| Background:Acute tubular necrosis (ATN) of the kidney is common injury affecting renal tubular function and it is responsible for the majority of cases of acute renal failure in the hospital setting. Acute ischemic renal injury, a major cause of ATN, often occurs in renal transplantation, poisoning and shock due to haemorrhage or sepsis. Following ischemic injury, tubular cells have been found to undergo frank necrosis, apoptosis, detachment, or dedifferentiation into a more mesenchymal phenotype. The dedifferentiated cells appear to spread out and to migrate into regions of denuded basement membrane, and they eventually proliferate to repopulate and repair the injured tubule. During the past several years, a great deal of attention has been focused on metanephric mesenchymal cells (MMCs). It was demonstrated that metanephric mesenchyme contains multipotent stem cells. Traditionally, stem cells were believed to be lineage restricted and organ specific. However, recent studies have shown that MMCs from rat have the ability to cross lineage boundaries and to form functional components of other tissues, expressing tissue-specific proteins in organs. The possibility that metanephric mesenchymal cells might functionally contribute to the renal tubule regeneration has been more controversial and suggests that approaches based on the delivery to the kidney of the appropriate MMCs should be investigated in our attempts to improve the outcome of ATN.Objective:The self-recovery of the renal tubule from acute tubular necrosis is that surviving cells from the areas bordering the injury must migrate into the injured tubule and proliferate to re-establish the normal tubular epithelium. However, such events are so limited and failed to alter the course of acute renal failure in human trials. In the present study, we aimed to describe the isolation and characterizations of MMCs derived from normal embryonic rat kidney and establish a method culturing MMCs primarily derived from metanephric tissue of embryonic rats. Also we explored the effects and mechanism of MMCs intravenously injected into rat suffered with ischemia/reperfusion injury induced tubulonecrosis, proposing that MMCs might repair the tubular injury, which was an entirely new approach for the treatment of acute tubular necrosis.Methods:(1) Primary metanephric mesenchymal cell cultures were prepared by explanting the pieces of metanephric tissue of embryonic kidney rudiments microdissected from gestation day 13 rat embryos (Sprague Dawley) into plastic flask,then cells grew out from the tissue pieces. Cells were identified by observation with inverted phase contrast microscope,as well as by phenotypic protein examination via flow cytometry (CD29, CD31, CD34, CD45, CD90, CD166) and immunochemistry staining (E-cadherin, Vimentin, Fibronectin). Also proliferation and renewal of cells was measured by means of methylthio tetrazole (MTT) and 5-bromodeoxyuridine (BrdU) and multi-direction differentiation including adipocytes and osteocytes was induced.(2) Cultured metanephric mesenchymal cells were labeled with 5-bromodeoxy- uridine. Acute renal tubular damage rat model was established by ischemia- reperfusion injury. SD rats were randomly divided into three groups which were MMCs injection group (MMCs), saline infusion (I/R) and sham operation group (sham). For experiments involving cell infusion after I/R, undifferentiated metanephric mesenchymal cells labelled with BrdU were intravenously implanted into SD rats after reperfusion as described. Cells were washed with phosphate-buffered saline, counted in a microcytometer chamber, and intravenously injected into the rats via a needle. After operation rats were sacrificed at 24h, 48h, 72h and 96h respectively, the changes of renal function and pathology were observed. Apoptosis was conformed by TUNEL assay,also the protein expression of Bcl-2 and Bax were detected by immunohistochemistry. The distribution of labeled MMCs in kidney was observed by immunofluorescence technique.Results:(1) The cultured MMCs showed shuttle shape with single nuclear in each one. Flow cytometry results showed that significant expression of CD29, CD90 and CD166 was positive over all of the cells and expression of CD31,CD34,CD45 was negative in contrast and immunochemistry staining results showed that significant expression of vimentin and fibronectin on the surface of MMCs, but E-cadherin was negative in contrast,which confirmed the mesenchymal cell and stem cell phenotype. Cultured cells could be propagated to 10 generations and remained the phenotype of mesenchymal cells. Also the cells could be induced to differentiate to adipocytes and osteocytes in special induction conditions.(2) MMCs infusion ameliorated the decline of renal function at each time point (P<0.05). Animals received both I/R and metanephric mesenchymal cells transplantation demonstrated a peak BUN of (44.08±5.63)mg/dl and Scr of (333.38±31.13)μmol/L on 48 hours after I/R with a subsequent decline to BUN of (30.08±6.16)mmol/L, Scr of (189.97±19.22)μmol/L on 72 hours and BUN of (15.05±2.66)mmol/L, Scr of (97.67±12.09)μmol/L on 96 hours. In contrast, animals that did not receive metanephric mesenchymal cells had a peak BUN of (66.85±7.65)mmol/L and Scr of (440.73±31.09)μmol/L on 48 hours and subsequently on 72 hours BUN of (42.22±5.61)mmol/L, Scr of (299.08±14.57)μmol/L, on 96 hours BUN of (31.32±4.15)mmol/L and Scr of (205.70±27.01)μmol/L.(3) HE staining showed pathological damage on MMCs injected kidney was less than I/R only kidneys at each time point (P<0.05). The peak of tubular injury was observed 48 hours after ischemia/reperfusion injury. The morphological alterations observed included widespread necrosis of tubular epithelial cells and tubular cast formation. Proximal and distal tubules showed loss of brush border, cytoplasmic vacuolization, and flattening of epithelial cells with aspect of apparent denudation of tubular basal membrane.(4) TUNEL results investigated that the number of apoptosis renal tubular epithelial cells on MMCs injected kidney was less than that of I/R only kidneys at each time point (P<0.05). Especially after 48 hours of MMCs injection, the number of apoptosis renal tubular epithelial cells on MMCs injected kidney (13.4±3.2/HPF) was significantly less than I/R only kidneys (25.4±5.2/HPF). And the immuno- histochemistry staining results showed that Bcl-2 positive cells in MMCs group were more than those in saline group, though Bax positive cells fewer. The ratio of Bcl-2/Bax increased significantly on the kidneys of MMCs injected group rats.(5) Immunofluorescence results showed that after 48 hours of labelled MMCs injection, only a few scattered 5-bromodeoxyuridine positive cells were detectable in proximal and distal tubules of the injured kidneys. And more and more 5-bromodeoxyuridine positive cells were found in the injured tubules of kidneys along with the prolonged MMCs injection time. At the 72h group, BrdU-labeled MMCs occurred in renal tissue [(59.6士6.4)/HP] and they significantly increased at the 96h [(142.8士8.0)/HP]. No localization of injected renal progenitor cells was detectable into the renal glomeruli.Conclusions:(1) Cultured MMCs from metanephric tissue of embryonic rats could be propagated and had strong proliferation ability and stability. They showed both the mesenchymal cell and stem cell phenotype. They also could be induced to differentiate to adipocytes and osteocytes in special conditions.(2) Cultured MMCs could migrate and repopulate in the ischemia/reperfusion injured renal tubules, contributing to the repair of the injured renal tubules of ischemia-reperfusion rats. They could ameliorate the decline of renal function and pathological damages, also inhibit renal cell apoptosis and regulate the expression of Bcl-2 and Bax. MMCs could protect rat renal function of suffered acute renal tubular damage induced by ischemia/reperfusion.
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