Critical role of complement in antibody mediated rejection in kidney transplantation

Abstract

Antibody-mediated rejection (AMR) represents a major challenge in kidney transplantation, significantly contributing to tissue injury and graft failure. AMR is primarily driven by donor-specific alloantibodies (DSAs), which recognize and bind to specific target antigens present within the transplanted kidney tissue. Upon binding, these DSAs commonly initiate activation of the complement system within the graft. The activation of the complement cascade sets off a powerful inflammatory response characterized by the recruitment and activation of immune cells, endothelial damage, and subsequent tissue injury. This inflammation underlies many clinical and histological manifestations of AMR, making complement activation a critical player in the disease process. Advancements in our understanding of how complement pathways contribute to kidney graft injury have opened new avenues for therapeutic intervention. Recent research has facilitated the development and application of novel therapies specifically designed to inhibit complement activation. Such targeted complement-inhibitory strategies have shown promise in improving graft outcomes by inhibiting complement-mediated damage and extending graft survival. This review comprehensively discusses the critical role of complement activation in inducing kidney graft injury with a focus on its role in AMR. By elucidating the detailed mechanisms and contributions of complement pathways, the review seeks to enhance the understanding necessary for developing targeted therapeutic interventions to prevent or treat AMR effectively.

Key Words: Complement; Donor-specific antibodies; Kidney; Allograft; Rejection; Graft failure

Core Tip: Antibody-mediated rejection (AMR) is a significant barrier to successful kidney transplantation, driven primarily by donor-specific alloantibodies. These antibodies activate complement pathways within the transplanted kidney, leading to severe inflammatory responses, endothelial injury, and tissue damage. Understanding the complement system’s critical role in AMR has led to innovative therapies aimed at inhibiting complement activation. Such complement-targeted strategies show potential in reducing inflammation, preventing graft injury, and improving overall graft survival. This review highlights the central contribution of complement activation in AMR, emphasizing therapeutic advancements and the importance of complement inhibition as a promising approach for preventing and managing rejection.

INTRODUCTION

Kidney transplantation (KT) is the treatment of choice for patients with end-stage kidney disease[1]. However, antibody-mediated rejection (AMR) continues to pose a major obstacle, often leading to graft failure despite progress in antibody detection methods and immunosuppressive therapies[2]. Currently, AMR is recognized as a leading cause of late or chronic graft rejection. Additionally, it is estimated that 30%-50% of acute rejection episodes are attributable to AMR[3]. Acute AMR is associated with poor graft outcomes after KT. Specifically, patients with repeated acute AMR episodes are at greater risk of progressing to chronic AMR, leading to graft loss. AMR is primarily driven by donor-specific antibodies (DSAs) that target either human leukocyte antigens (HLA) or non-HLA antigens within the transplanted kidney[4-6]. The complement system is a key player in this rejection process, which augments the immune response and contributes to tissue damage[7-9]. In addition the complement proteins play a central role in interlinking innate and adaptive immunity. In 1969, Terasaki and Patel described the phenomenon of immediate graft loss due to capillary thrombosis and necrosis in sensitized patients[10]. Using a clear and intuitive method, the authors showed that antibodies in a patient’s serum, which caused complement-dependent lysis of donor cells, led to immediate graft failure in most instances. This method became known as the complement-dependent cytotoxicity (CDC) crossmatch[11]. This immediate occurrence of rejection and poor graft outcomes across a positive CDC crossmatch highlighted the importance of preformed antibodies and complement activation in hyperacute rejection and graft loss[12]. Since then, positive CDC crossmatch has become contraindicated for KT. An unequivocal link between antibodies and rejection was established when Feucht et al[13] demonstrated the deposition of complement split product, C4d, and C3d in peritubular capillaries of kidney allograft biopsies[13]. This landmark study revealed that C4d is a durable biomarker for diagnosing AMR. This led to the development of consensus diagnostic criteria in kidney allograft biopsies for acute AMR at the 2001 Banff conference on allograft pathology[14]. The significance of complement activation as an effector mechanism of antibody-initiated allograft injury provides a promising therapeutic approach in the treatment of AMR[14]. Anti-human C5 monoclonal antibody (mAb), Eculizumab, has been used for the prevention of AMR in sensitized patients and in the treatment of established AMR[15].

In recent decades, the role of the complement system in AMR has attracted increasing attention. This review offers an in-depth analysis of the complement system’s role in transplant immunology. It highlights research showing that inhibiting terminal complement activation can help prevent AMR in sensitized kidney transplant recipients (KTRs). By shedding light on the specific mechanisms and pathways involved, the review aims to support the development of more precise therapies to effectively prevent or manage AMR in KTRs.

