| Renal tubulointerstitial fibrosis(RIF)is a common performance to end-stage renal failure occurring after a variety of primary and secondary chronic kidney disease (including chronic allograft nephropathy), is also the most important indicator to prognosis and the severity of renal function decline. As to the mechanism, now the excessive accumulation of the extracellular matrix (ECM) is considered to be one of the main reasons leading to RIF. Myofibroblasts (MyoFb) located in renal interstitium are the main source of ECM, its quantity is considered to be the best indicator to renal prognosis. As to the sources of MyoFb, now epithelial- mesenchymal transition(EMT) is thought to be one main source.Many factors can cause renal tubular epithelial cells undergo EMT, including injury, inflammation, a variety of cytokines act solo or in synergy,etc. TGF-β1 is found to be the most powerful factor leading to RIF, it is involved in all process of RIF. In recent years, numerous studies found that TGF-β1 initiates and regulates the whole process of EMT. IL-1βis a pro-inflammatory factor heavy secreted by macrophages who infiltrating in renal interstitium when RIF. IL-1βis participated in interstitial inflammation and fibrosis via various pathways. Even if some studies reports that IL-1βcan induce EMT on renal tubular epithelial cells, the results are different, the researches on the cytoskeleton are even rarer. As known,IL-1βand TGF-β1 are cytokines that have extensive functions, and effects on contrary at many times, in RIF, their expressions are significantly increased, although it has reported that both IL-1βand TGF-β1 could induce TECs undergoing EMT respectively in different degree, there potential interaction in the process of EMT still have not been reported.Cytoskeleton is constituted by microfilament, microtubule and intermediate filament. Microfilaments and microtubules are able to polymerizate/ depolymerizate rapidly under different stimulations inside and outside the cells, thus educe its biological role and participate in cell signal transduction. In the process of EMT, microfilaments can be reorganized, on the contrary, as microfilaments depolymerize, its effects on EMT induced by IL-1βand TGF-β1 respectively have not been reported at present.Objective Firstly, using TGF-β1 and IL-1βeffected on NRK52E cells respectively, observed and compared their effects on cell morphology, phenotypes of transition and cytoskeletons, analyzed the the similarities and differences in various links of EMT. Secondly, using TGF-β1 and IL-1βcombined effected on NRK52E cells,the aim was to observe the interaction between the factors in different sectors of the induction of EMT. Finally, we used cytochalasin B to damage microfilaments, aimed to observe its effects on EMT induced by IL-1βor TGF-β1 respectively. All of above works were aimed to enrich the theoretical basis of EMT on renal tubular epithelial cells and provide the theoretical basis for the therapy of RIF.Methods and Results1 The effects of TGF-β1 and IL-1βin EMT on NRK52E cells respectively Groups: (1) TGF-β1 induced group: the final concentration of TGF-β1was 10 ng / ml, control group: equal volume of 0.1M PBS; (2) IL-1βinduced group: the final concentration of IL-1βwas 30 ng / ml, control group: equal volume of 0.1M PBS. Cultured continuously for 72h after divided into groups, then undertook the following experiments respectively, methods and results were as follows:1.1 Morphology changesCells in control group showed typical cobblestone-like appearance all the time, the engagement between cells was tight. After induced by TGF-β1 for 72h, cells were confluenced,the cells in the center were showed short spindle appearance, the cells in the edges were showed long spindle appearance, radial alignment. Under high power microscope, there were many protrusions of varying lengths in the cells edge. After induced by IL-1βfor 72h, cells were mainly changed in long spindle, some cells have several long processes, anchoring on or wrapping in the surrounding cells, the phenomen of cell processes overlapped and acrossed were common.1.2 The mRNA expression of phenotype markers in NRK52E cellsRT-PCR results showed, after induced by TGF-β1 and IL-1βfor 72h, the mRNA expression of E-cadherin who was the marker of epithelial cells was