KLF4在2型糖尿病肾病蛋白尿发生中的作用及可能的机制

发布时间：2018-06-12 20:51

本文选题：KLF4 + 2型糖尿病肾病　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:糖尿病肾病(diabetic nephropathy,DN)是2型糖尿病的严重并发症。研究DN时蛋白尿的发生发展机制,对于预防DN,以及采取针对性的措施延缓DN的发展,从而提高2型糖尿病患者的生活质量和生活水平有着重要临床和社会意义。2型糖尿病时,足细胞之间的裂隙(slit diaphragm,SD)结构消失,足突融合(foot process effacement),最终导致蛋白尿的发生。足细胞裂孔膜上的nephrin、CD2AP,足细胞足突上的α-actin4,足细胞和肾小球基底膜之间的integrinβ1等蛋白分子的丢失都会造成足细胞损伤,导致蛋白尿的发生。Krüppel样转录因子(krüppel-like factors,KLFs)属于核转录因子超家族,调节细胞的多种生物过程,包括增殖、分化、生长、发育。近年来的研究发现,KLF4在肾脏的发育和维持其正常生理功能中也具有重要的作用。我们在前期的实验中,发现2型糖尿病小鼠肾脏中KLF4的mRNA的表达水平显著下降。结果初步提示,KLF4可能与2型糖尿病肾病的发生有关。阿霉素(adriamycin,ADM)诱导的肾病小鼠的尿蛋白水平显著升高,同时足细胞中,KLF4和nephrin的表达也是显著降低的。相反,增加肾病小鼠足细胞中KLF4的表达又可以升高nephrin的表达,降低尿蛋白水平。那么KLF4表达下降是否与2型糖尿病肾病蛋白尿的发生有关?其机制如何?针对这一问题,本实验以2型糖尿病的db/db小鼠以及高脂环境中的足细胞作为实验对象,研究KLF4在2型糖尿病肾病蛋白尿发生中的作用及其机制,为认识2型糖尿病肾病蛋白尿发生的分子机制提供实验数据。方法:1实验动物采用db/db小鼠作为2型糖尿病动物模型,用尾静脉注射过表达KLF4腺病毒(Ad-GFP-KLF4)的方法改变db/db小鼠肾内KLF4的表达水平。2细胞株条件性永生化小鼠足细胞。用棕榈酸(c16:0)模拟2型糖尿病的高脂环境。用过表达klf4腺病毒感染足细胞,改变klf4的表达水平。3标本收集分别于第8周、12周、16周收集尿液并取肾脏组织。肾脏组织分别用于westernblot、pcr、pas染色、免疫组化实验等。4细胞收集对数生长期足细胞经相应浓度bsa、c16:0、c16:0+ad-gfp、c16:0+ad-gfp-klf4处理48小时后,收集细胞进行westernblot及pcr分析实验。5lowrry法测定小鼠24小时尿蛋白总量。6实时定量pcr检测klf4在2型糖尿病小鼠肾脏中的mrna表达水平,检测nephrin和integrinβ1在高脂环境小鼠足细胞的mrna表达水平。7westernblot检测klf4在2型糖尿病小鼠肾脏中的蛋白表达水平,以及klf4、nephrin、α-actin4和integrinβ1在高脂环境小鼠足细胞的蛋白表达水平。8pas染色观察小鼠肾脏肾小球结构9免疫组织化学分析2型糖尿病小鼠肾小球中nephrin、cd2ap、α-actin4、integrinβ1和integrinα3的蛋白表达水平。10数据处理采用spss17.0软件进行统计学处理分析,对所测定结果进行正态性及方差齐性检验,数据用x±sd表示,两组间比较用双尾-t检验分析。p0.05为差异有显著性意义。结果:1klf4在db/db小鼠肾小球中表达下降1.12型糖尿病小鼠的尿蛋白水平与同周龄的对照组db/m小鼠(8周:5037.34±1037.73μg/24h;12周:2649.32±87.07μg/24h;16周:34605.02±16160.58μg/24h)相比,db/db小鼠在第8周(4777.18±1378.49μg/24h)时尿蛋白水平没有明显的变化,但随着周龄的增加,在第12周(18629.52±3885.96μg/24h,p0.01)和第16周(323798.60±67766.10μg/24h,p0.01)尿蛋白水平逐渐增高,显著高于对照组。1.2klf4在肾小球中的表达水平pcr的结果显示,与对照组db/m小鼠相比,db/db小鼠肾皮质中klf4的mrna表达水平显著下降。westernblot的结果显示,与对照组db/m小鼠相比,db/db小鼠肾皮质中klf4蛋白表达水平显著下降。免疫组化的结果显示,与对照组db/m小鼠(275.50±30.26)相比,db/db小鼠(150.25±15.97,p0.01)肾小球中klf4表达水平显著下降。以上结果提示,肾小球klf4表达下降可能与2型糖尿病肾病蛋白尿的发生有关。2肾小球过表达klf4后,db/db小鼠的尿蛋白水平显著降低2.1klf4在肾小球中的表达水平免疫组化的结果显示,与无过表达klf4的db/db小鼠(152.25±12.89)相比,过表达klf4的db/db小鼠肾小球中klf4表达水平(317.5±33.25,p0.01)显著上调。2.2肾小球过表达klf4后,db/db小鼠的尿蛋白水平过表达klf4后,db/db小鼠尿蛋白水平(264330.80±33824.15μg/24h,p0.05)较无过表达klf4的对照组(435255.6±10446.17μg/24h)明显降低。以上结果说明,肾小球klf4表达下降,是2型糖尿病肾病蛋白尿发生的原因之一,增加klf4的表达能够改善2型糖尿病肾病尿蛋白。3klf4可通过增加db/db小鼠肾小球中nephrin、integrinβ1和α-actin4的表达来改善2型糖尿病肾病的尿蛋白水平3.12型糖尿病小鼠肾小球的结构pas染色结果显示db/db小鼠与对照组db/m小鼠的肾小球结构没有明显差异。3.2肾小球蛋白滤过屏障关键蛋白,nephrin、integrinβ1、integrinα3、α-actin4和cd2ap的表达水平免疫组化的结果显示,除cd2ap和integrinα3外,db/db小鼠肾小球中nephrin(0.148±0.003,p0.01)、integrinβ1(0.109±0.009,p0.05)和α-actin4(0.136±0.001,p0.05)的表达水平均显著低于对照组db/m小鼠(nephrin:0.208±0.016;integrinβ1:0.180±0.008;α-actin4:0.159±0.007);当db/db小鼠过表达klf4后,肾小球中nephrin(0.178±0.006,p0.01)、integrinβ1(0.164±0.006,p0.01)和α-actin4(0.156±0.006,p0.05)的表达水平显著高于无过表达klf4的db/db小鼠(nephrin:0.148±0.003;integrinβ1:0.109±0.009;α-actin4:0.136±0.001)。与文献报道相符,integrinβ1、integrinα3、α-actin4和cd2ap在肾小管中也有表达。与对照组db/m小鼠相比,db/db小鼠肾小管中α-actin4的表达较低,integrinβ1、integrinα3、和cd2ap的表达无明显的差异;db/db小鼠过表达klf4后,肾小管中integrinβ1、integrinα3、α-actin4和cd2ap的表达无明显的变化。nephrin特异性表达在肾小球中,但在本实验中,在肾小管中也有少量阳性颗粒,可能是因为实验条件不够优化所致。以上结果说明,2型糖尿病小鼠肾小球中nephrin、integrinβ1和α-actin4的表达水平与klf4的表达水平有关,低表达水平的klf4有可能通过影响nephrin、integrinβ1和α-actin4的表达下降而与2型糖尿病肾病蛋白尿的发生相关联。另外,2型糖尿病肾病蛋白尿的发生可能还与肾小管中α-actin4的表达下降有关。4在高脂环境的足细胞中,klf4可以调节nephrin、integrinβ1的表达在不同浓度的c16:0环境下,足细胞中klf4呈浓度依赖性下降。并且发现,足细胞在c16:0浓度为250μm的高脂环境中48小时后,伴随着klf4的下降,nephrin和integrinβ1蛋白表达水平均显著下降,而α-actin4的蛋白表达水平没有明显的变化;对高脂环境的足细胞过表达klf4后,nephrin的mrna和蛋白表达水平显著升高;integrinβ1的mrna表达水平无明显的变化,蛋白表达水平显著升高。此结果说明,在高脂环境的足细胞中,klf4很可能从转录水平调节了nephrin的表达,进而上调了蛋白的表达;从尚未知的途径只影响了integrinβ1的蛋白表达。以上结果说明,2型糖尿病肾病蛋白尿的发生与klf4表达减少有关。其可能机制:(1)与足细胞中低水平klf4转录调节nephrin的表达下降有关;(2)与足细胞中低水平klf4通过未知途径使integrinβ1表达下降有关。结论:足细胞中klf4表达下降,进而使nephrin表达降低,导致了2型糖尿病肾病蛋白尿的发生。上调足细胞中KLF4的表达,可以减少蛋白尿。