HISTORICAL PERSPECTIVE

In 1896, Jules Bordet first described complement as a heat-sensitive component of fresh serum[16]. He demonstrated that if fresh serum containing an antibacterial antibody was added to the bacteria at physiologic temperature (37 ˚C), the bacteria were lysed. If, however, the serum was heated to 56 ˚C or higher, it lost its lytic capacity. This loss of lytic capacity was not due to decay of antibody activity because antibodies are heat stable, and heated serum was capable of agglutinating the bacteria. Bordet concluded that serum must contain another heat-labile component that assists or complements the lytic function of antibodies. This component was later given the name COMPLEMENT[16]. Since then, numerous clinical studies have investigated the role of complement in various human diseases, including kidney diseases.

OVERVIEW OF THE COMPLEMENT SYSTEM ACTIVATION

The complement system is activated by three distinct pathways based on the stimuli that activate their initiation proteins (Figure 1). These include: The classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). These three pathways differ in the inception stage of activity but ultimately lead to a common final pathway. The first pathway to be discovered was the CP, following the AP and, more recently, in 1990, the mannose-binding LP was described[17].

Figure 1 Complement activation pathways. Ag-Ab: Antigen-antibody; C1inh: C1 esterase inhibitor; MASP-1: Mannan-binding lectin-associated serine protease-1; MASP-2: Mannan-binding lectin-associated serine protease-2; MCP: Membrane cofactor protein; DAF: Decay accelerating factor.

The CP is initiated when plasma C1q interacts with the Fc segments of IgG and M. On C1q binding, C1r becomes activated, which subsequently activates C1s. Activated C1s cleaves complement protein C4 into C4a and C4b. The exposed thioester domain on C4b allows it to bind C2. C1s further cleaves C2, releasing the smaller fragment C2b and leaving the larger fragment C2a attached to C4b, forming the CP C3 convertase (C4b2a) (Figure 1).

The AP is uniquely activated through spontaneous hydrolysis of the complement component C3, a method known as tick-over. This lets the system be ready for rapid activation when required. During tick-over, a low level of C3 undergoes spontaneous hydrolysis and binds to Factor B (FB), which becomes a substrate for serine protease factor D. This leads to cleavage of FB to Bb and results in AP C3 convertase (C3bBb).

The LP of complement is activated by binding to specific carbohydrates or other ligands on the surface of microorganisms. In the mannose binding lectin (MBL) pathway, interaction with one of its two associated serine proteases (MASPs-1 or MASPs-2) promotes C3 cleavage, forming a C3 convertase identical to that of the CP (C4b2a).

Further interaction of C3b with the respective C3 convertases forms C5 convertases (C3bBbC3b and C4b2aC3b). These enzymes cleave C5 into C5a and C5b, initiating the membrane attack complex (MAC) assembly. This is formed Sequential binding of C5b with complement components C6, C7, C8, and multiple C9 molecules, forming a pore in the target cell membrane, resulting in cell lysis[17].

Functions of the complement system

As alluded to earlier, the complement system plays an important role as a link between the innate and the adaptive immune responses, supporting the processes of chemotaxis, opsonization, phagocytosis, and cell lysis of pathogens, as well as the removal of immune complexes and apoptotic cells. As a part of the innate immunity, the complement factors are specialized in recognizing and controlling pathogens; however, complement components are also involved in hemostasis, apoptosis, tumor pathogenesis, and autoimmune diseases.

Overall, the functions of the complement system can be categorized into three types: (1) Opsonization: Cleaved complement proteins opsonize cell surfaces identified for destruction by phagocytes (C3b, iC3b, and C3d); (2) Fragments formed in the process of activation of complement factors act as chemoattractants, recruiting phagocytes to the site of activation (C3a, C5a); and (3) The final common pathway results in the formation of a MAC, comprising C5b-C9, with the ability to form pores that lead to osmotic lysis of pathogens and damaged cells[18].

COMPLEMENT REGULATORY MECHANISMS

Since the complement system is a very potent pro-inflammatory system capable a number of regulatory mechanisms have evolved to restrict complement activity to designated targets. The regulatory proteins are divided into two types: Fluid-phase and membrane-bound molecules (Figure 1 and Table 1). Soluble regulators are distributed in plasma and other body fluids, and include factor H, factor H-like protein 1, properdin, carboxypeptidase N, C1 esterase inhibitor, C4BP, complement factor H (CFH)-related protein 1, clusterin, and vitronectin. Membrane-bound regulators include CR1, complement receptor type 2, complement receptor type 3, complement receptor type 4, membrane cofactor protein (MCP or CD46), decay-accelerating factor (or CD55), and CD59. Membrane-bound regulators are relatively nonspecific and control all three complement activation pathways; soluble regulators are more specific and control only the AP, CP, or LP and act exclusively on C3 or C4. Complement regulators act mainly by decay acceleration and cofactor activity. Since the C3 and C5 convertases play central roles in complement activation, many regulators act on these two proteins[19-21].

Table 1 List of regulatory proteins for complement system and their function.

