| Currently renal transplantation is the most effective method to treat end-stage renaldisease. Although the combined application of new immunosuppressive agents hasdramatically improved renal allograft survival rate, there were still some grafts developedirreversible loss of renal function after kidney transplantation. The defuctionalization ofrenal allograft generally occurs in two stages: a few cases occur immediately or withinseveral days (40days) post-transplant, and showed morphological manifestations of acuteantibody-mediated rejection; some cases occur years after transplantation (chronic loss ofallograft function), morphologically changes include renal interstitial sclerosis, tubularatrophy, loss of nephron, and thickening of arteriolar wall can be found in these cases, whichwas named renal allograft fibrosis.In renal fibrosis, activated fibroblasts are the most basic effector cells, fibroblastproliferation and production of extracellular matrix is the basis of renal fibrosis. Therewere several sources of activated fibroblasts according to newly reports. The pathway ofrenal tubular epithelial cells proceeding epithelial-mesenchymal transition (EMT) becomefibroblast has attracted much attention. The EMT of tubular epithelial cell means that therenal tubular epithelial cells reduced or even lost their epithelial cell phenotype, and acquiredphenotype of mesenchymal cell.Cellular rejection occupied high percentage of acute renal allograft rejection, whichshowed varying degrees of inflammatory cell infiltration in interstitial and accompanied by acharacteristic "tubulitis". The "tubulitis" refers to the phenomenon that CD8+Tlymphocyte infiltration into the renal tubular epithelial cells. There were some reportspoints out that the frequently attack of severity acute T cell-mediated rejection was closelyrelated with chronic renal allograft fibrosis. Whether or not T cells in the "tubulitis" hasparticipated in the process of renal allograft fibrosis and renal tubular epithelial cells EMT isconcerned, was not clear. In this study we obtained (T cells) thymus cells from rat thymustissues, then we observed the EMT of rat renal tubular epithelial cells (NRK-52E) inducedby thymus cells in vitro. We hope to provide experimental data for the study of mechanismto chronic renal allograft fibrosis. In addition, there were a few renal grafts failed in the early stage (within several days)fter transplantation, main reason for this situation is acute antibody mediated rejection. Thisype of rejection, although the incidence is very low (only0~8%), but will soon lead to theoss of graft function, and people have very limited view on it. In this study, we collected5ases of kidney allograft specimens which were resected early after transplantation becausef irreversible loss of function. By immunohistochemical staining using nine primaryntibodies, we classified the proportions of infiltrating immunocytes in intimal arteritis andlomerulitis in five allografts resected because of acute irreversible graft failure.ats thymus cells induced EMT in rat renal tubular epithelial cells (NRK-52E)NRK-52E co-cultured with rat thymocytes was named induced group, with normalultured NRK-52E cells as control group. After48h of incubation, we observed theorphological change in these two groups of NRK-52E cells, then real-time PCR andmmunofluorescence staining was performed to detect the phenotype change of NRK-52Eell induced by rat thymocytes.Rat thymocytes were obtained from Wistar rat thymus, fluorescence flow cytometryetection showed that the ratio of cell subpopulations of the thymocytes obtained wasonsistent with the distribution within thymic tissue.NRK-52E cells of the two groups were incubated respectively for48h, NRK-52E cellsn the control group always showed a typical cobblestone-like shape, and closely connectedetween adjacent cells; in the induced group, part of the cells turn to short spindle cells, andhe gap between cells increased. After statistics, spindle cell ratio per high power field (×00) of the control group cells was only8.2±2.5%; in the induced group, spindle cell ratio isigh up to71.2+15.8%, significantly higher than that in control group (P <0.01).By real-time PCR analysis, when NRK-52E cells was co-cultured with thymus cells for8hours, we found that although the mRNA of CK (epithelial cell marker) was notecreased significantly, but the mRNA of a-SMA and Vimentin (mesenchymal