Inhibition of urea transporter ameliorates uremic cardiomyopathy in chronic kidney disease.

Uremic cardiomyopathy, characterized by hypertension, cardiac hypertrophy, and fibrosis, is a complication of chronic kidney disease (CKD). Urea transporter (UT) inhibition increases the excretion of water and urea, but the effect on uremic cardiomyopathy has not been studied. We tested UT inhibition by dimethylthiourea (DMTU) in 5/6 nephrectomy mice. This treatment suppressed CKD-induced hypertension and cardiac hypertrophy. In CKD mice, cardiac fibrosis was associated with upregulation of UT and vimentin abundance. Inhibition of UT suppressed vimentin amount. Left ventricular mass index in DMTU-treated CKD was less compared with non-treated CKD mice as measured by echocardiography. Nephrectomy was performed in UT-A1/A3 knockout (UT-KO) to further confirm our finding. UT-A1/A3 deletion attenuates the CKD-induced increase in cardiac fibrosis and hypertension. The amount of α-smooth muscle actin and tgf-ß were significantly less in UT-KO with CKD than WT/CKD mice. To study the possibility that UT inhibition could benefit heart, we measured the mRNA of renin and angiotensin-converting enzyme (ACE), and found both were sharply increased in CKD heart; DMTU treatment and UT-KO significantly abolished these increases. Conclusion: Inhibition of UT reduced hypertension, cardiac fibrosis, and improved heart function. These changes are accompanied by inhibition of renin and ACE.

Asiaticoside inhibits TGF-ß1-induced mesothelial-mesenchymal transition and oxidative stress via the Nrf2/HO-1 signaling pathway in the human peritoneal mesothelial cell line HMrSV5.

BACKGROUND: Peritoneal fibrosis (PF) is a frequent complication caused by peritoneal dialysis (PD). Peritoneal mesothelial cells (PMCs), the first barrier of the peritoneum, play an important role in maintaining structure and function in the peritoneum during PD. Mesothelial-mesenchymal transition (MMT) and oxidative stress of PMCs are two key processes of PF. PURPOSE: To elucidate the efficacy and possible mechanism of asiaticoside inhibition of MMT and ROS generation in TGF-ß1-induced PF in human peritoneal mesothelial cells (HPMCs). METHODS: MMT and ROS generation of HPMCs were induced by TGF-ß1. To explain the anti-MMT and antioxidant role of asiaticoside, varied doses of asiaticoside, oxygen radical scavenger (NAC), TGF-ß receptor kinase inhibitor (LY2109761) and Nrf2 inhibitor (ML385) were used separately. Immunoblots were used to detect the expression of signaling associated proteins. DCFH-DA was used to detect the generation of ROS. Transwell migration assay and wound healing assay were used to verify the capacity of asiaticoside to inhibit MMT. Immunofluorescence assay was performed to observe the subcellular translocation of Nrf2 and expression of HO-1. RESULTS: Asiaticoside inhibited TGF-ß1-induced MMT and suppressed Smad signaling in a dose-dependent manner. Migration and invasion activities of HPMCs were decreased by asiaticoside. Asiaticoside decreased TGF-ß1-induced ROS, especially in a high dose (150 µM) for 6 h. Furthermore, ML385 partly abolished the inhibitory effect of asiaticoside on MMT, ROS and p-Smad2/3. CONCLUSIONS: Asiaticoside inhibited the TGF-ß1-induced MMT and ROS via Nrf2 activation, thus protecting the peritoneal membrane and preventing PF.

Astragalus Inhibits Epithelial-to-Mesenchymal Transition of Peritoneal Mesothelial Cells by Down-Regulating ß-Catenin.

