Establishment of gene therapy targeted for renal mesangial and proximal tubular cells

(1)抗肾小球系膜细胞单链单克隆抗体的制备为制备抗肾小球系膜细胞单链单克隆抗体，分别用大鼠和人肾小球系膜细胞免疫Balb/c小鼠。从这些免疫的小鼠脾脏中，建立了噬菌体展示型单克隆抗体的cDNA文库。目前，该文库正在进行二次筛选，但尚未获得特异性抗体。(2)IκBα基因转染肾损伤大鼠肾近端细胞的研究用重组腺病毒经肾动脉将抑制炎症关键分子核因子κB（NFκB）的截短型IκBα基因转移至大鼠肾组织。腺病毒载体可有效地将报告基因导入近端细胞，并转染截短型IκBα，可消除白蛋白负荷引起的肾小管间质改变。这些结果表明，抑制NFκB可防止该模型中的肾损伤，我们还研究了一种可能的治疗肾脏疾病的药物。我们发现，曲尼司特，这是已知的抑制NF κ B在几种细胞系，可以抑制NFκB的活化，表明这种药物可能是有用的临床设置。本研究表明，起始密码子上游105 bp的区域具有在近端肾小管细胞系LLC-PK 1中表达的启动子活性。此外，该区域的TCF-1结合基序在启动子活性中起重要作用。

(1) Development of single-chane monoclonoal antibody against mesangial cellsTo develop single-chain monoclonal antibody against mesangial cells, Balb/c mouse were immunized with rat and human mesangial cells. From these immunized mouse spleen, CDNA library of phage-display type monoclonal antibody was developed. Currently, second-screening of this library is being performed, although specific antibody has not been obtained, yet.(2) Gene transfer of truncated IκBα to renal proximal cells in renal impairment ratsAs renal interstitial injury model, albumin-loaded rats were used. Gene transfer of truncated IκBα, which inhibits nuclear factor κB (NFκB), key molecule for inflammation, was performed with recombinant adenovirus via renal artery. Adenovirus transferred reporter gene into proximal cells, efficiently, and transferred truncated IκBα abolished tubulo-interstitial changes by albumin loading. From these results, it was suggested that inhibition of NFκB prevented renal damage in this model, we also examined a possible drug for the treatment of renal diseases. We found that, tranilast, which is known to inhibit NFκB in several cell lines, could inhibit NFκB activation in mesangial cells, suggesting that this drug may be useful for clinical settings.To express transferred gene more efficiently, we analyzed promoter activity of vitamin D 1α-hydroxylase, which is predominantly expressed in proximal tubules. This study showed that region of 105 bp upstream of the initiation codon has promoter activity for expression in proximal tubular cell line, LLC-PK1. Furthermore, TCF-1 binding motif in this region is shown to play important roles in promoter activity.