Molecular and Cellular Pathogenesis of Kidney Disease in Sickle Cell Disorders

镰状细胞病肾病的分子和细胞发病机制

• 批准号: 10542842
• 负责人: Samit Ghosh
• 金额: $ 35.28万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542842
• 关键词: Acceleration; Acute; Acute Renal Failure with Renal Papillary Necrosis; Anabolism; Apoptosis; Binding; Biological Availability; Biological Markers; Caspase; Cell Death; Cells; Cessation of life; Characteristics; Chronic; Chronic Kidney Failure; Clinical; Data; Development; Drops; End stage renal failure; Epithelial Cells; Epithelium; Event; Exposure to; Genes; Genetic Diseases; HNF4A gene; Heme; Hemin; Hemoglobin; Hemolysis; Hemopexin; Hepatic; Hospitalization; Human; Impairment; In Vitro; Incidence; Individual; Infusion procedures; Injury to Kidney; Kidney; Kidney Diseases; Knock-out; Knockout Mice; Liver; Mediating; Mitochondria; Modeling; Molecular; Morbidity - disease rate; Mouse Strains; Mus; Pathogenesis; Pathway interactions; Patients; Phenocopy; Plasma; Production; Proteins; Reactive Oxygen Species; Refractory; Renal clearance function; Research; Risk; Risk Factors; Role; Sampling; Serum; Sickle Cell Anemia; Source; System; Testing; Toxic effect; Tubular formation; Urine; acute chest syndrome; adverse outcome; alpha-1-microglobulin; attenuation; biobank; clinical risk; clinically relevant; cohort; exhaust; experimental study; extracellular; heme 1; heme biosynthesis; heme oxygenase-1; hepatic nuclear factor 1; improved; in vivo; intravenous injection; knockout gene; mortality; mouse model; novel; overexpression; patient population; prognostic; promoter; prospective; sickling; targeted treatment; therapeutic target; transcription factor; vaso-occlusive pain; virtual

PROJECT SUMMARY/ABSTRACT Kidney disorders comprising acute kidney injury (AKI), chronic kidney disease (CKD) and end-stage renal disease (ESRD) account for significant morbidity and mortality in sickle cell disease (SCD). AKI, a potent risk factor for CKD and ESRD, develops primarily in SCD patients hospitalized with vasoocclusive pain crisis (VOC) or acute chest syndrome (ACS). These characteristic SCD events are associated with rapid drop in hemoglobin implying acute intravascular hemolysis releasing free circulating heme as a potential trigger for AKI. However, the precise mechanisms of this association have not been investigated per se, and therefore targeted therapies based on mechanistic models have not emerged for kidney injuries in SCD. Excess circulating heme is primarily scavenged by hemopexin (Hx) and delivered to liver for degradation by heme oxygenase-1 (HO-1). Due to chronic hemolysis, Hx is depleted in SCD. We reasoned that during acute intravascular hemolysis in SCD, excess extracellular heme will preferentially bind to alpha-1-microglobulin (A1M), a secondary plasma heme scavenger, which carries free heme to the kidneys. Consequently, renal proximal tubular epithelial cells (RPTECs) will be exposed to high amount of toxic heme. Induction of intracellular HO-1 normally protects RPTECs from heme toxicity and averts AKI. We have recently discovered that both patients and mice with SCD have elevated plasma A1M compare to normal controls. This discovery leads to the development of a clinically relevant model of AKI in humanized sickle mice by modest elevation of circulating heme through intravenous injection of purified heme (hemin). Pilot data suggests that SCD patients with higher A1M/Hx ratio posses the risk of developing AKI following VOC. Heme suppresses hepatocyte nuclear factor 4 alpha (HNF4a) expression associated with reduced hemopexin expression in liver following acute hemolysis. Preliminary data also showed that persistent exposure to excess heme renders RPTECs refractory to HO-1 induction during acute hemolysis in SCD. Moreover, we found that heme induces kruppel-like factor 9 (KLF9) associated with amplification of mitochondrial ROS (mtROS) that triggers renal tubular epithelial cell death. Based on these data we hypothesized that enhanced clearance of circulating heme to the kidneys and impaired induction of HO-1 in the renal tubular epithelium during intravascular hemolysis in SCD trigger tubular cell death and AKI development. We will test this hypothesis with three specific aims that integrate experiments with cultured and primary human RPTECs, murine models and clinical biorepository samples including serum, plasma and urine from multiple cohorts of SCD patients. Aim 1 will determine whether altered concentration of circulating heme scavenger proteins, can serve as risk factor for AKI in individuals with SCD. This aim will also determine if multiple hemolytic events develop CKD. Aim 2 will test the hypothesis that heme regulates the biosynthesis of Hx by down-regulating the expression of HNF4a. Aim 3 will utilize human RPTECs and specific gene knockout mouse strains to determine if heme induced KLF9 amplification accelerates cell death that involves overproduction of mtROS. This aim will use targeted HO-1 knockout or overexpression mice to determine whether amplified KLF9 blocks sufficient HO-1 induction and promotes heme induced AKI in SCD. This study will delineate the cellular and molecular pathogenesis of excess circulating heme mediated AKI in SCD during intravascular hemolysis, and identify potential therapeutic targets. This project will also elucidate a novel mechanism of heme-induced KLF9 mediated renal tubular epithelial cell death. Most importantly, rigorous analysis of clinical samples collected at baseline, during hospitalizations or following AKI incidences will establish whether A1M and Hx can serve as risk factors for AKI in SCD patients.

