| BackgroundWith the prevalence of tacrolimus-based immunosuppressive regimens after renal transplantation,long-term nephrotoxicity has become increasingly prominent.Traditional and conservative tacrolimus-based immunotherapy results in an incidence of acute and chronic nephrotoxicity at more than 90%,followed by a decline of renal function at 4%per year and a series of concurrent adverse comorbidities.The risk of secondary renal replacement therapy threatened long-term life quality and safety of renal transplant patients.Early identification and effective intervention of tacrolimus nephrotoxicity are necessaryapproachestoreducetacrolimustoxiceffects.The pharmacokinetics/pharmacodynamics profiles of tacrolimus vary from person to person,making it difficult to infer the relationship between drug exposure and toxic effects.In clinical practice,identification of tacrolimus nephrotoxicity relies on therapeutic drug monitoring,biochemical indicators of renal function,and pathological diagnosis,followed by clinical interventions including drug reduce,change or withdtraw.However,the non-specific/insensitivitive/injurious characteristics of the above diagnostic approches are less instructive in decision making,for which repeated toxic effects lead in irreversible damage to the transplanted kidney.Therefore,establishment of a targeted analysis method for key amino acid metabolic pathways and information nodes is necessary for biomarker identification.An integrated biomarker panel based on amino acid phenotyping can be established with the facility of data extraction,analysis,discrimination and integration methods in bioinformatics.The multi-level biomarker system helps with the identification of“high-risk”patients of tacrolimus nephrotoxicity after renal transplantation for early intervention.Huangkui Capsule is a Traditional Chinese Medicine preparation from the ethanol extract of Abelmoschus capitata,which is widely used in the treatment of immunological kidney disease.Previous studies have confirmed that its pharmacological activities including improving proteinuria/sodium retention,regulating blood sugar/blood lipids,relieving inflammation/immune response.Therefore,it is herein hypothesized that Huangkui Capsule may affect the efficacy of tacrolimus and alleviate tacrolimus nephrotoxic effects.Of note,there is limited studies focusing on the combined administration of Huangkui capsule and tacrolimus,and the clinical application of Huangkui Capsule after renal transplantation is less prevalent.MethodsA systematic review and meta-analysis for risk factors of CNI nephrotoxicity was performed on all retrieved studies through a comprehensive research of network database.Data was analyzed by Review Manager 5.2 with heterogeneity assessed using the Cochrane Q and I~2 test.CNI nephrotoxicity was primarily indicated with protocol biopsy or index-based clinical diagnosis,and the secondary outcome was defined as delayed graft function.This study presented a targeted metabolomic assay to quantify 23 amino acids in human plasma by ultra-high performance liquid chromatography coupled with tandem mass spectrometry.Chromatographic separation was carried out on an Agilent Zorbax SB-C18 column(3.0 mm×150 mm,5μm)with addition of an ion-pairing agent in the mobile phase,and MS/MS detection was achieved in the positive multiple reaction monitoring(MRM)mode.Blood samples from 25 apparently healthy subjects were collected into duplicate sets of EDTA-2K,EDTA-3K,coagulation,heparin and citrate tubes,and stored in capped vacutainer tubes at 4°C for 6 h,12 h and 24 h before sample analysis.ANOVA for repeated measurement was conducted based on the model of Mauchly’s test of Sphericity.The Pearson/Spearman correlation coefficients between amino acid concentrations and biochemical indexes were visualized with a heat map.Student’s t-test was applied for comparison between amino acid concentrations obtained from different vacutainer tubes,and consistency of the results was checked through Bland-Altman difference plots and Passing-Bablok regression analysis.Targeted amino acid phenotyping was conducted on a pre-validation set including 122plasma samples from 42 post-transplant patients with/without tacrolimus nephrotoxicity and well-matched healthy volunteers for biomarker identification,and an additional set of42 patients was enrolled for biomarker validation.A total of 23 amino acids was analyzed using the ultra-high performance liquid chromatography-tandem mass spectrometry system.The influential amino acid biomarkers identified through pattern recognition and cross-group comparison were incorporated into a biomarker panel together with genetic,operational and anthropometric parameters.A rat model was constructed to study tacrolimus nephrotocixity.The models were divided into blank control group(A),tacrolimus nephrotoxicity group(B)and combined administration of tacrolimus with Huangkui Capsules group(C).Blood and tissue samples of rats were collected at different time for examination of renal biochemical parameters,histopathology and drug concentration.Amino acid biomarkers at different sampling time were examined of sensitivity and specificity compared with conventional renal function indexes.ResultsTwelve observational studies containing a total of 2 849 cases were identified.Donor age(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 with no heterogeneity.Good correlation coefficients(r~2>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 FDA Guidance(2013)as the Chinese Phamacopedia(2015)Most amino acids showed a small concentration fluctuation with storage time in heparin plasma,followed by EDTA-3K.The detected amino acid concentrations were significantly lower in citrate plasma and slightly higher in serum,compared to those in heparin plasma.No fixed bias was observed of amino acid concentrations in EDTA and heparin plasma,but the differences for almost all the amino acids were of statistical significances.Amino acid concentrations in EDTA-3K plasma achieved a good consistency with those in heparin plasma by UHPLC-MS/MS analysis.A perturbed amino acid metabolic pattern was observed under tacrolimus nephrotoxicity from the without,denoting cysteine,isoleucine,methionine,symmetric dimethylarginine and tryptophan/kynurenine as influential biomarkers.Altered metabolic pathways concomitant with tacrolimus nephropathology included increased“arginine”,“methylation”,“kynurenine”metabolism and“isoleucine biosysthesis”.A logistic model incorporating the five amino acid parameters(AUC>0.7)and three primarily identified risk factors(donor age,recipient zero-time arteriosclerosis and CYP3A5 genotype)obtained the AUCs>0.8 in the pre-validation set(n=122),which remained influential in the validation set(n=42).Results on rat model proved that the combined administration of Huangkui Capsule and tacrolimus could effectively reduce and delay the onset of tacrolimus nephrotoxicity.The molecular mechanism was reckoned to be the reduction of the degree of renal interstitial fibrosis,change of the renal tubular phenotype and tubular dilation state as well as the infiltration of renal interstitial inflammatory cells.The blood concentration of tacrolimus was not influenced under Huangkui Capsule administration.Amino acid biomarkers exhibited significant changes at the early stage of tacrolimus nephrotoxicity.Biomarkers(symmetric dimethylarginine as the best)presented significant fluctuation than conventional renal function markers such as serum creatinine and urea nitrogen with better sensitivity.ConclusionsDonor age,recipient arteriosclerosis and receptor CYP3A5*3/*3 genotype were statistically significant risk factors for calcineurin inhibitor nephrotoxicity and could be applied as effective covariates for integrated biomarker system.The amino acid-targeted metabolomic phenotyping established in this study enabled an accurate and rapid quantification of 23 amino acids in human plasma.Efficient control of the two important variables in pre-analytical processes,namely blood collection tube and sample storage time,could effectively reduce measurement error.A biomarker system based on amino acid phenotyping was established through retrospective clinical study,and the robustness of the model was examined with high sensitivity in both the“pre-validation”and“validation”sets.The amino acid biomarkers were initially validated in an animal model.Combined administration of Huangkui Capsule and tacrolimus could effectively reduce renal damage,which could be applied in clinical practice. |
PDF Full Text Request