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: 10318643
• 负责人: Samit Ghosh
• 金额: $ 35.17万
• 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/10318643
• 关键词: 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; Epithelial Cells; 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; 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; base; biobank; clinical risk; clinically relevant; cohort; exhaust; experimental study; extracellular; heme biosynthesis; heme oxygenase-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.

项目概要/摘要 肾脏疾病，包括急性肾损伤 (AKI)、慢性肾脏病 (CKD) 和终末期肾病 镰状细胞病（ESRD）是镰状细胞病（SCD）发病率和死亡率显着的原因。 AKI，一种潜在的风险 CKD 和 ESRD 的因素，主要发生于因血管闭塞性疼痛危象 (VOC) 住院的 SCD 患者 或急性胸部综合症（ACS）。这些特征性 SCD 事件与血红蛋白快速下降有关 意味着急性血管内溶血释放游离循环血红素是 AKI 的潜在触发因素。然而， 这种关联的确切机制本身尚未得到研究，因此需要进行靶向治疗 目前尚未出现针对 SCD 肾损伤的基于机制模型。过量的循环血红素主要是 被血红素结合蛋白 (Hx) 清除并输送至肝脏由血红素加氧酶-1 (HO-1) 降解。由于 慢性溶血，SCD 中 Hx 耗尽。我们推断，在 SCD 急性血管内溶血期间， 过量的细胞外血红素将优先与 α-1-微球蛋白 (A1M)（一种次级血浆血红素）结合 清除剂，将游离血红素运送到肾脏。因此，肾近端肾小管上皮细胞 （RPTEC）将暴露于大量有毒血红素。细胞内 HO-1 的诱导通常可以保护 RPTEC 免受血红素毒性并避免 AKI。我们最近发现患有 SCD 的患者和小鼠 与正常对照相比，血浆 A1M 升高。这一发现导致了临床上的发展 通过静脉注射适度升高循环血红素，建立人源化镰状小鼠 AKI 相关模型 注射纯化血红素（血红素）。试点数据表明，A1M/Hx 比值较高的 SCD 患者具有 VOC 后发生 AKI 的风险。血红素抑制肝细胞核因子 4 α (HNF4a) 表达 与急性溶血后肝脏中血红素表达减少有关。初步数据还显示 持续暴露于过量血红素会使 RPTEC 在急性溶血期间难以诱导 HO-1 在SCD中。此外，我们发现血红素诱导与扩增相关的克鲁佩尔样因子 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到肾脏的清除并削弱了 HO-1 的诱导 SCD 血管内溶血期间肾小管上皮触发肾小管细胞死亡和 AKI 发展。 我们将通过三个具体目标来检验这一假设，这些目标将培养人类和原代人类的实验结合起来 RPTEC、小鼠模型和临床生物样本库样本，包括来自多个实验室的血清、血浆和尿液 SCD 患者队列。 目标 1 将确定循环血红素清除剂蛋白浓度的改变是否可以作为风险 SCD 患者 AKI 的影响因素。这一目标还将确定多次溶血事件是否会发展为 CKD。 目标 2 将检验血红素通过下调 Hx 的表达来调节 Hx 生物合成的假设 HNF4a。 目标 3 将利用人类 RPTEC 和特定基因敲除小鼠品系来确定血红素是否诱导 KLF9 扩增会加速细胞死亡，导致 mtROS 过量产生。该目标将使用定向HO-1 敲除或过表达小鼠以确定扩增的 KLF9 是否会阻断足够的 HO-1 诱导和 促进 SCD 中血红素诱导的 AKI。 这项研究将描述循环血红素过多介导的 AKI 的细胞和分子发病机制。 血管内溶血期间的 SCD，并确定潜在的治疗靶点。该项目还将阐明 血红素诱导 KLF9 介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 AKI 发生后收集的临床样本进行分析将确定 A1M 和 Hx 是否可以作为 SCD 患者发生 AKI 的危险因素。