العنوان البديل: The role of FoxO1 in renal podocyte injury and apoptosis induced by homocysteine. (English)

المؤلفون: 张宏红, 谢琳, 盛思琪, 卢冠军, 姜怡邓 1,2,3，, 杨安宁, 李桂忠

المصدر: Tianjin Medical Journal; Jan2022, Vol. 50 Issue 1, p53-58, 6p

الملخص (بالإنجليزية): Objective To discuss the role of forkhead box O1 (FoxO1) in the injury and apoptosis of podocytes induced by homocysteine (Hcy). Methods Ten Cbs^+/+ mice with normal cysteine β -synthase (Cbs) gene and 10 Cbs^+/- mice with single gene knockout were fed a high methionine diet. After 8 weeks, the mice were sacrificed to collect kidney tissues. Morphological changes of glomerulus were observed by PAS staining. Podocytes were cultured in vitro and divided into the control group and the Hcy group (treated with cell culture medium containing 80 μmol/L Hcy for 48 h). Podocytes were transfected with Ad-FoxO1 overexpressed adenovirus and Sh-FoxO1 interfering adenovirus carrying green fluorescent protein (GFP), and they were divided into the control group, the Ad-GFP group, the Ad-FoxO1 group, the Sh-NC group, the Sh-FoxO1 group, the Ad-GFP+Hcy group, the Ad-FoxO1+Hcy group, the Sh-NC+Hcy group and the Sh-FoxO1+Hcy group. Western blot assay was used to detect the expression of Podocin and Nephrin, FoxO1 and apoptosis-related B lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cystine proteinase 12 (Caspase12) proteins in renal tissues and podocytes of mice. The expression of FoxO1 mRNA in mouse kidney tissue and podocytes was detected by qPCR. Results PAS staining results showed that the glomerular structure was normal, the basement membrane was clear and distributed evenly in Cbs^+/+ mice, while the glomerular basement membrane presented intermittent thickening and the mesangial matrix increased in Cbs^+/- mice. Compared with Cbs^+/+ mice, the expression levels of Podocin, Nephrin and FoxO1 protein and FoxO1 mRNA were significantly decreased in Cbs^+/- mice (P＜0.01). Compared with the control group, the expression of FoxO1 protein and mRNA in podocytes were significantly decreased in the Hcy group after treated with Hcy (P＜0.01). After overexpression of FoxO1 in podocytes, compared with the Ad-GFP+Hcy group, the protein expression of Podocin and Nephrin were significantly increased, both the ratio of Bax/Bcl-2 and the protein expression of Caspase12 were significantly decreased in the Ad-FoxO1+Hcy group (P＜0.05). After FoxO1 interference, compared with the Sh-NC+Hcy group, Podocin and Nephrin protein expressions were significantly decreased, Bax/Bcl-2 ratio and Caspase12 protein expression were significantly increased in the Sh-FoxO1+Hcy group (P＜0.05). Conclusion FoxO1 can reduce Hcy- induced renal podocyte injury and apoptosis in mice. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 目的 探讨叉头状转录因子 O1（FoxO1）在同型半胱氨酸（Hcy）诱导的肾脏足细胞损伤及凋亡中的作用。 方法 采用高蛋氨酸饮食喂养胱硫醚 β-合成酶（Cbs）基因正常 Cbs^+/+小鼠和单基因敲除 Cbs+/-小鼠各 10 只。8 周后处 死，收集肾组织，PAS 染色观察小鼠肾小球形态学变化。体外培养足细胞，分为 Control 组和 Hcy 组（用含 80 μmol/L Hcy 的细胞培养液干预 48 h）。将携带绿色荧光蛋白（GFP）的过表达 FoxO1 腺病毒（Ad-FoxO1）和干扰 FoxO1 腺病毒 （Sh-FoxO1）转 染 足 细 胞 ，分 为 对 照 组 、Ad-GFP 组 、Ad-FoxO1 组 、Sh-NC 组 、Sh-FoxO1 组 、Ad-GFP+Hcy 组 、Ad- FoxO1+Hcy 组、Sh-NC+Hcy 组、Sh-FoxO1+Hcy 组。Western blot 检测小鼠肾组织和足细胞裂隙膜蛋白（Podocin 和 Nephrin）、FoxO1 及凋亡相关蛋白 B 淋巴细胞瘤-2（Bcl-2）、Bcl-2 相关 X 蛋白（Bax）、半胱氨酸蛋白酶 12（Caspase12） 蛋白表达；qPCR 检测小鼠肾组织和足细胞中 FoxO1 mRNA 的表达。结果 PAS 染色结果显示，Cbs^+/+组小鼠肾小球结 构正常，基底膜清晰且分布均匀，而 Cbs^+/-组小鼠肾小球基底膜则呈现节段性增厚，系膜基质增多；与 Cbs^+/+组小鼠比 较，Cbs^+/-组小鼠肾组织中 Podocin、Nephrin 和 FoxO1 蛋白、FoxO1 mRNA 的表达明显降低（P＜0.01）。与 Control 组比 较，Hcy 组 FoxO1 mRNA 和蛋白表达显著降低（P＜0.01）；过表达 FoxO1 后，与 Ad-GFP+Hcy 组相比，Ad-FoxO1+Hcy 组 Podocin 和 Nephrin 蛋白表达均明显升高，Bax/Bcl-2 比值、Caspase12 蛋白表达均明显降低（P＜0.05）；而干扰 FoxO1 后，与 Sh-NC+Hcy 组相比，Sh-FoxO1+Hcy 组 Podocin 和 Nephrin 蛋白表达均明显降低，Bax/Bcl-2 比值、Caspase12 蛋白 表达均明显增高（P＜0.05）。结论 FoxO1 可减轻 Hcy 诱导的小鼠肾脏足细胞损伤及凋亡。 [ABSTRACT FROM AUTHOR]

