Cold Ischemia Time in Kidney Transplantation: Pathophysiology, Clinical Impact, and Emerging Preservation Strategies – A Narrative Review

ABSTRACT

Chronic kidney disease remains a major global health burden, and kidney transplantation is the preferred treatment for patients with end-stage disease. Among the modifiable factors influencing graft outcomes, cold ischemia time (CIT) plays a central role in determining early allograft performance. Prolonged CIT exacerbates ischemia–reperfusion injury through metabolic depletion, oxidative stress, endothelial dysfunction, and innate immune activation, thereby increasing the risk of delayed graft function and subsequent graft impairment. This narrative review critically synthesizes current evidence on the pathophysiological mechanisms linking CIT to kidney graft dysfunction, as well as contemporary preservation strategies and emerging biomarkers of early injury. The literature consistently demonstrates a time-dependent association between prolonged CIT and adverse post-transplant outcomes, although effect magnitude varies according to donor profile and preservation method. In the context of expanding donor criteria and increasingly complex transplant logistics, optimization of CIT remains a key, potentially modifiable target to improve graft performance and resource utilization. Future studies integrating advanced preservation technologies and sensitive biomarkers are warranted to refine risk stratification and guide personalized graft management.

Descriptors
Kidney Transplantation; Cold Ischemia; Delayed Graft Function; Reperfusion Injury

RESUMO

A doença renal crônica permanece como um importante desafio global de saúde, e o transplante renal é o tratamento de escolha para pacientes com doença em estágio terminal. Entre os fatores modificáveis que influenciam os desfechos do enxerto, o tempo de isquemia a frio (TIF) desempenha um papel central na determinação do desempenho precoce do aloenxerto. Um TIF prolongado exacerba a lesão de isquemia-reperfusão por meio da depleção metabólica, estresse oxidativo, disfunção endotelial e ativação da imunidade inata, aumentando assim o risco de função tardia do enxerto e subsequente comprometimento do enxerto. Esta revisão narrativa sintetiza criticamente as evidências atuais sobre os mecanismos fisiopatológicos que ligam o TIF à disfunção do enxerto renal, bem como as estratégias contemporâneas de preservação e os biomarcadores emergentes de lesão precoce. A literatura demonstra consistentemente uma associação dependente do tempo entre o TIF prolongado e desfechos adversos pós-transplante, embora a magnitude do efeito varie de acordo com o perfil do doador e o método de preservação. No contexto da expansão dos critérios de doação e da logística de transplante cada vez mais complexa, a otimização do TIF permanece como um alvo fundamental e potencialmente modificável para melhorar o desempenho do enxerto e a utilização de recursos. Estudos futuros que integrem tecnologias avançadas de preservação e biomarcadores sensíveis são necessários para refinar a estratificação de risco e orientar o manejo personalizado do enxerto.

Descritores
Transplante Renal; Isquemia a Frio; Função Tardia do Enxerto; Lesão de Reperfusão

INTRODUCTION

Chronic kidney disease (CKD) is a complex clinical condition defined by persistent structural or functional kidney abnormalities lasting for more than three months, commonly characterized by a glomerular filtration rate below 60 mL/min/1.73 m².¹,² Epidemiological data indicate that CKD is associated with high prevalence, morbidity, and mortality worldwide.³ Kidney transplantation is considered the gold-standard therapy for patients with end-stage renal disease, providing superior survival and quality of life compared with dialysis.⁴ Nevertheless, successful transplantation depends on meticulous perioperative management, particularly adequate organ procurement, preservation, and transport.⁵

In the logistical framework of kidney transplantation, ischemia and reperfusion are critical processes that directly influence graft outcomes. Ischemia refers to the interruption of blood supply to tissues, resulting in reduced oxygen and nutrient delivery, whereas reperfusion denotes the restoration of blood flow. Although reperfusion is essential for graft viability, it paradoxically triggers ischemia–reperfusion injury, a complex pathophysiological process involving oxidative stress, inflammatory activation, cellular death, and progressive fibrogenesis.⁶ Ischemic injury in transplantation is classically divided into warm and cold phases. Warm ischemia occurs in the donor from the interruption of renal perfusion until initiation of cold preservation, representing a particularly vulnerable period for graft injury. Cold ischemia time (CIT) is defined as the interval between the initiation of cold perfusion in the donor and reperfusion in the recipient. Precise control of both ischemic intervals is crucial, as prolonged ischemia is consistently associated with impaired graft recovery. Extended CIT has emerged as a major modifiable risk factor for early and late graft dysfunction through mechanisms that include hypoxia, metabolite accumulation, adenosine triphosphate (ATP) depletion, microvascular impairment, and exacerbation of ischemia–reperfusion injury.⁷,⁸

Prolonged CIT is strongly associated with the development of delayed graft function (DGF), largely mediated by pathophysiological cascades culminating in acute tubular injury. Ischemia–reperfusion promotes intracellular acidosis, cellular edema, electrolyte imbalance, generation of reactive oxygen species (ROS), and release of damage-associated molecular patterns. These events activate the complement system and recruit inflammatory cells, amplifying tissue injury and microvascular dysfunction. Given these challenges, post-transplant management increasingly relies on sensitive and specific biomarkers capable of capturing early immunological and metabolic disturbances, thereby supporting risk stratification, therapeutic decision-making, and prevention of both acute rejection and chronic interstitial fibrosis.

