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Keywords:

  • Autophagy;
  • injury;
  • immunity;
  • kidney transplant;
  • stress

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

In response to ischemic, toxic or immunological insults, the more frequent injuries encountered by the kidney, cells must adapt to maintain vital metabolic functions and avoid cell death. Among the adaptive responses activated, autophagy emerges as an important integrator of various extracellular and intracellular triggers (often related to nutrients availability or immunological stimuli), which, as a consequence, may regulate cell viability, and also immune functions, both innate or adaptive. The aim of this review is to make the synthesis of the recent literature on the implications of autophagy in the kidney transplantation field and to discuss the future directions for research.


Abbreviations
ATG

autophagy-related gene

ATP

adenosine triphosphate

eIF2α

eukaryotic initiation factor 2α

ER

endoplasmic reticulum

GCN2

general control nonderepressible-2

IFNγ

interferon γ

Lamp

lysosomal associated membrane protein

LC3-PE

microtubule-associated protein light chain 3-phoshatidyl ethanolamine

mTORC1

mechanistic target of rapamycin complex 1

PERK

protein kinase RNA-like endoplasmic reticulum kinase

PtdIns3K

phosphatidyl inositol 3 kinase

ROCK

Rho-associated, coiled-coil containing protein kinase

ULK

Unc-51-like kinase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

Mounting experimental evidence has identified autophagy as a master adaptive program that responds to a considerable number of insults whose consequences far exceed life and death decisions [1, 2]. Kidney allografts are almost always hypoxic because of ischemia-reperfusion, arteriolar vasoconstriction, capillary destruction, rarefaction and interstitial fibrosis [3]. Moreover, the transplanted kidney is a proinflammatory milieu that promotes the release of danger-associated molecular patterns during ischemic insults, which are followed by the activation of humoral and cellular alloimmunity. Given that the main biological activators of autophagy are starvation and inflammatory mediators, which are also the princeps kidney transplant stressors [4, 5], there is little doubt that autophagy regulates kidney allograft tissue homeostasis to a far more extent than it is actually known, but experimental and clinical evidence implicating autophagy as an important stress response in the transplanted kidney is only emerging.

A Survey of Autophagy Biology

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

The process of autophagy is initiated by the formation and expansion of an isolation membrane, followed by a series of steps, including closure of the isolation membrane to form an autophagosome, docking and fusion of the autophagosome with a lysosome to form an autolysosome, breakdown and degradation of the cargo and the autophagosome inner membrane by lysosomal acid hydrolases, and recycling of the resulting products. Autophagy recognizes and degrades specific substrates, such as p62, polyubiquitinated protein aggregates, organelles, metabolic products or invasive microbes [6]

Autophagosome formation is the key, characteristic event in autophagy. The origin of the autophagosome membrane remains controversial and potential sources include the Golgi complex, the endosomes, the endoplasmic reticulum (ER), the mitochondria and the plasma membrane. In mammals, the core machinery of autophagy involves at least five major functional groups of proteins or protein complexes: (1) the Unc-51-like kinase (ULK) 1/2 complex is essential for autophagy induction; (2) the class III phosphatidyl inositol 3 kinase (PtdIns3K) complex is involved in vesicle nucleation; (3) the transmembrane protein ATG9L1 potentially contributes to the delivery of membrane from other sources to the forming autophagosome; (4) two conjugation systems, the microtubule-associated protein light chain 3-phoshatidyl ethanolamine (LC3-PE) and ATG12-ATG5-ATG16L complex, participate in vesicle elongation and completion; and (5) the proteins that mediate the late steps of autophagy including vesicle fusion and cargo degradation [5, 7, 8] (Figure 1). Upstream of the core machinery, autophagy is regulated by a complex signaling network. The mTOR pathway, specifically mechanistic target of rapamycin complex 1 (mTORC1), serves as a master regulator of autophagy. Many signaling pathways, which can be stimulated by the signals from nutrient, growth factors and energy, integrate and merge at mTORC1 to regulate autophagy [8]. Nonetheless, several mTOR-independent mechanisms have also been suggested. Notably, autophagy is induced by a variety of cellular stress such as ER stress, hypoxic stress, oxidative stress, DNA damage and pathogen invasion.

