WT1 and FAK in a pathological mechanism of daibetes nephropathy

1)初步数据显示，糖尿病肾病（DN）肾脏的肾小球上皮细胞（glomerular epithelial cells, GECs）数量减少，但p125 Focal adhesion kinase （FAK）和WT1在每个GEC中的表达与正常肾脏几乎相等。这些数据表明FAK和WT1在GECs中的表达水平在dn中没有显著降低。2)分析了27例新近开始血液透析（HD）治疗的终末期肾病（ESRD）患者基因组中WT1基因的突变。虽然在ESRD患者中仅内含子有突变增加的趋势，但没有统计学意义。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）。用巢式rt - pcr从患者尿液沉积物中获得WT1 cDNA。在大约40%的DN患者中检测到尿WT1 cDNA，而在没有蛋白尿的糖尿病（DM）患者中仅检测到2%（提交）。令人震惊的数据表明，肾小球肾小球（GEC）损害在肾小球肾小球肾病（DN）中更为明显，尿液WT I检测可能成为一种替代有创肾活检显示肾脏损害的新技术。利用尿WT1 cDNA广泛的突变搜索已在我的实验室进行。3) FAK和WT1在培养的GEC细胞系中的表达模式与在体GEC或新鲜分离的GEC（制剂）中的表达模式不同。通过在已知可降低WT1功能的化学物质中进行培养，建立了GEC的长期原代培养。对该方法制备的GEC进行了生物学研究。4)使用FAK阴性成纤维细胞，我们发现FAK家族激酶在肾脏中高表达的成员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信函，1998年)。5)正如我们所报道的，在自发性发展的糖尿病大鼠品系OLETF的肾小球中，FAK的表达和酪氨酸磷酸化在出现蛋白尿之前增加。这可能是由于超滤引起的GEC机械应力诱导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，只有高剂量ACE-I显著抑制OLETF肾小球中FAK表达的增加。这些数据表明，糖尿病引起的高滤过诱导FAK的表达和酪氨酸磷酸化可能至少是DN病理机制的一部分。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。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