Experiment Of Transplanting Metanephric Mesenchymal Stem Cells To Regenerate And Repair Acute Tubular Necrosis

Background: Acute renal failure (ARF) is a common severediseases with high mortality from 30% to 80% and occupies 5%～10% ofthe inpatients, though great progress had been made in supportivetreatment, nurse and replace therapy over the 30 years. Acutetubular necrosis (ATN) is responsible for the majority of ARF as high as75%～80%, with a series of manifestation such as tubular epithelial cellslose and basement membrane efluxion. But the survival cells besides thenecrosis can proliferate and migrate, then replace the necrotic cells.Although the renal tubule have such a capability to recover, the key pointis that there must enough survival cells. So during recent years somegrowth factors have been used to stimulate the tubular regeneration. Atthe same time, many experiments have reported that bone marrow stemcells(BMSC) as mesenchymal stem cells(MSCs) and hemopoietic stemcells(HSCs) could treat ARF or ATN and regenerate the injuried kidney,while the real mechanism is still obscure. Because many argues exist,BMSC is not considered to be the ideal choice to treat ARF. It is now clear that the embryonic metanephros mesenchymal contains stem cellswhich is multipotential and self-renewal and can replace the ageing orapoptotic cells during normal condition, participate the reparation andregeneration during pathologic state. The course of differentiation issimilar to the development of the embryonic kidney. So, we transplantedthe metanephros mesenchymal cells to ATN model rats by injection intothe caudal vein, traced the cells homing and differentiation, approachedthe possible mechanism of reparation and regeneration to providerationale and basis for cell therapy of ATN in clinic.Objective: To invest the reparation role and the possiblemechanism of transplanting embryonic metanephros mesenchymal cell toATN.Methods:1.Cultured and expanded the embryonic metanephros mesenchymal(RIMM-18) cells in vitro.①After resuscitation the cells were cultured inDMEM/F12 with 5% fetal bovine serum(FBS),10ng/mL FGF2 and100nmol/L 17β-estradiol(17β-E2);②Observed the cells'morphologyunder light microscope and electron microscope;③Identificated the cellsbiologic character by immunocytochemistry detection;④Induced thecells to differentiate to epithelia cells in vitro;⑤Labelled the cells withDAPI or Brdu before transplantation and calculate the labelling raterespectively;⑥Detected the cells' activity by MTT and trypan blue. 2. Made an ATN model induced by gentamicin: injected a total doseof 900mg/kg gentamicin subcutaneously within 3 days.3.Transplanted the cells: The rats were divided into four groups:①ATN with cells transplantation group: injected 1～6×10~7/ml cells intocaudal vein at the 3rd day of gentamicin injection;②ATN with culturemedium group: injected the same volume of culture medium without FBSat the 3rd day of gentamicin injection;③ATN group: injected gentamicinfor 3 days only;④control group: the normal rats without anyintervention. Rats in all groups were killed at 1d, 4d, 1w, 2w and 4wrespectively. Specimen as urine, blood and kidney were remained.4. Detected following parameters:①Traced the cells homing:observed the DAPI positive cells under fluorescence microscope,detected the Brdu positive cells in kidney by immunohistochemistrystaining.②Detected the function of renal tubule and glomeruli: BUN,SCr,urine protein,β2-MG,NAG etc.;③Observed the renal pathologicchange by HE and PAS staining, at the same time graded the Paller'sscore;④Detected the apoptosis by TUNEL;⑤Detected the Ki-67expression in renal tubule;⑥Detceted some relative cytokines:IL-1β, TNF-α, bFGF and Bcl-2 mRNA in renal cortex by RT-PCR;TNF-α, iNOS and Bcl-2 protein by immunohistochemistry;⑦Monitoredacute immunological rejection: monitored the CD3,CD4,CD8, CD4/CD8and IL-2,INF-γby flow cytometer and ELISA respectively. Results:1.Cell culture:①RIMM-18 cells grew adherencely asfibroblast-like;②The cells were vimentin positive and keratin negativeby immunocytochemistry;③The labeling rate of DAPI and Brdu was100% and 86% respectively;④The cells'activity was above 95% byMTT or trypan blue;⑤RIMM-18 cells could be differentiate to epitheliacells in vitro without 17β-E2 in the medium.2. ATN model induced by gentamicin: Total dose of 900mg/kgwithin 3 days could make ATN model successfully. A large first dosecould reduce the mortality rate.3. Embryonic metanephros mesenchymal transplantation: In celltransplantation group, we could find the results as following:①Themortality rate lowered obviously;②The renal function amelioratedvisiblely;③DAPI and Brdu positive cells could be detected in the tubuleat each time of 5 points;④The renal pathologic change relievedsignificantly;⑤The apoptosis in the tubule decreased and proliferationincreased;⑥The anti-inflammatory cytokines in the kidney wereup-regulated, and pro-inflammatory cytokines were down-regulated.4. Acute immunological rejection detection: The difference ofCD3, CD4, CD8, CD4/CD8 and IL-2,INF-γbetween each group was notsignificant. Conclusions:(1)RIMM-18 cells could remain it's stemness character after manypassages;(2) A large first dose of gentamicin could reduce the mortality rateduring making ATN model;(3)The transplanted RIMM-18 cells by injection into caudal veincould home to the damaged tubule and reduce the mortality rate,facilitate the renal function recovery and improve the renal pathology;(4)The possible mechanism of regeneration was that RIMM-18 cellscould up-regulate Bcl-2 and bFGF expression, down-regulateIL-1β, TNF-αand iNOS expression, decrease apoptosis and increase thetububles proliferation to regenerate the renal function;(5)RIMM-18 cells transplantation induced no acute immunologicalrejection.