Additional file 2: of Plasma receptor interacting protein kinase-3 levels are associated with acute respiratory distress syndrome in sepsis and trauma: a cohort study

Figure S1. Change in plasma RIPK3 concentration from presentation to 48 h by Acute Respiratory Distress Syndrome (ARDS) status excluding patients who met ARDS criteria ≤ 48 h after presentation among patients in the PETROS (trauma) cohort (No ARDS n = 143, ARDS n = 18 meeting criteria 55–140 h after presentation). Gray boxes represent interquartile range, with median designated by central line. Figure S2. Levels of mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) in whole lung homogenate are not significantly different between mice treated with ZVAD, LPS, or LPS-ZVAD. A. Immunoblot of whole lung homogenate showing similar MLKL and pMLKL regardless of treatment. B. Densitometry of whole lung homogenate. All comparisons between treatment groups are non-significant. Figure S3. Adjusted probability of acute kidney injury (AKI) across range of Δ receptor interacting protein kinase-3 (RIPK3) levels (RIPK3 change from presentation to 48 h) in each cohort. Estimated probabilities (line) with 95% confidence intervals (gray shading) determined using post-estimation marginal analysis after multivariable logistic regression modeling. A. MESSI cohort. Probabilities adjusted for age, red blood cell transfusions on the day of presentation, chronic kidney disease, diabetes mellitus, and shock at presentation. B. PETROS cohort. Probabilities adjusted for red blood cell transfusions in the first 6 h after presentation, trauma mechanism, abdominal injury severity, and shock prior to ICU admission. Figure S4. Plasma levels of Δ receptor interacting protein kinase-3 (ΔRIPK3, change from presentation to 48 h) by acute kidney injury (AKI) and acute respiratory distress syndrome (ARDS) category in human cohorts. Shaded portions of box plots show median concentrations (central line) and 25th and 75th percentiles (bottom and top lines of box). Brackets above the p values denote the two organ dysfunction categories being compared (Wilcoxon rank-sum test). A. MESSI cohort. Patients with AKI only, ARDS only, or AKI + ARDS had higher ΔRIPK3 than those with neither AKI nor ARDS. B. PETROS cohort. Patients with AKI + ARDS had higher ΔRIPK3 than those with AKI or ARDS only, groups which in turn had higher ΔRIPK3 than patients with neither. (ZIP 1021 kb)

补充图S1。针对PETROS（创伤）队列中患者，排除发病后48小时内符合急性呼吸窘迫综合征（Acute Respiratory Distress Syndrome, ARDS）诊断标准的病例，按ARDS状态分析从就诊至48小时时血浆RIPK3浓度的变化（无ARDS组n=143，ARDS组n=18，均为发病后55~140小时达到诊断标准）。灰色箱体代表四分位间距，中线为中位数。

补充图S2。经ZVAD、脂多糖（LPS）或LPS-ZVAD处理的小鼠，其全肺匀浆中混合谱系激酶结构域样蛋白（mixed lineage kinase domain-like protein, MLKL）及磷酸化MLKL（phosphorylated MLKL, pMLKL）水平无显著差异。A. 全肺匀浆免疫印迹结果：无论处理方式如何，MLKL及pMLKL表达水平均无明显差异。B. 全肺匀浆光密度定量分析：各处理组间比较均无统计学显著性。

补充图S3。各队列中，随受体相互作用蛋白激酶3（receptor interacting protein kinase-3, RIPK3）水平变化量（ΔRIPK3，即就诊至48小时的RIPK3变化值）变化的急性肾损伤（acute kidney injury, AKI）校正发病概率。经多变量logistic回归模型构建后，通过事后边际分析得到估计概率（曲线）及95%置信区间（灰色阴影区域）。A. MESSI队列：校正因素包括年龄、就诊当日红细胞输注量、慢性肾病、糖尿病及就诊时休克状态。B. PETROS队列：校正因素包括就诊后前6小时内红细胞输注量、创伤机制、腹部损伤严重程度及ICU入院前休克状态。

补充图S4。在人类队列中，按急性肾损伤（AKI）及急性呼吸窘迫综合征（ARDS）分类分析ΔRIPK3（即就诊至48小时的RIPK3变化值）的血浆水平。箱式图的阴影部分代表中位数浓度（中线）及25%、75%百分位数（箱体上下边线）。p值上方的方括号标注了正在进行比较的两类器官功能障碍组别（Wilcoxon秩和检验）。A. MESSI队列：仅AKI、仅ARDS或AKI+ARDS患者的ΔRIPK3水平均高于既无AKI也无ARDS的患者。B. PETROS队列：AKI+ARDS患者的ΔRIPK3水平高于仅AKI或仅ARDS患者，而后两组的ΔRIPK3水平又高于既无AKI也无ARDS的患者。（附件ZIP 1021 kb）