Effect Of Quercetin On Renal Interstitial Fibrosis In Rats

Background: Interstitial fibrosis and subsequent tubular atrophyare pivotal pathological changes that lead to end-stage kidneydiseases. Numbers of clinical pathology observations have shownthat tubulointerstitial injury plays more important role thanglomerular damage on renal failure. Nowadays, finding theprevention and cure methods for renal interstitial fibrosis is hottopic in research. Quercetin, one of flavonoids, showed strongeranti-oxidbility and anti-fibrosis function in drug-induce liver orlung fibrosis.Objective: This study adopted unilateral ureteral obstruction(UUO)animal model and observed the effects of quercetin on thepathological changes of renal interstitial fibrosis and theexpressions of TGF-β1, CTGF and α-SMA and the contents ofhydroxyproline in kidney of UUO rats, to discuss the therapeuticfunction and mechanism of Quercetin in renal interstitial fibrosis.Methods: 36 adult healthy Wistar rats randomly divided into 3groups (n=12): sham group,UUO group and Quercetin group. Rats inUUO group and Quercetin group underwent left unilateral ureteralligation as described previously. Rats in sham group had theirureters manipulated but not ligated. In Quercetin group, Quercetinwas administered daily at a dose 100mg/kg, day before UUO and dailyfor 10 days.Animal were euthanized at day 10, and the blood sample andobstructed kidneys were harvested. The sersum levels of BUN andcreatinine were measured. Kidney tissues were either fixed in 10%buffered formalin and embedded in paraffin, or store in -80℃. Thecontent of hydroxyproline in kidney tissue were measured bychemistry colorimetry methods. Paraffin sections (3-μm thick) werestained with HE or Masson. The expressions of TGF-β1, CTGF andα-SMA were assessed by immunohistochemistry. After washing inphosphate buffer saline, tissue sections were incubated in PBScontaining 5% normal goat serum and 0.3% TritonX-100 at roomtemperature for 30 min, followed by primary polyclonalrabbit-anti-TGF-β1 antibody (1:40), primary monoclonalmouse-anti-α-SMA (1:100) antibody or primary polyclonalrabbit-anti-CTGF antibody (1:50) at 4℃for 24 h. The sectionswere then incubated in biotinylated goat anti-rabbit or mouse IgG(1:200) at 37℃for 20min and in Strept-Avidin-Biotin Complex(1:100)at 37℃for 30min. Finally, the sections were reacted with DAB for5-10 min. Sections were rinsed in PBS to stop reaction, mountedon gelatin-coated slides, air dried, dehydrated with 70%-100 %alcohol, cleared with xylene, and cover-slipped for microscopicexamination. Stained interstitial fibrotic areas were assessed byusing a LUZEX-F image analyzer. The expressions of TGF-β1, CTGF andα-SMA were also assessed bywestern blot analysis. Tissue samples (100mg)were homogenized inlysis buffer ,the homogenates were vortexed for 30 s and thencentrifuged at 12000 ×g for 2 min at 4 ℃. Supernatants werecollected and Protein concentrations were determined using theBradford method, then the equivalent amounts(40μg) of proteinsamples were separated on 10% or 15% SDS polyacrylamide gelelectrophoresis and transferred onto PVDF membrane. The membraneswere incubated overnight at 4 ℃with the following primaryantibodies: polyclonal rabbit –anti -TGF-β1 antibody (1:100),primary monoclonal mouse-anti-α-SMA (1:200) antibody or primarypolyclonal rabbit-anti-CTGF antibody (1:100). The membranes wereextensively washed with TBST and incubated for 1 h with thesecondary antibody conjugated with AP at room temperature. Theimmune complexes were detected by DAB assay kit. The scanned imageswere imported into Adobe Photoshop software. Scanning densitometrywas used for semiquantitative analysis of the data. Data were presented as Mean±SD, statistical analysis wasperformed using t-test or one-way ANOVA, A P value <0.05 or P<0.01was considered significant. Results: 10d after UUO, the tumafaction and atrophy in renalparenchyma of obstructed kidneys were measured obviously. Fibrousmaterial and monocyte infiltration increased in the interstitialspace. Furthermore, thickening of interstitial space of the tubularbasement memberance and widening of the interstitial space of therenal cortex were noted. Hypertrophy or atrophy of juxtaglomerulartubules were also noted. There were cellular or albumin casts inpartly tubules. The immunohistochemistry and western blot analysisindicated that obstruction significantly increased the expressionsof TGF-β1,CTGF and α-SMA in renal interstitial tissue in UUOgroup and Quercetin group compared with sham group(P<0.01).Theexpressions of TGF-β1,CTGF and α-SMA in renal interstitialtissue in Quercetin group significantly decreased compared with UUOgroup(P<0.01 or P<0.05). The content of hydroxyproline in kidneytissue in UUO group and Quercetin group significantly increasedcompared with sham group(P<0.01). The content of hydroxyproline inkidney tissue in Quercetin group significantly decreased comparedwith UUO group(P<0.01 or P<0.05)。 Discussion: Unilateral ureteral obstruction (UUO) is awell-established experimental model of progressivetubulointerstitial fibrosis.There are many cellular factorscontribute to the development of renal interstitial fibrosis. A lotof studies suggest that TGF-β1 is the strongest cellular factorof inducing renal tubulointerstital fibrosis. TGF-β1 and itsdownstream factors-CTGF are able to stimulate cell proliferationand collagen synthesis . TGF-β1 and CTGF can induce normal tubularepithelial cells to transform into a myofibroblast phenotype andpromote the accumulation of excretion of extracellular matrix(ECM) ,contributing significantly to the progressivetubulointerstitial fibrosis in both experimental animals and humanrenal fibrosis. UUO is associated with increased plasmaα-SMA(asymbol of myofibroblast phenotype) activity and concentrationhydroxyproline in the experimental kidney. Quercetin , one offlavonoids, showed the stronger free radical scavenging capacityand anti-fibrosis function in drug-induce liver or lung fibrosis.Some outer experiments have proved that quercetin had anti-fibrosisability. In present study, UUO induced significant increasing on theexpression of TGF-β1,CTGF and α-SMA in obstructive kidney.Atthe same time ,the obstructive kidney represent pathologicaltubulointerstitial fibrosis .Treatment with Quercetin cansignificantly attenuated the expression of TGF-β1,CTGF and α-SMAand the content of hydroxyproline in kidney tissue, Thepathological injury in rats that treatment with Quercetin are alsolighter than that in UUO group.The results indicate that Quercetinexert the anti-fibrosis effect in renal interstitial fibrosis,maybe through inhibiting the activity of TGF-β1 and CTGF,preventing tubular epithelial cells to transform into amyofibroblast phenotype. Then alleviated the progress of renalintersitial fibrosis. Conclusion: Quercetin have the following function: 1. Alleviatethe pathological changes of renal interstitial fibrosis in UUO rats.2. Decreased the expressions of TGFβ-1,CTGF,α-SMA and contentsof hydroxyprolin in obstructed kidneys in UUO rats. Quercetinmaybe a new and available effective therapy means for renalinterstitial fibrosis.