Acyloxyacyl Hydrolase Protects against Kidney Injury via Inhibition of Tubular CD74-Macrophage Crosstalk.

Renal fibrosis is the common pathway in the progression of chronic kidney disease (CKD). Acyloxyacyl hydrolase (AOAH) is expressed in various phagocytes and is highly expressed in proximal tubular epithelial cells (PTECs). Research shows that AOAH plays a critical role in infections and chronic inflammatory diseases, although its role in kidney injury is unknown. Here, we found that AOAH deletion led to exacerbated kidney injury and fibrosis after folic acid (FA) administration, which was reversed by overexpression of Aoah in kidneys. ScRNA-seq revealed that Aoah-/- mice exhibited increased subpopulation of CD74+ PTECs, though the percentage of total PTECs were decreased compared to WT mice after FA treatment. Additionally, exacerbated kidney injury and fibrosis seen in Aoah-/- mice was attenuated via administration of methyl ester of (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), an inhibitor of macrophage inhibition factor (MIF) and CD74 binding. Finally, AOAH expression was found positively correlated with estimated glomerular filtration rate while negatively correlated with the degree of renal fibrosis in kidneys of CKD patients. Thus, our work indicates that AOAH protects against kidney injury and fibrosis by inhibiting renal tubular epithelial cells CD74 signaling pathways. Targeting kidney AOAH represents a promising strategy to prevent renal fibrosis progression.

Automated regional citrate anticoagulation based on online monitoring of ionized calcium concentration: Proof of concept.

BACKGROUND: Regional citrate anticoagulation (RCA), a complex and effective technique, is recommended as the anticoagulation of choice for continuous renal replacement therapy. One of its key objectives is to keep the ionized calcium in the targeted range. In this study, we aimed to develop an automated RCA based on online monitoring of the ionized calcium concentration and closed-loop feedback. METHODS: We constructed calcium-selective electrodes with liquid inner contact, which measured a potentiometric signal as the output. We tested the responses, stability, and selectivity of the electrodes in flowing fluid containing calcium chloride. We compared the measurement accuracy between the electrodes and an i-STAT system in vivo. Moreover, we established closed-loop feedback using a proportional-integral-derivative controller model. We performed simulated automated RCA both in vivo and in vitro. RESULTS: The electrode gave a Nernstian response to the variation of ionized calcium concentration. It showed high stability and a relatively short response time. Changes in the fluid flow rate, solution PH, and addition of metal ions including Mg2+ and K+ did not interfere with the measurements of ionized calcium. These measurements in whole blood by the electrode were very close to those assessed by the i-STAT system. The feedback control system responded quickly to an abnormal ionized calcium concentration and regulated the infusion rates of calcium or citrate to maintain the concentration of ionized calcium within the targeted range. CONCLUSIONS: We successfully trialed automated RCA, which may help simplify the complexities of RCA in the future.

Polyethyleneimine-anchored liposomes as scavengers for improving the efficiency of protein-bound uremic toxin clearance during dialysis.

Protein-bound uremic toxins (PBUTs) are significant toxins that are closely related to the prognosis of chronic kidney disease. They cannot be effectively removed by conventional dialysis therapies due to their high albumin binding affinity. Our previous research revealed that cationic liposomes (i.e., polyethyleneimine [PEI]-decorated liposomes) could enhance the clearance of PBUTs via electrostatic interactions. However, the poor biocompatibility (hemolysis) restricted their applications in clinical dialysis treatment. Herein, we produced PEI-anchored, linoleic acid-decorated liposomes (CP-LA liposomes) via the conjugation of PEI to cholesterol chloroformate (Chol-PEI, CP), and linoleic acid (LA) was added to provide liposomal colloidal stability. The CP-LA liposomes outperformed the plain liposomes, demonstrating significantly higher PBUT binding rates and removal rates. In addition, in vitro dialysis simulation verified that the CP-LA liposomes had a better capacity for PBUT clearance than the plain liposomes, especially for PBUTs with a strong negative net charge. Hemolysis and cytotoxicity tests revealed that the biocompatibility of the CP-LA liposomes was better than that of the physically-decorated PEI-liposome. CP-LA liposomes possess great potential for PBUT clearance in clinical dialysis therapy.

Linoleic acid-modified liposomes for the removal of protein-bound toxins: An in vitro study.