significantly reduced; the mRNA expressions ofα-SMA and FN who were the markers of mesenchymal cells were significantly increased, suggested that TGF-β1 and IL-1βhad respectively induced NRK52E cells undergo EMT. Unexpectedly, TGF-β1 and IL-1βalso could increase the mRNA expression of desmoplakin-1 (DPI) who was another epithelial cell marker, the mechanism remained unclear.1.3 The expression and distribution proteins of phenotype markers in NRK52E cellsThe immunocytochemical staining showed, E-cadherin positive staining intensity was middle in control group, smoothly,linearly and uniformly distributed in the cell membrane. After induced by TGF-β1 for 72h, E-cadherin protein expression was significantly reduced and interruptedly distributed in the cell membrane. After induced by IL-1βfor 72h, E-cadherin in the membrane was discontinuous distribution, the intensity of dyeing was increased in some area. Immunofluorescence staining showed, there was a weak positive staining about DPI protein, diffuse distribution in cell plasma. After induced by TGF-β1 and IL-1βfor 72h respectively, the fluorescence intensity of DPI was significantly increased, mainly clustered around the nucleus.Immunofluorescence staining also showed,α-SMA and FN staining were weak positive in control group, dispersed in the cytoplasm. After induced by TGF-β1 and IL-1βfor 72h respectively, the expressions of the both were significantly increased, showed strong positive staining and mainly gathered around the nucleus. The results of immunostaining were consistent with RT-PCR, further confirmed that TGF-β1 and IL-1βhad induced NRK52E cells undergo EMT.1.4β-actin andα-tubulin mRNA expressions and protein distribution and arrangement in NRK52E cellsRT-PCR results showed, TGF-β1 could increase the expression ofβ-actin mRNA, but had no significant effects onα-tubulin mRNA, while IL-1βcould increase the expression ofα-tubulin mRNA, had no impaction onβ-actin mRNA.Immunofluorescence staining showed that: (1)β-actin positive staining mainly accumulated below the cell membrane in control group. After induced by TGF-β1 for 72h,β-actin positive staining formed a large number of thick bundles of fiber-like structures in cytoplasm. the majority were arranged parallel to cell long axis. After induced by IL-1βfor 72h,β-actin positive staining formed filamentous fiber-like structures in the cytoplasm and cell processes and were arranged parallel to cell long axis.(2)α-tubulin positive fibers formed the microtubule organizing center in one side of the nucleus in control cells, and used it as the central, the fibers showed radial distribution reached the membrane. After induced by TGF-β1 for 72h,α-tubulin positive fibers in some cells changed from the radial distribution into arrangement parallel to cell long axis. After induced by IL-1βfor 72h,α-tubulin positive fibers increased in most cells, changed from the radial alignment into bundles, parallelled to cell long axis. The results above showed that after induced NRK52E cells undergo EMT, IL-1βand TGF-β1 also could affect the distribution and arrangement of cytoskeletons, TGF-β1 focused on affecting microfilament, while IL-1βfocused on affecting microtubules. 2 The EMT induced by IL-1βand TGF-β1 combinedly on NRK52E cells Groups:①c ontrol group: 0.1 MPBS (instead of IL-1βor TGF-β1)②IL-1βinduced group: the final concentration of IL-1βwas 30 ng / ml③TGF-β1 induced group: the final concentration of TGF-β1was 10 ng / ml④combinedly induced group: the final concentration of IL-1βand TGF-β1 was 30 ng / ml and 10 ng / ml respectively.Cultured continuously for 48h after divided into groups, then undertook the following experiments respectively, methods and results were as follows:2.1 Morphology changesControl group, IL-1βand TGF-βalone induced group the cell morphologys were similar to which induced for 72h. After induced by the both the cells morpholology changes were similar with induced group of IL-1βalone.2.2 The mRNA expression of phenotype markers: E-cadherin,α-SMA and FNRT-PCR results showed, the mRNA expression of E-cadherin was the most strong in the control group, decreased slightly in IL-1βinduced group, completely disappeared in TGF-β1 induced group and the combinated induced group. The mRNA expressions ofα-SMA and FN were the most in the combinated induced group, significantly