Evaluating urinary volatile organic compounds as a novel metabolomic biomarker to differentiate between acute tubular necrosis and acute graft rejection following kidney transplantation

Introduction: Acute tubular necrosis (ATN) and acute rejection (AR) are primary causes of early graft dysfunction following kidney transplantation. Timely differentiation between ATN and AR is critical as different management approaches are indicated. Currently, distinguishing between ATN and AR relies heavily on transplant biopsy findings for definitive diagnosis. It has been established that dysregulation in metabolic pathways occur from acute phases of graft dysfunction. Hence, there is a basis in utilizing metabolomic biomarkers to determine between the causes of acute post-transplant complications. A form of metabolomic biomarker, volatile organic compounds (VOCs) are gaseous products of metabolic processes in organisms which are typically released with greater abundance in disease when there is metabolic dysregulation. The extent in which urinary VOCs reflect the abnormal metabolic profiles between patients with ATN and AR is unknown. Our group aimed to address this.  
Methods: Kidney transplant recipients age 18-75 years diagnosed with ATN or AR following evaluation of transplant biopsy were included. Urine samples collected from study patients prior to biopsy were processed. Urine supernatant were extracted from residue, and sampled for stir bar sorptive extraction followed by Gas Chromatography–Mass Spectrometry (GC-MS) analysis. Post-processing of GC-MS data separated complex mixtures of VOCs based on their volatility & polarity. Mass-to-charge ratios and fragment patterns were measured for individual VOCs identification and quantification. Linear discriminant analysis (LDA) was performed to assess the ability of urinary VOCs in discriminating between ATN and AR. Linear regression analysis adjusting for age, sex, diabetes status, days between transplant and biopsy, and albumin:creatinine ratio was performed to determine significantly regulated urinary VOCs when comparing between the ATN versus AR group. 
Results: 47 study patients (26 with ATN, 21 with AR) were included. 27 VOCs total as identified from GC-MS were statistically associated with correct classification of ATN or AR as per transplant biopsy. However, 4 VOCs were detected in fewer than 20% of samples and were excluded from inferential analysis, leaving 23 VOCs for further analyses. Based on the 23 VOCs, LDA demonstrated distinct separation between the ATN and AR groups based on urinary VOCs profiling (Fig. 1) (p<0.001). Multivariate linear regression analysis reported 7 VOCs (caffeine; hexadecanamide; acetophenone; ethanone, 1-(4-methylphenyl)-; formamide, N-phenyl-; p-cresol; and disulfide, dimethyl) significantly upregulated in the ATN compared to the AR group (p<0.001) and 2 VOCs (isopropylbenzaldehyde; and benzoic acid, methyl ester) significantly downregulated in the ATN compared to the AR group (p<0.001).