Supplementary Material for: Candida Administration Worsens Neutrophil Extracellular Traps in Renal Ischemia Reperfusion Injury Mice: An Impact of Gut Fungi on Acute Kidney Injury

Because of gut-barrier defect (gut-leakage) after acute kidney injury (AKI) and higher abundance of Candida albicans in human intestines compared with mouse guts, Candida administration in renal ischemia reperfusion injury (I/R) mice possibly more closely resemble patients with AKI than non-Candida model. Fungi in feces were detectable only in mice with Candida administration. Candida renal-I/R mice, when compared with non-Candida I/R, demonstrated more profound injuries, including (i) gut-leakage; FITC-dextran assay and serum (1→3)-β-D-glucan (BG), (ii) systemic inflammation (serum cytokines), and (iii) neutrophil extracellular traps (NETs); gene expression of peptidyl arginase 4 (PAD4) and IL-1β, nuclear morphology staining by 4′,6-diamidino-2-phenylindole (DAPI) and co-staining of myeloperoxidase (MPO) with neutrophil elastase (NE) in peripheral blood neutrophils. Although renal excretory function (serum creatinine) and renal histology score were nondifferent between renal-I/R mice with and without Candida, prominent renal NETs (PAD4 and IL-1β expression with MPO and NE co-staining) was demonstrated in Candida renal-I/R mice. Additionally, neutrophil activation by lipopolysaccharide (LPS) plus BG (LPS + BG), when compared with LPS alone, caused (i) NETs formation; dsDNA, DAPI-stained nuclear morphology and MPO with NE co-staining, (ii) inflammatory responses; Spleen tyrosine kinase (Syk) and NFκB expression, and (iii) reduced cell energy status (maximal respiratory capacity using extracellular flux analysis). Also, LPS + BG-activated NETs formation was inhibited by a dectin-1 inhibitor, supporting an impact of BG signaling. In conclusion, Candida-renal I/R demonstrated more prominent serum BG and LPS from gut translocation that increased systemic inflammation and NETs through TLR-4 and dectin-1 activation. The influence of gut fungi in AKI should be concerned.

由于急性肾损伤（acute kidney injury, AKI）后可出现肠屏障缺陷（gut-barrier defect，即肠漏gut-leakage），且人类肠道内白色念珠菌（Candida albicans）的丰度高于小鼠肠道，因此在肾缺血再灌注损伤（renal ischemia reperfusion injury, I/R）小鼠模型中实施念珠菌定植，或许比非念珠菌模型更贴近AKI患者的真实病理状态。仅在经念珠菌定植的小鼠粪便中可检测到真菌。与非念珠菌I/R模型小鼠相比，念珠菌肾I/R模型小鼠表现出更严重的损伤，包括：(i) 肠漏：通过异硫氰酸荧光素-葡聚糖（FITC-dextran）实验与血清(1→3)-β-D-葡聚糖（(1→3)-β-D-glucan, BG）检测；(ii) 全身炎症反应：血清细胞因子水平变化；(iii) 中性粒细胞胞外陷阱（neutrophil extracellular traps, NETs）形成：包括肽基精氨酸酶4（peptidyl arginase 4, PAD4）与IL-1β的基因表达，外周血中性粒细胞的4',6-二脒基-2-苯基吲哚（4′,6-diamidino-2-phenylindole, DAPI）核形态染色，以及髓过氧化物酶（myeloperoxidase, MPO）与中性粒细胞弹性蛋白酶（neutrophil elastase, NE）的共染色。尽管有无念珠菌定植的肾I/R小鼠的肾脏排泄功能（血清肌酐水平）与肾脏组织学评分无显著差异，但念珠菌肾I/R模型小鼠可观察到显著的肾脏NETs形成：表现为PAD4与IL-1β表达上调，且存在MPO与NE的共染色。此外，与单独使用脂多糖（lipopolysaccharide, LPS）处理相比，脂多糖联合(1→3)-β-D-葡聚糖（LPS + BG）激活的中性粒细胞可引发：(i) NETs形成：包括双链DNA（dsDNA）检测、DAPI染色核形态观察以及MPO与NE的共染色；(ii) 炎症反应：脾酪氨酸激酶（Spleen tyrosine kinase, Syk）与核因子κB（NFκB）的表达变化；(iii) 细胞能量状态降低：通过细胞外流量分析检测的最大呼吸容量下降。同时，dectin-1抑制剂可抑制LPS + BG激活的NETs形成，这证实了BG信号通路的调控作用。综上，念珠菌定植的肾I/R模型小鼠可出现更高水平的血清BG与肠道易位产生的LPS，通过激活Toll样受体4（TLR-4）与dectin-1通路加重全身炎症反应与NETs形成。肠道真菌对急性肾损伤的影响值得关注。