Delayed Graft Function and the Renin-Angiotensin System

Abstract

Delayed graft function (DGF) is a form of acute kidney injury (AKI) and a common complication following kidney transplantation. It adversely influences patient outcomes increases the financial burden of transplantation, and currently, no specific treatments are available. In developing this form of AKI, activation of the renin-angiotensin system (RAS) has been proposed to play an important role. In this review, we discuss the role of RAS activation and its contribution to the pathophysiology of DGF following the different stages of the transplantation process, from procurement and ischemia to transplantation into the recipient and including data from experimental animal models. Deceased kidney donors, whether during cardiac or brain death, may experience activation of the RAS. That may be continued or further potentiated during procurement and organ preservation. Additional evidence suggests that during implantation of the kidney graft and reperfusion in the recipient, the RAS is activated and may likely remain activated, extrapolating from other forms of AKI where RAS overactivity is well documented. Of particular interest in this setting is the status of angiotensin-converting enzyme 2 (ACE2), a key RAS enzyme essential for the metabolism of Angiotensin II and abundantly present in the apical border of the proximal tubules, which is the site of predominant injury in AKI and DGF. Interventions aimed at safely downregulating the RAS using suitable shorter forms of ACE2 could be a way to offer protection against DGF.

Introduction

Delayed graft function (DGF) is a common complication wherein acute kidney injury (AKI) develops immediately post-transplantation ^1–3. Since it often necessitates dialysis treatment during the first week post-transplantation, this requirement for dialysis is usually included in the definition of DGF ^1–3. The importance of DGF, whether dialysis is implemented or not, must be recognized because it may influence the future outcome of the transplanted kidney ^1–3. Within the spectrum of DGF, early kidney function can vary from complete anuria or non-oliguric acute tubular necrosis (ATN), followed by a gradual return of function or even a rapid and immediate restoration of function ⁴. Extended periods of DGF are associated with unfavorable graft outcomes and decreased longevity of the transplanted kidney across a multitude of studies ^5–8. Moreover, the prolonged time on dialysis and increased hospital length of stay in DGF patients substantially increase the hospital expenditures and the financial impact of transplantation ^9–11.

The risk for DGF markedly increases in kidneys donated from deceased donors compared to living donors ⁴. While the incidence ranges from 4–10% in living donor kidney transplant recipients, it is observed in 30%–50% of deceased donor kidney transplant recipients ^12–14. Differentiating between donation after brain and circulatory death, the incidence is more pronounced when the donors have experienced circulatory death than brain death; however, long-term outcomes have been reported to be equivalent ^15–22. Because of organ scarcity, there is an increase in the utilization of expanded criteria donors and donations after cardiac death ^18,23. This rise has been accompanied by an increased incidence of DGF ^24–26. A range of factors can influence the occurrence of DGF. Notably, the duration of cold ischemia has been identified as a major significant contributor ^27,28. Additionally, the techniques utilized for preserving organs, such as static cold storage, significantly impact the occurrence of DGF ^29–31. This is primarily due to the generation of reactive oxygen species due to cellular energy depletion in an oxygen-deprived environment induced by cold storage ^29,32. Moreover, prolonged warm ischemia time also increases the risk for DGF, particularly when exceeding the 60-minute threshold ^33–35.

An important role in the pathophysiology of DGF can be attributed to ischemia-reperfusion injury (IRI) ^36,37. This injury is one of the pivotal mechanisms driving the onset of DGF and includes complex processes that already start pre-transplantation ^37–42. Over the preceding decade, notable advancements have been made in comprehending the molecular mechanisms of IRI. The renin-angiotensin-system (RAS) involvement in IRI and DGF has been proposed as a contributing mechanism. Several lines of evidence support activation of RAS in the pathophysiology of DGF ^39,43–46. The RAS can become activated either systemically or locally within the kidney, which may occur when the donor undergoes circulatory or brain death and during organ procurement. Subsequently, it may persist during organ implantation in the recipient and the following graft reperfusion. In this review, we explore the status of the RAS in both the donor and the recipient and its pathophysiological contribution to the development of DGF and postulate an important role of deficiency of a RAS enzyme, angiotensin-converting enzyme 2 (ACE2), in the pathogenesis of DGF. This hypothesis is prompted by our recent finding that this enzyme, abundantly present in the apical tubular border of the proximal tubule, is decreased in mouse models of DGF caused by prolonged cold ischemia time prior to kidney transplantation and AKI caused by IRI ^47,48.

