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USF2 Knockdown Downregulates THBS1 to Inhibit the TGF-Β Signaling Pathway and Reduce Pyroptosis in Sepsis-Induced Acute Kidney Injury

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USF2 Knockdown Downregulates THBS1 to Inhibit the TGF-Β Signaling Pathway and Reduce Pyroptosis in Sepsis-Induced Acute Kidney Injury USF2 Knockdown Downregulates THBS1 to Inhibit the TGF-β Signaling Pathway and Reduce Pyroptosis in Sepsis-Induced Acute Kidney Injury Jian Sun Shanghai Jiaotong University School of Medicine Xinhua Hospital Yang Wang Shanghai Jiaotong University School of Medicine Xinhua Hospital Lei Niu Shanghai Jiaotong University School of Medicine Xinhua Hospital Lujia Tang Shanghai Jiaotong University School of Medicine Xinhua Hospital Shuming Pan ( [email protected] ) Shanghai Jiaotong University School of Medicine Xinhua Hospital https://orcid.org/0000-0003-2518- 0290 Research Keywords: USF2, THBS1, TGF-β, Pyroptosis, Sepsis-induced acute kidney injury Posted Date: December 16th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-126763/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/33 Abstract Background Acute kidney injury (AKI) is a serious complication of sepsis. This study was performed to explore the mechanism that THBS1 mediated pyroptosis by regulating TGF-β signaling pathway in sepsis-induced AKI. Methods Gene expression microarray related to sepsis-induced AKI was obtained from the GEO database, and the mechanism in sepsis-induced AKI was predicted by bioinformatics analysis. qRT-PCR and ELISA were performed to detect expressions of THBS1, TNF-α, IL-1β, and IL-18 in sepsis-induced AKI patients and healthy volunteers. Mouse model of sepsis-induced AKI was established, with serum creatinine, urea nitrogen, 24-h urine output measured, and renal tissue lesions observed by HE staining. Cell model of sepsis-induced AKI was cultured in vitro, with expressions of TNF-α, IL-1β, and IL-18, pyroptosis, Caspase- 1 and GSDMD-N, and activation of TGF-β pathway detected. The upstream transcription factor USF2 was knocked down in cells to explore it effect on sepsis-induced AKI. Results THBS1 was highly expressed in patients with sepsis-induced AKI. Silencing THBS1 protected mice against sepsis-induced AKI, and signicantly decreased the expressions of NLRP3, Caspase-1, GSDMD-N, IL-1β, and IL-18, increased cell viability, and decreased LDH activity, partially reversing changes in cell morphology. Mechanistically, USF2 promoted oxidative stress response by transcriptionally activating THBS1 to activate TGF-β/NLRP3/Caspase-1 signaling pathway and stimulate pyroptosis, and nally exacerbated sepsis-induced AKI. Conclusions USF2 knockdown downregulates THBS1 to inhibit TGF-β signaling pathway and reduce pyroptosis and further to ameliorate sepsis-induced AKI. Background As a common complication of critically ill patient, sepsis-induced acute kidney injury (sepsis-induced AKI) is related to high risk of chronic complication, drastic physiologic changes and high mortality [1, 2]. About 35% of sepsis patients develop renal failure, and nearly half of critically ill patients with AKI also developed sepsis [1, 3]. In the past, sepsis-induced AKI was considered to be caused by renal hypoperfusion, but studies have found inammatory response and apoptosis may also be involved in the pathogenesis of sepsis-induced AKI [1, 4]. Oxidative stress has been reported to be related to sepsis and may contribute to AKI by generating reactive oxygen species (ROS) [5]. Existing evidence also reports that pyroptosis of renal tubular epithelial cells induced by Caspase-11 is a crucial event in the development of Page 2/33 sepsis-induced AKI [3]. Current treatment of sepsis-induced AKI mainly focuses on renal replacement therapy, uid resuscitation, vasoactive drugs and antibiotics [5]. Since the pathogenesis of sepsis-induced AKI involves multiple factors such as apoptosis, immune and inammatory processes, molecular mechanism targeting signaling pathways may have potential therapeutic value for sepsis-induced AKI. Thrombospondin1 (THBS1), also called TSP1, is a homotrimeric glycoprotein released by activated platelets and involved in brin clots reacting to injury [6]. Previous study demonstrates that THBS1 bound to CD47 to promote AKI and blocking of THBS1 signaling through CD47 improved renal interstitial brosis [7]. Pyroptosis, a new programmed cell death mode discovered in recent years, is characterized by dependence on cysteine-aspartic proteases 1 (caspase-1), NLRP3 inammasome activation and the release of a large number of proinammatory factors [8]. In human cells, TSP1 is an upstream inhibitor of IL-1β and caspase-1 mRNA, but not NLRP3 induced in the presence of LPS [9]. The involvement of THBS1 binding to the CD36 receptor in apoptosis has been reported in endothelial cells [10]. However, no study concerns the correlation between THBS1 and pyroptosis at present. Previous study reports that activation of TGF-β can be attenuated by suppressing THBS1 with the LSKL peptide [6]. TGF-β can induce the activation of NLRP3 inammasome and the cleavage of Gasdermin D (GSDMD) in rat with chronic kidney disease [11]. Those evidences support the association among