BACKGROUND/AIMS: The epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a crucial event in the induction of peritoneal fibrosis (PF), in which canonical Wnt/ß-catenin signaling participates. Smads signaling is reported to interact with ß-catenin and synergistically regulates EMT. This study was aimed to reveal the effect of Astragalus on ß-catenin in EMT of PMCs. METHODS: To obtain the role of ß-catenin in EMT, gene transfer into HMrSV5 cell line and rats has been achieved. After Astragalus treatment, EMT markers and signaling pathway-related indicators were detected by western blotting, immunofluorescence, immunohistochemistry, immunoprecipitation and real time-PCR. RESULTS: ß-catenin knockdown suppressed EMT of HMrSV5 cells. Astragalus alleviated EMT of PMCs characterized by increased E-cadherin and decreased α-SMA and Vimentin. In rat model of peritoneal dialysis (PD), Astragalus attenuated peritoneal thickening and fibrosis. Astragalus down-regulated ß-catenin by stabilizing the Glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin complex and further inhibited the nuclear translocation of ß-catenin. Meanwhile, Astragalus down-regulated ß-catenin by enhancing Smad7 expression. Silencing Smad7 antagonized the EMT-inhibitory effect of Astragalus. CONCLUSION: Astragalus inhibits EMT of PMCs by down-regulating ß-catenin. The modulation of ß-catenin in peritoneum can be a novel tool to prevent PF.

Exosomes: The New Mediator of Peritoneal Membrane Function.

Fibrosis and angiogenesis are the most common processes that result in progressive peritoneal tissue remodeling and, eventually, peritoneal membrane dysfunction. The role of exosomes, which contributes to intercellular communication, in these processes has been neglected. Various biomolecules, including DNA, mRNA, proteins, lipids, and particular certain miRNAs, can be transferred by exosomes to local, neighboring and distal cells. Upon stimulation by cytokines or other microenvironment stimuli, donor cells release a mass of exosomes to peritoneal mesothelial cells, further affecting fibrosis and angiogenesis. This important exosomes-mediated intracellular communication is thought to regulate peritoneal membrane function. Understanding the molecular mechanisms of these processes, targeting changes in exosomes and regulating exosomal miRNAs will advance therapeutic methods for protecting peritoneal membrane function.

Angiogenesis and Inflammation in Peritoneal Dialysis: The Role of Adipocytes.

Chronic inflammation and angiogenesis are the most common complications in patients undergoing maintenance peritoneal dialysis (PD), resulting in progressive peritoneum remolding and, eventually, utrafiltration failure. Contributing to the deeper tissue under the peritoneal membrane, adipocytes play a neglected role in this process. Some adipokines act as inflammatory and angiogenic promoters, while others have the opposite effects. Adipokines, together with inflammatory factors and other cytokines, modulate inflammation and neovascularization in a coordinated fashion. This review will also emphasize cellular regulators and their crosstalk in long-term PD. Understanding the molecular mechanism, targeting changes in adipocytes and regulating adipokine secretion will help extend therapeutic methods for preventing inflammation and angiogenesis in PD.

The Role of TLR4 in M1 Macrophage-Induced Epithelial-Mesenchymal Transition of Peritoneal Mesothelial Cells.

BACKGROUND/AIMS: Peritoneal fibrosis is a frequent complication of peritoneal dialysis that follows inflammation. It is recognized that epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs), plays a key role in fibrogenesis. However, the relationship between inflammatory macrophages and PMCs remains elusive. In this study, we investigated the effects of different polarized macrophages on EMT of HMrSV5 PMCs. METHODS: Monocytes were polarized to M1/M2 macrophages before being added to HMrSV5 in direct or indirect contact. Morphological changes of HMrSV5 were observed and toll-like receptors 4 (TLR4) on macrophage surfaces was detected using flow cytometry. EMT markers and intracellular signals of HMrSV5 cells were assessed using real time-PCR and WB. RESULTS: The typical epithelial cell morphology of HMrSV5 disappeared after co-culture with M1 macrophages and was accompanied by decreased E-cadherin and increased α-SMA, suggesting HMrSV5 undergo EMT. These effects depended on direct contact between the two cells, as indirect contact or co-culture with M2 macrophages had no effect. Intriguingly, we found TLR4 surface receptors were activated on sorted M1 cells in co-culture, and related signal adaptors, such as TRIF, were obviously upregulated. CONCLUSION: Direct contact with M1 macrophages induces EMT of PMCs, during which TRIF-dependent TLR4 signaling pathway was activated.

Intercellular communication in peritoneal dialysis.

Long-term peritoneal dialysis (PD) causes structural and functional alterations of the peritoneal membrane. Peritoneal deterioration and fibrosis are multicellular and multimolecular processes. Under stimulation by deleterious factors such as non-biocompatibility of PD solution, various cells in the abdominal cavity show differing characteristics, such as the secretion of different cytokines, varying protein expression levels, and transdifferentiation into other cells. In this review, we discuss the role of various cells in the abdominal cavity and their interactions in the pathogenesis of PD. An in-depth understanding of intercellular communication and inter-organ communication in PD will lead to a better understanding of the pathogenesis of this disease, enabling the development of novel therapeutic targets.

