Blockade of TGF-ß-activated kinase 1 prevents advanced glycation end products-induced inflammatory response in macrophages.

Advanced glycation end products (AGEs), inflammatory-activated macrophages are essential in the initiation and progression of diabetic nephropathy (DN). TGF-ß-activated kinase 1 (TAK1) plays a vital role in innate immune responses and inflammation. However, little information has been available about the effects of AGEs on the regulation of TAK1 expression and underlying mechanisms in AGEs-stimulated macrophage activation. We hypothesized TAK1 signal pathway in AGEs conditions could be a vital factor contributing to macrophage activation and inflammation. Thus, in the present study, we used bone marrow-derived macrophages (BMMs) to explore the functional role and potential mechanisms of TAK1 pathway under AGEs conditions. Results indicated that TAK1 played important roles in AGEs-induced mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B protein (NF-κB) activation, which regulated the production of monocyte chemo-attractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) in AGEs-stimulated macrophages. The results also suggested that TAK1 inhibitor (5Z-7-oxozeaenol) could inhibit AGEs-induced macrophage activation to down-regulate inflammatory cytokine production via MAPKs and NF-κB pathways, indicating that 5Z-7-oxozeaenol might be an immunoregulatory agent against AGEs-stimulated inflammatory response in DN.

High glucose induced-macrophage activation through TGF-ß-activated kinase 1 signaling pathway.

OBJECTIVE AND DESIGN: Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in innate immune responses and kidney disease, and is critically involved in macrophage activation. However, there is a paucity of data to explore the role of high glucose (HG) in the regulation of TAK1 signaling and its functional role in macrophage activation. We assume that TAK1 signaling in hyperglycemic condition could be a key factor leading to macrophage activation and inflammation response. METHODS: Mice macrophages were seeded on a 96-well cell culture plate; cell viability was tested after treatment with different concentration of TAK1 inhibitors. Cells were divided into groups (OZ300; MC; NC; HG; HG + OZ30, 100, 300 nM) and treated for given time course. Monocyte chemotactic protein1(MCP-1) and tumor necrosis factor-α (TNF-α) mRNA levels were evaluated by qRT-PCR. Flow cytometry and confocal microscopy are used to analyse the activated macrophage induced by HG. Expression levels of p-TAK1, TAB 1, p-JNK, p-p38MAPK, NF-κBpp65 were detected by western blot. Nuclear translocation of NF-κBp65 was assessed by confocal microscopy. RESULTS: Our data revealed that high glucose not only significantly increased macrophage activation and subsequently abnormal high-expression of MCP-1 and TNF-α, but likewise remarkably enhanced TAK1 activation, MAPK phosphorylation, NF-κB expression in macrophages. Furthermore, pharmacological inhibition of TAK1 attenuated high glucose-triggered signal pathways, macrophage activation and inflammatory cytokines in a simulated diabetic environment. CONCLUSION: Our findings suggested that high glucose activated macrophages mainly in TAK1/MAPKs and TAK1/NF-κB-dependent manners, which lead to the polarization of macrophages towards a pro-inflammatory phenotype, and finally lead to diabetic nephropathy. In sum, the study raises novel data about the molecular mechanisms involved in the high glucose-mediated inflammatory response in macrophages.

Compositional characteristics of the gut microbiome in patients with uremia.