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العنوان البديل: Mechanism of hyperhomocysteinemia induced renal injury in Cbs^+/- mice. (English)

المؤلفون: 吴 凯, 刘 昆, 谢 琳, 卢冠军, 马胜超, 李桂忠, 曹 军, 揭育祯1,2,3，, 姜怡邓, 郝银菊

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; 4/18/2021, Vol. 25 Issue 11, p1728-1732, 5p

مصطلحات موضوعية: TRANSMISSION electron microscopes, BASAL lamina, CHRONIC kidney failure, APOPTOSIS, PROTEIN expression, BCL genes

الملخص (بالإنجليزية): BACKGROUND: In chronic kidney disease, there is often an increase in the level of homocysteine, which can lead to podocyte apoptosis, but the specific mechanism is not clear. OBJECTIVE: To investigate the mechanism of hyperhomocysteinemia-induced renal injury in Cbs^+/- mice. METHODS: Cbs^+/+ mice (control group) and Cbs^+/- mice (model group) with similar body weight were selected, with 10 mice in each group, and were fed with high methionine diet. After 8 weeks, the mice were killed, the serum was separated and the kidney tissue was obtained. The levels of serum homocysteine, urea nitrogen and creatinine were measured by automatic biochemical analyzer. The renal injury was observed by Periodic Acid-Schiff staining and transmission electron microscope. TUNEL staining was used to observe the apoptosis of glomeruli. The protein expression levels of Bax, Bcl-2 and caspase12 were detected by western blot. RESULTS AND CONCLUSION: Compared with Cbs^+/+ mice, the level of serum homocysteine, urea nitrogen and creatinine in Cbs^+/- mice were significantly increased (P < 0.01). The Periodic Acid-Schiff staining results showed that the glomerular basement membrane of Cbs^+/+ mice was clear and the thickness was uniform, while the Cbs^+/- mouse glomerular basement membrane showed varying degrees of uneven thickness, widening of membrane area and thickening of matrix. Under the transmission electron microscope, the glomerular basement membrane of Cbs^+/+ mice was clear and the foot process was regular, while the glomerular basement membrane of Cbs^+/- mice was locally thickened and the foot process was irregular fusion. TUNEL staining showed that the number of apoptotic cells in glomeruli of Cbs^+/- mice was significantly increased compared with Cbs^+/+ mice; meanwhile, western blot detection showed that the protein levels of Bax/Bcl-2 and caspase12 were significantly increased (P < 0.05). To conclude, podocyte apoptosis plays an important role in hyperhomocysteinemiainduced renal injury in Cbs^+/- mice [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：在慢性肾脏病中常常伴有同型半胱氨酸水平升高，导致足细胞凋亡，但是其具体机制还尚未清楚。 目的：探讨高同型半胱氨酸血症诱导Cbs^+/-小鼠肾损伤的作用机制。 方法：选取体质量相近的Cbs+/+小鼠(对照组)和Cbs^+/-小鼠(模型组)，每组10只，两组小鼠均饲以高蛋氨酸饮食，8周后处死，分离血清，留 取肾脏组织。采用全自动生化分析仪检测血清同型半胱氨酸、尿素氮、肌酐水平；糖原染色法及透射电镜观察肾脏损伤情况；TUNEL染色 观察肾小球中细胞凋亡情况；Western blot检测Bax、Bcl-2和caspase12的蛋白表达水平。 结果与结论：①与Cbs^+/+小鼠比较，Cbs^+/-小鼠血清同型半胱氨酸、尿素氮及肌酐水平均明显升高(P < 0.01)；②糖原染色结果显示，Cbs^+/+小 鼠肾小球基底膜清楚，粗细均匀，而Cbs^+/-小鼠肾小球基底膜则呈现不同程度的粗细不均，膜区增宽、基质增厚；③透射电镜显示，Cbs^+/+ 小鼠肾小球基底膜清晰，足突规则，而Cbs^+/-小鼠肾小球基底膜局灶性增厚，足突不规则融合；④TUNEL染色结果显示，与Cbs^+/+小鼠比 较，Cbs^+/-小鼠肾小球内凋亡细胞数量明显增加；⑤Western blot检测结果显示，Bax/Bcl-2和caspase12的蛋白水平均升高(P < 0.05)；⑥结果 表明：足细胞凋亡在高同型半胱氨酸血症诱导Cbs+/-小鼠肾损伤中发挥着重要的作用。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

