Proteomics for rejection diagnosis in renal transplant patients: Where are we now?

Table 1 Proteomic studies on renal allograft rejection

Ref.	B/U	Training set	n	Validation set	n	Proteomic method	Performance	Identified molecules	Remarks
Akkina et al[35]	U	C (bx)	13	None		iTRAQ-	NR	None	Study included healthy individuals. Study concentrates on longitudinal stability of peptides in rejecting and non-rejecting patients
		BL	1			MALDI-
		IIa	1			MS/MS
		aABMR	1
Clarke et al[36]	U	C (st)	15	None		SELDI-	Accuracy 91% Sensitivity 83% Specificity 100% (2-marker classifier)	None
		AR	15			TOF-MS
Freue et al[37]	B	C (bx)	21	None		iTRAQ-	AUC 0.86 Sensitivity 80% specificity 90% (4-marker classifier)	Up-regulated: TTN, LBP, PI16, CFD, MBL2, SERPINA10, B2M Down-regulated: KNG1, AFM, SERPINA5, LCAT, SHBG	ELISA was performed on 4 of the identified markers (coagulation factor IX, SHBG, CFD, LCAT) in blood
		Ia	7			MALDI-
		Ib	1			MS/MS
		IIa	3
Günther et al[38]	B	C (st)	13	C (st)	7	iTRAQ-	AUC 0.76	21 peptides	Different statistical approaches to integrate proteomics and transcriptomic results are presented
		AR	13	AR	7	MALDI-	Sensitivity 57%
						MS/MS	specificity 86%
Jahnukainen et al[39]	U	C (st)	29	None		SELDI-	Sensitivity 81% Specificity 84% (100-marker classifier)	None	21 of the 28 rejection samples showed also signs of chronic rejection Article concentrates on differentiation of AR and BKV-NP
		Ia-IIb	28			TOF-MS
		BKV	21
Ling et al[40]	U	C (bx)	10	C (bx)	10	LC-MALDI-	AUC 0.96 (40-marker classifier)	COL1A2, COL3A1, UMOD, MMP-7, SERPING1, TIMP1	Study included healthy individuals and patients with native kidney disease (nephrotic syndrome). Results of proteomic analysis are related to mRNA expression profiling of corresponding biopsies
		AR	10	AR	10	TOF-MS
		BKV	10	BKV	4	LC-MS/MS
Loftheim et al[41]	U	C (st)	6	None		2D LC-	NR	Up-regulated: IGFBP7, VASN, EGF, LGALS3BP	Study collected sequential urines from the beginning after Tx. Analysed samples for rejection patterns were taken 7-11 d before biopsy
		BL	1			MS/MS
		Ia	4
		IIa	1
Mao et al[42]	U	C (bx)	22	C (bx)	14	SELDI-	Sensitivity 90% Specificity 71% (4-marker classifier)	None	All TCMR cases were subclinical rejections with grades ≥ Ia
		TCMR	27	TCMR	10	TOF-MS
Metzger et al[43]	U	C (bx)	23	C (bx)	36	CE-MS	AUC 0.91 Sensitivity 93% Specificity 78% (14-marker classifier)	3 fragments of COL1A1, 1 fragment of COL3A1	Rejections in the training set were all subclinical. The validation set contained 10 clinical and 18 subclinical rejection cases. Confounder like ATI in biopsies, urinary tract infection and CMV infection were considered
		Ia	13	Ia	23	LC-MS/MS
		Ib	3	Ib	5
O’Riordan et al[44]	U	C (st)	22	None		SELDI-	AUC 0.91 Sensitivity 91% Specificity 77% (2-marker classifier)	Up-regulated: SERPINA3 Downregulated: DEFB1	Study included healthy individuals
		AR	23			TOF-MS
O’Riordan et al[45]	U	C (st)	22	None		SELDI-	AUC 0.91 Sensitivity 91% Specificity 77% (2-marker classifier)	Up-regulated: SERPINA3 Downregulated: DEFB1
		BL	3			TOF MS
		Ia	6			LC-MS/MS
		Ib	4
		IIa	7
		IIb	3
Pisitkun et al[46]	U	C (bx)	2	None		LC-MS/MS	NR	Numerous molecules
		Ia	4
		Ib	1
		IIa	2
		ATI	7
Quintana et al[47]	U	C (st)	8	a/cABMR	8	MALDI-	IFTA vs cABMR AUC 1.0 Sensitivity 100% Specificity 100% (6-marker classifier)	None	Study included healthy individuals
		a/cABMR	10	IFTA	6	TOF-MS
		IFTA	8
Quintana et al[48]	U	C (st)	5	C (st)	9	LC-MS/MS	C vs IFTA/ABMR: AUC 0.82 IFTA vs ABMR 100% correct IFTA, 90% correct ABMR (2-markers)	Down-regulated: UMOD Differentiation between controls and IFTA/ABMR: KNG1	Study included healthy individuals Two unidentified peptides could differentiate between IFTA and ABMR, based on quantitative differences of the peptides (higher in ABMR)
		a/cABMR	10	a/cABMR	11
		IFTA	8	IFTA	8
Reichelt et al[49]	U	C (bx)	10	None		SELDI-	SAX2 protein chip: Sensitivity 90% Specificity 80% CM10 protein chip: Sensitivity 92% Specificity 85% (2-marker classifier)	None
		Ia	7			TOF-MS
		Ib	3
		IIa	1
		IIb	2