In this context, the present review aimed to critically evaluate the impact of CIT on kidney graft dysfunction, with emphasis on underlying pathophysiological mechanisms, damage-mitigation strategies, and emerging biomarker perspectives.

METHODOLOGY

This narrative review was conducted following principles of methodological transparency recommended by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, adapted to the narrative review design. A structured literature search was performed in the PubMed and ScienceDirect databases in October 2025.

The search strategy combined the following descriptors: “kidney transplantation,” “cold ischemia,” and “organ preservation,” using the Boolean operator AND. Studies published between January 2015 and October 2025 were considered, without language restrictions. Eligible studies included original clinical investigations evaluating the relationship between CIT and kidney graft outcomes in the context of renal transplantation. Articles were excluded if full text was unavailable, if they were not related to kidney transplantation, or if they consisted exclusively of experimental animal studies without clinical correlation.

Study selection was performed through title and abstract screening followed by full-text assessment for eligibility. Emphasis was placed on observational studies, which constituted most of the available evidence in this field, including retrospective and prospective cohort designs. The relative scarcity of randomized controlled trials in this domain likely reflects important ethical and logistical challenges inherent to randomizing preservation strategies in deceased-donor transplantation settings, in which established preservation methods are considered standard of care.

Data from the included studies were qualitatively synthesized with focus on the association between CIT and DGF, as well as on mechanistic insights related to ischemia–reperfusion injury and graft outcomes. This review has inherent limitations that should be acknowledged. First, the predominance of observational studies increases susceptibility to residual confounding. Second, heterogeneity exists in the operational definitions of DGF across studies, commonly defined either by dialysis requirement in the first seven days post-transplant or by early creatinine kinetics. Third, variability in CIT cut-off thresholds (typically ranging from 12 to 24 hours) and differences in preservation protocols across transplant centers may affect comparability. Finally, disparities among healthcare systems and organ allocation logistics may limit the external generalizability of the findings.

PATHOPHYSIOLOGY OF ISCHEMIA AND RENAL GRAFT REPERFUSION

During CIT, hypothermic preservation markedly reduces, but does not completely abolish, cellular metabolism in the renal allograft. Tubular epithelial cells, particularly those of the proximal tubule, remain metabolically vulnerable under hypoxic conditions, leading to progressive depletion of ATP, ionic imbalance, and intracellular acidosis.⁹ Mitochondrial oxidative phosphorylation is markedly impaired during ischemia due to oxygen deprivation, promoting electron leakage from the respiratory chain and low-level generation of ROS. Concurrently, activation of enzymatic sources such as NADPH oxidase further contributes to the oxidative burden in the ischemic tissue.¹⁰

A central and increasingly recognized event in ischemic injury is the accumulation of metabolic intermediates, most notably succinate, within mitochondria during oxygen deprivation. Upon reperfusion, the abrupt reintroduction of oxygen drives rapid oxidation of accumulated succinate through complex II, triggering reverse electron transport at complex I and resulting in a burst of mitochondrial ROS production.¹¹,¹² This oxidative surge represents a pivotal early event in ischemia–reperfusion injury and amplifies downstream inflammatory and microvascular dysfunction.

The reperfusion phase is characterized by sudden reoxygenation and restoration of physiological temperature, which together exacerbate oxidative stress and bioenergetic failure. Excessive ROS promote lipid peroxidation, protein oxidation, and DNA damage, while mitochondrial permeability transition pore (mPTP) opening leads to collapse of the mitochondrial membrane potential (ΔΨ_m), further ATP depletion, and initiation of regulated cell death pathways, including apoptosis and necrosis.^11,12 These events predominantly affect tubular epithelial cells and contribute to the structural substrate of DGF.

Endothelial dysfunction represents another key component of renal ischemia–reperfusion injury. Ischemic insult and oxidative stress disrupt the endothelial cytoskeleton and degrade the endothelial glycocalyx, compromising barrier integrity and microvascular homeostasis. The resulting increase in vascular permeability, interstitial edema, and peritubular capillary congestion contributes to the so-called “no-reflow” phenomenon, perpetuating regional hypoxia even after macroscopic reperfusion.⁶ Impaired endothelial repair capacity further sustains microcirculatory dysfunction and promotes fibrogenic signaling.

