Background: 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.Method: 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 in vitro 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.Results: 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.Conclusions: 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.

Original Article Transcriptomic analysis and laboratory experiments reveal potential critical genes and regulatory mechanisms in sepsis-associated acute kidney injury Boyang Liu, Shengxiang Ao, Fang Tan, Wei Ma, Haoru Liu, Huaping Liang, Xia Yang, Xinjin Chi

Background: Macrophages play important roles in diabetes and sepsis-related intestinal injury. Accumulating evidence suggests that microRNAs (miRNAs) act as the fundamental link between macrophage polarization and tissue injury. However, the underlying mechanisms of miRNAs in regulating macrophage polarization–related intestinal injury under diabetes and sepsis conditions remain unclear.Methods: The cecal ligation and puncture (CLP)–induced sepsis models were established in male wild-type (WT) and diabetic mice. Clodronate liposome was used to deplete macrophage. H&E staining, inflammatory cytokines [tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and IL-6], and intestinal mucosal barrier function markers [occludin, ZO-1, lipopolysaccharide (LPS), and intestinal fatty acid binding protein (iFABP)] were used to assess elevated intestinal damage. miRNA array, RNA-seq, and bioinformatic analysis were performed to detect the miRNA and messenger RNA (mRNA) expression and the potential regulation mechanism. In vitro, RAW264.7 cells were cultured in the absence or presence of high glucose and LPS, miR-3061 mimics, and Snail small interfering RNA stimulation, respectively, for further mechanism studies. Luciferase reporter assay was used to confirm the interplay between miRNA and its target genes.Results: Compared with WT CLP mice, the diabetic CLP mice showed severe intestinal damage characterized by significant increases in Chui’s scores, expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6), serum LPS and iFABP concentration, and significant reductions in tight junction protein occludin and ZO-1 levels. Macrophage depletion reversed the intestinal damage caused by CLP. The bioinformatic analysis revealed that miR-3061/Snail1 might be a potential regulation axis of macrophage polarization. Furthermore, high glucose and LPS stimulation increased M1 macrophage and reduced the levels of miR-3061, which was negatively associated with Snail1 in RAW264.7 cells. Mechanistic studies demonstrated that miR-3061 regulated macrophage polarization by targeting the Snail1 mRNA 3′‐untranslated region. Moreover, miR-3061 overexpression suppressed Snail1 expression and inhibited M1 macrophage and inflammatory cytokines.Conclusion: This study elucidated that diabetes exacerbated sepsis-induced intestinal injury by promoting M1 macrophage polarization and further demonstrated that the miR-3061/Sani1 axis may be the potential target of macrophage polarization.

Aberrant expression of G-protein-coupled receptor 158 (GPR158) has been reported to be inextricably linked to a variety of diseases affecting the central nervous system, including Alzheimer’s disease (AD), depression, intraocular pressure, and glioma, but the underlying mechanism remains elusive due to a lack of biological and pharmacological tools to elaborate its preferential cellular distribution and molecular interaction network. To assess the cellular localization, expression, and function of GPR158, we generated an epitope-tagged GPR158 mouse model (GPR158^Tag) that exhibited normal motor, cognitive, and social behavior, no deficiencies in social memory, and no anxiety-like behavior compared to C57BL/6J control mice at P60. Using immunofluorescence, we found that GPR158⁺ cells were distributed in several brain regions including the cerebral cortex, hippocampus, cerebellum, and caudate putamen. Next, using the cerebral cortex of the adult GPR158^Tag mice as a representative region, we found that GPR158 was only expressed in neurons, and not in microglia, oligodendrocytes, or astrocytes. Remarkably, the majority of GPR158 was enriched in Camk2a⁺ neurons whilst limited expression was found in PV⁺ interneurons. Concomitant 3D co-localization analysis revealed that GPR158 was mainly distributed in the postsynaptic membrane, but with a small portion in the presynaptic membrane. Lastly, via mass spectrometry analysis, we identified proteins that may interact with GPR158, and the relevant enrichment pathways were consistent with the immunofluorescence findings. RNA-seq analysis of the cerebral cortex of the GPR158^−/− mice showed that GPR158 and its putative interacting proteins are involved in the chloride channel complex and synaptic vesicle membrane composition. Using these GPR158^Tag mice, we were able to accurately label GPR158 and uncover its fundamental function in synaptic vesicle function and memory. Thus, this model will be a useful tool for subsequent biological, pharmacological, and electrophysiological studies related to GPR158.

Cyclodextrins (CyDs) in nano drug delivery systems have received much attention in pursuit of good compatibility, negligible toxicity, and improved pharmacokinetics of drugs. Their unique internal cavity has widened the application of CyDs in drug delivery based on its advantages. Besides this, the polyhydroxy structure has further extended the functions of CyDs by inter- and intramolecular interactions and chemical modification. Furthermore, the versatile functions of the complex contribute to alteration of the physicochemical characteristics of the drugs, significant therapeutic promise, a stimulus-responsive switch, a self-assembly capability, and fiber formation. This review attempts to list recent interesting strategies regarding CyDs and discusses their roles in nanoplatforms, and may act as a guideline for developing novel nanoplatforms. Future perspectives on the construction of CyD-based nanoplatforms are also discussed at the end of this review, which may provide possible direction for the construction of more rational and cost-effective delivery vehicles.