Role Of Interleukin17in TGF-β Signaling-mediated Renal Interstitial Fibrosis

BackgroundChronic kidney disease (CKD) is a condition in which renal excretory function progressively and irreversibly decreases as a consequence of renal tissue injury and nephron loss. Histologically end-stage renal disease manifests itself as glomerulosclerosis, vascular sclerosis, and tubulointerstitial fibrosis, with tubulointerstitial fibrosis having consistently been shown to be the best histological predictor of progression. Fibrotic disorders are commonplace, take many forms, and can be life-threatening. Fibrosis involves an excess accumulation of extracellular matrix (ECM)(primarily composed of collagen) and usually results in loss of function when normal tissue is replaced with scar tissue. Regardless of etiology, all patients with chronic renal disease show a progressive decline in renal function with time. The process is largely irreversible, inevitably leading to end-stage renal failure, a condition that requires life-long dialysis or renal transplantation, representing a heavy human, clinical and socioeconomic burden.IL-17, the hallmark cytokine of the newly defined T helper17(Thl7) cell subset, has important roles in protecting the host against extracellular pathogens, but also promotes inflammatory pathology in autoimmune disease. Involvement of IL-17in the pathogenesis of renal interstitial fibrosis is not clear except that IL-17+cells have been found in rejected renal graft displayed in a few studies. Therefore we focused on the contribution of IL-17to the renal fibrosis. Does IL-17play important role in other organ fibrosis? IL-17was identified as a critical element in TGF-β-driven chronic cardiac allograft fibrosis. IL-17can stimulates cardiac fibroblast proliferation and migration via negative regulation of the dual-specificity phosphate MKP-1/DUSP-1. IL-17A is required for BLM-induced pulmonary fibrosis and IL-1β-driven fibrosis is dependent on IL-17A, however, another research group found that IL-17-produing mediated lung inflammation but not fibrosis in experimental silicosis. Recently researchers showed blocking IL-17promoted the resolution of pulmonary inflammation and fibrosis via TGF-β-dependent and-independent mechanisms. In a study on liver fibrosis, IL-17signaling in Kupffer cells and hepatic stellate cells exacerbated liver fibrosis in mice. In addition, one work on scleroderma demonstrated that impaired IL-17signaling pathway contributed to the increased collagen expression in scleroderma fibroblasts.Taken together, the role of IL-17in tissue fibrosis is complicated and yet to know. It’s worthy to focus on the contribution of IL-17to the renal fibrosis and possible mechanism.ObjectiveThe present study aims to compare the progression of renal tubulointerstitial fibrosis in IL-17-/-mice with that in WT counterpart. Furthermore, impact of IL-17on activation and ECM synthesis of myofibroblasts will be studied. Finally we will investigate the signaling mechanism mediating the regulatory role of IL-17in TGF-P-driven renal interstitial fibrosis.Methods1. The overall designThis study through the vivo and vitro experiments to observe the role of IL-17A in renal fibrosis. In vivo, contrast the progression of renal fibrosis after UUO between the IL-17-/-mice (KO) and wild type mice (WT); In vitro, observe the effect of IL-17on renal myofibroblasts expressing alpha SMA, producing collagen and fibronectin and investigate their signaling mechanisms.2. In vivo2.1Preparation for UUO model and groupsUUO (unilateral ureteral obstruction) model group:The left ureterals of IL-17K0mice and Wild Type mice were ligatured. Sham operation group:Wild Type mice were opened the abdomens and then sutured. Mice were sacrificed3and7d after operation, and the renal tissues of the operated side (obstructed side for model group) were obtained.2.2Detection of IL-17mRNA in UUO group and Sham groupObtained the renal RNA of UUO model group and Sham operation group, and the mRNA expression level of IL-17mRNA.2.3Detection of fibrosis related index2.3.1Detected the pathological changes of renal tissueHE staining was used to observe the renal structure. PAS staining were used to evaluate the pathological changes of renal tissue. Masson staining was used to evaluate the accumulation of I-collagen.2.3.2The expression of relative fibrosis factorsObtained the renal RNA of UUO model group and Sham operation group, and the mRNA expression level of alpha smooth muscle actin (a-SMA), fibronectin, I-collagen, were detected by quantitative real-time PCR. Immunohistochemical staining was used to detect the level of a-SMA and Fibronectin.3. In VitroRenal fibroblasts of C57BL/6mouse were isolated and then cultivated with TGF-β in order to become myofibroblasts. Then IL-17was added into the culture to analyze the effect of IL-17on a-SMA, fibronectin、I-collagen expression of the myofibroblasts. a-SMA、 fibronectin and I-collagen were detected by quantitative real-time PCR. Western blotting was used to detect the a-SMA protein.At the same time, NRK-49F fibroblast from kidney and BALB/3T3from embryo were cultured for investigation of regulatory role of IL-17in activation and ECM synthesis of myofibroblast and involved signaling.Results1. In vivo1.1Detection of IL-17mRNA in UUO group and Sham groupThe mRNA levels of IL-17A mRNA were higher in WT mice after UUO. But it is almost no expression in IL-17-/-mice and Sham group.1.2The histopathological staining of renal1.2.1HE staining of renal revealed that with time of ureteral obstruction, there were amount of inflammatory cells infiltrating in the renal interstitium. But IL-17-/-mice was more serious than wild type. The Sham operation group was no obvious infiltration.1.2.2The PAS staining of renal revealed that with time of ureteral obstruction, renal tubular atrophy and interstitial area increased. But in the UUO model group, the fibrosis level of IL-17-/-mice was higher than WT mice. The Sham operation group was normal.1.2.3Masson staining of renal revealed that I type collagen deposition was obvious in UUO mice renal interstitium. Especially I type of collagen deposition area was bigger in IL-17-/-mice.1.3The expression of relative fibrosis factorsThe mRNA levels of a-SMA, Fibronectin and I-collagen were higher in IL-17-/-mice than in WT mice with time of ureteral obstruction by quantitative real-time PCR. a-SMA, Fibronectin protein levels were also consistent with the levels of mRNA by Immunohistochemical staining.2. In VitroTGF-β1could induce the renal fibroblasts of C57BL/6mouse to become myofibroblasts which expression higher a-SMA by quantitative real-time PCR. Quantitative real-time PCR analysis demonstrated that the relative expression of a-SMA, Fibronectin and collagen I by myofibroblasts under the stimulation of IL-17were lower than control. Western blotting result revealed that the expression of a-SMA protein was also lower than control.IL-17inhibited a-SMA protein produced by NRK-49F fibroblast, while no inhibition on non-renal fibroblast.3. Signaling mechanismTGF-P expression in renal was not affected in IL-17-/-mice after UUO surgery. The classical Smad-dependent signaling of NRK-49F fibroblast had no changes when stimulated by IL-17after TGF-P induction, whether the amounts of Smad2, Smad3, or activity of phosphorylated Smad2and Smad3. Regarding to Smad-independent signaling pathway, TGF-β/PI3K-Akt-TSC2-P70S6K has been shown to be inhibited by IL-17found in NRK-49F fibroblast.ConclusionIL-17-/-mice presented worse renal interstitial fibrosis compared with WT. Recombinant IL-17A inhibit renal primary fibroblast and fibroblast NRK-49F to express a-SMA, to produce fibronectin and collagen I. The regulatory role of IL-17on TGF-β-driven renal interstitial fibrosis after UUO was mediated at least by TGF-β/PI3K-Akt-TSC2-P70S6K signaling.