Overview of the RAS

The RAS was originally conceived as a hormonal system involved in the regulation of blood pressure and homeostasis of fluid and electrolyte balance ^49–52. It has moreover been recognized that the local RAS at the tissue level is also very important in disease pathophysiology, particularly at the kidney level where overactivity of this system plays a role in acute and chronic kidney injury ^43–45. Some of the many peptides and enzymes that form the complex RAS cascade are outlined in Figure 1. Starting from Angiotensinogen, which is primarily generated by hepatic cells and secreted into the circulation, Angiotensin I is formed by Renin secreted from the kidneys ^49–52. Renin is released from the cells of the juxtaglomerular apparatus (JGA) and its activity represents the first and rate-limiting step in the formation of angiotensin peptides ^50–52. Angiotensin I is converted to Angiotensin II by Angiotensin Converting Enzyme (ACE), either present in the circulation or on endothelial cells in many organs, including the kidney ^49–52. The octapeptide Angiotensin II is the major effector peptide of the RAS and exerts a variety of biological functions through activation of the Angiotensin II Receptor Type 1 (AT₁) as its main receptor ^53,54. Activation of this G-Protein coupled receptor that is ubiquitously expressed throughout the body systemically leads to vasoconstriction and vascular smooth muscle contraction; renal actions include stimulation of tubular reabsorption of sodium and water as well as Aldosterone secretion from the adrenal gland ^55–59. Activation of the AT₁ receptor also stimulates inflammation, fibrosis, and oxidative stress, which is particularly important when considering the pathophysiological consequences of persistent RAS activation ^60–62. This pathway, starting with the production of Angiotensinogen and concluding with the actions of Angiotensin II is often referred to as the classical RAS ⁵⁰.

Figure 1: Overview of the Renin-Angiotensin-System.

Overview of the Renin–Angiotensin-System including its peptides (blue), enzymes (green), receptors (purple) and targets for therapeutic interventions (red). Starting from Angiotensinogen as the substrate, Renin is a main enzyme to generate downstream angiotensin peptides. The classical arm of the RAS includes Angiotensin II, which is synthesized from Angiotensin I by Angiotensin Converting Enzyme (ACE) and exerts its effects by activating the Angiotensin II receptor type 1 (AT₁). While Angiotensin II is the main effector peptide, multiple other Angiotensin II-degrading and Angiotensin II-independent pathways exist including a variety of angiotensin peptides and enzymes like Angiotensin Converting Enzyme 2 (ACE2), Neprilysin (NEP), Chymase, Prolylendopeptidase (PEP), Prolylcarboxypeptidase (PRCP), Aminopeptidase A (APA) and Aminopeptidase N (APN). Apart from the AT₁ receptor, alternative receptors of RAS peptides include the Angiotensin II receptors typ 2 and 4 (AT₂ and AT₄) as well as the MAS receptor activated by Angiotensin 1–7. Therapeutic interventions targeted at Angiotensin II may inhibit upstream enzymes like Renin and ACE or block the AT₁ receptor as the main receptor of Angiotensin II. Recombinant ACE2 may enhance the degradation of Angiotensin II and its precursor Angiotensin I thereby reducing the levels of Angiotensin II and balancing its effects on Renin-Angiotensin-System. Created with biorender.com

Additional elements of the RAS comprise the non-classical RAS that includes pathways of alternative degradation of Angiotensin II and their products, which lead to largely opposing effects ⁶³. An important pathway of the alternative RAS includes ACE2, which degrades Angiotensin II to form Angiotensin 1–7 ^64–68. This peptide, by activation of the MAS receptor and possibly the Angiotensin II Receptor Type 2 (AT₂), has anti-inflammatory and anti-fibrotic properties that may counter the actions of the classical RAS ^69,70. ACE2 is located in the apical border of the proximal tubule, where it can metabolize Angiotensin II which is both locally formed and filtered from the circulation ⁷¹. Apart from this strategic localization, the two other RAS enzymes that also degrade Angiotensin II to Angiotensin 1–7, Prolylendopeptidase (PEP) and Prolylcarboxypeptidase (PRCP), are less potent which makes ACE2 a key RAS enzyme important for the degradation of Angiotensin II in the kidney ⁷². Other components of the non-classical RAS are Angiotensin 1–9 as an alternative peptide synthesized from Angiotensin II and the AT₂ receptor, which is more sparsely expressed and opposes functions mediated by the AT₁ receptor ^73–76.

