CYP3A5 polymorphism affects the increase in CYP3A activity after living kidney transplantation in patients with end stage renal disease

Abstract

Aims

It has been reported that cytochrome P450 (CYP)3A activity increases significantly in patients with end stage renal disease (ESRD) after kidney transplantation, with wide interindividual variability in the degree of increase. The aim of this study was to evaluate the influence of CYP3A5 polymorphism on the increase in CYP3A activity after living kidney transplantation, by measuring the plasma concentration of 4β‐hydroxycholesterol.

Methods

This prospective study recruited 22 patients with ESRD who underwent a first living kidney allograft transplantation, comprising 12 patients with CYP3A5*1 allele (CYP3A5*1/*1 or *1/*3) and 10 patients without CYP3A5*1 allele (CYP3A5*3/*3).

Results

No significant difference in estimated glomerular filtration rate over time was observed between patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele, suggesting that the degrees of recovery in renal function after living kidney transplantation were similar in the two groups. However, plasma concentrations of 4β‐hydroxycholesterol on days 90 (57.1 ± 13.4 vs. 39.5 ± 10.8 ng ml⁻¹) and 180 (55.0 ± 14.5 vs. 42.4 ± 12.6 ng ml⁻¹) after living kidney transplantation were significantly higher in the presence of the CYP3A5*1 allele than in the absence of the CYP3A5*1 allele [P = 0.0034 (95% confidence interval of difference 6.55, 28.6) and P = 0.043 (95% confidence interval of difference 0.47, 24.8), respectively], suggesting that CYP3A activity may increase markedly associated with recovery of renal function in patients with the CYP3A5*1 allele.

Conclusions

These findings suggest that the presence of the CYP3A5*1 allele contributes to marked elevation of CYP3A activity associated with recovery of renal function after kidney transplantation.

What is Already Known About this Subject

• It has been reported that CYP3A activity increases significantly in patients with end stage renal disease (ESRD) after kidney transplantation.

• However, wide interindividual variability in the degree of increase was observed.

What this Study Adds

• Plasma concentrations of 4β‐hydroxycholesterol were significantly higher in patients with the CYP3A5*1 allele than in patients without the CYP3A5*1 allele on days 90 and 180 after living kidney transplantation.

• CYP3A activity may increase markedly associated with recovery of renal function in patients with the CYP3A5*1 allele.

Introduction

Impaired renal function alters the elimination of many drugs mainly by decreasing their renal elimination. However, several studies in rats 1, 2, 3, 4, 5, 6 and in patients 7 have shown that renal failure also decreases the metabolic clearance of drugs, particularly those metabolized by cytochrome P450 (CYP) and substrates of some drug transporters. The underlying causes of altered functional expression of CYPs and drug transporters observed in kidney disease remain unclear, but several studies indicate that uremic toxins may play a role via transcriptional or translational modifications of CYP and drug transporter enzymes 8, 9, 10, 11.

In the CYP families, CYP3A4 is involved in the metabolism of 30–40% of currently prescribed drugs 12. CYP3A5 has genetic polymorphism and is expressed at high levels in people with at least one CYP3A5*1 allele 13. Therefore, phenotyping of cytochrome CYP3A seems to be of special importance. Some CYP3A substrates such as midazolam, erythromycin, alprazolam and nifedipine, urinary 6β‐hydroxycortisol to cortisol ratio; formation clearance of 6β‐hydroxycortisol and 4β‐hydroxycholesterol have been reported to be useful markers for CYP3A phenotyping 14, 15, 16, 17, 18, 19. Especially, 4β‐hydroxycholesterol may be useful in patients with renal failure because the kinetics of 4β‐hydroxycholesterol seem to be unaffected by renal function due to the slow elimination by 7α‐hydroxylation 20. We have previously reported a significant increase in plasma concentration of 4β‐hydroxycholesterol as an endogenous marker of CYP3A activity in patients with end stage renal disease (ESRD) after kidney transplantation 21. However, wide interindividual variability in the degree of increase was observed. Thus, it is important to identify the factors that account for this variability in order to support individualized drug therapy based on CYP3A activity after kidney transplantation. We have previously reported that CYP3A5 polymorphism and, to a lesser extent, accumulation of uraemic toxins, influence CYP3A activity in stable kidney transplant recipients 22. Then, wide interindividual variability in the degree of increase in CYP3A activity after kidney transplantation may be explained by CYP3A5 polymorphism. Based on this hypothesis, we evaluated the influence of CYP3A5 polymorphism on the increase in CYP3A activity after living kidney transplantation, by measuring the plasma concentration of 4β‐hydroxycholesterol.

