Ischemia/reperfusion injury (IRI) profoundly impacts graft survival following kidney transplantation. Epithelial injury is one of the earliest histological alterations of IRI and is especially observed in the corticomedullary junction. In this region oxygen tension is lowest whereas epithelial cells are metabolically very active. We hypothesized that urinary metabolomics could be a tool for non-invasive assessment of IRI-induced changes.

We used a rat IRI model in combination with the protective effect of therapeutic inhibition of Mannan-binding lectin (MBL). Samples (serum, urine, tissue) were collected at 2, 5 and 24 h post ischemia/reperfusion (I/R) and the urinary metabolic profiles were analysed using a GC-MS platform.

We demonstrate that this IRI model is characterized by early epithelial injury and an increased expression of KIM-1, NGAL, IL-6 and rise in serum creatinine. Moreover, already at 2 hours a strong reduction of GRP78 protein expression is observed, specifically in the corticomedullary junction. Loss of GRP78, a regulator of the ER-stress response, was accompanied by induction of downstream mediators spliced-XBP1 and CHOP. Inhibition of MBL in vivo protected tubular cells from rapid loss of GRP78 expression and consequent tubular injury. Exploratory data analysis of the urinary metabolic profiles showed a dominant effect of time, but not of the protective treatment. The use of PLS regression models in combination with all injury markers as response variables, only revealed a significant association between metabolic changes in urine and expression of GRP78. Exploring the variable importance of projection values we have identified a number of metabolites, including alpha-ketoglutarate, aconitic acid, uric acid, hippuric acid and desaminotyrosine which were significantly contributing to the model and were affected by protective anti-MBL treatment.

In conclusion, we show that loss of GRP78 and induction of the ER-stress response is a very early process in IRI, specifically taking place in the corticomedullary junction. Using a combination of statistical models and specific intervention we could link the metabolic trajectory to the recovery process and show that metabolomics is a valuable tool for the evaluation of IRI at cellular and tissue level.

CITATION INFORMATION: van Kooten C, Pacchiarotta T, van der Pol P, de Fijter J, Schlagwein N, van Gijlswijk D, Mayboroda O. ER Stress and Loss of GRP78 Expression Provides a Link Between Renal Ischemia/Reperfusion Injury and the Urinary Metabolome. Am J Transplant. 2016;16 (suppl 3).

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