PART Ⅰ：Metabolic Needs Of The Kidney Graft Undergoing Normothermic Machine Perfusion
Mar 21, 2022
ali.ma@wecistanche.com
PART Ⅰ：Metabolic needs of the kidney graft undergoing normothermic machine perfusion
Asel S. Arykbaeva, Dorottya K. de Vries & et al.
The key challenge in organ transplantation is the global shortage of donor organs. Because of the pressing demands, most transplant centers are progressively embracing organs from older and higher-risk donors. At the same time, many grafts are discarded because of a perceived risk that these organs may not function, or function suboptimally after transplantation.' One strategy to increase graft utility and improve transplant outcomes is the implementa-tion of more objective quality assessment tools by ex-situ perfusion of donor organs, thereby creating a window for functional testing and viability enhancement of the perfused graft, 4Rs: resuscitation, repair,' rejuvenation, and regeneration. Multiple trials have shown that ex-situ hypothermic machine perfusion for kidney grafts is feasible and safe, and improves clinical outcomes. An obvious next step was the introduction of (sub)normothermic machine perfusion (NMP). Although the feasibility of NMP has been proved in several trials, current kidney NMP proto-cols reflect pioneering proof-of-concept studies, studies that primarily concerned short periods of NMP. The specific metabolic prerequisites may, to some extent, vary with the specific aims of the perfusion; the 4 Rs. Shorter perfusion that aims at resuscitation, rejuvenation, and functional assessment should provide the metabolites that optimally sustain metabolic flexibility. Longer perfusions that aim at repair and rejuvenation come with the additional need for anabolic factors, such as essential amino acids and vitamins. Current protocols are all based on continuous perfusion with red blood cells or an alternate oxygen carrier, an isotonic or/and colloid solution, such as albumin, and glucose and amino acids2-5,10-1 as an energy source.4 A detailed overview of the published protocols(including the perfusate composition) is provided in Table 1.33,9,10
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Based on the observation that all NMP studies of human 5,12,13 kidneys so far report accruing lactate perfusate levels,-it could be argued that a physiological metabolic state is not established under the current NMP conditions.5" Although this phenomenon may reflect the use of discarded kidneys, lactate accumulation is also observed during NMP of kidneys that were accepted for transplantation. Hence, the current NMP perfusion protocols do not yet entail the optimal conditions required for induction of a physiological metabolic state in viable grafts, let alone that the protocols mimic the optimal conditions for viability testing or prolonged perfusions required for resuscitation and/or repair of so-called"marginal" kidney grafts.
The kidney is a metabolically highly active organ, with a per mass-energy requirement that is similar to the heart, and has specific substrate preferences and metabolic functions. The latter aspects are clearly illustrated by the kidney-specific patterns of metabolite uptake and release, shown by organ-specific arteriovenous concentration differences, and by its crucial role in body lactate disposal.5.Moreover, the kidney's functional diversity translates into heterogeneous metabolic profiles with distinct substrate preferences for each of its specialized functional subunits. As a consequence, an optimal NMP protocol should address the metabolic requirements of the kidney and compensate for the absence of the body's homeostatic system that would normally replenish nutrients and dispose of waste products.
NMP for viability testing or recovery of so-called "marginal" kidney grafts may come with even more demanding requirements. These"marginal" kidney grafts constitute a heterogeneous group that includes grafts from older donors, grafts from perceived higher-risk donors, and/or grafts that have sustained considerable procurement stress(such as pronged ischemia). These conditions all associate with an impaired resilience," as such"marginal" kidney grafts may present with impaired metabolic plasticity.1 Consequently, failure to meet the metabolic requirements of these compromised organs may lead to unjustified conclusions with respect to their viability. Similarly, better-tailored perfusion protocols are likely required to support an anabolic state and to meet the metabolic prerequisites for prolonged NMP aimed at ex-situ graft regeneration.
The focus of this review is to provide a theoretical framework of the metabolic aspects of renal NMP(i.e., how to provide optimal metabolic support during graft perfusion). Conclusions from the review may(partially) translate to other organs, Yet, profound organ-specific differences exist with regard to substrate preference. An evaluation of organ-specific differences(such as long-chain fatty acids as the preferred metabolic substrate of the myocardium-) was considered beyond the scope of the review. Similarly, aspects of oxygen delivery were considered beyond the scope of the review. This review is structured along with the 3 main metabolic dusters: carbohydrates, fatty acids, and amino acids followed by considerations concerning the provision of micronutrients. Finally, a practical overview of the options for monitoring metabolic homeostasis in the context of NMP is provided. Reference data with regard to aspects of the metabolic physiology of the kidney largely rely on human (living donors)and porcine studies that applied arteriovenous blood sampling over the kidney.
