*2.3. Biomarkers*

The included studies were split into diagnostic and prediction studies. To be considered a diagnostic study, the collection of a urine sample should be performed on the day that AR was suspected or when per-protocol biopsies were planned. There were a few exceptions to this rule where sample collection occasionally occurred up to seven days before biopsy. For prediction studies, urine samples were collected at any time point post-transplant and, in this analysis, these ranged from day one up to six months post-transplantation. Among the various techniques for targeted analysis of known urinary biomarkers, ELISA and RT-PCR were the most frequently utilized. Mass spectrometry, nuclear magnetic resonance spectroscopy, liquid chromatography, RNA expression, and transcriptome analysis by RNA-Seq were employed for unbiased metabolomics, proteomics, and genomic profiling and for detection and identification of urinary exosome proteins. All biomarkers are detailed per category in Table 2.

In accordance with previous studies, the most extensively assessed urinary biomarkers were C-X-C motif chemokine ligand 9 (CXCL9) and 10 (CXCL10), usually adjusted for urinary creatinine concentration. In detail, 12/38 (32%) studies either addressed CXCL9 and CXCL10 alone, in combination, or in the context of particular scores or formulas [16,17,24,26,34–36,44,47,50–52]. Other directly targeted cytokines and interleukins were chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 [22,31,43], CXCL13 [29], interleukin 10 (IL10) with interferon gamma (IFNγ) [46] and tumor necrosis factor alpha (TNFα) [32].


**Table 2.** Urinary biomarkers. Table illustrating all urinary biomarkers divided per category and in alphabetic order. Specific formulas and scores are also detailed.

Unbiased metabolomic analysis and untargeted profiling revealed multiple urinary metabolites as potential biomarkers of AR: nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), nicotinic acid, 1-methylnicotinamide (MNA), gamma-aminobutyric acid (GABA), cholesterol sulfate, homocysteine [18], the combination of alanine, citrate, lactate, and urea [21,27], and the combination of guanidoacetic acid, methylimidazoleacetic acid, dopamine, 4-guanidinobutyric acid, and l-tryptophan [25].

Urinary proteins of interest were neutrophil gelatinase-associated lipocalin (NGAL) [22,40], liver-type fatty acid-binding protein (LFABP) [22], human epididymis protein 4 (HE4) [22], matrix metalloproteinase 7 (MMP7) [47], soluble T cell immunoglobulin mucin domain 3 (sTIM3) [37]. The presence and diagnostic performance of urinary extracellular vesicle (exosome) proteins, derived from inflammatory cells and collected with the help of nano-membrane or immune-magnetic capture, was investigated by three studies [28,32,52].

Direct RT-PCR was used to identify targeted RNAs like programmed cell death protein1 (PD1) mRNA, forkhead box P3 (FOXP3) mRNA, and micro (mi) RNAs (miR-142-3p, miR-155-5p) [30,34,38,48], while genome-wide or transcriptome analysis were applied for the unbiased identification of circular and long noncoding RNAs [23,49].

The amount of urinary CD4+ and CD8+ T cells, tubular epithelial cells (TEC), podocalyxin (PDX)-positive, CD10+ or epithelial cell adhesion molecule (EPCAM)-positive cells was determined by flow cytometry and compared with biopsy results [20].

Two studies [35,44] investigated the diagnostic accuracy of the *CTOT4 formula* (CXCL10 mRNA, CD3ε mRNA, 18S rRNA), previously validated by the CTOT-4 (clinical trials in organ transplantation-4) multicenter study group [54]. CXCL10 was also included in newly derived scores such as the *Q score*, composed of six DNA, protein, and metabolite urinary biomarkers (cell-free DNA, methylated cell-free DNA, clusterin, total protein, creatinine, and CXCL10) [17], while CXCL9 and CXCL10 genes

were included in the *uCRM score* [24]. Finally, the *ABMR score* comprised more than 130 unique metabolites [41].
