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

肾病的发展是早产的最强预测因素 胰岛素依赖型糖尿病患者的死亡与心血管疾病 (IDDM)。肾病的风险，约25%的IDDM患者发生肾病 患者似乎至少在一定程度上是由基因决定的。一个 Na~+/H~+逆向转运蛋白活性增加与内源性生长 肾病。这项建议的总主题是审查 IDDM患者皮肤成纤维细胞生长表型的表达 及其与肾病的关系。由于Na+/H+的过度活性 逆向转运蛋白是培养的IDDM患者皮肤成纤维细胞的一个特征 肾病和该转运蛋白的NHE-1亚型 在细胞生长，Na+/H+逆向转运蛋白活性，相对丰度 NHE-1蛋白，并对其磷酸化水平进行评估。我们 NHE-1和细胞周期蛋白D1在皮肤中过度表达的假说 胰岛素依赖型糖尿病患者早期传代培养的成纤维细胞 肾病可能是生长表型的关键特征，这可能是 肾病的替代预测指标。 拟议中的研究还将有助于揭开DNA 胰岛素依赖型糖尿病患者成纤维细胞合成增强 肾病。将使用FACS分析来确定哪个阶段的 胰岛素依赖型糖尿病患者成纤维细胞周期异常 肾病。视网膜母细胞瘤蛋白(retinoblastoma Protein，PRB)在其非细胞周期中起作用。 磷酸化形式，在GS/1抑制细胞周期进展 边界。为了检查细胞周期中的G1转运，我们将评估pRb 细胞周期蛋白D_1、E和细胞周期蛋白D_1的丰度 基础阶段以pRb为底物的蛋白激酶(CD1K)活性 血管紧张素II或血清刺激后。因为细胞周期蛋白D1编码了 PRb磷酸化所需的全酶调节亚基 细胞周期的G1期，这个周期蛋白的相对丰度， 如果糖尿病肾病患者的细胞数量增加，可能是一种 G1期缩短的决定因素，从而增强DNA合成。我们 因此将表征细胞周期蛋白D1的表达和CD1K的活性 糖尿病肾病患者与非肾病患者成纤维细胞的比较 与年龄匹配的对照组[和患有 原发性高血压]以确定此和/或异常的位置 其他细胞周期蛋白和细胞周期蛋白依赖性激酶抑制蛋白(KIP‘s) 与观察到的IDDM患者生长表型改变有关 患有肾病。如果细胞周期蛋白D1水平和活性增加， 来自糖尿病肾病患者的成纤维细胞，我们将测试 改变细胞周期蛋白D1后细胞周期蛋白D1值明显升高的意义 通过微量注射可诱导的反义和感觉表达 我们最近描述了矢量的特征。

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.