Renal endothelium and the development of chronic kidney disease in sickle cell disorders

项目摘要/摘要 慢性肾脏病(CKD)广泛存在于镰状细胞病(SCD)患者中，是一种重要的 是发病率和早期死亡率的贡献者。SCD患者肾脏健康的进行性恶化与 慢性血管内溶血。然而，解释溶血驱动的机械方法 SCD中CKD的发病机制是有限的，因此基于机制模型的靶向治疗是 缺乏。进行性肾功能丧失，表现为蛋白尿增加和肾小球减少 滤过率(GFR)与人类和患有SCD的小鼠的微血管充血有关。我们假设 这种循环中的无细胞的血红素是溶血的产物，可能会引发继而发生的永久性内皮损伤。 CKD发展。内皮蛋白C受体(EPCR)维持内皮屏障的完整性。我们发现 SCD(SS)肾微血管内皮细胞EPCR的年龄依赖性及血红素诱导的缺失 老鼠。伴随而来的是，血浆中裂解的可溶性EPCR(SEPCR)升高。 蛋白尿。使用超分辨率超声成像，我们发现年轻的SS小鼠接受了 重复输注最小剂量的血红素和没有血红素挑战的老年SS小鼠引起大量 肾微血管疏松。血红素诱导内皮细胞P-选择素促进血管充血。我们的 Pilot数据显示，EPCR表达减少与肾微血管上P-选择素增加有关 SS小鼠的血管内皮细胞。中性粒细胞上表达的蛋白水解酶3(PR3)能降解EPCR。在我们反复强调的 与注射药物的年轻SS小鼠相比，我们注意到PR3的表达增加 在血浆和肾脏中积累的中性粒细胞中。此外，我们发现，亚铁血红素可以诱导 组蛋白4赖氨酸16位残基(H4K16ac)的乙酰化，这是已知的促进PR3的生物合成。基座 根据这些初步数据，我们假设血红素诱导的P-选择素和PR3同时导致了这种损失 在SCD中，EPCR导致内皮细胞瓦解、微血管充血和CKD的发展。我们会 用三个特定的目标来测试这一假设，这些实验结合了人肾内皮细胞和 体外中性粒细胞、小鼠模型和来自SCD患者多个队列的临床样本。 目的1将确定重复低剂量的血红素损伤促进肾内皮细胞P-选择素抑制 EPCR可导致SCD小鼠血管损伤和CKD的发生。目标2将测试是否有血红素- 在SCD中，激活的PR3促进了EPCR的切割，加速了CKD。目标3将确定sEPCR和 EPCR特异性基因变异可提示SCD患者发生CKD的风险。

PROJECT SUMMARY/ABSTRACT Chronic kidney disease (CKD), widespread among individuals with sickle cell disease (SCD), is a significant contributor to morbidity and early mortality. The progressive deterioration of renal health in SCD is associated with chronic intravascular hemolysis. However, the mechanistic approach to interpreting the hemolysis-driven pathogenesis of CKD in SCD is limited and therefore targeted therapies based on mechanistic models are lacking. Progressive loss of renal function, represented by increased albuminuria and reduced glomerular filtration rate (GFR), is associated with microvascular congestion in humans and mice with SCD. We postulated that circulating cell-free heme, a product of hemolysis, may instigate persistent endothelial damage that ensues CKD development. Endothelial protein C receptor (EPCR) maintains endothelial barrier integrity. We discovered an age-dependent as well as heme-induced loss of EPCR from renal microvascular endothelium in SCD (SS) mice. Concomitantly, the cleaved soluble form of EPCR (sEPCR) was elevated in the plasma associated with albuminuria. Using super-resolution ultrasound imaging, we found that younger SS mice challenged with the repetitive infusion of minimal doses of heme and the older SS mice without heme challenge incur substantial renal microvascular rarefaction. Heme induces endothelial P-selectin that promotes vascular congestion. Our pilot data show that reduced EPCR expression is associated with increased P-selectin on renal microvascular endothelium in the SS mice. Proteinase 3 (PR3), expressed on neutrophils, can degrade EPCR. In our repeated heme-challenged younger SS mice compared to vehicle-injected mice, we noticed increased expression of PR3 in plasma as well as in accumulated neutrophils from the kidneys. Moreover, we found that heme induces acetylation of histone 4 Lys 16 residues (H4K16ac) which are known to promote the biosynthesis of PR3. Based on these preliminary data, we hypothesize that heme-induced P-selectin and PR3 concurrently result in the loss of EPCR leading to endothelial disintegration, microvascular congestion, and CKD development in SCD. We will test this hypothesis with three specific aims that integrate experiments with human renal endothelial cells and neutrophils in vitro, murine models, and clinical samples from multiple cohorts of SCD patients. Aim 1 will determine that repeated low-dose heme insults promote renal endothelial P-selectin that suppresses EPCR leading to vascular damage and development of CKD in mice with SCD. Aim 2 will test whether heme- activated PR3 facilitates EPCR cleavage and expedite CKD in SCD. Aim 3 will determine whether sEPCR and specific genetic variants of EPCR can indicate the risk of CKD in SCD patients.