Mechanistic Analysis of Nonoxygenated Hypothermic Machine Perfusion’s Protection on Warm Ischemic Kidney Uncovers Greater eNOS Phosphorylation and Vasodilation

Abstract

Protection of endothelial cell function may explain the benefits of nonoxygenated hypothermic machine perfusion (MP) for marginal kidney preservation. However, this hypothesis remains to be tested with a preclinical model. We postulated that MP protects the nitric oxide (NO) signaling pathway, altered by static cold storage (CS), and improves renal circulation recovery compared to CS. The endothelium releases the vasodilator NO in response to flow via either increased endothelial NO synthase (eNOS) expression (KLF2-dependent) or activation of eNOS by phosphorylation (via Akt, PKA or AMPK). Using a porcine model of kidney transplantation, including 1 h of warm ischemia and preserved 24 h by CS or MP (n ¼ 5), we reported that MP did not alter the cortical levels of KLF2 and eNOS at the end of preservation, but significantly increased eNOS activating phosphorylation compared to CS. eNOS phosphorylation appeared AMPK-dependent and was concomitant to an increased NO-dependent vasodilation of renal arteries measured, ex situ, at the end of preservation. In vivo, laser Doppler showed that cortical microcirculation was improved at reperfusion in MP kidneys. In conclusion, we demonstrate for the first time, in a large-animal model, that MP protects the NO signaling pathway, confirming the value of MP for marginal kidney preservation.Abbreviations: Akt, protein kinase B; AMPKa, 5 0 -adenosine monophosphate-activated protein kinase alpha; ANOVA, analysis of variance; CS, cold storage; CTL, control; DCD, donation after circulatory death; eNOS, endothelial nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; KLF2, Kruppel-like factor 2; L19, ribosomal protein L19; L-NAME, NGnitro-L-arginine methyl ester; MP, machine perfusion; MPS, machine perfusion solution; NO, nitric oxide; PKA, protein kinase A; RPLPO, ribosomal phosphoprotein large P0 subunit; RT-qPCR, reverse transcriptase quantitative polymerase chain reaction; SEM, standard error of the mean; UW, University of Wisconsin; WI-60, 1 h warm ischemia

Defining the optimal duration for normothermic regional perfusion in the kidney donor: A porcine preclinical study

“…This suggests that NRP is a beneficial reconditioning allowing improved kidney perfusion. 59,60 To evaluate the impact of observed effects and to validate the protective effect of NRP, we allotransplanted the kidneys previously macrophage invasion signals during NRP suggests that a threshold has been reached and that a shorter NRP duration is preferable. Indeed, eNOS activity is an important factor for organ preservation and conditioning, [56][57][58] and our team demonstrated its involvement in HMP benefits.…”

Combined liver‐kidney perfusion enhances protective effects of normothermic perfusion on liver grafts from donation after cardiac death

“…Furthermore, endothelial cells are constantly exposed to flow. Flow‐related shear stress is one of the most potent stimuli for NO release, which is a potent vasodilator . We demonstrated that combined liver‐kidney NMP might protect the NO signaling pathway by up‐regulation of eNOS through an AMPK‐α–dependent mechanism.…”

Should Pulsatile Preservation Be the Gold Standard in Kidney Transplantation?

“…The actual cellular mechanisms behind how this protection occurs are currently poorly understood. Of interest, pulsatile flow, alongside reproducing a more physiological setting, has also been associated with the expression of flowdependent vasoprotective endothelial genes [2,11] and reduced expression of inflammatory genes [12]. In particular, Kruppel-like factor 2 (KLF2) seems to play a critical role through the inhibition of pro-inflammatory responses, the production of vasodilators, specifically endothelial nitric oxide, and the expression of anti-thrombogenic mediators (e.g., thrombomodulin) [11].…”

“…Of interest, pulsatile flow, alongside reproducing a more physiological setting, has also been associated with the expression of flowdependent vasoprotective endothelial genes [2,11] and reduced expression of inflammatory genes [12]. In particular, Kruppel-like factor 2 (KLF2) seems to play a critical role through the inhibition of pro-inflammatory responses, the production of vasodilators, specifically endothelial nitric oxide, and the expression of anti-thrombogenic mediators (e.g., thrombomodulin) [11]. The shear-dependent stimulation of the vasculature in the preserved organ can also be obtained by continuous flow, but studies have shown improved circulation and organ function when pulsatile perfusion was used (with evidence collected for both liver and kidney) [13, 14•].…”