The gut-lung axis in critical illness: microbiome composition as a predictor of mortality at day 28 in mechanically ventilated patients.

BACKGROUND: Microbial communities are of critical importance in the human host. The lung and gut microbial communities represent the most essential microbiota within the human body, collectively referred to as the gut-lung axis. However, the differentiation between these communities and their influence on clinical outcomes in critically ill patients remains uncertain. METHODS: An observational cohort study was obtained in the intensive care unit (ICU) of an affiliated university hospital. Sequential samples were procured from two distinct anatomical sites, namely the respiratory and intestinal tracts, at two precisely defined time intervals: within 48 h and on day 7 following intubation. Subsequently, these samples underwent a comprehensive analysis to characterize microbial communities using 16S ribosomal RNA (rRNA) gene sequencing and to quantify concentrations of fecal short-chain fatty acids (SCFAs). The primary predictors in this investigation included lung and gut microbial diversity, along with indicator species. The primary outcome of interest was the survival status at 28 days following mechanical ventilation. RESULTS: Sixty-two mechanically ventilated critically ill patients were included in this study. Compared to the survivors, the diversity of microorganisms was significantly lower in the deceased, with a significant contribution from the gut-originated fraction of lung microorganisms. Lower concentrations of fecal SCFAs were detected in the deceased. Multivariate Cox regression analysis revealed that not only lung microbial diversity but also the abundance of Enterococcaceae from the gut were correlated with day 28 mortality. CONCLUSION: Critically ill patients exhibited lung and gut microbial dysbiosis after mechanical ventilation, as evidenced by a significant decrease in lung microbial diversity and the proliferation of Enterococcaceae in the gut. Levels of fecal SCFAs in the deceased served as a marker of imbalance between commensal and pathogenic flora in the gut. These findings emphasize the clinical significance of microbial profiling in predicting the prognosis of ICU patients.

Lipopolysaccharide Mediates the Destruction of Intercellular Tight Junction among Renal Tubular Epithelial Cells via RhoT1/SMAD-4/JAM-3 Pathway.

Background: The morbidity of sepsis induced acute kidney injury remains unacceptable high and the mechanisms of that disease remains unclear. For urine backleak and intercellular tight junction among tubular epithelial cells (TECs) destruction often occur during sepsis induced acute kidney injury, we examined whether lipopolysaccharide could damage intercellular tight junction among TECs and associated mechanisms in our present study. Methods: HK-2 cells were cultured, transfected with different SiRNAs and stimulated with LPS and PYR-41. Transepithelial Permeability Assay and Transepithelial Electrical Resistance Assay were used to evaluate intercellular tight junction destruction and Western Blot and Immunofluorescence were used to evaluate proteins expression. Results: Transepithelial Permeability increased significantly (P<0.05) and Transepithelial Electrical Resistance reduced remarkably (P<0.05) of the monolayer TECs stimulated with LPS. The expression of JAM-3 and RhoT1 decreased significantly (P<0.05) in TECs stimulated with LPS, while the level of SMAD-4 increased significantly (P<0.05). Downregulation of the expression of SMAD-4 with RNA interference could increase the expression of JAM-3 in LPS treated TECs. Moreover, upregulation of RhoT1 level by decreased the degradation of RhoT1 could decrease the expression of SMAD-4 and increase the JAM-3 level in TECs treated with LPS, while downregulation of RhoT1 level with RNA interference had the opposite effects. Conclusion: LPS mediates intercellular tight junction destruction among TECs and RhoT1/SMAD-4/JAM-3 is a pivotal pathway to mediate the phenomenon.

AGEs induce high expression of Dll4 via endoplasmic reticulum stress PERK signaling-mediated internal ribosomal entry site mechanism in macrophages.

