#2716 Selenoprotein-P1 (SEPP-1) in human proximal tubule cells after ischemic-reperfusion injury: an in-vitro model
Authors: D'agostino Mario , Celano Marilena , Musolino Michela , Tripodi Loredana , Zicarelli Mariateresa , Russo Emilio , Andreucci Michele , Bolignano Davide , Coppolino Giuseppe 
Published: May 2024

Abstract

Background and Aims
Novel biomarkers, anticipating serum creatinine rise, are eagerly needed to have a speedy diagnosis of acute kidney injury (AKI) and thus to mitigate and to preserve kidney function with well-timed therapeutic plans. Recently, in a pilot study on patients undergoing elective major cardiac surgery with cardiopulmonary bypass (CPB), selenoprotein-p1 (SEPP-1) has emerged as a promising candidate plasmatic biomarker for an early AKI risk stratification. SEPP-1 increased particularly between 4th to 8th hour after CPB and with a strict relation with ischemia duration (Bolignano et Al. Rev. Cardiovasc. Med. 2022). SEPP1 is primarily secreted from liver and acts as a selenium transporter supplying tissues and organs with this trace mineral which elicits the activity of specific glutathione peroxidase selenoenzymes. Additionally, renal tubular cells are able to synthetize this protein but remain unknown if, during AKI, SEPP1 increases as defeat answer to renal cells damage or if it's hepatic SEPP1 that accumulates for a reduced renal glomerular filtration rate. The primary aim of this study was to investigate the release of SEPP-1 by renal tubular cells in an experimental model of AKI induced by ischemia-reperfusion and secondarily evaluate the effect of selenium supplying on renal cells injury.

Method
Human proximal tubular cells (PTC), HK2 cells and human embryonic kidney 293 cells (HEK293) were cultured in Keratinocyte-SFM a complete serum-free medium supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) (LGC Standards s.r.l., Milan, Italy) and in Eagle's Minimum Essential Medium (E-MEM) (LGC Standards s.r.l., Milan, Italy) containing 10% fetal bovine serum, respectively. The cells were pre-incubated with medium containing Sodium Selenite 100 nM for 24 h and then were treated for 24 hours with CoCl2 (Sigma Aldrich S.r.l., Milan, Italy) a chemical hypoxia inducer (500 µM for HK-2 and 250 µM for HEK-293). After that cell viability was evaluated by MTT and trypan bleu dye exclusion assay, while, protein expression of HIF (hypoxia marker) and SEPP1 was analyzed by western blot analysis.

Results
Ischemia-reperfusion was simulated by the exposure of HK-2 and HEK-293 to CoCl2 and confirmed by HIF expression. The expression of SEPP-1 increased significantly in HK-2 and HEK-293 after hypoxia stress (Fig. 1). Furthermore, the treatment with CoCl2 determined a decrease of cell viability (about 50% less) and Sodium selenite alone increased cell proliferation (about 15% more) in both cell lines. Interestingly cell growth after hypoxia augmented significantly if cell lines were pre-treated with sodium-selenite, highly in HK-2 cells (p &lt; 0.001).

Conclusion
These preliminary data evidenced, in vitro, that AKI is able to induce renal tubular expression of SEPP-1 and probably SEPP-1 release in human after renal injury is not only a result of a reduced filtration of the hepatic released protein. Furthermore, SEPP-1 showed a possible protective role in AKI. In fact, AKI induced by ischemia-reperfusion, in our in vitro cellular model, was able to induce an increased expression of SEPP-1 in renal tubular cells suggesting a shielding mechanism against injury related to selenium transport. This hypothesis was supported by an exalted SEPP-1 expression after Sodium-Selenite adding to cells’ culture. Supplementary experiments, including functional molecular assays in vitro and in vivo studies will allow to better characterize the role of SEPP-1 in AKI.