ABSTRACT: During acute or chronic uremia, the cumulative harmful effects of uremic toxins result in numerous health problems and, ultimately, mortality. Previous research has identified that uremic retention solutes originate from the gut microbiome, indicating that uremia may be closely associated with gut microbiome dysbiosis. To deepen our understanding of the compositional characteristics of the gut microbiome in patients with uremia and thereby promote precision medicine in the treatment of uremia, we conducted a study of the compositional characteristics of the gut microbiome in 20 patients with uremia. The gut microbiome diversity of uremic patients and the control group showed certain differences. Nonmetric multidimensional scaling analysis showed that the beta diversity of the gut microbiome of uremic patients was significantly different from that of the healthy control individuals, with a distinct clustering effect in the uremic patient group, and it also showed a similarly distinct clustering effect in the healthy control group. The Chao1 index and Sobs index were significantly lower in the uremic patient group than in the healthy control group ( P < 0.05). By analyzing the composition and abundance distribution of the gut microbiome in the uremic patient group and healthy control group, we found that the relative abundance of the gut microbiome constituents Fusobacteriota , Enterobacteriaceae, Oscillospirales, Ruminococcaceae, and Lachnospiraceae was significantly increased in the intestines of uremic patients. We also detected the rare taxa Erysipelotrichaceae, which was present only in the uremic patient group. Predictive functional analysis suggested that an increased abundance of Ruminococcaceae and Lachnospirales, which are associated with indoxyl sulfate and phenylacetyl glutamine, and an increased abundance of Oscillospirales, which is associated with pyruvate metabolism, in uremic patients may strongly influence the gut environment according to renal function, resulting in dysbiosis associated with uremic toxin production. Rare taxa such as Erysipelotrichaceae have been suggested to be detrimental to intestinal disease. Further research into these gut microbiomes may provide new ideas for the prevention and treatment of uremia with the gut microbiome.

Immune-metabolic marker of albumin-to-fibrinogen ratio based prognostic nomogram for patients following peritoneal dialysis.

Background: The nutritional status and coagulation function of peritoneal dialysis (PD) patients are closely associated with their prognosis. This study aims to investigate the prognostic value of the albumin-to-fibrinogen ratio (AFR) on mortality in PD patients and to establish a prognostic prediction model based on AFR. Methods: We retrospectively collected data from 148 PD patients treated at our hospital between Oct. 2011 and Dec. 2021. Using the "survminer" package in R, we determined the optimal cutoff value for AFR and divided the patients into low-AFR and high-AFR groups. The primary endpoint of this study was overall survival (OS). Univariate and multivariate Cox analyses were used to assess the impact of AFR and other factors on prognosis, and a corresponding prognostic prediction model was constructed using a nomogram, which was evaluated through ROC curves, the c-index, and calibration plots. Results: The optimal cutoff value for AFR was 9.06. In the entire cohort, 30 patients (20.2%) were classified into the low-AFR group. Compared to the high-AFR group, patients in the low-AFR group were older, had lower total urine output over 24 h, higher blood urea nitrogen, higher total protein and urinary microalbumin levels, and longer remission times (p < 0.05). They also had a poorer OS (HR: 1.824, 95%CI: 1.282-2.594, p < 0.05). Multivariate Cox analysis indicated that AFR was an independent prognostic factor for OS (HR: 1.824, 95% CI: 1.282-2.594, p < 0.05). A prognostic prediction model based on AFR, age, and cause of ESRD was successfully validated for predicting OS in PD patients. Conclusion: AFR represents a potential prognostic biomarker for PD patients. The prognostic prediction model based on AFR can provide accurate OS predictions for PD patients, aiding clinicians in making better-informed decisions.

Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts.

Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA-protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

Monocyte to high-density lipoprotein ratio based prognostic nomogram for patients following allogeneic vascular replacement pancreaticoduodenectomy.

Background: Preoperative immune-inflammatory condition influencing the metabolism of malignancies. We sought to investigate the prognostic value of a novel immune-inflammatory metabolic marker, the monocyte-to-high-density lipoprotein ratio (MHR), in patients with locally advanced pancreatic cancer. Methods: A retrospective analysis was conducted on the clinical data of 118 patients with locally advanced pancreatic cancer and obstructive jaundice who underwent allogeneic vascular replacement pancreaticoduodenectomy in our hospital from Apr. 2011 to Dec. 2023. To assess the predictive capacity of immune-inflammatory metabolic marker, we utilized the area under the receiver operating characteristic curve (AUC-ROC) and assessed the predictive potential of MHR in forecasting outcomes through both univariate and multivariate Cox proportional hazard analyses. Results: The area under AUC for MHR in predicting 1-year postoperative survival was 0.714, with an optimal cutoff value of 1.184, yielding a sensitivity of 78.9% and specificity of 66.2%. Based on this cutoff value, patients were divided into a low MHR group (MHR ≤1.184, n = 61) and a high MHR group (MHR >1.184, n = 57). The median survival times for the low and high MHR groups were 27.0 months and 12.0 months, respectively (χ2 = 30.575, p < 0.001), and the median DFS were 18.0 months and 8.0 months, respectively (χ2 = 26.330, p < 0.001). Univariate and multivariate analyses indicated that preoperative MHR, preoperative creatinine, operation duration, and TNM stage were independent predictors of postoperative mortality, while preoperative MHR, preoperative creatinine, and TNM stage were independent predictors of postoperative recurrence risk. Conclusion: MHR, as an independent immune-inflammatory metabolic predictor of OS and DFS in patients with advanced PC after pancreaticoduodenectomy. Early monitoring and reduction of MHR may be of great significance in improving prognosis.