Carbohydrates
In general and especially for the kidney, current NMP protocols mainly rely on glucose as a metabolic substrate for the perfused graft. However, absent uptake or release of glucose from the kidney, as determined by arteriovenous measurements in the human kidney, could imply minimal renal glucose catabolism(Figure 1'). This gross observation ignores the particular complex and spatially diverse organization of renal carbohydrate metabolism, with some areas relying on glycolysis and others actively involved in glucose-
22 This diversity follows the functional hetneogenesis.15, heterogeneity of the kidney with broad regional variations in cellular metabolic rates and profound differences in local oxygen tension. Cortical glomeruli represent well-oxygenated vascular structures that principally function as"passive" filters. The medullary tubules, on the other hand, represent a series of highly active, metabolically demanding pumps27,2 However, as an inevitable consequence of the countercurrent concentration mechanism of the loops of Henle, aspects of the deeper medulla are exposed to profound hypoxia. Hence, cells in this area are obligatory glycolytic (acetate producing), and reported as relatively resistant to anoxia (Figure 2-).
Absent lactate release, or even net lactate uptake from the circulation under physiologic conditions5,16,18 implies that the lactate formed in the deeper medulla is efficiently cleared within the organ. Recent studies identified the proximal tubules as the primary site of lactate disposal.
Although the renal gluconeogenetic capacity may imply glucose independence, it is important to point out that gluconeogenesis is an anabolic process, and thus imposes an avoidable energy burden on the graft. In addition, inadequate availability of glucose may profoundly interfere with the graft's metabolic plasticity(i.e., the physiologic ability to switch between different metabolic substrates to maintain metabolic competence). As a consequence, the provision of an adequate glucose supply is a critical requisite for NMP. In this context, it is important to consider that supraphysiological glucose concentrations will impose an avoidable metabolic burden on proximal convoluted tubules (local burden)within a perfused kidney because blood(perfusate)glucose is filtered into the glomerular ultrafiltrate, and subsequently actively reabsorbed in the proximal convoluted tubule. This process of glucose reabsorption is among the primary energy-demanding processes in the kidney. Consequently, from the perspective of minimizing energy requirements during NMP, perfusate physiologic glucose concentration of 3.5 to 5.5 mmol/L should be maintained. It could be speculated that pharmaceutical interference with the active glucose transporters, such as sodium-glucose cotransporter 2 inhibitor, could be ad. advantageous in the context of NMP as it minimizes the energy requisite/burden associated with glucose reabsorption by the distal tubules. 5,36
A yet unresolved question is whether insulin should also be provided during NMP. Glucose entry is controlled by the family of glucose transporters (GLUTs), most of which are insulin-independent. An exception is the insulin-dependent GLUT4, which controls postprandial glucose disposal. Because GLUT4 is also expressed in the kidney,' insulin supplementation during NMP should be considered.
Table 1 | Overview of the hardware (perfusion control mode, pressure, and gas supply), the perfusate composition, and supplements provided during NMP of the kidney for the different protocols
Apart from the lactate uptake, the arteriovenous concentration differences show that the kidney also clears organic acids, such as citrate and malate, from the circulation. Both are direct intermediates in the citric acid cycle. For citrate, it has been speculated that this clearance reflects a urinary excretion mechanism as part of a citrate disposal system. Yet, tracer studies in mice and persistent citrate clearance by transiently anuric deceased donor grafts in the phase immediately following transplantation imply that citrate is, at least partially, metabolized by the kidney rather than just secreted in the urine."Consequently, citrate could be considered a carbon source in the context of renal NMP.
Figure 1 | Arteriovenous measurements of glucose and creatinine concentrations in prerenal (arterial [A]; red) and postrenal (venous [V]; blue) blood samples of (healthy) living kidney donors (n [ 5) in samples collected as described by Lindeman et al., 16 measured in a standard manner by the Clinical Chemistry Laboratory. (a) The lower venous creatinine levels illustrate renal creatinine clearance. (b) Varying glucose uptake among the different donors reflects a heterogeneous carbohydrate turnover. Adapted from Lindeman JH, Wijermars LG, Kostidis S, et al. Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia-reperfusion injury. K
The above observations imply a central role for the kidney in the physiological control of carbohydrate and organic acid homeostasis. In fact, clinical studies show that acute kidney injury is associated with profound derangements in carbohydrate metabolism that include a profoundly impaired lactate clearance. Along similar lines, differences in postreperfusion lactate dynamics (arteriovenous concentration differences)discriminate between the different levels of postreperfusion metabolic competence following kidney transplantation (Figure 3'). Accordingly, one could speculate that lactate metabolism can be used as a readout of graft metabolic competence. In this respect, progressive perfusate lactate accumulation was observed in NMP experiments using human discarded kidney grafts51.15challenges the reinstatement of physiological metabolic homeostasis under the current NMP conditions.
Although the lactate accumulation during NMP could reflect the disposal of red blood cell-produced lactate (either through direct lactate oxidation and/or alternatively lactate conversion to glucose [Cori cyde]), it is likely that the lactate accumulation largely relates to ongoing renal lactate production as a result of impaired oxidative phosphorylation under the current NMP conditions. Accruing perfusate lactate levels will result in metabolic acidosis. Although this acidosis can be efficiently corrected by titrating bicarbonate to the perfusate, lactate accumulation may result in product-inhibition of renal and erythrocyte lactate dehydrogenase activity, and as a consequence an impaired reduction of formed pyruvate to lactate. This final step in the glycolytic pathway is critical for maintaining oxidation-reduction neutrality by regenerating nicotinamide adenine dinucleotide positive (NAD+)from the reduced NAD formed during glycolysis. As a consequence, lactate dehydrogenase product inhibition resulting from the accruing lactate levels may result in cellular oxidation-reduction stress.
