A novel method for monitoring glucocorticoid-induced changes of the glucocorticoid receptor in kidney transplant recipients.
BACKGROUND
Initial high dose glucocorticosteroid (GC) treatment of transplant recipients induces changes in the glucocorticoid receptor (GR) that may affect the mode of action of the drug. We developed a TaqMan one-step RT-PCR quantification method to investigate the effect of bolus GC treatment on the structure of the GR complex by measuring the levels of GR isoform expression and FK binding protein 5 (FKBP5) transcription.


METHODS
Peripheral blood mononuclear cells (PBMCs) were obtained before renal transplantation and immediately after the initial methylprednisolone treatment. Gene expression of the GR-alpha, -beta, -P isoforms and FKBP5 were quantified using a simplex TaqMan relative quantification assay. Genotyping was performed using the TaqMan allele discrimination assay.


RESULTS
The gene expression assay was validated and is efficient to detect extremely low quantity of GR-beta expression. Increased expression of FKBP5 (14.01+11.52 folds), GR-alpha (1.47+1.00 folds), GR-beta (6.21+5.71 folds) and GR-P (2.72+1.90 folds) were observed after steroid bolus. The GR haplotype A-T-G is significantly related to elevation of GRbeta transcription (p=0.002). Patients' hospitalization time after transplantation correlated with increment of GR-alpha (p=0.01) and FKBP5 (p=0.007) gene expression. The GR-beta expression level is extremely low compared with the GR-alpha isoform. By contrast, the GR-P isoform is relatively abundantly expressed in PBMCs.


CONCLUSIONS
The method to measure GR isoform enabled us to determine that acute high-dose GC treatment alters the structure of the GR. The initial therapy with high dose steroid may induce a GC resistance phenotype. The modulatory role of the GR-P isoform is not fully defined but may provide another dimension in understanding the function of GR and the role of steroid in transplant immunosuppression.
Chen
Yan
Y
University of Southern California, Los Angeles, CA, USA. chenyan@usc.edu
Burckart
Gilbert J
GJ
Shah
Tariq
T
Pravica
Vera
V
Hutchinson
Ian V
IV
D006084:Graft Rejection / Q000276:immunology* / Q000517:prevention & control*; D006239:Haplotypes; D006801:Humans; D016030:Kidney Transplantation; D007902:Length of Stay; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D008775:Methylprednisolone / Q000627:therapeutic use*; D020641:Polymorphism, Single Nucleotide; D011237:Predictive Value of Tests; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000099159:Tacrolimus Binding Protein 5
D008775:Methylprednisolone; D020033:Protein Isoforms; D011965:Receptors, Glucocorticoid; D022021:Tacrolimus Binding Proteins; D000099159:Tacrolimus Binding Protein 5