Apart from the systemic RAS in the circulation, angiotensin peptides are also synthesized locally at the tissue level. The kidney proximal tubule has key RAS components, comprising a local independent RAS such that Angiotensin II can be synthesized locally by the proximal tubule ^77–81. Both ACE and ACE2 are abundantly present in the apical border of the proximal tubule, and whereas ACE promotes the formation of Angiotensin II, ACE2 fosters its degradation, thereby controlling the formation and degradation of both locally formed and filtered Angiotensin II. ⁷¹ Alterations in RAS components are involved in the pathophysiology of kidney disease ^66,82–84. They can occur both systemically and locally, with changes in RAS enzymes such as ACE2 usually being more pronounced on the tissue level than in the circulation where the level of ACE2 is very low ⁴⁹. Of note, the circulating form of soluble ACE2 is too large to pass the glomerular filtration barrier. Our lab has generated shorter forms of soluble ACE2 that are short enough to pass the glomerular filtration barrier and, therefore, more suitable for downregulating the kidney RAS (84). In the subsequent sections, we will delve into the existing understanding of the role played by the RAS in experimental and clinical DGF.

The RAS in DGF: Experimental data:

Activation of the RAS has been shown in experimental animal models of IRI ^85–92. Although we did not find studies investigating the RAS in specific experimental models of DGF, the findings from animal models of IRI are relevant to DGF. Indeed, the transient reduction of blood flow and subsequent reperfusion seen in IRI are also part of the pathophysiology of DGF ^36,37. Table 1 summarizes studies investigating the RAS in animal models of IRI. Kidney Angiotensin II has been reported to be elevated 24 hours after reperfusion in rats following 60 min of bilateral ischemia and returned to the levels of sham-operated controls five days after reperfusion ⁸⁵. Allred et al. also showed increased levels of kidney Angiotensin II 24 hours after reperfusion following 60 min of unilateral ischemia ⁸⁶. Plasma Angiotensin II at this time point was not different from sham-operated controls ⁸⁶. Kidney Angiotensin II has also been shown to be increased as early as four hours after reperfusion following 45 min of unilateral ischemia, whereas kidney Angiotensin 1–7 was reduced ⁸⁷.

Table 1:

Data on RAS components evaluated on experimental models of AKI

Author	Species	Model	Key Findings
Allred et al. 86	Rat	Unilateral ischemia (60 min)	• Kidney Ang II increased • AT1 receptor density decreased • Urinary Ang I and Ang 1–7 increased • ACE and Neprilysin activity decreased • Renin activity increased
da Silveira et al. 87	Rat	Unilateral ischemia (45 min)	• Kidney Ang II increased • Kidney Ang 1–7 decreased • AT1 mRNA decreased • ACE activity decreased • Renin mRNA decreased • ACE2 mRNA decreased • MAS mRNA increased
Villanueva et al. 88	Rat	Bilateral ischemia (30 min)	• Renin protein and staining inreased
Weber et al. 89	Rabbit	Unilateral ischemia (90 min)	• Renin activity in plasma and JGA increased
Csohany et al. 90	Rat	Unilateral ischemia (50 min)	• Production and release of Renin from collecting duct increased
Studies including therapeutic interventions
Kontogiannis et al. 85	Rat	Bilateral ischemia (60 min)	• Kidney Ang II increased • AT1 mRNA and receptor binding decreased • Angiotensinogen mRNA decreased • Administration of Losartan at reperfusion improved kideny function
Molinas et al. 91	Rat	Unilateral ischemia (40 min)	• Losartan pretreatment improved kidney function and reduced inflammation
Rodríguez-Romo et al. 92	Rat	Bilateral ischemia (45 min)	• Losartan pretreatment improved kidney function and tubular/glomerular architecture after 9 months
Fouad et al. 96	Rat	Bilateral ischemia (45 min)	• Telmisartan pretreatment improved kidney function and tissue injury; reduced inflammation, oxidative stress and apoptosis
Hammad et al. 98	Rat	Unilateral Ischemia (40 min)	• Administration of Aliskiren one day prior and five days after ischemia improved kidney function and reduced expression of injury markers
Hammad et al. 97	Rat	Bilateral Ischemia (20 min)	• Administration of Aliskiren six days prior and two days after ischemia improved kidney function and tissue injury; reduced inflammation, apoptosis and fibrosis • Combined inhibition of Neprilysin increased effectiveness
Ziypak et al. 99	Rat	Unilateral Ischemia (45 min)	• Plasma Ang II increased • Aliskiren pretreatment reduced plasma Ang II, improved kidney function and tissue injury, reduced oxidative stress and inflammation
Efrati et al. 100	Rat	Unilateral Ischemia (60 min)	• Kidney Ang II increased • Plasma Ang II increased • Adminstration of Captopril after ischemia reduced kidney/plasma Ang II, improved kidney function and tissue injury
Habibey et al. 101	Rat	Unilateral Ischemia (30 min)	• Administration of Captorpil one hour prior to ischemia reduced tissue injury
Krishan et al. 102	Rat	Bilateral Ischemia (30 min)	• Administration of Captopril and Lisinopril (seperatly) improved kidney function and tissue injury
Kocak et al. 103	Rat	Bilateral Ischemia (60 min)	• Administration of Captopril and Telmisartan (seperatly) improved kidney function and reduced inflammation, oxidative stress and apoptosis
Altunoluk et al. 104	Rat	Unilateral Ischemia (60 min)	• Administration of Zofenopril for one day prior and one day after ischemia reduced tissue injury and oxidative stress

