Abstract
Subclinical acute rejection (SCAR) in kidney transplantation, defined by histologic lesions without clinical dysfunction, remains a major cause of allograft injury and is currently detectable only by invasive protocol biopsies. We conducted a multicenter study in which transcriptomic profiling of Formalin-Fixed, Paraffin-Embedded biopsies from SCAR and control patients revealed a distinct signature with upregulation of NFKBIZ, TNFSF14, SLAMF8, and CD247, validated by qRT-PCR and immunohistochemistry but not detectable in urine. Focusing on secreted cytokines, CXCL10 and FasL emerged as candidate urinary biomarkers and were first measured in 12 SCAR patients and 12 controls, showing a significant increase in SCAR. Validation in an independent cohort of 86 kidney transplant recipients, after excluding patients with confounders, confirmed higher CXCL10 and FasL levels in SCAR. When combined as a composite biomarker signature (CXCL10 + FasL), ROC analysis yielded an AUC of 0.711 (95% CI, 0.549–0.874), with 50% sensitivity and 84% specificity at the optimal cutoff. In the still poorly studied context of SCAR, this work is a proof-of-concept approach linking intragraft transcriptomics to urinary cytokine levels. Our findings support the utility of urinary CXCL10 and FasL in assisting clinicians in identifying patients who may benefit from further evaluation, including consideration of a graft biopsy, thereby contributing to improved long-term allograft outcomes.
Data availability
The microarray data generated and analyzed in this study are MIAME-compliant and have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE294632. These data are publicly accessible at [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE294632]. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request.
References
Rush, D. N., Jeffery, J. R. & Gough, J. Sequential protocol biopsies in renal transplant patients Clinico-pathological correlations using the Banff schema. Transplantation 59, 511–514 (1995).
Mehta, R., Sood, P. & Hariharan, S. Subclinical rejection in renal transplantation: Reappraised. Transplantation 100, 1610–1618 (2016).
Ibernón, M. et al. Subclinical rejection impairs glomerular adaptation after renal transplantation. Kidney Int. 70, 557–561 (2006).
Nankivell, B. J. et al. Natural history, risk factors, and impact of subclinical rejection in kidney transplantation. Transplantation 78, 242–249 (2004).
Loupy, A. et al. Subclinical rejection phenotypes at 1 year post-transplant and outcome of kidney allografts. J. Am. Soc. Nephrol. 26, 1721–1731 (2015).
Shapiro, R. et al. An analysis of early renal transplant protocol biopsies - the high incidence of subclinical tubulitis. Am. J. Transplant. 1, 47–50 (2001).
Jain, S., Curwood, V., White, S. A., Furness, P. N. & Nicholson, M. L. Sub-clinical acute rejection detected using protocol biopsies in patients with delayed graft function. Transpl. Int. 13(Suppl 1), S52–S55 (2000).
Kurtkoti, J. et al. The utility of 1- and 3-month protocol biopsies on renal allograft function: A randomized controlled study. Am. J. Transplant. 8, 317–323 (2008).
Sis, B. et al. Banff ‘09 meeting report: Antibody mediated graft deterioration and implementation of Banff working groups. Am. J. Transplant. 10, 464–471 (2010).
Nankivell, B. J. & Chapman, J. R. The significance of subclinical rejection and the value of protocol biopsies. Am. J. Transplant. 6, 2006–2012 (2006).
Rush, D. et al. Beneficial effects of treatment of early subclinical rejection. J. Am. Soc. Nephrol. 9, 2129–2134 (1998).
Seifert, M. E. et al. Impact of subclinical borderline inflammation on kidney transplant outcomes. Transplant. Direct 9, E663 (2021).
Mengel, M. et al. Protocol biopsies in renal transplantation: Insights into patient management and pathogenesis. Am. J. Transplant. 7, 512–517 (2007).
Ferguson, C., Winters, S., Jackson, S., McToal, M. & Low, G. A retrospective analysis of complication and adequacy rates of ultrasound-guided native and transplant non-focal renal biopsies. Abdom. Radiol. 43, 2183–2189 (2018).
Song, Y. et al. Advancements in noninvasive techniques for transplant rejection: From biomarker detection to molecular imaging. J. Transl. Med. https://doi.org/10.1186/s12967-024-05964-4 (2025).
Menon, M. C., Keung, K. L., Murphy, B. & O’Connell, P. J. The use of genomics and pathway analysis in our understanding and prediction of clinical renal transplant injury. Transplantation 100, 1405–1414 (2016).
Vitalone, M. J. et al. Transcriptional perturbations in graft rejection. Transplantation 99, 1882–1893 (2015).
Mueller, T. F. & Mas, V. R. Microarray applications in nephrology with special focus on transplantation. J. Nephrol. 25, 589–602 (2012).
Hayde, N. et al. Increased intragraft rejection-associated gene transcripts in patients with donor-specific antibodies and normal biopsies. Kidney Int. 86, 600–609 (2014).
Halloran, P. F. et al. Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: An international prospective study (INTERCOM). Am. J. Transplant. 13, 2865–2874 (2013).
