In vitro FK506 kidney tubular cell toxicity

Nephrotoxicity is the most prominent side effect of the new immunosuppressive drug FK506. Some of the histopathological changes associated with cyclosporine (CyA) nephrotoxicity such as tubular vacuolization and glomerular thrombosis have also been reported with FK506 therapy. In this study we used kidney tubular cells in culture to address the issue of FK506- and CyA-induced tubular damage. Exposure of tubular cells to high concentrations of FK506 or CyA (10, 50 and 100 µM) induced a time-and dose-dependent cell injury in vitro characterized by a direct cytotoxic effect on tubular cells as expressed by release of ³H-thymidine from prelabelled cells, N-acetyl-β-D-glucosaminidase (NAG) release and cell detachment. Ultrastructural changes (vacuolization, swelling and mitochondrial enlargement) and inhibition of the growth (DNA and RNA synthesis) of cultured tubular cells were also observed at high concentrations of FK506 and CyA. These concentrations are higher than those reached in clinical situations, but close to the concentrations that may be reached by FK506 or CyA in tissues. Low concentrations of FK506 and CyA (1, 0.1 and 0.01 µM) were not cytotoxic and induced only a minimal inhibitory effect on the growth of tubular cells in vitro. At the same concentration CyA induced more cell detachment, more NAG release and a stronger inhibitory effect on cell growth than FK506 (P < 0.01). Since an evident cytotoxic effect was observed only at high concentrations, we can speculate that tubular toxicity is due to the accumulation of drug in the cells inducing cell disruption and death.     

Cisplatin toxicity reduced in human cultured renal tubular cells by oxygen pretreatment

Abstract

Cisplatin is an effective and widely used chemotherapy agent and its side effects, particularly nephrotoxicity, limit its usage and related platinum-based drugs. Cisplatin nephrotoxicity is mainly due to extremely increase in reactive oxygen species (ROS) generation leading to kidney tubular cell death. Preconditioning with oxidative stress has been demonstrated to stimulate the cellular adaptation to subsequent severe oxidative stress. Short term oxygen pre-exposure as a mild oxidative stress may enhance some endogenous defense mechanisms, so its effect on Cisplatin induced cell death was investigated in present research. We studied the effects of hyperoxic environment pre-exposure on Cisplatin toxicity in an in-vitro model of cultured human embryonic tubular epithelial cells (AD293). Viability of AD293 cells, as evaluated by MTT-assay, was affected by Cisplatin in a time (1–4?h) dependent model. Biochemical markers of cell apoptosis were evaluated using immunoblotting. Pretreatment with nearly pure oxygen (≥90%) for 2?h significantly reduced the level of cell damage. Activated caspase 3 and Bax/Bcl-2 ratio were significantly increased in Cisplatin-treated cells. Oxygen pretreatment inhibited caspase 3 activation and decreased Bax/Bcl-2 ratio. Oxygen pre-treatment itself not showed any cytotoxicity in exposure times up to 3?h. Our data indicate that hyperoxic preconditioning reduces Cisplatin toxicity in cultured human tubular epithelial cells. The exact mechanism of protection is unclear, though enhancement of some endogenous defense mechanisms and subsequently scavenging of free oxygen radicals may play an important role.     

Analysis and isolation of renal tubular cells by flow cytometry.

