| Background Acute renal failure (ARF) is a common severedisease with many complicated etiologies in clinical practice. Themortality associated with this syndrome is high and still ranges between50％and 80％. Acute tubular necrosis (ATN) is the the main cause ofARF, as high as 75％～80％. Despite tremendous advances in supportivetreatment and renal replacement therapy (RRT) over the past twenty years,treatment options for this life-threatening disease are non-specific. Inrecent years, excitement has grown over the therapeutic potential ofcellular transplantation instead of organ transplantation. Stem cell therapyhas been used successfully in many fields, such as injecting skeletalmuscle cells directly to post-myocardial infarction tissue; making nervecells to Parksing patient's brain. They have gained some evidence ofclinical improvement. Especially in the neurology field, many trials havedemonstrated that celluar transplantation contributed to rebuiding,repairing and improving injured nervous pathway. As to renal tubularepithelial cells, they are known for limited capacity to repair themslevesfollowing acute injury. However, the recovery is dependent on the abilityof the remaining tubular cells to dedifferentiate, enter the cell cycle,proliferate, reline the damaged areas along the nephron, andredifferentiate. It is universal believed tubular epithelial cells regress toprimitive/embryonic mesenchymal phenotype in response to injury. Thisreverse embryogenesis is a key step in regeneration of the tubularepithelium. However, the better recovery depends on the replacement orregeneration of dead cells by adequate new cells. For this reason, wedevised, through renal subcapsular transplantation technology, suppliedadequate exogenous renal stem/progenitor cells to substitute injuredtubular cells, repair the renal tubulus and improve renal function, namely cellular regeneration strategy, which had most prominent clinicalapplication. Embryonic metanephron mesochymal stem cells aregenerally defined as clonogenic cells that are capable of both self-renewaland multilineage differentiation. Foreign researchers have demonstrated asingle metanephric mesenchymal cell can differentiate into all tubularstructures of the adult nephron with the exception of the collecting system,indicating that the kidney contains epithelial stem cell, namelymetanephron mesochymal cell is MM-Ep. The subrenal capsulartechnique has reviously mainly been applied in studies ofchemotherapeutics sensibility. A number of studies have demonstratedthat the kidney tunic microenvironment is suitable for metanephrosdevelopment and maturation. Because of abundant vascular bed beneaththe renal capsule, the grafted cells can be absorbed to renal parenchymaquickly. The subcapsular transplantation of MMCs may be a newtherapeutic approach to ATN treatment. In order to verified our thesis andprovide technical basis for clinical treatment, we carded out the presentstudy to evaluate the nephroprotective effects of renal subcaspsulartransplanting MMCs on nephrotoxic ATN induced by gentamicin in rats,by the means of tracing experiment in vivo, detecting function,morphology, tubular proliferation and apotosis.Objective After culture-expanded MMCs in vitro, explored thenephroprotective effects of renal subcaspsular transplanting MMCs onnephrotoxic ATN induced by gentamicin; also explored the role of celluarapotosis to injured tubular epithelial cells.Methods (1)The experiment of culture-expanded MMCs in vitro.The RIMM-18 renal stem cells, a kind gift from Ph.D Levashova(Laboratory of Comparative Carcinogenesis, National Cancer Institute,National Institutes of Health, Frederick, Maryland) were demonstrated tohave the characteristics of undefferentiated mesenchymal cell.①Revival, culture and propagation for RIMM-18 cells Revival: rewarmingquickly in 37℃waterbath, washing cells——transformed cells tocentrifuge tube to centrifuge, added media and centrifuged again,repeated above. Culture: RIMM-18 cells were cultured in Dulbecco'smodified Eagle's media (DMEM)/F12 (1:1) supplemented with 5% fetalbovine serum (FBS), bFGF(10ng/mL) and 1713-estradiol (100nmol/L)(the complete defined media). The cells were maintained at 37℃in ahumidified atmosphere containing 5% CO2. The media were changedregularly every two or three days. Passage: Cells were washed inD-Hank's fluid twice, digested with 1ml 0.25% trypsin/0.02%EDTA(1:1)for 30 seconds at 37℃until most of cells were detached. RIMM-18 cellswere mechanically dissociated by gentle aspiration through repeatedpippeting and plated at the density of 10~4 cells/cm~2.