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JUL 01, 2017 8:00 AM PDT

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Abstract

Acute kidney injury (AKI) is a serious public health problem worldwide, with ischemia and reperfusion (IR) being the main lesion-aggravating factors contributing to the evolution to a chronic kidney disease (CKD). The major intervention approaches available, hemodialysis and renal transplantation, are considered palliative options for the treatment of the disease. Face to this restricted scenario, the use of embryonic stem cells is suggested as an alternative, offering the possibility of replacement of affected cells and/or the release of paracrine factors crucial for tissue repair. Bioactive lipids such as sphingosine 1-phosphate (S1P) have significant effects on the modulation of signaling pathways which are important for tissue regeneration, such as cell survival, proliferation, differentiation, and migration. Thus, the main objective of this work is to explore the protective potential of S1P and the activation pathway of its receptors (S1Pr1-5) in human embryonic stem cells (H9) and human kidney proximal tubule cells (HK-2) submitted to models of IR lesion, via depletion of ATP with Antimycin A (AA). S1P treatment decreased cell death after AA in both cell lines, and also decreased caspase-3 labeling in H9 cells. Nevertheless, S1P increased fluorescence intensity of Ki67 proliferation marker in HK-2 cells. HK-2 and H9 showed unaltered endogenous production of lipids, such as phosphatidylcholine (PC), phosphoethanolamine (PE) and lysophosphatidic acid (LPA), in the different conditions. However, the activity of the sphingosine kinase enzyme (SK), responsible for the production of S1P, was shown to be increased in H9 cells submitted to S1P + AA treatment compared to AA treatment. H9 showed expression of all five S1P receptors whereas HK-2 did not express S1Pr3. In addition, HK-2 showed increased fluorescence intensity for S1Pr1 after S1P + AA, and also for S1Pr2 after S1P. Inhibition of SK played an important role in both cell lines and a clear involvement of the JAK2 / STAT3 pathway was observed in H9 cells, using the AG-490 pathway inhibitor. In vivo treatment of male Wistar rats with a dose of 106 H9 cells via renal capsule injection showed improvement in renal parenchymal filling seen by SPECT after IR lesion. Mass spectrometric analysis of renal tissue was able to identify specific molecules in the IR renal cortex of treated animals compared to sham. Thus, we conclude that S1P, through the modulation of its receptors, seems to play an effective role in cellular protection in IR models of both H9 and HK-2 cells


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