Astragalus polysaccharides augment BMSC homing via SDF-1/CXCR4 modulation: a novel approach to counteract peritoneal mesenchymal transformation and fibrosis.

PURPOSE: This study aimed to evaluate the potential of astragalus polysaccharide (APS) pretreatment in enhancing the homing and anti-peritoneal fibrosis capabilities of bone marrow mesenchymal stromal cells (BMSCs) and to elucidate the underlying mechanisms. METHODS: Forty male Sprague-Dawley rats were allocated into four groups: control, peritoneal dialysis fluid (PDF), PDF + BMSCs, and PDF + APSBMSCs (APS-pre-treated BMSCs). A peritoneal fibrosis model was induced using PDF. Dil-labeled BMSCs were administered intravenously. Post-transplantation, BMSC homing to the peritoneum and pathological alterations were assessed. Stromal cell-derived factor-1 (SDF-1) levels were quantified via enzyme-linked immunosorbent assay (ELISA), while CXCR4 expression in BMSCs was determined using PCR and immunofluorescence. Additionally, a co-culture system involving BMSCs and peritoneal mesothelial cells (PMCs) was established using a Transwell setup to examine the in vitro effects of APS on BMSC migration and therapeutic efficacy, with the CXCR4 inhibitor AMD3100 deployed to dissect the role of the SDF-1/CXCR4 axis and its downstream impacts. RESULTS: In vivo and in vitro experiments confirmed that APS pre-treatment notably facilitated the targeted homing of BMSCs to the peritoneal tissue of PDF-treated rats, thereby amplifying their therapeutic impact. PDF exposure markedly increased SDF-1 levels in peritoneal and serum samples, which encouraged the migration of CXCR4-positive BMSCs. Inhibition of the SDF-1/CXCR4 axis through AMD3100 application diminished BMSC migration, consequently attenuating their therapeutic response to peritoneal mesenchyme-to-mesothelial transition (MMT). Furthermore, APS upregulated CXCR4 expression in BMSCs, intensified the activation of the SDF-1/CXCR4 axis's downstream pathways, and partially reversed the AMD3100-induced effects. CONCLUSION: APS augments the SDF-1/CXCR4 axis's downstream pathway activation by increasing CXCR4 expression in BMSCs. This action bolsters the targeted homing of BMSCs to the peritoneal tissue and amplifies their suppressive influence on MMT, thereby improving peritoneal fibrosis.

Shen-yan-yi-hao oral solution ameliorates IgA nephropathy via intestinal IL-17/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world, it is one of the most common causes of kidney disease and can lead to end-stage kidney disease, however, its pathogenesis is still complicated. The Shen-yan-yi-hao oral solution (SOLI) is an effective prescription for the clinical treatment of IgAN while its specific mechanism remains to be further elucidated. AIM OF THE STUDY: This study investigates SOLI's effects on IgAN in rats, particularly on the intestinal mucosal barrier, and identifies potential therapeutic targets through network pharmacology and molecular docking, validated experimentally. MATERIALS AND METHODS: Target genes for SOLI in IgAN were identified and analysed through molecular docking and KEGG pathway enrichment. An IgAN rat model examined SOLI's effect on renal biomarkers and cytokines involved in specific pathways, ileum mucosal lesions, and the intestinal immune system. The IL-17 pathway's role was studied in IEC-6 cells with SOLI in vitro. RESULT: Rats developed increased proteinuria and kidney damage marked by IgA deposition and inflammation. SOLI treatment significantly ameliorated these symptoms, reduced galactose-deficient Ig A1 (Gd-IgA1), and decreased cytokines like IL-17, TNF-α, IL-6 and IL-1ß etc. SOLI also normalized intestinal tight junction protein expression, ameliorated intestinal damage, and regulated intestinal immune response (focused on IL-17/NF-κB signal pathway). SOLI moderated the abnormally activated IL-17 pathway, which damages intestinal epithelial cells, suggesting IgAN treatment potential. CONCLUSION: SOLI reduces proteinuria and enhances intestinal mucosal function in IgAN rats, kidney protection in the IgAN rat model may initiate from modulating the intestinal IL-17/NF-κB pathway and subsequent Gd-IgA1 accumulation.