العنوان البديل: Increased FoxO1 DNA methylation level in homocysteine-induced podocyte apoptosis. (English)

المؤلفون: 刘 昆, 谢 琳, 曹 军, 丁 宁, 徐灵博, 马胜超, 李桂忠, 姜怡邓, 卢冠军

المصدر: Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu; Jan2021, Vol. 25 Issue 2, p269-273, 5p

مصطلحات موضوعية: HOMOCYSTEINE, BAX protein, BCL-2 proteins, DNA methylation, APOPTOSIS, PROTEIN expression

الملخص (بالإنجليزية): BACKGROUND: The increase of homocysteine can lead to renal injury and podocyte apoptosis, but the specific mechanism is not clear. OBJECTIVE: To investigate the effect of Forkhead box O1 (FoxO1) and its DNA methylation in podocyte apoptosis induced by homocysteine. METHODS: Mouse renal podocytes (MPC-5) were cultured in vitro and divided into control group (0 μmol/L homocysteine) and homocysteine group (80 μmol/L homocysteine). After 48 hours of intervention, the expression of podocyte apoptosis-related proteins Bax, caspase12 and Bcl-2 was detected by immunofluorescence technique; the expression level of FoxO1 mRNA was detected by real-time fluorescence quantitative PCR; the protein expression levels of FoxO1 and DNMT1 were detected by western blot; DNA methylation level of FoxO1 was detected by nested methylation-specific PCR. RESULTS AND CONCLUSION: Compared with the control group, the expression levels of Bax and caspase12 protein in podocytes of the homocysteine group were significantly increased, while the expression of Bcl-2 protein was significantly decreased. The expression levels of FoxO1 mRNA and protein were significantly decreased in the homocysteine group compared with the control group (P < 0.01). At the same time, the methylation level of FoxO1 DNA in the homocysteine group was significantly higher than that in the control group (P < 0.01), and the expression of DNMT1 protein in podocytes in the homocysteine group was significantly higher than that in the control group (P < 0.01). To conclude, FoxO1 DNA hypermethylation plays a significant role in podocyte apoptosis induced by homocysteine, whereas DNMT1 participates in homocysteine-induced podocyte apoptosis. [ABSTRACT FROM AUTHOR]

Abstract (Chinese): 背景：同型半胱氨酸增多会引起肾损伤并导致足细胞凋亡，但是其具体机制还尚不清楚。 目的：探讨叉头框转录因子 O1 (forkhead box O，FoxO1) 及其 DNA 甲基化在同型半胱氨酸致足细胞凋亡中的作用。 方法：体外培养小鼠肾脏足细胞 (MPC-5)，将其分为对照组(0 μmol/L同型半胱氨酸)和同型半胱氨酸组 (80 μmol/L 同型半胱氨酸)。干预细胞 48 h后，采用免疫荧光技术检验足细胞凋亡相关蛋白Bax、caspase12 和 Bcl-2 的表达情况；采用实时荧光定量 PCR(qRT-PCR) 检测 FoxO1 mRNA 水平；采用 Western blot 检测 FoxO1和DNMT1蛋白表达水平；采用巢式降落式特异性 PCR(nMS-PCR) 测验 FoxO1 的 DNA甲基化水平。 结果与结论：①与对照组相比，同型半胱氨酸组足细胞中 Bax 和 caspase12 表达明显增高，Bcl-2 表达明显降低；②FoxO1的mRNA和蛋白表达 水平明显降低(P < 0.01)；③与对照组相比，同型半胱氨酸组 FoxO1 DNA 甲基化水平明显升高 (P < 0.01)，同型半胱氨酸组足细胞中DNMT1蛋 白表达明显增高 (P < 0.01)；④结果表明：FoxO1 DNA 高甲基化在同型半胱氨酸致足细胞凋亡中作用显著，而 DNMT1 参与同型半胱氨酸诱导 的足细胞凋亡过程。 [ABSTRACT FROM AUTHOR]

: Copyright of Chinese Journal of Tissue Engineering Research / Zhongguo Zuzhi Gongcheng Yanjiu is the property of Chinese Journal of Tissue Engineering Research and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)