Biomarkers in renal transplantation: An updated review

Table 1 Definition and principal characteristics of biomarkers

Biomarker	A characteristic objectively measured as an indicator of a biological process or a response to a pharmacological intervention
Proteomics	The systematic analysis of proteins for their identity, quantity and function
Genomics	The study of the genome for estimating the risk for an individual to develop a disease
Transcriptomics	The study of expression patterns of all gene transcript
Metabolomics	The quantitative analysis of all the metabolites of a specific biological sample

Table 2 Biomarker candidates in the context of ischemia reperfusion injury and delayed graft function

Symbol	Gene description	Cytoband
ACTA2	Actin, alpha 2, smooth muscle, aorta	10q23.31
UMOD	Uromodulin	16p12.3
LGALS3	Lectin, galactoside-binding, soluble, 3	14q22.3
SAT1	Spermidine/spermine N1-acetyltransferase 1	Xp22.11
HAVCR1	Hepatitis A virus cellular receptor 1	5q33.3
CXCL1	Chemokine (C-X-C motif) ligand 1	4q13.3
ANXA2	Annexin A2	15q22.2
S100A6	S100 calcium binding protein A6	1q21.3
CYR61	Cysteine rich angiogenic inducer 61	1p22.3
S100B	S100 calcium binding protein B	21q22.3
AMBP	Alpha-1-microglobulin/bikunin precursor	9q32
LCN2	Lipocalin 2	9q34.11
C3	Complement component 3	19p13.3
FABP1	Fatty acid binding protein 1, liver	2p11.2
ATF3	Activating transcription factor 3	1q32.3
NTN1	Netrin 1	17p13.1
ENG	Endoglin	9q34.11
GUCY2G	Guanylate cyclase 2G	10q25.2
BID	BH3 interacting domain death agonist	22q11.21
BCL2	B-Cell CLL/lymphoma 2	18q21.33
BAX	BCL2 associated X protein	19q13.33
PTGS2	Prostaglandin-endoperoxide synthase 2	1q31.1
ADAMTS1	ADAM metallopeptidase with thrombospondin type 1 motif 1	21q21.3
CDKN1A	Cyclin dependent kinase inhibitor 1A	6p21.2

Table 3 Unbiased proteomic studies for acute rejection

Ref.	Biomarker candidate	Sample type	Sample numbers	Outcome
Freue et al[69]	TTN, LBP, CFD, MBL2, SERPINA10, AFM, KNG1, LCAT, SHBG	Plasma	32	AR
Sigdel et al[70]	UMOD, PEDF, CD44	Urine	60	AR
Wu et al[71]	NF-κB, STAT1, STAT3 and 63 other proteins	Plasma	13	AR
Loftheim et al[72]	IGFBP7, VASN, EGF, LG3BP	Urine	12	AR
Sigdel et al[73]	HLA-DRB1, FGB, FGA, KRT14, HIST1H4B, ACTB, KRT7, DPP4	Urine	154	AR

AR: Acute rejection; TTN: Titin; LBP: Lipid binding protein; MBL2: Mannose binding lectin 2; SERPINA 10: Protein Z-dependent protease inhibitor; AFM: Atomic force microscopy; KNG1: Kininogen1 protein; LCAT: Lecithin–cholesterol acyltransferase; SHBG: Sex hormon binding protein; UMOD: Uromodulin; PEDF: Pigment epithelium derived factor; NFκB: Nuclear factor kappa B; STAT1: Signal transducer and activator of transcription 1; STAT3: Signal transducer and activator of transcription 3; IGFBP7: Insulin like growth factor binding protein 7; VASN: Vasorin; EGF: Epidermal growth factor; LG3BP: Galectin-3-binding protein; FGB: Fibrinogen beta chain precursor; FGA: Fibrinogen alpha chain precursor; KRT14: Keratin14; HIST1H4B: Histone cluster 1 H4 family member b; ACTB: Actin beta; KRT7: Keratin 7; DPP4: Dipeptidil-peptidasi 4.

