Protease-Activated Receptor-2 and Phospholipid Metabolism Analysis in Hyperuricemia-Induced Renal Injury.

Interstitial inflammation is an important mechanism of pathological damage in renal injury caused by hyperuricemia. Protease-activated receptor-2 (PAR2) is a class of targets that act upstream of the PI3K/AKT/NF-κB pathway and is involved in various inflammatory diseases. We induced a hyperuricemia model in rats by adenine and ethambutol gavage in an in vivo experiment. We demonstrated that PAR2 and PI3K/AKT/NF-κB pathway expression were significantly upregulated in renal tissues, with massive inflammatory cell infiltration in the renal interstitium and renal tissue injury. Treating hyperuricemic rats with AZ3451, a selective metabotropic antagonist of PAR2, we demonstrated that PAR2 antagonism inhibited the PI3K/AKT/NF-κB pathway and attenuated tubular dilation and tubulointerstitial inflammatory cell infiltration. The phospholipid metabolism profiles provided a perfect separation between the normal and hyperuricemic rats. In addition, we also found that AZ3451 can affect phospholipid metabolism. Our work suggests that PAR2 may mediate hyperuricemia-mediated renal injury by activating the PI3K/AKT/NF-κB pathway. The PAR2 antagonist AZ3451 may be a promising therapeutic strategy for hyperuricemia-induced inflammatory responses.

Ultra-sensitive electrochemiluminescent biosensor for miRNA based on CRISPR/Cas13a trans-cleavage-triggered hybridization chain reaction and magnetic-assisted enrichment.

The great selectivity and trans-cleavage activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a had been coupled with high amplification efficiency of hybridization chain reaction (HCR) and magnetic-assisted enrichment, high sensitivity of electrochemiluminescence (ECL) detection to develop an ultra-sensitive biosensor for microRNA-21 (miRNA-21). The CRISPR/Cas13a was used to recognize target RNA with high specificity and performed the trans-cleavage activity. An initiation strand was generated to bind to the probe on the surface of nanomagnetic beads and then trigged HCR to produce long double-strand DNAs (dsDNAs) to realize signal amplification. Ru(phen)32+ can be inserted in the groove of the dsDNAs and acts as the ECL indicator, which can be separated through magnetic enrichment and allowed the platform to reduce the signal background. Under the optimized conditions, there is a good linear correlation between the ECL intensity and the logarithm of miRNA-21 concentration in the range 1 fM-10 nM; the limit of detection (LOD) was 0.53 fM. The proposed system was applied to detect miRNA-21 from the urine of acute kidney injury (AKI) patients with good results.

Crosstalk Between Histone and m6A Modifications and Emerging Roles of m6A RNA Methylation.

RNA, like DNA and proteins, has been discovered to undergo dynamic and reversible chemical alterations, increasing the diversity and functional complexity of the molecule. N-6-methyladenosine (m6A) RNA methylation serves as a bridge between transcription and translation and is critical for many diseases' progression. There is a complex interrelationship between m6A modifications and other epigenetic modifications. Their crosstalk significantly affects transcriptional outputs, translation, recruitment of chromatin modifiers, as well as the deployment of the m6A methyltransferase complex at target sites. This article outlines the potential function of m6A RNA methylation in epigenetics and summarizes its interactions with histone modifications.

Heme Oxygenase 1/Peroxisome Proliferator-Activated Receptor Gamma Pathway Protects Intimal Hyperplasia and Mitigates Arteriovenous Fistula Dysfunction by Regulating Oxidative Stress and Inflammatory Response.

