Vitamin D supplementation reduced blood inflammatory cytokines expression and improved graft function in kidney transplant recipients.

Background: Chronic allograft dysfunction(CAD) is the leading cause of graft loss in kidney transplant recipients (KTRs). Inflammatory process is believed to be one of the major contributors to CAD. The aim of this study is to explore the anti-inflammatory effect of vitamin D (VD) supplementation in KTRs and its role in the graft function improvement(protection). Methods: A retrospective cohort of 39 KTRs with chronic antibody mediated rejection(CAMR)or stable renal function and a prospective cohort of 42 KTRs treated or untreated with VD were enrolled. Serum levels of vitamin D metabolism and serum inflammatory cytokines, renal graft function, and routine blood biomarkers were tested and dynamically tracked within 12 months post-transplant. Results: Compared with the stable group, the CAMR group exhibited significantly elevated serum levels of inflammatory cytokines IL-1ß, IFN-γ, IL-2, IL-10, IP-10, and HMGB1 (P <0.05). The supplementation of vitamin D effectively increased the serum concentration of vitamin D in kidney transplant recipients (KTRs) in the treated group. During the course of treatment, the treated group exhibited a gradual increase in eGFR levels, which were significantly higher than those observed in the untreated group at 12 months post-transplant (p<0.05). Notably, as eGFR improved, there was a significant decrease in levels of IL-1ß, IFN-γ, IL-2, IL-10, IP-10 and HMGB1 in the treated group compared to the untreated group (P<0.05). Conclusion: This study confirmed that immune-inflammation is a crucial factor in the development of CAD in KTRs.VD deficiency impairs its anti-inflammatory activity. By assisting in the regulation of excessive immune inflammation and restoration of immune homeostasis, effective VD supplementation contributes to protection and maintenance of graft function in KTRs.

Highly sensitive and rapid determination of azathioprine metabolites in whole blood lysate by liquid chromatography-tandem mass spectrometry.

Individualized therapy involves genetic test of drug metabolism, which provides information about the initial dose and therapeutic drug monitoring for adjusting the subsequent dose. Consequently, toxic side effects are expected to be minimized and therapeutic effects to be maximized. In this study, an ultra-performance liquid chromatography tandem mass spectrometry method that was specific, accurate and sensitive was developed to simultaneously determine azathioprine two metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methyl-mercaptopurine riboside (6-MMPr) in the whole blood lysate. We precipitated the sample by trifluoroacetic acid under the protection of dithiothreitol, with 6-MMPr and 6-TGN being hydrolyzed to produce 6-methymercaptopurine and 6-thioguanine. In the chromatographic separation, Waters ACQUITY BEH C18 (2.1 × 100 mm, 1.7 µm) chromatographic column was applied and gradient elution was conducted with 0.02 mol/L ammonium acetate buffer (which contains 0.3% formic acid) and acetonitrile at a flow rate of 0.4 ml/min. Tandem mass spectrometry in multiple reaction monitoring mode was applied for detection via electrospray ionization source in positive ionization mode. The analyzing process lasted for no more than 2 min. The calibration curve for each metabolite fitted a least squares model (weighed 1/X) from 1.25 to 5000 ng/ml (r2 > 0.99). The ion pairs were detected as 6-MMP m/z 167.07 â†’ 152.15, 6-TG m/z 168.06 â†’ 134.13, and internal standard m/z 171.07 â†’ 137.14. Under the guidance of FDA guidelines for bioanalytical method validation, we carried out validation and obtained satisfactory results. The method was successfully utilized for monitoring the concentrations of each metabolite from 65 affected patients who had received azathioprine maintenance therapy and achieved optimal results.

Correlation of CYP2C19 genotype with plasma voriconazole exposure in South-western Chinese Han patients with invasive fungal infections.

The aim of this study was to investigate the correlation between CYP2C19 genotype and dose-adjusted voriconazole (VCZ) trough concentrations (C0/dose).We analyzed the correlation between CYP2C192(681G>A), CYP2C193(636G>A), and CYP2C1917(-806C>T) genetic polymorphisms and the dose-corrected pre-dose concentration (C0/dose) in 106 South-western Chinese Han patients.The frequencies of variant alleles of CYP2C192, 3, and 17 were 29.7%, 4.25%, and 0.92%. For 49.3% of the VCZ samples, the therapeutic window between 1.5 and 5.5 µg/ml was reached. Following the first dose VCZ measurement, in subsequent samples the proportion of VCZ C0 within the therapeutic window increased, suggesting effective therapeutic drug monitoring (TDM) (P = .001). The VCZ C0 was significantly different (P = .010) between patients with normal metabolism (NMs), intermediate metabolism (IMs), and poor metabolism (PMs). The VZC C0/dose was 12.2 (interquartile range (IQR), 8.33-18.2 µg·ml/kg·day), and 7.68 (IQR, 4.07-16.3 µg·ml/kg·day) in PMs and IMs patients, respectively, which was significantly higher than in NMs phenotype patients (4.68; IQR, 2.51-8.87 µg·ml/kg·day, P = .008 and P = .014).This study demonstrated that the VCZ C0/dose was significantly influenced by the CYP2C19 genotype in South-western Chinese Han patients. In this patient population, more over-exposure was observed in patients with a CYP2C19 genotype associated with poor or intermediate metabolism. CYP2C19 genotype-based dosing combined with TDM will support individualization of VCZ dosing, and potentially will minimize toxicity and maximize therapeutic efficacy.