Methods

Patients

This prospective study recruited 22 patients with ESRD (creatinine clearance less than 15 ml min⁻¹) who underwent a first living kidney allograft transplantation between January 2011 and August 2014 in the Department of Urology, Faculty of Medicine at Oita University. After living kidney transplantation, all patients were followed for 180 days. Morning blood samples were collected in tubes containing EDTA anticoagulant before and 7, 14, 30, 90 and 180 days after living kidney transplantation. All blood samples were centrifuged and plasma samples frozen at −40°C within 2 h of peripheral venipuncture. The following clinical data were collected: gender, age, body weight, prescribed drugs and laboratory data including white blood cell count, haemoglobin, C‐reactive protein, alanine aminotransaminase (ALT), total bilirubin, serum creatinine, blood urea nitrogen and total cholesterol. Estimated glomerular filtration rate (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD‐EPI) equation for Japanese 23. Before living kidney transplantation, all patients were haemodialyzed three times a week. No patient had an episode of rejection and none received medications known to inhibit or induce CYP3A during the study 24. All patients were treated with a triple therapy immunosuppression protocol consisting of tacrolimus, mycophenolate mofetil and methylprednisolone. Furthermore, all patients received induction therapy with basiliximab before and 4 days after living kidney transplantation. This study was approved by the ethics committee of Oita University (approval number: 568). Each subject received prior explanations about the scientific purpose of the study, and gave written informed consent.

Determination of CYP3A5 genotype

A venous blood sample (5 ml) was obtained from each patient and DNA was prepared using the Maxwell® 16 DNA Purification Kit (Promega, Tokyo, Japan). All samples were analyzed for the single nucleotide polymorphism A6986G (CYP3A5*3). Allelic discrimination reaction was performed using TaqMan genotyping assays (C_26201809_30) in a LightCycler® Nano System (Roche Applied Science, Penzberg, Germany). When the CYP3A5*3 allele was not detected, the test allele was designated CYP3A5*1.

