Discovery and validation of a urinary extracellular vesicle protein signature for the diagnosis of renal allograft fibrosis

Abstract

Interstitial fibrosis is the best indicator of irreversible or ongoing renal injury after kidney transplantation and faces considerable diagnostic challenges. Owing to the direct connection between urine and kidney, small urinary extracellular vesicles (suEVs) are promising candidates for developing non-invasive and highly sensitive diagnostic biomarkers for allograft fibrosis. Herein, we established an optimized method for separating suEVs from renal transplant recipients with high yield and purity. Through unbiased proteomic and in-depth bioinformatic analyses, we delineated an extensive protein landscape of suEVs and identified sorting nexin 3 (SNX3), vacuolar protein sorting-associated protein 4B (VPS4B) and smoothened (SMO) proteins as potential biomarkers for accurate and consistent diagnosis of fibrosis. Extensive validation across three independent cohorts demonstrated their excellent diagnostic performance in both transplant recipients and chronic kidney disease patients, achieving an outstanding AUC value of 0.9909 and accuracy of 90.6%, respectively. Moreover, this model demonstrated a prognostic value in a 3-month follow-up assessment of renal allograft recipients. Mechanically, we indicated that SNX3 promotes fibroblast activation through the regulation of Wnt secretion. Our study is the first to report the suEV protein biomarkers for the diagnosis of allograft fibrosis, offering a non-invasive alternative to renal biopsy and enabling improved risk stratification for transplant patients, which aids in better management of transplant recipients to improve long-term allograft survival.

Keywords

biomarkers / interstitial fibrosis / kidney transplantation / non-invasive diagnosis / urinary extracellular vesicles

References

[1] T. Chen, X. Sun, S. Tsuei, R. Yang, W. Yip, H. Fu, Lancet Reg. Health West. Pac. 2025, 54, 101268.

[2] J. Y. Yang, R. D. Sarwal, T. K. Sigdel, I. Damm, B. Rosenbaum, J. M. Liberto, C. Chan-On, J. M. Arreola-Guerra, J. Alberu, F. Vincenti, M. M. Sarwal, Sci. Transl. Med. 2020, 12, eaba2501.

[3] S. Hariharan, J. R. Ingelfinger, A. K. Israni, G. Danovitch, N. Engl. J. Med. 2021, 385, 729.

[4] E. Hysi, J. Baek, A. Koven, X. He, L. Ulloa Severino, Y. Wu, K. Kek, S. Huang, A. Krizova, M. Farcas, M. Ordon, K. Fok, R. Stewart, K. T. Pace, M. C. Kolios, K. J. Parker, D. A. Yuen, Nat. Med. 2025, 31, 970.

[5] T. Saritas, R. Kramann, Transplantation 2021, 105, e114.

[6] K. Kalantar-Zadeh, T. H. Jafar, D. Nitsch, B. L. Neuen, V. Perkovic, Lancet 2021, 398, 786.

[7] L. Li, H. Fu, Y. Liu, Nat. Rev. Nephrol. 2022, 18, 545.

[8] E. Hysi, J. Baek, A. Koven, X. He, L. Ulloa Severino, Y. Wu, K. Kek, S. Huang, A. Krizova, M. Farcas, M. Ordon, K. H. Fok, R. Stewart, K. T. Pace, M. C. Kolios, K. J. Parker, D. A. Yuen, Nat. Med. 2025, 31, 970.

[9] H. J. Jeong, Korean J. Nephrol. 2020, 39, 17.

[10] A. Loupy, M. Mengel, M. Haas, Kidney Int. 2022, 101, 678.

[11] I. Sedej, M. Štalekar, M. Tušek Žnidarič, K. Goričar, N. Kojc, P. Kogovšek, V. Dolžan, M. Arnol, M. Lenassi, J. Extracell. Vesicles 2022, 11, e12268.

