Elevated Glycolytic Markers in Urinary Extracellular Vesicles in Kidney Transplant T Cell-Mediated Rejection

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Background: Urinary extracellular vesicles (UEV), containing components of their parent cells, are released from cells lining the kidney and urinary tract. Changes in kidney protein expression are reflected in the proteome of UEV and its use as a non-invasive marker of allograft function is of particular interest in kidney transplantation. Under stress, renal energy metabolism switches from fatty acid oxidation to glycolysis. We evaluated changes in glycolytic control proteins in UEV at the time of clinically-indicated kidney transplant biopsies and correlated this with histological lesions described in the Banff classification. Methods: This prospective observational study involved collection of pre-biopsy urine samples from kidney transplant recipients at the time of indication biopsy. UEV were isolated by differential ultracentrifugation. Western blots were performed to confirm vesicle markers (CD9, TSG101 and Tamm-Horsfall protein) and then probed for glycolytic markers. Results: The cohort of 25 subjects (mean age 50.6 years, 60% male) included 5 cases of T-cell mediated rejection (TCMR), 4 cases of borderline TCMR and 16 normal biopsies. TCMR and borderline TCMR were analysed as one group (n = 9) and the following glycolytic markers were detected in this group: phosphofructokinase-liver, isoenzymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2, phosphorylated ser461 PFKFB2, phosphorylated ser483 PFKFB2, PFKFB3, PFKFB4) and pyruvate kinase (PK-M2, PK-LR). There was significantly increased expression of PFKFB3+ UEV in 3/9 (33%) of TCMR samples compared to 0/16 of normal biopsy samples (p=0.04). PFKFB4+ UEV was expressed in 6/9 (66%) of TCMR samples compared to 1/16 of normal biopsy samples (p=0.003). Conclusions: This study demonstrates elevated expression of PFKFB3 and PFKFB4 in UEV in kidney transplant recipients with biopsy-proven TCMR. This may represent altered energy metabolism in the form of increased renal glycolysis in association with cell mediated rejection.

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