Transplantation/Immunology
Rapid sampling microdialysis as a novel tool for parenchyma assessment during static cold storage and hypothermic machine perfusion in a translational ex vivo porcine kidney model

https://doi.org/10.1016/j.jss.2015.07.004Get rights and content

Abstract

Background

Viability assessment during preservation is imperative to avoid unnecessary discard of marginal organs maximizing graft outcomes in kidney transplantation. To address this need, we have developed a novel system based on a rapid sampling microdialysis (rsMD) analyzer allowing continuous tissue monitoring and measurement of metabolic markers of cell damage. Our aim was to develop a tool that allows for accurate assessment of tissue metabolism and organ viability in the preservation period.

Methods

Twenty-two porcine kidneys subjected to 15 min of warm ischemia underwent either 24 h of static cold storage (SCS) or 10 h of hypothermic machine perfusion (HMP). After preservation, tissue temperature was allowed to passively increase to ambient temperature as an ischemic challenge. Cortical and medullary metabolism was monitored throughout with online measurements of lactate concentrations made every 60 s.

Results

On commencement of monitoring, lactate concentrations were successfully detected within 15 mins. During the initial 1.5 h, lactate concentrations were similar during SCS (65 μM) and HMP (124 μM, P > 0.05) but lower after 10 h of SCS (SCS: 68 μM versus HMP: 230 μM, P < 0.001). Warming data suggest a resilience of HMP kidneys to subsequent temperature induced ischemia compared to SCS kidneys.

Conclusions

This preliminary study provides the baseline ischemic profile for porcine kidneys while validating the technique of rsMD as a tool for organ viability assessment during preservation. The data characterize metabolic differences between SCS and HMP preserved allografts and can help elucidate why HMP is clinically superior to SCS allowing development of interventions to augment these benefits.

Introduction

Almost 100,000 patients are registered on the United States organ transplant waiting list awaiting a suitable kidney [1]; in the United Kingdom, the number of patients awaiting a kidney transplant has risen by 26% from a decade ago, with 6348 patients currently on the national list [2]. With these numbers ever increasing, alternative sources of organs have been sought, and there has been increasing use of extended criteria donor organs and organs from donation after cardiac death (DCD) to meet demands.

Hypothermic machine perfusion (HMP) has proved advantageous for the preservation of such kidneys compared to static cold storage (SCS) [3], [4], [5], [6], and its use is advocated by the British Transplantation Society [7]. A recent meta-analysis of 18 studies incorporating 2203 kidneys has suggested HMP results in improved immediate post-transplant graft function, with potentially better renal graft survival rates compared to SCS [8]. Such benefits have to date been attributed to superior preservation of microcirculatory integrity and inflammation [9]. At present, however, there are no accepted indicators that can determine organ viability and potential postoperative function before an organ is transplanted [10], [11], and 12–18% percent of kidneys are discarded because of concerns about their suitability for transplantation [2]. An assessment of renal tissue condition during preservation would facilitate a greater level of discrimination in identifying those organs at higher risk of delayed function and potentially nonfunction and would enable a more accurate and informed selection of kidneys suitable for transplantation.

Microdialysis is a minimally invasive technique that allows the interstitial composition of a target tissue to be sampled, followed by ex vivo analysis, providing an accurate method of monitoring tissue biochemistry and metabolism over time. The use of conventional offline microdialysis for in vivo tissue viability assessment has been validated in a variety of research and clinical scenarios [12], including monitoring of cerebral ischemia [13], [14], [15], [16], [17], bowel ischemia [18], [19], [20], free flap surgery [21], [22], [23], and postoperatively in kidney [24], [25] and liver [26], [27], [28], [29] transplantation. Microdialysis techniques can be translated easily from an experimental to a clinical setting because of the availability of Food and Drug Administration–approved and CE-marked probes [12]. There are limited reports of the use of microdialysis as a tool for organ assessment during SCS [24], [30], [31] or HMP [32]; however, the clear need for greater tissue assessment compels us to propose that microdialysis can be used to effectively quantify the development of ischemic injury during this period.

We have developed a novel online rapid sampling microdialysis (rsMD) analyzer capable of continuously measuring the concentration of markers of tissue ischemia at 30 s intervals. To date, no such system has been used to directly characterize and compare the effects of SCS and HMP on renal biochemical activity and the ischemic injury sustained during preservation in real time. If capable of accurately monitoring tissue ischemia during organ preservation, rsMD may have a promising future role in graft assessment.

Our objectives were two-fold: (1) to assess the feasibility of using rsMD to monitor changes in glycolytic metabolites in the renal cortex and medulla during SCS and HMP in a porcine model and (2) to compare the effects of SCS and HMP on renal cellular metabolism and ischemia during the preservation period.

Section snippets

Methods

An experimental porcine DCD model was used to determine the feasibility of using the rsMD system for tissue assessment during ex vivo hypothermic preservation. This research was conducted at laboratories of Imperial College London. Local guidelines and policies were adhered to.

SCS and HMP dynamics

Ten kidneys underwent 24 h of SCS and a further 12 underwent 10 h of HMP. Perfusion parameters were similar between HMP kidneys with systolic pressures of 36 ± 8.5 mmHg, flow rates of 27.7 ± 6.1 mL/min/100g, PFIs of 0.75 ± 0.27 mL/min/100 g/mmHg, and resistances of 0.39 ± 0.16 mmHg/ml/min by the end of perfusion (Fig. 3), with a trend of improving perfusion metrics.

Microdialysis

Overall medullary lactate levels were greater than cortical levels throughout preservation and passive warming. RsMD data from two

Discussion

We have used rsMD to identify the differing effects on renal energy metabolism brought about by SCS and HMP, both during hypothermic preservation and subsequent warming. Both modalities show contrasting effects, and these differences may help elucidate the reported clinical superiority of HMP that is observed to afford metabolic resilience on the renal parenchyma to warm ischemia after initial cold preservation.

Associations between delayed graft function (DGF) and increased risk of graft loss

Conclusion and future work

Continuous online microdialysis has provided an important insight into the chemical processes active during the ex vivo phase of a donor kidney during SCS and HMP, offering, for the first time, a possible biochemical explanation regarding the protective role of HMP for kidneys. Further studies would need to be conducted to confirm this. Determining the potential of this technology in assessing allografts before transplantation requires an assessment of organ function in grafts with variable

Acknowledgment

This research was supported by a grant from the Live Life Then Give Life Transplantation Charity.

We gratefully acknowledge the contribution of Nick Bullock for his assistance with the experiments in this work.

Authorship: K.H. and S.G. participated in research design, performance of the work, data analysis and writing the article. S.D. participated in research design and performance of the research. M.R. and C.L. participated in data analysis and contributed analytical expertise and tools. G.H.

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