The Role of Bone Morphogenetic Protein-7 in Renal Endothelial Cell Biology

描述(申请人提供)：据估计，到2010年，终末期肾病(ESRD)的年增量将超过17万例，患病率超过66万人。据进一步估计，以卡特里娜飓风前的美元计算，治疗ESRD患者的年成本将超过390亿美元。大多数研究认为，肾间质显微镜下形态的改变与导致终末期肾病的肾功能下降有关。肾间质中研究最少的部分是管周毛细血管的内皮细胞。骨形态发生蛋白-7(BMP-7)具有肾脏保护和肾再生的作用，延缓了终末期肾病的进展。正在测试的一般假设是，BMP-7的肾脏保护和再修复作用的一部分是由于这种生长因子具有内皮保护和内皮修复作用。据推测，BMP-7直接作用于肾内皮细胞，并促进肾脏三维细胞外基质中的血管生成。{通过制定保存/恢复肾小管周围毛细血管的战略；该项目可能有助于延长肾功能，从而最大限度地减少与治疗终末期肾病相关的成本。本研究的具体目的是：1)体外检测外源性BMP-7对内皮细胞生物学的影响；2)体内检测外源性BMP-7对肾内皮细胞生物学的影响。这些实验将利用永生化的小鼠内皮细胞系，在二维和三维细胞培养中使用1型和IV型胶原基质。培养中的细胞将被BMP-7和/或转化生长因子-β(TGF-β)处理或不处理，并评估其迁移、增殖、凋亡和毛细血管形成情况。将确定不同的蛋白激酶途径和对这些蛋白激酶的阻断在这些过程中的作用。体内研究将利用一种可复制的肾纤维化动物模型，即急性和慢性单侧输尿管梗阻(UUO)的小鼠。{UUO模型中与肾小管周围毛细血管完整性相关的发现将在出现高血糖的肥胖小鼠模型中进一步测试。肥胖模型将对慢性肾脏疾病有更广泛的影响，而急性UUO模型将确定BMP-7具有内皮保护作用的机制。双标记免疫细胞化学将用于识别肾内皮细胞，以及BMP-7治疗和转化生长因子-β调节对内皮细胞内和邻近内皮细胞蛋白激酶活性的影响。体外和体内研究将结合起来，以最大限度地发挥BMP-7治疗的效果，以保护肾内皮细胞，进而保护肾上皮细胞，并最终保护肾功能。如果患有肾脏疾病的联邦医疗保险患者没有进展到透析，估计每个患者在联邦医疗保险方面的节省是250.000美元。这项提案审查了一种旨在延缓或阻止肾脏疾病进展的治疗方法，从而节省了年度医疗保险预算的很大一部分。

DESCRIPTION (provided by applicant): It is estimated that by 2010, the annual increase in end-stage renal disease (ESRD) will exceed 170,000 cases with a prevalence of over 660,000 patients. It is further estimated that the yearly cost of treating patients with ESRD will be over 39 billion in pre-Katrina dollars. By most accounts, changes in the microscopic appearances in the interstitium of the kidney correlate with the decline of renal function leading to ESRD. A part of the renal interstitium that has been minimally investigated are the endothelial cells of the peritubular capillaries. Bone Morphogenetic Protein-7 (BMP-7) has renoprotective and renoregenarative properties blunting progression of ESRD. The general hypothesis being tested is that part of the renoprotective and renorestorative effects of BMP-7 are due to this growth factor being endothelial-protective and endothelial restorative. It is postulated that BMP-7 directly effects renal endothelial cells and promotes angiogenesis in the 3-dimensional extracellular matrix of the kidneys. {By developing strategies to preserve/restore renal peritubular capillaries; this project may contribute to prolonging renal function thereby minimizing the costs associated with treating ESRD}. The specific aims of this proposal are to; 1) Determine the effects of exogenous BMP-7 on endothelial cell biology in vitro; and 2) Determine the effects of exogenous BMP-7 on renal endothelial cell biology in vivo. These experiments will utilize an immortalized mouse endothelial cell line in both 2-dimentional and 3- dimensional cell culture with collagen 1 and collagen IV matrices. The cells in culture will be treated or not with BMP-7 and/or transforming growth factor-beta (TGF-¿) and assessed for migration, proliferation, apoptosis and capillary tube formation. The contribution of various protein kinase pathways and blockade of the kinases on these processes will be determined. The in vivo studies will utilize a reproducible animal model of renal fibrosis the mouse with acute and chronic unilateral ureteral obstruction (UUO). {The findings relevant to peritubular capillary integrity in the UUO model will be further tested in an obese mouse model that develops hyperglycemia. The obese model will have broader implications to chronic kidney disease while the acute UUO model will identify mechanisms by which BMP-7 is endothelial protective}. Dual-label immunocytochemistry will be employed to identify renal endothelial cells and the impact of BMP-7 treatment along with TGF-¿ modulation on protein kinase activities within and adjacent to the endothelial cells. The in vitro and in vivo studies will be integrated to maximize the effects of BMP-7 treatment that preserve renal endothelial cells and in turn, renal epithelial cells and ultimately renal function. If a Medicare patient with kidney disease does not progress to dialysis the estimated savings to Medicare is $250.000 for each patient. This proposal examines a treatment designed to delay or halt the progression of renal disease thereby saving a significant portion of the yearly Medicare budget.