Glycolytic control proteins in urinary extracellular vesicles are elevated during kidney transplant T cell-mediated rejection.

BACKGROUND: A priority in kidney transplant management is the ability to monitor allograft health accurately, frequently and less-invasively. Metabolic reprogramming from fatty acid oxidation to glycolysis has been associated with kidney injury. Given the histological localisation of T cell-mediated rejection (TCMR) to the tubulointerstitium, we hypothesised that expression of glycolytic control proteins contained in urinary extracellular vesicles (UEV) may increase during TCMR. METHODS: In this prospective observational study, urine samples were collected from kidney transplant recipients prior to indication biopsy. UEV were separated by differential ultracentrifugation. Vesicle markers, glycolytic control proteins and CD3 were assayed by immunoblotting. Differences in protein detection were compared across biopsy diagnoses (TCMR versus not) and Banff lesion scores. RESULTS: 51 paired urine and biopsy samples from 43 subjects were included. The TCMR group comprised of 6 cases of TCMR and 1 borderline TCMR. The remaining 44 samples comprised a "No TCMR" group. There was significant increase in phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) (p = 0.018) in TCMR compared to No TCMR, and similarly when tubulitis (p = 0.037) and interstitial inflammation (p = 0.047) were present. Total inflammation score ≥ 1 was associated with increases in PFKFB2 (p = 0.027), PFKFB3 (p = 0.090) and PFKFB4 (p = 0.0098). Interstitial fibrosis was associated with increased PFKFB2 (p = 0.0045) and PFKFB3 (p = 0.045). CD3 + UEV did not correlate with TCMR diagnosis. When combining the four glycolytic control proteins governing the phosphofructokinase-1 step of glycolysis (PFK-L, PFKFB2, PFKFB3 and PFKFB4), presence of ≥ 3 markers discriminated TCMR with ROC AUC of 0.73 (95% CI 0.50-0.96). CONCLUSION: Increased rate-limiting enzymes of glycolysis in UEV were detected in association with tubulointerstitial inflammation and fibrosis. This suggests altered energy metabolism in the form of increased renal glycolysis occurring in the tubular epithelium, consistent with findings in native kidney injury. Further work is required to evaluate whether this could serve as a non-invasive strategy to study pathology in kidney transplantation.