项目摘要/摘要 肾脏疾病包括急性肾损伤(AKI)、慢性肾脏疾病(CKD)和终末期肾脏 在镰状细胞病(SCD)中，疾病(ESRD)占很大的发病率和死亡率。阿琪，这是一个巨大的风险 CKD和ESRD的因素，主要发生在有血管闭塞性疼痛危象(VOC)的SCD患者中 或急性胸腔综合征(ACS)。这些特征性的SCD事件与血红蛋白的迅速下降有关 提示急性血管内溶血释放游离循环中的血红素是AKI的潜在触发因素。然而， 这种联系的确切机制本身还没有被研究，因此有针对性的治疗。 SCD中肾脏损伤的机制模型尚未出现。过量的循环中的血红素主要是 被血凝素(HX)清除，并被血红素加氧酶-1(HO-1)降解到肝脏。由于 慢性溶血，HX在SCD中被耗尽。我们推测在SCD的急性血管内溶血过程中， 过量的胞外血红素将优先与次级血浆血红素α-1-微球蛋白(A1M)结合 清道夫，它把游离的血红素带到肾脏。因此，肾近端小管上皮细胞 (RPTECs)将暴露在高剂量的有毒血红素中。诱导细胞内HO-1通常具有保护作用 RPTECs从血红素毒性和避免AKI。我们最近发现患有SCD的患者和小鼠 与正常对照组相比，血浆A1M升高。这一发现导致了临床上一种 静脉注射适度升高循环血红素建立人源化镰刀鼠AKI的相关模型 注射提纯的亚铁血红素(亚铁血红素)。Pilot数据显示，A1M/HX比率较高的SCD患者具有 VOC后发生AKI的风险。血红素抑制肝细胞核因子4a的表达 与急性溶血后肝脏中的血凝素表达减少有关。初步数据还显示， 持续暴露于过量的血红素使RPTECs在急性溶血时难以诱导HO-1 在SCD。此外，我们还发现，血红素诱导Kruppel样因子9(KLF9)与KLF9的扩增有关。 线粒体ROS(MtROS)触发肾小管上皮细胞死亡。基于这些数据，我们 假说增加了循环中的血红素到肾脏的清除，并损害了HO-1的诱导 SCD患者血管内溶血过程中肾小管上皮细胞死亡和AKI的发生。 我们将用三个特定的目标来验证这一假设，这三个目标结合了培养的和原代人类的实验 RPTECs，小鼠模型和临床生物库样本，包括来自 SCD患者的队列。 目标1将确定循环中的血红素清道夫蛋白的浓度变化是否会成为危险因素 SCD患者AKI的相关因素。这一目标也将决定是否有多个溶血事件发展为慢性肾脏病。 目的2将验证以下假设：血红素通过下调HX的表达来调节HX的生物合成 HNF4a。 AIM 3将利用人类RPTECs和特定基因敲除小鼠品系来确定血红素是否会诱导KLF9 扩增会加速细胞死亡，这涉及到mtROS的过度生产。这一目标将使用靶向HO-1 基因敲除或过度表达小鼠以确定扩增的KLF9是否阻断了足够的HO-1诱导和 促进血红素在SCD中诱导的AKI。 本研究将阐明过量循环血红素介导的AKI在细胞和分子水平上的发病机制。 SCD在血管内溶血过程中的作用，并确定潜在的治疗靶点。该项目还将阐明 血红素诱导KLF9诱导肾小管上皮细胞死亡的新机制最重要的是，严谨 对基线、住院期间或急性心肌梗死事件后收集的临床样本的分析将确定 A1M和HX是否可作为SCD患者AKI的危险因素。