markers)ere increased to1.56times and3.89times of the control group respectively (P <0.05).Immunofluorescence staining showed that, in the control group NRK-52E cells,K-19and E-Ca (epithelial cell markers) staining was positive, whereas the mesenchymalell markers а-SMA was negative, Vimentin and Desmin were weakly positive, thisemonstrated that NRK-52E cells in control group still remained epithelial cellharacteristics. While in the induced group, although the CK-19positive staining was notecreased, but the E-Ca staining was almost entirely negative. At the same time in the cytoplasm we can found strong positive staining of а-SMA, Desmin and Vimentin. We alsofound that Vimentin staining showed grossus fiber structure, radially arranged in theperinuclear cytoplasm.In short, after co-cultured with thymus cell for48hours, the ratio of spindle cells ofNRK-52E cell increased. We observed the phenotypic changes in the induced NRK-52Ecell: include visible decrease of epithelial marker expression, and increase of mesenchymalmarker expression, which occurred in epithelial-mesenchymal cell transition.Thymus cell induction medium induced NRK-52E cell EMTPrimary cultured rat thymus cells were stimulated with phytohemagglutinin (PHA) andconcanavalin A (ConA). Then we observed the cell morphology, we also detected cellcycle end lymphocyte subsets by flow cytometry, at last we performed ELISA assay to detectcytokines IL-2in the supernatant of stimulated thymocyte. We collected the serum freeculture supernatants of activated thymocytes as the induction medium, then inducedNRK-52E cells for48hours (induced group); H-DMEM medium used to wash thymocytesat the third time was used as control medium, NRK-52E cells cultured with control mediumwas control group. After48h of incubation, cells were observed by inverted microscope todetect morphology changes. We use real-time PCR, immunocytochemistry andimmunofluorescence staining, and Western Blot to detect NRK-52E cell phenotypic changein the mRNA and protein levels.The results show that after stimulation, rat thymocytes became suspended large cellclusters, and results of cell cycle analysis showed that the proliferation index of stimulatedthymocyte was27.45±1.77%, significantly higher than that in the control group (19.23±2.37%), P<0.05; at the same time, mature T lymphocyte ratio, especially the ratio of CD8+toxicity T cell (stimulated group is18.06±1.89%, the control group was only7.22±1.01%)increased significantly (P<0.05); ELISA detection showed that thymus cells in stimulatedgroup had gained secretion ability of cytokine IL-2. In short, with PHA and ConA combinedstimulation, rat thymus cell has been activated, and gained secretory function.48h after NRK-52E cells induced by induction medium, NRK-52E cells in the controlgroup showed the typical slabstone like morphology of epithelial cells, adjacent cells areclosely linked. While most of NRK-52E cell in induced group became radially arranged,long spindle-shaped cells, there were sharp protrusions in the cell membrane, the gapbetween cells increases obviously. After counting and statistics, spindle cell ratio per field(×400) of the control group NRK-52E cells was only12.8±7.2%, significantly lower than the spindle cell ratio of the induction group (62.2±13.1%), P <0.05.Real-time PCR showed that48hours after the NRK-52E cell was induced by inductionmedium, the mRNA of epithelial marker CK was reduced to0.23times of the control group(P<0.05), at the same time, mRNA of mesenchymal cell markers a-SMA and Vimentin wereincreased1.7times and1.68times of the control group, the difference was statisticallysignificance (P<0.05).The Immunocytochemical staining showed that, NRK-52E cells in the control groupshowed CK-19and E-Ca positive stain, CK-19diffusely distributed in the cell plasm, andE-Ca is mainly distributed on the cell membrane; mesenchymal cell markers like a-SMA,Vimentin and Desmin were all negative. While in NRK-52E cells in the induced group,CK-19and E-Ca staining were almost completely disappeared; whereas the mesenchymalcell markers a-SMA, Vimentin and Desmin showed strongly positive staining, whichdiffusely distributed throughout the cytoplasm.Results of Immunofluorescence staining showed a similar trend, in the control group,NRK-52E cells showed CK-19and E-Ca strong positive; a-SMA staining was completelynegative, Vimentin and Desmin showed weak positive staining; in the induced