SFN promotes renal fibrosis via binding with MYH9 in chronic kidney disease.

Chronic kidney disease (CKD) is a clinical syndrome characterized by persistent renal function decline. Renal fibrosis is the main pathological process in CKD, but an effective treatment does not exist. Stratifin (SFN) is a highly-conserved, multi-function soluble acidic protein. Therefore, this study explored the effects of SFN on renal fibrosis. First, we found that SFN was highly expressed in patients with CKD, as well as in renal fibrosis animal and cell models. Next, transforming growth factor-beta 1 (TGF-ß1) induced injury and fibrosis in human renal tubule epithelial cells, and SFN knockdown reversed these effects. Furthermore, SFN knockdown mitigated unilateral ureteral obstruction (UUO)-induced renal tubular dilatation and renal interstitial fibrosis in mice. Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS), co-immunoprecipitation (Co-IP), and immunofluorescence co-localization assays demonstrated that SFN bound the non-muscle myosin-encoding gene, myosin heavy chain 9 (MYH9), in the cytoplasm of renal tubular epithelial cells. MYH9 knockdown also reduced Col-1 and α-SMA expression, which are fibrosis markers. Finally, silencing SFN decreased MYH9 expression, alleviating renal fibrosis. These results suggest that SFN promotes renal fibrosis in CKD by interacting with MYH9. This study may provide potential strategies for the treatment of CKD.

The Prognostic Value of PERK in Cancer and Its Relationship With Immune Cell Infiltration.

Background: Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a type I transmembrane protein that functions as an endoplasmic reticulum (ER) stress sensor to regulate global protein synthesis. Recent research studies suggest that PERK, as an important receptor protein of unfolded protein response, is involved in the pathogenesis of many cancers. This study aimed to investigate PERK expression and its relationship with prognosis in pan-cancer and attempted to explore the relevant mechanism of PERK involved in the regulation of cancer pathogenesis. Methods: The Oncomine and TIMER databases were used to analyze the expression of PERK between pan-cancer samples and normal samples. Survival analysis was performed using the PrognoScan, Kaplan-Meier (K-M) plotter, and UALCAN databases. Gene set enrichment analysis (GSEA) was used to perform the functional enrichment analysis of the PERK gene in breast invasive carcinoma (BRCA), head and neck squamous cell carcinoma (HNSC), and thyroid carcinoma (THCA). The TIMER database was used to investigate the correlation between PERK expression and tumor-infiltrating immune cells and analyze the relationship of PERK with marker genes of immune cells which were downloaded from the CellMarker database in BRCA, HNSC, and THCA. Results: PERK was differentially expressed in various cancers, such as breast cancer, liver cancer, lung cancer, gastric carcinoma, lymphoma, thyroid cancer, leukemia, and head and neck squamous cell carcinomas. The high expression of PERK was associated with a poor prognosis in KIRP, LGG, BRCA, and THCA and with a favorable prognosis in HNSC. The results of GSEA indicated that PERK was mainly enriched in immune-related signaling pathways in BRCA, HNSC, and THCA. Moreover, PERK expression was significant positively correlated with infiltrating levels of macrophages and dendritic cells and was strongly associated with a variety of immune markers, especially macrophage mannose receptor 1 (MRC1, also called CD206) and T-helper cells (Th). Conclusion: The high expression of PERK could promote the infiltration of multiple immune cells in the tumor microenvironment and could deteriorate the outcomes of patients with breast and thyroid cancers, suggesting that PERK as well as tumor-infiltrating immune cells could be taken as potential biomarkers of prognosis.

Paeoniflorin Ameliorates Macrophage Infiltration and Activation by Inhibiting the TLR4 Signaling Pathway in Diabetic Nephropathy.

