Mechanisms of Hypothermic Machine Perfusion to Decrease Donation After Cardiac Death Graft Inflammation: Through the Pathway of Upregulating Expression of KLF2 and Inhibiting TGF-β Signaling

Abstract

Hypothermic machine perfusion (HMP) has been known as an efficient way to improve kidney graft function, but the underlying mechanisms remain unclear. Here, we adopt a rabbit reperfusion mode to investigate the upstream mechanisms of end-ischemic HMP of kidneys from donors after cardiac death (DCD), with static cold storage (CS) as a control. Eighteen New Zealand healthy male rabbits (12 weeks old, with a weight of 3.0 ± 0.2 kg) were randomly divided into three groups: HMP group, CS group, and Normal group (n = 6). The left kidney of rabbits underwent warm ischemia for 25 min through clamping the left renal pedicle and then reperfusion for 1 h. Then the left kidneys were preserved by CS or HMP (4°C for 4 h) ex vivo respectively, after they were autotransplanted and rabbits were submitted to a right nephrectomy. Twenty-four hours after reperfusion, all left renal specimens were collected. Finally, the expression of Krüppel-like factor 2 (KLF2), transforming growth factor-β (TGF-β) and SMAD4 protein in renal cortical tissue were detected by immunoblotting, and the TGF-β and SMAD4 expressions were further confirmed by immunohistochemistry analysis. We found that expression of KLF2 in HMP group was significantly higher than CS group (P = 0.011), while expression of TGF-β and SMAD4 in HMP group were significantly lower than CS group (P = 0.002, P = 0.01, respectively); Compared with normal group, the expression of TGF-β and SMAD4 in HMP and CS group significantly increased (P<0.05). Compared with CS group, TGF-β and SMAD4 protein were equally down-regulated in glomerular and the tubular epithelial cells in HMP group confirmed by immunohistochemistry. In conclusion, HMP may decrease DCD kidneys inflammation through the pathway of upregulating expression of KLF2 and inhibiting TGF-β signaling after transplantation.

Hypothermic oxygenated perfusion (HOPE) attenuates ischemia/reperfusion injury in the liver through inhibition of the TXNIP/NLRP3 inflammasome pathway in a rat model of donation after cardiac death

“…Studies of DCD kidney preservation found flow cessation during CS results in a significant reduction of several endothelial vasoprotective pathways, which leads to cell activation and apoptosis, whereas the flow stimulation provided by HOPE can regulate the shear stress-sensitive factors and provide vasodilation and endothelial protection (33)(34)(35). Shear stress is a parallel frictional drag force that is linearly proportional to the flow (63).…”

“…Previous studies have confirmed that HOPE could resuscitate DCD livers by renewing the metabolically depleted energy, altering the antioxidative status, relieving the downstream activation of sterile inflammation (29)(30)(31)(32), and regulating shear stress-sensitive factors in the endothelium (33)(34)(35). Current multicenter trials are underway to evaluate the use of portable HOPE devices to maximize organ preservation duration (10).…”

Pretreatment Donors after Circulatory Death with Simvastatin Alleviates Liver Ischemia Reperfusion Injury through a KLF2-Dependent Mechanism in Rat

“…Liver samples were processed from −80°C storage and Western blotting performed as described [ 20 ]. The primary antibodies used are as follows: KLF2 (1 : 400, rabbit anti-KLF2 antibody, Biosynthesis Biotechnology, Beijing, China), phosphorylated eNOS at Ser1177 (1 : 1000, rabbit anti-phosphorylated eNOS antibody, Cell Signaling, Danvers, MA), total eNOS (1 : 1000, rabbit anti-eNOS antibody, Cell Signaling, Danvers, MA), Bax (1 : 1000, rabbit anti-Bax antibody, Proteintech, Manchester, UK), and Bcl-2 (1 : 1000, rabbit anti-Bcl-2 antibody, Proteintech, Manchester, UK).…”

In Vitro/Ex Vivo Models for the Study of Ischemia Reperfusion Injury during Kidney Perfusion

“…KLF2 acts selectively as an important mechano-activated transcription factor in multiple endothelial functions, like the regulation of endothelial transcriptional programs controlling vascular tone, blood vessel development, thrombosis/hemostasis, and inflammation [58]. Shear stress was demonstrated to be one of the protective mechanisms of perfusion, through a mechanism which was thought to involve KLF2 and eNOS/NO expression in large animal models (porcine kidney) [72], small animal models (rabbit kidney) [73], and in vitro models of endothelial cells [74]. These results are in association with decreased KLF2 and eNOS expression, both in vitro and in vivo, due to flow cessation in the ischemia model [57,75].…”