Transplantation Of Genetically Engineered Mesangial Cells To Detect Acute Glomerulonephritis Ex Vivo And In Vivo

Background and AimGlomerulonephritis is one of common causes of chronic renal failure.Cur-rently,technologies are not available for continuous,non -invasive assessment of glomerulonephritis.Monocytes/macrophages play a crucial role in the genera-tion of various glomerulonephritis¹.Under pathologic situations,activated mac-rophages elaborate a variety of inflammatory mediators and stimulate resident cells,leading to initiation and progression of glomerular injury².Monitoring of macrophage-mesangial cell cross-talk is important for evaluation of activity of glomerulonephritis as well as for understanding of mechanisms underlying glo-merular injury.We stably introduced the secreted alkaline phosphatase(SEAP) gene into mesangial cells under the control of theκB enhancer elements.The aim of this study is to confirm whether this established system can monitor mac-rophage -mesangial cell cross -talk in vitro,whether it can detect local glo-merulonephritis or not when the genetically engineered cells are transplanted in vivo.MethodsClonal mesangial cells(SM43)were established from isolated renal glo-meruli of a male Sprague-Dawley rat⁹.Healthy male adult SD rats were used. Main devices including:Gene Light 55 luminometer,clean bench,CO2 incuba-tor.Great EscAPe SEAP detection kit,anti- Thy 1.lantibody¹¹,pNFκB-SEAP plasmid,IL-1β,TNF-αand MG132 were used. Using a calcium-phosphate co-precipitation method,mesangial cells (passage 10th-15th;5×10⁵ cells)were transfected with pNFκB-SEAP (5μg)together with pcDNA3.1(1μg)that encodes a neomycin phosphotrans-ferase gene.pNFκB-SEAP encodes SEAP under the control of 4 copies of theκB enhancer element.Stable transfectants were selected by 330μg/ml G418, and a clone with the highest SEAP inducibility was selected and used for stud-ies.We designated this sensor clone as SM/NFκB-SEAP5.Using the same method,SM/CRE-SEAP15 and SM/SV-SEAP28 cells were established. Stably transfected cells between passage 20th and 35th were used for experi-ments.Establishment of glomerulonephritis model:For this purpose,anti-Thy 1. 1 glomerulonephritis was induced in rats by intravenous administration with an anti-Thy 1.1 monoclonal antibody 1-22-3(1ml)from tail vein.Isolation of glomeruli:Cut and spin kidneys from normal or nephritic rats, pass through three sieves with diameter of holes as 180,125,106μm,collect glo-meruli with 0-4℃PBS.Cell transplantation:1×10⁶SM/NFκB-SEAP5 cells suspended in 1 ml of medium containing 1%FBS were injected into the left renal artery of normal rats or nephritic rats subjected to anti-Thy 1.1 glomerulonephritis(day 1).Detection of glomerulonephritis:For this purpose,rats were divided into 5 groups;normal rats(group A),normal rats injected with sensor cells(group B),nephritic rats(group C)and nephritic rats injected with sensor cells (group D),nephritic rats with the cells injected intraperitoneally(group.E). Sera were collected before and 1,3,6,8,10 days after the cell injection and subjected to SEAP assay.Each experimental group included at least 4 rats.In experiment 1,first characterize the sensor cells by immunofluorescent method,then monitor macrophages-mesangial cell interaction in vitro by the established sensor cells with Northern blot analysis and SEAP assay.In experiment 2,the cells were transplanted in vivo to detect glomerulone-phritis.Northern blot analysis and SEAP assay were used to monitor SEAP mR-NA and SEAP activity.In experiment 3,co-culture and cross-feeding studies were used to in- vestigate whether NF-κB signal pathway is activated spontaneously in explated normal isolated glomeruli and the relationship between NF-κB signal pathway and MAPK signals.Statistical analysis:Assays were performed in triplicate or quadruplicate. For animal experiments,one group include 4 to 6 rats.Data were expressed as means±SE.Statistical analysis was performed using the non-parametric Mann -Whitney U test to compare data in different groups,p＜0.05 was considered to be a statistically significant difference.ResultsExperiment 1:The mesangial cell phenotype of this clone was confirmed by positive staining forα-smooth muscle actin and Thy 1.1 using an anti-α-smooth muscle actin monoclonal antibody(1:100 dilution)and an anti-Thy 1.1 monoclonal antibody 1-22-3(1:100).The established sensor cell line expressed and secreted SEAP in response to a macrophage cytokine IL-1βin dose-and time-dependent manners.The kinetics of SEAP activity in condi-tioned media was closely correlated with the level of SEAP mRNA.Similar re-sponses were also observed in the sensor cells exposed to