Determination of Tacrolimus Concentration and Protein Expression of P-Glycoprotein in Single Human Renal Core Biopsies
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Published on: December 23, 2015
Veronica Krogstad1, Nils T Vethe2, Ida Robertsen1
1Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo.
Background:
Tacrolimus (TAC) is currently the cornerstone of immunosuppressive protocols for renal transplant recipients. Despite therapeutic whole blood monitoring, TAC is associated with nephrotoxicity, and it has been hypothesized that intrarenal accumulation of TAC and/or its metabolites are involved. As TAC is a substrate of P-glycoprotein (P-gp), the expression and activity of this efflux transporter could influence the levels of TAC in renal tissue. The primary aim of this study was to develop and validate a method for quantification of TAC in tissue homogenates from single human renal core biopsies. The secondary aim was to provide measures of P-gp expression and of the demethylated metabolites of TAC in the same renal biopsy.
Methods:
Human renal tissue, with and without clinical TAC exposure, was used for method development and validation. Homogenates were prepared with bead-beating, and concentrations of TAC and its demethylated metabolites were analyzed with liquid chromatography tandem mass spectrometry after protein precipitation. A Western blot method was used for semiquantification of P-gp expression in the homogenates. The final methods were applied to renal core biopsies from 2 transplant patients.
Results:
The TAC assay showed within- and between-run mean accuracy between 99.7% and 107% and coefficients of variation ≤6.7%. Matrix effects were nonsignificant, and samples were stable for 3 months preanalytically when stored at -80°C. TAC concentrations in the renal core biopsies were 62.6 and 43.7 pg/mg tissue. The methods for measurement of desmethyl-TAC and P-gp expression were suitable for semiquantification in homogenates from renal core biopsies.
Conclusions:
These methods may be valuable for the elucidation of the pharmacokinetic mechanisms behind TAC-induced nephrotoxicity in renal transplant recipients.
Insights
This study developed a validated method to measure Tacrolimus (TAC) and its metabolites in renal tissue, aiding research into TAC-induced nephrotoxicity in kidney transplant patients.
Area of Science:
- Pharmacology
- Nephrology
- Analytical Chemistry
Background:
- Tacrolimus (TAC) is crucial for immunosuppression in kidney transplant recipients but can cause nephrotoxicity.
- Intrarenal accumulation of TAC and its metabolites, influenced by P-glycoprotein (P-gp), is a suspected cause of toxicity.
- Current monitoring relies on whole blood levels, not intrarenal concentrations.
Purpose of the Study:
- To develop and validate a method for quantifying TAC in human renal core biopsies.
- To measure P-glycoprotein (P-gp) expression and TAC metabolites in the same biopsies.
- To investigate pharmacokinetic mechanisms of TAC-induced nephrotoxicity.
Main Methods:
- Developed and validated a liquid chromatography-tandem mass spectrometry assay for TAC and its demethylated metabolites in renal tissue homogenates.
- Employed Western blotting for semiquantification of P-gp expression.
- Applied methods to single human renal core biopsies from transplant patients.
Main Results:
- The TAC assay demonstrated high accuracy (99.7-107%) and precision (CV ≤6.7%).
- Quantified TAC concentrations in renal biopsies were 62.6 and 43.7 pg/mg tissue.
- Methods were suitable for semiquantifying desmethyl-TAC and P-gp in small biopsy samples.
Conclusions:
- Validated methods enable direct measurement of TAC and related markers in renal biopsies.
- These techniques can elucidate the role of intrarenal TAC pharmacokinetics in nephrotoxicity.
- Provides a tool for personalized immunosuppressive therapy monitoring in kidney transplant recipients.
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