The cells of the renal cortex have rich heterogeneity of structure and function. Flow cytometry, the technique of rapid laser-based single cell analysis, can give information about cellular mixtures not obtainable by any other means. We examined a variety of fluorescent markers to identify populations of renal cells by flow cytometry. Cellular digests of rat cortex were fluorescently stained with either enzymatic activity probes, or polyclonal antibodies. Fluorescent staining for the proximal marker gamma-glutamyl-transpeptidase (tau-GT) was an order of magnitude brighter than autofluorescence, and stained 71 +/- 11% of the cells. Second, we colocalized enzymatic and antibody markers. There was tight colocalization of tau-GT enzyme activity, detected with fluorogenic substrates, with specific surface binding of tau-GT antibodies. Third, populations of fluorescently labelled cells can be rapidly isolated by flow cytometry sorting. Flow cytometry sorting isolated 10(7) cells positive for the proximal tubular marker tau-GT in a little under one hour. The sorted cells were viable with 99 +/- 2% trypan blue exclusion (N = 8). Sodium-dependent phloridzin-inhibitable glucose uptake was present in sorted cells, with greater uptake/mg protein than in unsorted controls. The sorted cells grew in culture as a monolayer of tightly adherent cuboidal cells. Hence, flow cytometry allows us to quantitate the heterogeneity in mixed renal cellular digests. Flow cytometry allows us to rapidly isolate millions of cells according to fluorescently tagged markers. The isolated cells are viable, retain sodium-dependent transport properties, and grow in culture.     

1-Methylhydantoin cytotoxicity on renal proximal tubular cells in vitro

1-Methylhydantoin is produced by bacterial creatinine deaminase in the intestinal tract of uremic patients and retaken up into the body. The present study was designed to explore the toxic effect of 1-methylhydantoin on renal proximal tubular cells in vitro. HK-2 (Human renal proximal tubular cell line) was used as the subject. The cell viability was assessed by MTT assay. The cytotoxicity of 1-methylhydantoin to HK-2 was determined by NAG release test. Apoptosis of cultured HK-2 was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33258. Cells were exposed to 1-methylhydantoin (0.25mMol/L, 0.5mMol/L, or 1mMol/L), or creatinine (1mMol/L) for 24 h. 1-methylhydantoin induced a significant (p < 0.01) dose-dependent loss of cell viability. 1-methylhydantoin-treated HK-2 displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated that 1-methylhydantoin induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by flow cytometry, increased significantly (p < 0.01) after 1-methylhydantoin (0.5mMol/L ) for 24 h. The results of the present study clearly demonstrate that both 1-methylhydantoin and creatinine are toxic for proximal tubular cells but that the damage resulting from the 1-methylhydantoin is more severe.     

Antigen presentation by renal tubular epithelial cells

The interaction between immune effector cells such as T lymphocytes and parenchymal cells in organ-specific immune injury is dynamic. It is now appreciated that the specificity, intensity, and eventual destructive effects of such interactions can be greatly influenced by responses of the target issue. Renal tubular cells are particularly well suited to participate in such immune collaborations. (1) They are exposed to innumerable potentially immunogenic peptides from blood and glomerular filtrate and have pathways to further process these peptides; (2) they express surface molecules which facilitate their engagement to T cells; and (3) they can produce proinflammatory cytokines. In the models of immune-mediated tubulointerstitial injury that are currently studied, there has been a great interest in defining the T lymphocytes that initiate, accelerate, or suppress disease. Surprisingly, there has been relatively little attention on defining the tubular cell responses that regulate these immune-mediated processes. This review will therefore focus on this intriguing aspect of immune tubular injury and relate what is known about antigen presentation by tubular cells in autoimmune renal disease.     

Effects of mycophenolic acid on human renal proximal and distal tubular cells in vitro.