Measurement of plasma concentration of 4β‐hydroxycholesterol

Plasma concentration of 4β‐hydroxycholesterol was determined using a gas chromatography/mass spectrometry (GC/MS) system according to the procedures that we reported previously 21. One hundred μl of plasma sample was mixed with 20 μl of internal standard (1000 nm 4β‐hydroxycholesterol‐d7 in 2‐propanol) and 200 μl of 2 m sodium methoxide solution in ethanol, and the mixture was left at room temperature for 20 min to convert esterified to free 4β‐hydroxycholesterol. Then, 500 μl of water and 2 ml of n‐hexane were added and extraction was performed by vortexing for 1 min. After centrifugation at 3000 g for 5 min at 20°C, the organic phase was transferred to a glass tube and evaporated to dryness by a stream of nitrogen at 40°C. The residue was reconstituted with 100 μl of tert‐butyldimethylsilylimidazole‐dimethylformamide and incubated at room temperature for 12 h to convert 4β‐hydroxycholesterol to tert‐butyldimethylsilyl ether. Subsequently, 1 ml of water and 2 ml of ethyl acetate were added, and the mixture was vortexed for 1 min. After centrifugation at 3000 g for 5 min at 20°C, the organic phase was transferred to a glass tube and evaporated to dryness by a stream of nitrogen at 40°C. The residue was reconstituted with 100 μl of n‐hexane. The sample was immediately transferred to auto‐sampler vials, and 1 μl of sample was splitless injected into a GC/MS system. An Agilent 7890GC gas chromatograph equipped with an HP‐5MS capillary column (30 m × 0.25 mm, 0.25 μm phase thickness) was used, which was connected to an HP 5975 mass selective detector and an HP 7693 automatic sample injector (Agilent Technologies, IL, USA). The oven temperature program was as follows: 180°C for 1 min, increase at a rate of 35°C/min to 270°C, and then 20°C/min to 310°C, followed by 310°C for 15 min. Helium was used as a carrier gas at a flow rate of 1 ml min⁻¹. The detector temperature was 270°C and the detector transfer line temperature was set at 280°C. The mass spectrometer was used in the selected ion monitoring mode, and the following ions (m/z) were monitored: 573.5 for 4β‐hydroxycholesterol and 580.6 for 4β‐hydroxycholesterol‐d7 (internal standard). The electron ionization energy was 70 eV. Inter‐assay coefficients of variation for 20 and 200 ng ml⁻¹ samples were 3.8 and 4.2%, respectively (n = 6). Intra‐assay coefficients of variation for 20 and 200 ng ml⁻¹ samples were 3.4 and 4.7%, respectively (n = 6). The lower limit of quantification was 1 ng ml⁻¹ and the accuracy of the analysis ranged from 94.6 to 105.8%.

Data analysis and statistics

Data are expressed as mean ± standard deviation (SD). Variables before and after living kidney transplantation were compared by Dunnett’s test. Differences and 95% confidence interval (CI) of differences between patients with the CYP3A5*1 allele (CYP3A5*1/*1 or *1/*3) and patients without the CYP3A5*1 allele (CYP3A5*3/*3) were compared using Fisher's exact test, paired t‐test, two‐sided Student's t‐test or Welch's t‐test. Correlation between variables was analyzed by Pearson's product–moment correlation coefficient. A P value less than 0.05 was considered statistically significant. Statistical analyses were performed using the R software version 3.1.1 (http://www.r‐project.org) and the Predictive Analysis Software (PASW) Statistics version 21.0 (SPSS Inc., IL, USA).

Results

Table 1 summarizes the clinical data of the 22 patients with ESRD, comprising 12 patients with the CYP3A5*1 allele (CYP3A5*1/*1 or *1/*3) and 10 patients without the CYP3A5*1 allele (CYP3A5*3/*3) before and 180 days after living kidney transplantation. As expected, blood urea nitrogen decreased significantly after living kidney transplantation in both groups. On the other hand, no significant differences in ALT and total bilirubin were observed before and after living kidney transplantation, suggesting that hepatic function was stable during the study. Similarly, no significant difference in total cholesterol was observed before and after living kidney transplantation. Furthermore, no significant differences in all clinical data were observed between patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele before and 180 days after living kidney transplantation.

Table 1.