image

Figure 1. Autophagosome formation and membrane dynamics in mammal cells The major membrane source of autophagosomes is thought to be the endoplasmic reticulum (ER). After induction of autophagy, the Unc-51-like kinase (ULK1) complex downstream of the inhibitory mTOR signaling complex, translocates to the ER and transiently associates with VMP1, resulting in activation of the class III phosphatidylinositol-3-OH kinase (PI(3)K) complex. The PI(3)K complex produces phosphatidylinositol-3-phosphate (PtdIns(3)P), which recruits effectors such as double FYVE-containing protein 1 (DFCP1) and WD repeat domain phosphoinositide-interacting (WIPI) family proteins. DFCP1 translocate to the autophagosome formation site in a PtdIns(3)P-dependent manner to generate ER-associated Ω-like structures termed omegasomes. At the final step of autophagosome formation, elongation of the isolation membrane and/or completion of enclosure require two ubiquitin-like conjugates. The first is the ATG12–ATG5 conjugate, which functions as a dimeric complex together with ATG16L1. The second is the phosphatidyl ethanolamine (PE)-conjugated microtubule-associated protein light chain 3 (LC3). Cargo are sequestrated within the autophagosome, and LC3 serves as an adaptor for selective substrates such as P62, which recruits damages organelles or protein aggregate for autophagy-mediated degradation.

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Autophagy occurs at a basal level in most cells to maintain cellular homeostasis, whereas stress-induced autophagy primarily serves as an adaptive and defensive strategy for cell survival. Upon metabolic stress, autophagy generates amino acids and lipids that can be reused for protein synthesis and adenosine triphosphate (ATP) production. As a cellular housekeeper, autophagy can clear protein aggregates, damaged organelles and intracellular pathogens. Autophagy may also limit DNA damage and chromosomal instability. Despite the cytoprotective role of autophagy for cell survival, autophagy has also been suggested to be a mechanism of cell death. However, there is little convincing evidence that autophagy can directly kill cells under physiological or pathological conditions [9], although it appears logical that the degradation of cellular organelles by excessive autophagy would be detrimental [10].

The relations between autophagy and cell death and survival remains highly complex and controversial [11, 12]. Autophagy and apoptosis are not mutually exclusive, and they share many of the same regulators. As a consequence, accumulating evidence reveals that autophagy and apoptosis can cooperate, antagonize or assist each other, thus influencing differentially the fate of the cell (Figure 2). There is a controversy regarding the role of autophagy in promoting cell death (autophagic cell death). This controversy is fueled by the absence of a rigorous definition of autophagic cell death, and nowadays, autophagic cell death should be clearly defined with the following criteria: (A) when cell death occurs without caspase activation; (B) when there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (C) when suppression of autophagy is able to rescue or prevent cell death. In mammals (but not in model organisms such as Caenorhabditis elegans and Drosophila melanogaster) [12], there is no clear evidence indicating the presence of such autophagic cell death under physiological settings, based on the phenotypes of various mouse models in which some of the key autophagy-related genes (ATGs) are genetically altered or deleted. Most of the experiments showing autophagic cell death in mammalian cells were mainly conducted under in vitro cell culture conditions and in cells with defective apoptosis machinery such as Bax−/− Bak−/− double-knockout cells [13]. These considerations led to actually consider a reality in which nongenetically modified mammal cells under physiological conditions die with autophagy but not by autophagy.

image

Figure 2. Interrelations between autophagy and apoptosis Schematic representation of the cross-talk between autophagy and apoptosis. (A) Autophagy antagonizes apoptotic cell death by promoting cell survival through, for example, the removal of damaged organelles that are source of genotoxic reactive oxygen species, or by catabolizing cellular macromolecules to provide a source of energy and nutrients for the starved cell; or by limiting endoplasmic reticulum (ER) stress through the degradation of unfolded protein aggregates. These functions block a stimulus that would trigger an apoptotic response. (B) Autophagy, although not leading to cell death itself, enables the apoptotic program by participating in certain morphological changes, such as adenosine triphosphate (ATP)-dependent events such as phosphatidyl serine exposure and membrane blebbing. (C) Autophagy and apoptosis lead to cell death, either in parallel, or autophagy may influence apoptosis.

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Autophagy is involved in physiology such as animal development and cellular differentiation [14]. Remarkably, autophagy contributes to the pathogenesis of an increasing number of diseases including neurodegenerative disease, cancer, aging, infectious and inflammatory disease, cardiovascular disease, metabolic disease, pulmonary disease and kidney disease [15, 16].

Autophagic Flux Induction and Outputs in the Injured Kidney

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

Ischemia and reperfusion injuries

Ischemia and reperfusion injuries are the most investigated injuries that challenge the transplanted kidney and during which autophagy is involved.