The inflammatory response is rapidly activated following endothelial injury. Upregulation of adhesion molecules, including P-selectin and intercellular adhesion molecule-1 (ICAM-1), facilitates leukocyte rolling, adhesion, and transmigration into the renal interstitium. Infiltrating neutrophils and mononuclear cells amplify tissue injury through additional ROS generation, protease release, and cytokine production, establishing a self-propagating inflammatory loop.¹³,¹⁴

At the molecular level, ischemic and necrotic cells release damage-associated molecular patterns, such as high-mobility group box 1 (HMGB1), heat shock proteins, hyaluronic acid fragments, and fibronectin. These endogenous danger signals are recognized primarily by Toll-like receptors (TLR2 and TLR4) expressed on tubular epithelial cells and antigen-presenting cells, including dendritic cells and macrophages.¹⁵-¹⁷ Engagement of these receptors activates intracellular signaling pathways involving nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs), culminating in robust production of pro-inflammatory cytokines (e.g., interleukin-1, interleukin-6, tumor necrosis factor-α) and chemokines that further recruit immune effector cells.¹⁷,¹⁸

Collectively, the interplay between mitochondrial dysfunction, oxidative stress, endothelial injury, and innate immune activation establishes the pathophysiological basis of DGF and contributes to adverse long-term graft outcomes. Clinically, post-transplant graft function is commonly categorized as immediate graft function, DGF, or primary nonfunction, with acute tubular injury secondary to ischemia–reperfusion representing the principal substrate for DGF.¹⁴ Therefore, strategies aimed at minimizing CIT and mitochondrial and microvascular injury remain central targets for improving renal allograft performance.

COLD ISCHEMIA TIME

CIT is conventionally defined as the interval between the initiation of cold preservation following vascular clamping and the restoration of graft perfusion in the recipient. Acceptable ischemic thresholds vary by organ type, typically remaining below 6 hours for heart and lung grafts and ideally within 24 hours for kidneys, although preservation up to approximately 36 hours has been reported in selected circumstances.¹⁹

Extensive evidence indicates that prolonged CIT is associated with an increased risk of DGF, acute tubular injury, and inferior early graft performance. However, the relationship between CIT and post-transplant outcomes is not strictly linear and is substantially modified by donor characteristics, preservation strategies, and recipient factors. Kidneys from living donors typically exhibit superior outcomes compared with those from deceased donors, partly due to markedly shorter CIT. Nevertheless, attributing improved graft survival solely to reduced CIT would be overly simplistic, as living donation is also associated with more favorable donor health status, absence of brain death–related inflammatory injury, shorter warm ischemia time, and frequently better immunologic matching.²⁰

Despite the well-established biological plausibility linking prolonged hypothermic storage to ischemia–reperfusion injury, some observational studies have reported attenuated or inconsistent long-term effects of CIT. For example, a retrospective single-center study including 117 recipients found no significant association between CIT exceeding 36 hours and one-year graft outcomes when serum creatinine was used as the primary marker.²¹ However, the interpretability of these findings is limited by the narrow CIT range of the comparator group (30–35.9 hours), the modest sample size, and the recognized insensitivity of serum creatinine to detect early or subclinical graft dysfunction. These limitations substantially weaken the ability to exclude clinically meaningful effects.

Conversely, another study evaluating 113 recipients of deceased-donor kidneys across CIT intervals ranging from 13 to 37 hours demonstrated a positive correlation between longer CIT and higher serum creatinine at six months post-transplant, alongside reduced graft survival in the shorter CIT strata.²⁰ Notably, this association lost statistical significance at one year, suggesting that the deleterious impact of prolonged CIT may be more evident in the early post-transplant period, potentially mediated through DGF and early tubular injury, while longer-term outcomes may become increasingly influenced by immunologic and recipient-related factors.²¹ These findings underscore the importance of longitudinal analyses incorporating more sensitive functional and structural biomarkers.

Large registry data further reinforce the clinical and economic relevance of CIT. An analysis of 81,945 kidney transplant recipients demonstrated that longer CIT was independently associated with a higher risk of DGF (odds ratio—OR = 1.41; 95% confidence interval – 95% CI 1.38–1.44), prolonged length of hospital stay (OR = 1.04; 95% CI 1.02–1.05), and increased transplant-related costs.²² These observations highlight that the impact of CIT extends beyond immediate graft biology, influencing healthcare resource utilization and system-level efficiency.