Other RAS alterations that have been described in experimental studies of IRI include increased cortical renin activity and protein levels 24 hours after reperfusion ^86,88. Moreover, increased renin secretion from the juxtaglomerular apparatus and the collecting duct was shown in rats and rabbits early after reperfusion ^89,90. Renin may drive Angiotensin II synthesis, particularly since ACE activity may be decreased in IRI ^86,87. Increased urinary levels of angiotensinogen and other angiotensin peptides like Angiotensin I and Angiotensin 1–7 have been reported in IRI as well ^86,93. Angiotensinogen can be produced locally in the proximal tubule and may be transcriptionally upregulated ^80,94,95. When the glomerular filtration barrier is altered, angiotensinogen may also be filtered from the circulation and activate the kidney RAS ^94,95.

While the mechanism of RAS activation in IRI is not fully understood, it has been demonstrated that downregulation of the RAS can ameliorate kidney dysfunction in experimental models of IRI ^{85,91,92,96–104}. Administration of Losartan, an antagonist of the AT₁ receptor, to rats at the time of reperfusion lowered serum creatinine 72 hours after ischemia ⁸⁵. When given prior to ischemia for three days, it exerted a renoprotective effect in rats 24 hours after ischemia ⁹¹. Pretreatment with Telmisartan, another antagonist of the AT₁ receptor, has also been shown to improve kidney function and injury as early as three hours after ischemia ⁹⁶. In addition, Rodriguez-Romo et al. reported that pretreatment with Losartan prevented the development of chronic kidney disease (CKD) following AKI in rats ⁹². Apart from interventions blocking the AT₁ receptor, inhibition of Renin also protected against IRI in experimental rodent models ^97–99. Ziypak et al. found elevated plasma Angiotensin II in rats 24 hours after ischemia. They demonstrated that two doses of Aliskiren, a direct inhibitor of Renin, administered before ischemia lowered plasma Angiotensin II and improved kidney function ⁹⁹. When given one day prior and up to five days after ischemia, kidney function in rats receiving Aliskiren was also improved ⁹⁸. Hammad et al. showed a protective effect of Aliskiren pretreatment for six days and continued up to two days after ischemia ⁹⁷. Interestingly, the authors demonstrated additional protection using combined Neprilysin and Renin inhibition. The increased efficiency was attributed to natriuretic peptides, normally degraded by Neprilysin ⁹⁷. Inhibition of ACE, the primary enzyme that generates Angiotensin II, has also been shown to be protective in experimental rodent models of IRI ^100–104. Efrati et al. found elevated kidney Angiotensin II in rats two days after ischemia and elevated plasma Angiotensin II up to seven days after ischemia ¹⁰⁰. Both were reduced by administration of the ACE inhibitor Captopril along with improved kidney function and reduced tissue injury and inflammation ¹⁰⁰. Other investigators also demonstrated protective effects of the ACE inhibitors Captopril, Lisinopril, and Zofenopril ^101–104. Administration of these drugs to rats improved kidney function and reduced tissue injury, inflammation, oxidative stress, and apoptosis two and 24 hours after ischemia ^101–104.