Soluble	Function	Pathway affected
C1 inhibitor	Dissociate C1r and C1s from C1q	Classical
C4 binding protein	Blocks formation of C3 convertase by binding C4b, Cofactor for cleavage of C4b by factor I	Classical, lectin
Factor H	Bind C3b, displacing Bb, Cofactor for factor I	Alternate
Factor I	Cleaves C4b and C3b aided by factor H, MCPC4BP or CR1	All 3 pathways
S protein	Binds soluble C567 and prevents its insertion into cell membrane	Terminal pathway
Anaphylatoxin inactivator	Inactivates anaphylatoxin activity of C3a and C5a by carboxypeptidase N-catalyzed removal of C-terminal Arg	Effector
Membrane bound
Membrane cofactor protein	Block formation of C3 convertase by binding C3b or C4b	All 3 pathways
Decay-accelerating factor	Accelerates dissociation of C4b2a and C3bBb	All 3 pathways
Complement receptor type 1	Bind C4b, displacing C2b or C3b displacing Bb; cofactor for I	All 3 pathways
Homologus restriction factor	Bind to C5b678 on autologous or allogenic cell blocking binding of C9	Terminal pathway

MECHANISMS OF COMPLEMENT-MEDIATED KIDNEY ALLOGRAFT INJURY

The complement system plays a significant and multifaceted role in kidney allograft tissue injury. Its involvement begins even before the transplant takes place and continues throughout the lifespan of the allograft. Beyond its role in adaptive immunity, complement is a key player in innate immune responses directed at the transplanted tissue. It also serves as a central effector mechanism in AMR. Interestingly, recent research has also highlighted the complement system's potential role in promoting immune tolerance of the allograft. In the sections that follow, we explore the specific contributions of complement to various forms of allograft injury as well as its emerging role in fostering tolerance[22-24].

ISCHEMIA-REPERFUSION INJURY OF KIDNEY ALLOGRAFT TISSUE

The importance of the complement system in renal ischemia-reperfusion injury (IRI) is widely recognized; however, its contribution to the pathogenesis of tissue damage in the donor remains underexposed. During KT, the donated organ often undergoes a period of ischemia, when it is deprived of oxygen and nutrients (Figure 2). This interruption in blood flow disrupts cellular metabolism, leading to ischemic injury and necrosis. Two key factors influencing long-term graft survival are the quality of the donor organ and the duration of ischemia. Kidneys from living donors generally perform better and last longer than those from deceased donors, whether brain-dead or after cardiac death. Complement activation during cold ischemia is believed to influence how well the transplanted kidney functions afterward[25,26].

Figure 2 The role of the classical complement pathway in acute antibody-mediated rejection in kidney transplant recipients. HLA class: Human leucocyte antigen class; DSA: Donor specific antibody; PTC: Peritubular capillaritis; NK cells: Natural killer cells; APC: Antigen presenting cell; TCR: T cell receptor.

Research by Damman et al[27] showed that kidneys from brain-dead donors exhibit higher expression of the C3 gene and more C3d deposits compared to kidneys from living donors. Further findings by the same group[28] revealed that both complement and coagulation pathways become active in donor kidneys after brain death, even before the organs are retrieved for transplantation. Troise et al[29] described that deceased donors have increased levels of C5a and C5b-9 in blood, correlated with increased risk of rejection compared to living donors.

When blood flow is restored to the transplanted kidney, a process known as reperfusion, additional injury occurs. The return of circulation triggers innate immune responses, as necrotic and apoptotic cells attract leukocytes and macrophages to the site. This cascade involves the release of proinflammatory cytokines, chemokines, and reactive oxygen species, which amplify the inflammatory response. As IRI progresses, damage-associated molecular patterns sustain inflammation and stimulate complement activation[30,31].

Pratt et al[32] demonstrated that complement component C3 is activated during reperfusion and plays a central role in initiating IRI, leading to subsequent graft damage and potential rejection. Supporting this, studies using mice lacking certain complement components show that deficiency in C3, C5, or C6 provides protection against IRI, whereas a C4 deficiency does not[33].

Two key byproducts of complement activation, the anaphylatoxins C3a and C5a, intensify inflammation. These fragments are generated through proteolytic cleavage, with C5a acting as a chemotactic agent that draws neutrophils and macrophages to the injury site. C3a also attracts monocytes and mast cells[34]. C5a binds to receptors C5aR1 and C5aR2, both present on immune cells, which trigger multiple downstream effects, including cell migration, activation of signaling pathways, increased calcium levels, and degranulation. Notably, the C5a/C5aR signaling axis is also implicated in promoting kidney fibrosis[35].

Beyond activating the innate immune system, IRI also enhances adaptive immune responses. The inflammatory environment caused by complement activation encourages the maturation of antigen-presenting cells (APCs) and the activation of B and T lymphocytes. Complement receptor 2 signaling, in particular, boosts B cell responses to antigens presented on C3d-opsonized cells. Moreover, complement fragments like C3b can activate receptors on various immune cells, increasing cytokine production and phagocytic activity[21,36].