Table 4 Selected promising molecules and pathways evaluated as biomarkers in acute rejection[7]

Biomarker	Sample (assay method)	Patients/samples	Rejection/no rejection	Sensitivity/specificity (%)	PPV/NPV(%)	AUC
Granzyme B, perforin and FasL[74]	PBL (PCR)	25/31	11/20	100/95	100/95	NA
FOXP3[88]	PBL, urine (PCR)	65/78	20/58	94-100/95/100	94-100/95/100	0.95-1.00
Granzyme B, perforin[75]	Urine (PCR)	85/151	24/127	79-83/77-83	NA	NA
OX40, OX40L, PD-1 and FOXP3[76]	Urine (PCR)	46/46	21/25	95/92	NA	0.98
CD3ε,CXCL10, 18S rRNA[77]	Urine (PCR)	485/4300	43/1,70	79/78 (71/72)	NA	0.85 (0.74)
TIM-3[81]	PBL, urine (PCR)	115/160	65/95	87-100/95-100	87-100/93-100	0.96-1.00
CXCL9, CXCL10[78]	Urine (multiplex bead assay)	156/156	25/131	80-86/76-80	NA	0.83-0.87
CXCL9 mRNA and protein[79]	PBL, urine (PCR, ELISA, SELDI-TOF-MS	280/2770	37/113	66.7-85.2/ 79.6/80.7	61.5/67.6/83-92	0.78-0.85
miR-142-5p	Biopsy sample (PCR)	32/33	12/21	92-100/90-95	NA	0.96-0.99
miR-155
miR-223[83]
miR-210[85]	Urine (PCR)	81/88	68/20	52/74	NA	0.7
IFNγ-producing memory T cells[89]	PBL (ELISPOT)	23/23	12/10	80/83	NA	0.8

All the studies include a validation set. PPV: Positive predictive value; NPV: Negative predictive value; AUC: Area under the curve; PBL: Peripheral blood lymphocytes; PCR: Polymerase chain reaction; NA: Not available; PD-1: Programmed death 1; CXCL10: Interferon-inducible cytokine IP-10; 18S rRNA: 18S ribosomal RNA; TIM-3: T-cell immunoglobulin and mucin-domain containing-3; CXCL9: C-X-C motif chemokine 9; ELISA: Enzyme-linked immunosorbent assay; SELDI-TOF-MS: Surface-enhanced laser desorption/ionization time-of-flight MS; miRNA: microRNA; IFNγ: Interferon gamma; ELISPOT: Enzyme-linked immunoSpot.

Table 5 Seventeen genes involved in the study kidney solid organ response test

Symbol	Gene name	Cytoband
Genes derived from the NIH SNSO1 study
DUSP1	Dual-specificity phosphatase 1	5q35.1
NAMPT	Nicotinamide phosphoribosyltransferase	7q22.3
PSEN1	Presenilin 1	14q24.2
MAPK9	Mitogen-activated protein kinase 9	5q35.3
NKTR	Natural killer cell triggering receptor	3p22.1
CFLAR	CASP8 and FADD like apoptosis regulator gene	2q33.1
IFNGR1	Ligand binding chain of the gamma interferon receptor gene	6q23.3
ITGAX	Integrin alphaXchain protein encoding gene	16p11.2
RNF130	Ring finger motif encoding gene	5q35.3
RYBP	RING1 and YY1 binding protein encoding gene	3p13
Genes added to improve the accuracy of kSORT
CEACAM4	Carcinoembryonic antigen related cell adhesion molecule 4	19q13.2
EPOR	Erythropoietin receptor encoding gene	19p13.2
GZMK	Granzyme K encoding gene	5q11.2
RARA	Retinoic acid receptor encoding gene	17q21.2
RHEB	Ras homolog enriched in brain encoding gene	7q36.1
RXRA	Retinoic X receptor alpha encoding gene	9q34.2
SLC25A37	Solute carrier family 25 number 37 encoding gene	8p21.2

The 17 gene set was selected in 143 samples for acute rejection classification and predicted AR up to 3 mo prior to detection by the current gold standard (biopsy). kSORT: Kidney solid organ response test; SNSO1: Steroid-Free vs Steroid-Based Immunosuppression in Pediatric Renal (Kidney) Transplantation.