Beyond its established association with delayed graft function, accumulating evidence suggests that prolonged CIT may adversely affect several clinically relevant outcomes.²³ Large registry-based analyses have demonstrated associations between increasing CIT and inferior death-censored graft survival, higher rates of primary non-function, and impaired renal function recovery. Furthermore, prolonged ischemic exposure has been associated with greater healthcare utilization, including longer hospitalization and increased transplant-related costs. Evidence regarding acute rejection remains heterogeneous, but large cohort studies have reported an increased risk of acute rejection among recipients exposed to prolonged CIT, supporting the biological plausibility that ischemia–reperfusion injury may enhance alloimmune activation through endothelial dysfunction and innate immune signaling.²⁴ In contrast, the independent impact of CIT on patient survival remains less established, as donor quality and recipient-related factors appear to exert a stronger influence on mortality than ischemic exposure by itself.²⁵ Collectively, these findings support the concept that CIT should be regarded not only as a determinant of DGF but also as a broader prognostic marker of kidney transplant outcomes.

Taken together, current evidence supports CIT as a critical but context-dependent determinant of kidney transplant outcomes. Although preservation times approaching 36 hours may be acceptable under selected conditions, risk appears to increase progressively with longer ischemic exposure, particularly among higher-risk grafts. Future studies should prioritize refined risk stratification, incorporation of sensitive biomarkers of graft injury, and evaluation of mitigation strategies—such as machine perfusion—to better define safe ischemic thresholds in contemporary transplantation practice.

ASSOCIATION BETWEEN COLD ISCHEMIA TIME AND DELAYED GRAFT FUNCTION

Assessment of kidney graft viability and early performance commonly relies on urine output, serum creatinine kinetics, and the presence or absence of metabolic instability.¹⁴ DGF is classically defined as the need for dialysis in the first seven days after transplantation and remains one of the most frequent early complications following kidney transplantation.²⁰

Despite advances in donor management, preservation techniques, and recipient care, the incidence of DGF has remained relatively stable over time. Reported rates range from 20–50% in deceased-donor transplantation and 4–10% in living-donor procedures. Importantly, untreated or severe DGF is associated with increased risk of graft loss and reduced patient survival. Beyond the conventional dialysis-based definition, alternative criteria have been proposed to capture milder phenotypes or severity gradients, including failure of serum creatinine to decline by > 10% in the first three days, serum creatinine > 7 mg/dL on postoperative day 7, and prolonged time to achieve creatinine clearance > 10 mL/min.²⁰ However, heterogeneity in definitions continues to limit cross-study comparability.

Among the modifiable perioperative variables, CIT has consistently emerged as a major risk factor for DGF. A prior meta-analysis including 59,089 kidney transplant recipients reported an overall DGF incidence of 28.13%, identifying prolonged CIT as the principal contributor to this outcome.²⁶ Although short-term graft survival has improved in recent decades, graft dysfunction, particularly among deceased-donor recipients, remains common, with DGF affecting up to half of these patients in some cohorts.²⁷

Mechanistically, prolonged CIT amplifies ischemia–reperfusion injury by intensifying mitochondrial dysfunction, ATP depletion, and oxidative stress, ultimately promoting acute tubular injury. Experimental and clinical evidence indicates that extended hypothermic storage increases the burst of ROS and pro-inflammatory cytokines upon reperfusion, thereby exacerbating renal injury and delaying functional recovery.²⁸ Consistent with this biological rationale, observational studies have suggested clinically relevant CIT thresholds. For instance, CIT exceeding 14.3 hours has been reported as a predictor of DGF, while a large study of deceased-donor recipients (n = 7,542) demonstrated that CIT > 14 hours significantly increased the risk of both DGF and graft loss, particularly in kidneys from older donors and those obtained after circulatory death.²⁹

Nevertheless, the strength of the association between CIT and DGF should be interpreted according to the broader donor–recipient context. The effect of CIT is strongly modified by donor quality, procurement logistics, preservation modality, and recipient comorbidity burden. Kidneys from expanded-criteria donors and donation after circulatory death donors appear especially vulnerable to prolonged ischemic exposure, reinforcing the concept that CIT acts synergistically with baseline graft risk rather than as an isolated determinant.

In the Brazilian context, emerging observational data provide additional nuance. A retrospective analysis of medical records from 2007 to 2020 demonstrated a significant positive correlation between CIT and serum creatinine at six months post-transplant, but this association was no longer significant at 12 months, suggesting that shorter CIT predominantly influences early graft performance. Moreover, a national study comparing expanded-criteria donors (ECD) and standard-criteria donors (SCD) during the first post-transplant year reported significantly lower estimated glomerular filtration rate and reduced graft survival among ECD recipients, findings partly attributed to the low utilization of pre- and post-transplant frozen-section biopsies in this group. Furthermore, a Brazilian cohort evaluating determinants of transplant success identified a higher prevalence of infectious complications among recipients of deceased-donor grafts, which were also characterized by longer CIT. Collectively, these data suggest that prolonged preservation time may contribute not only to ischemia–reperfusion injury but also to increased susceptibility to post-transplant complications that adversely affect graft performance.

