Transcriptomic, therapeutic and genetic investigations of sickle cell nephropathy

摘要 镰状细胞病是一种遗传性疾病，对家庭和医疗保健具有深远的影响 基础设施。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依赖的ESRD易感性受遗传因素的影响。 我们以前证明了22号染色体上两个物理上非常接近的基因座Myh9和APOL1， 是SCD患者蛋白尿的独立预测因子。这一区域，特别是两个主要的风险等位基因(命名为G1 和G2)，已在非SCD肾病中广泛复制。然而，相互矛盾的研究表明 APOL1或Myh9中的等位基因可能通过一种尚不清楚的机制参与了病理过程。 最近，我们通过活体研究来了解该基因座在终末期肾病中的作用。我们发现了一个 APOL1在斑马鱼肾脏发育中的作用，并发现了一个复杂的遗传结构，其中 APOL1的G1风险等位基因为功能性零，而G2风险等位基因为显性负效应。关键的是，我们 还发现，在贫血应激的背景下，APOL1和Myh9在遗传上相互作用，潜在地协调 关于每个基因在ESRD中的作用的不同观察。这些进步为我们提供了机会 探讨该领域的关键问题，包括：病理中涉及的时空背景；潜在的 可作为治疗干预靶点的细胞机制和途径；以及在多大程度上 其他遗传因素导致SCD肾病。配备强大的、多学科的工作工具， 包括斑马鱼突变体，它概括了一种实验上容易处理的、与体内生理相关的 病理学方面，我们提出了三个目标。首先，为了检查APOL1/Myh9基因座的细胞病理学，我们将 在我们的APOL1斑马鱼模型中，研究多种相关细胞类型中转录网络的变化。 通过这种方法，我们将能够查询两种建议的途径，如自噬和改变 氯通道功能，并以不偏不倚的方式发现新的。第二，没有 SCD肾病的机制知识有限，治疗选择有限。尽管血管紧张素转换 酶抑制剂(ACEI)似乎可以减少蛋白尿，并被广泛使用，其有效性在 APOL1和SCD肾病仍未得到证实。我们将在体内筛选相关的APOL1模型 FDA批准的可迅速过渡到临床领域的先导治疗化合物。最后， 尽管APOL1和Myh9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，对这些因素的识别可能会揭示其发病机制。我们将利用我们的大型 SCD肾病队列和我们的体内工具来识别其他易感基因，从而提供 进入这种表型的生物学的潜在的正交入口点。总而言之，我们的研究将向 Myh9/APOL1基因支持的病理机制，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，可以确定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.