IRI activates all three complement pathways[34], and the presence of complement split products such as C3c and C3d in kidney biopsies confirms this activation, regardless of the initiating pathway. In a study of 44 patients, Bobka et al[37] found these components in time-zero biopsies taken at the time of transplantation, before circulation was established. C3c was detected in glomeruli, tubular cells, and peritubular endothelial cells, with more intense staining in patients who later experienced complications such as delayed graft function or immune rejection. However, even patients without complications showed some degree of staining, indicating subclinical complement activity[38].

ACTIVATION OF THE COMPLEMENT SYSTEM IN AMR

The role of complement in AMR is critical, as AMR is the major cause of acute rejection episodes and long-term graft loss. AMR is mediated by antibodies, but antibodies do not kill allograft cells directly. Allograft destruction occurs via the activation of the complement system and the actions of other effector cells, like NK cells and cytotoxic CD8-positive T cells. In KT, the recipient’s alloantibodies can bind to antigens expressed on the graft’s endothelial cells. These DSAs may be present before transplantation (preformed) due to prior sensitization events such as blood transfusions, pregnancies, previous transplants, or they may develop after transplantation (de novo). When DSAs bind to the endothelial surface of the allograft, they lead to a cascade of events that contribute to tissue injury. Immune complexes activate the classical complement pathway by binding to C1q (Figure 3). DSAs of IG1 and IG3 subclasses bind to donor antigens and recruit C1q, initiating the CP cascade. C1q activation leads to cleavage of C4 and C2, forming C3 convertase, which cleaves C3 into C3a (anaphylatoxin) and C3b (opsonin). Further cleavage of C3 Leads to the formation of C5 convertase, which activates C5 into C5a (anaphylatoxin) and C5b. Complement fragments C3a and C5a act as potent anaphylatoxins, promoting neutrophil, monocyte, and macrophage infiltration, leading to graft damage. Complement component C5b recruits C6, C7, C8, and multiple C9 molecules to form the MAC (C5b-9), which causes direct endothelial cell swelling, detachment, and loss of barrier function, resulting in graft inflammation and thrombosis. Endothelial cell damage triggers coagulation pathways, leading to thrombotic microangiopathy (TMA), a hallmark of severe AMR. Complement activation leads to the production and deposition of the C4d fragment within the kidney’s peritubular capillaries, serving as a key marker for AMR[38]. Complement activation also promotes inflammation and thrombosis in the graft’s microvasculature, which can result in ischemia, cell death, and eventual graft failure[39].

Figure 3 The role of complement in the pathogenesis of ischemic reperfusion injury in renal transplantation. MASP-1: Mannan-binding lectin-associated serine protease-1; MASP-2: Mannan-binding lectin-associated serine protease-2; MAC: Membrane attack complex; ROS: Reactive oxygen species; NADPH oxidase: Nicotinamide adenine dinucleotide phosphate oxidase; DAMP: Damage-associated molecular patterns; PAMP: Pathogen-associated molecular patterns.

Complement also plays an important role in B-cell maturation and antibody production. C3d opsonized antigens from damaged cells bind to complement receptor 2 on B lymphocytes and promote B cells’ activation and production of antibodies[40]. The anaphylatoxins, C3a and C5a, play an important role in AMR by activating B cells and polarization of T cells[41].

Modern diagnostic techniques, including highly sensitive assays for anti-HLA antibodies and DSAs, as well as staining for C4d in biopsy samples, have improved the diagnosis of AMR. C4d presence in peritubular capillaries is a critical component of the Banff classification, helping distinguish between acute and chronic cellular and humoral rejection[42].

Diagnosing and categorizing AMR involves both serological testing for DSAs targeting HLA or non-HLA antigens and thorough histological evaluation of kidney biopsies. Biopsies are assessed for immunomorphological features, such as linear C4d deposits in peritubular capillaries, an indicator of active AMR, as well as microvascular inflammation, arteritis, and chronic changes like transplant glomerulopathy or basement membrane multilayering in peritubular capillaries[38].

Evaluation for C4d deposition is a routine part of biopsy analysis, using immunofluorescence on frozen tissue or immunohistochemistry on paraffin-embedded samples. Despite its specificity, C4d staining has limited sensitivity. Studies have shown that only about 40% of kidney transplants displaying histological features of AMR show positive C4d staining[38,39]. Moreover, in vitro experiments reveal that alloantibody-exposed endothelial cells can produce inflammatory molecules without complement activation[43]. Additionally, endothelial injury may occur through direct engagement with NK cells or macrophages via Fc receptors recognizing bound alloantibodies[44].

Recently, the ability of DSAs to bind C1q, the first component of the classical complement pathway, has emerged as a useful biomarker for AMR[39]. C1q-binding DSAs are considered more cytotoxic and associated with a higher risk of rejection. In a study of 1016 transplant recipients, Loupy et al[44] showed that C1q-binding DSAs were linked with C4d deposition and could also identify cases of AMR even in the absence of C4d staining, leading to the concept of C4d-negative but C1q-positive (C4d-/C1q-DSA+) rejection.

