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

本研究采用基因转移技术研究MAP激酶和AP-1在肾小球损伤中的作用。(1)探讨MAP激酶和AP-1在培养的系膜细胞生长的分子机制中的可能意义。PDGF-BB刺激显著激活ERK、JNK和AP-1，随后DNA合成和细胞数量显著增加。ERK、JNK或c-Jun的显性阴性突变体显著抑制PDGF-BB诱导的系膜细胞生长。这些结果表明ERK、JNK和AP-1参与了系膜细胞的生长。(2)肾小球细胞中TGF-b1的过度表达与肾小球硬化的发生有关。我们通过将TGF-b1启动子基因转染系膜细胞，研究TGF-b1过表达的分子机制。PDGF-BB刺激显著增加肾小球细胞中TGF-b1启动子活性，而这种增加被ERK或c-Jun显性阴性突变体显著抑制。因此，ERK和AP-1似乎参与了肾小球细胞中TGF-b1的过表达。(3)观察MAP激酶和AP-1在抗thy1抗体诱导的大鼠肾小球肾炎中的作用。急性肾小球肾炎患者肾小球ERK、JNK、AP-1快速显著激活。ERK、JNK或c-Jun显性阴性突变体经肾动脉在肾脏内的基因转移未能抑制肾小球肾炎中肾小球TGF-b1或细胞外基质或肾小球细胞生长的增加。显性阴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未能充分抑制MAP激酶或AP-1的激活。然而，我们使用基因转移技术的体外研究支持MAP激酶和AP-1参与肾小球硬化的病理生理。

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.