Friedewald, J. J. et al. Development and clinical validity of a novel blood-based molecular biomarker for subclinical acute rejection following kidney transplant. Am. J. Transplant. 19, 98–109 (2019).
Goldfarb, D. A. Urinary-Cell mRNA profile and acute cellular rejection in kidney allografts. J. Urol. 190, 2175–2176 (2013).
Tajima, S. et al. Urinary human epididymis secretory protein 4 as a useful biomarker for subclinical acute rejection three months after kidney transplantation. Int. J. Mol. Sci. 20 (2019).
Cox, S. N. et al. Formalin-fixed paraffin-embedded renal biopsy tissues: an underexploited biospecimen resource for gene expression profiling in IgA nephropathy. Sci. Rep. 10 (2020).
Teng, L. et al. SLAMF8 Participates in acute renal transplant rejection via TLR4 pathway on pro-inflammatory macrophages. Front. Immunol. 13, (2022).
Reeve, J. et al. Molecular diagnosis of T cell-mediated rejection in human kidney transplant biopsies. Am. J. Transplant. 13, 645–655 (2013).
Famulski, K. S. et al. Kidney transplants with progressing chronic diseases express high levels of acute kidney injury transcripts. Am. J. Transplant. 13, 634–644 (2013).
Chung, K. W. et al. Involvement of NF-κBIZ and related cytokines in age-associated renal fibrosis. Oncotarget 8, 7315–7327 (2017).
Baniyash, M. TCR ζ-chain downregulation: Curtailing an excessive inflammatory immune response. Nat. Rev. Immunol. 4, 675–687 (2004).
Krepsova, E. et al. Effect of induction therapy on the expression of molecular markers associated with rejection and tolerance. BMC Nephrol. 16, (2015).
Li, Y. et al. Tumor necrosis factor superfamily 14 is critical for the development of renal fibrosis. Aging (Albany. NY) 12, 25469–25486 (2020).
Xiang, W. et al. Pre-transplant transcriptional signature in peripheral blood mononuclear cells of acute renal allograft rejection. Front. Med. 8, (2022).
Robertson, H. et al. Decoding the hallmarks of allograft dysfunction with a comprehensive pan-organ transcriptomic atlas. Nat. Med. 30, 3748–3757 (2024).
Uhlén, M. et al. Tissue-based map of the human proteome. Science (80). 347 (2015).
Hirt-Minkowski, P. & Schaub, S. Urine CXCL10 as a biomarker in kidney transplantation. Curr. Opin. Organ Transplant. 29, 138–143 (2024).
Yannaraki, M. et al. Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection. Transpl. Int. 19, 759–768 (2006).
Gao, J., Wu, L., Wang, S. & Chen, X. Role of chemokine (C–X–C Motif) Ligand 10 (CXCL10) in renal diseases. Mediators Inflamm. 2020, 6194864 (2020).
Masteller, E. L. & Bluestone, J. A. Chemokines: Directing leukocyte infiltration into allografts. Curr. Opin. Immunol. 14, 562–568 (2002).
Dufour, J. H. et al. IFN-γ-inducible protein 10 (IP-10; CXCL10)-Deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J. Immunol. 168, 3195–3204 (2002).
Heng, B. et al. Diagnostic performance of Fas ligand mRNA expression for acute rejection after kidney transplantation: A systematic review and meta-analysis. PLoS One 11, (2016).
Legendre, C. et al. Histologic features of chronic allograft nephropathy revealed by protocol biopsies in kidney transplant recipients. Transplantation 65, 1506–1509 (1998).
Rush, D. N., Henry, S. F., Jeffery, J. R., Schroeder, T. J. & Gough, J. Histological findings in early routine biopsies of stable renal allograft recipients. Transplantation 57, 208–211 (1994).
Mehta, R. et al. Short-term adverse effects of early subclinical allograft inflammation in kidney transplant recipients with a rapid steroid withdrawal protocol. Am. J. Transplant. 18, 1710–1717 (2018).
Nankivell, B. J. et al. The clinical and pathological significance of borderline T cell–mediated rejection. Am. J. Transplant. 19, 1452–1463 (2019).
Kee, T. Y. S. et al. Treatment of subclinical rejection diagnosed by protocol biopsy of kidney transplants. Transplantation 82, 36–42 (2006).
Heilman, R. L. et al. Impact of Subclinical Inflammation on the development of interstitial fibrosis and tubular atrophy in kidney transplant recipients. Am. J. Transplant. 10, 563–570 (2010).
Gloor, J. M. et al. Subclinical rejection in tacrolimus-treated renal transplant recipients. Transplantation 73, 1965–1968 (2002).
Anglicheau, D. et al. Discovery and validation of a molecular signature for the noninvasive diagnosis of human renal allograft fibrosis. Transplantation 93, 1136–1146 (2012).
Feng, Y. et al. The central inflammatory regulator IκBζ: Induction, regulation and physiological functions. Front. Immunol. 14, (2023).
Groten, S. A. et al. Targeting synergetic endothelial inflammation by inhibiting NFKB and JAK-STAT pathways. iScience 28 (2025).
