GROWTH FACTOR EXPRESSION IN RENAL ENDOTHELIAL CELLS

血管内皮细胞的破坏性增殖和硬化 肾微血管通过以下途径不可逆转地损害肾功能 管腔狭窄、肾小球结构破坏和肾小球缩小 过滤表面。这些研究的目的是定义机制 调节肾内皮细胞产生的生长因子是 与内皮下的增殖和硬化症有关 一层。 尤其与破坏性肾小球疾病有关的是增殖 系膜细胞、成纤维细胞和基质沉积，所有这些都是 由血小板衍生生长因子(PDGF)刺激。我们的数据，使用 原代培养的人肾微血管内皮细胞 PDGF B/c-sis信使RNA(MRNA)的表达和活性释放 由血管部位的相关制剂调节。血小板衍生生长因子的转录 B/c-sis mRNA由血小板(转化生长因子β)和 凝血产物(凝血酶)，并被升高的激素抑制 CAMP水平(儿茶酚胺)。最新数据显示，肾内皮细胞 不同结构形式的PDGF B mRNA的表达。建议数 研究将确定不同PDGF B mRNA的相对贡献 利用体外和非洲爪哇翻译PDGF活性蛋白产物 每种分离和合成结构形式的卵母细胞翻译 PDGF B/c-cic基因的表达。其他实验将确定是否表达 不同的PDGF B/c-sis mRNAs受转录调控的差异 和稳定机制。 参与肾脏调节的基因组PDGF B/c-cic序列 将通过DNA足迹技术识别内皮细胞的表达 和瞬时表达的一系列侧翼序列缺失 构造。参与cAMP介导的PDGF抑制作用的DNA序列 B/c-sis的转录将通过凝胶位移进行鉴定和表征 紫外光交联和AS鉴定DNA结合蛋白 CAMP依赖的激酶底物。PDGF B/c-sis的完整组织作用 将通过PDGF B/c-sis的原位杂交来鉴定其表达 肾活检标本中的mRNA。 这些研究将确定调节微血管的分子机制 内皮细胞产生PDGF并可能提供潜在的靶点 对破坏性内皮下增殖过程的干预。

Destructive proliferation and sclerosis at sites underlying endothelium in the kidney microvasculature irreversibly impair renal function through luminal narrowing, destruction of glomerular architecture and reduction of filtration surface. The objective of these studies is to define mechanisms regulating renal endothelial cell production of growth factors that are implicated in proliferation and sclerosis subjacent to the endothelial layer. Especially relevant in destructive glomerular diseases are proliferation of mesangial cells, fibroblasts and matrix deposition, all of which are stimulated by platelet-derived growth factor (PDGF). Our data, using primary cultured human renal microvascular endothelial cells, have shown PDGF B/c-sis messenger RNA (mRNA) expression and activity release are regulated by agents relevant at vascular sites. Transcription of PDGF B/c-sis mRNA is induced by platelet (transforming growth factor beta) and coagulation products (thrombin) and suppressed by hormones which elevate cAMP levels (catecholamines). New data has shown renal endothelial expression of distinct structural forms of PDGF B mRNA. The proposed studies will determine the relative contribution of different PDGF B mRNA's to translation of active PDGF protein product, using in vitro and xenopus oocyte translation of isolated and synthesized structural forms of each PDGF B/c-cic mRNA. Additional experiments will determine if expression of different PDGF B/c-sis mRNAs is differentially regulated by transcriptional and stabilization mechanisms. Genomic PDGF B/c-cic sequences participating in regulation of renal endothelial expression will be identified by DNA footprinting techniques and transient expression of a series of flanking sequence deletion constructs. DNA sequences participating in cAMP-mediated repression of PDGF B/c-sis transcription will be identified and characterized by gel shift assays, and DNA binding proteins identified by UV cross-linking and as substrates for cAMP dependent kinase. Intact tissue roles for PDGF B/c-sis expression will be identified using in situ hybridization of PDGF B/c-sis mRNA in renal biopsy samples. These studies will define molecular mechanisms regulating microvascular endothelial production of PDGF and may provide potential targets for intervention in destructive subendothlial proliferative processes.