Purpose: An arteriovenous fistula (AVF) is the preferred vascular access mode for maintenance hemodialysis, and access stenosis and thrombosis are the primary causes of AVF dysfunction. This study is aimed at exploring the molecular mechanisms underlying AVF development and the roles of the heme oxygenase 1/peroxisome proliferator-activated receptor gamma (HO-1/PPAR-γ) pathway in AVF. Method: AVF model mice were established, and the vascular tissues from the arteriovenous anastomosis site were sent for mRNA sequencing. Differentially expressed mRNAs (DEmRNAs) were screened and subjected to functional analysis. Thereafter, the mice with HO-1 knockdown and coprotoporphyrin IX chloride (COPP) pretreatment were used to investigate the roles of the HO-1/PPAR-γ pathway in AVF. Results: By sequencing, 2514 DEmRNAs, including 1323 upregulated and 1191 downregulated genes, were identified. These DEmRNAs were significantly enriched in the PPAR signaling pathway, AMPK signaling pathway, glucagon signaling pathway, IL-17 signaling pathway, and Toll-like receptor signaling pathway. High expression of HO-1 and PPAR-γ reduced endothelial damage and intimal hyperplasia during AVF maturation. After AVF was established, the levels of transforming growth factor-ß (TGF-ß), interleukin-1ß (IL-1ß), interleukin-18 (IL-18), and reactive oxygen species (ROS) were significantly increased (P < 0.05), and HO-1 normal expression and COPP pretreatment evidently decreased their levels in AVF (P < 0.05). Additionally, AVF significantly upregulated HO-1 and PPAR-γ and downregulated MMP9, and COPP pretreatment and HO-1 normal expression further upregulated and downregulated their expression. Conclusion: The HO-1/PPAR-γ pathway may suppress intimal hyperplasia induced by AVF and protect the intima of blood vessels by regulating MMP9 and ROS, thus mitigating AVF dysfunction.

Correlation between histone H3K4 trimethylation and DNA methylation and evaluation of the metabolomic features in acute rejection after kidney transplantation.

OBJECTIVE: To investigate the difference between trimethylation of monocyte histone H3K4 and DNA methylation in acute rejection (AR) after renal transplantation in rats and reveal the epigenetic mechanism of the AR rats based on metabolomics. METHOD: Peripheral blood mononuclear cells (PBMCs) were isolated, and CD4 +CD25 + Treg cells were sorted by flow cytometry. The Foxp3 mRNA and protein levels of CD4 +CD25 + Treg cells were detected by real-time RT-PCR and Western blotting, respectively. High-throughput screening was applied to evaluate the H3K4 methylation of monocytes using chromatin immunoprecipitation with DNA microarray (ChIP-chip) and verified by ChIP with real-time PCR (ChIP-qPCR). Methylated DNA immunoprecipitation sequencing was combined with real-time PCR (MeDIP-qPCR) to detect the DNA methylation level of positive genes (ABCC4, Mef2d, Tbx1 and Eif6). Real-time quantitative PCR (qRT-PCR) and Western blotting were used to detect the mRNA and protein levels of positive genes. The difference in lipid metabolism in the blood of (non) acute rejection rats was analysed by HPLC/MS. RESULTS: AR rats showed an apparent increase in BUN and Cr levels, as well as IL-2, IL-10 and IFN-γ. Compared with non-AR rats, the expression of CD4 +CD25 + Treg cells and Foxp3 mRNA and protein were significantly lower in AR rats. AR rats also showed an increase in H3K4 trimethylation of CD4 +CD25 + Treg and decrease in DNA methylation. There were significant differences in the DNA methylation level of four positive genes between AR and non-AR rats (P<0.05), in addition to differences in the expression levels of mRNA and protein. Pathological examination of the transplanted kidney indicated that AR rats had more severe pathological injury of the kidney than the non-AR rats. There were significant increase in the contents of several phosphatidylcholines, lysophosphatidylcholine, free fatty acids and carnitine in AR rats which detected by HPLC/MS. CONCLUSION: H3K4 trimethylation increased in PBMCs in AR rats, while DNA methylation decreased, indicating the presence of epigenetic differences between AR and non-AR rats. Metabolomic studies indicated a significant increase in AR rats in the contents of several metabolites, such as phosphatidylcholines, lysophosphatidylcholine, free fatty acids and carnitine, suggesting an increasein phospholipase activity and leading to an energy metabolism imbalance with intensification of ß-oxidation. DNA methylation may be associated with an increase in phosphatidylcholines, lysophosphatidylcholine and free fatty acids in AR rats, which may further affect energy metabolism by enhancing the tricarboxylic acid cycle in AR rats.