Table 6 Performance of kidney solid organ response test in the acute rejection in renal transplantation AART143, AART124, and AART100 cohorts

kSORT predictions
	AART143 (training set)		AART124 (validation set)		AART100 (cross-validation set)
	AR	No AR	AR	No AR	AR	No AR
Real results						3
AR	39	8	21	2	36	43
No AR	9	87	1	100	3
Sensitivity (95%CI)	82.98% (69.19%-92.35%)		91.30% (71.96%-98.38%)		92.31% (79.13%-98.38%)
Specificity (95%CI)	90.63% (82.95%-95.62%)		99.01% (94.61%-99.97%)		93.48% (82.1%-96.63%)
PPV (95%CI)	81.25% (68.06%-89.81%)		95.46% (78.20%-99.19%)		93.21% (79.68%-97.35%)
NPV (95%CI)	91.58% (84.25%-95.67%)		98.04% (93.13%-99.46%)		93.48% (82.45%-97.76%)
AUC (95%CI)	0.94 (0.91-0.98)		0.95 (0.88-1.00)		0.92 (0.86-0.98)

kSORT: Kidney solid organ response test; AART: Assessment of acute rejection in renal transplantation; AR: Acute rejection; PPV: Positive predictive value; NPV: Negative predictive value; AUC: Area under the curve.

Table 7 Eleven genes overexpressed in the common rejection module[103]

Symbol	Gene name	Cytoband
BASP1	Brain abundant membrane attached signal protein 1	5p15.1
CD6	CD6 molecule	11q12.2
CXCL10	C-X-C Motif chemokine ligand 10	4q21.1
CXCL9	C-X-C Motif chemokine ligand 9	4q21.1
INPP5D	Inositol polyphosphate-5-phosphatase D	2q37.1
ISG20	Interferon stimulated exonuclease gene 20	15q26.1
LCK	LCK protooncogene, SRC family tyrosine kinase	1p35.2
NKG7	Natural killer cell granule protein 7	19q13.41
PSMB9	Proteasome subunit beta 9	6p21.32
RUNX3	Runt related transcription factor 3	1p36.11
TAP1	Transporter 1, ATP binding cassette subfamily B member	6p21.32

These genes were overexpressed in acute rejection across all transplanted organs and could diagnose acute rejection with high specificity and sensitivity.

Table 8 Analysis of pooled urine proteins collected from patients with acute rejection, BK virus nephropathy, and chronic allograft nephropathy when compared to STA urine with the criteria of > 1.5 fold change of each transplant injury phenotype (acute rejection, BK virus nephropathy, and chronic allograft nephropathy), compared to STA pooled urine and with a P-value of ≤ 0.05[131]

Increased in AR	Increased in BKVN	Increased in CAN
HLA-DRB1, FGB, FGA, FGG, KRT14, HIST1H4B, KRT7, DPP4	KRT18, SUMO2, STMN1, CFHR2, KRT8, KRT19, RPL18, KRT75, FAM3C, HIST1H2BA	CALR, FAM151A, SERPINA2P, FAM3C, DAG1, KITLG, LUM, FABP4, AGT, LRG1