INTRODUCTION: Protein-bound uremic toxins (PBUTs) and liver failure-related cholestatic solutes are associated with adverse outcomes in patients with chronic kidney disease (CKD) and liver failure, respectively, and are not easily removed by traditional dialysis therapies. We constructed linoleic acid-modified liposomes (LA-liposomes) as indirect adsorbent in the dialysate, and evaluated their effects on the clearance of the representative PBUTs and cholestatic solutes. METHODS: The LA-liposomes were prepared by the thin-film hydration method. The binding rates of liposomes and protein-bound solutes were detected by the ultrafiltration column. The in vitro dialysis experiments were performed using both non-current and current devices to assay the clearing efficiency of the dialysate supported by LA-liposomes. RESULTS: The LA-liposomes exhibited good binding properties to the PBUTs, bilirubin and bile acids. The LA-liposome dialysate showed higher solute reduction rates of the representative PBUTs and cholestatic solutes than the traditional dialysate or dialysate supported by the unmodified plain liposomes. Also, albumin binding of the PBUTs was significantly inhibited by the addition of linoleic acid (LA), and the removal efficiency of PBUTs was greatly enhanced by the combination of indirect adsorbent LA-liposomes and LA as the competitive displacer. CONCLUSION: LA-liposomes were efficient in the clearance of the representative PBUTs and liver failure-related solutes. Moreover, the combination of indirect adsorbent LA-liposomes and competitive displacer suggested a potential application for the extremely highly-bound solutes.

Pharmacokinetics of N-ethylpentylone and its effect on increasing levels of dopamine and serotonin in the nucleus accumbens of conscious rats.

N-Ethylpentylone (NEP) is one of the most confiscated synthetic cathinones in the world. However, its pharmacology and pharmacokinetics remain largely unknown. In this study, the pharmacokentics of NEP in rat nucleus accumbens (NAc) was assessed via brain microdialysis after the intraperitoneal (ip) administration of NEP (20 or 50 mg/kg). The concentrations of dopamine (DA) and serotonin (5-HT) and their metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and 5-hydroxyindoleacetic acid (5-HIAA), were simultaneously monitored to elucidate the pharmacological effect of NEP. In addition, the plasma levels of NEP were also assessed. The pharmacokinetics of NEP showed a dose-related pattern, with NEP rapidly passing through the blood-brain barrier and reaching a maximum concentration (Cmax ) at approximately 40-minutes postdose. Approximately 4% of plasma NEP was distributed to the NAc, and considering a homogeneous brain distribution, over 90% of plasma NEP was potentially distributed to the brain. High values of area under curve (AUC) and mean residence time (MRT) of NEP were observed in both the NAc and plasma, indicating large and long-lasting effects. NEP elicited dose-related increases in microdialysate DA and 5-HT and increased the concentration of 3-MT in a dose-related manner. However, the rate of DA converted into 3-MT was unaffected. NEP had a negative effect on the rates of which DA and 5-HT were transformed into DOPAC and 5-HIAA, respectively. In summary, NEP rapidly entered the NAc and showed a long-lasting effect. In addition, DA increased more significantly than 5-HT, indicating a large potential for NEP abuse.

An LC-MS/MS method for comparing the stability of ethanol's non-oxidative metabolites in dried blood spots during 90 days.

Problems of stability were found for biomarkers of alcohol consumption: ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths), and fatty acid ethyl esters (FAEEs) in whole blood. The purpose of this study was to establish a method for the determination of these four kinds of ethanol's non-oxidative metabolites in dried blood spots (DBS) by liquid chromatography tandem mass spectrometry (LC-MS/MS), and to evaluate their stability. In this method, 50 µL of human blood was spotted onto a filter paper for DBS analysis. Samples were extracted by methanol, reconstituted by 2-propanol, and injected into the LC-MS/MS system. Limits of detection were among 0.5-50 ng/mL, and deviations in accuracy and precision were all lower than 15% at three quality control levels. The stability of the four kinds of ethanol non-oxidative metabolites in DBS was investigated during a 90-day range under three temperatures, -20 °C, 4 °C, and 25 °C. EtG and EtS showed a high level of stability in DBS in the 90-day range, regardless of the temperature. FAEEs were unstable after three days. PEths showed stability within 15 days in postmortem DBS and 60 days in antemortem DBS, respectively, at all temperatures.

Simultaneous determination of N-ethylpentylone, dopamine, 5-hydroxytryptamine and their metabolites in rat brain microdialysis by liquid chromatography tandem mass spectrometry.

N-Ethylpentylone (NEP) is a popular synthetic cathinone abused worldwide. To obtain more information about its pharmacokinetics and pharmacodynamics, a rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method was developed for the determination of NEP, two important neurotransmitters, dopamine and serotonin, and their metabolites, including 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine and 5-hydroxyindole-3-acetic acid, in rat brain microdialysate. The analytes were separated on a Phnomenex Polar C18 column, with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) under gradient elution to shorten the total chromatographic run time. A triple quadruple mass spectrometer coupled with an electrospray ionization source in both positive and negative ion mode was used to detect the analytes. This method showed excellent accuracy (87.4-113.5%) and precision (relative standard deviation <15%) at three quality control levels. The limits of detection were 0.2 ng/mL for NEP and 0.2-50 nm for the others and good linearity was obtained. This study pioneered a method to integrate exogenous drugs and endogenous neurotransmitters as the drugs act on the same determination system, which means that this innovation can provide support for further study of the addictive effects of NEP or other synthetic cathinones on extracellular levels of dopamine and 5-hydroxytryptamine.