BACKGROUND:Mycophenolic acid has been shown to be effective for the prevention and treatment of renal allograft rejection. Rejection episodes were found to be associated with an infiltration of lymphocytes and macrophages/monocytes into the diseased kidney. Expression of RANTES, HLA-DR and ICAM-1 may be important for the pathogenesis of this leukocyte infiltration. Therefore the aim of this study was to evaluate the effect of the antiproliferative and immunosuppressive agent mycophenolic acid (MPA) on cell growth and cytokine-induced expression of RANTES, HLA-DR and ICAM-1 of highly purified proximal (PTC) and distal tubular cells (DTC) from human kidney. METHODS:Human PTC and DTC were cultured in the presence of different concentrations of MPA (0.25-50 microM) or MPA plus guanosine (100 microM). Total cell number (DNA content) was determined after 4 days of cell culture by a non-radioactive fluorescence assay. Cells were stimulated by a combination of cytokines (IL1beta+gammaIFN+TNFalpha=cytomix) or cytomix plus MPA. Secretion of RANTES protein was evaluated with an enzyme-linked-immunosorbent assay. Cell surface expression of HLA-DR and ICAM-1 was assessed by flow cytometric analysis. RESULTS:MPA inhibited cell growth of PTC and DTC in a dose-dependent manner. This effect was totally abolished by the addition of guanosine. Cytokine-induced RANTES expression was synergistically increased in the presence of MPA, an effect that was partially prevented by the addition of guanosine. Cytokine stimulation resulted in de novo expression of HLA-DR and a marked increase of ICAM-1 expression, which was partially inhibited by dexamethasone. Addition of MPA did not influence this stimulated expression. CONCLUSIONS:We demonstrate that MPA has an effect on cell growth and chemokine release of tubular epithelial cells, and that these effects are dependent on the inhibition of cellular guanosine production. The clinical consequences of this possible pro-inflammatory effect of MPA on RANTES release may be abolished by a concomitant treatment with steroids.     

Cytokine regulation of ICAM-1 expression on human renal tubular epithelial cells in vitro.

Regulation of the intercellular adhesion molecule-1 (ICAM-1) expression on human renal tubular epithelial cells in culture (hKEC-1) was investigated. A large proportion of hKEC-1 cells from the primary cultures expressed the ICAM-1 antigen. Supernatants from mixed lymphocyte reaction (MLR) of both specific and third-party combinations augmented the expression of the ICAM-1 antigen, in a dose-dependent manner. A kinetic study revealed maximal augmentation by MLR supernatant on the first day, with a gradual decrease thereafter. Among several recombinant human cytokines tested, i.e., interferon-gamma, tumor necrosis factor-alpha, interleukin 1 alpha and beta, and IL-4, IFN-gamma, TNF-alpha, and IL-1 alpha/beta were shown to augment the expression of ICAM-1. MLR supernatants and IFN-gamma were more effective in augmenting ICAM expression than TNF-alpha and IL-1 alpha/beta. IFN-gamma upregulated ICAM-1 expression in a dose-dependent manner, and maximal augmentation was achieved on the first day. The MLR supernatants were shown to contain IFN-gamma and TNF-alpha, and the activity of the MLR supernatant was partially inhibited by neutralizing antibody against IFN-gamma. These data suggest that cytokines, especially IFN-gamma, TNF-alpha, and IL-1 alpha/beta, released by T cells and antigen-presenting cells upon recognition of alloantigens upregulate ICAM-1 expression on renal tubular epithelial cells. This may result in an increase in the attachment of graft-infiltrating T cells to the renal tubular cells, by the ICAM-1-LFA-1 interaction.     

Reactive oxygen molecule-mediated injury in endothelial and renal tubular epithelial cells in vitro