Further evidence from Viglietti et al[45] indicated that patients with C1q-binding DSAs after an acute AMR episode had a higher risk of graft failure. Even after standard AMR treatment, including plasma exchange, intravenous immunoglobulin (IVIG), and rituximab, about one-third of patients still had detectable complement-binding DSAs. In some cases, non-complement-binding DSAs present at diagnosis later acquired complement-binding capability. The likelihood of complement binding correlated with higher DSA concentrations, as measured by increased mean fluorescence intensity[46]. These findings suggest that incorporating C1q-binding assessments into routine evaluation may allow earlier detection and more targeted immunosuppressive therapy.

In another study by Sicard et al[47], detection of C3d-binding DSAs was found to be an even more sensitive and specific marker of early graft injury. Among 69 transplant patients, the presence of C3d-binding DSAs outperformed both C4d staining and C1q-binding DSA detection in diagnosing AMR at one and three years post-transplant. Orandi et al[48] reported that patients who developed AMR had a 4.61-fold higher risk of graft loss when compared to controls. Loupy et al[49] reported that glomerular and peritubular inflammation, transplant glomerulopathy, and posttransplant C1q-binding DSA (pathologic and immunologic factors of AMR) on 1-year protocol biopsies were independently associated with graft loss[49]. Gloor and colleagues reported a threefold increase in graft loss in AMR patients[50]. Kikić et al[51] describe that complement activation is associated with a poorer prognosis and is generally considered an important contributor to graft injury in AMR.

Interestingly, older transplant recipients tend to exhibit a weaker humoral immune response and are less likely to develop DSAs[45]. Therefore, assessing complement-binding DSA activity in elderly patients could help guide more personalized immunosuppressive strategies.

Finally, it is important to recognize that antibodies against non-HLA targets can also mediate AMR. In cases where C4d deposits are observed but no anti-HLA antibodies are detected in the blood, the possibility of non-HLA antibody-induced complement activation should be considered[39,52].

ROLE OF THE COMPLEMENT SYSTEM IN T-CELL-MEDIATED REJECTION

Although the complement system plays a critical role in AMR, recent research indicates an increasing role of the complement system in regulating T-cell-mediated alloimmune responses (Figure 3)[53]. APCs locally produce complement components C3a and C5a, which are essential for effective T-cell co-stimulation. In addition, T-cells themselves generate C3a and C5a, which act in an autocrine manner to support the activation of Th1 Lymphocytes. Dendritic cells also contribute by producing properdin and factor H, which influence T-cell function. Animal models have demonstrated that a deficiency in the recipient’s C3 component impairs the production of chemokines and cytokines, thereby reducing the activation, expansion, and infiltration of donor-specific T-cells into heart transplants following IRI. These findings establish a connection between IRI-induced damage and T-cell-driven rejection, mediated through complement activation by MBL. Conversely, some complement components, such as C1q, C3, C4, and C5a, have been shown to exert immunomodulatory effects on APCs, promoting the development of immature, tolerogenic dendritic cell phenotypes[4,21,54].

ROLE OF THE COMPLEMENT SYSTEM IN POST-TRANSPLANT TMA

TMA is a clinical and pathological syndrome characterized by microvascular thrombosis, hemolytic anemia, thrombocytopenia, and fragmented red blood cells on peripheral blood film. Among KTRs, TMA is relatively common, though it often presents without systemic symptoms and may only be detected through biopsy of the transplanted kidney. TMA can either develop anew in the transplanted organ or represent a recurrence of the original diseases[55]. Recurrent cases are frequently linked to genetic mutations in complement-related genes, such as CFH, complement factor I (CFI), MCP, and C3, commonly associated with atypical hemolytic uremic syndrome (aHUS). Certain complement gene haplotypes are known to increase susceptibility to TMA across different populations[56]. However, the causes of TMA in transplant recipients can be diverse and complex.

In KTRs, TMA can be triggered by complement activation due to factors such as autoimmune disorders, severe hypertension, infections (especially hepatitis C), and AMR. It can also result from endothelial injury caused by immunosuppressive drugs like calcineurin inhibitors (CNIs) or mammalian target of rapamycin inhibitors. While high doses or combined use of these drugs are associated with a greater risk of TMA, the condition still only affects a small subset of patients, suggesting that additional predisposing factors play a role[57,58].

Notably, in a small study involving 24 patients with de novo TMA after KT, mutations in CFH and CFI, compatible with aHUS, were found in 29% of cases[59]. A recent retrospective study by Broecker et al[60] found that CNI therapy and AMR were the most frequently identified causes, accounting for 22% and 11% of cases, respectively[60]. Yet, in 63% of patients, the underlying cause remained uncertain or unknown. In over half of the patients (56%), at least one potential trigger was identified, including prothrombotic conditions like antiphospholipid syndrome, malignant hypertension, tuberculosis treatment, de novo post-infectious glomerulonephritis, acute cytomegalovirus infection, lung transplant, pancreatic surgery, sepsis, and histiocytic glomerulopathy.