Schaub et al[13]	U	C (bx)	22	None		SELDI-	Sensitivity 94% Specificity 82% (3-marker classifier)	Cleaved B2M Cleaved B2M	Study included healthy individuals. The clinical confounder CMV viremia was assessed. Longitudinal evaluation of urine proteome patterns differentiated between patients with stable course and rejection
		Ia	7			TOF-MS
		Ib	8
		IIa	3
		ATI	5
		GL	5
Schaub et al[15]	U	C (bx)	22	None		SELDI-	NR		Study included healthy individuals. Study concentrated on cleavage mechanisms for b2-microglobulin
		Ia	7			TOF-MS,
		Ib	8			LC-MALDI-
		IIa	3			MS
		ATI	5
		GL	5
Sigdel et al[14]	U	C (bx)	10	None		LC-MALDI-	NR	List of 73 candidates, incl. fragments of collagens, UMOD, B2M, PTGDS	Study included healthy individuals
		AR	10			MS/MS
Sigdel et al[50]	U	C (bx)	10	None		LC-MS/MS	AUC 0.84-0.97 for 3 single molecules (by ELISA)	Upregulated: SERPINF1 Down-regulated: UMOD, CD44	Study included healthy individuals and patients with native kidney disease (proteinuria)
		AR	10
Sigdel et al[51]	U	C (bx)	30	None		iTRAQ-	AUC 0.8 for 3 single molecules (by ELISA)	HLA-DRB1, KRT14, HIST1H4B, FGG, ACTB, FGB, FGA, KRT7, DPP4, cleaved B2M	In ELISA studies, FGG could also segregate AR from BKV-nephropathy Validation set for detection of FGG, HLA DRB1, FGB by ELISA included 44 stable transplant patients and 44 patients with rejection
		Ia-IIb	30			LC-MS/MS
		aABMR	2
		IFTA	30
		BKV	18
Sigdel et al[52]	U	C (bx)	20	None		iTRAQ-	NR	Enriched in exosomal fraction in AR: A2M, APOA2, APOM, CD5L, CLCA1, FGA, FGB, IGHM, DEFA5, PROS1, KIAA0753 Exclusively in the exosomal fraction in AR: CLCA1, PROS1, KIAA0753	Study concentrated on differences between the whole proteome in urine (non-fractionated) and the exosomal fraction
		≥ Ia	20			LC-MS/MS
Stubendorff et al[53]	U	C (st)	16	C (st)	16	SELDI-	Sensitivity 94% Specificity 44% (4-marker classifier) Sensitivity 80% Specificity 81% for 2 molecules (by ELISA)	Up-regulated: A1MG, HP	Results on longitudinally collected samples suggest that alpha-1-microglobulin and haptoglobin indicate upcoming AR early
		AR	16	AR	16	TOF MS
Sui et al[54]	B	C (bx)	12	None		MALDI-	Recognition capability for AR 90%	None	Study included healthy individuals. Sample clean-up was performed with magnetic beads
		AR	12			TOF-MS
		CR	12
Wang et al[55]	B	C (bx)	19	C (bx)	10	SELDI-	C vs subclinical ≥ Ia Sensitivity 100% Specificity 90% (3-marker classifier) C vs TCMR Sensitivity 90% Specificity 90% (7-marker classifier) AR vs subclinical Sensitivity 100% Specificity 100% (4-marker classifier)	None	≥ Ia refers to subclinical rejections only. All (non-graded) TCMR cases were clinical rejections
		≥ Ia	14	≥ Ia	10	TOF-MS
		TCMR	28
		ATI	10
Wittke et al[56]	U	C (bx)	29	C (bx)	10	CE-MS,	Sensitivity 67% Specificity 80% (17-marker classifier)	COL4A5	Transplant patients with urinary tract infection were included, with biopsy-confirmed absence of rejection. Of the rejection cases, 13 were subclinical and 6 clinical
		Ia	11	Ia	6	LC-MS/MS
		Ib	6	Ib	3
		IIa	1
		IIb	1	UTI	7
		UTI	10
Wu et al[57]	B	C (st)	8	None		iTRAQ-	NR	Numerous molecules belonging to different pathways: e.g., inflammatory response, complement, defence response, protein maturation and processing, humoral immune response
		Ib	1			2D LC-
		IIa	2			MS/MS
		IIb	1
		III	1
Yang et al[58]	U	C (bx)	36	C (bx)	14	SELDI-	C vs TCMR/ABMR Sensitivity 100% Specificity 78% (3-marker classifier) ABMR vs TCMR Sensitivity 80% Specificity 95% (5-marker classifier)	None
		TCMR	30	TCMR	10	TOF-MS
		aABMR	25	aABMR	10
		ATI	10
Zhang et al[59]	U	C (bx)	41	None		MALDI-	Different classifier combinations: Sensitivity 73%-88% Specificity 53%-62%	Up-regulated: B2M, SERPINA1. Down-regulated: PSAP	Study included healthy individuals and patients with native kidney disease (nephrotic syndrome). Saposin B was high in transplant patients with stable course over 280 d and low in patients with subsequent graft failure
		CR/(AR)	90			TOF-MS
						MALDI-
						MS/MS
Ziegler et al[60]	B	C	48	None		SELDI-	Sensitivity 100% Specificity 94% for 2 molecules (by ELISA)	Out of 22 candidates decreased: APOA1, SERPINA3	Two patients with TCMR had also signs of additional ABMR. The 2 markers for rejection were not informative in samples collected a few days before the rejection
		Ia	10			TOF-MS
		Ib	7			MALDI-
						MS/MS