A Low Fixed Tacrolimus Starting Dose Is Effective and Safe in Chinese Renal Transplantation Recipients.

BACKGROUND We investigated whether a low fixed Tac starting dose regimen could lead to a better achievement of Tac target concentrations, as well as an effective immunosuppressive treatment, in Chinese kidney transplant recipients (KTRs). MATERIAL AND METHODS We collected whole-blood and serum samples from 189 KTRs and the Tac starting dose was 2, 2.5, or 3 mg/day. Information on Tac C0, dose, body weight, body mass index (BMI), Scr, eGFR, and CYP3A5 genotypes were collected from a routine therapeutic drug monitoring database. The correlation between Tac C0 and body weight (or BMI) was investigated by calculating the goodness of fit. Multivariable logistic regression was performed to estimate the independent associated factors. RESULTS The patients with 3 mg per day of Tac had higher C0 at day 7 compared to those with 2 or 2.5 mg. For patients receiving the same Tac starting dose, no significant difference was found in Tac C0 at day 7 among different body weight or BMI groups. There was no significant difference in Scr or eGFR at 1 year after transplant, nor was there a significant difference in the rates of DGF or AR at post-transplant day 30 among different Tac starting dose groups or among the 3 Tac C0 range groups. CYP3A5 genotype and Tac initial dose were independently associated with Tac C0. CONCLUSIONS CYP3A5 genotype and Tac initial dose were independently associated with Tac C0 in renal transplant recipients. Our results suggest that a low Tac target C0 range (5-8 ng/ml) with a low fixed starting dose (3 mg/day) would be safe and effective among Chinese KTRs.

Urinary 8-oxo-7,8-dihydroguanosine as a Potential Biomarker of Aging.

Background: A molecular biomarker of physiologic age, as opposed to chronologic age, is needed in clinical medicine. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGsn) and 8-oxo-7, 8-dihydroguanosine (8-oxoGsn) are two promising aging biomarkers. Methods: A total of 1,228 healthy Chinese residents (613 males and 615 females) 2-90 years of age were randomly selected. Spot urine samples were collected, and the concentrations of 8-oxodGsn and 8-oxoGsn were measured using ultra-high-performance liquid chromatography with a triple quadrupole mass spectrometer (UPLC-MS/MS). Method validation, including accuracy, precision, linearity and quantification limit, was performed. The relationship between oxidized guanosine and age/gender was evaluated. Results: 8-oxodGsn and 8-oxoGsn were eluted at 1.61 and 1.30 min, respectively. The calibration curve was linear in the range of 0.2-500 ng/ml for both analytes. The lowest limit of quantification (LLOQ) was 0.2 ng/ml for 8-oxodGsn and 0.1 ng/ml for 8-oxoGsn. There was an age-dependent increase in the biomarkers from the 21- to 30-year-old group to the 81- to 90-year-old group in both genders. In the subjects older than 61 years of age, the levels of 8-oxodGsn as well as 8-oxoGsn in urine were much higher in females than in males. The content of 8-oxoGsn correlated more closely with age and was higher (approximately 2-fold) than that of 8-oxodGsn for a given individual. Conclusions: 8-oxodGsn and 8-oxoGsn can be easily measured by UPLC-MS/MS. Urinary 8-oxoGsn may be a potential biomarker to determine a person's physiologic age and identify individuals at high risk of developing age-associated disease.

Soluble Tim-3 and Gal-9 are associated with renal allograft dysfunction in kidney transplant recipients: A cross-sectional study.

