Lipoxins Regulate the Egr-1 Network and Reverse Diabetic Kidney Disease

The failure of spontaneous resolution underlies chronic inflammatory conditions including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules which promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators which promote the resolution of inflammation. Here we investigated the potential of LXA4 and a synthetic LX analogue [Benzo-LXA4] as therapeutics in a murine model of diabetic kidney disease. Diabetes was induced with streptozotocin in ApoE-/- mice and kidney disease was observed. The development of diabetes-induced albuminuria, mesangial expansion and collagen deposition was attenuated by LXs. It is noteworthy that LXs also attenuated established kidney disease in diabetic mice administered LXs 10 weeks after disease onset, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature [725 genes; FDR P=0.05]. Comparison of this murine gene signature with human DKD identified shared renal pro-inflammatory/pro-fibrotic signals (TNF-a, IL-1ß, NF-?B). In diabetic mice we identified 20 and 51 transcripts regulated by LXA4 and Benzo-LXA4, respectively, and pathway analysis identified established (TGF-ß1, PDGF, TNF-a, NF-?B) and novel (Early growth response-1 - EGR-1) networks activated in diabetes and regulated by LXs. Using human renal epithelial cells, we demonstrate that LXs attenuate TNF-a driven Egr-1 activation, with Egr-1 important for cellular responses to TGF-ß1 and TNF-a. These data demonstrate for the first time that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation. Overall design: RNA-seq was performed to understand gene expression changes caused by injection of lipoxin and benzo-lipoxin a compared to the ethanol vehicle in diabetic and non-diabetic mice. N=6 replicates were profiled.自发消退失败是慢性炎症性疾病的核心病理基础，其中包括糖尿病微血管并发症，例如糖尿病肾病（diabetic kidney disease, DKD）。内源性产生的可促进炎症生理性消退的分子的发现，提示这类生物活性物质在慢性炎症场景中具有治疗潜力。脂氧素（Lipoxins, LXs）是一类可促进炎症消退的脂质介质。本研究旨在探究脂氧素A4（LXA4）及其合成类似物[苯甲酸酯修饰LXA4（Benzo-LXA4）]在糖尿病肾病小鼠模型中的治疗潜力。 本研究采用链脲佐菌素（streptozotocin）诱导载脂蛋白E基因敲除（ApoE-/-）小鼠构建糖尿病模型，并观察其肾脏病变情况。脂氧素可抑制糖尿病诱导的蛋白尿、系膜扩张及胶原沉积的进展。值得注意的是，在糖尿病小鼠肾病发病10周后给予脂氧素干预，仍可减轻已成型的肾脏病变，并改善肾脏功能。 肾脏转录组测序（RNA-seq）分析确定了糖尿病相关基因特征[共725个基因；错误发现率（False Discovery Rate, FDR）P=0.05]。将该小鼠基因特征与人类糖尿病肾病（DKD）数据进行比对，发现二者共享肾脏促炎/促纤维化信号通路（肿瘤坏死因子α（Tumor Necrosis Factor α, TNF-α）、白细胞介素1β（Interleukin 1β, IL-1β）、核因子κB（Nuclear Factor κB, NF-κB））。在糖尿病小鼠中，我们分别鉴定出受LXA4和Benzo-LXA4调控的20个和51个转录本；通路分析发现，糖尿病状态下激活且受脂氧素调控的通路既包括已被报道的转化生长因子β1（Transforming Growth Factor β1, TGF-β1）、血小板衍生生长因子（Platelet Derived Growth Factor, PDGF）、TNF-α、NF-κB通路，也包括新发现的早期生长反应因子1（Early Growth Response 1, EGR-1）调控网络。 通过人肾上皮细胞实验，我们证实脂氧素可抑制TNF-α介导的Egr-1激活，而Egr-1对于细胞应答TGF-β1和TNF-α具有关键作用。本研究首次证实，脂氧素可逆转已成型的糖尿病并发症，为以促进炎症消退为目标的治疗策略提供了实验依据。 实验整体设计：本研究通过RNA-seq分析，对比糖尿病与非糖尿病小鼠分别经脂氧素、苯甲酸酯修饰脂氧素A4干预，与乙醇溶剂对照组相比的基因表达变化情况。每组设置6个生物学重复样本进行测序分析。