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 包括急性肾脏损伤（AKI），慢性肾脏疾病（CKD）和末期肾脏的肾脏疾病 疾病（ESRD）解释了镰状细胞病（SCD）的显着发病率和死亡率。 AKI，有效的风险 CKD和ESRD的因素，主要是在患有血管判断性疼痛危机（VOC）住院的SCD患者中发展 或急性胸部综合征（ACS）。这些特征性SCD事件与血红蛋白的快速下降有关 意味着急性血管内溶血释放自由循环血红素，作为AKI的潜在触发因素。然而， 该关联的确切机制本身尚未研究，因此针对疗法 基于机械模型，SCD中的肾脏受伤并未出现。多余的循环血红素主要是 通过血红素（HX）清除，并通过血红素加氧酶-1（HO-1）递送至肝脏降解。由于 慢性溶血，HX在SCD中耗尽。我们认为在SCD中急性血管内溶血期间， 过多的细胞外血红素将优先结合α-1-微球蛋白（A1M），次级等离子体血红素 Scavenger，可以将血红素自由到肾脏。因此，肾近端管状上皮细胞 （RPTEC）将暴露于大量的有毒血红素。诱导细胞内HO-1通常保护 来自血红素毒性并避免AKI的RPTEC。我们最近发现患者和SCD的患者和小鼠 与正常对照相比，血浆A1M升高。这一发现导致临床上的发展 通过静脉内循环血红素的适度升高，人为化镰状小鼠AKI的相关模型 注射纯化的血红素（Hemin）。试点数据表明，具有较高A1M/HX比率的SCD患者具有 有VOC后发展AKI的风险。血红素抑制肝细胞核因子4α（HNF4A）表达 与急性溶血后肝脏中肝中的血红素表达降低有关。初步数据也显示 在急性溶血期间，这种持续暴露于过量的血红素使RPTEC对HO-1诱导难治性。 在SCD中。此外，我们发现血红素诱导了类似Kruppel的因子9（KLF9） 线粒体ROS（MTROS）触发肾小管上皮细胞死亡。根据这些数据，我们 假设增强了循环血红素对肾脏的清除率，并损害了HO-1在 SCD中血管内溶血期间的肾小管上皮触发管状细胞死亡和AKI发育。 我们将以三个特定的目的测试该假设，将实验与培养和原代人集成 RPTEC，鼠模型和临床生物疗法样品，包括血清，血浆和尿液。 SCD患者队列。 AIM 1将确定循环血红素清除蛋白的浓度改变是否可以作为风险 SCD患者AKI的因素。该目标还将确定多个溶血事件是否发展为CKD。 AIM 2将测试血红素通过下调的表达来调节HX的生物合成的假设 HNF4A。 AIM 3将利用人类RPTEC和特定的基因基因敲除小鼠菌株来确定血红素诱导的KLF9是否 扩增加速了涉及MTROS过量生产的细胞死亡。这个目标将使用针对目标的HO-1 敲除或过表达小鼠，以确定扩增的KLF9是否足够HO-1诱导和 在SCD中促进血红素诱导的AKI。 这项研究将描述过量循环血红素介导的AKI的细胞和分子发病机理 血管内溶血期间的SCD，并鉴定潜在的治疗靶标。该项目还将阐明 血红素诱导的KLF9介导的肾小管上皮细胞死亡的新型机制。最重要的是，严格 分析在基线时收集的临床样本，住院期间或之后的AKI事件发生分析 A1M和HX是否可以作为SCD患者AKI的风险因素。