Taken together, the current body of evidence supports prolonged CIT as a robust and biologically plausible risk factor for DGF, particularly in higher-risk donor profiles. However, its impact is context-dependent and modulated by donor characteristics, preservation practices, and perioperative management. Future investigations should prioritize standardized DGF definitions, integration of mechanistic biomarkers, and stratified analyses according to donor risk and preservation technology to define clinically actionable CIT thresholds in contemporary kidney transplantation.

COLD ISCHEMIA TIME VERSUS EXPANDED-CRITERIA DONORS

The persistent mismatch between organ supply and demand remains a major challenge in kidney transplantation. One widely adopted strategy to mitigate waiting list mortality has been the expansion of donor acceptance criteria, including the use of organs from older deceased donors and those with relevant comorbidities. Although this approach has increased transplant rates, concerns remain regarding the durability and functional performance of these grafts and the factors that may modify their prognosis.³⁰,³¹

ECD are traditionally defined as donors aged ≥ 60 years old, or those aged 50–59 who present at least two or more risk factors, commonly including death due to cerebrovascular accident, history of systemic hypertension, and pre-retrieval serum creatinine > 1.5 mg/dL. The incorporation of ECD kidneys into allocation systems has substantially increased transplant activity and has been associated with a three–nine-year survival advantage for recipients compared with remaining on dialysis.³⁰ Nevertheless, ECD grafts exhibit greater biological vulnerability to ischemia–reperfusion injury, raising concern regarding their tolerance to prolonged CIT.

Brazilian data provide important real-world insight into this interaction. In a cohort of 255 kidney transplant recipients, 90.6% (n = 231) received SCD kidneys and 9.4% (n = 24) received ECD grafts. Mean CIT was comparable between groups (21.17 ± 3.88 hours in ECD versus 21.82 ± 4.97 hours in SCD), and no statistically significant differences were observed in DGF incidence, number of early post-transplant hemodialysis sessions, donor terminal creatinine, acute rejection, or immunosuppressive therapy. Notably, the overall mean CIT in this Brazilian cohort (23.2 hours) exceeded typical values reported in North American (14.2 hours) and European (18 hours) series, highlighting potential logistical challenges in the national transplant system and underscoring the need for context-specific analyses.

Despite its historical utility, the binary SCD/ECD classification has important limitations, primarily due to its coarse risk stratification and inability to capture the continuous spectrum of donor quality. To address these shortcomings, the kidney donor profile index (KDPI) was developed as a more granular predictive tool. The KDPI integrates multiple donor demographic and clinical variables into a continuous score ranging from 0 (the highest quality) to 100% (the lowest quality), enabling more refined risk assessment and allocation decisions.³¹

Large contemporary analyses have further clarified the interaction between donor quality and CIT. In a study of 69,490 kidney transplant recipients in the United States of America (2008–2014), both KDPI and CIT independently influenced post-transplant renal function and estimated glomerular filtration rate. Interestingly, the adverse impact of prolonged CIT appeared more pronounced in grafts with low-to-intermediate KDPI, suggesting that higher-quality kidneys may lose part of their functional advantage when exposed to extended preservation times.³² Conversely, very high-KDPI kidneys may already possess limited functional reserve, potentially attenuating the relative incremental effect of CIT.

Collectively, current evidence indicates that CIT should not be interpreted in isolation but rather in a multidimensional donor risk framework. While expanded donor utilization remains essential to address organ shortage, grafts from higher-risk donors, particularly ECD or high-KDPI kidneys, may require stricter ischemic time management and optimized preservation strategies. Future studies should prioritize integrated risk models combining CIT, KDPI, and preservation modality to better individualize allocation decisions and improve graft outcomes in contemporary kidney transplantation.

