 2011 1223

Fig. 2. Western Blot and Semi-quantitative Results of the FN and a-SMAExpression in Kidneys of Sham, UUO 3 d and UUO 7 d with or withoutRhein Treatment

The results show that rhein can reduce a-SMA expression and extracellular matrixaccumulation in kidneys of unilateral ureteral obstruction model mice. ∗ p and # p�0.01versus sham, ∗∗ p and ## p�0.05 versus UUO 3 d and UUO 7 d, respectively.

Fig. 3. TGF-b1 Protein Levels Determined by ELISA in the Kidneys at 3or 7 d after UUO with or without Rhein Treatment and Sham Group (A)

The protein levels of TGF-b1 type I receptor at at 3 or 7 d after UUO with or withoutrhein treatment (B, C). Rhein reduced the TGF-b1 and its type I receptor levels of uni-lateral ureteral obstruction mice renal tissues. ∗ p and # p�0.01 versus sham, ∗∗ p and## p�0.05 versus UUO 3 d and UUO 7 d, respectively.

tion. In addition, induction of inhibitory Smad proteins couldlead to suppression of R-Smad signaling by competitivelybinding to TGF-b1 receptors.25—27) We found that neither thephosphorylation of Smad-2/3 nor the expression of in-hibitory Smad-6 and -7 were significantly altered by rhein(data not shown) and further research is needed.

We also examine the effect of rhein on the TGF-b1 and itstype I receptor expression in vivo and in vitro. TGF-b1 playsa determinant role in the pathological accumulation of extra-cellular matrix in normal tissues after various injurious in-sults. Up-regulation of TGF-b1 expression is found in most,if not all, forms of chronic renal fibrotic diseases in animalmodels and in patients.28) Consistent with early study, theprotein expression of TGF-b1 increased significantly afterUUO operation (Fig. 3). TGF-b1 not only induces myofi-broblastic activation from interstitial quiescent fibroblasts,but also initiates myofibroblastic transition from tubular epi-thelial cells at the advanced stage of chronic renal diseases.

Over-expression of TGF-b1 in transgenic mice induced in-creased expression of fibrotic matrix protein. Conversely,suppression of TGF-b1 signaling either by truncated, solublereceptor or by an antisense approach significantly retards theprogression of renal interstitial fibrosis in animals.29,30) Inpresent study, we observed that rhein therapy can reduce theTGF-b1 and Tb1R-I levels of renal tissues in unilateralureteral obstruction mice. But we did not observe the similarphenomenon in vitro. The protein expression of Tb1R-I inNRK-49F cells was not significantly altered by rhein. So theeffect of rhein on TGF-b1 axis expression is more compli-cated than what we have thought. The decreased TGF-b1 andits type I receptor expression in vivo may be due to an indi-rect effect of rhein or a secondary consequence resultingfrom the antifibrotic action of rhein. Many other cell typesand cytokines must be involved, and more researches areneeded. Together with the early study, rhein can indirectly re-duce renal interstitial fibroblast activation and epithelial-to-

1224 Vol. 34, No. 8

Fig. 4. Rhein Abrogates the Expression of a-SMA in Renal Interstitial Fibroblast NRK-49F CellsWestern blot and semi-quantitative analysis show rhein inhibit a-SMA expression of NRK-49F cells induced by TGF-b1 in a time- and dose-dependent manner. NRK-49F cells

incubated with TGF-b1 (2 ng/ml) and rhein(1 ng/ml) for various periods of time as indicated (A, B), or increasing amounts of rhein for 48 h (C, D). Immunofluorescence stainingshows the a-SMA expression in NRK-49F cells after various treatments for 48 h. C, H: control; F, I: 2 ng/ml of TGF-b1; G, J: 2 ng/ml of TGF-b1 and 1 ng/ml of rhein. ∗ p, ∗∗ p, # pand ## p�0.05.

mesenchymal transition tubular by inhibit the expressionTGF-b1 through an unknown mechanism. These observa-tions highlight the effectiveness and efficacy of rhein as a po-tential therapeutic agent for inhibiting myofibroblast accumu-lation and interstitial fibrogenesis in the diseased kidneys.

In summary, this study suggests that rhein acts as a nega-tive regulator of renal interstitial fibrosis that reduces renal fi-brosis induced by UUO. And its therapeutic mechanism is, at

least in part, blocking interstitial fibroblasts cells activation.In this regard, the present study not only underscores that theblockade of the activation of interstitial fibroblast cells is anovel strategy for prevention of fibrotic diseases but also setsa foundation for the rational utilization of rhein in combatingrenal fibrosis.

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Fig. 5. Rhein Inhibits the Expression of FN in Renal Interstitial Fibroblast NRK-49F Cells

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