Department of Anesthesiology, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
Front Immunol. 2023 Jan 13;13:1053754. doi: 10.3389/fimmu.2022.1053754. eCollection 2022.
Acute kidney injury is a common and severe complication of sepsis. Sepsis -induced acute kidney injury(S-AKI) is an independent risk factor for mortality among sepsis patients. However, the mechanisms of S-AKI are complex and poorly understand. Therefore, exploring the underlying mechanisms of S-AKI may lead to the development of therapeutic targets.
A model of S-AKI was established in male C57BL/6 mice using cecal ligation and puncture (CLP). The data-independent acquisition (DIA)-mass spectrometry-based proteomics was used to explore the protein expression changes and analyze the key proteomics profile in control and CLP group. The methodology was also used to identify the key proteins and pathways. S-AKI was established by treating the HK-2 cells with lipopolysaccharide (LPS). Subsequently, the effect and mechanism of Cathepsin B (CTSB) in inducing apoptosis in HK-2 cells were observed and verified.
The renal injury scores, serum creatinine, blood urea nitrogen, and kidney injury molecule 1 were higher in septic mice than in non-septic mice. The proteomic analysis identified a total of 449 differentially expressed proteins (DEPs). GO and KEGG analysis showed that DEPs were mostly enriched in lysosomal-related cell structures and pathways. CTSB and MAPK were identified as key proteins in S-AKI. Electron microscopy observed enlarged lysosomes, swelled and ruptured mitochondria, and cytoplasmic vacuolization in CLP group. TUNEL staining and CTSB activity test showed that the apoptosis and CTSB activity were higher in CLP group than in control group. In HK-2 cell injury model, the CTSB activity and mRNA expression were increased in LPS-treated cells. Acridine orange staining showed that LPS caused lysosomal membrane permeabilization (LMP). CA074 as an inhibitor of CTSB could effectively inhibit CTSB activity. CCK8 and Annexin V/PI staining results indicated that CA074 reversed LPS-induced apoptosis of HK-2 cells. The JC-1 and western blot results showed that LPS inhibited mitochondrial membrane potential and activated mitochondrial apoptosis pathway, which could be reversed by CA074.
LMP and CTSB contribute to pathogenesis of S-AKI. LPS treatment induced HK-2 cell injury by activating mitochondrial apoptosis pathway. Inhibition of CTSB might be a new therapeutic strategy to alleviate sepsis-induced acute kidney injury.
急性肾损伤是脓毒症的常见且严重的并发症。脓毒症引起的急性肾损伤（S-AKI）是脓毒症患者死亡的独立危险因素。然而，S-AKI 的机制复杂且了解甚少。因此，探索 S-AKI 的潜在机制可能会导致治疗靶点的开发。
采用盲肠结扎穿刺（CLP）法建立雄性 C57BL/6 小鼠 S-AKI 模型。采用数据非依赖性采集（DIA）-质谱蛋白质组学技术，对对照组和 CLP 组的蛋白表达变化进行分析，寻找关键蛋白质组学谱。该方法还用于鉴定关键蛋白和途径。用脂多糖（LPS）处理 HK-2 细胞建立 S-AKI 模型。随后观察并验证组织蛋白酶 B（CTSB）在诱导 HK-2 细胞凋亡中的作用和机制。
与非脓毒症小鼠相比，脓毒症小鼠的肾脏损伤评分、血清肌酐、血尿素氮和肾损伤分子 1 水平更高。蛋白质组学分析共鉴定出 449 个差异表达蛋白（DEPs）。GO 和 KEGG 分析显示，DEPs 主要富集在溶酶体相关细胞结构和途径中。CTSB 和 MAPK 被鉴定为 S-AKI 的关键蛋白。电子显微镜观察到 CLP 组溶酶体增大、线粒体肿胀破裂和细胞质空泡化。TUNEL 染色和 CTSB 活性检测显示 CLP 组细胞凋亡和 CTSB 活性均高于对照组。在 LPS 处理的 HK-2 细胞损伤模型中，LPS 处理组的 CTSB 活性和 mRNA 表达增加。吖啶橙染色显示 LPS 引起溶酶体膜通透性（LMP）。CTSB 抑制剂 CA074 可有效抑制 CTSB 活性。CCK8 和 Annexin V/PI 染色结果表明 CA074 逆转了 LPS 诱导的 HK-2 细胞凋亡。JC-1 和 Western blot 结果表明 LPS 抑制了线粒体膜电位并激活了线粒体凋亡途径，CA074 可逆转这一作用。
LMP 和 CTSB 参与 S-AKI 的发病机制。LPS 通过激活线粒体凋亡途径诱导 HK-2 细胞损伤。抑制 CTSB 可能是缓解脓毒症引起的急性肾损伤的一种新的治疗策略。
