| Objective:To investigate if bone marrow derived stem cells can differentiate into renal parenchymal cells in mouse ischemia-reperfusion renal injury model, explore the role of bone marrow stem cells in the recovery of acute kidney injury. Moreover, the trans-differentiation of bone marrow stem cells into renal progenitor cells, the mobilization of bone marrow stem cell by G-CSF, and the contribution to the recovery of renal injury of bone marrow derived stem cells are also to be examined.Methods:Six-week-old transgenic C57BL/6J mice labeled with green fluorescent protein (GFP) as bone marrow donors, C57BL/6 mice without fluorescence label as recipients of bone marrow transplantation were used. All recipients received lethal irradiation,8.5Gy total body y ray irradiation of 137Cs before bone marrow transplantation, and the transplantation of bone marrow mononuclear cells 2×105by retrobulbar injection was done two hours later after irradiation. Bone marrow reconstruction after transplantation was proved by flow cytometry five weeks after transplantation. After the bone marrow reconstruction was completed, left renal pedicles of all mice were cross-clasped for 30 minutes followed by reperfusion to establish the ischemia-reperfusion animal model at week 6. Mice were divided into two groups:(1) saline control group (n=10):chimeric mice were injected s.c. with saline 0.2ml/day for a week from three days before surgery. (2) G-CSF mobilization group (n=10):chimeric mice were injected s.c. with recombinant human G-CSF, 200μg/kg/day, once a day for a week from three days before surgery. On the 1st day after mobilization, we examined the percentage of Sca-1+, c-Kit+and CD34+ stem cell in nonerythroid cells of peripheral blood by flow cytometry. One week after ischemia, pathological staining of renal sections conducted with Hemotoxylin and Eosin staining method, and the degree of renal tubular injury was analyzed by semi-quantitative method of Vyacheslav. The differences of pathological renal injury between the G-CSF mobilization group and the saline control group were compared. By collagenase digestion, we obtained the scattered single renal cells. And we used different stem cell antigen markers such as Sca-1, c-Kit and CD34 to investigate the differences of percentage of stem cells between the two groups by flow cytometry. Four weeks after ischemia, we observed the trans-differentiation of bone marrow derived stem cell in kidney by immunohistochemistry. We also compared the differences of recovery of renal injury between the two groups by analysis the numbers of vascular endothelial cells in the kidney and bone marrow derived renal progenitor cells.Results:After G-CSF mobilization, The percentage of stem cells with Sca-1+, c-Kit+ and CD34-antigen in peripheral blood of G-CSF mobilization group (3.16%±0.11%, 3.78%±0.08%,6.09%±0.13%) were higher than those of control group (1.67%±0.10%.2.19%±0.06%,2.85%±0.14%). This result revealed that bone marrow derived stem cells were effectively mobilized by G-CSF to peripheral. One week after ischemia, mice of mobilization group (0.86%±0.05%,0.78%±0.11%. 0.90%±0.08%) showed higher percentage of Sca-1+, c-Kit+and CD34+bone marrow derived stem cells (GFP and stem cell antigen double positive) in the kidney compared to control group (0.51%±0.06%,0.47%±0.08%,0.43%±0.03%). and the difference was statistically significant (P<0.05). The percentage of bone marrow derived GFP positive cells in kidney in the G-CSF-treated mice was 13.05%±1.14% while in the control mice was 7.75%±0.58% which was also statistically significant (P<0.05). The results demonstrated the homing of bone marrow derived stem cells to kidney after ischemia mobilized by G-CSF. Four weeks after ischemia, we observed the integration of bone marrow derived stem cells into tubular interstitium and glomerular vascular cluster by immunohistochemistry. And the GFP+ bone marrow derived stem cells can transdifferentiate into CD31 or SMA positive vascular endothelial cells and participated in the neovascularization after injury. We further detected GFP positive bone marrow derived cells in renal tubules in injured regions, so this result revealed that bone marrow derived cells transformed into tubular epithelial cell and participated in recovery of tubular injury. We also observed GFP and Sca-1 double positive bone marrow derived renal progenitor cells in renal interstitium. One week after ischemia, the extent of tubular epithelial damage score of mobilization group (1.46±0.05) was significantly (P<0.05) lower than that of the control group (2.32±0.16) by Hemotoxylin and Eosin staining. Four weeks after ischemia, mice of G-CSF mobilization group (31.3±0.42) showed more CD31 positive cells in the kidney compared to control group (20.05±0.58), and the difference was statistically significant (P<0.05). By counting the numbers of bone marrow derived renal progenitor cells, the number of GFP and Sca-1 double positive cells in the mobilization group (6.00±0.82) was significantly (P<0.05) more than that of the control group (3.50±0.58). And the percentage of GFP and Sca-1 double positive renal cells was also higher compared to control group by flow cytometry. All these results demonstrated that G-CSF contributes to the recovery of renal injury.Conclusion:(1) Bone marrow derived stem cells participate in the renal neovascularization and tubular regeneration after injury.(2) G-CSF can effectively mediate the mobilization of bone marrow derived stem cells to peripheral and homing to kidney.(3) G-CSF mobilization can alleviate the degree of renal histopathological injury after ischemia. (4) G-CSF mobilization can accelerate renal recovery by promoting renal neovascularization and the differentiation of bone marrow derived cells into renal progenitor cells.(5) Bone marrow derived cells can transdifferentiate into renal progenitor cells. This result confirms the presumption that renal progenitor cells maybe partly come from bone marrow.
|
PDF Full Text Request