Urine immune profiling by measurement of multiple cytokine/chemokine mRNA levels in renal allograft dysfunction

Rubina Naqvi, Salma Batool Jafri, Zahabia Imani, Mohammad Mubarak, Rana Muzaffar

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DOI: 10.4236/oji.2011.13011                

Abstract

Background: An accurate diagnosis of cause of acute renal graft dysfunction is crucial for the optimal management of transplant recipients. Currently available tests are either insensitive or nonspecific, or are invasive, such as allograft biopsy. During last decade, attempts have been made in search of non invasive markers for the evaluation of cause of graft dysfunction. We studied a set of genes expressed on cytotoxic T Lymphocytes and those related to functioning of regulatory or helper T cells. Methods: We obtained 108 urine samples from 108 renal allograft recipients at the time of graft biopsy done for the evaluation of cause of graft dysfunction. RNA was extracted from urinary cells and messenger RNA (mRNA) encoding perforin, granzyme B (GB), FoxP3, CD3?, CXCR3, TGF-?, CTLA4, PI-9, IL-10, TNF?, T-bet and 18SrRNA measured with the use of quantitative real time polymerase chain reaction (RT-PCR). The levels of expression of genes were correlated with the biopsy findings and the results compared among different groups. Renal allograft biopsies at this institution are performed when there is unexplained rise in serum creatinine of >20% from the baseline value and reported according to Banff classification. SPSS v10.0 used for analy-sis.Results: The mRNA copy numbers of GB, Perforin, FoxP3, CD3, CXCR3, TGF-?, CTL A4, PI9, IL-10, TNF?, and T-bet were log transformed and mean (± SD) levels studied. The expression of all studied genes were compared between ‘nonspecific biopsy findings’ and other specific diagnoses. GB, Perforin, FoxP3, TGF-?, CD3?, CTLA4 CXCR3 and T-bet were higher in acute cellular rejection (ACR), whereas, TGF-? was also found higher in infection, and PI-9 in chronic allograft nephropathy (CAN) and borderline rejection group. Conclusion: Measurement of mRNA levels for genes like GB, Perforin, FoxP3, TGF-β, CD3?, CTLA4, CXCR3 and T-bet in urine samples offers a non invasive means of diag-nosing cause of graft dysfunction.

