WT1 and FAK in a pathological mechanism of daibetes nephropathy

WT1和FAK在糖尿病肾病病理机制中的作用

• 批准号: 09671149
• 负责人: MIMURA Toshihide
• 金额: $ 2.05万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671149/
• 关键词: FAK; WT1; Progressive renal failure; Diabetes nephropathy; ACE-I; Glomerular epithelial cells; nested-RT-PCR; focal adhesion kinase; progressive renal damage; glomerularepithelial cell

1) The number of glomerular epithelial cells (GECs) was reduced in diabetes nephropathy (DN) kidney as preliminary data shown, however, the expression of p125 Focal adhesion kinase (FAK) and WT1 was almost equal in each GEC to normal kidneys. These data suggest that the expression level of FAK and WT1 in GECs was not significantly reduced in DN.2) The mutation in genomic WT1 gene was analyzed in 27 end-stage renal disease (ESRD) patients who recently started hemodialysis (HD) treatment. Although there were tendency of increased mutations only in introns in ESRD patients, it was not statistically significant. To analyze mutations in mRNA of WT1, a new technique was developed in my laboratory (submitted). Briefly WT1 cDNA was obtained from patient urine sediments using nested-RT-PCR.Urine WT1 cDNA was detected in approximately 40% DN patients, on the other hand in only 2% diabetes mellitus (DM) patients without proteinuria (submitted). The striking data suggest that GEC damage was develo … More ped in DN and the detection of urine WT I could be the novel technique to show renal damage replacing invasive renal biopsy. Utilizing urine WT1 cDNA the extensive mutation search has been in progress in my laboratory.3) The expression pattern of FAK and WT1 in cultured GEC line was different from that in in vivo-GEC or freshly isolated GEC (in preparation). Long term primary culture of GEC has been established by culturing in the presence of a chemical which is known to reduce WT1 function. Biology of GEC obtained by this method has been studied.4) Using FAK negative fibroblasts, we showed that Cake, a member of FAK family kinase highly expressed in kidney, could play a similar role of FAK in integrin siganl transduction (Ueki, Mimura et al. FEBS letter, 1998).5) As we have reported, the expression and tyrosine phosphorylation of FAK increase in glomeruli of OLETF, spontaneous developing DM rat strain, before presence of proteinuria. It could suggest that mechanical stress of GEC due to hyperfiltration induces the FAK expression and tyrosine phosphorylation. It was of interest whether angiotensin converting enzyme inhibitor (ACE-I) could suppress the elevation of FAK in OLETF by reducing intraglomeruli hypertension. Only high dose ACE-I significantly suppressed the increase of expression of FAK in OLETF glomeruli comparing to that of controls (in preparation). These data suggested that hyperfiltration due to diabetes induced expression and tyrosine phosphorylation of FAK which could be at least a part of the pathological mechanism of DN.The support of this grant has brought to our laboratory and to the medical biology Field several new research results as well as new questions. Less

1)糖尿病肾病（DN）患者肾小球上皮细胞（GEC）数量减少，但p125黏着斑激酶（FAK）和WT 1的表达与正常肾脏几乎相同。2）对27例近期开始接受血液透析（HD）的终末期肾病（ESRD）患者进行WT1基因突变检测。虽然ESRD患者仅内含子突变有增加的趋势，但无统计学意义。为了分析WT1 mRNA的突变，我的实验室开发了一种新技术（已提交）。简而言之，使用巢式RT-PCR从患者尿沉淀物中获得WT1 cDNA。在大约40%的DN患者中检测到尿WT1 cDNA，而在另一方面，仅2%的无蛋白尿的糖尿病（DM）患者中检测到尿WT1 cDNA（已提交）。令人震惊的数据表明，GEC损伤是在细胞凋亡的过程中发生的。 ...更多信息 DN患者尿WT I的检测可替代有创性肾活检，成为显示肾损害的新技术。3）FAK和WT1在体外培养的GEC中的表达模式与在体GEC或新鲜分离的GEC（制备中）不同。GEC的长期原代培养已经通过在已知降低WT1功能的化学物质存在下培养而建立。4）以FAK阴性的成纤维细胞为研究对象，我们发现在肾脏中高表达的FAK家族激酶Cake在整合素信号转导中具有与FAK相似的作用（Ueki，Mimura等，FEBS letter，1998）。5）正如我们所报道的，在OLETF的肾小球中FAK的表达和酪氨酸磷酸化增加，自发发展的DM大鼠品系，在蛋白尿存在之前。提示超滤引起的机械应力诱导了FAK的表达和酪氨酸磷酸化。血管紧张素转换酶抑制剂（ACE-I）是否能通过降低肾小球内高血压来抑制OLETF中FAK的升高，值得关注。与对照组相比，仅高剂量ACE-I显著抑制OLETF肾小球中FAK表达的增加。这些数据表明，由于糖尿病引起的超滤诱导FAK的表达和酪氨酸磷酸化，这可能至少是DN的病理机制的一部分。该基金的支持给我们的实验室和医学生物学领域带来了一些新的研究成果，也带来了新的问题。少

项目成果

Ueki, K., Mimura, T., Nakamoto, T., Sasaki, T., Aizawa, S., Hirai, H., Yano, S., Naruse, T., and Nojima, Y.: "Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125FAK." FEBS letters. 432. 197-201 (1998)

Ueki, K.、Mimura, T.、Nakamoto, T.、Sasaki, T.、Aizawa, S.、Hirai, H.、Yano, S.、Naruse, T. 和 Nojima, Y.：“整合素介导

Kanda, H., Mimura, T., Morino, N., Hamasaki, K., Nakamoto, T., Hirai, H.Morimoto, C., Yazaki, Y., and Nojima, Y.: "Ligation of the T cell antigen receptor induces tyrosine phosphorylation of p105CasL,member of the p130Cas-related docking protein family, a

Kanda, H.、Mimura, T.、Morino, N.、Hamasaki, K.、Nakamoto, T.、Hirai, H.Morimoto, C.、Yazaki, Y. 和 Nojima, Y.：“T 的结扎

Mimura,T.,Minota,S.,et al.: "Constitutive tyrosine phosphorylation of the vav proto-oncogene product in MRL/Mp-lpr/lpr mice." J.Immunol.158. 2977-2983 (1997)

Mimura,T.,Minota,S.,et al.：“MRL/Mp-lpr/lpr 小鼠中 vav 原癌基因产物的组成型酪氨酸磷酸化。”

Hamasaki, K., Mimura, T., Kanda, H., Morino, N., Yazaki, Y., and Nojima, Y.: "Development of systemic lupus erythematosus in a rheumatoid arthritis patient with anti-ribosomal P protein antibody." Lupus. 6. 734-736 (1997)

Hamasaki, K.、Mimura, T.、Kanda, H.、Morino, N.、Yazaki, Y. 和 Nojima, Y.：“类风湿性关节炎患者使用抗核糖体 P 蛋白抗体发展为系统性红斑狼疮。