more than IL-1βand TGF-β1 alone induced group. Demonstrated that IL-1βand TGF-β1 had synergistic effects on inducing NRK52E cells undergo EMT.2.3β-actin andα-tubulin mRNA expressions and protein distribution and arrangement in NRK52E cellsRT-PCR results showed, the mRNA expression ofβ-actin in the combinedly induced group was significantly higher than IL-1βand TGF-β1 alone induced group, the mRNA expression ofα-tubulin in the combinedly induced group was same with IL-1βalone induced group, higher than TGF-β1 induced group, suggested that the both factors had synergistic effect onβ-actin mRNA expression but had no synergistic effect on theα-tubulin mRNA expression. Immunofluorescence staining results showed,β-actin andα-tubulin positive staining, distribution and arrangement in control group, IL-1βand TGF-β1 alone induced group were similar with which was induced for 72h. The distribution and arrangement ofβ-actin in combinedly induced group were similar with which in TGF-β1 alone induced group, The distribution and arrangement ofα-tubulin in combinedly induced group were similar with which in IL-1βalone induced group. These results suggested, after IL-1βand TGF-β1 combinedly induced NRK52E cells undergo EMT, there was no obvious antagonism on the distribution and arrangement of the cytoskeleton.2.4 Changes to cell adhesion and migration When cells were after cultured for 48h, CCK-8 test and cell scratch were used to assay the cell adhesion in Matrigel gel and cell migration respectively. The results showed: after inoculation for 2h, cell adhesion was the highest in the control group, secondly in IL-1βinduced group and TGF-β1 induced group, lowest in the combinedly inducted group. The results indicated that after IL-1βand TGF-β1 induced NRK52E cells undergo EMT respectively, IL-1βand TGF-β1 also could reduce the ability to cell adhesion, the both factor had synergistic effect.The relative migration distance was same in control group and IL-1βinduced group, significant increased in TGF-β1 induced group and combinedly induced group, but there was no difference in the last two groups. Suggested that on cell migration IL-1βhad no significant effect, while TGF-β1 had significantly stimulated effect, the two combined application had no obvious antagonism.3. The effect of microfilament depolymerization on IL-1βand TGF-β1 induced EMT in NRK52E cellsGroups:①control group: 0.1 MPBS (instead of IL-1βor TGF-β1).②CB alone group: the final concentration of CB was 0.2μg/ml.③IL-1βinduced group: the final concentration of IL-1βwas 30 ng / ml.④CB+IL-1βinduced group: the final concentration of CB and IL-1βwas 0.2μg/ml and 30 ng / ml respectively,⑤TGF-β1 induced group: the final concentration of TGF-β1 was 10 ng / ml.⑥CB + TGF-β1 induced group: the final concentration of CB and TGF-β1 was 0.2μg/ml and 10 ng / ml.Cultured continuously for 48h after divided into groups, then undertook the following experiments respectively, methods and results were as follows:3.1 The distribution and arrangement ofβ-actin andα-tubulin protein in NRK52E cells (immunofluorescence staining)3.1.1 (The distribution and arrangement ofβ-actin in control group, IL-1βinduced group, TGF-βinduced group was similar with which was induced for 72h).β-actin staining in the CB alone group was very weak, no significantβ-actin positive fibers, many dual-core cells could be seen. In CB + IL-1βinduced group,β-actin formed many filamentous fiber-like structures in the cytoplasm near the nuclei. In CB + TGF-β1 induced group,β-actin expression was weak, mainly near the nucleus, they were short-beam-shaped radially arranged, theβ-actin staining was significantly reduced near the membrane area. Suggested that CB had inhibited the microfilament polymerization and made the function of microfilament abnormal. IL-1βcould significantly antagonize the ability of CB which inhibites the microfilament polymerization, while TGF-β1 only could weakly antagonize it.3.1.2 (The distribution and arrangement ofα-tubulin in control group, IL-1βinduced group, TGF-βinduced group was similar with which was induced for 72h).