The role of macrophage-derived Exosomes in reversing peritoneal fibrosis: Insights from Astragaloside IV.

BACKGROUND: Peritoneal dialysis (PD) is a successful renal replacement therapy for end-stage renal disease. Long-term PD causes mesothelial-mesenchymal transition (MMT) of peritoneal mesothelial cells (PMCs), leading to peritoneal fibrosis (PF), which reduces the efficiency of PD. Macrophages are thought to play a role in the onset and perpetuation of peritoneal injury. However, the mechanisms by which macrophages-PMCs communication regulates peritoneal fibrosis are not fully understood resulting in a lack of disease-modifying drugs. Astragaloside IV (AS-IV) possessed anti-fibrotic effect towards PF in PD whereas the mechanistic effect of AS-IV in PD is unknown. METHODS: The primary macrophages were extracted and treated with LPS or AS-IV, then co-cultured with primary PMCs in transwell plates. The macrophage-derived exosomes were extracted and purified by differential centrifugation, then co-cultured with primary PMCs. Small RNA-seq was used to detect differential miRNAs in exosomes, and then KEGG analysis and q-PCR were performed for validation. In vivo PD rat models were established by inducing with high-glucose peritoneal dialysis fluid and different concentrations of AS-IV and exosomes were intraperitoneal injection. Through qRT-PCR, western blotting, and luciferase reporting, candidate proteins and pathways were validated in vivo and in vitro. The functions of the validated pathways were further investigated using the mimic or inhibition strategy. PF and inflammatory situations were assessed. RESULTS: We found AS-IV reversed the MMT of PMCs caused by LPS-stimulated macrophages and the improving effect was mediated by macrophage-derived exosomes in vitro. We also demonstrated that AS-IV significantly reduced the MMT of PMCs in vitro or PF in a rat PD model via regulating exosome-contained miR-204-5p which targets Foxc1/ß-catenin signaling pathway. CONCLUSION: AS-IV attenuates macrophage-derived exosomes induced fibrosis in PD through the miR-204-5p/Foxc1 pathway.

TMT quantitative proteomics and network pharmacology reveal the mechanism by which asiaticoside regulates the JAK2/STAT3 signaling pathway to inhibit peritoneal fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine, Centella asiatica (L.) Urb., has been extensively utilized in clinics to treat a variety of fibrotic disorders. Asiaticoside (ASI), as an important active ingredient, has attracted much attention in this field. However, the effect of ASI on peritoneal fibrosis (PF) is still unclear. Therefore, we evaluated the benefits of ASI for PF and mesothelial-mesenchymal transition (MMT) and revealed the underlying mechanisms. AIM OF STUDY: The objective of this investigation was to anticipate the potential molecular mechanism of ASI against peritoneal mesothelial cells (PMCs) MMT employing proteomics and network pharmacology, and to confirm it using in vivo and in vitro studies. MATERIALS AND METHODS: The mesentery of peritoneal fibrosis mice and normal mice were analyzed quantitatively for proteins that were differentially expressed using a technique tandem mass tag (TMT). Next, the core target genes of ASI against PF were screened through network pharmacology analysis, and PPI and C-P‒T networks were constructed by Cytoscape Version 3.7.2. According to the findings of a GO and KEGG enrichment analysis of differential proteins and core target genes, the signaling pathway with a high correlation degree was selected as the key signaling pathway of ASI inhibiting the PMCs MMT for further molecular docking analysis and experimental verification. RESULTS: TMT-based quantitative proteome analysis revealed the identification of 5727 proteins, of which 70 were downregulated and 178 were upregulated. Among them, the levels of STAT1, STAT2, and STAT3 in the mesentery of mice with peritoneal fibrosis were considerably lower than in the control group, indicating a role for the STAT family in the pathogenesis of peritoneal fibrosis. Then, a total of 98 ASI-PF-related targets were identified by network pharmacology analysis. JAK2 is one of the top 10 core target genes representing a potential therapeutic target. JAK/STAT signaling may represent a core pathway mediating PF effects by ASI. Molecular docking studies showed that ASI had the potential to interact favorably with target genes involved in the JAK/STAT signaling pathway, such as JAK2 and STAT3. The experimental results showed that ASI could significantly alleviate Chlorhexidine Gluconate (CG)-induced peritoneal histopathological changes and increase JAK2 and STAT3 phosphorylation levels. In TGF-ß1-stimulated HMrSV5 cells, E-cadherin expression levels were dramatically reduced whereas Vimentin, p-JAK2, α-SMA, and p-STAT3 expression levels were considerably increased. ASI inhibited the TGF-ß1-induced HMrSV5 cell MMT, decreased the activation of JAK2/STAT3 signaling, and increased the nuclear translocation of p-STAT3, which was consistent with the effect of the JAK2/STAT3 pathway inhibitor AG490. CONCLUSION: ASI can inhibit PMCs MMT and alleviate PF by regulating the JAK2/STAT3 signaling pathway.