Clinical characteristics of patients in the study

Characteristic	Before living kidney transplantation		180 days after living kidney transplantation
	Patients with the CYP3A5*1 allele	Patients without the CYP3A5*1 allele	Patients with the CYP3A5*1 allele	Patients without the CYP3A5*1 allele
	(CYP3A5*1/*1 or *1/*3)	(CYP3A5*3/*3)	(CYP3A5*1/*1 or *1/*3)	(CYP3A5*3/*3)
Number of patients	12	10
Males/females	9/3	8/2
Cause of kidney disease
Glomerulonephritis	4	5
Immunoglobulin A nephropathy	1	3
Thin basement membrane disease	1	0
Nephrosclerosis	0	1
Unknown nephritis	6	1
Age (years)	47.0 ± 14.3 [24–65]	46.2 ± 14.8 [27–66]
Body weight (kg)	63.5 ± 16.0 [45.0–91.6]	62.9 ± 14.2 [40.1–92.1]	60.4 ± 18.1 [44.2–104.0]	59.9 ± 14.2 [38.0–90.6]
White blood cell count (/μl)	6433 ± 1965 [3740–9300]	6329 ± 2009 [3290–9860]	5237 ± 2082 [3480–9270]	5392 ± 1765 [2420–8110]
Haemoglobin (g dl−1)	11.5 ± 1.4 [9.6–14.0]	11.9 ± 1.7 [10.5–16.1]	11.4 ± 0.9 [9.8–13.3]	11.8 ± 1.6 [8.4–13.6]
C‐reactive protein (mg dl−1)	0.04 ± 0.03 [0.01–0.09]	0.08 ± 0.06 [0.02–0.18]	0.03 ± 0.03 [0.01–0.10]	0.06 ± 0.06 [0.01–0.18]
ALT (IU l−1)	13.6 ± 10.6 [5.1–44.6]	10.7 ± 6.3 [4.6–27.2]	12.3 ± 7.0 [5.5–23.5]	14.9 ± 6.8 [8.6–29.2]
Total bilirubin (mg dl−1)	0.47 ± 0.16 [0.25–0.70]	0.53 ± 0.16 [0.33–0.92]	0.51 ± 0.14 [0.32–0.74]	0.47 ± 0.33 [0.18–1.04]
Blood urea nitrogen (mg dl−1)	51.4 ± 18.0 [28.9–100.7]	52.3 ± 14.3 [25.4–72.0]	27.3 ± 5.9† [18.8‐37.4]	23.8 ± 4.0† [17.1‐29.7]
Total cholesterol (mg dl−1)	191.1 ± 62.0 [106.0–310.0]	163.5 ± 43.9 [126.0–243.0]	192.8 ± 24.7 [143.0–231.0]	179.5 ± 19.1 [142.0–205.0]

ALT, alanine aminotransaminase; CYP, cytochrome P450. Data are expressed as numbers, or mean ± SD [range].

^† P < 0.01 vs. before transplantation.

Figure 1A shows the change in serum creatinine level over time in ESRD patients after living kidney transplantation (n = 22). Serum creatinine level decreased significantly on day 7 after living kidney transplantation, and thereafter remained almost stable until day 180 after living kidney transplantation. Figure 2A shows the changes in eGFR over time after living kidney transplantation in patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele. No significant difference in eGFR over time was observed between patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele.

Figure 1.

Changes in serum creatinine level (A) and plasma concentration of 4β‐hydroxycholesterol (B) over time in patients with end stage renal disease after living kidney transplantation. Data are presented as mean ± SD. *P < 0.05 and ^† P < 0.01 vs. before transplantation, by Dunnett’s test

Figure 2.

Changes in estimated glomerular filtration rate (eGFR) (A) and plasma concentration of 4β‐hydroxycholesterol (B) over time in patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele after living kidney transplantation. Data are presented as mean ± SD. *P < 0.05 and ^† P < 0.01 by Student's t‐test. ^# P < 0.05 and ^‡ P < 0.01 by Dunnett test. Patients with CYP3A5*1 allele, patients without CYP3A5*1 allele

