Ras guanine nucleotide-releasing protein-4 promotes renal inflammatory injury in type 2 diabetes mellitus
Ras guanine nucleotide-releasing protein-4 promotes renal inflammatory injury in type 2 diabetes mellitus
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DOI：
10.1016/j.metabol.2022.155177
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发表时间：
2022-03-23
影响因子：
9.8
通讯作者：
Yu, Pei
中科院分区：
文献类型：
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作者：
Huang, Shuai;Wang, Junmei;Yu, Pei
简介：Ras鸟嘌呤核苷酸释放蛋白4（Ras guanine nucleotide-releasing protein-4，RasGRP 4）是Ras蛋白的激活剂，在炎症反应和免疫激活中起重要作用。这项研究确定了RasGRP 4在糖尿病肾病（DKD）进展中的作用。方法：采用CRISPR/Cas9技术建立RasGRP 4基因敲除（KO）小鼠。在C57 BL/6 J野生型（WT）小鼠和RasGRP 4 KO小鼠中，通过高脂肪饮食结合连续5次每日腹膜内注射链脲佐菌素（60 mg/kg）诱导糖尿病。采用苏木素伊红染色、过碘酸希夫染色、马松三色染色观察病理损伤的组织学变化。免疫组化染色观察炎性细胞浸润情况。采用定量PCR和Western blotting检测肾组织炎症介质的表达和信号通路的激活情况。体外细胞共培养实验探讨外周血单个核细胞（PBMCs）与肾小球内皮细胞（GEnCs）之间的相互作用。结果如下：与WT小鼠相比，RasGRP 4 KO小鼠发生较不严重的糖尿病肾损伤，表现出较低的蛋白尿，减少的CD 3（+）T淋巴细胞和F4/80（+）巨噬细胞浸润，较少的炎性介质表达，包括白细胞介素6、肿瘤坏死α、细胞间粘附分子-1和血管细胞粘附分子-1，以及糖尿病肾脏中含有NLR家族pyrin结构域的3炎性体和丝裂原活化蛋白激酶（MAPK）/核因子κ B（NF-κ B）信号通路中的关键信号转导分子的较低表达水平。体外实验表明，与WT小鼠相比，RasGRP 4 KO小鼠PBMC的粘附功能降低。此外，GEnC中NLRP 3炎性体和MAPK/NF-κ B信号通路中的粘附分子和关键信号转导分子的表达被PBMC的上清液刺激，所述PBMC来源于用高糖处理的RasGRP 4 KO小鼠，并且与来源于WT小鼠的PBMC相比也显著降低。结论：RasGRP 4可能通过调节PBMC与GEnCs的相互作用，进而激活NLRP 3炎性小体和MAPK/NF-κ B信号通路，促进糖尿病患者PBMC介导的炎症损伤。
Introduction: Ras guanine nucleotide-releasing protein-4 (RasGRP4) is an activator of Ras protein, which plays significant roles in both the inflammatory response and immune activation. This study determined the role of RasGRP4 in diabetic kidney disease (DKD) progression. Methods: CRISPR/Cas9 technology was used to establish RasGRP4 knockout (KO) mice. Diabetes was induced by a high-fat diet combined with five consecutive daily intraperitoneal injections of streptozotocin (60 mg/kg) in C57BL/6J wild-type (WT) mice and RasGRP4 KO mice. Hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome staining were used to observe the histology of pathological injury. Immunohistochemical staining was used to analyze inflammatory cell infiltration. Quantitative PCR and Western blotting were used to detect the expression of inflammatory mediators and the activation of signaling pathways in renal tissues. In vitro cell co-culture experiments were performed to explore the interactions between peripheral blood mononuclear cells (PBMCs) and glomerular endothelial cells (GEnCs). Results: RasGRP4 KO mice developed less severe diabetic kidney injury compared to WT mice, exhibiting lower proteinuria, reduced CD3(+) T lymphocyte and F4/80(+) macrophage infiltration, less inflammatory mediator expression including interleukin 6, tumor necrosis alpha, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, and lower expression levels of critical signal transduction molecules in the NLR family pyrin domain-containing 3 inflammasome and mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-kappa B signaling pathways in the diabetic kidney. In vitro experiments showed that the adhesion function of PBMCs of RasGRP4 KO mice was reduced compared to that of WT mice. Moreover, the expression of adhesion molecules and critical signal transduction molecules in the NLRP3 inflammasome and MAPK/NF-kappa B signaling pathways in GEnCs was stimulated by the supernatant of PBMCs, which were derived from RasGRP4 KO mice treated with high glucose and were also significantly reduced compared to those derived from WT mice. Conclusion: RasGRP4 promotes the inflammatory injury mediated by PBMCs in diabetes, probably by regulating the interaction between PBMCs and GEnCs and further activating the NLRP3 inflammasome and MAPK/NF-kappa B signaling pathways.