Causal Effects of Specific Gut Microbiota on Chronic Kidney Diseases and Renal Function-A Two-Sample Mendelian Randomization Study.

BACKGROUND: Targeting the gut microbiota may become a new therapeutic to prevent and delay the progression of chronic kidney disease (CKD). Nonetheless, the causal relationship between specific intestinal flora and CKD is still unclear. MATERIALS AND METHOD: To identify genetically predicted microbiota, we used summary data from genome-wide association studies on gut microbiota in 18340 participants from 24 cohorts. Furthermore, we genetically predicted the causal relationship between 211 gut microbiotas and six phenotypes (outcomes) (CKD, estimated glomerular filtration rate (eGFR), urine albumin to creatinine ratio (UACR), dialysis, rapid progress to CKD, and rapid decline of eGFR). Four Mendelian randomization (MR) methods, including inverse variance weighted (IVW), MR-Egger, weighted median, and weighted mode were used to investigate the casual relationship between gut microbiotas and various outcomes. The result of IVW was deemed as the primary result. Then, Cochrane's Q test, MR-Egger, and MR-PRESSO Global test were used to detect heterogeneity and pleiotropy. The leave-one method was used for testing the stability of MR results and Bonferroni-corrected was used to test the strength of the causal relationship between exposure and outcome. RESULTS: Through the MR analysis of 211 microbiotas and six clinical phenotypes, a total of 36 intestinal microflora were found to be associated with various outcomes. Among them, Class Bacteroidia (=-0.005, 95% CI: -0.001 to -0.008, p = 0.002) has a strong causality with lower eGFR after the Bonferroni-corrected test, whereas phylum Actinobacteria (OR = 1.0009, 95%CI: 1.0003-1.0015, p = 0.0024) has a strong causal relationship with dialysis. The Cochrane's Q test reveals that there is no significant heterogeneity between various single nucleotide polymorphisms. In addition, no significant level of pleiotropy was found according to MR-Egger and MR-PRESSO Global tests. CONCLUSIONS: Through the two-sample MR analysis, we identified the specific intestinal flora that has a causal relationship with the incidence and progression of CKD at the level of gene prediction, which may provide helpful biomarkers for early disease diagnosis and potential therapeutic targets for CKD.

Isolation and characterization of peritoneal microvascular pericytes.

As a potential source of myofibroblasts, pericytes may play a role in human peritoneal fibrosis. The culture of primary vascular pericytes in animals has previously been reported, most of which are derived from cerebral and retinal microvasculature. Here, in the field of peritoneal dialysis, we describe a method to isolate and characterize mouse peritoneal microvascular pericytes. The mesenteric tissues of five mice were collected and digested by type II collagenase and type I DNase. After cell attachment, the culture fluid was replaced with pericyte-conditioned medium. Pericytes with high purity (99.0%) could be isolated by enzymatic disaggregation combined with conditional culture and magnetic activated cell sorting. The primary cells were triangular or polygonal with protrusions, and confluent cell culture could be established in 3 days. The primary pericytes were positive for platelet-derived growth factor receptor-ß, α-smooth muscle actin, neuron-glial antigen 2, and CD13. Moreover, they promoted formation of endothelial tubes, and pericyte-myofibroblast transition occurred after treatment with transforming growth factor-ß1. In summary, we describe here a reproducible isolation protocol for primary peritoneal pericytes, which may be a powerful tool for in vitro peritoneal fibrosis studies.

A Novel Nomogram Based on a Competing Risk Model Predicting Cardiovascular Death Risk in Patients With Chronic Kidney Disease.

