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.

项目总结/摘要 包括急性肾损伤（阿基）、慢性肾病（CKD）和终末期肾病的肾病 在镰状细胞病（SCD）中，ESRD占显著发病率和死亡率。阿基，潜在风险 CKD和ESRD的主要危险因素，主要发生在因血管闭塞性疼痛危象（VOC）住院的SCD患者中 或急性胸部综合征（ACS）。这些特征性SCD事件与血红蛋白快速下降相关 提示急性血管内溶血释放游离循环血红素是阿基的潜在触发因素。然而，在这方面， 这种关联的确切机制本身尚未研究，因此靶向治疗 尚未出现SCD中肾损伤的机制模型。过量的循环血红素主要是 经血红素结合蛋白（Hx）清除后，转运至肝脏，被血红素加氧酶-1（HO-1）降解。由于 慢性溶血，SCD中Hx耗尽。我们推断在SCD的急性血管内溶血期间， 过量的细胞外血红素将优先结合α-1-微球蛋白（A1 M），一种次级血浆血红素 清道夫，携带游离血红素到肾脏。因此，近端肾小管上皮细胞 （RPTEC）将暴露于大量的有毒血红素。细胞内HO-1的诱导通常保护 RPTEC避免血红素毒性并避免阿基。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A1 M升高。这一发现导致了一种临床上 人源化镰状小鼠中阿基的相关模型，通过静脉内注射适量升高循环血红素 注射纯化的血红素（氯化血红素）。初步数据表明，A1 M/Hx比值较高的SCD患者， VOC后发生阿基的风险。血红素抑制肝细胞核因子4 α（HNF 4a）表达 与急性溶血后肝脏中血红素结合蛋白表达减少相关。初步数据还显示， 持续暴露于过量血红素使RPTEC在急性溶血期间对HO-1诱导不敏感， 在SCD。此外，我们发现血红素诱导kruppel样因子9（KLF 9）与扩增的 线粒体ROS（mtROS），其触发肾小管上皮细胞死亡。根据这些数据，我们 假设，增加血液中血红素对肾脏的清除率，并削弱HO-1的诱导， SCD中血管内溶血期间的肾小管上皮细胞触发肾小管细胞死亡和阿基发展。 我们将用三个具体的目标来检验这个假设，这些目标是将培养的和原代的人类 RPTEC、鼠模型和临床生物储存样品，包括来自多个 SCD患者的队列。 目的1将确定是否改变浓度的循环血红素清除蛋白，可以作为风险 SCD患者的阿基因素。这一目标也将确定是否多次溶血事件发展为CKD。 目的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。 HNF 4a。 目的3将利用人RPTEC和特定基因敲除小鼠品系来确定血红素是否诱导KLF 9 扩增加速了涉及mtROS过度产生的细胞死亡。这一目标将使用针对HO-1 敲除或过表达小鼠，以确定扩增的KLF 9是否阻断足够的HO-1诱导， 促进SCD中血红素诱导的阿基。 这项研究将描述过量循环血红素介导的阿基的细胞和分子发病机制， 血管内溶血过程中的SCD，并确定潜在的治疗靶点。该项目还将阐明 血红素诱导KLF 9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、住院期间或阿基发生后采集的临床样本进行分析， A1 M和Hx是否可作为SCD患者阿基的危险因素。