Effects of N-ethylpentylone on locomotor activity and anxiety-like behavior in rats.

N-ethylpentylone (NEP), a new synthetic cathinone, has been rising to be one of the most popular cathinone derivatives in recent years. However, research on NEP is rather limited. In this study, locomotor stimulation and sensitization, as well as anxiety-like behavior induced by NEP were studied in Sprague-Dawley rats, using the open field and elevated plus maze respectively. Rats were administered NEP (5, 20 or 50 mg/kg, intraperitoneal), with saline as the negative control and methamphetamine (5 mg/kg) as a positive control. Acute administration of NEP at all the doses tested significantly promoted locomotor activity, presenting an inverted U-shaped dose-effect curve. The highest activity was observed at the 20 mg/kg dose group, with the average distance traveled 18 times higher than the saline group. Repeated administration of NEP enhanced locomotor activity only at the 5 mg/kg dose group. After a week's withdrawal, re-challenge of NEP failed to induce marked behavioral sensitization. In elevated plus maze experiments, both acute and repeated administration of 20 mg/kg NEP induced anxiolytic-like effects, while no significant alteration was observed in the 5 and 50 mg/kg dose groups. In summary, acute administration of NEP caused significantly enhanced locomotor activity in rats at all the tested doses, while repeated NEP administration enhanced locomotor activity only at a low dose (5 mg/kg), while a high dose (20 mg/kg) of NEP induced anxiolytic-like effects after both acute and repeated administration.

Esculin ameliorates cognitive impairment in experimental diabetic nephropathy and induces anti-oxidative stress and anti-inflammatory effects via the MAPK pathway.

Esculin is a derivative of coumarin, which is also an active ingredient of ash bark, and has antibacterial, anti-inflammatory, anti­allergy and skin protective effects. The underlying mechanism and protective effects of esculin on cognitive impairment in experimental diabetic nephropathy (DN) was investigated in the present study. Male C57BL/6J 6­week­old mice were injected intravenously with a single dose of streptozotocin (STZ; 30 mg/kg). At 2 weeks after the STZ injection, mice received intravenous injection with 5, 10 or 20 mg/kg esculin for 2 weeks. In the present study, the results of the Morris water maze test demonstrated that esculin significantly improved behavior and recognition memory in STZ­induced diabetic rats. Furthermore, treatment of STZ­induced diabetic rats with esculin significantly inhibited tumor necrosis factor­α, interleukin­6, malondialdehyde, monocyte chemoattractant protein­1 and intracellular adhesion molecule­1 activity levels, and increased the activity of superoxide dismutase, in the kidney, which was determined by ELISA. In addition, esculin treatment significantly suppressed the renal protein expression of activator protein 1, phosphorylated (p)­p38 mitogen activated protein kinase (MAPK) and p­c­Jun N­terminal kinase, and increased p­extracellular signal regulated kinase 1/2 protein expression, in STZ­induced diabetic rats, as determined by western blotting. These results indicate that esculin may ameliorate cognitive impairment in experimental DN, and exert anti­oxidative stress and anti­inflammatory effects, via the MAPK signaling pathway. Thus, it may serve as a potential target for cognitive impairment of DN in the future.

Stability of Ethyl Glucuronide, Ethyl Sulfate, Phosphatidylethanols and Fatty Acid Ethyl Esters in Postmortem Human Blood.

The lack of systematic studies on the stability of ethanol's non-oxidative metabolites in postmortem specimens restricts their use in forensic cases. This study aimed to compare the stability of ethyl glucuronide (EtG), ethyl sulfate (EtS), phosphatidylethanols (PEths) and fatty acid ethyl esters (FAEEs) in postmortem human blood. Three groups were established based on the level and source of ethanol: the blank group, the ethanol-spiked group and the ethanol-positive group. Each group contained six blood samples from different corpses. The samples in each group were placed at 37, 25, 4 and -20°C. Every 24 h for 7 days, 50 µL was collected from each sample. The levels of EtG, EtS, PEths and FAEEs were determined by liquid chromatography-mass spectrometry, and their stability was evaluated. EtG was not detected in the blank group, but it was found in samples in the ethanol-spiked group placed at 37°C, and it was degraded in the ethanol-positive group at 37 and 25°C. EtS showed no change in any of the groups. PEths were not detected in the blank group, but formation was found in the ethanol-spiked group at all temperatures. In the ethanol-positive group, PEth levels fluctuated at 37°C, decreased at 25°C and increased at -20°C. FAEEs were generated in the blank group and in the ethanol-spiked group at all temperatures. In the ethanol-positive group, FAEEs were degraded at 37 and 25°C but were generated at 4 and -20°C. EtS is a reliable biomarker of ethanol consumption, and EtG could be used as a biomarker at low temperatures (4 and -20°C), but PEths and FAEEs are not appropriate biomarkers of ethanol consumption.