Kasibhatla, S. et al. DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-κB and AP-1. Mol. Cell 1, 543–551 (1998).
Jia, H. M. et al. FASLG as a key member of necroptosis participats in acute myocardial infarction by regulating immune infiltration. Cardiol. Res. 15, 262–274 (2024).
Zhong, Y. et al. LIGHT aggravates sepsis-associated acute kidney injury via TLR4-MyD88-NF-κB pathway. J. Cell. Mol. Med. 24, 11936–11948 (2020).
Rabant, M. et al. Urinary C-X-C motif chemokine 10 independently improves the noninvasive diagnosis of antibody-mediated kidney allograft rejection. J. Am. Soc. Nephrol. 26, 2840–2851 (2015).
Ho, J. et al. Validation of urinary CXCL10 As a marker of borderline, subclinical, and clinical tubulitis. Transplantation 92, 878–882 (2011).
Ho, J. et al. Urinary CXCL10 chemokine is associated with alloimmune and virus compartment-specific renal allograft inflammation. Transplantation 102, 521–529 (2018).
Handschin, J. et al. Technical considerations and confounders for urine CXCL10 chemokine measurement. Transplant. Direct 6, E519 (2020).
Tinel, C. et al. Development and validation of an optimized integrative model using urinary chemokines for noninvasive diagnosis of acute allograft rejection. Am. J. Transplant. 20, 3462–3476 (2020).
Van Loon, E. et al. Automated urinary chemokine assays for noninvasive detection of kidney transplant rejection: A prospective cohort study. Am. J. Kidney Dis. 83, 467–476 (2024).
Tinel, C. et al. Transforming kidney transplant monitoring with urine CXCL9 and CXCL10: Practical clinical implementation. Sci. Rep. 14, (2024).
Rabant, M. et al. Early low urinary CXCL9 and CXCL10 might predict immunological quiescence in clinically and histologically stable kidney recipients. Am. J. Transplant. 16, 1868–1881 (2016).
Blydt-Hansen, T. D., Gibson, I. W., Gao, A., Dufault, B. & Ho, J. Elevated urinary CXCL10-to-creatinine ratio is associated with subclinical and clinical rejection in pediatric renal transplantation. Transplantation 99, 797–804 (2015).
Tinel, C. et al. Deciphering the prognostic and predictive value of urinary CXCL10 in kidney recipients with BK virus reactivation. Front. Immunol. 11, (2020).
Hirt-Minkowski, P. et al. Detection of clinical and subclinical tubulo-interstitial inflammation by the urinary CXCL10 chemokine in a real-life setting. Am. J. Transplant. 12, 1811–1823 (2012).
Strasser, A., Jost, P. J. & Nagata, S. The many roles of FAS receptor signaling in the immune system. Immunity 30, 180–192 (2009).
Aquino-Dias, E. C. et al. Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function. Kidney Int. 73, 877–884 (2008).
Dong, G. H. et al. Intercellular adhesion molecular-1, Fas, and Fas ligand as diagnostic biomarkers for acute allograft rejection of pancreaticoduodenal transplantation in pigs. Dig. Dis. Sci. 59, 778–786 (2014).
Sarwal, M. M. & Naesens, M. Urine trumps the protocol biopsy for subclinical rejection surveillance. Kidney Int. 104, 432–439 (2023).
De CardosoAguiar, R., Willicombe, M. & Roufosse, C. Noninvasive diagnosis of kidney allograft rejection. J. Am. Soc. Nephrol. https://doi.org/10.1681/ASN.0000000842 (2025).
Acknowledgements
The authors are deeply grateful to the kidney transplant recipients for their participation in this study.
Funding
This study has been supported by a grant of the Ministry of Health (RF-2010-2318732 to FPS) and by the National Center for Gene Therapy and Drugs Based on RNA Technology, Italian Ministry for Universities and Research—MUR (Project No. CN_00000041 to G.P.). The authors are also grateful for support from the following National Research Centers: High-Performance Computing, Big Data and Quantum Computing (Project No. CN_00000013 to G.P.); Additional support was provided by ELIXIR-IT through the empowering project ELIXIRNextGenIT (Grant Code: IR0000010 to G.P.); Life Science Hub Regione Puglia (LSH-Puglia, Grant No. T4-AN-01 H93C22000560003 to G.P.); INNOVA—Italian Network of Excellence for Advanced Diagnosis (Grant Code: PNC-E3-2022–23683266, PNC-HLS-DA to G.P.).
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F.P.S and SNC. conceived and designed the study. S.N.C. F.P.S. G.P wrote and edited the manuscript. S.N.C., S.C. and E.P. performed experiments, processed data, and contributed to statistical analyses. L.B., D.D., V.C., A.A., I.G., U.M., N.B., and M.R. collected patient samples and curated clinical data. All authors contributed to data interpretation, reviewed the manuscript, and approved the final version.
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Cox, S.N., Chiurlia, S., Pasculli, E. et al. Integration of intragraft transcriptomics and urinary cytokines identifies CXCL10 and FasL signature in subclinical acute rejection. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35923-6
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DOI: https://doi.org/10.1038/s41598-026-35923-6