Objective: Chronic kidney disease (CKD) patients are more likely to die from cardiovascular disease (CVD) than develop renal failure. This study aimed to develop a new nomogram for predicting the risk of cardiovascular death in CKD patients. Methods: This study enrolled 1656 CKD patients from NHANES 2003 to 2006 survey. Data sets from 2005 to 2006 survey population were used to build a nomogram for predicting the risk of cardiovascular death, and the nomogram was validated using data from 2003 to 2004 survey population. To identify the main determinants of cardiovascular death, we performed univariate analysis and backward-stepwise regression to select the key factors. The probability of cardiovascular death for each patient in 5, 7, and 9 years was calculated using a nomogram based on the predictors. To assess the nomogram's performance, the area under receiver operating characteristic curve (AUC) and the calibration curve with 1,000 bootstraps resamples were utilized. The prediction model's discrimination was examined using cumulative incidence function (CIF). Results: Age, homocysteine, potassium levels, CKD stage, and anemia were included in the nomogram after screening risk factors using univariate analysis and backward-stepwise regression. Internal validation revealed that this nomogram possesses high discrimination and calibration (AUC values of 5-, 7-, and 9-years were 0.79, 0.81, and 0.81, respectively). External validation confirmed the same findings (AUC values of 5-, 7- and 9-years were 0.76, 0.73, and 0.73, respectively). According to CIF, the established nomogram effectively differentiates patients at a high risk of cardiovascular death from those at low risk. Conclusion: This work develops a novel nomogram that integrates age, homocysteine, potassium levels, CKD stage, and anemia and can be used to more easily predict cardiovascular death in CKD patients, highlighting its potential value in clinical application.

Astragalus Total Saponins Ameliorate Peritoneal Fibrosis by Promoting Mitochondrial Synthesis and Inhibiting Apoptosis.

Peritoneal fibrosis (PF) is a disease caused by prolonged exposure of the peritoneum to high levels of dialysis fluid. Astragalus total saponins (ATS) is a phytochemical naturally occurring in Radix Astragali that has anti-inflammatory and anti-oxidant properties. In this study, we constructed an in vivo model of PF using 4.25% glucose-containing administered intraperitoneally to rats and incubated peritoneal mesothelial cells (PMCs) with 4.25% glucose-containing peritoneal dialysis fluid to construct an in vitro model of PF. Furthermore, siRNA of PGC-1[Formula: see text] was used to inhibit the expression of PGC-1[Formula: see text] to further investigate the mechanism of the protective effect of ATS on PF. In both in vivo and in vitro models, ATS treatment showed a protective effect against PF, with ATS reducing the thickness of peritoneal tissues in PF rats, increasing the viability of PMCs, increasing the mitochondrial membrane potential and reducing apoptosis ratio. ATS treatment also reduced the expressions of peritoneal fibrosis markers (Smad2, p-Smad2 and [Formula: see text]-SMA) and apoptosis markers (Caspase3, cleaved-Caspase3 and Bax) and restored the expressions of mitochondrial synthesis proteins (PGC-1[Formula: see text], NRF1 and TFAM) in ATS-treated peritoneal tissues or PMCs. Furthermore, in the presence of PGC-1[Formula: see text] inhibition, the protective effect of ATS on PF was blocked. In conclusion, ATS treatment may be an effective therapeutic agent to inhibit high glucose-induced in peritoneal fibrosis through PGC-1[Formula: see text]-mediated apoptosis.

Akkermansia muciniphila attenuates LPS-induced acute kidney injury by inhibiting TLR4/NF-κB pathway.

Acute kidney injury (AKI) is a global public health hazard with high morbidity and mortality. Sepsis accounts for nearly half of all causes of AKI. Scientists have made a great effort to explore effective therapeutic agents with limited side effects in the treatment of AKI, but have had little success. With the development of gut flora study, Akkermansia muciniphila (A. muciniphila) has been proven to prevent different organs by regulating the inflammatory response. However, the reno-protective function is still unknown. Here, the AKI model was induced using lipopolysaccharide (LPS) in mice with or without pretreatment of A. muciniphila. Renal function and histological change were measured. Inflammatory factors were detected by ELISA and rt-PCR. TLR4/NF-κB signaling factors and NLRP3 inflammasome were tested by western blot and immunohistochemistry. Pretreatment of A. muciniphila markedly inhibited inflammatory response and ameliorated kidney histopathological changes. Furthermore, the TLR4, p-NF-κB p65, and downstream IκBα were notably activated in the model group and inhibited by A. muciniphila. A similar effect was found in the regulation of NLRP3 inflammasome. In conclusion, pretreatment with A. muciniphila could protect against LPS-induced AKI by inhibition of the TLR4/NF-κB pathway and NLRP3 inflammasome activation. It may be a new therapeutic strategy for AKI prevention and treatment in the future.