[12] C. J. Blijdorp, D. Burger, A. Llorente, E. S. Martens-Uzunova, U. Erdbrügger, J. Am. Soc. Nephrol. 2022, 33, 467.

[13] R. Tenchov, J. M. Sasso, X. Wang, W.-S. Liaw, C.-A. Chen, Q. A. Zhou, ACS Nano 2022, 16, 17802.

[14] U. Erdbrügger, C. J. Blijdorp, I. V. Bijnsdorp, F. E. Borràs, D. Burger, B. Bussolati, J. B. Byrd, A. Clayton, J. W. Dear, J. M. Falcón-Pérez, C. Grange, A. F. Hill, H. Holthöfer, E. J. Hoorn, G. Jenster, C. R. Jimenez, K. Junker, J. Klein, M. A. Knepper, E. H. Koritzinsky, J. M. Luther, M. Lenassi, J. Leivo, I. Mertens, L. Musante, E. Oeyen, M. Puhka, M. E. van Royen, C. Sánchez, C. Soekmadji, V. Thongboonkerd, V. van Steijn, G. Verhaegh, J. P. Webber, K. Witwer, P. S. T. Yuen, L. Zheng, A. Llorente, E. S. Martens-Uzunova, J. Extracell. Vesicles 2021, 10, e12093.

[15] M. A. Kumar, S. K. Baba, H. Q. Sadida, S. A. Marzooqi, J. Jerobin, F. H. Altemani, N. Algehainy, M. A. Alanazi, A.-B. Abou-Samra, R. Kumar, A. S. Al-Shabeeb Akil, M. A. Macha, R. Mir, A. A. Bhat, Sci. Transl. Med. 2024, 9, 27.

[16] M. H. van Heugten, C. J. Blijdorp, S. Arjune, H. van Willigenburg, K. Bezstarosti, J. A. Demmers, U. Musterd-Bhaggoe, E. Meijer, R. T. Gansevoort, R. Zietse, J. Am. Soc. Nephrol. 2023, 10, 1681.

[17] R. El Fekih, J. Hurley, V. Tadigotla, A. Alghamdi, A. Srivastava, C. Coticchia, J. Choi, H. Allos, K. Yatim, J. Alhaddad, S. Eskandari, P. Chu, A. B. Mihali, I. T. Lape, M. P. Lima Filho, B. T. Aoyama, A. Chandraker, K. Safa, J. F. Markmann, L. V. Riella, R. N. Formica, J. Skog, J. R. Azzi, J. Am. Soc. Nephrol. 2021, 32, 994.

[18] S. Du, L. Zhai, S. Ye, L. Wang, M. Liu, M. Tan, Sci. China Life Sci. 2023, 66, 2587.

[19] B. Konety, N. Shore, A. K. Kader, S. Porten, S. Daneshmand, T. Lough, Y. Lotan, Eur. Urol. 2019, 76, 238.

[20] T. Dreyer, S. Brandt, K. Fabrin, N. Azawi, J. L. Vásquez, A. Ernst, L. Dyrskjøt, J. B. Jensen, Eur. Urol. 2025, 88, 23.

[21] R. Tutrone, B. Lowentritt, B. Neuman, M. J. Donovan, E. Hallmark, T. J. Cole, Y. Yao, C. Biesecker, S. Kumar, V. Verma, G. R. Sant, J. Alter, J. Skog, Prostate Cancer Prostatic Dis. 2023, 26, 596.

[22] X. Liu, J. Miao, C. Wang, S. Zhou, S. Chen, Q. Ren, X. Hong, Y. Wang, F. F. Hou, L. Zhou, Y. Liu, Kidney Int. 2020, 97, 1181.

[23] J. M. McDaniels, A. C. Shetty, C. Kuscu, C. Kuscu, E. Bardhi, T. Rousselle, C. Drachenberg, M. Talwar, J. D. Eason, T. Muthukumar, D. G. Maluf, V. R. Mas, Kidney Int. 2023, 103, 1077.

