CELL GROWTH, NA/H EXCHANGE, AND CYCLINS IN IDDM PATIENTS

肾病的发展是早产儿的最强预测因素。 胰岛素依赖型糖尿病患者的死亡和心血管疾病 （胰岛素依赖型糖尿病）。肾病的风险，发生在约25%的胰岛素依赖型糖尿病 患者，似乎，至少部分，是由基因决定的。一个 增加Na+/H+逆向转运蛋白的活性和内在生长 肾病本提案的总主题是审查 胰岛素依赖型糖尿病患者皮肤成纤维细胞的生长表型 及其与肾病的关系。由于Na+/H+的过度活跃， 反向转运蛋白是培养的皮肤成纤维细胞的一个特征 与肾病和NHE-1亚型的这种转运蛋白参与 在细胞生长中，Na+/H+逆向转运蛋白活性， 将评价NHE-1蛋白及其磷酸化水平。我们 推测NHE-1和cyclin D1蛋白在皮肤中过度表达 生长在早期传代培养物中的成纤维细胞， 肾病可能是生长表型的关键特征， 肾病的替代预测指标 拟议中的研究还将有助于揭示DNA 在患有糖尿病的IDDM患者的成纤维细胞中， 肾病将使用FACS分析来确定哪一个阶段的 细胞周期异常的胰岛素依赖型糖尿病患者的成纤维细胞 肾病视网膜母细胞瘤蛋白（pRB），在其非- 磷酸化形式，在GS/1抑制细胞周期进程 边界为了检查细胞周期中的G1转运，我们将评估pRB 磷酸化，细胞周期蛋白D1和E以及细胞周期蛋白D1的丰度 在基础阶段使用pRB作为底物的CD 1 K活性， 用血管紧张素II或血清刺激后。当细胞周期蛋白D1编码 pRB磷酸化所需的全酶的调节亚基， 细胞周期的G1期，这种细胞周期蛋白的相对丰度， 如果在伴有肾病的胰岛素依赖型糖尿病患者的细胞中增加， 缩短G1转运的决定子，从而增强DNA合成。我们 因此，将表征细胞周期蛋白D1表达和CD 1 K活性， 伴有和不伴有肾病的IDDM患者的成纤维细胞， 与年龄匹配的对照组[和 原发性高血压]，以确定这种异常和/或 其它G1细胞周期蛋白和细胞周期蛋白依赖性激酶抑制蛋白（KIP 有助于在IDDM患者中观察到的生长表型改变 患有肾病如果细胞周期蛋白D1的水平和活性增加， 从患有肾病的IDDM患者的成纤维细胞中，我们将检测 通过改变细胞周期蛋白D1明确增加细胞周期蛋白D1的意义 通过显微注射可诱导的反义和感觉的表达 我们最近描述的向量。

The development of nephropathy is the strongest predictor of premature death and cardiovascular disease in insulin-dependent diabetic patients (IDDM). The risk of nephropathy, which occurs in about 25% of IDDM patients, appear, at least in part, to be genetically determined. An increase in the activity of the Na+/H+ antiporter and intrinsic growth nephropathy. The overall theme of this proposal is the examination of the growth phenotype expressed in skin fibroblasts from IDDM patients and its relationship to nephropathy. Since overactivity of the Na+/H+ antiporter is a feature of cultured skin fibroblasts from IDDM patients with nephropathy and the NHE-1 isoform of this transporter is involved in cell growth, Na+/H+ antiporter activity, the relative abundance of NHE-1 protein, and its phosphorylation levels will be evaluated. We hypothesize that over-expression of NHE-1 and cyclin D1 protein in skin fibroblasts grown in early passage in culture from IDDM patients with nephropathy may be key features of a growth phenotype which may serve as a surrogate predictor for nephropathy. The proposed studies will also help unravel the mechanism(s) whereby DNA synthesis is enhanced in fibroblasts from IDDM patients with nephropathy. FACS analysis will be used to determine which phase of the cell-cycle is abnormal in fibroblasts from IDDM patients with nephropathy. The retinoblastoma protein (pRB), acting in its un- phosphorylated form, inhibits cell-cycle progression at the GS/1 boundary. To examine G1 transit in the cell cycle, we will assess pRB phosphorylation, the abundance of cyclins D1 and E as well as cyclin D1 kinase (CD1K) activity using pRB as the substrate in the basal stage and after stimulation with angiotensin II or serum. As cyclin D1 encodes the regulatory subunit of a holoenzyme required for pRB phosphorylation in the G1 phase of the cell cycle, the relative abundance of this cyclin, if increased in cells from IDDM patients with nephropathy, could be a determinant of shortened G1 transit and thus enhanced DNA synthesis. We will therefore characterize cyclin D1 expression and CD1K activity in the fibroblasts of IDDM patients with and without nephropathy as compared to those from age-matched controls [and individuals with essential hypertension] to determine where abnormalities of this and/or other G1 cyclins and cyclin dependent kinase inhibitor proteins (KIP's contribute to the altered growth phenotype observed in IDDM patients with nephropathy. If cyclin D1 levels and activity are increased in the fibroblasts from IDDM patients with nephropathy, we will test the significance of the increased cyclin D1 explicitly by altering cyclin D1 expression through the microinjection of inducible antisense and sensory vectors we have recently characterized.