Taken together, data from experimental models show that activation of the RAS in kidney IRI occurs early on and may persist for the first days after injury. Angiotensin II, by activation of the AT₁ receptor, influences the severity of AKI and the AKI to CKD transition, as shown by the nephroprotective effect of pharmacologic blockade of Angiotensin II synthesis and receptor binding. This shows the important role of RAS overactivity in the pathogenesis of DGF and AKI and supports interventions to downregulate the RAS as a therapeutic target to improve transplant outcomes. In this regard, our group has found that in a mouse model of DGF, kidney Angiotensin II levels are increased ~2-fold 48 hours after kidney transplant surgery caused by cold ischemia ⁴⁸. In this model, DGF is produced by an extended period of cold ischemia of 3 hours. Importantly, we found that ACE2 in kidney proximal tubules was reduced at the protein and enzymatic activity level. This loss of ACE2 from the proximal tubular border can only potentiate the accumulation of Angiotensin II and its decremental effects. We postulate, therefore that administration of soluble recombinant ACE2 proteins that can pass the glomerular filtration barrier may have a protective effect to prevent/attenuate DGF.

The RAS in DGF: Clinical data

Castro Filho et al. utilized biopsies from 356 adult recipients of kidney transplants who were clinically suspected to have DGF and found that 42.1% of these cases exhibited indications aligning with AKI by ATN ¹⁰⁵. While the precise molecular mechanisms remain not fully elucidated, a growing body of evidence proposes an important role for an acute inflammatory response and activation of the RAS in the pathogenesis of ATN and AKI ^106,107. Angiotensin II upregulates the expression of pro-inflammatory and pro-fibrotic mediators, such as transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) which underscores the potential role of RAS activation in the pathophysiology of DGF ¹⁰⁸. Furthermore, urinary angiotensinogen has been reported as a biomarker of AKI indicative of RAS overactivity in the kidney ^94,95. This further suggests that an overactive RAS and the release of Angiotensin II may play a role in the pathogenesis and contribute to AKI and DGF. It’s important, however, to appreciate that DGF is a complex condition influenced by various factors, with the RAS representing only one facet of the pathophysiology. For the purpose of clarity, below we are reviewing what is known about the RAS in the different stages of the human transplantation process from the kidney donor, the removal of the graft, the ischemia time and subsequent reperfusion in the transplant recipient (Figure 2).

Figure 2: Overview of potential steps of RAS activation during deceased donor kidney transplantation.

The RAS in kidney donors:

In this section, we will review the activation of RAS in the context of the kidney donor. Kidneys from living donors, including related or non-related and with or without good human leukocyte antigen matching, demonstrate better survival rates than those from deceased donors and the lowest rates of DGF ^109–111. This observation may be attributed to lower rates of shock, ischemia, and agonal events adversely affecting the donor kidney before organ procurement ^110,111. An objective before and during organ procurement is to minimize the cellular damage induced by ischemia, including warm and cold ischemia, and optimize organ viability. In deceased donor kidney transplantation, recipients of kidneys donated after cardiac death have a two-fold risk of developing DGF compared to recipients receiving a kidney donated after brain death ^18,112,113. Cardiac death donors typically experience longer warm ischemia time than brain death donors. which could account for the increased risk of DGF ¹¹⁴. Warm ischemia time in kidneys donated after cardiac death begins during the preceding phase of cardiorespiratory collapse, when systolic arterial blood pressure and arterial oxygen saturation markedly drop and ends with the initiation of organ cold perfusion during procurement for transplantation ¹¹⁴. The pronounced hemodynamic alterations may result in early ischemic damage to the kidneys that may trigger activation of the RAS, aiming to maintain renal perfusion ^115–117. This suggests that the RAS may already be activated in cardiac death donors preceding organ procurement.

