Table shows the clinical data of recipients
Table 2 shows the clinical data of 23 recipients with CYP3A5*1 allele classified by the presence or absence of CYP3A phenoconversion. Phenoconversion of CYP3A was observed in 10 recipients with CYP3A5*1 allele. No significant differences in eGFR, ALT and total bilirubin were observed between recipients with and those without CYP3A phenoconversion, suggesting that renal and hepatic functions were similar in the two groups. Similarly, no significant difference in total cholesterol was observed between two groups. Furthermore, no significant differences in all clinical data except for sex was observed between two groups.
Plasma concentrations of indoxyl sulfate, intact-PTH, IL-6 and TNF-α in recipients with and those without CYP3A phenoconversion were compared (Fig. 1). Plasma indoxyl sulfate concentration was significantly higher in recipients with CYP3A phenoconversion compared to those without phenoconversion (p < 0.05, 95% CI of difference 1.33–20.6). On the other hand, Nuclear/Cytosol Fractionation Kit concentrations of intact-PTH, IL-6 and TNF-α were not significantly different between the two groups. In addition, plasma concentrations of indoxyl sulfate, intact-PTH, IL-6 and TNF-α did not differ between recipients with CYP3A5*1 allele and those without CYP3A5*1 allele (Fig. 2). Furthermore, plasma concentrations of 4β-hydroxycholesterol, indoxyl sulfate, intact-PTH, IL-6 and TNF-α did not correlate significantly with eGFR or elapsed time after transplantation (data not shown).
Discussion In this study, phenoconversion of CYP3A, defined as a genotypic extensive/intermediate metabolizer (with CYP3A5*1 allele) exhibiting CYP3A activity below the cutoff value that discriminates between extensive/intermediate and poor metabolizers, was observed in stable kidney transplant recipients, and plasma indoxyl sulfate concentration may have been involved in the phenomenon. This is the first report showing that accumulation of indoxyl sulfate may partially explain the gap in CYP3A activity unexplained by genetic contribution in patients with chronic renal failure, which is the greatest strength in this study. The allele frequency of CYP3A5*3 was 79.7% in our study, which agreed with previous studies in the Japanese population [, , ]. Plasma 4β-hydroxycholesterol concentration was significantly different between recipients with and those without CYP3A5*1 allele as we reported previously [23,30]. 4β-hydroxycholesterol is formed by CYP3A4 and 3A5 [31,32] and is slowly eliminated from the circulation due to slow 7α-hydroxylation by CYP7A1 , which is not affected by renal failure . These findings suggest that plasma 4β-hydroxycholesterol concentration is a suitable biomarker for CYP3A activity in patients with renal failure. Phenoconversion of CYP3A was found in 43.5% of the recipients with CYP3A5*1 allele. To the best of our knowledge, this is the first report showing the occurrence of CYP3A phenoconversion in humans. In this study, we measured only CYP3A5*3 as a genetic marker to judge the presence or absence of CYP3A phenoconversion, because CYP3A5*3 is the major single nucleotide polymorphism in CYP3A  and a number of Japanese have CYP3A5*3 allele [, , ]. There are other single nucleotide polymorphisms of CYP3A5 including CYP3A5*2, CYP3A5*4, and CYP3A5*5, as well as polymorphisms of CYP3A4 including CYP3A4*1B, CYP3A4*4, CYP3A4*16, CYP3A4*18, and CYP3A4*22. However, these polymorphisms are not frequent in the Japanese population [28,, , , , ]. Thus, they were not genotyped in this study. The cutoff plasma 4β-hydroxycholesterol concentration for phenoconversion of CYP3A was set at 40.0 ng/mL in this study. This value was similar to the intermediate value between recipients with and without CYP3A5*1 allele, which we reported previously [23,30]. We measured plasma concentrations of indoxyl sulfate, intact-PTH, IL-6 and TNF-α as candidate molecules involved in phenoconversion of CYP3A because they have been reported to cause downregulation of CYP3A in renal failure, and only plasma indoxyl sulfate concentration was significantly different between recipients with and those without CYP3A phenoconversion. Uremic toxins have been shown to alter CYP expression and function via transcriptional or translational modifications of CYP enzymes and direct inhibition of CYP-mediated metabolism [, , ]. Especially, indoxyl sulfate is implicated in decreased CYP3A activity in patients with chronic renal failure . The detailed mechanism by which indoxyl sulfate downregulates CYP3A is unclear, but indoxyl sulfate is known to upregulate nuclear factor-κB (NF-κB) activity , and upregulation of NF-κB may decrease histone 4 acetylation in the CYP3A promoter , leading to downregulation of CYP3A expression. The detailed mechanisms by which PTH, IL-6, and TNF-α downregulate CYP3A are also unclear. PTH has been reported to downregulate CYP3A via activation of NF-κB, similar to indoxyl sulfate . IL-6 and TNF-α may induce alteration of the subcellular location of retinoid X receptor-α [21,42], which heterodimerizes with the pregnane X receptor to regulate CYP3A expression . Fifty-three of the 63 recipients (84.1%) in this study had renal failure as indicated by eGFR below 60 ml/min/1.73m2, and their plasma indoxyl sulfate concentrations were above the normal range  with large inter-individual variability (12.0 ± 11.8 μM). On the other hand, plasma concentrations of intact-PTH, IL-6, and TNF-α were low in the recipients participating in this study, which could have contributed to the result of no association with CYP3A phenoconversion.