Meprin Metalloproteases in Kidney Injury

摘要 急性肾损伤（阿基）与高发病率和死亡率相关，并显著影响患者的生活质量。 患者及其家属的生活质量。在美国治疗急性肾病的费用 以每年数百亿美元的规模。然而，阿基的治疗结果很差，部分原因是 根本机制尚未完全了解。缺血/再灌注（IR）是阿基的主要原因， 在起始、组织修复和慢性阶段与炎症相关。然而， 调节IR中的炎症还没有完全确定。近端肾小管最易发生阿基 IR引起的损伤。Meprins，属于虾红素家族的锌金属蛋白酶，是最丰富表达的 近端肾小管刷状缘膜（BBM）中的蛋白质。迈普林由两个 亚基，α和β，形成两种高度相似的蛋白质同种型; meprin A（α-α亚基或 α-β亚基的异源寡聚体）和meprin B（β-β亚基的同源寡聚体）。干扰甲氨蝶呤 基因和给予meprin抑制剂可保护小鼠免受IR诱导的阿基，这表明meprin 加重肾损伤。meprins增强IR肾损伤的机制尚不完全清楚 明白meprin蛋白亚型具有共同和不同的底物。研究这些相互作用 meprins和它们在肾脏中的靶点之间的联系将增加对它们如何影响细胞功能的理解 和肾脏疾病的病理学。已知的meprin底物包括几种炎症介质， 作为（i）促炎细胞因子，例如白介素（IL-1β、IL-6、IL-18）和趋化因子，例如单核细胞趋化因子， 引诱蛋白-1（MCP-1）。通过meprins的蛋白水解加工使IL-6失活，但激活IL-1β和IL-18。 meprin在炎症中的作用的额外支持来自最近的研究，表明meprin α 介导抗炎肽N-乙酰基-丝氨酰-乙酰基-赖氨酰-脯氨酸（Ac-SDKP）从 胸腺素β4。尚不清楚Ac-SDKP释放是否在调节IR诱导的肾损伤中起作用。一 炎症的长期后果是纤维化，这表明细胞外基质失衡 (ECM)蛋白质代谢由于meprins切割和/或降解几种ECM蛋白（例如巢蛋白-1， 层粘连蛋白、纤连蛋白和胶原蛋白），它们可以减轻与IR诱导的肾损伤相关的纤维化。 Meprins还通过蛋白水解处理介导ECM代谢相关细胞信号通路的蛋白质 (e.g.蛋白激酶A途径）。通过meprins的蛋白水解处理降低了三种激酶的活性， PKA催化亚基的同种型（PKA Cα、Cβ1和Cβ2）。拟议的研究将使用meprin 敲除小鼠，以确定meprins调节炎症和炎症进展的机制。 IR诱导的肾损伤的起始、修复和慢性阶段。这些目标将通过以下方式实现： 三个密切相关的具体目标;（i）确定如何以及哪些meprin同种型影响炎性 在IR诱导的肾损伤的起始、修复和慢性阶段的反应，（ii）确定如何 蛋白水解酶对PKA催化亚基（PKA C）异构体的蛋白水解加工影响下游 IR诱导的急性肾损伤中PKA信号通路的靶点，（iii）确定纤维化相关的 IR诱导的肾损伤中受meprin活性影响的基因。结合蛋白质组学、实时PCR、流式细胞术 将使用流式细胞术和免疫组织化学分析。这些研究的数据将增强 了解IR诱导的肾损伤进展的潜在机制，并告知 开发更有效的治疗方法。

ABSTRACT Acute kidney injury (AKI) is associated with high morbidity and mortality rates, and significantly impacts the quality of life for patients and their families. The cost of treating acute kidney disease in the United States runs in the tens of billions of dollars annually. However, treatment outcomes for AKI are poor in part because underlying mechanisms are not fully understood. Ischemia/reperfusion (IR) is the leading cause of AKI and is associated with inflammation in the initiation, tissue repair, and chronic phases. However, the genes which modulate inflammation in IR are not fully defined. The proximal kidney tubules are the most susceptible to AKI IR-induced injury. Meprins, zinc metalloproteases of the astacin family, are the most abundantly expressed proteins in the brush border membranes (BBMs) of proximal kidney tubules. Meprins are made up of two subunits, α and β, which form two highly similar protein isoforms; meprin A (a homooligomer of α-α subunits or a heterooligomer of α-β subunits) and meprin B (a homooligomer of β-β subunits). Disruption of the meprin genes and administration meprin inhibitors protect mice from IR-induced AKI, suggesting that meprins exacerbate kidney injury. The mechanisms by which meprins enhance IR-induced kidney injury are not fully understood. The meprin protein isoforms have common and distinct substrates. Studying the interactions between meprins and their targets in the kidney will increase understanding of how they impact cell function and the pathology of kidney disease. Known meprin substrates include several mediators of inflammation such as (i) proinflammatory cytokines e.g. interleukins (IL-1β, IL-6, IL-18) and chemokines e.g. monocyte chemo- attractant protein-1 (MCP-1). Proteolytic processing by meprins inactivates IL-6, but activates IL-1β and IL-18. Additional support for a role for meprins in inflammation came from recent studies demonstrating that meprin α mediates the release of the anti-inflammatory peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) from thymosin β4. It is not known if the Ac-SDKP release plays a role in modulating IR-induced kidney injury. A long-term consequence of inflammation is fibrosis, suggesting that there is an imbalance in extracellular matrix (ECM) protein metabolism. Since meprins cleave and/or degrade several ECM proteins (e.g. nidogen-1, laminin, fibronectin, and collagen), they could alleviate the fibrosis associated with IR-induced kidney injury. Meprins also proteolytically process proteins that mediate cell signaling pathways involved in ECM metabolism (e.g. the protein kinase A pathway). Proteolytic processing by meprins reduces the kinase activity of three isoforms of the catalytic subunit of PKA (PKA Cα, Cβ1, and Cβ2). The proposed studies will utilize meprin knock out mice to determine the mechanisms by which meprins modulate inflammation and the progression of IR-induced renal injury in the initiation, reparative, and chronic phases. The goals will be achieved by pursuing three closely related specific aims; (i) determine how and which meprin isoforms impact the inflammatory response in the initiation, reparative, and chronic phases of IR-induced kidney injury, (ii) determine how proteolytic processing of isoforms of the catalytic subunit of PKA (PKA C) by meprins impacts downstream targets of the PKA signaling pathway in IR-induced acute kidney injury, (iii) determine fibrosis-associated genes impacted by meprin activity in IR-induced renal injury. A combination of proteomics, real-time PCR, flow cytometry, and immunohistochemical analysis will be used. Data from these studies will enhance understanding of the mechanisms underlying the progression of IR-induced kidney injury and inform the development of more effective therapies.