Keywords

Renal Allograft Dysfunction; Rejection; Cytokines; Chemokines

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Akhtar, F., Rana, T.A., Kazi, J., Zafar, N., Hashmi, A., Bhatti, S., et al. (1998) Correlation between Biopsies and Noninvasive Assessment of Acute Graft Dysfunction. Transplantation Proceeding, 30, 3069. doi:10.1016/S0041-1345(98)00933-6
[2]	Gwinner, W. (2007) Renal transplant rejection markers. World Journal of Urology, 25, 445-455. doi:10.1007/s00345-007-0211-6
[3]	Mannon, R.B. and Kirk, A.D. (2006) Beyond histology: Novel tools to diagnose allograft dysfunction. Clinical Journal of the American Society of Nephrology, 1, 358-366.
[4]	Yannaraki, M., Rebibou, J.M., Ducloux, D., Saas, P., Duperrier, A., Felix, S., et al. (2006) Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection. Transplant International, 19, 759-768. doi:10.1111/j.1432-2277.2006.00351.x
[5]	Strom, T.B. (2005) Rejection—more than the eye can see. New England Journal of Medicine, 353, 2394-2396. doi:10.1056/NEJMe058257
[6]	Anglicheau, D. and Suthanthiran, M. (2008) Noninvasive prediction of organ graft rejection and outcome using gene expression patterns. Transplantation, 86, 192-199. doi:10.1097/TP.0b013e31817eef7b
[7]	Hu, H. and Knechtle, S.J. (2006) Elevation of multiple cytokines/chemokines in urine of human renal transplant recipients with acute and chronic injuries: Potential usage for diagnosis and monitoring. Transplantation Review, 20, 165-171. doi:10.1016/j.trre.2006.07.003
[8]	Suthanthiran M. (1998) Human renal allograft rejection: molecular characterization. Nephrology Dialysis Transplantation, 13, 21-24. doi:10.1093/ndt/13.suppl_1.21
[9]	Li, B., Hartono, C., Ding, R., Sharma, V.K., Ramaswamy, R., Qian, B., et al. (2001) Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. New England Journal of Medicine, 344, 947-954. doi:10.1056/NEJM200103293441301
[10]	Graziotto, R., Del Prette, D., Rigotti, P., Anglani, F., Baldan, N., Furian, L., et al. (2006) Perforin, granzyme B, and fas ligand for molecular diagnosis of acute renal allograft rejection: Analyses on serial biopsies suggest methodological issues. Transplantation, 81, 1125-1132. doi:10.1097/01.tp.0000208573.16839.67
[11]	Kotsch, K., Mashreghi, M.F., Bold, G., Tretow, P., Beyer, J., Matz, M., et al. (2004) Enhanced granulysin mRNA expression in urinary sediment in early and delayed acute renal allograft rejection. Transplantation, 77, 1866-1875. doi:10.1097/01.TP.0000131157.19937.3F
[12]	Matz, M., Beyer, J., Wunsch, D., Mashreghi, M.F., Seiler, M., Pratschke, J., et al. (2006) Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function. Kidney International, 69, 1683-1690. doi:10.1038/sj.ki.5000343
[13]	Kim, S.H., Oh, E.J., Ghee, J.Y., Song, H.K., Han, D.H., Yoon, H.E., et al. (2009) Clinical significance of monitoring circulating CD4+CD25+ regulatory T cells in kidney transplantation during the early posttransplant period. Journal of Korean Medical Science, 24, S135-142. doi:10.3346/jkms.2009.24.S1.S135
[14]	Kingsley, C.I., Karim, M., Bushell, A.R., Wood, K.J. (2002) CD25+CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. Journal of Immunology, 168, 1080-1086.
[15]	Ding, R., Li, B., Muthukumar, T., Dadhania, D., Medeiros, M., Hartono, C., et al. (2003) CD103 mRNA levels in urinary cells predict acute rejection of renal allografts. Transplantation, 75, 1307-1312. doi:10.1097/01.TP.0000064210.92444.B5
[16]	Tatapudi, R.R., Muthukumar, T., Dadhania, D., Ding, R., Li, B., Sharma, V.K., et al. (2004) Noninvasive detection of renal allograft inflammation by measurements of mRNA for IP-10 and CXCR3 in urine. Kidney International, 65, 2390-2397. doi:10.1111/j.1523-1755.2004.00663.x
[17]	Hoffmann, U., Segerer, S., Rümmele, P., Krüger, B., Pietrzyk, M., Hofst?dter, F., et al. (2006) Expression of the chemokine receptor CXCR3 in human renal allografts—a prospective study. Nephrology Dialysis Transplantation, 21, 1373-1381. doi:10.1093/ndt/gfk075
[18]	Muthukumar, T., Ding, R., Dadhania, D., Medeiros, M., Li, B., Sharma, V.K., et al. (2003) Serine proteinase inhibitor-9, an endogenous blocker of granzyme B/perforin lytic pathway, is hyperexpressed during acute rejection of renal allografts. Transplantation, 75, 1565-1570. doi:10.1097/01.TP.0000058230.91518.2F
[19]	Muthukumar, T., Dadhania, D., Ding, R., Snopkowski, C., Naqvi, R., Lee, J.B., et al. (2005) Messenger RNA for FOXP3 in the urine of renal-allograft recipients. New England Journal of Medicine, 353, 2342-2351. doi:10.1056/NEJMoa051907
[20]	Wang, S., Jiang, J., Guan, Q., Lan, Z., Wang, H,, Nguan, C.Y.C, et al. (2008) Reduction of Foxp3-expressing regulatory T cell infilterates during the progression of renal allograft rejection in a mouse model. Transplant Immunology, 19, 93-102. doi:10.1016/j.trim.2008.03.004