Clinical and experimental studies have moreover reported activation of the RAS in the context of brain death. While warm ischemia time is shorter in donation after brain death, brain death typically comes with generalized ischemia due to the hyperactivity of the sympathetic system ^118,119. Brain death constitutes a stressful state, triggers a systemic response, and creates a proinflammatory state even before the occurrence of brain death itself, causing notable disturbances in hemodynamics, hormones, and immunological homeostasis ^120–122. An interesting study by Lopau et al. showed an initial rise in plasma angiotensin II and plasma renin activity upon diagnosing brain death ¹²². Through a series of sequential measurements, it was observed that this increase returned to normal within a span of six hours ¹²². Amado et al. similarly identified a rise in plasma renin activity in samples obtained immediately following the confirmation of brain death ¹²³. Furthermore, an experimental investigation conducted on pigs also demonstrated a swift rise in plasma angiotensin II levels subsequent to the initiation of brain death ¹²⁴. Throughout the 4-hour monitoring period, these levels remained elevated. These observations suggest that in kidneys from brain-dead donors, the RAS may be activated before procurement as well, but the state of RAS activation may depend on the timing of organ procurement. In the context of brain death, RAS activation may be influenced by the hemodynamic collapse occurring before kidney retrieval, an activated sympathetic system, and treatment of patients with vasopressors ⁴⁶. All of these potentially result in increased levels of Angiotensin II by an augmented release or external administration of epinephrine and norepinephrine, leading to a temporary surge in renin secretion ¹²⁵.

In the context of living-related kidney donors, a recent study by Ohashi et al. found an increase in plasma and urinary angiotensinogen levels seven days after the kidney donation procedure compared to one day before the actual transplant surgery ¹²⁶. As these measurements were taken a week after the kidney donation, the data indicates that the surgical extraction of the kidney, even when performed under ideal circumstances, induces activation of the RAS that persists for at least a span of seven days. Furthermore, the elevation in urinary angiotensinogen suggests the activation of the intrarenal RAS since urinary angiotensinogen predominantly originates within the kidney rather than being derived from circulation ¹²⁷. Experimental data moreover supports that surgical manipulation during nephrectomy may trigger increased angiotensinogen levels, potentially leading to heightened Angiotensin II production ¹²⁸. Klett et al. found that the liver rapidly produces acute phase reactants after nephrectomy. In an experimental model, they observed a 2.6-fold rise in plasma angiotensinogen levels 12 hours after bilateral nephrectomy, linked to the inflammatory response ¹²⁸. This parallels the behavior of other acute-phase reactant proteins like c~-acid glycoprotein (AGP) and c~2-macroglobulin (AMG). During acute inflammation, IL-6, AGP, and AMG notably boosted angiotensinogen synthesis and secretion. However, the RAS status remains uncertain without direct measurement of other RAS peptides since angiotensinogen alone needs to be cleaved by renin or kallikrein lead to the formation of Angiotensin II (Figure 1).

That activation of the RAS in the donor’s kidney negatively impacts the transplant outcome, and the occurrence of DGF was shown in 1990 by Koller et al. ¹²⁹. The authors measured Renin and Angiotensin II levels in 22 consecutive organ donors 60 minutes before initiating the organ harvesting procedure. When correlating these results with the initial graft function observed after transplantation, the study showed that utilizing kidneys from donors with elevated Renin and Angiotensin II levels was associated with a 41% increase in the risk of DGF ¹²⁹.

Although we didn’t come across more recent studies with measurements of RAS components in donors, an interesting older study by Huland et al. reinforces the idea that heightened RAS activity negatively impacts transplant outcomes ¹³⁰. The authors administered the Angiotensin II receptor blocker Saralasin during the donor nephrectomy intravenously 10 min before clamping the aorta. Impressively, this intervention substantially decreased the postoperative occurrence of acute renal failure in the recipients from 58% to 25% in two groups of 24 patients each ¹³⁰.

The studies mentioned above indicate activation of the RAS in kidney donors and its negative impact on transplant outcomes and the occurrence of DGF. It is reasonable to conclude that a significant portion of deceased donor kidneys may exhibit an activated RAS, possibly due to cardiac or brain death processes and surgical manipulation like aortic clamping and temporary renal ischemia before and during the procurement. Modulating and downregulating this system could potentially offer protection against negative post-transplant outcomes, including DGF. An alternative perspective on RAS as a potential risk factor leading to DGF would be considering outcomes from donors who received RAS blockers. As far as our current knowledge extends, this specific information isn’t available. Nonetheless, this aspect will be discussed below in the context of recipients.