AR: Acute rejection; BKVN: BK virus nephropathy; CAN: Chronic allograft nephropathy; FGB: Fibrinogen beta chain; FGA: Fibrinogen alpha chain; FGG: Fibrinogen gamma chain; KRT14: Keratin 14; HIST1H4B: Histone cluster 1 H4 family member b; KRT7: Keratin 7; DPP4: Dipeptidyl peptidase 4; KRT18: Keratin 18; SUMO2: Small ubiquitin-like modifier 2; STMN1: Stathmin1; CFHR2: Complement factor H related 2; KRT8: Keratin 8; KRT19: Keratin 19; RPL18: Ribosomal protein L18; KRT75: Keratin 75; FAM3C: Family with sequence similarity 3 member C; HIST1H2BA: Histone cluster 1 H2B family member a; CALR: Calreticulin; FAM151A: Family with sequence similarity 151 member A; SERPINA2P: Serpin family A member 2; FAM3C: Family with sequence similarity 3 member C; DAG1: Dystroglycan 1; KITLG: KIT ligand; LUM: Lumican; FABP4: Fatty acid binding protein 4; AGT: Angiotensinogen; LRG1: Leucine rich alpha-2-glycoprotein 1.

Table 9 Genes higher (fold change higher than 6.00) expressed in renal tissue of patients affected by interstitial fibrosis/tubular atrophy[119]

Symbol	Gene name	Cytoband
IGHA1	Immunoglobulin heavy constant alpha 1	14q32.33
IGHG1	Immunoglobulin heavy constant gamma 1	14q32.33
CCR2	Chemokine C-C motif receptor 2	3p21.31
DFFB	DNA fragmentation factor 40 Da beta subunit	1p36.32
CD44	CD44 antigen	11p13
IFNA1	Interferon alpha 1	9p21.3
GZMK	Granzyme K	5q11.2
MMP9	Matrix metallopeptidase 9	20q13.12
TNFRSF17	Tumor necrosis factor receptor superfamily, member 17	16p13.13
CXCR4	Chemokine C-X-C motif receptor 4	2q22.1

Table 10 Thirteen genes associated with chronic allograft dysfunction identified by biopsy transcriptome expression[121]

Symbol	Gene description	Cytoband	CADI 12 mo correlation	P value
CHCHD10	Coiled-coil-helix-coiled- coil helix domain containing 10	22q11.23	0.404	2.85 × 10-5
KLHL13	Kelch-like family member 13	Xq23-q24	0.369	1.49 × 10-4
FJX1	Four jointed box 1	11p13	0.367	1.60 × 10-4
MET	Met proto-oncogene	7q31	0.352	3.01 × 10-4
SERINC5	Serine incorporator 5	5q14.1	0.318	0.0012
RNF149	Ring finger protein 149	2q11.2	0.28	0.0046
SPRY4	Sprouty homolog 4	5q31.3	0.27	0.0062
TGIF1	TGF-β induced factor homeobox 1	18p11.3	0.244	0.0140
KAAG1	Kidney associated antigen 1	6p22.1	0.24	0.0154
ST5	Suppressor of tumorigenicity 5	11p15	0.232	0.0197
WNT9A	Wingless-type MMTV integration site family member 9A	1q42	0.212	0.0332
ASB15	Ankirin repeat and SOCS box-containing 15	7q31.31	-263	0.0079
RXRA	Retinoid X receptor alpha	9q34.3	-0.3	0.0023

CADI: Chronic allograft dysfunction index.

Table 11 International research consortia in rejection/tolerance

Acronym	Description	Year
ITN	Immune tolerance network	Since 2002
IOC	Indices of tolerance	2003-2007
RISET	Reprogramming the immune system for establishment of tolerance	2005-2010
GAMBIT Study	Genetic analysis and monitoring of biomarkers of immunological tolerance	2010
The One Study	A unified approach to evaluating cellular immunotherapy in solid organ transplantation	2011
Bio-DRIM	Personalized minimization or immunosuppression after solid organ transplantation by biomarker driven stratification of patients to improve the long-term outcome and health-economic data of transplantation	2012
BIOMARGIN	Biomarkers of renal graft injuries in kidney allograft recipients	2013

GAMBIT: Genetic Analysis and Monitoring of Biomarkers of Immunological Tolerance.