STRATEGIES FOR MINIMIZING THE EFFECTS OF COLD ISCHEMIA

Static cold storage is the method used to preserve grafts in solid organ transplantation, with the aim of reducing ischemia–reperfusion injury caused by the transplantation process. It is understood that cold storage reduces ischemia/reperfusion injury by decreasing cellular activity and the production of toxic metabolites prior to transplantation, thereby preserving graft quality and reducing long-term complications. However, prolonged cold storage, known as cold ischemia, may be associated with a poorer prognosis for the renal graft.³³

In this regard, a study conducted in Brazil involving 106 kidney transplant recipients concluded that a CIT exceeding 20 hours is a risk factor for the development of DGF; it is therefore necessary to develop strategies to reduce this time.²⁴ Furthermore, a Spanish study aimed to characterize the relationship between CIT and graft survival in renal patients, as well as the main associated events. Based on an analysis of 378 kidney transplant recipients, it was found that for every additional hour during the cold ischemia period, the risk of DGF increased by 10% and was associated with a higher incidence of rejection.³⁴

Preservation solutions may be able to reduce cellular swelling, acidosis and imbalances in ionic homeostasis.³⁵ University of Wisconsin and histidine-tryptophan-ketoglutarate (HTK) solutions are the most commonly used in kidney transplants, with University of Wisconsin having been considered the standard for the preservation of abdominal organs since 1987.³⁵ Experimental studies suggest that the superior cooling properties of University of Wisconsin may explain the reduced damage to renal allografts when compared with HTK. Furthermore, the preservation of renal allografts in University of Wisconsin solution has shown superior results, particularly when CIT is prolonged.³⁵

Recently, with the aim of limiting the negative impact of cold ischemia, a new preservation technique known as machine hypothermic perfusion has been introduced.³⁶ This technique keeps renal grafts in a preservation solution at 4 °C prior to kidney transplantation, but it creates a flow through the kidney generated by a peristaltic pump in a closed circuit, allowing recirculation through the renal vasculature. Continuous perfusion allows for a better supply of oxygen and nutrients, and the elimination of toxic metabolites. Despite the various advantages, the necessary equipment is more complex and expensive and requires continuous monitoring and assessment of the graft during the preservation period.³⁶,³⁷

New studies are exploring alternatives for the preservation of renal grafts. Machine-mediated normothermic perfusion (MNP) of the renal graft at 35–37 °C has been the subject of studies due to its potential benefits.³⁸ Authors observe improved renal graft function following MNP, when compared to grafts subjected to static cold storage. It is clear that NMP is an innovative technology, with clear importance as a complement to the renal graft preservation process, as it provides a protective environment, ensuring optimal oxygen supply and metabolic support to the graft.³⁹ Conversely, this technology requires a specific setup and refined technical expertise, and may not be widely adopted worldwide.³⁷

Considering the problems associated with PNM and cold ischemia, a new option has emerged that combines the two forms of preservation, known as subnormothermic machine perfusion (PSM). Experimentally tested at temperature ranges of 20–22 and 8–10 °C, PSM is a modified form of mechanical hypothermic perfusion with the potential to positively impact the final outcome of the transplant.³⁷ Experiments in animal models have shown improved structural integrity of the renal graft, as well as a significant improvement in renal functional parameters.⁴⁰ As with PNM, the complex setup and associated costs remain barriers to the worldwide adoption of this preservation method. The ideal preservation temperature is essential to minimize transplant-induced ischemia–reperfusion injury. However, in addition to this, pharmacological innovations are emerging with the aim of complementing existing preservation methods, particularly hypothermic and subnormothermic preservation.³⁷

All preservation processes are based on the use of preservation solutions to control factors such as osmolarity, pH, metabolic support and the maintenance of cell volume. Their composition is varied, containing electrolytes, buffering systems, polymers such as polyethylene glycol, and nutrients and antioxidants such as glucose, glutathione, and adenosine, which provide fuel and protection. Among the most studied components, there are amino acids, molecules that act as metabolic substrates in cells. Due to their antioxidant properties, amino acids can reduce renal damage caused by transplant-induced ischemia–reperfusion injury.⁴¹

A study was conducted on kidneys obtained from mongrel dogs that had undergone a 72-hour ischemic injury. The animals were divided into two groups: the first one received grafts stored in a conventional preservation solution, whilst the second group received grafts treated with a solution supplemented with L-arginine, an essential amino acid. Considering this, it was observed that L-arginine, a substrate for nitric oxide synthesis, has a beneficial and protective effect on long-term hypothermic ischemic damage.⁴² Although there are promising findings, further research is needed to ensure a safe and reproducible process for humans.