Figure 1B shows the change in plasma concentration of 4β‐hydroxycholesterol over time in ESRD patients after living kidney transplantation. Compared with before living kidney transplantation (37.8 ± 13.0 ng ml⁻¹), plasma 4β‐hydroxycholesterol concentrations were significantly elevated on days 90 and 180 after living kidney transplantation [49.1 ± 15.0 ng ml⁻¹, P = 0.042 (95% CI of difference 0.29, 22.2) and 49.3 ± 14.8 ng ml⁻¹, P = 0.038, (95% CI of difference 0.47, 22.4), respectively]. Figure 2B shows the changes in plasma concentration of 4β‐hydroxycholesterol over time after living kidney transplantation in the two groups. In patients with the CYP3A5*1 allele, plasma 4β‐hydroxycholesterol concentrations were significantly elevated on days 90 and 180 after living kidney transplantation [57.1 ± 13.5 ng ml⁻¹, P = 0.0072 (95% CI of difference 4.17, 34.1) and 55.0 ± 14.5 ng ml⁻¹, P = 0.019, (95% CI of difference 2.12, 32.1), respectively] compared with before living kidney transplantation (37.9 ± 12.9 ng ml⁻¹), although no differences were observed in patients without the CYP3A5*1 allele. Furthermore, plasma concentrations of 4β‐hydroxycholesterol were significantly higher in patients with CYP3A5*1 allele than in patients without the CYP3A5*1 allele on days 90 and 180 after living kidney transplantation [P = 0.0034 (95% CI of difference 6.55, 28.6) and P = 0.043 (95% CI of difference 0.47, 24.8), respectively]. Especially, a mean difference of 1.5 fold was observed on day 90 after living kidney transplantation (Figure 3A). On the other hand, no significant correlation was observed between eGFR and plasma concentration of 4β‐hydroxycholesterol on day 90 after living kidney transplantation (Figure 3; r = 0.29, P = 0.19).

Figure 3.

Plasma concentrations of 4β‐hydroxycholesterol in patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele (A) and correlation between eGFR and plasma concentration of 4β‐hydroxycholesterol (B) on day 90 after living kidney transplantation

Discussion

In this study, we investigated the effect of CYP3A5 polymorphism on the increase in CYP3A activity after living kidney transplantation by measuring the plasma concentration of 4β‐hydroxycholesterol. A recent review has shown 35–75% decreases in liver CYP3A activity and 45–91% decreases in liver CYP3A protein expression in experimental circumstances in rats or mice with renal failure 25. Human studies of CYP3A activity using midazolam as a probe yielded controversial results, which reported a decrease 7 and no difference 26, 27 in CYP3A activity in ESRD patients compared with healthy controls. This paradox may be caused by alternation of midazolam pharmacokinetics in patients with renal failure. Midazolam is normally 96% protein bound and thus the unbound fraction may increase in patients with renal failure, resulting in increased hepatic clearance. On the other hand, 4β‐hydroxycholesterol is slowly eliminated from the circulation due to slow 7α‐hydroxylation by CYP7A1 20 and CYP7A1 is not affected by renal failure 28, suggesting that the kinetics of 4β‐hydroxycholesterol are not affected by renal function. Hence, 4β‐hydroxycholesterol is suitable for the evaluation of CYP3A activity in ESRD patients.

Serum creatinine levels decreased significantly from day 7 after living kidney transplantation and remained stable up to the end of study. This indicates successful living kidney transplantations in the patients who participated in this study. No significant differences in eGFR over time were observed between patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele. These data suggest that renal function recovered to similar degrees after living kidney transplantation in the two groups. On the other hand, plasma 4β‐hydroxycholesterol concentrations were significantly elevated on days 90 and 180 after living kidney transplantation, indicating recovery of CYP3A activity with improvement in renal function in ESRD patients after living kidney transplantation. Plasma 4β‐hydroxycholesterol concentrations 180 days after kidney transplantation were slightly higher compared with the concentrations in stable kidney transplant recipients reported previously (39.5 ± 11.7 ng ml⁻¹), which may be due to the change in renal function over time after kidney transplantation (180 vs. 1398 ± 1315 days) 22. Furthermore, plasma concentrations of 4β‐hydroxycholesterol on days 90 and 180 after living kidney transplantation were significantly higher in patients with the CYP3A5*1 allele than in patients without the CYP3A5*1 allele. These results suggest that CYP3A activity may increase markedly associated with recovery of renal function in patients with the CYP3A5*1 allele, or that CYP3A activity may not decrease remarkably in association with renal failure in patients without the CYP3A5*1 allele. To the best of our knowledge, this is the first report of the association of a genetic factor with change in CYP3A activity accompanying renal failure. On the other hand, no significant correlation was observed between eGFR and plasma concentration of 4β‐hydroxycholesterol on day 90 after living kidney transplantation, supporting the finding in stable kidney transplant recipients reported previously 22.