Efficient determination of six fatty acid ethyl ethers in human whole blood by gas chromatography-mass spectrometry.

Fatty acid ethyl esters (FAEEs) have been widely studied as specific markers of ethanol intake and mediators of ethanol-induced diseases. In the present study, a simple and rapid gas chromatography-mass spectrometry (GC-MS) method was established for the qualitative and quantitative analysis of six fatty acid ethyl esters (FAEEs), including ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate, and ethyl arachidonate, in human whole blood. FAEEs were extracted from 200 µL of human whole blood by a modified liquid-liquid extraction, and the hexane layer was injected directly into GC-MS with ethyl heptadecanoate as the internal standard. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 5-50 ng/mL and 15-200 ng/mL, respectively. Linearity ranged up to 10 µg/mL with r2 higher than 0.998. Accuracy was in the range of 90.3-109.7%, while intra-day and inter-day precision were 0.7-9.3% and 3.4-12.5%, respectively. This method was then applied to 38 real samples from forensic cases. Differences in the most common FAEEs between Chinese and Western subjects were discussed. The relationship of FAEE concentrations with age and gender was also investigated.

Simultaneous determination of ethanol's four types of non-oxidative metabolites in human whole blood by liquid chromatography tandem mass spectrometry.

The importance of ethanol non-oxidative metabolites as the specific biomarkers of alcohol consumption in clinical and forensic settings is increasingly acknowledged. Simultaneous determination of these metabolites can provide a wealth of information like drinking habit and history, but it was difficult to achieve because of their wide range of polarity. This work describes development and validation of a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for 4 types of ethanol non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters and phosphatidylethanols) in 50 µL of human whole blood. Pretreatment method, column and MS conditions were optimized. For the first time, the four types of ethanol non-oxidative metabolites with enormous discrepancies of property were simultaneously extracted and analyzed in one run within 40 min. The limits of detections (LODs) were among 0.1-10 ng/mL, and good linearity was obtained. Deviations in precision and accuracy were all lower than 15% at three QC levels. This method was then applied to two forensic samples, resulting in information on drinking habits and drinking time which were very useful for the interpretation of the blood alcohol results.

Ultrasound-assisted dispersive liquid-liquid microextraction for the determination of seven recreational drugs in human whole blood using gas chromatography-mass spectrometry.

Recreational drugs have large impact on public health and security, and to monitor them is of urgent demand. In the present study, ultrasound-assisted dispersive liquid-liquid microextraction combined with the detection of gas chromatography-mass spectrometry was applied to the determination of seven common recreational drugs, including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, meperidine, methadone and ketamine in 200µL of human whole blood. A series of factors which would affect the extraction efficiency were systematically investigated, including the nature and the volume of extraction and dispersing solvents, ultrasonication time, salting-out effect and pH value. The method consumed small amount of sample. The limits of detection and limits of quantification for each analyte were 10 and 40ng/mL, respectively, and the linearity was in the range of 0.04-25µg/mL (R2 higher than 0.99). Good specificity, precision (1.5-8.2% for the intra-day study and 2.6-12.8% for the inter-day study), satisfactory accuracy (85.0-117.1%) and extraction recovery (77.0-92.4%) were obtained, which makes it a high performance method for the determination of recreational drugs in human whole blood samples.

Rapid determination of nine barbiturates in human whole blood by liquid chromatography-tandem mass spectrometry.

A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the qualitative and quantitative analysis of nine barbiturates (barbital, phenobarbital, pentobarbital, amobarbital, secobarbital, thiopental, butalbital, butabarbital, and hexobarbital) in human whole blood. Barbiturates were extracted from 100 µL of human whole blood samples using a simple liquid-liquid extraction (LLE) procedure, and detected by LC-MS/MS. An UPLC C18 (2.1 mm × 100 mm, 1.7 µm) column was used at 40 °C for the separation and acetonitrile/water system was used as the mobile phase with gradient elution. This method showed excellent accuracy (86-111%) and precision (relative standard deviation <15%). The limits of detection (LODs) were 0.2 ng/mL for barbital and secobarbital and 0.5 ng/mL for the other barbiturates. The linearity ranged from 2 ng/mL to 2000 ng/mL, with r2 > 0.99 over the range. This method achieved the separation and detection of pentobarbital and amobarbital at the same time in a convenient way. Moreover, it was both simple and sensitive for the determination of nine most commonly used barbiturate drugs, which was meaningful in the field of clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd.