#957 The immunometabolic profile of human peritoneal macrophages during peritoneal dialysis and its relation to systemic cardiovascular effects
Authors: Karsten Micky , Hahn Nico , Vree Puck , Vervloet Marc , Van den Bossche Jan , Jakulj Lily 
Published: May 2024

Abstract

Background and Aims
Patients with end-stage kidney disease (ESKD) treated with peritoneal dialysis (PD) exhibit a high risk of heart failure (HF). This largely concerns HF with a preserved ejection fraction (HFpEF). Uremic toxins, oxidative stress and systemic inflammation related to ESKD contribute to this HFpEF phenotype, which may be aggravated by the daily administration of glucose-rich PD-fluids. These induce insulin resistance and formation of advanced glycaemic end products, resulting in microvascular injury and arteriosclerosis.
A hallmark of HFpEF is the presence of profibrotic cardiac macrophages that aggravate fibrosis. In the peritoneum, the non-physiological high glucose concentrations trigger local oxidative stress, fibrosis and inflammation, processes alike those seen in HFpEF. While the role of macrophages in HFpEF is established, the involvement of human peritoneal macrophages (HPMs) in PD-induced peritoneal injury is largely unknown.
We hypothesize that HPMs, chronically exposed to the glucose-rich fluids, exhibit an inflammation-driven immunometabolic profile. This may contribute to cardiovascular (CV)-risk in PD-treated patients. We present preliminary results on the isolation of HPMs from PD effluent (PDE) and a detailed protocol to establish the immunometabolic profile of HPMs. These profiles will subsequently be related to cardiac function (left ventricular global longitudinal strain (LV-GLS), an echocardiographic measure of HFpEF) and parameters of systemic inflammation.

Method
In 15 adult PD-treated patients we will prospectively perform cardiac echography, draw a blood sample and collect PDE for HPM isolation. To obtain control HPMs a peritoneal saline flush will be collected from 5 adult patients with non-metastasized colon cancer during a routine diagnostic laparoscopy. HPMs will be isolated from the PDE and saline by centrifugation, followed by freezing for storage. We compared fresh and thawed cells of a PDE-sample to evaluate the impact of storage on HPM phenotype and functionality, respectively using flow cytometry and measurement of IL-6 production, with and without a lipopolysaccharide (LPS) challenge.
In-depth HPM characterization will be performed by measuring glucose and lactate, conducting metabolic flux analysis, assessing glycolysis and mitochondrial characteristics using Seahorse®, and employing cytometry to evaluate reactive oxygen species and metabolic fuel preference. A phenotypical profile will be established using fluorescent probes and antibodies. To assess functionality, HPMs will be exposed in vitro to “M1” and “M2” inducers and we will use human monocyte-derived macrophages from healthy adult controls as a comparison.
We will relate the immunometabolic, phenotypical and functional HPM characteristics to LV-GLS, blood cytokine (obtained with Bio-Plex multiplex) and blood immune profiles (obtained with cytometry) in PD-treated patients. Furthermore, we will compare the HPM characteristics of the PD-treated patients with the control group.

Results
Preliminary results of 6 patients show that we can isolate HPMs from the PDE with a yield of &gt;5M cells. The phenotype and functionality of HPMs was evaluated both directly after isolation and after a freeze-thaw cycle. Flow cytometry showed high percentages of CD14+HLA-DR+CD64+ macrophages in both fresh and biobanked HPMs. IL-6 production was observed in both fresh and biobanked HPMs even without LPS-stimulation (Figure).

Conclusion
By successfully isolating and storing HPMs from the easily accessible PDE, we optimized the conditions to assess their immunometabolic characteristics. The proposed protocol not only aims to provide valuable insights into HPM phenotype and functionality, but also allows us to relate these to cardiac function and systemic inflammation. With this strategy, we aim to gain insight in the immunometabolic effects of PD on peritoneal immune cells and how these relate to CV-effects.