Understanding the mechanisms behind TMA, particularly those involving complement activation after KT, requires a thorough diagnostic evaluation. In certain cases, where the cause is identifiable, preventive treatments may be considered before transplantation[61].

ROLE OF THE COMPLEMENT SYSTEM IN RECURRENT GLOMERULONEPHRITIS

Complement activation in a transplanted kidney can also be associated with the recurrence of the original kidney disease that affected the recipient[62]. Genetic mutations in genes encoding soluble complement regulatory proteins, such as factor H, CFHR 1-3 and 5, and factor I, as well as components that activate the complement system, like C3 and FB, can lead to abnormal activation of the alternative complement pathway in the allograft. This dysregulation contributes to glomerular injury and may lead to disease recurrence, sometimes accompanied by TMA[63].

A rare glomerular disease known as C3 glomerulopathy (C3G) arises from impaired regulation of the alternative complement pathway. It is characterized by the presence of C3 deposits in the glomeruli, as seen under immunofluorescence, without associated immunoglobulin or immune complex deposits. After KT, C3G has a high recurrence rate, affecting approximately 70% of patients[64,65].

In contrast, immune complex-associated membranoproliferative glomerulonephritis (MPGN) appears to be driven by immune complex formation, which then activates the complement system. The recurrence of MPGN in kidney allografts, marked by polyclonal immunoglobulin deposits, is less frequent than that of C3G. Additionally, the absence of C3 or C4d deposits is associated with a lower risk of MPGN recurrence[66].

Importantly, the severity of glomerulonephritis, whether C3G or MPGN, has been linked to mutations in genes involved in complement regulation and activation, further highlighting the role of complement dysregulation in disease progression and recurrence[67,68].

ROLE OF THE COMPLEMENT SYSTEM IN CNI-INDUCED NEPHROTOXICITY

Acute CNI-induced nephrotoxicity is dose-dependent and typically reversible with dose reduction. It usually occurs early after treatment initiation and is associated with vasoconstriction of the afferent and efferent arterioles, endothelial dysfunction, and a resulting decrease in renal blood flow[69]. As previously discussed, CNIs are also implicated in the development of TMA.

Chronic CNI-induced nephrotoxicity was once thought to be a major contributor to late graft failure. However, more recent studies highlight the central role of chronic AMR, which is often associated with poor medication adherence or subtherapeutic immunosuppressant levels. Histological findings traditionally associated with chronic CNI toxicity, such as arteriolar hyalinosis, interstitial fibrosis, tubular atrophy, and focal or global glomerulosclerosis, are nonspecific, and the exact mechanisms driving these changes remain unclear[70].

Emerging evidence from animal models and in vitro studies suggests that complement activation plays a role in the pathogenesis of CNI-induced nephrotoxicity. In mice, treatment with subcutaneous cyclosporin A (CyA) has been shown to cause tubular injury and interstitial fibrosis, accompanied by increased deposition of complement components C4d, C3, and the MAC (including C9) in renal tissue[71,72].

In vitro studies have demonstrated that CyA stimulates endothelial cells to release complement-activating microparticles. Similar microparticles have been identified in the blood of KTRs. In research by Renner et al[73], these CyA-induced microparticles were shown to activate the alternative complement pathway and cause endothelial damage in vitro. When injected into mice, the microparticles triggered local mesangial complement activation and mesangial cell proliferation[73].

Additionally, CNIs have been found to directly induce complement activation and reduce the expression of complement regulatory proteins in cultured human renal tubular cells. This suggests a multifaceted role of complement dysregulation in both acute and chronic CNI-induced kidney injury[74].

CLINICAL AND HISTOPATHOLOGICAL FEATURES OF COMPLEMENT-MEDIATED AMR

Complement-mediated AMR typically presents with signs of graft dysfunction, most commonly elevated serum creatinine, reduced glomerular filtration rate (GFR), proteinuria, and occasionally hypertension[2]. These features may emerge early after transplantation or later in the post-transplant course, depending on whether the recipient harbors preformed DSAs or develops them de novo. In many cases, patients may be asymptomatic, and evidence of AMR is only uncovered during routine surveillance biopsies or investigations triggered by subtle functional decline. The presence of complement-fixing DSAs, especially those that bind to C1q or C3d, has been strongly associated with more aggressive forms of AMR and worse clinical outcomes, including higher rates of treatment resistance, chronic injury, and graft loss[37,39].

