Normal Saline solutions cause endothelial dysfunction through loss of membrane integrity, ATP release, and inflammatory responses mediated by P2X7R/p38 MAPK/MK2 signaling pathways

Abstract

Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1β (IL-1β) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1β led to impaired endothelial relaxation. IL-1β treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.

A cell permeant phosphopeptide mimetic of Niban inhibits p38 MAPK and restores endothelial function after injury

“…This produced the maximal force tension relationship as previously described. 4 After equilibration, the rings were primed with 110 mM of potassium chloride (with equimolar replacement of sodium chloride in bicarbonate buffer) to determine functional viability. Viable rings were then tested for contractile response to a dose of phenylephrine (PE) to yield submaximal contraction (approximately 60%-70% of maximum KCl; 5 × 10 −6 M for HSV and 1-5 × 10 −7 M for RA) and relaxed with carbachol (CCH, 5 × 10 −7 M), an acetylcholine analogue, to determine endothelial-dependent relaxation responses.…”

“…The inner lining of the vascular wall consists of a monolayer of endothelial cells. Mechanical forces (blood flow disturbances, mechanical stretch), 1,2 chemical stressors (glycemic, oxidative, osmotic, acidosis), [3][4][5][6][7][8][9] inflammation, 10 and aging 11 are associated with dysfunction of this fragile endothelial monolayer. A common physiologic sequela of endothelial injury is impaired endothelial-dependent relaxation, often referred to as "endothelial dysfunction.…”

“…17 Exposure of veins to acidic saline solutions such as Normal Saline (NS) that is widely used as a resuscitation fluid, or mechanical stretch during surgical harvest leads to endothelial injury and release of ATP, LDH, and decreased TEER. 4 Prolonged exposure to high concentrations of ATP activates the purinergic receptor, P2X7R. P2X7R activation is one of the most potent activators of the inflammasome.…”

“…3 HSV is injured by mechanical stretch during harvest and pressure distention, storage in acidic NS solution, and orientation marking with surgical skin markers. 1,4,9,22,[26][27][28] Previously, to understand the response to surgical injury, segments of HSV removed atraumatically were compared to cognate segments after harvest and preparation injury. Injured HSV segments demonstrated impaired endothelial-dependent relaxation which was associated with a decrease in Niban phosphorylation.…”

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression

“… 26 Further studies indicated that P2X7R activation promotes the neuroinflammatory response through the p38 MAPK pathway. 27 – 29 EA attenuates SNL-induced microglial activation mediated by p38 MAPK. 30 Therefore, we hypothesized that the analgesic effect of EA may be achieved by reducing the expression of P2X7R and thus inhibiting the phosphorylation of p38 MAPK.…”