ProfKin: A comprehensive web server for structure-based kinase profiling.

Protein kinases are central mediators of signal-transduction cascades and attractive drug targets for therapeutic intervention. Since kinases are structurally and mechanistically related to each other, kinase inhibitor selectivity is often investigated by kinase profiling and considered as an important index for drug discovery. We here describe a versatile web server termed ProfKin for structure-based kinase profiling, which is based on a kinase-ligand focused database (KinLigDB). It provides all ready-to-use 3D structure coordinates of 4219 kinase-ligand complex structures covering 297 human kinases and the associated information, particularly including binding site type, binding ligand type, interaction fingerprints, downstream molecules and related human diseases. The web server works via predicting possible binding modes for the query molecule, prioritizing the binding modes guided by an interaction fingerprint analysis method, and giving a list of ranked kinases by a comprehensive index. Users can freely select entire or part of the KinLigDB database, e.g. via subfamily and binding site type, to customize the profiling contents. The superimpositions of the predicted binding poses of the query molecule with reference binding modes can be visually inspected on the website. The additional classification attributes and phylogenetic tree are also given for each top-ranked kinase.

Effects of p-Cresol on Oxidative Stress, Glutathione Depletion, and Necrosis in HepaRG Cells: Comparisons to Other Uremic Toxins and the Role of p-Cresol Glucuronide Formation.

The toxicological effects of p-cresol have primarily been attributed to its metabolism products; however, very little human data are available in the key organ (i.e., liver) responsible for the generation of these metabolites. Experiments were conducted in HepaRG cells utilizing the following markers of cellular toxicity: 2'-7'-dichlorofluorescein (DCF; oxidative stress) formation, total cellular glutathione (GSH) concentration, and lactate dehydrogenase (LDH; cellular necrosis) release. Concentrations of p-cresol, p-cresol sulfate, and p-cresol glucuronide were determined using validated assays. p-Cresol exposure resulted in concentration- and time-dependent changes in DCF (EC50 = 0.64 ± 0.37 mM at 24 h of exposure) formation, GSH (EC50 = 1.00 ± 0.07 mM) concentration, and LDH (EC50 = 0.85 ± 0.14 mM) release at toxicologically relevant conditions. p-Cresol was also relatively more toxic than 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, and hippuric acid on all markers. Although the exogenous administration of p-cresol sulfate and p-cresol glucuronide generated high intracellular concentrations of these metabolites, both metabolites were less toxic compared to p-cresol at equal-molar conditions. Moreover, p-cresol glucuronide was the predominant metabolite generated in situ from p-cresol exposure. Selective attenuation of glucuronidation (without affecting p-cresol sulfate formation, while increasing p-cresol accumulation) using independent chemical inhibitors (i.e., 0.75 mM l-borneol, 75 µM amentoflavone, or 100 µM diclofenac) consistently resulted in further increases in LDH release associated with p-cresol exposure (by 28.3 ± 5.3%, 30.0 ± 8.2% or 27.3 ± 6.8%, respectively, compared to p-cresol treatment). These novel data indicated that p-cresol was a relatively potent toxicant, and that glucuronidation was unlikely to be associated with the manifestation of its toxic effects in HepaRG cells.

Metabolomics Evaluation of Patients With Stage 5 Chronic Kidney Disease Before Dialysis, Maintenance Hemodialysis, and Peritoneal Dialysis.

OBJECTIVE: Current treatment options for patients with stage 5 chronic kidney disease before dialysis (predialysis CKD-5) are determined by individual circumstances, economic factors, and the doctor's advice. This study aimed to explore the plasma metabolic traits of patients with predialysis CKD-5 compared with maintenance hemodialysis (HD) and peritoneal dialysis (PD) patients, to learn more about the impact of the dialysis process on the blood environment. METHODS: Our study enrolled 31 predialysis CKD-5 patients, 31 HD patients, and 30 PD patients. Metabolite profiling was performed using a targeted metabolomics platform by applying an ultra-high-performance liquid chromatography-tandem mass spectrometry method, and the subsequent comparisons among all three groups were made to explore metabolic alterations. RESULTS: Cysteine metabolism was significantly altered between predialysis CKD-5 patients and both groups of dialysis patients. A disturbance in purine metabolism was the most extensively changed pathway identified between the HD and PD groups. A total of 20 discriminating metabolites with large fluctuations in plasma concentrations were screened from the group comparisons, including 2-keto-D-gluconic acid, kynurenic acid, s-adenosylhomocysteine, L-glutamine, adenosine, and nicotinamide. CONCLUSION: Our study provided a comprehensive metabolomics evaluation among predialysis CKD-5, HD, and PD patients, which described the disturbance of metabolic pathways, discriminating metabolites and their possible biological significances. The identification of specific metabolites related to dialysis therapy might provide insights for the management of advanced CKD stages and inform shared decision-making.