[Abstract]:Objective: diabetic nephropathy (DN) is a serious complication of type 2 diabetes. The study of the pathogenesis of proteinuria in DN, for the prevention of DN, and to take specific measures to delay the development of DN, so as to improve the quality of life and the living level of type 2 diabetes patients have important clinical and social significance of.2 type diabetes, The slit diaphragm (SD) structure between the podocytes disappears and the foot process fusion (foot process effacement) leads to the occurrence of proteinuria. The nephrin, CD2AP, the alpha -actin4 on the foot cell of the podocytes, the loss of the integrin beta 1 and other protein molecules between the podocytes and the basement membrane of the glomeruli will cause the foot cell damage and lead to the injury of the podocyte. The occurrence of.Kr u ppel like transcription factor (KR u ppel-like factors, KLFs) is a nuclear transcription factor superfamily, which regulates a variety of biological processes in cells, including proliferation, differentiation, growth, and development. Recent studies have found that KLF4 also plays an important role in the development of the kidney and the maintenance of its normal physiological functions. It was found that the expression level of KLF4 mRNA in the kidneys of type 2 diabetic mice decreased significantly. The results suggested that KLF4 may be associated with the occurrence of type 2 diabetic nephropathy. The urinary protein level of nephrotic mice induced by adriamycin (adriamycin, ADM) was significantly increased, and the expression of KLF4 and nephrin in the podocytes was also significantly reduced. The expression of KLF4 in the podocytes of nephrotic mice can also increase the expression of nephrin and reduce the level of urinary protein. Is the decline of KLF4 expression related to the pathogenesis of type 2 diabetic nephropathy? How is the mechanism of this problem? In this study, the experiment was conducted on the db/db mice of type 2 diabetes mellitus and the podocytes in the high fat environment as the experimental object. The role of KLF4 in the pathogenesis of type 2 diabetic nephrotic proteinuria and its mechanism provide experimental data for understanding the molecular mechanism of proteinuria in type 2 diabetic nephropathy. Method: 1 experimental animals used db/db mice as animal model of type 2 diabetes mellitus, and KLF4 adenovirus (Ad-GFP-KLF4) was injected into the tail vein to change the kidney of db/db mice. KLF4 expression level.2 cell line conditioned immortalized mouse foot cells. Use palmitic acid (c16:0) to simulate the high fat environment of type 2 diabetes. Using KLF4 adenovirus to infect podocytes, and change the expression level of KLF4,.3 specimens were collected for eighth weeks, 12 weeks, 16 weeks to collect urine and take kidney tissue. The renal tissue was used for Westernblot, PCR, respectively. PAS staining, immunohistochemistry and other.4 cells collected logarithmic growth stage podocytes for 48 hours after the corresponding concentration of BSA, c16:0, c16:0+ad-gfp, c16:0+ad-gfp-klf4, and collected cells for Westernblot and PCR analysis experiments..5lowrry method was used to determine