The RAS during organ procurement and preservation:

Organ preservation, whether through static cold storage or advanced methods like machine perfusion, aims to minimize reperfusion injury and optimize graft outcomes ¹³¹. Extended periods of preservation and ischemia cause vascular changes in organs to be transplanted, resulting in vasoconstriction, and the release of vasoactive agents that may negatively influence early graft function post-transplantation ^132,133. Angiotensin II is a potent vasoconstrictor and may substantially induce vasoconstriction during ischemia ^54,55,57. This way, downregulating an overactive RAS before and during organ procurement may pose a clinically relevant therapeutic target to improve kidney preservation for transplantation. Moreover, using additives in preservation solutions, such as prostaglandin, a potent renal vasodilator, could antagonize the vasoconstrictive effects during ischemia ¹³³. However, prostaglandin directly stimulates renin secretion, which may promote RAS overactivity ⁴⁶. Perfusion of kidneys with preservation solutions before static cold storage can also influence the activity of the RAS. Sulikowski et al. showed that perfusion of rat kidneys with University of Wisconsin (UW) and Euro Collins (EC) solution led to an increased gene expression of Angiotensinogen, Renin, and ACE as compared to kidneys perfused with saline solution ¹³⁴. After static cold storage for 24 hours in the respective solution, gene expression in kidneys perfused with UW solution was reduced to baseline levels while it stayed elevated in EC-perfused kidneys ¹³⁴. Even with the progress made in creating improved preservation solutions to reduce organ damage and DGF, the overall duration of cold ischemia remains the main factor influencing the occurrence of DGF since each additional hour of cold storage time heightens the risk of graft failure or post-transplant mortality ¹³⁵.

The RAS during kidney implantation:

Once the kidney is implanted and blood flow restored, ischemia-reperfusion injury unfolds when oxygen levels increase, and the pH normalizes ^37,136. The intensity of renal damage during the procurement and ischemia time is strongly linked with early failures of renal grafts ¹³⁷. The RAS plays a role in most forms of AKI. While blocking its principal effector, Angiotensin II is a cornerstone of therapy for progressive CKD, blockade of this system in AKI and DGF is potentially detrimental. Because of the potentially detrimental effects on blood pressure and renal hemodynamics, most clinicians avoid RAS blockers in the early transplantation period. However, RAS blockade may be potentially beneficial also in this setting ¹³⁸.

The transplant surgery itself is a major surgery during which RAS activation may occur. Efrati el at. Reported that intrarenal inflammation and RAS activation were independently initiated by the onset of anesthesia, regardless of the specific anesthetic agent used or the anesthesia protocol. They observed a notable increase in intrarenal Angiotensin II, TGF-β, IL-6, and IL-10 in rats. Levels of Cystatin C to assess kidney function were observed to be significantly elevated in the blood of anesthetized rats when compared to conscious controls and ‘stress control’ animals ¹³⁹. As highlighted earlier, Angiotensin II promotes the expression of proinflammatory and profibrotic agents and, as the primary effector peptide of the RAS, therefore may drive the deleterious role of RAS activation in the pathogenesis of DGF¹⁰⁸.

The RAS in the transplant recipient:

RAS activity in kidney transplant recipients, particularly in the early post-transplant phase, is not well-studied. Kidney transplant recipients may experience low blood pressure perioperatively, which increases the risk of AKI, contributing to the development of DGF ^140,141. This could explain the impact of the dialysis method used before transplantation on the occurrence of DGF ⁴⁶. Vanholder et al. reported a higher incidence of DGF in patients undergoing hemodialysis than peritoneal dialysis ¹⁴². Furthermore, hemodialysis with ultrafiltration within 24 hours before transplantation has been shown to increase the risk of post-transplantation AKI ¹⁴³. With or without hypotension arising from hemodialysis, hypovolemia can directly stimulate Renin secretion in response to low arterial blood pressure and sympathetic nervous system activity ¹⁴⁴. The newly transplanted kidney may be particularly susceptible to this hypoperfusion, given its prior exposure to ischemic damage during procurement and ischemia.

