Male or female renal transplant recipients, aged 18 or over, who qualified for a renal biopsy as a routine evaluation at 3 month or because of an impaired renal function, and for whom blood and urine collection was possible, were considered for eligibility. Patients could not be included if they received nephrotoxic drugs other than those administered as standard care for renal transplant, within 1 month prior to screening until the last sample collection. Drugs known to alter tubular secretion of creatinine (e.g., cimetidine, amiodarone, dronedarone) but not cotrimoxazole, taken within 7 days prior to screening until the last sample collection, were additional exclusion criteria.
Inclusion started on July the 6th 2011 and ended on March the 11th 2015.
This prospective, single-center, open label study was conducted in the nephrology department of Bicêtre University Hospital, APHP. It was part of the European Innovative Medicines Initiative Joint Undertaking (IMI JU) Safer and Faster Evidence based Translation (SAFE-T) Consortium aimed at the clinical biomarker qualification for drug-induced injury to kidney, liver and the vascular system. All the drugs and their doses were left to the treating nephrologists, using “real world” treatment options.
All the patients gave their written informed consent to participate.
The study was approved by the French drug agency (ANSM) the 22 September 2010 (ID RCB : 2010-A01050-39) and a French ethical research committee (CPP Ile de France VII, ID : 11-004).
The duration of participation per patient (from screening to end-of-study) was 1 or 2 days depending on the time interval between blood and urine collection (Day 1), and the graft biopsy (Day 1 or Day 2).
Blood and urine samples were collected just prior graft biopsy.
Fasting blood samples were collected for plasma (on Li-Heparin and EDTA) and serum preparation (in a dry tube). The blood samples were centrifuged at +4°C at 2600 g for 10 min, within 30 min of collection for plasma samples, and after 30-60 min of clotting at +37ºC further 30 min at +4ºC for serum sample. Immediately following centrifugation, 3 aliquots (0.5 mL each) of the supernatant were transferred into 3 labeled polypropylene cryovials and immediately frozen at -80°C.
Fasting urine sample (spot urine) was collected in a dry container, after that he/she had emptied his/her bladder (overnight urine discarded) and immediately placed in an ice bath, then centrifuged at +4°C at 3000 g for 5 min, within 30 min of collection. Aliquots (1 mL each) of the supernatant were transferred into 12 labeled polypropylene cryovials, and they were immediately frozen at -80°C.
- Study endpoints
a) Histological study endpoints:
All kidney samples were reviewed by the same trained pathologist who classified the renal biopsies according to Banff 2007 (17)(18) classification using the quantitative criteria for glomerulitis (“g”), tubulitis (“t”), intimal arteritis (“v”), interstitial infiltrate of mononuclear cell (“i”), and interstitial fibrosis (“ci”).
The same pathologist also classified patients in 11 groups based on his histological conclusion.
Classical BM, serum creatinine, BUN, and urinary total proteins were measured as routine parameters at Bicêtre Hospital, eGFR was calculated according to MDRD modified formula (19).
The novel urinary BM cystatin C, NGAL, clusterin, calbindin, KIM-1, osteopontin, and MCP1 were quantified by Natural Medical Sciences Institute (NMI) using multiplex Luminex immunoassays. Urinary NAG was measured at Sanofi with an enzymatic assay, urinary microalbumin and plasma cystatin C with immunoturbidimetric assays. Urinary RBP4 was measured by NMI using immunoprecipitation LC/MS assay as previously described (20)(21) with amendment (see Additional file 1). Urinary aquaporin 2, podocin, podocalyxin, nephrin, and kirrel-1 were quantified at Sanofi by using an HPLC-MRM-MS multiplex method derived from previously developed singleplex LC/MS methods for aquaporin-2 (22), podocin (23) and podocalyxin (24) (see Additional file 1).
- Statistical methods (see Additional file 2)
Descriptive statistics were presented as mean ± SD, median ± interquartile range.
All serum or blood renal BM were analyzed as raw data. All urinary renal biomarkers were analyzed as raw data corrected by urinary creatinine.
Empirical ROC curves were constructed for each BM using patients belonging to a clinical group (e.g. ATN, GP, ACR…) as patients belonging to the event group versus patients belonging to the SMRP (or all the other patients) as patients belonging to the non-event group; if the number of patients in the event group was lower or equal than 5 then no ROC curves were constructed.
For all cases, combination of biomarkers (after log transformation of BM values, except for eGFR) were explored by selection of the best BM combination (in terms of lowest Akaike criteria) containing 3 BM. AUROC bootstrap estimates and 95%CIs were provided for all ROC curves of each individual BM and combination of BM.
For individual and combination of BM (when AUROC>0.7), bootstrap best BM’s diagnostic threshold and associated 95%CIs were estimated by looking for the pair of estimated sensitivity/specificity closest to perfect sensitivity and specificity. Sensitivity and specificity bootstrap estimates and 95% CIs associated with this best BM’s diagnostic threshold were also calculated.
Estimates were calculated using bootstrap methods in order to lighten the overfitting issue related to the search of the best biomarker among several ones.