[24] M. Puhka, M. E. Nordberg, S. Valkonen, A. Rannikko, O. Kallioniemi, P. Siljander, T. M. af Hällström, Eur. J. Pharm. Sci. 2017, 98, 30.

[25] L. Wang, C. Qi, H. Cao, Y. Zhang, X. Liu, L. Qiu, H. Wang, L. Xu, Z. Wu, J. Liu, S. Wang, D. Kong, Y. Wang, Small 2023, 19, e2301019.

[26] H. Cao, H. Gao, L. Wang, Y. Cheng, X. Wu, X. Shen, H. Wang, Z. Wang, P. Zhan, J. Liu, Z. Li, D. Kong, Y. Shi, D. Ding, Y. Wang, ACS Nano 2022, 16, 13992.

[27] Y. Cheng, Y. Yue, H. Cao, W. Geng, L. Wang, X. Hu, H. Li, Q. Bian, X. Kong, J. Liu, D. Kong, D. Guo, Y. Wang, J. Nanobiotechnol. 2021, 19, 451.

[28] H. Cao, Y. Cheng, H. Gao, J. Zhuang, W. Zhang, Q. Bian, F. Wang, Y. Du, Z. Li, D. Kong, D. Ding, ACS Nano 2020, 14, 4014.

[29] B. Dhondt, E. Geeurickx, J. Tulkens, J. Van Deun, G. Vergauwen, L. Lippens, I. Miinalainen, P. Rappu, J. Heino, P. Ost, N. Lumen, O. De Wever, A. Hendrix, J. Extracell. Vesicles 2020, 9, 1736935.

[30] F. Braun, M. Rinschen, D. Buchner, K. Bohl, I. Plagmann, D. Bachurski, M. Richard Späth, P. Antczak, H. Göbel, C. Klein, J. W. Lackmann, O. Kretz, V. G. Puelles, R. Wahba, M. Hallek, B. Schermer, T. Benzing, T. B. Huber, A. Beyer, D. Stippel, C. E. Kurschat, R. U. Müller, J. Extracell. Vesicles 2020, 10, e12026.

[31] Q. Sun, M. Baues, B. M. Klinkhammer, J. Ehling, S. Djudjaj, N. I. Drude, C. Daniel, K. Amann, R. Kramann, H. Kim, J. Saez-Rodriguez, R. Weiskirchen, D. C. Onthank, R. M. Botnar, F. Kiessling, J. Floege, T. Lammers, P. Boor, Sci. Transl. Med. 2019, 11, eaat4865.

[32] S. Chen, M. Zhang, J. Li, J. Huang, S. Zhou, X. Hou, H. Ye, X. Liu, S. Xiang, W. Shen, J. Miao, F. F. Hou, Y. Liu, L. Zhou, J. Extracell. Vesicles 2022, 11, e12203.

[33] A.-R. Shen, L. Lv, EBioMedicine 2022, 86, 104360.

[34] S. B. Arya, S. P. Collie, C. A. Parent, Trends Cell Biol. 2024, 34, 90.

[35] X. Chen, H. Tan, J. Xu, Y. Tian, Q. Yuan, Y. Zuo, Q. Chen, X. Hong, H. Fu, F. F. Hou, L. Zhou, Y. Liu, Kidney Int. 2022, 102, 506.

[36] P. Romagnani, R. Agarwal, J. C. Chan, A. Levin, R. Kalyesubula, S. Karam, M. Nangaku, B. Rodríguez-Iturbe, H. Anders, Nat. Rev. Dis. Primers. 2025, 11, 8.

[37] J. Liu, Q. Xiao, J. Xiao, C. Niu, Y. Li, X. Zhang, Z. Zhou, G. Shu, G. Yin, Signal. Transduct. Target. Ther. 2022, 7, 3.

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2026 The Author(s). Interdisciplinary Medicine published by Wiley-VCH GmbH on behalf of Nanfang Hospital, Southern Medical University.