Additional insight was provided by Lorenz et al. by comparing the early graft function in 260 cadaveric kidney transplant recipients with or without peri-transplantation ACE inhibitor (ACEi) or Angiotensin receptor blocker (ARB) therapy ¹⁴⁵. Patients that received ACEi/ARB therapy before and after the kidney transplantation showed a quicker decline of serum creatinine levels. Among patients developing DGF in this study, those on ACEi/ARB therapy had faster graft function recovery as well. In a recent study moreover, Cockfield et al. identified significant findings that underscore the broader benefits of RAS inhibition on renal grafts, extending beyond their hemodynamic effects ¹⁴⁶. Interstitial fibrosis and tubular atrophy, with or without inflammation, may serve as adverse prognostic factors for renal graft survival ^147–150. Coadministration of ACEi/ARB therapy with low-dose tacrolimus, mitigated the progression of interstitial fibrosis and tubular atrophy with or without inflammation over a 24-month period compared to low‐dose tacrolimus combined with other antihypertensive therapy and high-dose tacrolimus combined with either ACEi/ARB or other antihypertensive therapy. The study also demonstrated that addition of ACEi/ARB therapy attenuated the risk of T-cell mediated rejection while using a low dose of Tacrolimus compared to patients receiving low-dose tacrolimus combined with other antihypertensive therapy. These observations support a role of Angiotensin II in regulating inflammation and fibrosis in the pathogenesis of kidney disease by upregulation pro-inflammatory and pro-fibrotic mediators, such as TGF-β and TNF-α ¹⁰⁸. Moreover, the beneficial effects of ACEi/ARB therapy combined with low-dose tacrolimus highlight possible overlapping pathways in which the two medications might interact, like direct inhibition of allograft specific T cells. Nataraj et al. demonstrated that autocrine signaling via AT₁ receptor activated the phosphatase calcineurin in murine T cells which led to the upregulation of genes associated with T cell proliferation and activation ¹⁵¹. Conversely, genetic deletion of the AT₁ receptor in knock-out mice enhanced the immunosuppressive effect of the calcineurin inhibitor cyclosporin A in an in-vivo heart transplantation model ¹⁵¹. A possible interaction of inhibitory signals from ACEi/ARB and tacrolimus therapy may imply that antagonizing the RAS could potentially enhance the immunosuppressive effects of calcineurin inhibition. If resulting in a decreased requirement for higher doses to achieve effective immunosuppression this approach may serve to mitigate dose-related toxicity.

Conclusion and Future Perspective:

Activation of the RAS in the context of kidney transplantation and DGF can occur at the different steps of the transplantation procedure. Donor intrinsic factors like immunologic or hemodynamic processes during brain or cardiac death may influence RAS activation already before organ procurement. The surgical procedure followed by organ preservation moreover influences the RAS due to the surgery itself, the duration of ischemia and use of preservation solutions. Once the kidney is implanted into the transplant recipient perioperative hypotension or hypovolemia may further activate the RAS. An activated RAS in combination with the ischemic damage and subsequent reperfusion injury may then adversely influence transplant outcomes as well as early graft function and contribute to the pathophysiology of DGF. Downregulation of the RAS has been shown to exert a protective effect in experimental animal models of IRI. Clinical studies have moreover suggested that the use of RAS blockers in the peri-transplantation setting is safe and may have potential benefits regarding early graft function. Despite these findings, RAS blockers are usually not started, and often discontinued in preparation for kidney transplants due to concerns about hypotension and adverse hemodynamic effects.

Further research is needed to fully understand the contribution of RAS overactivity to early graft function and the pathophysiology of DGF as well as the potential benefits and complications of pharmacologic RAS blockade in kidney transplantation. Modern techniques should be used to evaluate the many components of the rich RAS, its several peptides and enzymes (Fig 1) and identify new targets for possible therapeutic intervention targeted at reducing RAS overactivity. Given the potential role of RAS overactivity in the pathogenesis of DGF and our recent findings that kidney ACE2 is markedly reduced in mouse models of AKI and DGF (47,48), we surmise that as an alternative approach to RAS blockers, RAS downregulation may be achieved more safely with soluble forms of ACE2 that are short enough to pass the glomerular filtration barrier. Their potential kidney protective effect should be tested experimentally as a way to dissipate Angiotensin II and prevent its accumulation within the kidney.

Abbreviations:

DGF

Delayed Graft Function

RAS

Renin-Angiotensin System

ACE2

Angiotensin Converting Enzyme 2

Ang II

Angiotensin II