BACKGROUND: T cell immunoglobulin mucin-3 (Tim-3) has been reported to participate in the regulation of immune response and the induction of allograft tolerance. However, the association between Tim-3 and renal allograft dysfunction is unclear. We studied the expression of cellular and soluble Tim-3 (sTim-3), soluble galectin-9 (sGal-9) and carcinoembryonic antigen-related cell adhesion molecule-1 (sCEACAM-1) in kidney transplantation recipients (KTRs) to explore their roles in allograft dysfunction. METHODS: 96 KTRs (53 with stable graft and 43 with graft dysfunction) and 30 healthy controls (HC) were enrolled. Among the KTRs, 55 used Tacrolimus (TAC) and 41 used Sirolimus (SRL). In the dysfunction group, 29 recipients have undergone graft biopsy and 14 were classified as biopsy-proven rejection (BPR). Cellular Tim-3 was determined by flow cytometry. sTim-3 was determined by ELISA. sGal-9 and sCEACAM-1 were determined by Bio-Plex® suspension array system. RESULTS: KTRs with renal dysfunction showed significantly higher levels of sTim-3 and sGal-9 but similar levels of cellular Tim-3 and sCEACAM-1 compared with stable recipients. Correlation analysis revealed that estimated glomerular filtration rate (eGFR) was negatively associated with sTim-3 and sGal-9. Both BPR and non-BPR groups showed comparable levels of Tim-3, Gal-9 and CEACAM-1. Moreover, SRL group showed significantly higher levels of sCEACAM-1 than TAC and HC groups. CONCLUSIONS: sTim-3 and sGal-9 were promising biomarkers for allograft dysfunction, but unable to differentiate allograft rejection from other causes of renal dysfunction in KTRs. Moreover, long-term administration of sirolimus would up-regulate sCEACAM-1 level, while exert similar regulatory effects on Tim-3 and Gal-9 compared to tacrolimus.

[Association of CYP3A5 and MDR1 genetic polymorphisms with the blood concentration of tacrolimus in Chinese liver and renal transplant recipients].

OBJECTIVE: To investigate the association of CYP3A5 and MDR1 genetic polymorphisms with the concentration/ dose (C/D) ratio of tacrolimus for the feasibility of individualized medication. METHODS: The concentration of tacrolimus was detected by enzyme-multiplied immunoassay technique, and was adjusted by weight and dosage to C/D ratios. The single nucleotide polymorphisms of CYP3A5 A6986G and MDR1 C3435T, G2677T/ A, T1236C were determined by TaqMan RT-PCR. The differences of C/D ratio were compared among all of the genotype groups. RESULTS: There were 5 cases with CYP3A5 *1/*1, 22 cases with CYP3A5 *1/*3, and 33 cases with CYP3A5 *3/*3. The C/D ratios of the patients with at least one CYP3A5 *1 allele (130.40 +/- 53.94) was significantly lower than those with CYP3A5 *3/*3 (198.12 +/- 90.80) (P < 0.01). For MDR1, there were 22, 23 and 15 recipients carried C/C, C/T and T/T respectively in C3435T, and 8, 32 and 20 recipients carried T/T, T/ C and C/C respectively in T1236C. The carriers with G/G, G/T, G/A, T/A, T/T were 9, 24, 5, 8 and 14 respectively in G2677T/A. No significant difference was found in the C/D ratios of tacrolimus among different MDR1 genotypes. CONCLUSIONS: Determination of CYP3A5 genotype could help individualize tacrolimus dose regimen prospectively. The patients with CYP3A5 *3 *3 require less dose of tacrolimus to reach the same concentrations comparing with the patients with at least one CYP3A5 * 1 allele.

[Regulatory function of tacrolimus and CsA on CD4/CD8 T lymphocyte subgroups and costimulators on them in allo-liver recipients].

AIM: To explore the regulatory function of FK506 and CsA on CD4/CD8 T lymphocyte subgroups and co-stimulators on them. METHODS: The fluorescein-labelled monoclonal antibodies and flowcytometer were used to determine the T-lymphocyte subgroups and the expression of CD28, CD152 and ICOS on them in allo-liver recipients treated with FK506 or CsA at the end of 2 months after transplantation and treatment. Healthy volunteers and the patients who suffered from severe hepatic diseases and would receive liver transplantation were used as controls. RESULTS: In disease-control group, the balance of T cell subgroups was disturbed and the expression of co-stimulators was abnormal. In liver recipients receiving immunosuppressive therapy, the expression of T-cell subgroups returned to the normal level, the expressions of CD28 and ICOS on T cells decreased significantly (P<0.05), while the expression of CD152 on T cells increased significantly (P<0.05). Between two treatment group, the expression of CD4(+)T cells and the expression of CD28 and ICOS on CD8(+)T cells in CsA-treated group were much higher than those in FK506-treated group (P<0.05), and there was no significant difference between two treatment groups in other indexes. CONCLUSION: At routine blood concentration, there is some difference in the regulatory effect of FK506 and CsA on T-cell subgroups and the expression of co-stimulators on T cells. The regulatory effect of FK506 on T-cell subgroups is stronger than that of CsA. FK506 can not only inhibit the expression of positive co-stimulatory molecules CD28 and ICOS but also promote the expression of negative co-stimulatory molecule CD152, while CsA can exert its immunosuppressive effect mainly through promoting the expression of CD152.