Figure 2 | Schematic overview of the metabolic substrates utilized by the kidney in their simplified pathways. Under normal conditions, lactate is used for gluconeogenesis, and glucose, fatty acids, glutamine (amino acids), and possibly citrate are used to fuel oxidative phosphorylation. Under hypoxic conditions, or under conditions of high-energy demand, glycolysis is activated, resulting in a net release of lactate from the kidney. NAD, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NH3, ammonia.
The kidney not only shares this gluconeogenesis capability with the liver, but it also has the capacity to store glycogen, albeit less equipped with a glycogenolytic system compared with that of the liver. As a consequence, the role of renal glycogenolysis in maintaining blood glucose levels during intermittent fasting is reported to be limited.24 Still, because inverse associations are found between kidney glycogen stores and the extent of ischemic kidney injury, glycogen stores may serve as an endogenous energy buffer under extreme conditions, such as graft procurement and transplantation. Consequently, it could be speculated that the preservation, or even reestablishment, of renal glycogen stores by NMP is beneficial.
Figure 3 | Lactate dynamics illustrating the different levels of renal metabolic competence following kidney transplantation. Curves reflflect the relative renal arterial (red) and venous (blue) lactate levels. Initial washout of lactate accumulated during storage (indicated by the gray bar) and immediate (living donor) or delayed (deceased donor graft without delayed graft function [DGF]) suppression of glycolysis (indicated by the green bar). Transient persistent normoxic glycolysis in deceased donor grafts without and with DGF (indicated by the red bar). Adapted from Lindeman JH, Wijermars LG, Kostidis S, et al. Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia-reperfusion injury
Fatty acids
Although the current perfusion protocols are essentially glucose-centered, in view studies show that the kidney, like the heart, relies on fatty acids as a primary fuel source.7Yet, although the heart has a dear preference for long-chain(C16 and longer)fatty acids, the kidney essentially fuels on medium-chain fatty acids (MCFAs)(Figure 46 and Supplementary Table S2).
Figure 4 | Arteriovenous (AV) concentration differences in (healthy) living donor kidneys for middle-chain fatty acids (MCFAs) and long-chain fatty acids (LCFAs), illustrating the preference of the kidney for MCFAs.
The renal preference for MCFAs has several advantages over long-chain fatty acids, as they do not rely on specific transmembrane transporters to cross the plasma and mitochondrial membranes by specific transporters and carnitine shuttling. So, MCFAs can simply diffuse through the inner mitochondrial membrane, where the oxidation of the fatty acids occurs. With the aim of optimally mimicking renal metabolic physiology during NMP provision of MCFAs during NMP should be considered. In this context, most parenteral lipid emulsion formulations are essentially long-chain fatty acid-based and are therefore not optimally suited for renal NMP. However,MCFA-enriched formulas are available: Lipofundin(MCFA 10%) and Smoflipid(MCFA 6%). Given the apparent minimal oxidation of long-chain fatty acids by the kidney, further enrichment of MCFAs to these solutions might be preferable.
Lipid delivery during NMP comes with several challenges. The vast majority of plasma fatty acids are carried by albumin, with unbound fatty acids representing only a minor fraction(<0.01%)of the total. More important, clinical-grade albumin, most often used in the NMP protocols, has not undergone any pretreatment to free fatty acid binding sites. As a result, binding sites will be occupied mainly by the naturally dominating long-chain fatty acids. Furthermore, NMP is performed in an isolated, closed, and limited volume setup. As a consequence, the system has a limited lipid buffering capacity (because no adipose tissue, muscle, and liver are included), which imposes challenges with respect to the maintenance of MCFA supply and potential lipotoxicity. In the light of the limited free fatty acid carrying capacity of plasma or its derivates, it could be argued that preference should be given to lauric acid(C12:0)enrichment because this MCFA has the highest per molecule energy content.
Figure 5 | Gauging metabolic status. Schematic overview illustrating a cluster of liquid biomarkers that can be applied to monitor metabolic status during renal normothermic machine perfusion. ATP, adenosine triphosphate; GTP, guanosine triphosphate.
Apart from their role as an energy source, provision of other lipid classes critical to cellular function, such as essential unsaturated fatty acids and cholesterol, should be considered during prolonged NMP, in particular in protocols that aim at organ regeneration. However, the inclusion of unsaturated fatty acids in the NMP protocols carries specific risks. Unsaturated fatty acids may promote oxidative stress and lipid peroxidation, particularly in the presence of excess free hemoglobin/heme released as a consequence of hemolysis7during the perfusion or when using heme-based oxygen carriers