Association between Nod-like receptor protein 3 inflammasome and gouty nephropathy.

Crystalized deposits of monosodium urate activate the Nod-like receptor protein 3 (NLRP3) inflammasome, resulting in kidney damage. The present study investigated whether the NLRP3 inflammasome is associated with the progression of hyperuricaemia and gouty nephropathy. Adult male patients were recruited at the Affiliated Baoan Hospital of Shenzhen and divided into three groups of 15 patients each: The control group, the hyperuricaemia group and the gouty nephropathy group. General characteristics and organ function indicators were also measured for each patient. NLRP3, apoptosis-associated speck like protein (ASC) and caspase-1 mRNA and protein expressions in peripheral blood mononuclear cells were detected. The expression of certain downstream inflammatory factors, including interleukin (IL)-1ß and IL-18 were also assessed in plasma. The results demonstrated that the concentration of uric acid and creatinine were increased in the hyperuricaemia and gouty nephropathy groups compared with the control group. NLRP3, ASC and caspase-1 mRNA and protein expression, and IL-1ß and IL-18 expression were increased in the hyperuricaemia and gouty nephropathy groups compared with the control group. In addition, ASC and caspase-1 mRNA and protein expression, and IL-1ß expression were higher in the gouty nephropathy group compared with the hyperuricaemia group. In conclusion, the present results supported the hypothesis that the NLRP3 inflammasome signalling pathway is associated with gouty nephropathy leading to initiation of the inflammatory response and causing renal damage.

NLRP3 inflammasome and lipid metabolism analysis based on UPLC-Q-TOF-MS in gouty nephropathy.

To determine the differences in plasma metabolism between healthy patients and patients with hyperuricaemia and gouty nephropathy, the present study identified differentially expressed metabolites associated with gouty nephropathy. Furthermore, the NLRP3 inflammasome signalling pathway in gouty nephropathy was explored, and the mechanism of hyperuricaemia­induced renal damage. Adult male patients examined between July 2016 and June 2017 were selected as the patient cohort for the present study from the Affiliated Bao'an Hospital of Shenzhen, Southern Medical University (Shenzhen, China). These patients were divided into three groups of 30 patients each: Control, hyperuricaemia and gouty nephropathy groups. The expression levels of NLRP3, ASC and caspase­1 mRNA and protein were detected in peripheral blood mononuclear cells, and the plasma levels of IL­1ß and IL­18. Ultra­performance liquid chromatography coupled with quadrupole time­of­flight mass spectrometry was used to determine differential levels of metabolites between patients from different groups, in order to identify potential biomarkers. The expression of the NLRP3 inflammasome in peripheral blood mononuclear cells, and the levels of IL­1ß and IL­18 in the plasma were increased in the gouty nephropathy group compared with the control and hyperuricaemia groups. In addition, 46 metabolites were identified as potential plasma metabolic biomarkers that were able to distinguish gouty nephropathy from hyperuricaemia. The majority of these metabolites were involved in lipid metabolism, in particular the activity of phospholipase Α2 and ß­oxidation. These data indicated that lipid metabolism and the NLRP3 inflammasome serve a pivotal role in gouty nephropathy. In addition, the results suggested that lipids may mediate the progression of gouty nephropathy through the activity of phospholipase A2, ß­oxidation and activation of the NLRP3 inflammasome.

Monocytes, endoplasmic reticulum stress and metabolomics in dogs with multiple organ dysfunction syndrome treated by continuous venovenous hemodiafiltration.