Ischemia-reperfusion injuries

Ischemia-reperfusion injury potentates the activation of autophagy induced by cold preservation. Indeed, oxidative stress up-regulates the expression of autophagic regulators, including LC3 and Beclin1, and facilitates the ATG4-dependent lipidation of LC3, leading to autophagosomes maturation. Inhibition of autophagy using 3-methyladenine increases ischemia-reperfusion injury in mice, which indicate that autophagy may be nephroprotective in the setting [17]. However, other authors found inverse results: autophagy inhibition by 3-methyladenine reduced the extent of injury [18]. This highlights the limitations of the chemical approach for autophagy inhibition because chemical drugs do not specifically target autophagy, and molecules with broad-spectrum biological functions may interfere with autophagy. Recently, the use of ATG-deficient murine models for the study of the implications of autophagy in ischemia-reperfusion helped to more definitively demonstrate the protective role of autophagy on the integrity of the tubular compartment during ischemia-reperfusion. Atg5 depletion in both distal and proximal tubule cells results in impaired kidney function at the basal level, and dramatically sensitizes the kidneys to ischemic injury, resulting in impaired kidney function, accumulation of damaged mitochondria as well as increased tubular cell apoptosis and proliferation [19, 20]. These results have been confirmed by similar findings in tubule specific ATG7-null mice, in which the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates [21]. Overall, these recent studies have highlighted the critical role that autophagy plays in maintaining tubular cell integrity during ischemia-reperfusion.

Nonconventional secretion during ischemic stress

Current evidence suggests that autophagy is involved in the establishment of intercellular communication networks, especially during apoptosis, and could be of great importance in modulating the microenvironmental and functional consequences of cell death. The inhibition of conventional secretion (the classical pathway for a protein to be secreted requires a N-terminal signal peptide, which allows retrotranslocation in the ER lumen after translation, folding and quality control, and traffic through the Golgi network, followed by vesicular transport to the plasma membrane, vesicle fusion and secretion) that occurs during stress conditions like starvation might activate unconventional secretion pathways that become the principal mode of intercellular communication. How these unconventionally secreted proteins and immunogenic molecules are released into the extracellular space is not yet understood. Autophagic components, including LC3, ATG16L1 and lysosomal associated membrane protein (Lamp) 2, can also be released from serum-starved endothelial cells in which autophagosomes accumulate [22]. This process depends on the activation of executioner caspases, indicating that the initiation of apoptosis promotes the release of these autophagic components. We have identified and characterized membrane vesicles that are released by serum-starved human endothelial cells with engaged apoptotic and autophagic programs [23]. These vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase (ROCK) 1 activation. Instead, they contain autophagosomes at different stages of maturation, as well as mitochondria. These membrane vesicles are also enriched in various danger signals, contain ATP and could be involved in the modulation of innate immunity during ischemic stress.

Autophagy as a therapeutic target for ischemia-reperfusion injuries

An important challenge for the design of autophagy-modulating drugs and their preclinical and clinical application for protection against ischemia-reperfusion injury is the specificity of the drug for the biological target. The best example to date is sirolimus, which induces autophagy by inhibiting mTORC1, and which is naturally appealing for being tested in this indication, but for which there is now strong experimental and clinical evidence that it impairs the kidney's ability to recovery from injury [24, 25]. Indeed, the recovery of delayed graft function, which is related to acute tubular necrosis, relies on the ability of sublethally injured tubular cells to enter the cell cycle and proliferate, and it is widely demonstrated that mTOR inhibition inhibits cell growth, and therefore, reduces the ability of the cells to proliferate [24, 26]. The same concerns can be opposed to the other widely used chemicals that modulate autophagy, such as bafilomycin, chloroquine or 3-methyladenine, which have wide biological effects that far extend autophagy regulation, and for which the net clinical effect may paradoxally aggravate tissue injuries. As an example, 3-methyladenine promotes cell death by inhibiting class I PI3K signaling, and inhibits cell death, by inducing autophagy, through inhibition of class III PI3K. A more specific approach is required that would involve fine-tuning of autophagy, and candidate are emerging. As an example, a peptide, Tat-Beclin1, derived from a region of the autophagy protein, Beclin1, which binds human immunodeficiency virus (HIV)-1 Nef, is a potent inducer of autophagy [2]. Tat is an HIV-derived protein that facilitates virus entry within the cell. When fused with Beclin1, Tat allows Beclin1 delivery in the cell, without the need of transfection. This peptide specifically activates autophagy and promotes protein aggregates and viruses clearance, and is devoid of “off-targets” effects associated with the broad-spectrum chemical that inhibit autophagy.