②RIMM-18 cellslabeling experiment in vitro (MMCs labeled by DAPI): RIMM-18cells were cultured in DAPI (50μg/mL) media for 40 minutes beforetransplantation. After quickly washing the cells in seven changes ofD-Hank's to remove unincorporated DAPI, we detached RIMM-18 cellsfrom the bottom of the flasks with 0.25%trypsin/0.02% EDTA(1:1) 1mLand then harvested cells for detecting by fluorescence microscopeon 40 minutes, 5d, 8d, 11d and 14d.③Identification of RIMM-18 cells:The identity of the cultural cells was confirmed by immunocytochemistrydetection with specific antibody and observation of profile under invertedmicroscope.(2)The experiment of renal subcapsular transplantation ofmetanephric mesenchyme cells. One hundred and sixty seven femaleSprague-Dawley rats (aged 8-12 weeks) were divided into five differentgroups randomly as follows:①Control group(Control): done nothing toit;②Acute tubular necrosis group(ATN): gentamicin (200mg/(kg.d))injected subcutaneouly for four days;③Acute tubular necrosis plus sham operation group (ATN+Sham): gentamicin (200mg/(kg.d)) injectedsubcutaneouly for four days and injected saline(0.2ml) to right subrenalcapsular at the first day;④Acute tubular necrosis plus media group(ATN+Med): gentamicin (200mg/(kg.d)) injected subcutaneouly for fourdays and injected media (0.2mL, fresh serum-free) to right subrenalcapsule at the first day;⑤Acute tubular necrosis plus celluartransplantation group(ATN+Cell): gentamicin (200mg/(kg.d)) injectedsubcutaneouly for four days and injected cells(1×10~7 of cells in 0.2mL offresh free-serum) to right subrenal capsule at the first day. Rats in allgroups were killed at day of 5,8,11,14 after injection gentamycin. At eachtime point, parameters were detected as follow:①The tracing trial invivo;②Detection 24 hours urinary protein, urinary NAG, serum ureanitrogen, serum creatinin.③Histopathologic examination by lightmicroscopy and severity scores.④Immunohistochemistry for Ki-67 ofrenal tubular epithelial cells and counting proliferation index.(3) The effection of metanephric mesenchyme cells on apotosis inATN rats. The arrangement of rats in this section was as same as thesecond part.①TUNEL(TDT-mediated dUTP-biotin Nick End-Labeling) assay for detecting apotosis cells;②RT-PCR for detecting renalBcl-2 (B-cell lymphoma/leukemia-2)mRNA;③Immunohistochemistryfor detecting Bcl-2protein expression level.Results (1)The experiment of culture-expanded MMCs in vitro.We have culture-expanded RIMM-18 cells in vitro successfully.RIMM-18 cells were characterized by a number of features, includingtheir round nuclei with DAPI staining resulted in green fluorescence(detecting by fluorescent microscope under 370nm), the bigspindle-shaped profiles (revealed by staining with hematoxylin and eosin,Giemsa), their growth in a "scattered colony" pattern. The phenotype ofcultured RIMM-18 cells was vimentin positive and keratin negative by immuocytochemistry.(2)The experiment of renal subcapsular transplantation ofmetanephric mesenchyme cells. Compared with other groups, theright kidneys of acute tubular necrosis plus celluar transplantation groupdisplayed:①Renal function was improved obviously;②The right kidneydamages were relatively gentle and histopathologic lesion scores wererelatively lower;③The degree of proliferation of renal tubular epithelialcells were improved;④The mortality rate was decreased.(3)The effection of metanephric mesenchyme cells on apotosis inATN rats. Compared with other groups, the right kidneys of acutetubular necrosis plus celluar transplantation group displayed:①Theapoptotic degree of renal tubular epithelial cells descented.②Theexpression level of Bcl-2 mRNA was upregulated.③The expression levelof Bcl-2 protein was relatively stronger.Conclusion (1)RIMM-18 cells can be culture-expanded in vitrowith stable mesenchymal characteristics. (2)After renal subcapsulartransplantation RIMM-18 cells on ATN rats, we demonstrated the graftedcells can migrate to the injured renal tubule, and may transform totubular epithelial cells, accelerate tubular proliferation, improvehistopathologic lesion, promote structural and functional repair of renaltubule, and decrease mortality ultimately. (3)Bcl-2 mRNA and its proteinwere upregulated after tansplantation of RIMM-18 cells into the renalsubcapusle, which may serve as an mechanism for the tubularregeneration.
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