Paeoniflorin (PF) is the primary component of total glucosides of paeony (TGP). It exerts multiple effects, including immunoregulatory and anti-inflammatory effects. Our previous study has found that PF has a remarkable renal-protective effect in diabetic mice, but exact mechanism has not been clarified. This study mainly explores whether PF affects macrophage infiltration and activation in diabetic kidney through TLR4 pathway. Thus, this study was conducted to investigate the effect of PF on a streptozotocin (STZ)-induced experimental DN model. The results suggested that the onset and clinical symptoms of DN in mice were remarkably ameliorated after the administration of PF. Moreover, the number of infiltrating macrophages in the mouse kidneys was also markedly decreased. Instead of inhibiting the activation of macrophages directly, PF could influence macrophages by suppressing iNOS expression as well as the production of TNF-α, IL-1ß, and MCP-1 both in vivo and in vitro. These effects might be attributable to the inhibition of the TLR4 signaling pathway. The percentage of M1-phenotype cells as well as the mRNA levels of iNOS, TNF-α, IL-1ß, and MCP-1 were downregulated when PF-treated polarized macrophages were cultured under conditions of high glucose (HG) levels. In addition, the expression of TLR4, along with that of downstream signaling molecule proteins, was also reduced. Our study has provided new insights into the potential of PF as a promising therapeutic agent for treating DN and has illustrated the underlying mechanism of PF from a new perspective.

Paeoniflorin prevents TLR2/4-mediated inflammation in type 2 diabetic nephropathy.

Paeoniflorin is an effective Chinese traditional medicine with anti-inflammatory and immune-regulatory effects. The aim of this study was to investigate the underlying renoprotective mechanism of Paeoniflorin. In vivo, db/db mice were intraperitoneally injected with Paeoniflorin at a dose of 15, 30, or 60 mg/kg respectively. The immunostaining of TLR2, TLR4, CD68, NF-kB p65 and the mRNA level of inflammatory factors, together with the protein expression of TLR2/4 signaling were evaluated. Our data demonstrated that Paeoniflorin could decrease the urinary albumin excretion rate and inhibit macrophage infiltration and activation through blockage of the TLR2/4 signaling pathway compared with the db/db group in vivo. In vitro, RAW264.7 cells were categorized into control, bovin serum albumin (BSA)-stimulated, advanced glycation end products (AGEs)-stimulated, Paeoniflorin intervention and oxidized phospholipid (OxPAPC)-inhibited groups. The cell viability, the optimal stimulated time and concentration were measured as well as the TLR2/4 signaling activation determined by RT-PCR, Western blot and ELISA. Our data demonstrated that Paeoniflorin reduced the AGEs-induced TLR2/4 activation and inflammatory responses, which was consistent with the TLR2/4 inhibitor group. These findings indicate that Paeoniflorin prevents macrophage activation via inhibition of TLR2/4 signaling expression in type 2 diabetic nephropathy.

TGP attenuates endoplasmic reticulum stress and regulates the expression of thioredoxin-interacting protein in the kidneys of diabetic rats.

Recent evidence suggests that the endoplasmic reticulum stress (ERS)-thioredoxin-interacting protein (TXNIP)-inflammation chain contributes to diabetic renal injury. The aim of the current study was to investigate whether total glucosides of peony (TGP) could inhibit ERS and attenuate up-regulation of TXNIP in the kidneys of rats with streptozotocin-induced diabetes. TGP was orally administered daily at a dose of 50, 100, or 200 mg/kg for 8 weeks. The expression of glucose-regulated protein 78 (GRP78), phospho-protein kinase RNA-like ER kinase (p-PERK), phosphor- eukaryotic translation initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), and TXNIP was assessed. Results indicated that TGP significantly decreased diabetes-induced albuminuria and it acted by down-regulating activation of the ERS-TXNIP-inflammation chain in the kidneys of diabetic rats. These findings indicate that renoprotection from TGP in diabetic rats possibly contributed to inhibition of ERS and decreased expression of TXNIP. These findings also offer a new perspective from which to study the molecular mechanisms of diabetic nephropathy and prevent its progression.

Paeoniflorin attenuates incipient diabetic nephropathy in streptozotocin-induced mice by the suppression of the Toll-like receptor-2 signaling pathway.