Reports have indicated that 4β‐hydroxycholesterol is formed by CYP3A4 and 3A5 19, 29, although the ratio is unknown. The increase in CYP3A activity after living kidney transplantation only in patients with the CYP3A5*1 allele in this study suggests that change in CYP3A5 activity, and not CYP3A4 activity, contributes predominantly to variation in CYP3A activity in ESRD patients. In addition, the finding of no difference in plasma 4β‐hydroxycholesterol concentration before kidney transplantation between ESRD patients with the CYP3A5*1 allele and patients without the CYP3A5*1 allele may imply that ESRD patients, even those with the CYP3A5*1 allele, have mainly CYP3A4 activity and little CYP3A5 activity before transplantation. The involvement of CYP3A5 polymorphism in CYP3A activity would explain the controversy of CYP3A activity in ESRD patients compared with healthy controls in previous studies 7, 26, 27, none of which genotyped the CYP3A5 gene. Furthermore, this may partially explain the paradox in the FDA review highlighting that approximately half of new molecular entities metabolized by CYP3A are markedly altered in kidney disease whereas the other half are unchanged 30. Racial differences in the frequency of the CYP3A5*3 allele have been reported 13, 31. In typical racial groups with high frequencies of the CYP3A5*1 allele, special attention may be required to the decrease in CYP3A activity associated with renal failure.

The mechanism of increase in CYP3A activity after kidney transplantation is unknown. Decrease in uremic toxins after kidney transplantation may play a role. It has been reported that indoxyl sulfate inhibits CYP3A activity and 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furan‐propanoic acid inhibits organic anion transport polypeptide 2 11. Thus, decrease in indoxyl sulfate after kidney transplantation may be involved in the increase in CYP3A activity after kidney transplantation. Parathyroid hormone is also known to mediate downregulation of CYP3A 32. Evaluation of plasma concentrations of indoxyl sulfate and parathyroid hormone may help to reveal the mechanism in patients with ESRD. Furthermore, a recent study suggests that CYP3A down‐regulation in renal failure may be mediated by decreased binding with the pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates CYP3A 33. Recovery of PXR binding after kidney transplantation may also be involved in the increase in CYP3A activity after kidney transplantation.

There are some limitations in this study. First, CYP3A activity was not measured in healthy volunteers. Thus the magnitude of change in CYP3A activity in ESRD patients is not clear. However, CYP3A activity in patients in this study appeared to be lower compared with Japanese healthy volunteers reported previously (77.0 ± 40.0 ng ml⁻¹) 34. Second, it remains uncertain whether CYP3A5 activity actually decreases in renal failure. Further in vitro studies are needed to resolve this issue. Finally, we were not able to compare the differences in plasma concentration of 4β‐hydroxycholesterol between three genotypes, CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3, because there were only three patients with CYP3A5*1/*1. A further large scale study is needed to determine whether there is a relationship between the number of *1 alleles and recovery of CYP3A activity after kidney transplantation.

In conclusion, we demonstrated significant differences in plasma concentration of 4β‐hydroxycholesterol after living kidney transplantation between patients with CYP3A5*1 allele and patients without CYP3A5*1 allele. The findings suggest that the presence of the CYP3A5*1 allele contributes to a marked increase in CYP3A activity associated with recovery of renal function after kidney transplantation.

Competing Interests

All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare YS had support from The Research Foundation for Pharmaceutical Sciences, The Nakatomi Foundation and Japan Society for the Promotion of Science (JSPS) for the submitted work. There are no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work.

Suzuki, Y. , Fujioka, T. , Sato, F. , Matsumoto, K. , Muraya, N. , Tanaka, R. , Sato, Y. , Ohno, K. , Mimata, H. , Kishino, S. , and Itoh, H. (2015) CYP3A5 polymorphism affects the increase in CYP3A activity after living kidney transplantation in patients with end stage renal disease. Br J Clin Pharmacol, 80: 1421–1428. doi: 10.1111/bcp.12733.