Risk factors for calcineurin inhibitor nephrotoxicity after renal transplantation: a systematic review and meta-analysis.

BACKGROUND: Nephrotoxicity of calcineurin inhibitors (CNIs) is the major concern for long-term allograft survival despite its predominant role in current immunosuppressive regime after renal transplantation. CNI nephrotoxicity is multifactorial with demographic, environmental, and pharmacogenetic flexibility, whereas studies indicating risk factors for CNI nephrotoxicity obtained incomplete or conflicting results. METHODS: A systematic review and meta-analysis of risk factors for CNI nephrotoxicity was performed on all retrieved studies through a comprehensive research of network database. Data were analyzed by Review Manager 5.2 with heterogeneity assessed using the Cochrane Q and I2 tests. CNI nephrotoxicity was primarily indicated with protocol biopsy or index-based clinical diagnosis, and the secondary outcome was defined as delayed graft function. RESULTS: Twelve observational studies containing a total of 2,849 cases were identified. Donor age (odds ratio [OR], 1.01; 95% CI, 1.01-1.03; p=0.02), recipient zero-time arteriosclerosis (OR, 1.44; 95% CI, 1.04-1.99; p=0.03), and CYP3A5*3/*3 genotype (OR, 2.80; 95% CI, 2.63-2.98; p=0.00) were confirmed as risk factors for CNI nephrotoxicity. Subgroup and sensitivity analysis claimed donor age as a significant contributor in Asian and Caucasian areas. CONCLUSION: Older donor age, recipient zero-time arteriosclerosis, and CYP3A5*3/*3 genotype might add up the risk for CNI nephrotoxicity, which could be interpreted into a robust biomarker system.

Targeted metabolomic analysis of 33 amino acids and biogenic amines in human urine by ion-pairing HPLC-MS/MS: Biomarkers for tacrolimus nephrotoxicity after renal transplantation.

Calcineurin inhibitor nephrotoxicity, especially for the widely used tacrolimus, has become a major concern in post-transplant immunosuppression. Multiparametric amino acid metabolomics is useful for biomarker identification of tacrolimus nephrotoxicity, for which specific quantitative methods are highlighted as a premise. This article presents a targeted metabolomic assay to quantify 33 amino acids and biogenic amines in human urine by high-performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was carried out on an Agilent Zorbax SB-C18 column (3.0 × 150 mm, 5 µm) with addition of an ion-pairing agent in the mobile phase, and MS/MS detection was achieved in both the positive and negative multiple reaction monitoring modes. Good correlation coefficients (r2 > 0.98) were obtained for most analytes. Intra- and inter-day precision, stability, carryover and incurred sample reanalysis met with the acceptance criteria of the guidance of the US Food and Drug Administration. Analysis on urine from healthy volunteers and renal transplantation patients with tacrolimus nephrotoxicity confirmed symmetric dimethylarginine and serine as biomarkers for kidney injury, with AUC values of 0.95 and 0.81 in receiver operating characteristic analysis, respectively. Additionally, symmetric dimethylarginine exhibited a tight correlation with serum creatinine, and was therefore indicative of renal function. The targeted metabolomic assay was time and cost prohibitive for amino acid analysis in human urine, facilitating the biomarker identification of tacrolimus nephrotoxicity.

Isolation and characterization of putative mesenchymal stem cells from mammalian gut.

Here, we provide a protocol for reliable isolation and subculture of putative mesenchymal stem cells from mice colons. This method provides a good approach to cultivate and characterize putative colonic mesenchymal stem cells (cMSCs). A high purity of cMSCs can be obtained according to this protocol. The whole isolation processes of cMSCs take about 2 h with two important digestion steps involved. Only with common culture medium, maturation of cMSCs in culture proceeds approximately 2 weeks to allow relevant researches to be conducted. This protocol sheds light on better cultivation of MSCs in vitro from post-natal colon tissues. These putative cMSCs share common phenotypic property with those in vivo reported, and contain potential lineage differentiation capacity. The successful culture of cMSCs in vitro provides an ideal model for study of MSCs biology in the intestine.