The functional implications of common genetic variation in CYP3A5 and ABCB1 in human proximal tubule cells.

BACKGROUND: Calcineurin inhibitors (CNIs) are the primary immunosuppressive drugs used in solid organ transplantation but are associated with the development of histological lesions leading to kidney failure. CNIs are metabolized by CYP3A and excreted by not only P-glycoprotein (P-gp) (ABCB1) in the gut and liver, but also by proximal tubule cells (PTCs) in the kidney. Multiple studies have demonstrated the importance of genetic variation in CYP3A5 and ABCB1 for CNI disposition and nephrotoxicity. The present study was designed to study the functional implication of variation in these two genes in human PTCs. METHODS: A technique was developed to culture cells from renal tissue obtained from renal graft recipients by routine kidney biopsy. Primary cells were immortalized, subcloned, and then characterized for specific PTC markers (AQP1, CD13, brush border morphology) and donor CYP3A5(rs776746)/ABCB1(rs1045642) genotype. We then selected specific sets of confirmed conditionally immortalized PTCs (ciPTC) according to different combinations of the aforementioned genetic variants. Quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry were performed for studying CYP3A5 and ABCB1 expression. CYP3A5 activity was assessed by differential midazolam (MDZ) hydroxylation and P-gp (ABCB1 product) activity by a calcein efflux assay. Differential drug metabolism between cell lines was assessed by tacrolimus disappearance over 24 h. RESULTS: Cell lines were generated from 27 out of 38 tissue samples. On the basis of genotype and PTC biomarkers, 11 subclones were selected. In vitro PTC morphology with brush border microvilli was confirmed. CYP3A5*1 carriers had increased 1-OH/4-OH MDZ formation versus homozygous *3 carriers (mean: 2.36 (95% CI:1.11-3.40) vs 0.88 (95% CI:0.48-1.27); p < 0.05). P-gp activity was confirmed by calcein accumulation (mean 38.6%; 95% CI:32.8-44.4%), which was higher in cell lines with the ABCB1 3435TT than the 3435CC/CT genotype (46.2% vs 35.5%; 95% CI:28.7-42.2%). Tacrolimus disappearance was about two-fold higher in cell lines with the combined CYP3A5*1/ABCB1 3435TT genotype versus other genotype combinations. CONCLUSION: Biopsy-derived and immortalized human PTC cell lines demonstrate functional expression of genes involved in CNI metabolism. Differences in functional expression were detected according to common genetic variants in CYP3A5 and ABCB1. The studied genetic variants had a significant impact on in vitro tacrolimus metabolism. In particular, ciPTC with the combined CYP3A5*1/ABCB1 3435TT genotype demonstrated higher tacrolimus disappearance versus ciPTCs with a different pharmacogenetic profile. This in vitro model stresses the importance of the incorporation of pharmacogenetic variation in studies involved in (renal) drug disposition.

Mutational Spectrum of the CTNS Gene in Egyptian Patients with Nephropathic Cystinosis.

BACKGROUND: Nephropathic cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene, encoding for cystinosin, a carrier protein transporting cystine out of lysosomes. Its deficiency leads to cystine accumulation and cell damage in multiple organs, especially in the kidney. In this study, we aimed to provide the first report describing the mutational spectrum of Egyptian patients with nephropathic cystinosis and their genotype-phenotype correlation. METHODS: Fifteen Egyptian patients from 13 unrelated families with infantile nephropathic cystinosis were evaluated clinically, biochemically, and genetically. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the ten coding exons, exon-intron interfaces, and promoter region. RESULTS: None of the 15 Egyptian patients had the 57-kb deletion. Twenty-seven mutant alleles and 12 pathogenic mutations were detected including six novel mutations: two frameshift (c.260_261delTT; p.F87SfsX36, c.1032delCinsTG; p.F345CfsX19), one nonsense (c.734G>A; p.W245fsX), two missense (c.1084G>A; pG362R, c.560A>G; p.K187R), and one intronic splicing mutation (IVS3+5g>t). A novel promoter region mutation (1-593-41C>T) seemed to be detected but was excluded as a pathogenic mutation by quantitative real-time PCR analysis. CONCLUSIONS: This study could be the basis for future genetic counseling and prenatal diagnosis of patients with nephropathic cystinosis in Egyptian and surrounding populations. The screening for the 57-kb deletion is not recommended anymore outside its geographical distribution, especially in the region of the Middle East. A common Middle Eastern mutation (c.681G>A; E227E) was pointed out and discussed.