Total extract of Abelmoschus manihot L. alleviates uric acid-induced renal tubular epithelial injury via inhibition of caspase-8/caspase-3/NLRP3/GSDME signaling.

Purpose: The incidence of uric acid (UA)-induced kidney injury is increasing owing to the high incidence of hyperuricemia in recent years. The flower of Abelmoschus manihot (Linneus) Medik is a traditional Chinese medicinal herb widely used in the treatment of some kidney diseases. In our previous study, we reported that the total extract of A. manihot L. flower (TEA) attenuated adriamycin-induced renal tubular cell injury. In this study, we aimed to evaluate the role of TEA in UA-induced tubular cell injury. Methods: Normal rat proximal epithelial NRK-52E cells were incubated with UA to mimic hyperuricemia conditions. The role of TEA in the renal tubular cells was also assessed. The cellular morphology was observed using phase-contrast microscopy, and cell viability was analyzed using the Cell Counting kit-8. Living and dead cells were stained using a Calcein-AM/PI double stain kit. The release of lactate dehydrogenase (LDH) was analyzed by LDH cytotoxicity Assay Kit. The expression of target proteins was analyzed using western blot analysis. Results: UA triggered NRK-52E cell injury, as evidenced by morphological changes, detachment of cells from the bottom, cell swelling, large bubbles blowing from cell membrane and loss of cell viability. UA increased release of LDH. UA induced the expression of p-ERK1/2 and the subsequent activation of caspase-8, caspase-3, and NLRP3 inflammasomes. Pyroptosis was elicited by UA after gasdermin E N-terminal (GSDME-NT) was cleaved from gasdermin E (GSDME). Z-DEVD-FMK, a caspase-3 inhibitor, suppressed the expression of both NLRP3 and GSDME-NT, but not that of caspase-8. INF39, an NLRP3 inhibitor, altered the expression of GSDME-NT expression, but not that caspase-3 and caspase-8. TEA alleviated UA-induced cell injury by suppressing ERK1/2/caspase-8/caspase-3/NLRP3/GSDME signaling. Conclusion: GSDME-mediated pyroptosis was involved in UA-induced renal tubular cell injury. This is the first study to report that TEA protects renal tubular epithelial cells against UA by inhibiting the ERK/1/2/caspase-8/caspase-3/NLRP3/GSDME pathway.

Updating Reports of Phase 3 Clinical Trials for Cancer.

IMPORTANCE: Phase 3 randomized clinical trials (RCTs) are usually reported after a predetermined number of events (death or disease progression) have occurred, when survival curves remain poorly defined. Updated reports are important in providing mature data. OBJECTIVES: To evaluate the proportion of phase 3 RCTs for cancer that are updated and the factors that are associated with updating them and, for updated trials, to compare initial and updated results. DESIGN, SETTING, AND PARTICIPANTS: This study identified reports of 2-group RCTs with a sample size of at least 100, published in 6 major journals between 1990 and 2010, that evaluated drug treatments for breast, lung, or prostate cancer. PubMed and abstracts of large cancer conferences were searched to identify updated (or earlier) reports of the same trials published up to 2019. Logistic regression was used to identify factors associated with the provision of updated reports. The hazard ratios defining the relative treatment effects for the primary and secondary end points between the initial and updated reports were compared. MAIN OUTCOMES AND MEASURES: Proportion of RCTs whose results are updated, factors associated with updating, and change in hazard ratio for the primary end point between initial and updated reports. RESULTS: A total of 207 RCTs met the inclusion criteria, and 41 (20%) were found to have updated reports. The factors significantly associated with an update included positive trial results (odds ratio [OR], 8.7 [95% CI, 3.3-23.3]), larger trial size (OR, 1.0006 [95% CI, 1.0000-1.0012]), evaluation of hormonal agents (OR, 5.8 [95% CI, 1.6-21.8]) or targeted agents (OR, 4.3 [95% CI, 1.3-14.6]) compared with chemotherapy, and evaluation of adjuvant therapy rather than therapy for advanced disease (OR, 8.0 [95% CI, 2.9-21.9]). For 31 trials for which initial and updated hazard ratios for the primary end point were available, the median hazard ratio increased from 0.66 (95% CI, 0.22-1.20) to 0.74 (95% CI, 0.32-1.19) (P < .001), indicating a decreased level of effectiveness. CONCLUSIONS AND RELEVANCE: Only 20% of reports of phase 3 clinical trials for breast, lung, and prostate cancer were updated. Original reports of such trials are based on relatively few events, and their results are immature; more mature data indicate a decreased level of effect in updated trials. Updated reporting to provide mature, long-term results of clinical trials should be mandated.