α-tubulin positive staining in CB alone group was significantly increased compared with the control group, its distribution and arrangement was slightly disordered, but still radially arranged from the nucleus to the membrane, suggested that the distribution and arrangement of microtubules had not been obviously impacted after microfilament depolymerization. In CB + IL-1βinduced group, the distribution and arrangement of microtubules was similar with it in IL-1βinduced group, implied that the distribution and arrangement ofα-tubulin induced by IL-1βwasn't been significantly interfered by microfilament depolymerization. In CB + TGF-β1 induced group, the distribution and arrangement ofα-tubulin was slightly disordered, but still radially arranged from the nucleus to the membrane,α-tubulin positive fibers paralleling to cell long axis were disappeared, indicated that microfilament depolymerization could mildly inhibit the effects induced by TGF-β1 on the distribution and arrangement ofα-tubulin.3.2 The mRNA expressions ofβ-actin andα-tubulin in NRK52E cellRT-PCR results showed,β-actin mRNA expression in the CB, CB + IL-1βand CB + TGF-β1 induced group were significantly higher than it in the control group, IL-1βand TGF-β1 alone induced group respectively.α-tubulin mRNA expression in the CB, CB + TGF-β1 induced group was significantly higher than it in the control group and the TGF-β1 alone induced group, there had no significant difference in the other groups.3.3 Morphology changesCells in CB alone group still showed a typical cobblestone-like morphology. In CB + IL-1βinduced group the proportion of long axis and short axis was increased in the majority cells, some cells were spindle changed, but the cell processes were significantly reduced, cells were closely arranged. In CB + TGF-β1 induced group cells at the border although were radially arranged, they were closely arranged, had no long thin protuberances. Illustrated that the morphology changes induced by IL-1βand TGF-β1 were significantly interfered by microfilament depolymerization.3.4 mRNA expressions of E-cadherin,α-SMA and FNRT-PCR results showed: Compared with the control group, the E-cadherin mRNA expression was significantly increased,α-SMA mRNA expression was significantly decreased in CB alone group,suggested that microfilament depolymerization might be against the EMT. Compared with IL-1βinduced group, E-cadherin,α-SMA and FN mRNA expression in CB + IL-1βinduced group were all increased significantly, suggested that the effect of CB on IL-1βinduced NRK52E cells transition might be complex. Compared with TGF-β1 induced group, E-cadherin mRNA expression was increased,α-SMA mRNA expression was decreased in CB+TGF-β1 induced group, suggested that CB could significantly inhibited TGF-β1 induced cell transition.3.5 Changes to cell adhesion and migrationThe methods were as before, the results showed: CB could increase the adhesion of cells significantly after inhibited microfilament polymerization, and had the obvious antagonism on IL-1βand TGF-β1 induced reduction of cell adhesion respectively. Scratch test results showed: compared with IL-1βinduced group and CB alone group, the migration distance in CB+IL-1βinduced group was reduced significantly. Compared with TGF-β1 induced group, the relative migration distance in CB+TGF-β1 induced group reduced significantly and was similar with CB alone group, illustrated that after CB inhibited microfilament polymerization, cell migration promoted by TGF-β1 was inhibited completely.Conclusions1 Both TGF-β1 and IL-1βcould induce NRK52E cells undergo EMT respectively.2 TGF-β1 had the synergistic effect with IL-1βon inducing NRK52E cells transition, affecting the cytoskeleton and inhibiting the cell adhesion.3 After induced NRE52E cells transition, TGF-β1 might lead to cytoskeletal reorganization and focus on the microfilament, when microfilament polymerization was obstaclesd, the effects of TGF-β1 on EMT was significantly inhibited, on the same time, the effect of TGF-β1 on cell adhesion and migration was also significantly inhibited.Thus, we speculated that the structure and function of microfilament had an important impact on TGF-β1 induced NRE52E cells EMT.4 After induced NRK52E cells transition, IL-1βfocused on effecting the microtubules, as well, When the cell microfilament polymerization was blocked, the effectes on IL-1βinduced EMT were also different from TGF-β1, suggested that the signal transduction to IL-1βwas different from TGF-β1.
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