the total amount of 24 hour urine protein in mice and.6 real time PCR detection KLF4 was in the kidney of type 2 diabetic mice. Na expression level, detection of mRNA expression level of nephrin and integrin beta 1 in high fat environment mouse foot cells.7westernblot detection KLF4 in type 2 diabetic mice kidney protein expression level, and KLF4, nephrin, alpha -actin4 and integrin beta 1 in the high fat environment mouse foot cells egg white expression level.8pas staining mouse kidney glomeruli Structure 9 immunohistochemical analysis of the protein expression level of nephrin, CD2AP, alpha -actin4, integrin beta 1 and integrin alpha 3 in the glomeruli of type 2 diabetic mice. The.10 data processing was analyzed by SPSS17.0 software. The results were tested with normality and homogeneity of variance. The data were expressed in X + SD, and the two groups were compared with double tail -t. The test and analysis of.P0.05 showed significant difference. Results: 1klf4 was expressed in db/db mice glomeruli to decrease the urine protein level of type 1.12 diabetic mice and db/m mice of the same week age group (8 weeks: 5037.34 + 1037.73 mu g/24h; 12 weeks: 2649.32 + 87.07 mu g/24h; 16 weeks: 34605.02 + 16160.58 Mu g/24h), db/db mice in eighth weeks (4777.18 + 1) There was no significant change in urine protein level at 378.49 g/24h), but with the increase of week age, the level of urine protein increased gradually at twelfth weeks (18629.52 + 3885.96, P0.01) and sixteenth weeks (323798.60 + 67766.10, g/24h, P0.01), which was significantly higher than that of.1.2klf4 in the small kidney balls of the control group. The result showed that it was with the control group of db/m mice. The mRNA expression level of KLF4 in the renal cortex of db/db mice decreased significantly by.Westernblot, and the results showed that the expression of KLF4 protein in the renal cortex of db/db mice decreased significantly compared with the control group db/m mice. The results of immunohistochemistry showed that compared with the control group db/m mice (275.50 + 30.26), db/db mice (150.25 + 15.97, P0.01) were KLF4 in the glomeruli. The above results suggest that the decrease of KLF4 expression in glomeruli may be related to the occurrence of.2 glomerular overexpression of KLF4 in type 2 diabetic nephropathy. The urinary protein level in db/db mice significantly decreased the expression level of 2.1klf4 in the glomeruli, and the db/db mice without overexpression of KLF4 (152.25 + 12) .89) compared with the expression level of KLF4 in the glomeruli of db/db mice expressing KLF4 (317.5 + 33.25, P0.01), the.2.2 glomerular overexpression KLF4 was significantly up-regulated, and the urine protein level of db/db mice was overexpressed in KLF4, and the urinary protein level of db/db mice (264330.80 + 33824.15 mu g/24h, P0.05) was significantly higher than that of the control group (435255.6 + 10446.17 mu). These results suggest that the decrease of KLF4 expression in glomeruli is one of the causes of proteinuria in type 2 diabetic nephropathy. The increase of KLF4 expression can improve the urinary protein.3klf4 of type 2 diabetic nephropathy, which can improve the urinary protein level of type 2 diabetic nephropathy by increasing nephrin, integrin beta 1 and alpha -actin4 in the glomeruli of mice. The glomerular structure PAS staining results showed that the glomerular structure of db/db mice and the control group db/m mice had no significant difference in the.3.2 glomerular protein filter barrier key protein, nephrin, integrin