Tetramethylpyrazine Ameliorates Peritoneal Angiogenesis by Regulating VEGF/Hippo/YAP Signaling.

Angiogenesis of human peritoneal vascular endothelial cells (HPVECs), linked to vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling, is a complication of peritoneal fibrosis (PF). Hippo/YAP signaling interacts with VEGF/VEGFR2 signaling, but the effect on peritoneal angiogenesis and PF has not been studied. We tested VEGF/Hippo/YAP inhibition by tetramethylpyrazine (TMP) in PF mice and HPVECs. This treatment ameliorated peritoneal dialysis (PD)-induced angiogenesis and PF. In mice, PF was associated with upregulation of VEGF, and TMP ameliorated submesothelial fibrosis, perivascular bleeding, and Collagen I abundance. In HPVECs, angiogenesis occurred due to human peritoneal mesothelial cells (HPMCs)-conditioned medium, and TMP alleviated HPVECs migration, tube formation, and YAP nuclear translocation. YAP knockdown PF mouse and HPVEC models were established to further confirm our finding. YAP deletion attenuated the PD-induced or VEGF-induced increase in angiogenesis and PF. The amount of CYR61 and CTGF was significantly less in the YAP knockdown group. To study the possibility that TMP could benefit angiogenesis, we measured the HPVECs migration and tube formation and found that both were sharply increased in YAP overexpression; TMP treatment partly abolished these increases. As well, the amount of VEGFR localized in the trans-Golgi network was lower by double immunofluorescence; VEGFR and its downstream signaling pathways including p-ERK, p-P38, and p-Akt were more in HPVECs with YAP overexpression. Overall, TMP treatment ameliorated angiogenesis, PF, and peritoneum injury. These changes were accompanied by inhibition of VEGF/Hippo/YAP.

Downregulation of let-7 by Electrical Acupuncture Increases Protein Synthesis in Mice.

BACKGROUND: Our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) prevents muscle atrophy by attenuation of protein degradation in mice. The current study examines the impact of Acu/LFES on protein synthesis. METHOD: C57/BL6 mice received Acu/LFES treatment on hindlimb for 30 min once. Acu/LFES points were selected by WHO Standard Acupuncture Nomenclature and electric stimulation applied using an SDZ-II Electronic acupuncture instrument. Muscle protein synthesis was measured by the surface-sensing of translation (SUnSET) assay. Exosomes were isolated using serial centrifugation and concentration and size of the collected exosomes were measured using a NanoSight instrument. The mature microRNA library in serum exosomes was validated using a High Sensitivity DNA chip. RESULTS: Protein synthesis was enhanced in the both hindlimb and forelimb muscles. Blocking exosome secretion with GW4869 decreased the Acu/LFES-induced increases in protein synthesis. MicroRNA-deep sequencing demonstrated that four members of the Let-7 miRNA family were significantly decreased in serum exosomes. Real time qPCR further verified Acu/LFES-mediated decreases of let-7c-5p in serum exosomes and skeletal muscles. In cultured C2C12 myotubes, inhibition of let-7c not only increased protein synthesis, but also enhanced protein abundance of Igf1 and Igf1 receptors. Using a luciferase reporter assay, we demonstrated that let-7 directly inhibits Igf1. CONCLUSION: Acu/LFES on hindlimb decreases let-7-5p leading to upregulation of the Igf1 signaling and increasing protein synthesis in both hindlimb and forelimb skeletal muscles. This provides a new understanding of how the electrical acupuncture treatment can positively influence muscle health.