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Item does not contain fulltext, BACKGROUND: Common genetic variants of the enzymes and efflux pump involved in tacrolimus disposition have been associated with calcineurin inhibitor nephrotoxicity, but their importance is unclear because of the multifactorial background of renal fibrosis. This study explores the pro-fibrotic response of tacrolimus exposure in relation to the differential capacity for tacrolimus metabolism in proximal tubule cells (PTCs) with a variable (pharmaco)genetic background. METHODS: PTCs were obtained from protocol allograft biopsies with different combinations of CYP3A5 and ABCB1 variants and were incubated with tacrolimus within the concentration range found in vivo. Gene and protein expression, CYP3A5 and P-glycoprotein function, and tacrolimus metabolites were measured in PTC. Connective tissue growth factor (CTGF) expression was assessed in protocol biopsies of kidney allograft recipients. RESULTS: PTCs produce CTGF in response to escalating tacrolimus exposure, which is approximately 2-fold higher in cells with the CYP3A5*1 and ABCB1 TT combination in vitro. Increasing tacrolimus exposure results in relative higher generation of the main tacrolimus metabolite {13-O-desmethyl tacrolimus [M1]} in cells with this same genetic background. Protocol biopsies show a larger increase in in vivo CTGF tissue expression over time in TT vs. CC/CT but was not affected by the CYP3A5 genotype. CONCLUSIONS: Tacrolimus exposure induces a pro-fibrotic response in a PTC model in function of the donor pharmacogenetic background associated with tacrolimus metabolism. This finding provides a mechanistic insight into the nephrotoxicity associated with tacrolimus treatment and offers opportunities for a tailored immunosuppressive treatment.

Item does not contain fulltext, Background: Cognitive functions are altered in patients with chronic kidney disease. However it is suggested that cognitive functions, at least partially, improve after kidney transplantation. A possible cause for this improvement could be the reduction of uremic retention solutes after transplantation. This study assessed the association between the changes in uremic toxin concentration with the changes in cognitive function in patients after kidney transplantation. Methods: 10 kidney transplant recipients were included and compared to 18 controls (9 patients on hemodialysis, and 9 patients with CKD stage 4 or 5 (eGFR < 30ml/min/1.73m2 not on dialysis). An extensive neuropsychological assessment, covering the five major cognitive domains, that is, memory, attention and concentration, information processing speed, abstract reasoning, and executive function, was done before, at one week and three months after transplantation. Similarly assessments of the 18 matched control patients were performed longitudinally over a period of three to five months. Concentrations of sixteen uremic retention solutes namely indoxylglucuronide, p-cresylglucuronide, phenylglucuronide, CMPF, indoxyl sulphate, p-cresyl sulphate, hippuric acid, phenyl sulphate, kynurenine, tryptophan, kynurenic acid, tyrosine, indole-3-acetic acid, phenylalanine, trimethylamine N-oxide (TMAO), and phenylacetylglutamine were measured in serum samples collected at the time of the neuropsychological assessments. Results: A significant improvement in cognitive function was only found in the processing speed domain, and observed in both transplant patients as well as CKD patients. No significant differences between transplant patients and the control groups were seen in the other cognitive domains. As expected, the serum concentration of most uremic toxins decreased significantly within one week after kidney transplantation. Conclusions: There was no significant improvement in cognitive function that could be s

Item does not contain fulltext, 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 g