WT1 and FAK in a pathological mechanism of daibetes nephropathy

1)初步数据显示，糖尿病肾病(DN)肾组织中肾小球上皮细胞(GECs)数量减少，而p125粘着斑激酶(FAK)和WT1在各GEC中的表达与正常肾脏基本相同。2)对27例新近开始血液透析(HD)治疗的终末期肾病(ESRD)患者进行了WT1基因突变分析。虽然ESRD患者只有内含子突变增加的趋势，但差异无统计学意义。为了分析WT1的mRNA突变，我的实验室开发了一项新技术(提交)。用套式RT-PCR法从患者尿沉渣中获得WT1基因，在大约40%的糖尿病患者中检测到尿WT1基因，而在无蛋白尿的糖尿病患者中只有2%的患者检测到尿蛋白WT1基因。惊人的数据表明，GEC的损害是由…发展而来的糖尿病肾病和尿WT I检测有可能取代有创肾活检而成为显示肾损害的新技术。本实验室利用尿WT1基因进行了广泛的突变搜索。3)FAK和WT1在培养的GEC中的表达模式不同于在体GEC和新鲜分离的GEC(制备中)。GEC的长期原代培养是通过在一种已知会降低WT1功能的化学物质的存在下培养而建立的。4)利用FAK阴性的成纤维细胞，我们发现在肾脏中高表达的FAK家族成员Cake在整合素信号转导中可以发挥与FAK相似的作用(Ueki，Mimura等)。Febs Letter，1998)5)正如我们所报道的，在蛋白尿出现之前，OLETF，自发发育的DM大鼠系，肾小球中FAK的表达和酪氨酸磷酸化增加。提示高滤过致肾小管上皮细胞机械应激可诱导FAK表达和酪氨酸磷酸化。血管紧张素转换酶抑制剂(ACE-I)是否可通过降低肾小球内高压来抑制OLETF中FAK的升高。与对照组相比，只有高剂量的ACE-I能显著抑制肾小球FAK的表达。这些数据表明，糖尿病引起的高滤过诱导FAK的表达和酪氨酸磷酸化，这可能是糖尿病肾病的病理机制之一。这项拨款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和新的问题。较少

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