datasheet1_Angiotensin AT2 Receptor is Anti-inflammatory and Reno-Protective in Lipopolysaccharide Mice Model: Role of IL-10.docx

Acute kidney injury (AKI) due to endotoxemic insult is predicted by the infiltration of neutrophils, monocytes and macrophages, and the release of pro-and anti-inflammatory cytokines to the site of injury. Earlier, we have demonstrated the role of angiotensin-II type 2 receptor (AT2R) stimulation in reno-protection in lipopolysaccharide (LPS)-induced inflammation and AKI in C57BL6/NHsd mice. Moreover, AT2R activation has been shown to increase the anti-inflammatory cytokine interleukin-10 (IL-10), its role in AT2R-mediated anti-inflammation and reno-protection is unknown. To address this question, in the present study mice were treated with the AT2R agonist C21 (0.3 mg/kg, intraperitoneally), LPS (5 mg/kg, intraperitoneally), or LPS with C21 pre-treatment with or without neutralizing IL-10 antibody. Treatment with C21 alone caused an increase in the plasma and kidney IL-10 levels, which peaks at 2-h, and returned to baseline at 6-h. The C21-induced IL-10 increase was blocked by the AT2R antagonist PD123319 suggesting AT2R’s involvement. LPS treatment caused a profound increase in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the LPS-induced increase in these cytokines was attenuated by the C21 pre-treatment (1-h prior LPS) both in the plasma and kidney. Neutralizing IL-10 antibody treatment abrogated the C21-lowering of TNF-α and IL-6 in the kidney but not in the plasma. Similar results as related to the cytokines profiles in all the groups were also observed in the heart and spleen. The alteration in early cytokine profile prompted us to measure the markers of renal function (blood urea nitogen and urinary creatinine) in order to analyze the effect of IL-10 neutralization. However, it was too early to observe changes in renal function. Therefore, the renal function and injury markers were again measured at 24 h. Treatment with neutralizing IL-10 antibody attenuated the C21-mediated improvement in indices of the kidney function, but not the biomarkers of renal injury (kidney injury molecule-1 and neutrophil-gelatinase associated lipocalin). Collectively, our data suggest that the involvement of IL-10 in AT2R-mediated anti-inflammation and reno-protection against LPS is complex, mediating the renal cytokine profile and kidney filtration function, but not the plasma cytokine profile and renal injury markers.

急性肾损伤（Acute kidney injury, AKI）因内毒素性损伤所致的病情，可通过中性粒细胞、单核细胞与巨噬细胞的浸润，以及促炎、抗炎细胞因子向损伤部位的释放进行预测。本团队此前已证实，血管紧张素II 2型受体（angiotensin-II type 2 receptor, AT2R）激动可在脂多糖（lipopolysaccharide, LPS）诱导的C57BL6/NHsd小鼠炎症及AKI模型中发挥肾保护作用。此外，已有研究表明AT2R激活可升高抗炎细胞因子白细胞介素-10（interleukin-10, IL-10），但AT2R介导的抗炎及肾保护作用中IL-10所扮演的角色尚未明确。

为解答该科学问题，本研究将小鼠分别予以以下处理：AT2R激动剂C21（0.3 mg/kg，腹腔注射）、脂多糖（LPS，5 mg/kg，腹腔注射），或在脂多糖给药前1小时予以C21预处理，并联合或不联合IL-10中和抗体。单独给予C21可使血浆及肾脏组织中的IL-10水平升高，该升高于2小时达峰，6小时恢复至基线水平；AT2R拮抗剂PD123319可阻断C21诱导的IL-10升高，提示AT2R参与了该过程。脂多糖处理可显著升高肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）及白细胞介素-6（interleukin-6, IL-6）的水平，而C21预处理（脂多糖给药前1小时）可减弱血浆及肾脏组织中上述细胞因子的脂多糖诱导性升高。IL-10中和抗体处理可消除C21对肾脏组织中TNF-α及IL-6的下调作用，但对血浆中的该效应无影响。所有组别细胞因子谱的相似变化亦在心脏与脾脏组织中被观测到。

早期细胞因子谱的变化促使我们检测肾功能标志物（血尿素氮及尿肌酐），以分析IL-10中和的效应，但彼时尚未能观测到肾功能变化。因此研究团队于造模后24小时再次检测肾功能及肾损伤标志物。IL-10中和抗体处理可减弱C21介导的肾功能指标改善，但对肾损伤生物标志物（肾损伤分子-1及中性粒细胞明胶酶相关脂质运载蛋白）无显著影响。

综上，本研究数据表明，IL-10在AT2R介导的抗脂多糖炎症及肾保护作用中的参与机制较为复杂：其可调控肾脏细胞因子谱与肾脏滤过功能，但不影响血浆细胞因子谱及肾损伤标志物。