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

描述（申请人提供）：预计到2010年，终末期肾病（ESRD）每年新增病例将超过17万例，患病人数将超过66万。据进一步估计，在卡特里娜飓风之前，每年治疗 ESRD 患者的费用将超过 390 亿美元。大多数情况下，肾间质微观外观的变化与肾功能下降导致终末期肾病相关。肾间质的部分研究最少的是肾小管周围毛细血管的内皮细胞。骨形态发生蛋白 7 (BMP-7) 具有肾脏保护和肾脏再生特性，可减缓 ESRD 的进展。正在测试的一般假设是，BMP-7 的部分肾脏保护和肾脏恢复作用是由于该生长因子具有内皮保护和内皮恢复作用。据推测，BMP-7 直接影响肾内皮细胞并促进肾脏 3 维细胞外基质中的血管生成。 {通过制定保护/恢复肾小管周围毛细血管的策略；该项目可能有助于延长肾功能，从而最大限度地降低与治疗 ESRD 相关的成本}。该提案的具体目标是： 1) 体外确定外源BMP-7对内皮细胞生物学的影响； 2)确定外源BMP-7对体内肾内皮细胞生物学的影响。这些实验将利用永生化小鼠内皮细胞系，在二维和三维细胞培养物中使用胶原蛋白 1 和胶原蛋白 IV 基质。培养中的细胞将接受或不接受BMP-7和/或转化生长因子-β(TGF-¿)处理，并评估迁移、增殖、凋亡和毛细管形成。将确定各种蛋白激酶途径的贡献以及激酶对这些过程的阻断。体内研究将利用患有急性和慢性单侧输尿管梗阻（UUO）的小鼠的可重复肾纤维化动物模型。 {UUO 模型中与管周毛细血管完整性相关的发现将在发生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产生更广泛的影响，而急性 UUO 模型将确定 BMP-7 的内皮保护机制}。双标记免疫细胞化学将用于鉴定肾内皮细胞以及BMP-7治疗和TGF-¿调节对内皮细胞内和邻近内皮细胞的蛋白激酶活性的影响。体外和体内研究将被整合，以最大限度地发挥 BMP-7 治疗的效果，从而保护肾内皮细胞，进而保护肾上皮细胞并最终保护肾功能。如果患有肾病的 Medicare 患者没有进展到透析，则每位患者预计可为 Medicare 节省 250,000 美元。该提案研究了一种旨在延缓或阻止肾脏疾病进展的治疗方法，从而节省每年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.