group, theepithelial cell marker CK-19was weakly positive, fluorescence intensity decreasedsignificantly compared with the control group, the other epithelial cell marker E-Ca isnegative; at the same time mesenchymal cell markers a-SMA, Vimentin and Desmin showedstrong positive staining. Vimentin staining showed grossus fiber structure, radially arrangedin the perinuclear cytoplasm.The Western Blot results demostrated that,48hours after NRK-52E cells was culturedwith induction medium, expression of the epithelial cell marker CK-18protein wassignificantly decreased (P<0.05); while the expression of mesenchymal marker a-SMAprotein was significantly higher than which of the control group, after the gray value analysisand data statistics, P <0.05.In conclusion, when NRK-52E cells induced by thymic cell induction medium for48hours, morphological change occurred, phenotypic changes in NRK-52E cells was alsodetected (loss of epithelial cell phenotype and acquire mesenchymal cell phenotypes), whichdemonstrated that NRK-52E cells had occurred epithelial-mesenchymal cell transition. Inshort, the in vitro activated T lymphocytes can induce EMT of renal tubular epithelial cell.Observation of infiltrating immunocytes in acute antibody-mediated rejectionWe obtained five kidney allografts removed from renal transplant recipients who developed irreversibly graft dysfunction early after transplantation. Then for the aim ofdiagnosis, the specimens were performed HE staining and detected C4d deposition byimmunohistochemistry. Based on the diagnosis of antibody mediated rejection we clarifiedthe immunophenotype of the cells in the intimal arteritis and glomerulitis byimmunohistochemistry in serial sections of the resected kidney. Moreover, in aim to clarifythe relationship of position between the immunocytes and endothelial cells, double stainingof immunohistochemistry was performed with the antibodies of macrophages (lysozyme)and endothelial cells (CD34).These five kidney allografts showed classical pathological features ofantibody-mediated rejection,we found infarction at different extension, and we can seeintimal arteritis, glomerulitis, and peritubular capillaritis in tissue beside the infarction area.Immunohistochemistry for C4d showed that all of these five cases were positive for C4dstaining, according to the latest diagnose standard, finally all these cases were diagnosed assuffered acute antibody mediated rejection.The result of immunohistochemistry for the immunocytes showed that in the intimalarteritis, the most important cellular infiltration came from macrophages and T-lymphocyteswhich took percentage of40.4±9.2%and30.6±13.3%respectively. There was nosignificant difference between the two kinds of cells, but their percentages were higher thanthose of B cells, plasma cells and neutrophils.45.4%of the T-cells were CD8+cytotoxicT-cells. Neutrophils were also present but accounted for a relatively low proportion of thecells (8.8±8.6%). B-cells and plasma cells all accounted for <5%of the immunocytes. NKcells were readily detected (4.2±4.2%). When we compared types of arteritis, the CD15+neutrophils accounted for as many as27.8±15.1%of immunocytes in V3vasculitis and only1.0±1.4%in V2vasculitis. CD3+T-lymphocytes accounted for25.8±7.3%of immunocytesin V3vasculitis and41.5±7.9%in V2vasculitis. The results of double staining ofimmunohistochemistry showed that lysozyme-positive macrophages were located beneaththe CD34-positive endothelial cells indicating that the immunocytes indeed infiltrated in thevessel wall in the intimal arteritis rather than being located in the lumen. In glomerulitis,the immunocytes were mainly macrophages (53.1±9.1%) and neutrophils (34.6±9.9%).In conclusion, Macrophages and T-lymphocytes accounted for the highest percentage ofimmunocytes in the intimal arteritis of irreversible renal failure associated withantibody-mediated rejection.45.4%of the T-cells were CD8+cytotoxic T-cells.Neutrophils and NK cells were also present in these lesions. The proportion of neutrophils in V3vasculitis was much higher than in V2vasculitis. These observations suggest thatbesides macrophages and T-lymphocytes, neutrophils may also play a role in the more severearterial lesion. To our knowledge this is the first report of this observation. Macrophagesand neutrophils were the main inflammatory cells in the glomerulitis of acute rejection.
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