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Urine Angiotensin II Signature Proteins as Markers of Fibrosis in Kidney Transplant Recipients

Mohammed-Ali, Zahraa, PhD1; Tokar, Tomas, PhD2; Batruch, Ihor, MSc3; Reid, Shelby, MSc4; Tavares-Brum, Alexandre, BSc5; Yip, Paul, PhD, FCACB1; Cardinal, Héloïse, MD, PhD, FRCPC5; Hébert, Marie-Josée, MD, PhD, FRCPC5; Li, Yanhong, PhD1; Kim, S. Joseph, MD, PhD, FRCPC1,6; Jurisica, Igor, PhD2,7; John, Rohan, MD8; Konvalinka, Ana, MD, PhD, FRCPC1,6

doi: 10.1097/TP.0000000000002676
Original Basic Science—General
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Background. Interstitial fibrosis/tubular atrophy (IFTA) is an important cause of kidney allograft loss; however, noninvasive markers to identify IFTA or guide antifibrotic therapy are lacking. Using angiotensin II (AngII) as the prototypical inducer of IFTA, we previously identified 83 AngII-regulated proteins in vitro. We developed mass spectrometry–based assays for quantification of 6 AngII signature proteins (bone marrow stromal cell antigen 1, glutamine synthetase [GLNA], laminin subunit beta-2, lysophospholipase I, ras homolog family member B, and thrombospondin-I [TSP1]) and hypothesized that their urine excretion will correlate with IFTA in kidney transplant patients.

Methods. Urine excretion of 6 AngII-regulated proteins was quantified using selected reaction monitoring and normalized by urine creatinine. Immunohistochemistry was used to assess protein expression of TSP1 and GLNA in kidney biopsies.

Results. The urine excretion rates of AngII-regulated proteins were found to be increased in 15 kidney transplant recipients with IFTA compared with 20 matched controls with no IFTA (mean log2[fmol/µmol of creatinine], bone marrow stromal cell antigen 1: 3.8 versus 3.0, P = 0.03; GLNA: 1.2 versus −0.4, P = 0.03; laminin subunit beta-2: 6.1 versus 5.4, P = 0.06; lysophospholipase I: 2.1 versus 0.6, P = 0.002; ras homolog family member B: 1.2 versus −0.1, P = 0.006; TSP1_GGV: 2.5 versus 1.9; P = 0.15; and TSP1_TIV: 2.0 versus 0.6, P = 0.0006). Receiver operating characteristic curve analysis demonstrated an area under the curve = 0.86 for the ability of urine AngII signature proteins to discriminate IFTA from controls. Urine excretion of AngII signature proteins correlated strongly with chronic IFTA and total inflammation. In a separate cohort of 19 kidney transplant recipients, the urine excretion of these 6 proteins was significantly lower following therapy with AngII inhibitors (P < 0.05).

Conclusions. AngII-regulated proteins may represent markers of IFTA and guide antifibrotic therapies.

1 Toronto General Hospital Research Institute, Advanced Diagnostics Division, University Health Network, Toronto, ON, Canada.

2 Data Science Discovery Centre for Chronic Diseases, Krembil Research Institute, Toronto Western Hospital, University Health Network, Toronto, ON, Canada.

3 Department of Laboratory Medicine and Pathobiology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada.

4 Institute of Medical Science, University of Toronto, Toronto, ON, Canada.

5 Department of Medicine, Division of Nephrology, Centre Hospitalier de l’Université de Montréal, Université de Montréal, Montréal, QC, Canada.

6 Division of Nephrology, Department of Medicine, Toronto General Hospital, University Health Network, University of Toronto, Toronto, ON, Canada.

7 Departments of Medical Biophysics and Computer Science, University of Toronto, Toronto, Canada.

8 Department of Pathology, Toronto General Hospital, University Health Network, Toronto, ON, Canada.

Received 26 June 2018. Revision received 28 January 2019.

Accepted 2 February 2019.

Z.M.-A. participated in research design, performance of experiments, data analysis, and writing of the paper. T.T. performed the statistical analysis, generated figures, and provided relevant methods. I.B. provided technical assistance in mass spectrometry experiments. S.R. provided technical assistance in urine sample processing. P.Y. provided clinical chemistry laboratory measurements of total urine protein and creatinine. H.C., M.-J.H., and A.T.-B. provided all the urine and biopsy samples and clinical information for the study and participated in the study design. H.C. and M.-J.H. reviewed the manuscript. Y.L. performed case-control matching. S.J.K. contributed to research design and analysis. I.J. was consulted on data analysis and participated in the manuscript preparation. R.J. participated in the assessment of renal pathology and immunohistochemistry staining and was consulted on research design. A.K. conceived and designed the project and participated in writing of the manuscript.

The authors declare no conflicts of interest.

Z.M.-A. was supported by the Toronto General Hospital Research Institute Fellowship and the Canadian Society of Transplantation Fellowship. A.K. received operating funds from the Canadian National Transplant Research Program Personalized Medicine Grant for the current project. A.K. also holds a biomedical research grant from the Kidney Foundation of Canada and a Kidney Research Scientist Core Education and National Training Program New Investigator salary Award, the Canadian Institutes of Health Research Catalyst Grant (2018), and the Predictive Biomarker Kidney Foundation of Canada operating Grant (2019). Computational analysis was supported in part by Ontario Research Fund (#34876), Natural Sciences Research Council (#203475), Canada Foundation for Innovation (#29272, #225404, and #30865), Krembil Foundation, and IBM.

Correspondence: Zahraa Mohammed-Ali, PhD. (z.mohammedali@outlook.com)

Ana Konvalinka, MD, PhD, FRCPC, Toronto General Hospital, 11-PMB-189, 585 University Avenue, Toronto, M5G 2N2, ON, Canada. (ana.konvalinka@uhn.ca).

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