TIMP2 protects against sepsis-associated acute kidney injury by cAMP/NLRP3 axis-mediated pyroptosis.

The tissue inhibitor of metalloproteinases 2 (TIMP2) has emerged as a promising biomarker for predicting the risk of sepsis-associated acute kidney injury (SA-AKI). However, its exact role in SA-AKI and the underlying mechanism remains unclear. In this study, we investigated the impact of kidney tubule-specific Timp2 knockout mice on kidney injury and inflammation. Our findings demonstrated that Timp2-knockout mice exhibited more severe kidney injury than wild-type mice, along with elevated levels of pyroptosis markers NOD-like receptor protein 3 (NLRP3), Caspase1, and gasdermin D (GSDMD) in the early stage of SA-AKI. Conversely, the expression of exogenous TIMP2 in TIMP2-knockout mice still protected against kidney damage and inflammation. In in vitro experiments, using recombinant TIMP2 protein, TIMP2 knockdown demonstrated that exogenous TIMP2 inhibited pyroptosis of renal tubular cells stimulated by lipopolysaccharide (LPS). Mechanistically, TIMP2 promoted the ubiquitination and autophagy-dependent degradation of NLRP3 by increasing intracellular cyclic adenosine monophosphate (cAMP), which mediated NLRP3 degradation through recruiting the E3 ligase MARCH7, attenuating downstream pyroptosis, and thus alleviating primary tubular cell damage. These results revealed the renoprotective role of extracellular TIMP2 in SA-AKI by attenuating tubular pyroptosis, and suggested that exogenous administration of TIMP2 could be a promising therapeutic intervention for SA-AKI treatment.NEW & NOTEWORTHY Tissue inhibitor of metalloproteinase 2 (TIMP-2) has been found to be the best biomarker for predicting the risk of sepsis-associated acute kidney injury (SA-AKI). However, its role and the underlying mechanism in SA-AKI remain elusive. The authors demonstrated in this study using kidney tubule-specific knockout mice model of SA-AKI and primary renal tubule cells stimulated with lipopolysaccharide (LPS) that extracellular TIMP-2 promoted NOD-like receptor protein 3 (NLRP3) ubiquitination and autophagy-dependent degradation by increasing intracellular cyclic adenosine monophosphate (cAMP), thus attenuated pyroptosis and alleviated renal damage.

SAP130 released by ferroptosis tubular epithelial cells promotes macrophage polarization via Mincle signaling in sepsis acute kidney injury.

The pathological mechanism of sepsis-associated acute kidney injury (SA-AKI) is complex and involves tubular epithelial cell (TEC) death and immune cell activation. However, the interaction between tubular cell death and macrophage-mediated inflammation remains unclear. In this study, we uncovered that TEC ferroptosis was activated in SA-AKI. Increased levels of ferroptotic markers, including ferroptosis-related proteins, lipid peroxidation, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), reactive oxygen species (ROS), and mitochondrial damage, were observed in the kidney tissue of cecum ligation and puncture (CLP) and Lipopolysaccharide (LPS)-induced SA-AKI mouse models, which were subsequently suppressed by Ferrostatin-1 (Fer-1). In vitro experiments showed that Fer-1 inhibits LPS-induced mitochondrial damage, Fe2+ accumulation, and cytosolic ROS production. Moreover, it was found that TEC ferroptosis induced by promoted macrophage-inducible C-type lectin (Mincle) and its downstream expression and M1 polarization, which was mediated by the release of spliceosome-associated protein 130 (SAP130), an endogenous ligand of Mincle, from TEC. It was confirmed in vitro that the supernatant from LPS-stimulated TECs promoted Mincle expression and M1 polarization in macrophages. Further experiments revealed that M1 macrophages aggravated TEC ferroptosis, which was offset by neutralizing SAP130 or inhibiting Mincle expression. In addition, neutralizing the circulatory SAP130 blunted kidney ferroptosis and Mincle expression, as well as macrophage infiltration in the kidney of SA-AKI mice. In conclusion, the release of SAP130 from ferroptotic TECs promoted M1 macrophage polarization by triggering Mincle/syk/NF-κB signaling, and M1 macrophages, in turn, aggravated TEC ferroptosis.

[Transcriptome changes upon Toll-like receptor 9 pathway activation in primary renal tubular epithelial cells].

OBJECTIVE: To explore the effect of Toll-like receptor 9 (TLR9) signaling pathway activation on the transcriptome in the renal tubular cells. METHODS: Mouse primary renal tubular epithelial cells were extracted and cultured. When the degree of cell fusion reached 80%, they were divided into two groups, which were added with 10 µL phosphate buffered saline (PBS, PBS control group) and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide (CpG-ODN) with a final concentration of 5 µmol/L (CpG-ODN treatment group). The RNA sequencing was performed on the Illumina platform after extraction. DEGseq software was used to analyze the differential expression of genes between the two groups. Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways. Homer software was used to predict transcription factors. RESULTS: Compared with the PBS control group, there were a total of 584 differentially expressed genes in the CpG-ODN treatment group, of which 102 were up-regulated and 482 were down-regulated. The most significantly enriched gene ontology (GO) terms of differentially expressed genes included response to interferon-ß, defense response to virus and other inflammatory pathway. The most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways included 2'-5'-oligoadenylate synthase activity, regulation of ribonuclease activity, negative regulation of virus life cycle, cellular response to interferon-ßand defense response to protozoan. The results of transcription factor prediction showed that interferon regulatory factor 3 (IRF3) was the most significantly enriched transcription factor in the promoter sequence of differential genes; the most significant transcription factor downstream of TLR9 was IRF3, and other predicted transcription factors such as transcription factor 21 (TCF21), zinc finger protein 135 (ZNF135), and PR domain containing 4 (PRDM4) might be new candidates for TLR9 signaling pathway. CONCLUSIONS: CpG-ODN activates TLR9 signaling pathway, and primary renal tubular epithelial cells can directly respond to CpG-ODN stimulation and undergo transcriptome changes, which provides a basis for further research on the molecular mechanism of TLR9 pathway in sepsis induced acute kidney injury.