Another additive used in renal preservation solutions is hemoglobin-based oxygen carriers, which replace donor blood in graft preservation. Known as Hemopure, this is a bovine hemoglobin that transports oxygen in a similar way to human hemoglobin.⁴³ Recent studies have explored the feasibility of using Hemopure in human normothermic renal perfusion, a preservation model that requires an adequate oxygen supply. An experiment involving 14 discarded human kidneys was perfused for 6 hours at 37 °C using a pressure-controlled system; seven kidneys were perfused with Hemopure and the remainder with red blood cell concentrate, with the aim of increasing oxygen-carrying capacity. During this process, renal resistance, oxygen extraction, metabolic activity, energy reserves, and histological characteristics were assessed. This experiment demonstrated the feasibility of using the Hemopure solution as a logistically more convenient and ready-to-use alternative to PNM.⁴⁴

Alternatively, gas signaling molecules produced in mammalian tissues, such as nitric oxide and carbon monoxide, have been used to mitigate prolonged kidney failure. These molecules are involved in physiological processes crucial for the maintenance of tissue homeostasis, participating in cell signaling and vasodilation.⁴⁵,⁴⁶ Previous studies evaluated perfused kidneys under warm preservation conditions following 10 minutes of ischemia and 16 hours of refrigerated storage across four groups, assessing renal function and viability. The nitric oxide donor, sodium nitroprusside, and the carbon monoxide-releasing molecule increased renal blood flow during warm preservation. Furthermore, following reperfusion, renal blood flow improved significantly in the group perfused with carbon monoxide, demonstrating beneficial vasodilatory effects during warm preservation, thereby improving renal function during reperfusion; nitric oxide exerted similar effects, albeit less pronounced.⁴⁷

FUTURE OUTLOOK AND NEW TECHNOLOGIES

The pursuit of earlier and more accurate detection of acute kidney injury (AKI) in the transplant setting has intensified in recent years, driven by the recognized limitations of conventional markers such as serum creatinine and urine output. Contemporary research has focused on the identification of sensitive and specific molecular biomarkers measurable in urine or plasma that may enable earlier detection of graft injury, improved risk stratification, and more timely therapeutic intervention. Among the most extensively investigated candidates, there are neutrophil gelatinase–associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), interleukin-18 (IL-18), liver-type fatty acid–binding protein (L-FABP), tissue inhibitor of metalloproteinases-2 (TIMP-2), insulin-like growth factor binding protein-7 (IGFBP7), and calprotectin.⁴⁸

Neutrophil Gelatinase–Associated Lipocalin

NGAL is a small protein that exists in humans as monomeric, homodimeric, and heterodimeric forms conjugated to gelatinase and predominantly associated with neutrophils. Renal tubular epithelial cells rapidly secrete the monomeric form following injury.⁴⁹ Although constitutively expressed at low levels in multiple tissues, NGAL is markedly upregulated at both mRNA and protein levels after ischemic renal injury.⁵⁰ Its concentration rises within approximately 3 hours of insult and typically peaks between 6 and 12 hours, making it one of the earliest detectable biomarkers of AKI. In severe injury, elevated levels may persist for up to five days.⁵⁰ NGAL remains the most extensively studied AKI biomarker across clinical scenarios, but interpretation may be influenced by demographic and inflammatory variables, including age and sex.⁵¹

Kidney Injury Molecule-1

KIM-1 is a transmembrane glycoprotein minimally expressed in the normal kidney but strongly upregulated following tubular injury.⁴⁸ Its expression in proximal tubular cells is associated with epithelial repair and regeneration, particularly through the phagocytosis of apoptotic bodies and cellular debris.⁵² These properties support its utility as a biomarker of nephrotoxicity in both preclinical and early-phase clinical studies. Notably, experimental models have demonstrated that sustained KIM-1 expression may contribute to progressive interstitial inflammation and fibrosis in murine kidneys.⁵³ As with NGAL, demographic factors such as age and sex may influence measured levels, representing an important limitation for clinical interpretation.⁵¹

Liver-Type Fatty Acid–Binding Protein

L-FABP is a lipid-binding protein involved in intracellular fatty acid trafficking and lipid metabolism.⁵⁴ It is expressed in the liver, intestine, stomach, and kidney, and it facilitates transport of fatty acids to mitochondria and peroxisomes for β-oxidation and energy production in tubular cells.⁵⁵,⁵⁶ In the kidney, L-FABP is localized primarily to proximal tubular cells and is excreted into the tubular lumen bound to toxic peroxisomal byproducts. Elevated urinary L-FABP has been associated with increased susceptibility to renal stress and may serve as an early indicator of tubular injury in the transplant context.⁴⁸

Interleukin-18

IL-18 is a pro-inflammatory cytokine belonging to the interleukin-1 superfamily. It is synthesized as an inactive precursor in monocytes and macrophages and requires cleavage by caspase-1 for activation and secretion.⁴⁸ In addition to immune cells, IL-18 is produced by collecting duct cells in the healthy kidney and by injured tubular epithelial cells.⁵⁷,⁵⁸ Experimental studies in wild-type mice have demonstrated significant elevation of IL-18 levels during AKI,⁵⁹ supporting its role as both an early biomarker and a potential therapeutic target in ischemic renal injury.

