A simple ex vivo model of human renal allograft preservation using the gonadal vein

Abstract

Introduction: Hypothermic machine perfusion, an organ preservation modality, involves flow of chilled preservation fluid through an allograft's vasculature. This study describes a simple, reproducible, human model that allows for interrogation of flow effects during ex vivo organ perfusion.

Materials and methods: Gonadal veins from deceased human renal allografts were subjected to either static cold storage or hypothermic machine perfusion for up to 24 hours. Caspase-3, Krüppel-like factor 2 expression and electron microscopic analysis were compared between 'flow' and 'no-flow' conditions, with living donor gonadal vein sections serving as negative controls.

Results: The increase in caspase-3 expression was less pronounced for hypothermic machine-perfused veins compared with static cold storage (median-fold increase 1.2 vs 2.3; P < 0.05). Transmission electron microscopy provided ultrastructural corroboration of endothelial cell apoptosis in static cold storage conditions. For static cold storage preserved veins, Krüppel-like factor 2 expression diminished in a time-dependent manner between baseline and 12 hours (P < 0.05) but was abrogated and reversed by hypothermic machine perfusion (P < 0.05).

Conclusions: Our methodology is a simple, reproducible and successful model of ex vivo perfusion in the context of human organ preservation. To demonstrate the model's utility, we establish that two widely used markers of endothelial health (caspase-3 and Krüppel-like factor 2) differ between the flow and no-flow conditions of the two predominant kidney preservation modalities. These findings suggest that ex vivo perfusion may mediate the induction of a biochemically favourable endothelial niche which may contribute tohypothermic machine perfusion's association with improved renal transplantation outcomes.

Keywords: Kidney transplantation; Krüppel-like transcription factors; Organ preservation.

Figure 1

The intracellular Krüppel-like factor 2 (KLF2) pathway, its upstream modulation and downstream sequelae. Shear stress is mechanotransduced across the apical endothelial cell membrane by a number of receptors which induce cascading activation of intracellular messengers that culminate in transcription of the KLF2 gene. KLF2 mediates pleiotropic endothelial effects – vasodilatory, anti-thrombotic, anti-angiogenic and anti-inflammatory.^,,,

Figure 2

Gonadal vein apparatus. (A) 3-mm polycarbonate T-piece (Organ Recovery Systems, IL). (B) Gonadal vein segment fixed to T-piece with a 2/0 Vicryl tie. (C) Bulldog clip. (D) 22-gauge cannula cut obliquely to a length of 20 mm (BD, NJ). (E) Evidence of venotomy closure using Prolene polypropylene 5/0 (Ethicon, NJ).

Figure 3

Gonadal vein apparatus in circuit with a modified LifePort Kidney Transporter 1.0 (Organ Recovery Systems, IL) perfusion device. (A) Standard LifePort® inlet attached to T-piece terminus to serve as inflow end. (B) Vein outflow end fixed so as to be submerged on the KPS-1® reservoir (Organ Recovery Systems, IL). (C) KPS-1® reservoir in LifePort 1.0 perfusion cassette (Organ Recovery Systems, IL).

Figure 4

Representative high-power caspase-3 immunohistological photomicrographs from both living and cadaveric gonadal vein segments taken from sampling at T0 and T24 (shown at 40 times magnification). (A) A contiguous confluent endothelium, present as a monolayer, is evident in sections of living donor gonadal vein segments taken at baseline. (B) At T0, cadaveric gonadal vein sections exhibit a similar morphology although demonstrate a higher positivity for caspase-3 by cell count. (C) Sections of living donor gonadal vein segments after 24 hours of static cold storage manifest loss of the confluent endothelial cell monolayer and cell contraction. (D) Cadaveric gonadal vein segments after 24 hours of static cold storage exhibit a high caspase-3 positivity with concomitant losses of large swathes of endothelial integrity. Caspase-3 positivity can also be seen in the underlying cells of the subintimal region.

Figure 5

Electrophotomicrographs from sections of cadaveric gonadal veins segments sampled at baseline and after 12 hours of static cold storage and hypothermic machine perfusion. (A) There is evidence of cell denudation (black arrows) and a corrugated endothelium at baseline in this cadaveric gonadal vein section. (B) After 12 hours of static cold storage, this cadaveric gonadal vein section manifests more dramatic swathes of denudation (black arrows) than at baseline and also appears condensed (white arrow).

Figure 6

Percentage change in KLF2 expression from baseline to six hours of static cold storage compared with hypothermic machine perfusion. Individual marks represent each datum with horizontal lines depicting the median and interquartile range. After six hours, there is no significant difference in median percentage change in Krüppel-like factor 2 (KLF2) expression from baseline between static cold storage (SCS) and hypothermic machine perfused (HMP) paired samples.

Figure 7

Percentage change in KLF2 expression from baseline to 12 hours of static cold storage compared with hypothermic machine perfusion (HMP). Individual marks represent each datum, with horizontal lines depicting the median and interquartile range (IQR). After 12 hours, median Krüppel-like factor 2 (KLF2) expression had decreased by 59.4% (IQR –86.4 to –18.4) in static cold storage (SCS) designated gonadal vein samples. However, matched hypothermic machine perfused gonadal vein samples exhibited a median increase in KLF2 expression of 13.4% (IQR –52.7 to 57.9). This alteration from baseline was found to be statistically significant (P = 0.031).