another macrophage cytokine TNF-α,but not by other stimuli including 12-O-tetrade-canoylphorbol-13-acetate,forskolin or platelet-derived growth factor.When activated rat macrophages were co-cultured with the sensor cells,rapid and dramatic induction of SEAP was observed.The induction was detectable within 4 h,peaked to maximum after 24 h and returned to the basal level within several days,the induction could be abrogated by NF-κB inhibitor MG132.Experiment 2:Fluorescent microscopic analysis of glomeruli isolated from cell-injected(left)and uninjected(right)kidneys showed marked accumula-tion of sensor cells in glomeruli isolated from cell-injected left kidneys,but not in glomeruli isolated from uninjected right kidneys.The percentage of DiI-pos-itive glomeruli was 90.8±2.9%(mean±SE).DiI-positive sensor cells were never detected in glomeruli from uninjected kidneys.In contrast to untransfected mesangial cells(SM43),SM/SV-SEAP28 cells exhibited very high levels of SEAP activity in culture media and expression of SEAP mRNA.When SM/SV -SEAP28 cells were transplated into normal rats,substantial increase in the level of SEAP was observed in a cell number-dependent manner,confirming that SEAP is a useful reporter molecule that can be detected in vivo.After the transplantation of SM/NFκB-SEAP5 sensor cells in vivo,significant elevation of Serum SEAP was observed within 24 h(2.26±0.34 fold;p＜0.05), peaked to maximum at day 3-6(3.46±0.44 fold at 3 day,3.47±0.85 fold at day 6;p＜0.05)and declined thereafter.When sensor cells were injec-ted intraperitoneally into nephritic rats,elevation of SEAP activity was not ob-served.This result indicated that locally created microsensor perceived and re-sponded only to the local inflammation.Experiment 3:We found that ex vivo incubation of isolated rat glomeruli spontaneously expressed a NF-κB-dependent gene,monocyte chemoattractant protein-1(MCP-1).The induction of MCP-1 was also observed in cultured mesangial cells exposed either to isolated normal glomeruli or medium condi-tioned by normal glomeruli,and it was abrogated in the presence of MG132,an inhibitor of NF-κB.The activation of NF-κB by glomerulus-derived parac-rine factors was further confirmed using reporter mesangial cells that produce SEAP under the control of NF-κB.The reporter mesangial cells exhibited up-regulation of SEAP mRNA and SEAP activity when exposed to isolated glomeruli or glomerular conditioned medium.Furthermore,isolated glomeruli adoptively transferred with the reporter mesangial ceils also showed upregulation of SEAP mRNA and SEAP activity.To examine the upstream events involved,we fo-cused on the MAP kinase pathways.Reporter mesangial cells were exposed to i-solated glomeruli in the presence of inhibitors for extracellular signal-regulated kinase(ERK)and p38 MAP kinase,and activation of NF-κB was tested. Suppression of ERK by PD098059 or inhibition of p38 MAP kinase by SB203580 significantly attenuated activation of NF-κB in reporter mesangial cells.Inter-estingly,activation of NF-κB in reporter cells by glomerular conditioned medi-um was not affected by the treatment with PD098059 or SB203580,suggesting that activation of MAP kinases is involved in glomerular production of paracrine, NF-κB activators. ConclusionsThe established sensor cell line expressed and secreted SEAP in response to a macrophage cytokine IL-1βin dose-and time-dependent manners.The kinetics of SEAP activity in conditioned media was closely correlated with the level of SEAP mRNA.Similar responses were also observed in the sensor cells exposed to another macrophage cytokine TNF-α,but not by other stimuli inclu-ding 12-O-tetradecanoylphorbol-13-acetate,forskolin or platelet-de-rived growth factor.When activated rat macrophages were co-cultured with the sensor cells,rapid and dramatic induction of SEAP was observed.The induction was detectable within 4 h,peaked to maximum after 24 h and returned to the basal level within several days.These data demonstrated that the established system provides simple and useful tools for continuous monitoring of cross-talk between macrophages and mesangial cells in vitro and ex vivo.2.When the established mesangial cell line is transplanted in vivo via kid-ney artery,the serum SEAP level elevated significantly.The dynamics of serum SEAP is closely associated with the activity of glomerulonephritis.After drug in-tervention the serum level of SEAP decreased significantly.So it is feasible to detect acute glomerulonephritis by the genetically engineered biosensor system.3.These data demonstrate for the first time that NF-κB is spontaneously activated in isolated glomeruli.This activation is mediated by MAPK.