To investigate renal tubular epithelial cell injury mediated by reactive oxygen molecules and to explore the relative susceptibility of epithelial cells and endothelial cells to oxidant injury, we determined cell injury in human umbilical vein endothelial cells and in four renal tubular epithelial cell lines including LLC-PK1, MDCK, OK and normal human kidney cortical epithelial cells (NHK-C). Cells were exposed to reactive oxygen molecules including superoxide anion, hydrogen peroxide and hydroxyl radical generated by xanthine oxidase and hypoxanthine. We determined early sublethal injury with efflux of 3H-adenine metabolites and a decline in ATP levels, while late lytic injury and cell detachment were determined by release of 51chromium. When the cells were exposed to 25, 50, and 100 mU/ml xanthine oxidase with 5.0 mM hypoxanthine, ATP levels were significantly lower (P less than 0.001) in LLC-PK1, NHK-C and OK cells compared to MDCK cells while ATP levels were significantly lower (P less than 0.01) in endothelial cells compared to all tubular cell lines. A similar pattern of injury was seen with efflux of 3H-adenine metabolites. When the cells were exposed to 50 mU/ml xanthine oxidase with 5.0 mM hypoxanthine for five hours, total 51chromium release was significantly (P less than 0.001) greater in LLC-PK1, NHK-C and OK cells compared to MDCK cells, while total 51chromium release was significantly (P less than 0.001) greater in endothelial cells compared to all tubular cells. However, lytic injury was the greatest in LLC-PK1 cells and NHK-C cells while cell detachment was the greatest in endothelial cells. MDCK cells were remarkably resistant to oxidant-mediated cell detachment and cell lysis. In addition, we determined ATP levels, 3H-adenine release and 51chromium release in LLC-PK1, NHK-C and endothelial cells in the presence of superoxide dismutase to dismute superoxide anion, catalase to metabolize hydrogen peroxide, DMPO to trap hydroxyl radical and DMTU to scavenge hydrogen peroxide and hydroxyl radical. We found that catalase and DMTU (scavengers of hydrogen peroxide) provided significant protection from ATP depletion, prevented efflux of 3H-adenine metabolites and cell detachment while DMPO (scavenger of hydroxyl radical) prevented lytic injury. In addition, we found that the membrane-permeable iron chelator, phenanthroline, and preincubation with deferoxamine prevented cell detachment and cell lysis, confirming the role of hydroxyl radical in cell injury.(ABSTRACT TRUNCATED AT 400 WORDS)     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

目的 观察不同条件下骨髓间充质干细胞（MSC）体外诱导分化为肾小管上皮样细胞的差异。 方法 抽取SD大鼠的骨髓,经密度梯度离心分离,联合贴壁筛选法获取纯化的MSC。以流式细胞仪鉴定间充质干细胞表面标志。取扩增3代的MSC分组培养：（1）对照组：用含胎牛血清培养基;（2）全反式维甲酸（ATRA）组：胎牛血清+缺血再灌注肾脏匀浆上清+ATRA;（3）联合诱导组：胎牛血清+缺血再灌注肾脏匀浆上清+ATRA+表皮生长因子（EGF）+骨形成蛋白（BMP-7）。诱导7 d后,倒置显微镜下观察细胞形态变化;化学染色检测细胞碱性磷酸酶表达;免疫细胞化学法检测细胞角蛋白18（cytokeratin-18）、E钙黏蛋白(E-cadherin)的表达。 结果 流式细胞仪显示,体外分离培养的第3代MSC,CD44阳性细胞表达率为97.8%±0.9%;CD90阳性细胞表达率为96.8%±1.4%;CD29阳性细胞表达率为97.6%±2.4%;而CD11b/c阳性细胞表达率为13.2%±0.6%; CD34阳性细胞表达率为1.2%±0.5%。诱导7 d后,与对照组长梭形细胞相比,ATRA组部分细胞为圆形、短梭形单层排列;联合诱导组的大部分细胞为圆形、短梭形,细胞密集处呈鹅卵石样排列。碱性磷酸酶染色显示,对照组细胞为阴性;ATRA组部分细胞阳性;联合诱导组阳性细胞数明显增多。免疫细胞化学显示,ATRA组和联合诱导组细胞cytokeratin-18阳性表达率分别为29.47%±1.08%和47.52%±2.13%,显著高于对照组（P ＜ 0.05）;E-cadherin阳性表达率分别为14.88%±2.46%和36.15%±1.13%,也显著高于对照组（P ＜ 0.05）。 结论 在体外模拟的急性肾衰竭微环境中加入ATRA可诱导MSC部分分化为肾小管上皮样细胞。联合EGF、BMP-7共同诱导能进一步促进MSC向肾小管上皮样细胞分化。     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.     

体外诱导骨髓间充质干细胞向肾小管上皮样细胞分化

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%,respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increassed by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.