OBJECTIVES: We tried to investigate the mechanism of continuous venovenous hemodiafiltration (CVVHDF) treatment in monocytes function, endoplasmic reticulum (ER) stress signaling pathways, metabolomics and histopathological changes of MODS dogs, and aimed to enhance the understanding of pathogenesis and provide novel avenues to potential therapies. METHODS: 12 male Beagle dogs were used to develop the stable models of MODS by using hemorrhagic shock plus resuscitation and endotoxemia, and assigned randomly to CVVHDF group (n=6) and MODS group (n=6). The dogs in CVVHDF group were given the typical CVVHDF treatment for 24h after the completion of endotoxin intravenous infusion, while those in MODS group were offered the i.v heparin instead only. Serum sample were collected at five time points, i.e. before anesthesia, 0h, 6h, 12h and 24h after the endotoxin injection (T1~T5, respectively), and meanwhile, the changes of mRNA, protein and human umbilical vein endothelial cells (HUVECs) apoptosis rates in JNK, CHOP and Caspase-12 were observed before and after interfered by RNA interference technology. RESULTS: The levels of DLA-DR, IL-1ß and IL-4 were higher than those in MODS group after the CVVHDF treatment, and the early and late apoptosis rates showed downward trend compared with MODS group. In vitro and prior to RNA interference (RNAi), the levels of mRNA and protein expression and HUVECs apoptosis rates of JNK, CHOP and Caspase-12 in CVVHDF group were significantly lower compared to T1 and MODS group respectively. However, the levels of mRNA and protein expression and HUVECs apoptosis rates were significantly lower than those before interfered by RNAi in both two groups. The serum levels of LPCs, ornithine, proline, methionine, etc. were down-regulated while carnitines, FFAs, PC, etc. were increased significantly in MODS (T4), and the serum levels of methionine, proline, arginine and lysine were increased while carnitine, LPCs, PCs, SMs and orthophosporic acid were decreased after 12 hours CVVHDF treatment (T4). CONCLUSION: CVVHDF treatment could reduce the apoptosis of the cells by enhancing the antigen presentation, improving the anti-inflammatory and proinflammatory imbalance and even correcting the metabolic disorder of amino acids and phospholipids.

Improvement of immune dysfunction in dogs with multiple organ dysfunction by high-volume hemofiltration.

INTRODUCTION: Observing the effects of high-volume hemofiltration (HVHF) treatment on the monocytes apoptosis, antigen presentation, and secretion function, this study investigated the mechanism of HVHF effect on immunity homeostasis in multiple organ dysfunction syndrome (MODS) in an animal model. MATERIALS AND METHODS: Lipopolysaccharides were administered in 12 Beagle dogs in order to induce MODS. Six dogs were randomly assigned to receive HVHF treatment for 12 hours (HVHF group) and the rest did not receive any treatment (the MODS group). The expression of DLA-DR, apoptosis, and cytokine levels were measured at 7 time points: normal condition (T1), after operation (T2), and zero, 3, 6, 9, and 12 hours after endotoxin injection (T3 to T7, respectively). RESULTS: Apoptosis of CD14+ mononuclear cell increased in early and late stages gradually in the MODS group and began to decline gradually after the HVHF treatment. There was a significant difference between the two groups at time points T2 to T7 (P < .01). After HVHF, the impaired expression of dog leukocyte antigen-DR showed an improvement trend in the HVHF group, which was significant better at T5 and T7 than that in the MODS group (P < .05). Interleukin-4 secretion increased significantly with HVHF and was significantly higher at time points T4 to T7 as compared with the MODS group (P < .01).  Conclusions. High-volume hemofiltration can alleviate the mononuclear cell apoptosis, improve antigen-presenting function and secretion function, inhibit the release of inflammatory factors, and maintain immune homeostasis, and consequently alleviate symptoms of MODS effectively.