Immunosuppressive Drugs

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

Cyclosporine nephrotoxicity

Cyclosporine induces an ER stress in tubular cells [27]. Cells that are subject to ER stress activate the unfolded protein response, a protective mechanism that reduces protein load and increases nascent protein folding, contributing to cell protection [28]. Recent evidence suggests that ER stress drives autophagy, through ill-defined mechanisms that include protein kinase RNA-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) signaling, could regulate autophagic machinery expression and isolation membrane formation and IRE1 activation (JNK and XBP1 may regulate autophagy [5]). Particularly, under ER stress conditions, autophagy has been shown to alleviate ER stress and to reduce cell death [29]. ER stress induced by cyclosporine is a cellular regulator of autophagy. ER stress activates autophagy in response to cyclosporine exposure in human epithelial cells in culture. Autophagy inhibition by targeting Beclin1 increases cyclosporine-induced tubular cell cytotoxicity, suggesting that autophagy serves as a protective mechanism against cyclosporine toxicity [30]. These findings were confirmed in a rat model of cyclosporine nephrotoxicity, in which cyclosporine treatment increased the expression of LC3-II and Beclin1 in the kidney in a dose-dependent manner. Concurrent pravastatin or N-acetylcysteine treatment reduced urinary excretion of 8-hydroxy-2′-deoxyguanosine and subsequently decreased LC3-II expression and the number of p62-positive cells compared with the cyclosporine group, suggesting that cyclosporine-induced oxidative stress could activate autophagy [31].

Sirolimus side effects

Sirolimus is another immunosuppressive drug that is used to prevent acute rejection. Sirolimus is known to induce autophagy by inhibiting mTOR signaling. Sirolimus does not inhibit calcineurin; thus, it was anticipated that sirolimus would lack the nephrotoxic profile of the calcineurin inhibitors. In clinical practice, sirolimus was initially introduced as an adjunct to calcineurin inhibitors, and it is now frequently used in regimens that minimize or avoid these nephrotoxic drugs. However, sirolimus is associated with a wide array of adverse events, including proteinuria, systemic inflammation, edema, cutaneous rash, aphthous, pneumonia, dyslipidemia or diabetes [32]. Even if most of the side effects induced by sirolimus are deemed to be related to mTOR inhibition, their precise mechanisms are not characterized, and the respective roles of autophagy versus the other mTOR-related biological effects (e.g. translation initiation and elongation) are not known. There is actually no direct evidence that autophagy promotes these side effects, but given the importance of mTOR and autophagy in podocyte responses to stress in one hand [33, 34], and in regulating immune responses on the other hand [35, 36], one can imagine that autophagy mediated by pharmacological mTOR inhibition is involved in such side effects. Only few examples support this point of view. Among the sirolimus-induced side effects, sirolimus-induced β cell injury could involve autophagy [37]. This study, which was based on a murine model of islet transplantation, showed that the up-regulation of autophagy after sirolimus treatment resulted in a significant impairment of β cell insulin function, and this effect may contribute to islet graft dysfunction observed in islet recipients. These results also indicated that 3-methyladenine ameliorated rapamycin-related β cell dysfunction both in vitro and in vivo. Even if preliminary, and not focused on the model of kidney transplantation, this study indicates that autophagy may be directly involved in a sirolimus-related side effect.

Inflammation and Immunity

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

Autophagy regulates both innate and adaptive immunity (Table 1), with apparently opposite consequences in terms of modulation. Autophagy tones down inflammation. Autophagy degrades protein aggregates and altered mitochondria, which promote oxidative stress and may activate inflammasomes [4, 38], and modulates nonconventional secretion of cytokines; but it facilitates the clonal expansion of T cells (by providing nutrient supply), promotes thymic education and provides peptides for MHC class I and II presentation (and crosspresentation) [39, 40]. Overall, autophagy increases intracellular pathogen clearance. Moreover, numerous immune-related triggers, like Toll-like or nod-like receptors ligands or proinflammatory cytokines, activate autophagy [38, 41].

