Transcriptomic, therapeutic and genetic investigations of sickle cell nephropathy

摘要 镰状细胞病（SCD）是一种遗传性疾病，对家庭和医疗保健有着深远的影响。 基础设施演进值得注意的是，SCD可引发肾病，导致终末期肾病（ESRD）， 结果和没有有效的治疗。SCD依赖性ESRD易感性受遗传因素影响。 我们以前证明了MYH 9和APOL 1，22号染色体上两个物理上非常接近的基因座， 是SCD患者蛋白尿的独立预测因子。该区域，特别是两个主要的风险等位基因（命名为G1 和G2），在非SCD肾病中被广泛复制。然而，相互矛盾的研究表明， APOL 1或MYH 9中的等位基因可能通过一种尚不清楚的机制导致病理学。 最近，我们采用体内研究来了解该位点对ESRD的贡献。我们发现了一 APOL 1在发育中的斑马鱼肾脏中的作用，并揭示了一个复杂的遗传结构，其中 APOL 1 G1危险等位基因是功能性无效，而G2危险等位基因发挥显性负效应。关键是，我们 还发现APOL 1和MYH 9在贫血应激的背景下在遗传上相互作用， 关于每个基因在ESRD中的作用的不同观察。这些进步使我们有机会 探讨该领域的关键问题，包括：病理学中涉及的时空背景;潜在的 可以作为治疗干预目标的细胞机制和途径;以及 其他遗传因素也导致SCD肾病。配备了强大的多学科工作工具， 包括斑马鱼突变体，其重现了实验上易于处理的、生理上相关的体内 病理学，我们提出三个目标。首先，为了检查APOL 1/MYH 9基因座的细胞病理学，我们将 在我们的APOL 1斑马鱼模型中研究多种相关细胞类型中改变的转录网络。 通过这种方法，我们将能够查询这两个建议的途径，如自噬和改变 氯离子通道功能，并以公正的方式发现新的氯离子通道。第二，缺席 SCD肾病的机制知识限制了治疗选择。虽然血管紧张素转化 酶抑制剂（ACEi）似乎可以减少白蛋白尿并被广泛使用，其功效是 APOL 1和SCD肾病尚未得到证实。我们将筛选相关的APOL 1体内模型， FDA批准的先导治疗化合物，可以迅速过渡到临床竞技场。最后， 尽管APOL 1和MYH 9传递SCD肾病的关键风险，但有明确的证据表明， 遗传风险因素，其识别可能会告知病理机制。我们将利用我们的大 SCD肾病队列和我们的体内工具，以确定其他易感基因座，从而提供 潜在的正交切入点进入这种表型的生物学。综合起来，我们的研究将告知 由MYH 9/APOL 1基因座支持的病理机制，确定SCD肾病的新驱动因素，并提供 这是一个令人兴奋的机会，可以识别出具有研究和治疗潜力的先导化合物。

ABSTRACT Sickle cell disease (SCD) is a genetic disorder with profound consequences to families and the health care infrastructure. Notably, SCD can trigger nephropathy leading to end-stage renal disease (ESRD) with poor outcomes and no effective treatment. SCD-dependent susceptibility to ESRD is influenced by genetic factors. We demonstrated previously that MYH9 and APOL1, two loci in close physical proximity on chromosome 22, are independent predictors of proteinuria in SCD. This region, particularly two major risk alleles (named G1 and G2), has been replicated widely in non-SCD nephropathy. However, conflicting studies have suggested that alleles in either APOL1 or MYH9 might contribute to the pathology through an as yet unclear mechanism. Recently, we employed in vivo studies to understand the contribution of this locus to ESRD. We discovered a role for APOL1 in the developing zebrafish kidney and uncovered a complex genetic architecture, wherein the APOL1 G1 risk allele is a functional null while the G2 risk allele exerts a dominant negative effect. Critically, we also found that APOL1 and MYH9 interact genetically, in the context of anemic stress, potentially reconciling diverse observations about the roles of each gene in ESRD. These advances afford us the opportunity to probe key questions in the field, including: the spatiotemporal context involved in the pathology; the underlying cellular mechanisms and pathways that could be targeted for therapeutic intervention; and the extent to which other genetic factors contribute the SCD nephropathy. Equipped with potent, multidisciplinary working tools, including zebrafish mutants that recapitulate an experimentally tractable, physiologically relevant in vivo pathology, we propose three Aims. First, to examine the cellular pathology of the APOL1/MYH9 locus we will investigate the altered transcriptional networks in multiple relevant cell types in our APOL1 zebrafish models. Through this method, we will be able to query both suggested pathways, such as autophagy and altered chloride channel functionality and discover new ones in an unbiased fashion. Second, the absence of mechanistic knowledge in SCD nephropathy has limited therapeutic options. Although angiotensin-converting enzyme inhibitors (ACEi) appear to reduce albuminuria and are used widely, their efficacy in the context of APOL1 and SCD nephropathies remains unproven. We will screen our relevant APOL1 in vivo models for FDA-approved lead therapeutic compounds that could be transitioned rapidly into the clinical arena. Finally, although APOL1 and MYH9 convey critical risk for SCD nephropathy, there is clear evidence for additional genetic risk factors, the identification of which will likely inform pathomechanism. We will leverage our large SCD nephropathy cohort and our in vivo tools to identify additional susceptibility loci and, thus, provide potentially orthogonal entry points into the biology of this phenotype. Taken together, our studies will inform the pathomechanism underpinned by the MYH9/APOL1 locus, identify new drivers for SCD nephropathy and offer an exciting opportunity to identify lead compounds that will have both investigative and therapeutic potential.