THBS1, inammatory response, pyroptosis and AKI. We further searched the upstream transcriptional regulators of THBS1 in the TTRUST database, including E2F1, NR1I2, SNAI1, TP53, USF2 and WT1. Overexpression of USF2 is found in previous study to result in renal injury [12]. Hence, in this study, we aim to explore the mechanism that transcriptional regulator USF2 regulates THBS1 and pyroptosis via the TGF-β signaling pathway in sepsis-induced AKI. Methods Ethics statement This study was performed with the approval of the Clinical Ethical Committee of Xinhua Hospital Aliated to Shanghai Jiao Tong University School of Medicine. Written informed consents were obtained from all patients and healthy volunteers. The animal experiments were conducted based on minimized animal number and the least pains. Bioinformatics analysis GSE60088 chip (loading in GPL1261 platform le) related to sepsis-induced AKI was obtained from GEO database (https://www.ncbi.nlm.nih.gov/geo/), including 5 renal tissue control samples (control) and 5 sepsis-induced AKI samples (disease). Differential expression analysis of genes was performed by R language “limma” package (http://www.bioconductor.org/packages/release/bioc/html/limma.html), with |log2FC| > 1 and P < 0.05 as the screening criteria. Related genes were obtained by “searching sepsis- induced kidney injury” in GeneCards database (https://www.genecards.org/). Intersection between differentially expressed genes and the genes related to sepsis-induced AKI was performed by jvenn Page 3/33 (http://jvenn.toulouse.inra.fr/app/example.html). The heatmap of candidate gene expressions was plotted by using R language “pheatmap” package (https://cran.r- project.org/web/packages/pheatmap/index.html). Enrichment analysis on candidate genes was performed by WebGestalt (http://www.webgestalt.org/). Upstream transcriptional regulators of genes were found by TTRUST database (https://www.grnpedia.org/trrust/). Clinical samples Fifteen patients with sepsis-induced AKI were enrolled in this study. Inclusion criteria were: aged 18 ~ 80 years, male or female, and the diagnosis of all patients was in accordance with the International Sepsis Guidelines Diagnostic Criteria [13] and Acute Kidney Injury Diagnostic Criteria [14]. Exclusion criteria were: chronic kidney disease or kidney transplantation or AKI caused by non-sepsis, or the presence of autoimmune diseases. Fifteen healthy volunteers (conrmed absence of renal impairment, autoimmune diseases, or malignant tumors) were also enrolled in this study. Peripheral blood was collected from all subjects, and the samples were centrifuged within 2 h and stored at -80 °C. Establishment of animal model A total of 120 C57BL/6J mice (aged 6 ~ 8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China with animal license number (SYXK (Jing) 2016-0011). Mice were housed in separate cages at 20–26 °C, with free access to food and water and a 12-hlight/dark cycle. After adaptive feeding for 6 days, mice were assigned into 5 groups randomly, with 24 mice in each group: sham group, AKI group, AKI + sh-NC group, AKI + sh-THBS1 group and AKI + sh-USF2 group. Mice in all groups (except the sham group) were injected intraperitoneally with lipopolysaccharide (LPS) 10 mg/kg. The mice in AKI + sh-THBS1 group and AKI + sh-NC group were injected with adenovirus low expression vector sh-THBS1 and its control adenovirus in the tail vein, respectively, and the mice in AKI + sh-USF2 group and AKI + sh-NC group were injected with adenovirus low expression vector sh-USF2 and its control adenovirus in the tail vein, respectively, with a dose of 0.1 mL. Adenovirus and control vectors were synthesized and packaged by Hanheng Biological Company (Shanghai, China), and the adenovirus virus was 1010/mL. After injection of lipopolysaccharide or phosphate buffered saline (PBS) for 24 h, 6 mice in each group were placed in sealed metabolic cages and fasted. The 24-h natural urine output of mice was collected and accurately weighed by an electronic balance. The remaining mice in each group were sacriced after injection of LPS or PBS for 48 h, and serum samples were separated. Contents of serum creatinine and urea nitrogen were measured using an Automated Chemistry Analyzer (Shenzhen Rayto Life Science Co., Ltd, Shenzhen, Guangdong, China). Hematoxylin-eosin (HE) staining Mouse kidney tissues were collected and xed with 4% formaldehyde for 6 h and embedded in paran. Paran-embedded tissue sections were deparanized in xylene and successively soaked in 100%, 95%, 80%, and 75% ethanol, and then washed with distilled water. Then sections were stained with hematoxylin staining solution for 10 min, rinsed with distilled water, differentiated tissues in differentiation solution for 30 s, soaked in distilled water for 15 min, stained with eosin staining solution Page 4/33 for 2 min and then rinsed with distilled water, successively soaked in 95%, 95%, 100%, and 100% ethanol for 1 min. Next, sections were placed in xylene carbonic acid (3:1) for 1 min, soaked in xylene (I) for 1 min and soaked in xylene (II) for 1 min. Finally, sections were mounted with neutral resin and observed under a microscope (Olympus, Japan) with images randomly collected.
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