Table 1. Immune functions of autophagy
Innate immunityAdaptive immunity
Regulation of inflammatory cytokine productionThymic selection
Regulation of type I interferon productionAntigen presentation
Regulation of inflammatory transcriptional responseApoptotic bodies clearance
Apoptotic bodies clearanceLymphocytes homeostasis
Pathogen degradation
Phagosome maturation

A fascinating aspect of autophagy that lies besides its well-known effects on survival, and which is not yet explored, is its intricate role in the tuning of immunological functions, both innate and adaptive, and which could be of great relevance if applied to the inflamed transplanted kidney and alloimmunity. Recently, arguments have emerged for a role of autophagy in the modulation of the immune kidney allograft in response to injury [20, 42]. For example, mice kidneys that are ATG5 deficient in the tubule, and which are subjected to ischemia-reperfusion injury, display less macrophagic infiltration than their control littermate [20]. This indicates that autophagy may dampen sterile inflammation, but it is not known if this is a consequence of a reduced dead cell (which release intracellular contents that active danger receptors), or more specific functions of autophagy on inflammation. This last hypothesis is supported by the observation made that more altered mitochondria, which constitute powerful proinflammatory agents, accumulate in ATG5-deficient tubules [20]. Besides ischemic stress, cytokinic stress may also activate autophagy in the kidney. Interferon γ (IFNγ) is a master regulator of the homeostasis of the kidney transplant during rejection. IFNγ plays important roles in inflammation, making it particularly relevant to transplantation, with diverse and potentially contradictory effects on organ allograft survival [43]. Autophagy and IFNγ interfere reciprocally. IFNγ promotes autophagy in immune and nonimmune mice cells by mechanisms that involve Beclin1 expression, and autophagy is required for IFNγ-mediated antimicrobial efficacy, a process that involves immunity-related guanosine triphosphatases [44, 45]. Autophagy is activated in human renal epithelial cells in response to IFNγ. Mechanistically, IFNγ induces tryptophan metabolism, which then activates the general control nonderepressible-2 (GCN2) kinase, leading to the phosphorylation of eIF2α, an activator of autophagy [42]. Autophagy negatively regulates inflammatory cytokine secretion in response to IFNγ, which corroborates with previous studies demonstrating that autophagy reduces IL-1β production, as a consequence by a mechanism that involves the destruction of inflammasomes and altered mitochondria [46].

Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

Data related to autophagy are really scarce in transplant immunity, but there is no doubt that it could provide a better understanding of the modulation of immune-related process. Exciting research avenues are numerous, and would, for example, provide clues into how autophagy can be integrated in the danger model, which links sterile inflammation (like ischemia-reperfusion injury) to adaptive immunity. For example, ATP, which is produced by mitochondria, acts as an danger signal by activating purinergic receptors on the surface of dendritic cells, and it has been shown that autophagy was required for the release of ATP by early apoptotic cell, before the permeabilization of the plasma membrane [47]. Other appealing aspects of autophagy lie on how autophagosomes, which are produced in response to stress, and within which macromolecules are processed and digested by lysosomal hydrolases, modify the immunopeptidome of the antigen-presenting cells, and how these modified peptides would alter the efficacy of peptides presentation and the profile of the presented peptides by antigen-presenting cells, as it has been described with mTORC1 inhibitors [48]. Besides inflammation, other biological functions of autophagy in the regulation of tissue homeostasis are just emerging, like fibrogenesis, which is of paramount importance in the structural deterioration of the kidney transplant [49].

A technical issue that remains to be resolved is the ability to monitor autophagy in clinical practice (in other words, for diagnosis and therapeutic counseling), and to date, conflicting results have been published on the ability of immunohistochemistry to detect autophagy in human tissues, and recent ones seriously challenged the feasibility [50]. Given that autophagy is a dynamic process, it must be appreciated in terms of flux and requires chemical modulators for inhibiting autophagosomes degradation by lysosomes, and the characterization of this flux on biopsy specimen is virtually impossible because it is essentially static. An increased number of autophagosomes in a cell does not mean that autophagosomes biogenesis is activated because lysosomal degradation can be inhibited. The great challenge is that the autophagic machinery is not transcriptionally regulated, and no direct quantitative markers can be used. Some candidate, like p62/sequestosome 1, can be quantitatively modulated during autophagy, but its expression suffers from a lack of specificity because numerous biological processes, like oxidative stress, can regulate its expression, in addition to autophagy.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. A Survey of Autophagy Biology
  5. Autophagic Flux Induction and Outputs in the Injured Kidney
  6. Immunosuppressive Drugs
  7. Inflammation and Immunity
  8. Perspectives
  9. Disclosure
  10. References