Histopathologically, complement-mediated AMR is defined by a combination of morphological changes and immunopathological markers[75]. A central feature is the linear deposition of the C4d fragment in peritubular capillaries, detectable through immunofluorescence on frozen tissue or immunohistochemistry on paraffin-embedded sections. C4d serves as a footprint of classical complement pathway activation[13]. In addition, microvascular inflammation is commonly observed, with glomerulitis and peritubular capillaritis indicating ongoing endothelial injury. These lesions are accompanied by the presence of neutrophils and mononuclear cells within glomerular and capillary lumens (Figure 4). In chronic or repeated episodes of AMR, structural alterations such as transplant glomerulopathy, characterized by duplication of the glomerular basement membrane and multilayering of the peritubular capillary basement membrane, reflect chronic endothelial injury and persistent complement activation[76]. Despite its diagnostic value, C4d staining is not always present; therefore, biopsy interpretation requires correlation with serologic DSA profiling and histologic indicators of antibody activity[77,78].

Figure 4 Histopathology of acute antibody-mediated rejection. A: Acute glomerulitis. Some of the glomerular capillary lumens are obliterated by inflammatory cell infiltration and/or endothelial cell swelling (arrow). [hematoxylin and eosin (HE), × 400]; B: Foci of mild peritubular capillaritis with up to 4 inflammatory cells in capillary lumens (ptc1) (arrows) (HE, × 400); C: An interlobar sized artery showing transmural arteritis (v3) (HE, × 400); D: Diffuse C4d positivity (C4d) (C4d3) in peritubular capillaries. Glomerular mesangial positivity serves as the internal control (Flourescein isothiocyanate, immunofluorescence for C4d, × 200).

Advances in understanding the role of complement in AMR have led to greater emphasis on evaluating the complement-binding capacity of DSAs (e.g., C1q or C3d assays) and integrating these findings into the diagnostic algorithm. Such tools not only help in diagnosing C4d-negative AMR but also aid in risk stratification and therapeutic decision-making. Ultimately, recognizing the clinical and pathological signatures of complement-mediated AMR is essential for early intervention and improving long-term graft survival[39].

ROUTINE TESTING OF COMPLEMENT IN KT

There are various assays to evaluate complement system activation in KTRs. Elevated levels of C5b-9 in plasma indicate complement activation in AMR, where complement plays a role, suggesting that the candidate may be suitable for complement inhibition. However, complement may also be activated locally in tissues without resulting in increased activation products in plasma; thus, a normal serum C5b-9 Level does not rule out complement activation in tissues. Therefore, graft biopsy may ultimately serve as the definitive test for diagnosing AMR, as activation products can be readily detected using immunofluorescence microscopy[79,80]. In KT, detecting C4d deposition in kidney allograft biopsies is a crucial diagnostic tool for AMR. While not always essential for diagnosing AMR, C4d deposition indicates complement activation and correlates with an increased risk of allograft loss. Complement-binding DSAs also significantly raise the risk of AMR and decrease graft survival. Additionally, complement activation produces several soluble fragments that can act as noninvasive clinical biomarkers of inflammation. During vascular injury, damaged cells release microvesicles into the plasma, and endothelial microvesicles increase in diseases associated with endothelial injury[81]. Tower et al[82] reported that the number of C4d-opsonized endothelial microvesicles rises in AMR, likely reflecting complement-mediated endothelial injury.

MONITORING TESTS TO TRACK LONG-TERM OUTCOMES IN KT

Long-term monitoring in KTRs is essential to assess graft function, detect early signs of rejection, and manage complications. Serum creatinine and eGFR are routinely measured to evaluate kidney function, with rising levels indicating potential graft damage or chronic allograft nephropathy. Urinalysis and proteinuria monitoring help identify protein loss, infections, or damage to the glomerular filtration barrier. Regular assessment of immunosuppressive drug levels, such as tacrolimus or CyA, ensures adequate immunosuppression while minimizing toxicity. Imaging studies, particularly renal ultrasound with Doppler, are used to assess kidney size, perfusion, and detect structural or vascular complications. Emerging tools like donor-derived cell-free DNA provide a non-invasive means to detect subclinical rejection. In certain cases, especially when there is unexplained graft dysfunction, a kidney biopsy is performed to confirm rejection or other pathological changes. Additionally, routine monitoring of blood pressure, blood glucose, and serum electrolytes is vital to address cardiovascular risk factors and maintain metabolic stability, which are crucial for better long-term graft and patient outcomes[4,6].

THERAPEUTIC TARGETING OF COMPLEMENT IN AMR

Therapeutic targeting of the complement system in AMR involves using drugs to block or modulate complement activation, aiming to reduce inflammation and tissue damage. This approach has shown promise in preventing and treating AMR, a major cause of graft failure. Given the pivotal role of the complement system in the pathogenesis of AMR, particularly in cases involving DSAs that activate the complement system, therapeutic strategies aimed at inhibiting complement activation have gained significant attention in recent years. Traditional treatments for AMR, such as plasmapheresis, IVIG, rituximab, and anti-thymocyte globulin, focus on reducing circulating antibodies or depleting B cells, but often fail to fully prevent complement-mediated tissue injury[83]. Targeting the complement cascade directly offers a more specific approach to interrupting the downstream effects of DSA-induced injury, including inflammation, endothelial damage, and thrombosis[84,85].