Background and aim: Advanced glycation end products (AGEs)- exposed macrophages was characterized by Delta-like ligand 4 (Dll4) high expressed and has been shown to participate in diabetes-related atherosclerosis. This study was aimed to investigate the translational regulatory mechanism of Dll4 high expression in macrophages exposed to AGEs. Methods: Human Dll4 5' untranslated region (5'UTR) sequence was cloned and inserted into a bicistronic reporter plasmid. Human THP-1 macrophages transfected with the bicistronic reporter plasmids were exposed to AGEs. Dual-luciferase assay was used to detect internal ribosome entry site (IRES) activity contained in Dll4 5'UTR. Small interference RNA transfection was used to knock-down specific gene expression. Localization of protein was analyzed. Results: AGEs exposure significantly induced IRES activity in Dll4 5' UTR in human macrophages. Internal potential promoter and ribosome read-through mechanisms were excluded. Inhibition of endoplasmic reticulum stress and specific silencing of protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) signaling pathway activation reduced IRES activity in Dll4 5' UTR in human macrophages. Dll4 5' UTR IRES activity was also inhibited by targeted silencing of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Moreover, specific inhibition of PERK/eIF2α signaling pathway led to deactivation of hnRNPA1, resulting to reduction of AGEs- induced Dll4 5' UTR IRES activity in human macrophages. Conclusions: AGEs induced Dll4 5' UTR IRES activity in human macrophages which was dependent on endoplasmic reticulum stress PERK/eIF2α signaling pathway. hnRNPA1 acted the role as an ITAF was also indispensable for AGEs-induced Dll4 5'UTR IRES activity in human macrophages.

Nrp-1 Mediated Plasmatic Ago2 Binding miR-21a-3p Internalization: A Novel Mechanism for miR-21a-3p Accumulation in Renal Tubular Epithelial Cells during Sepsis.

The mechanism underlying sepsis-associated acute kidney injury (SAKI), which is an independent risk factor for sepsis-associated death, is unclear. A previous study indicates that during sepsis miR-21a-3p accumulates in renal tubular epithelial cells (TECs) as the mediator of inflammation and mediates TEC malfunction by manipulating its metabolism. However, the specific mechanism responsible for the accumulation of miR-21a-3p in TECs during sepsis is unrevealed. In this study, a cecal ligation and puncture- (CLP-) induced sepsis rat model and rat TEC line were used to elucidate the mechanism. Firstly, miR-21a-3p and Ago2 levels were found out to increase in both plasma and TECs during sepsis, and the increase of intracellular Ago2 and miR-21a-3p could be mitigated when Ago2 was either inactivated or downregulated in septic plasma. Moreover, membrane Nrp-1 expression of TECs was increased significantly during sepsis and Nrp-1 knockdown also mitigated the rise of both the intracellular Ago2 and miR-21a-3p levels in TECs incubated with septic plasma. Furthermore, it was revealed that Ago2 can be internalized by TECs mediated with Nrp-1 and this process had no effect on the intracellular content of miR-21a-3p. Both Ago2 and miR-21a-3p could bind to TECs derived Nrp-1 directly. Finally, it was determined that miR-21a-3p was internalized by TECs via Nrp-1 and Ago2 facilitated this process. Taken together, it can be concluded from our results that Ago2 binding miR-21a-3p from septic plasma can be actively internalized by TECs via Nrp-1 mediated cell internalization, and this mechanism is crucial for the rise of intracellular miR-21a-3p content of TECs during sepsis. These findings will improve our understanding of the mechanisms underlying SAKI and aid in developing novel therapeutic strategies.

MiR-21-3p Plays a Crucial Role in Metabolism Alteration of Renal Tubular Epithelial Cells during Sepsis Associated Acute Kidney Injury via AKT/CDK2-FOXO1 Pathway.