beta 1, integrin alpha 3, alpha -actin4 and CD2AP expressed in the immunohistochemical results, except CD2AP and integrin alpha 3, db/db mice. The expression level of nephrin (0.148 + 0.003, P0.01) in glomeruli, integrin beta 1 (0.109 + 0.009, P0.05) and alpha -actin4 (0.136 + 0.001, P0.05) were significantly lower than that of the control group db/m mice (nephrin:0.208 + 0.016; integrin beta 1:0.180 + 0.008; alpha -actin4:0.159 + 0.007). The expression level of RIN beta 1 (0.164 + 0.006, P0.01) and alpha -actin4 (0.156 + 0.006, P0.05) was significantly higher than that of db/db mice without overexpression of KLF4 (nephrin:0.148 + 0.003; integrin beta 1:0.109 + 0.009; alpha -actin4:0.136 + 0.001). The same as the literature reported, integrin beta 1, integrin alpha 3, and the expression in renal tubules. In contrast, the expression of alpha -actin4 in the renal tubules of db/db mice was lower, integrin beta 1, integrin alpha 3, and CD2AP expression were not significantly different. After db/db mice overexpressed KLF4, the expression of integrin beta 1, integrin alpha 3, alpha -actin4 and CD2AP in renal tubules had no significant changes in the glomeruli, but in this experiment, the renal tubules were also in the renal tubules. The results suggest that the expression level of nephrin, integrin beta 1 and alpha -actin4 in the glomeruli of type 2 diabetic mice is related to the level of KLF4 expression. The low expression level of KLF4 may be associated with the decrease of the expression of nephrin, integrin beta 1, and alpha -actin4 and type 2 diabetes. In addition, the occurrence of proteinuria in type 2 diabetic nephropathy may also be associated with a decrease in the expression of alpha -actin4 in renal tubules related to.4 in the hyperlipidemic podocytes, KLF4 can regulate nephrin, the expression of integrin beta 1 in the c16:0 environment of different concentrations, KLF4 in a concentration dependent decrease in the podocytes, and found, After 48 hours in the high fat environment with the concentration of 250 c16:0 m, the expression level of nephrin and integrin beta 1 protein was significantly decreased with the decrease of KLF4, while the protein expression level of alpha -actin4 was not significantly changed; the mRNA and protein expression level of nephrin was significantly increased after the hyperlipidemic podocyte KLF4 was overexpressed; integrin beta 1. There is no obvious change in the expression level of mRNA and a significant increase in protein expression level. This results suggest that in the hyperlipidemic podocytes, KLF4 is likely to regulate the expression of nephrin from the transcriptional level, and then up-regulated the expression of the protein; the unknown pathway only affects the protein expression of integrin beta 1. The above results indicate that type 2 diabetic nephropathy is indicated. The occurrence of proteinuria is related to the decrease of KLF4 expression. The possible mechanism is: (1) it is related to the decline in the expression of low level KLF4 transcriptional regulation of nephrin; (2) the low level of KLF4 in the Poda is associated with the decline in the expression of integrin beta 1 in the unknown pathway. Conclusion: the KLF4 table in the podocytes decreases, and the expression of nephrin is reduced, resulting in type 2 diabetes. The onset of proteinuria in patients with disease nephropathy can increase proteinuria by up regulating the expression of KLF4 in podocytes.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【相似文献】