Patient group definitions: C (bx): Control patients with biopsy-confirmed absence of rejection; C (st): Control patients without biopsy to exclude rejection; AR: Acute rejection without further histologic grading; CR: Chronic rejection without further histologic grading; TCMR: T cell-mediated without further histologic grading; ABMR: Antibody-mediated rejection with prefix “a” (acute) and “c” (chronic); BL: Borderline rejection (suspicious for rejection); IFTA: Interstitial fibrosis and tubular atrophy; BKV: BK virus nephropathy; ATI: Acute tubular injury; GL: De novo or recurrent glomerulopathy; UTI: Urinary tract infection with biopsy-confirmed absence of rejection; Ia, Ib: T cell-mediated tubulointerstitial (rejection specified as “mild” (a) and “severe” (b); IIa, IIb: T cell-mediated vascular rejection specified as “mild” (a) and “severe” (b); III: T cell-mediated vascular rejection with transmural arteritis; CMV: Cytomegalovirus; AUC: Area under the curve; CE: Capillary electrophoresis; iTRAQ: Isobaric Tags for Relative and Absolute Quantification; LC: Liquid chromatography; MALDI: Matrix-assisted laser desorption ionization; MS: Mass spectrometry; MS/MS: Tandem mass spectrometry; SELDI: Surface-enhanced laser desorption ionization; TOF: Time of flight; B/U: Examined matrix (blood: B, urine: U); n: Number of patients in each category; NR: Not reported.

Table 2 Ongoing proteomic studies on rejection in renal transplant patients

Study identifier and title	Aim	Institution/PI	Single/multi-centre	Patients	Study start	Estimated primary completion	Status of the study
NCT01515605	Analysis of GATA3, GATA4, GAPDH, TRPC3, TRPC6, granzyme B, perforin, FOXP3, ISG15, Mx1, MMP-3, MMP-9 in blood cells, proteomic analysis of urine, tissue analysis in a longitudinal fashion. Correlation of these parameters to the outcome	Odense University Hospital, Denmark	NR	1000	January 2011	March 2014	Unknown
Molecular biological and molecular genetic monitoring of therapy after kidney transplantation
NCT01315067	Phase III in-place validation of a pre-defined, published urinary peptide panel for acute TCMR against the current standard allograft biopsy[43]	Hannover Medical School, Germany	Multi	600	October 2011	December 2015	Active, not recruiting
Non-invasive diagnosis of acute rejection in renal transplant patients using mass spectrometry of urine samples - a multicentre diagnostic phase III trial[62]
NCT01531257	Validation of a set of candidate molecules by urine proteomics, gene expression analysis of blood cells and graft biopsies in a longitudinal fashion with respect to AR and IFTA	Northwestern University, Chicago, Illinois, United States	Single	250	April 2010	April 2016	Recruiting
Proteogenomic monitoring and assessment of kidney transplant recipients
NCT01289717	Discovery and validation of candidate molecules by urine proteomics, gene expression analysis of blood cells and allograft biopsies in a longitudinal fashion with respect to AR and IFTA	National Institute of Allergy and Infectious Diseases; Northwestern University, Chicago, Illinois, United States	Multi	307	March 2011	June 2016	Active, not recruiting
Discovery and validation of proteogenomic biomarker panels in a prospective serial blood and urine monitoring study of kidney transplant recipients - transplant proteogenomics
NCT02463253	Analysis of proteogenomic and proteomic biomarkers in relation to the biopsy diagnosis of acute rejection in a longitudinal fashion	University of California, Sacramento, California, United States	Single	50	April 2015	December 2016	Recruiting
Correlation of molecular biomarkers with biopsy findings and outcomes in renal transplant recipients

All studies are prospective, observational cohort studies in adult patients. Preliminary reports have not been published yet. Except study NCT 01315067, all studies collect samples in a longitudinal fashion and examine additional markers obtained by genomic analysis of blood cells. PI: Principal investigator site; AR: Acute rejection; IFTA: Interstitial fibrosis and tubular atrophy; NR: Not reported.