间充质干细胞通过调节天然免疫系统减轻急性肾损伤的机制研究

Mesenchymal stem cells attenuate acute kidney injury via regulation of natural immune system

目的：探讨骨髓来源的间充质干细胞（MSCs）减轻缺血/再灌注损伤（IRI）诱导急性肾损伤（AKI）的可能机制。方法雄性C57/BL6小鼠45只，按随机数字表法分为假手术（Sham）组、IRI组及IRI＋MSCs组3组，每组15只。采用夹闭双侧肾蒂35min的方法建立小鼠IRI诱导的AKI模型；Sham组只开腹分离肾蒂，但不夹闭双侧肾动脉。于再灌注6h，IRI＋MSCs组经尾静脉注射1×104/μLC57/BL6小鼠骨髓来源的MSCs100μL；IRI组尾静脉注射等量生理盐水。再灌注48h后取血检测各组血清肌酐（SCr）、尿素氮（BUN）水平；过碘酸雪夫染色（PAS染色）后显微镜下观察肾脏病理改变并计算肾小管损伤评分。再灌注24h和72h用流式细胞仪检测浸润至肾脏的白细胞总数（CD45＋），用免疫荧光技术检测浸润至肾脏的中性粒细胞（Ly-6G＋）及巨噬细胞（F4/80＋）数量。结果与Sham组比较，IRI组48hSCr、BUN显著升高〔SCr（μmol/L）：180.3±8.8比9.7±3.5，BUN（mmol/L）：112.1±8.3比9.4±2.3，均P＜0.01〕，肾小管损伤评分升高（分：4.80±0.55比0）；24h和72h肾脏白细胞浸润数显著升高（×105个/g：60.50±2.56比19.46±4.83，42.00±1.87比14.70±3.74，均P＜0.01），同时免疫荧光染色可观察到肾脏中性粒细胞和巨噬细胞浸润数明显增加，但再灌注72h细胞数量较24h时明显减少。与IRI组相比，IRI＋MSCs组可显著降低48hSCr、BUN水平〔SCr（μmol/L）：99.0±8.0比180.3±8.8，BUN（mmol/L）：84.5±7.6比112.1±8.3，均P＜0.01〕，肾脏中坏死肾小管明显减少，肾小管有所修复，肾小管损伤评分明显降低（分：2.60±0.55比4.80±0.55，P＜0.05）；24h和72h时浸润至肾脏的白细胞总数显著减少（×105个/g：24.20±4.53比60.50±2.56，31.70±3.15比42.00±1.87，均P＜0.01），肾脏的中性粒细胞数量明显减少，巨噬细胞数量明显增加；但72h白细胞浸润数较24h时显著增加（×105个/g：31.70±3.15比24.20±4.53，P＜0.05）。结论 MSCs可能通过减少浸润至肾脏的炎性细胞总数，特别是中性粒细胞数量，增加浸润至肾脏的巨噬细胞数量，从而减轻IRI诱导的AKI。

Objective To explore the potential mechanisms of mesenchymal stem cell （MSC） therapy in ischemia/reperfusion injury （IRI）-induced acute kidney injury （AKI）. Methods Forty-five C57/BL6 male mice were randomly divided into three groups： sham group, IRI group, and IRI＋MSCs group, with 15 mice in each group. The IRI-induced AKI model in mice was reproduced by clamping both renal pedicles for 35 minutes. In the sham group, both kidneys were exposed, but their pedicles were not clamped. Six hours after reperfusion, mice in IRI＋MSCs group received 100 μL of MSCs （1×104 /μL） isolated from the bone marrow from C57/BL6 mice via tail vein, while the mice in the IRI group received same amount of normal saline. Blood samples were harvested at 48 hours after reperfusion, and levels of serum creatinine （SCr） and blood urea nitrogen （BUN） were determined. The changes in renal pathology were observed by microscopy with PAS staining, and the tubular injury and acute tubular necrosis （ATN） scores were calculated. The number of leukocytes （CD45＋） infiltrated in kidney at 24 hours and 72 hours after reperfusion was measured with flow cytometry. The number of neutrophils （Ly-6G＋） and macrophages （F4/80＋） infiltrated in kidneys at 24 hours and 72 hours after reperfusion was determined by immunofluorescence. Results There was significant increase in the related parameters in IRI group compared with those of sham group. The levels of SCr （μmol/L） and BUN （mmol/L） were 180.3±8.8 vs. 9.7±3.5, and 1 121.1±8.3 vs. 9.4±2.3, both P 〈 0.01. The score of tubular injury was 4.80±0.55 vs. 0 at 48 hours after reperfusion. The quantity of leukocyte （CD45＋） infiltration in kidney at 24 hours and 72 hours after reperfusion was increased （×105 cells/g： 60.50±2.56 vs. 19.46±4.83, 42.00±1.87 vs. 14.70±3.74, both P 〈 0.01）, and the number of neutrophils （Ly-6G＋） and macrophages （F4/80＋） infiltrated in kidney at 24 hours and 72 hours after reperfusion was also increase although the number of leukocytes infiltrated in kidney was significantly lower at 72 hours after reperfusion than that at 24 hours. There was significant lowering of the levels of SCr and BUN [SCr （μmol/L）： 99.0±8.0 vs. 180.3±8.8, BUN （mmol/L）： 84.5±7.6 vs. 112.1±8.3, both P 〈 0.01] in IRI＋MSCs group, compared to IRI group. For the degree of tubular necrosis in two groups, the tubular injury scores were 2.60±0.55 vs. 4.80±0.55 （P 〈 0.05）. The number of leukocytes infiltrated in kidney at 24 hours and 72 hours after reperfusion （×105 cells/g） were 24.20±4.53 vs. 60.50±2.56, 31.70±3.15 vs. 42.00±1.87 （both P 〈 0.01）. The number of neutrophils was lowered despite （the number of macrophages was increased）. However, the number of infiltrated leukocytes was significantly more in IRI＋MSCs group at 72 hours than that at 24 hours （×105 cells/g： 31.70±3.15 vs. 24.20±4.53, P 〈 0.05）. Conclusion MSCs could protect against IRI induced AKI by reducing the total number of leuckocytes, especially that of the neutrophils infiltrating into ischemic kidney and by recruiting macrophages into ischemic kidney.