Study on molecular mechanism and therapy of chromic renal failure using gene transfer technique

本研究通过基因转移技术探讨MAP-1和AP-1在肾小球损伤中的作用。(1)探讨MAP-1和AP-1在系膜细胞生长的分子机制中的可能意义。PDGF-BB刺激可显著激活ERK、JNK和AP-1，使DNA合成和细胞数显著增加。ERK、JNK和c-Jun的显性负性突变可显著抑制PDGF-BB诱导的系膜细胞的生长，提示ERK、JNK和AP-1参与了系膜细胞的生长。(2)肾小球细胞过度表达转化生长因子-β1参与了肾小球硬化的发生发展。我们通过将转化生长因子-β1的启动子基因导入系膜细胞，研究了转化生长因子-β1过表达的分子机制。PDGF-BB刺激可显著增强肾小球细胞中转化生长因子-β1的启动子活性，这种作用可被显性的ERK或c-jun负性突变体显著抑制。因此，ERK和AP-1可能参与了肾小球细胞中转化生长因子-β1的过度表达。(3)探讨MAP-1和AP-1在抗Th1抗体诱导的大鼠肾小球肾炎中的作用。急性肾小球肾炎时，肾小球ERK、JNK和AP-1迅速显著激活。显性负突变ERK、JNK或c-jun基因经肾动脉肾内转移不能抑制肾小球转化生长因子-β1或细胞外基质的增加或肾小球细胞的生长。显性负性突变体的基因转移未能阻止肾小球肾炎的发生，可能是由于基因转移技术对MAPK或AP-1活性的抑制不足所致。然而，我们使用基因转移技术进行的体外研究支持MAP Kinae和AP-1参与肾小球硬化的病理生理学。

This study was undertaken to examine the role of MAP kinases and AP-1 in glomerular injury using the gene transfer technique. (1) We examined the possible significance of MAP kinases and AP-1 in the molecular mechanism of growth of cultured mesangial cells. PDGF-BB stimulation significantly activated ERK, JNK and AP-1, followed by the significant increase in DNA synthesis and cell number. Dominant negative mutants of ERK, JNK or c-Jun significantly suppressed the growth of mesangila cells induced by PDGF-BB.These results show that ERK, JNK and AP-1 are involved in mesangial cell growth. (2) Overexpression of TGF-b1 in glomerular cells is shown to be responsible for the development of glomerulosclerosis. We examined the molecular mechanism of TGF-b1 overexpression, by transfecting the promoter gene of TGF-b1 into mesangial cells. PDGF-BB stimulation significantly increased the promoter activity of TGF-b1 in glomerular cells, and this increase was significantly suppressed by dominant negative mutants of ERK or c-Jun. Thus, ERK and AP-1 seem to participate in overexpression of TGF-b1 in glomerular cells. (3) We examined the role of MAP kinases and AP-1 in rat glomerulonephritis induced by anti-thy1 antibody injection. Glomerular ERK, JNK and AP-1 were rapidly and significantly activated in acute glomerulonephritis. Gene transfer of dominant negative mutants of ERK, JNK or c-Jun within the kidney via the renal artery failed to inhibit the increase in glomerular TGF-b1 or extracellular matrix or glomerular cell growth seen in glomerulonephritis. The failure of gene transfer of dominant negative mutants to prevent the development of glomerulonephritis possibly was due to the insufficient suppression of MAP kinase or AP-1 activation by the gene transfer technique. However, our in vitro study using gene transfer technique supports that MAP kinae and AP-1 are involved in the pathophysiology of glomerulosclerosis.