A Preliminary Study On The Role Of IL-17A In Progression Of Renal Tubulointerstitial Fibrosis

BackgroundFibrosis is an important result of many organs with chronic inflammation andleads organs to have losting the functions. Therefore, it is very important forpreventing fibrosis to research the mechanism of the development of fibrosis and lookfor the key target to block fibrosis.However, the mechanism of fibrosis has not fully understood. It was clear thatfibrosis was close to the chronic inflammation. Many inflammation factors might beinvolved in the development of fibrosis. For example, transforming growthfactor-beta1,(TGF-β1) could promote the fibroblasts to hyperplasia anddifferentiation, and transformated to myofibroblasts, and induced the expression ofextra cellular matrix (collagen protein, fibronectin and so on). In addition,inflammation factors IL-β1, TNF-α, IL-6play an important role in the developmentof fibrosis.IL-17is also an important pro-inflammation factor. IL-17family including IL-17A, B, C, D, E, F subtypes. IL-17A is the most main subtype,Followed by IL-17F.There was regulation fuction between them. Th17cells mainly expressed IL-17.Th17plays an important role in development of inflammation through releasing IL-17. So,what the role of IL-17A in fibrosis? There are some research suggests that inmyocardial fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, and renal fibrosiscaused by chronic rejection after kidney transplantation,IL-17Acould promote thefibrosis.But, the role of IL-17A in renal fibrosis is not clear.ObjectiveTo investigate the effection of IL-17A on progression of renal tubulointerstitial fibrosis, Including effection of IL-17A on related matrix proteins, such as alphasmooth muscle Actin (α-SMA)、Fibronectin、 I-collagen.Methods:1. The overall design.This study through the vivo and vitro experiments to observe the effection ofIL-17A on renal fibrosis. In vivo, contrast the effection of renal fibrosis after UUObetween the IL-17A knockout (IL-17AKO) and wild type mice; In vitro, observe theeffection of IL-17on renal myofibroblasts expressing alpha SMA, collagen andfibronectin and investigate the role of IL-17in renal fibrosis.2. In vivo2.1Preparation for UUO model and groupsUUO(unilateral ureteral obstruction) model group：The left ureterals of IL-17KOmice and Wild Type mice were ligatured. Sham operation group: Wild Type micewere opened the abdomens and then sutured.Mice were sacrificed1，3and7d afteroperation，and the renal tissues of the operated side (obstructed side for model group)were obtained．2.2Detection of IL-17A mRNA in UUO group and Sham groupObtained the renal RNA of UUO model group and Sham operation group, andthe mRNA expression level of IL-17A mRNA.2.3Detection of fibrosis related index2.3.1Detected the pathological changes of renal tissueHE staining were used to observe the renal structure. PASM staining were usedto evaluate the pathological changes of renal tissue. Masson staining were used toevaluate the accumulation of I-collagen.2.3.2The expression of relative fibrosis factorsObtained the renal RNA of UUO model group and Sham operation group, andthe mRNA expression level of Alpha smooth muscle Actin (α-SMA),Fibronectin, I-collagen,were detected by quantitative real-time PCR. Immunohistochemicalstaining was used to detect the level of α-SMA and Fibronectin.2.4The expression of the inflammatory factor, adhesion molecule, chemokinesObtained the renal RNA of UUO model group and Sham operation group, andthe mRNA expression level of IL-1β, TNF-α, IL-6, ICAM-1, MCP-1, TGF-β, IL-6were detected by quantitative real-time PCR.3. In VitroRenal fibroblasts of C57BL/6mouse were isolated and then cultivated withTGF-β1in order to become myofibroblasts. Then IL-17A was added into the cultureto analyse the effect of IL-17A on α-SMA、Fibronectin、 I-collagen expression of themyofibroblasts. α-SMA、Fibronectin and I-collagen were detected by quantitativereal-time PCR. Western blotting was used to detect the α-SMA protein.Results1. In vivo1.1Detection of IL-17A mRNA in UUO group and Sham groupThe mRNA levels of IL-17A mRNA were higher in WT mice after UUO.But it isalmost no expression in IL-17AKO mice and Sham group.1.2The histopathological staining of renal1.2.1HE staining of renal revealed that with time of ureteral obstruction, there wereamount of inflammatory cells infiltrating in the renal interstitium. But IL-17AKOmice was more serious than wild type. The Sham operation group was no obviousinfiltration.1.2.2The PASM staining of renal revealed that with time of ureteral obstruction,renal tubular atrophy and interstitial area increased. But in the UUO model group, thefibrosis level of IL-17AKO mice was higher than WT mice. The Sham operationgroup was normal. 1.2.3Masson staining of renal revealed that Ⅰtype collagen deposition was obviousin UUO mice renal interstitium. Especially Ⅰt ype of collagen deposition areawasbigger in IL-17AKO mice.1.3The expression of relative fibrosis factorsThe mRNA levels of α-SMA, Fibronectin and I-collagen were higher inIL-17AKO mice than in WT mice with time of ureteral obstruction by quantitativereal-time PCR. α-SMA, Fibronectin protein levels were also consistent with the levelsof mRNA by Immunohistochemical staining.1.4The expression of the inflammatory factor, adhesion molecule, chemokinesThe mRNA levels of IL-1β,TNF-α,ICAM-1,MCP-1were higher in UUO mice.But IL-1β, TNF-α,ICAM-1,MCP-1mRNA levels in IL-17AKO mice were higher thanWT mice. TGF-β1and IL-6mRNA expression levels were no significant differencesbetween IL-17AKO mice and WT mice.2. In VitroTGF-β1could induce the renal fibroblasts of C57BL/6mouse to becomemyofibroblasts which expression higher α-SMA by quantitative real-time PCR.Quantitative real-time PCR analysis demonstrated that the relative expression ofα-SMA, Fibronectin and collagenⅠ by myofibroblasts under the stimulation of IL-17were lower than control. Western blotting result revealed that the expression ofα-SMA protein also lower than control.Conclusion:Unilateral ureteral obstruction operation can lead to renal tissue fibrosis.IL-17konck down can lead to higher degree of renal fibrosis. Under the stimulation ofIL-17A, the relative expression of α-SMA, Fibronectin and collagen I bymyofibroblasts were lower than control. These results point out that IL-17may notpromote the fibrosis in renal and may be down regulate the renal fibrosis.