OBJECTIVE: Sepsis and associated acute kidney injury (SAKI) are determined to be closely related to poor prognosis. Because the metabolic alterations of tubular epithelial cells (TECs) are crucial for the occurrence and development of SAKI, we carried out this present study to identify the metabolism changes of TECs during SAKI and relevant mechanisms. METHODS: Rat SAKI model and rat tubular epithelial cell line were used in our study. ELISA was used to determine the serum cytokines levels. Protein expressions were examined with Western-Blotting and the transcriptions of RNAs were determined with qRT-PCR. ADP/ATP assay and Oil Red O staining were used to examine the energy and lipid metabolism, respectively. Dual-luciferase reporter assay was carried out to determine the interactions between miRNA and specific proteins. Cell cycle arrest and apoptosis were determined with flow cytometry. RESULTS: Sepsis and AKI were induced 12 h after CLP. Energy and lipid metabolism reduced significantly while FOXO1 levels increased remarkably in TECs during SAKI. The expressions of both AKT and CDK2 and the transcriptions of relevant mRNAs reduced significantly in TECs during SAKI while miR-21-3p expression increased remarkably. Both AKT and CDK2 were determined as the direct targets of miR-21-3p. Furthermore, by in vitro experiments, it was demonstrated that FOXO1 levels were regulated by miR-21-3p in TECs via AKT/CDK2 and AKT/CDK2-FOXO1 pathway was crucial in the regulations of miR-21-3p on lipid metabolism, cell cycle arrest, and apoptosis of TECs. CONCLUSIONS: MiR-21-3p mediates metabolism and cell fate alterations of TECs via manipulating AKT/CDK2-FOXO1 pathway, and that is crucial in the regulation of energy metabolism of TECs during SAKI.

IQGAP1 mediates podocyte injury in diabetic kidney disease by regulating nephrin endocytosis.

Diabetic kidney disease (DKD) is a complication associated with diabetes and is a major public health problem in modern society. Podocyte injury is the central target of the development of DKD, and the loss or dysregulation of nephrin, a key structural and signalling molecule located in the podocyte slit diaphragm (SD), initiates potentially catastrophic downstream events within podocytes. IQGAP1, a scaffold protein containing multiple protein-binding domains that regulates endocytosis, can interact with nephrin in podocytes. It is hypothesized that IQGAP1 contributes to nephrin endocytosis and may participate in the pathogenesis of DKD. The dramatically increased histo-nephrin granularity score in DKD glomeruli showed a significant positive correlation with increased IQGAP1-nephrin interaction without changes in the total protein content of nephrin and IQGAP1. In cultured human podocytes, hyperglycaemia induced the intracellular translocation of IQGAP1 from the cytosol to the vicinity of the cytomembrane, reinforced the IQGAP1-nephrin interaction, and augmented nephrin endocytosis. Moreover, impaired podocyte function, such as migration, extensibility and permeability, were further aggravated by wild-type IQGAP1 plasmid transfection, and these effects were partially restored by siRNA-mediated IQGAP1 downregulation. Collectively, these findings show that IQGAP1, an intracellular partner of nephrin, is involved in nephrin endocytosis and the functional regulation of podocytes in DKD.

Novel ASK1 inhibitor AGI-1067 improves AGE-induced cardiac dysfunction by inhibiting MKKs/p38 MAPK and NF-κB apoptotic signaling.

Heart failure has been identified as one of the clinical manifestations of diabetic cardiovascular complications. Excessive myocardium apoptosis characterizes cardiac dysfunctions, which are correlated with an increased level of advanced glycation end products (AGEs). In this study, we investigated the participation of reactive oxygen species (ROS) and the involvements of apoptosis signal-regulating kinase 1 (ASK1)/mitogen-activated protein kinase (MAPK) kinases (MKKs)/p38 MAPK and nuclear factor κB (NF-κB) pathways in AGE-induced apoptosis-mediated cardiac dysfunctions. The antioxidant and therapeutic effects of a novel ASK1 inhibitor, AGI-1067, were also studied. Myocardium and isolated primary myocytes were exposed to AGEs and treated with AGI-1067. Invasive hemodynamic and echocardiographic assessments were used to evaluate the cardiac functions. ROS formation was evaluated by dihydroethidium fluorescence staining. A terminal deoxynucleotidyl transferase dUTP nick end labelling assay was used to detect the apoptotic cells. ASK1 and NADPH activities were determined by kinase assays. The association between ASK1 and thioredoxin 1 (Trx1) was assessed by immunoprecipitation. Western blotting was used to evaluate the phosphorylation and expression levels of proteins. Our results showed that AGE exposure significantly activated ASK1/MKKs/p38 MAPK, which led to increased cardiac apoptosis and cardiac impairments. AGI-1067 administration inhibited the activation of MKKs/p38 MAPK by inhibiting the disassociation of ASK1 and Trx1, which suppressed the AGE-induced myocyte apoptosis. Moreover, the NF-κB activation as well as the ROS generation was inhibited. As a result, cardiac functions were improved. Our findings suggested that AGI-1067 recovered AGE-induced cardiac dysfunction by blocking both ASK1/MKKs/p38 and NF-κB apoptotic signaling pathways.

Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

The aim of the present study was to investigate the increase in serum and urine levels of high mobility group box protein 1 (HMGB1) during sepsis and the effect of blood purification treatments on HMGB1 levels and patient prognosis. A total of 40 intensive care patients with sepsis were randomly assigned to different groups (n=10 per group): A control group (sepsis group), a continuous renal replacement treatment (CRRT) group, a hemoperfusion (HP) group and an HP+CRRT group. The blood purification treatments using HP and/or CRRT were performed immediately after the diagnosis of sepsis. HMGB1 levels were measured using ELISA, and Acute Physiology and Chronic Health Evaluation (APACHE) II scores and 30-day survival rates were evaluated. Relative to a healthy control group (n=10), HMGB1 levels were observed to be significantly upregulated during sepsis (P<0.05). Following the initiation of sepsis, serum HMGB1 continued to increase in the sepsis group and was significantly elevated at 24 h (P<0.05), whereas urine HMGB1 levels decreased significantly at 12 and 24 h (P<0.05). Serum HMGB1 levels were significantly positively correlated with APACHE II scores (r=0.7284, P<0.01) and significantly negatively correlated with urine HMGB1 levels (r=-0.5103, P=0.026). Serum HMGB1 levels were significantly reduced in the HP and HP+CRRT groups by 12 and 24 h following the initiation of treatment (both P<0.05). Changes in the urine HMGB1 level differed in each group. Relative to the sepsis group, the APACHE II scores of all blood purification groups were significantly reduced (P<0.05) and the 30-day survival rate of the HP+CRRT group was significantly increased (P=0.0107). The results of the present study indicate that blood purification initiated at the point of diagnosis in patients with sepsis may attenuate serum HMGB1 upregulation, promote urinary excretion of HMGB1 and improve the prognosis of sepsis.

HMGB1 Turns Renal Tubular Epithelial Cells into Inflammatory Promoters by Interacting with TLR4 During Sepsis.

Our study was undertaken to investigate whether the inflammatory mediator high-mobility group box 1 (HMGB1) can enter the renal tissue and urine and what is the functional change of renal tubular epithelial cells (TECs) interacting with HMGB1 during sepsis. We found that the transcription levels of interleukin 1 (IL-1) and interleukin 6 (IL-6) mRNA in TECs increased significantly during sepsis and these processes can be blocked by splenectomy. We also found out HMGB1 accumulated in the renal tissue and entered urine during sepsis and toll-like receptor 4 (TLR4) was expressed by TECs. In vitro, we demonstrated that HMGB1 induced MAPK and NF-κB activation and G1 cell cycle arrest in TECs. We also found that the mRNA transcription levels of IL-1, IL-6, and tissue inhibitor of metalloproteinases 2 (TIMP2) increased significantly and the IL-1, IL-6, and TIMP2 can be secreted by TECs stimulated by HMGB1. In contrast, LPS RS can block all of the processes above in vitro. In vivo, the increase of the mRNA transcription level of TIMP2 was also observed. These data indicate that HMGB1 accumulates in renal tissue and enters the urine and the interaction between HMGB1 and TLR4 turns TECs into inflammatory promoters during sepsis.