Toll-like receptors (TLRs) may be involved in diabetic nephropathy (DN). Paeoniflorin (PF) is an effective Chinese traditional medicine with anti-inflammatory and immunoregulatory effects that may inhibit the TLR2 signaling pathway. In this study, we investigated the effects of PF on the kidneys of mice with streptozotocin-induced type 1 diabetes mellitus using TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT). After 12 weeks of intraperitoneal injection of PF at doses of 25, 50, and 100 mg/kg once a day, diabetic mice had significantly reduced albuminuria and attenuated renal histopathology. These changes were associated with substantially alleviated macrophage infiltration and decreased expression of TLR2 signaling pathway biomarkers. These data support a role of TLR2 in promoting inflammation and indicate that the effect of PF is associated with the inhibition of the TLR2 pathway in the kidneys of diabetic mice. PF thus shows therapeutic potential for the prevention and treatment of DN.

The role of TGF-ß-activated kinase 1 in db/db mice and high glucose-induced macrophage.

Accumulating evidence reveals that inflammation plays a vital part in the development of diabetic nephropathy (DN), little information is available about the TGF-ß-activated kinase 1 (TAK1) signal pathway activating inflammatory response in DN. We used bone marrow-derived macrophages (BMMs) and db/db mice to investigate the potential protective effects and mechanisms of TAK1 inhibitor (5Z-7-oxozeaenol) on diabetic kidney disease. The study showed that pretreatment with 5Z-7-oxozeaenol not only remarkably decreased high glucose (HG) stimulated excessive release of MCP-1 and TNF-α, but also significantly down-regulated ERK1/2, p38MAPK phosphorylation, and NF-κB activation in macrophages. In consistent, 5Z-7-oxozeaenol markedly reduced diabetes-induced albuminuria, histological changes, macrophage infiltration, and renal inflammatory cytokines expression and exerted its function through down-regulating ERK1/2, p38MAPK, NF-κB activation in the kidneys of db/db mice. Our findings may provide a novel direction to study the molecular mechanism and a perspective intervention to halt the progression of DN.

Paeoniflorin inhibits high glucose-induced macrophage activation through TLR2-dependent signal pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin(PF), extracted from the root peeled of Paeonia lactiflora Pall(Family: Ranunculaceae), has therapeutic potential in many animal models of inflammatory diseases. AIM OF THE STUDY: Although the anti-inflammatory efficacy of PF has been well illustrated in several animal models, whether it could attenuate diabetic nephropathy and detailed mechanisms are still obscure. Till now, accumulating evidence has proposed the pivotal role of toll-like receptors (TLRs) in renal inflammation in diabetic patients. In this setting, the current study aimed to investigate the effects and underlying mechanism of PF on high glucose-induced activation of toll like-receptor 2 (TLR2) signaling in macrophages. MATERIALS AND METHODS: Bone marrow-derived macrophages (BMDM) were isolated from male Tlr2tm1kir (TLR2-/-) mice and wild-type littermates (C57BL/6JWT). The level of TLR2 and activation of downstream signaling were evaluated in response to 30mmol/L high glucose (HG)-containing medium. Macrophages behaviors, which include cell viability, migration and inflammatory cytokines production, were also determined. RESULTS: PF suppressed HG-induced production of TLR2, activation of downstream signaling and synthesis of inducible nitric oxide synthase (iNOS). PF could further inhibit MyD88-dependent pathway in HG-induced models in which TLR2 was knocked out. Moreover, deletion of TLR2 inhibited the HG-induced activation of MyD88-dependent pathway, but not TIR domain containing adapter inducing interferon-ß (Trif) signal pathway in BMDMs. As HG stimulation polarizes macrophages into M1 phenotype, treatment of PF or knockout of TLR2 significantly reduces M1 markers on the membrane of macrophages. Additionally, levels of inflammatory cytokines and iNOS were remarkably reduced in response to PF or TLR2 deficiency. CONCLUSION: Collectively, these data demonstrated that HG activated macrophages primarily through TLR2-dependent mechanisms which aggravated the severity of renal inflammation and eventually contributed to DN. Additionally, PF might be applied as a potential therapeutic agent in the battle against progressive DN.