#897 Peritoneal inflammation in PD-related peritonitis induces systemic eryptosis: in vitro and in vivo assessments
Authors: Virzi Grazia Maria , Morisi Niccolò , Manani Sabrina Milan , Tantillo Ilaria , Ronco Claudio , Zanella Monica 
Published: May 2024

Abstract

Background and Aims
Erythrocytes (RBCs) have a highly specialized and organized membrane structure and undergo programmed cell death, known as eryptosis ( = stress-induced RBC death mechanism). Triggers of eryptosis include oxidative stress, inflammation and several uremic toxins. Our preliminary data show a significant increase of eryptosis during peritoneal dialysis (PD) -associated peritonitis in PD patients. The aims of this study were: assessment of incrementation of eryptosis in PD patients with peritonitis, evaluation of the relationship between systemic eryptosis in peritonitis and specific peritonitis biomarkers in PD effluent (PDE), and confirmation of the induction of eryptosis by peritonitis in a vitro setting.

Method
We enrolled 34 PD patients in stable condition (without systemic inflammation nor PD-related peritonitis in the last 3 months), 31 PD patients with acute peritonitis, and 17 healthy subjects (CTR). For PD stable patients, blood samples for eryptosis evaluation were drawn during the regular outpatient. For PD patients with peritonitis, blood samples and PDE samples were collected at the time of peritonitis diagnosis. PDE samples were used for peritoneal White Blood Cell count (pWBC), peritoneal Neutrophil Gelatinase-associated Lipocalin (pNGAL) and peritoneal cytokines levels (IL-1β and IL-6) measurements.
For in vitro study, healthy RBCs were exposed to plasma of 31 PD patients with peritonitis and plasma of CTR group for 2, 4 and 24 hours. Morphological markers of eryptosis (cell membrane scrambling, cell shrinkage) and eryptosis percentage (based on PS exposure at RBC surface and AnnexinV-binding) were evaluated by flow cytometric analyses in vivo and in vitro.

Results
Totally, 65 chronic PD patients were included in this study. 57% of patients were treated with continuous ambulatory PD (CAPD) and 43% with automated PD (APD). The median PD vintage was 38 months (IQR 3.6–124.9 months). Table 1 reports clinical data for our PD population (Table 1). The percentage of AnnexinV-binding RBCs was significantly higher in PD patients than in CTR (2.7%; IQR 1.6–3.9 vs CTR: 0.8; IQR 0.7–1.3 P &lt; .05). Eryptosis levels did not differ significantly between PD pts with and without diabetes, with and without hypertension, with and without cardiovascular disease. Eryptosis showed no significant differences between patients treated by CAPD/APD, and with Kt/Vurea values ≤ 1.7 and &gt; 1.7. Generally, RBCs of all 65 PD patients were dramatically deranged in their morphology. In particular, RBCs from PD patients with peritonitis were characterized by dramatically deranged morphology and increased median cell volume in comparison with PD stable patients (P &lt; .001). The percentages of AnnexinV-binding RBCs were significantly increased in the PD-associated peritonitis group (9.6%; IQR 4.2–16.7 versus 2.7%; IQR 1.6–3.9) (P&lt;.0001) (Fig. 1). The percentage of AnnexinV-binding RBCs was significantly higher in PD patients with peritonitis than in healthy CTR (P &lt; .001).
We confirmed these in vivo data by in vitro results: healthy RBCs incubated with plasma from PD patients with peritonitis demonstrated a significant increase of eryptosis compared to healthy RBCs exposed to plasma from control group at all times (Fig. 2).
Furthermore, significant positive strong correlations were observed between eryptosis level and all analysed peritoneal biomarkers of peritonitis (Spearman's rho pWBC = 0.77, P&lt;.001, Spearman's rho pNGAL = 0.64, P = .001, Spearman's rho IL-6 = 0.61, P = .003 and Spearman's rho IL-1β = 0.64, P = .001,) (Fig. 3).

Conclusion
We investigated a potential connection between systemic eryptosis and peritoneal biomarkers of peritonitis. In particular, we theorized that the eryptosis enhancement is directly connected with peritonitis, and, based on the results, we hypothesized that the peritoneal membrane injury could be related to eryptosis. Upregulation of inflammatory markers could explain the increased rate of systemic eryptosis during PD-related peritonitis. In particular, we corroborated this point with our observations about in vitro induction of eryptosis by plasma from PD patients on the first day of peritonitis.