[21]	Veronese, F., Rotman, S., Smith, R.N., Pelle, T.D., Farrell, M.L., Kawai, T., et al. (2007) Pathological and clinical correlates of FOXP3+ cells in renal allograft during acute rejection. American Journal of Transplantation, 7, 914-922. doi:10.1111/j.1600-6143.2006.01704.x
[22]	Navarro, J.F., Mora, C. and Muros, M. (2006) Urinary tumor necrosis factor-? excretion independently correlates with clinical markers of glomerular and tubulointerstitial injury in type 2 diabetic patients. Nephrology Dialysis Transplantation, 21, 3428-3434. doi:10.1093/ndt/gfl469
[23]	Cottrez, F. and Groux, H. (2001) Regulation of TGF-beta response during T cell activation is modulated by IL-10. Journal of Immunology, 167, 773-778.
[24]	Pribylova-Hribova, P., Kotch, K., Lodererova, A., Vik icky, O., Vitko, S., Volk, H.D., et al. (2006) TGF-? 1 mRNA upregulation influences chronic renal allograft dysfunction. Kidney International, 69, 1872-1879. doi:10.1038/sj.ki.5000328
[25]	Ibrahim, S., Saadi, G. and Al-Ansary, M. (2007) Estimation of serum and urinary profibrotic cytokines in renal allograft recipients. The Internet Journal of Nephrology, 4.
[26]	Helantera, I., Teppo, A.M. and Koskinen, P. (2006) Increased urinary excretion of transforming growth factor ?1 in renal transplant recipients during cytomegalovirus infection. Transplant Immunology, 15, 217-221. doi:10.1016/j.trim.2005.11.001
[27]	Pearce, E.L., Mullen, A.C., Martins, G.A., Krawczvk, C.M., Hutchins, A.S., Zediak, V.P., et al. (2003) Control of effector CD8+ T cell function by the transcription factor Eomesodermin, Science, 302, 1041-1043. doi:10.1126/science.1090148
[28]	Szabo, S.J., Kim, S.T., Costa, G.L., Zhang, X., Fathman, C.G. and Glimcher, L.H. (2000) A Novel Transcription Factor, T-bet, detects Th1 Lineage Commitment, Cell, 100, 655-659. doi:10.1016/S0092-8674(00)80702-3
[29]	Mullen, A.C., Hutchins, A.S., High, F.A., Lee, H.W., Sykes, K.J., Chodosh, L.A., et al. (2002) Hlx is induced by and genetically interacts with T-bet to promote heritable T(H)1 gene induction. Nature Immunology, 3, 652-658.
[30]	Callard, R.E. (2007) Decision making by the immune response. Immunology & Cell Biology, 85, 300-305. doi:10.1038/sj.icb.7100060
[31]	Linsley, P., Bradshaw, J., Greene, J., et al. (1996) Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity, 4, 535-543. doi:10.1016/S1074-7613(00)80480-X
[32]	Finn, P.W., He, H., Wang, Y., Wang, Z., Guan, G., Listman, J., et al. (1997) Synergistic induction of CTLA-4 expression by costimulation with TCR plus CD28 signals mediated by increased transcription and messenger ribonucleic acid stability. Journal of Immunology, 158, 4074-4081.
[33]	Racusen, L.C., Solez, K., Colvin, R.B., et al. (1999) Banff 97 working classification of renal allograft pathology. Kid International, 55, 713-723. doi:10.1046/j.1523-1755.1999.00299.x
[34]	Martin, L., Funes de la Vega, M., Bocrie, O., Harzallah, A., Justrabo, E., Rifle, G., et al. (2007) Detection of Foxp3+ cells on biopsies of kidney transplants with early acute rejection. Transplantation Proceedings, 39, 2586-2588. doi:10.1016/j.transproceed.2007.08.037
[35]	Zeller, J.C., Panoskaltsis-Mortari, A., Murphy, W.J., Ruscetti, F.W., Narula, S., Roncarolo, M.G., et al. (1999) Induction of CD4+ T cell alloantigen-specific hyporesponsiveness by IL-10 and TGF-β. Journal of Immunology, 163, 3684.
[36]	Mansour, H., Homs, S., Desvaux, D., Badoul, C., Dahan, K., Matignon, M., et al. (2008) Intragraft levels of Foxp3 mRNA predict progression in renal transplants with borderline change. Journal of the American Society of Nephrology, 19, 2277-2281. doi:10.1681/ASN.2008030254
[37]	Bunnag, S., Allanach, K., Jhangri, G.S., Sis, B., Einecke, G., Mengel, M., et al. (2008) Foxp3 expression in human kidney transplant biopsies is associated with rejection and time post transplant but not with favorable outcome. American Journal of Transplantation, 8, 1423-11433. doi:10.1111/j.1600-6143.2008.02268.x
[38]	Zheng, Y., Josefowicz, S.Z., Kas, A., Chu, T.T., Gavin, M.A. and Rudensky, A.Y. (2007) Genome wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature, 445, 936-940. doi:10.1038/nature05563
[39]	Letterio, J.J. and Roberts, A.B. (1998) Regulation of Immune response by TGFβ. Annual Review of Immunology, 16, 137. doi:10.1146/annurev.immunol.16.1.137
[40]	Naqvi, R., Muthukumar, T., Dhadania, D., Ding, R., Snopkowski, C., Li, B. et al. (2005) The Yin and Yang of Allograft rejection: overexpression of T-bet as well as CTLA 4 during acute rejection of human renal allografts. Journal of the American Society of Nephrology, 34A.