Several complement inhibitors have been explored in the context of AMR. One of the most studied agents is eculizumab, a mAb that binds to complement component C5, preventing its cleavage and subsequent formation of the MAC. Eculizumab has shown promise in preventing AMR in highly sensitized KTRs, particularly in reducing early rejection episodes and preserving allograft function. However, its high cost, the need for ongoing administration, and limited efficacy in treating established AMR have prompted further investigation into alternative targets. Other emerging therapies include C1 INHs, which block the initiation of the classical complement pathway, and compstatin-based drugs (such as pegcetacoplan), which inhibit C3 activation and thereby disrupt all downstream complement activity[86-89].

Despite encouraging results in preclinical studies and early-phase clinical trials, complement-targeted therapies are not yet part of standard care and are typically reserved for refractory or high-risk cases. Future strategies may involve combining complement inhibitors with conventional immunosuppressive agents to improve efficacy while minimizing toxicity. Personalized treatment approaches, guided by the presence of complement-fixing DSAs and biomarkers such as C4d or C3d, may further enhance outcomes. Continued research is essential to define optimal timing, dosing, and long-term safety of complement inhibitors in AMR, as well as their role in preventing chronic rejection and preserving long-term graft survival[21,33,54,90-94].

Therapeutic targeting of the complement system offers a promising approach to managing AMR in KTRs, with the potential to improve graft outcomes and reduce the risk of late graft failure. Continued research is exploring new complement inhibitors and strategies to optimize these therapies and minimize potential side effects.

ROLE OF COMPLEMENT IN INDUCING TRANSPLANT TOLERANCE

To prevent organ transplant rejection and improve graft survival, immunosuppressive therapy with minimal side effects is essential. However, despite advancements, the average lifespan of a kidney transplant has not seen major improvements in recent years. As a result, achieving transplant tolerance remains a key goal for researchers and transplant physicians[91].

CD4(+) Foxp3(+) regulatory T-cells (Tregs) play a crucial role in maintaining immune balance and promoting tolerance. Traditionally, the complement system was thought to defend against foreign antigens by marking them for destruction or directly killing cells carrying these antigens. Complement activation triggers inflammation and enhances adaptive immune responses. However, recent studies suggest that, in certain cases, complement can also influence immune reactions in both ways, either promoting pathology or inducing tolerance[92].

Interestingly, genetic deficiencies or drug-induced blocking of C3aR/C5aR1 receptors on induced regulatory T-cells (iTregs) has been shown to boost the production of both mouse and human iTregs. This finding suggests that targeting the interaction between complement components (such as C3a/C3aR and C5a/C5aR1) could help enhance iTreg-mediated tolerance to transplanted organs in humans[93,94].

FUTURE DIRECTIONS

The complement system is increasingly recognized as a central player in the immunopathology of KT, particularly in the development of AMR, IRI, and chronic allograft dysfunction[89]. Future research and clinical applications are likely to focus on refining our understanding of complement activation pathways, not only as effectors of tissue injury but also as potential biomarkers and therapeutic targets[90]. As tools for detecting complement-fixing DSAs, such as C1q- and C3d-binding assays, become more accessible and standardized, they may allow for better stratification of transplant recipients based on risk and more tailored immunosuppressive regimens[89].

One promising area is the development of precision complement inhibition therapies, which aim to selectively block specific components of the complement cascade, such as C1, C3, or C5, without compromising the entire immune response. These targeted approaches could minimize systemic immunosuppression and reduce the risk of infections and malignancies associated with broad immunosuppressive therapies. The advent of novel agents like compstatin analogs, C1 INHs, and small-molecule complement blockers offers exciting prospects for both prophylactic and therapeutic use in high-risk or sensitized transplant recipients[94,95].

Moreover, the integration of complement-related biomarkers into routine transplant monitoring could enable earlier detection of subclinical rejection, improving long-term outcomes. There is also growing interest in exploring the role of complement beyond acute rejection, such as its involvement in chronic allograft injury, fibrosis, and transplant tolerance. Understanding these roles could open doors to new strategies for promoting long-term graft survival. Ultimately, the future of complement research in KT lies in combining molecular diagnostics, targeted therapy, and individualized patient care to enhance outcomes and move closer to precision transplant medicine[94].

CONCLUSION

In conclusion, the complement system plays a vital and multifaceted role in coordinating both innate and adaptive immune responses against kidney allograft tissue. Its involvement is particularly significant in the pathogenesis of AMR, where complement activation often directly contributes to graft injury and failure. The intricate interactions between complement components and immune cells influence not only the immediate immune response but also long-term graft outcomes. Therefore, gaining a deeper and more nuanced understanding of the complement cascade in the context of KT is imperative. Such insights will pave the way for the development of more accurate diagnostic tools and targeted therapies. These advancements hold the promise of improving the precision and effectiveness of clinical strategies in managing KTRs, ultimately enhancing graft survival and patient quality of life of these patients.