Insulin-Like Growth Factor Binding Protein-7 and Tissue Inhibitor of Metalloproteinases-2

IGFBP7 and TIMP-2 belong to the class of cell-cycle arrest biomarkers and act predominantly during the G1 phase, allowing cells to avoid replication under conditions of potential DNA damage.⁶⁰ Injured renal epithelial cells upregulate these proteins, thereby halting cell-cycle progression as a protective response,⁶¹ In a multicenter observational study involving patients at risk for AKI, the combined [TIMP-2]•[IGFBP7] signal outperformed urinary KIM-1, serum NGAL, and urinary IL-18 in risk prediction, providing incremental clinical information.⁶² Despite their promise, these biomarkers remain relatively novel, and further studies are required to clarify their pathophysiological roles and optimal clinical thresholds.⁴⁸

Calprotectin

Calprotectin is a heterodimer composed of S100A8 and S100A9 subunits. Intracellularly, it interacts with cytoskeletal components to modulate cellular organization, whereas extracellularly, when released by activated immune cells, it functions as a damage-associated molecular pattern capable of promoting monocyte differentiation into macrophages.⁶³,⁶⁴ Calprotectin expression has been identified in collecting duct epithelial cells in experimental models of renal injury, supporting its involvement in AKI pathophysiology.⁶⁵ Clinical studies using immunoassays have suggested that urinary calprotectin may help differentiate prerenal from intrinsic graft injury, positioning it as a promising adjunct biomarker in the transplant setting.⁶⁶

CRITICAL OUTLOOK AND TRANSLATIONAL CHALLENGES

Despite substantial progress, the clinical integration of novel AKI biomarkers in kidney transplantation remains incomplete. Key barriers include inter-assay variability, lack of universally accepted cutoff values, influence of systemic inflammation, and limited validation in large, multicenter transplant cohorts. Importantly, most biomarkers reflect tubular stress rather than irreversible injury, which complicates their interpretation in the immediate post-transplant period.

Future directions should emphasize multimarker panels, integration with machine-perfusion viability metrics, and incorporation into predictive algorithms that combine donor, preservation, and recipient variables. From a Brazilian and broader middle-income country perspective, cost-effectiveness, assay availability, and laboratory standardization will be decisive factors for real-world implementation. Prospective trials specifically designed in transplant populations are urgently needed to determine whether biomarker-guided management can meaningfully reduce DGF and improve long-term graft survival.

CONCLUSION

Taken together, the available evidence supports a consistent association between prolonged CIT and both early and late renal allograft dysfunction, largely mediated by exacerbation of ischemia–reperfusion injury and oxidative stress driven by ROS. However, the strength of this inference remains limited by the predominantly observational and narrative nature of the existing literature, which is inherently subject to heterogeneity, residual confounding, and limited causal resolution. Nonetheless, CIT is widely recognized as an independent risk factor for both short- and long-term transplant outcomes.

Beyond ischemia duration, graft vulnerability is a multifactorial process influenced by organ preservation quality, inflammatory cytokine release, donor and recipient characteristics, and the specific ischemic context. These converging insults promote mitochondrial dysfunction, endothelial activation, interstitial edema, and acute tubular necrosis, ultimately increasing the risk of delayed graft function and impairing long-term graft survival. Current evidence indicates that CIT directly impacts renal function and may influence both patient and graft survival, as well as hospitalization rates.

Advances in the understanding of ischemia–reperfusion biology have driven the development of mitigation strategies, including optimized preservation solutions and both hypothermic and normothermic machine perfusion. When appropriately implemented, these approaches have the potential to attenuate injury, improve early graft performance, and enhance long-term transplant outcomes.

Despite these advances, important knowledge gaps remain. Future research should prioritize prospective studies integrating mechanistic biomarkers, advanced preservation technologies, and standardized clinical endpoints to better establish causality and refine risk stratification. Multicenter studies with longitudinal follow-up and histopathological assessment are particularly needed, especially in the context of ECD. In addition, the incorporation of novel perfusion systems and systematic use of emerging biomarkers may further clarify their prognostic value. Such efforts will be essential to translate mechanistic insights into precision preservation strategies and evidence-based clinical decision-making in kidney transplantation, including improvements in allocation policies and compatibility assessment.

ACKNOWLEDGEMENT

Not applicable.

• DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE TOOLS
  The authors declare that no artificial intelligence tools were used in the preparation, writing, data analysis, or review of this manuscript.
• FUNDING
  Not applicable.

DATA AVAILABILITY STATEMENT

All datasets were generated or analyzed in the current study.

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Edited by

• Section editor:
  Ilka de Fátima Santana F. Boin https://orcid.org/0000-0002-1165-2149

Publication Dates

• Publication in this collection
  07 Aug 2026
• Date of issue
  2026

History

• Received
  26 Mar 2026
• Accepted
  17 June 2026