Table 12 Immune/inflammatory molecules among the 357 gene signatures of tolerance

Categories	Diseases or functions annotation	Molecules	No. of molecules
Cell-to-cell signaling and interaction, cellular function and maintenance, hematological system development and function, inflammatory response	Phagocytosis of leukocyte cell lines	FGR, MRC1, TLR4	3
Cell-to-cell signaling and interaction, hematological system development and function, immune cell trafficking, inflammatory response, tissue development	Binding of neutrophils	FGR, LSP1, TLR4	3
Antimicrobial response, inflammatory response	Antibacterial response	CARD9, FGR, LYST, NLRC4, TLR4	5
Cell-to-cell signaling and interaction, hematological system development and function, inflammatory response	Binding of professional phagocytic cells	FGR, LSP1, NOTCH2, TLR4	4
Inflammatory response	Immune response of cells	CARD9, CLEC7A, ETS2, FGR, MRC1, SCARF1, MYO7A, TLR4	8
Antimicrobial response, inflammatory response	Antimicrobial response	CARD9, CLEC7A, FGR, LYST, NLRC4, TLR4	6
Inflammatory response	Innate immune response	CARD9, CLEC7A, TLR4, TRIM59	4
Cellular function and maintenance, inflammatory response	Phagocytosis	CLEC7A, ETS2, FGR, MRC1, MYO7A, TLR4, TPCN2	7
Cell-to-cell signaling and interaction, cellular growth and proliferation, hematological system development and function, inflammatory response	Stimulation of phagocytes	IL4R, TLR4	2
Antimicrobial response, humoral immune response, inflammatory response	Antifungal response	CARD9, CLEC7A	2
Cell-to-cell signaling and interaction, cellular function and maintenance, inflammatory response	Phagocytosis of cells	CLEC7A, ETS2, FGR, MRC1,MYO7A, TLR4	6

These genes potentially predict those patients that can be successfully weaned off immunosuppression[133]. FGR: Tyrosine-protein kinase Fgr; MRC1: Mannose receptor, C type 1; TLR4: Toll-like receptor 4; FGR: Tyrosine-protein kinase Fgr; LSP1: Lymphocyte-specific protein 1; CARD9: Caspase recruitment domain family member 9; LYST: Lysosomal-trafficking regulator; NLRC4: NLR family CARD domain-containing protein 4; NOTCH2: Neurogenic locus notch homolog protein 2; CLEC7A: C-type lectin domain family 7 member A; ETS2: Protein C-ets-2; SCARF1: Scavenger receptor class F member 1; MYO7A: Unconventional myosin-VIIa; TRIM59: Tripartite motif-containing protein 59; TPCN2: Two pore calcium channel protein 2; IL4R: Interleukin 4 receptor.

Table 13 Immunosuppression-independent gene signatures predicting tolerance[134]

Symbol	Gene name	Molecular function	Biological processes
ATXN3 ↓	Ataxin 3	Ubiquitin-specific protease activity	Protein metabolism
BCLA1 ↓	BCL2-related protein A1	Receptor signaling complex scaffold activity	Apoptosis
EEF1A1 ↓	Eukaryotic translation elongation factor 1 alpha 1	Transcription regulator activity	Regulation of cell cycle
GEMIN7 ↑	Gem associated protein 9	Ribonucleoprotein	Regulation of nucleobase, nucleosides, nucleotide and nucleic acid metabolism
IGLC1 ↑	Immunoglobulin lambda constant 1	Antigen binding	Immune response
MS4A4A ↑	Membrane-spanning 4-domains, subfamily A, member 4A	- - -	- - -
NFκBIA ↑	Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha	Transcription regulator activity	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism
RAB40C ↑	RAB40C, member of RAS oncogene family	GTPase activity	Cell communication, signal transduction
TNFAIP3 ↓	Tumor necrosis factor, alpha-induced protein 3	Transcription regulator activity	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism

↓Immunosuppression-free gene expression downregulated in tolerant patients; ↑Immunosuppression-free gene expression upregulated in tolerant patients; BCL2: B-cell lymphoma 2.