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Integrated Kidney Exosome Analysis (iKEA) For The Detection Of Kidney Transplant Rejection Jongmin Park, Hsing-Ying Lin, Jean Pierre Assaker, Sangmoo Jeong, Chen-Han Huang, Ahmed Kurdi, Kyungheon Lee, Kyle Fraser, Changwook Min, Siawosh Eskandari, Sujit Routray, Bakhos A. Tannous, Reza Abdi, Leonardo Riella, Anil Chandraker, Cesar M. Castro, Ralph Weissleder, Hakho Lee, and Jamil R. Azzi ACS Nano, Just Accepted Manuscript • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017
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ACS Nano
Integrated Kidney Exosome Analysis (iKEA) For The Detection Of Kidney Transplant Rejection
Jongmin Park1,2†, Hsing-Ying Lin1,2†, Jean Pierre Assaker3†, Sangmoo Jeong1,2, Chen-Han Huang1,2, Ahmed Kurdi3, Kyungheon Lee1,2, Kyle Fraser1,2, Changwook Min1,2, Siawosh Eskandari3, Sujit Routray3, Bakhos Tannous4, Reza Abdi3, Leonardo Riella3, Anil Chandraker3, Cesar M. Castro1,5, Ralph Weissleder1,6, Hakho Lee1,2*, Jamil R. Azzi3* 1. Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 2. Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 3. Transplantation Research Center, Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115 4. Experimental Therapeutics and Molecular Imaging Laboratory and Department of Neurology, NeuroOncology Division, Massachusetts General Hospital, Boston, MA, 02129 5. Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 6. Department of Systems Biology, Harvard Medical School, Boston, MA 02115
† These authors contributed equally
Corresponding authors Jamil R. Azzi, MD (
[email protected]) Hakho Lee, PhD (
[email protected])
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ABSTRACT Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are non-invasive alternatives, but are late markers with low specificity. We here report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as surrogate marker for inflammation. We optimized iKEA to detect T-cell derived EVs, and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients, and achieved high detection accuracy (91.1%). Fast, non-invasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.
Keywords: biosensor; urine exosomes; acute cellular rejection; kidney transplant; proteomics
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Kidney transplantation is the preferred and most successful therapy for end stage renal disease. Nonetheless, long-term allograft survival remains suboptimal, as transplant recipients eventually develop acute or chronic rejection.1 While biopsy remains the gold standard for diagnosis of renal graft rejection,2, 3 it has many drawbacks including inherent procedural risk, complications and comparatively high costs. In addition, histopathological analyses are often confounded by inter-observer variability and sampling error.3, 4 As a non-invasive alternative, serum creatinine (SCr) and urinary protein excretion are used, but they lack sensitivity and specificity for allograft rejection.5 An increase in SCr is a late marker of kidney injury, and often fails to reflect the early inflammatory processes revealed by histopathology. The push for accurate non-invasive biomarkers to achieve sensitive, frequent, and cost effective monitoring of disease activity seeks to decrease early and late clinical rejection episodes.6 Extracellular vesicles (EVs), including exosomes and microvesicles, are actively secreted by cells into biofluids.7 EVs carry molecular constituents of cells, including transmembrane and intracellular proteins, and nucleic acids.8–10 A growing number of studies have shown that i) EVs can reflect parental cells, and ii) with their abundance and structural stability, these vesicles can be used as a surrogate biomarker.11–13 EVs have been extensively studied for cancer detection;14, 15 they often reflect global tumor burden and heterogeneity, overcoming sampling biases.16 Moreover, the amount and molecular profiles of cancer-derived EVs highly correlate with tumor burden and treatment efficacy.17, 18 We reasoned that EVs within urine can be used to monitor kidney transplant rejection. During acute cellular rejection (ACR) of kidney allografts, T cells infiltrate the kidney tubules and in close proximity to forming urine; this increases the chance of T cell-derived EVs entering urine (Fig. 1a). Capturing T-cell specific EVs can thus serve as a surrogate biomarker for ACR. Here, we report an EV-based diagnostic platform for kidney rejection. Termed iKEA (integrated kidney exosome assay), it detects urine EVs (uEVs) from T cell lymphocytes. We adapted a magneto-electrochemical strategy for EV detection:19 Tcell specific EVs were immuno-magnetically enriched and detected via electrochemistry. Combining magnetic enrichment and enzymatic signal amplification, the iKEA was highly sensitive (~104 EVs) and fast (2 hours), and the detection system could be implemented as a portable device. Applying iKEA to cell culture and clinical urine samples, we found that CD3 is a potent marker to detect T-cell derived uEVs. In further clinical tests, the CD3-based iKEA achieved the diagnostic accuracy of 91.1% in a discovery cohort (30 patients) and 83.7% in a validation cohort (14 patients). iKEA is an effective tool to perform non-invasive, serial monitoring, offering clinicians with actionable knowledge to improve patient outcomes and reduce complications in renal transplantation. RESULTS AND DISCUSSION Integrated kidney exosome analysis (iKEA) Kidney histology of an ACR patient shows a distinct pattern of tubulitis, wherein lymphocytes attack tubular cells (Fig. 1b). Presence of T cells in tubules increases the chance of T-cell derived EVs entering urine, which could be used as a surrogate marker for ACR. Indeed, uEVs from a rejection patient were stained positive for CD3 (Fig. 1c). We devised the iKEA assay adopting magnetic selection with electrochemical detection.19 The assay first enriches target EVs via with immunomagnetic capture. Collected EVs are then labeled with an oxidizing enzyme (horseradish peroxidase, HRP) through a second antibody. Mixing beads with a chromogenic electron mediator (3,3’,5,5’- tetramethylbenzidine, TMB) generates electrical currents 3 ACS Paragon Plus Environment
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measured by an electrode (Fig. 2a). Using magnetic beads affords several advantages: i) large surface area of beads enhances the enrichment efficiency; ii) assay steps are simplified with magnetic manipulation (e.g., washing); and iii) beads can be magnetically concentrated on top of the electrode to enhance the analytical signal. Portable detection system To facilitate uEV analysis, we implemented a compact iKEA device for electrochemical readout (Fig. 2b). The device contained a custom-designed potentiostat (Fig. 2c and Fig. S1) that measures electrical current between working (W) and counter (C) electrodes. A constant potential was applied between working and reference (R) electrodes during the measurement. The device operated standalone, and communicated with external devices for data logging (Bluetooth or USB). A small magnet was placed under the electrode location to concentrate magnetic beads. When benchmarked against a benchtop system (SP-200, Bio-Logic), the iKEA device offered comparable performance (Fig. 2d) despite being much cheaper (
