Kidney diseases are a growing health problem, considered to have a direct and indirect impact on morbidity and mortality worldwide, by increasing the risks associated with the major killers, such as cardiovascular diseases, diabetes, hypertension, and infection (1). The timely identification and management of kidney diseases represent the most effective strategy to sustainably address the growing global burden and prevent the progression to end-stage renal disease (ESRD).

In 2010, 2.62 million people received dialysis worldwide, and the need for dialysis was projected to double by 2030. Despite being a life-saving treatment, dialysis is merely a supportive measure. Indeed, the life expectancy and the quality of life in dialysis patients is much lower compared to the general population (2). Basic research studies have identified many targets to delay the progression of kidney disease; however, only few of these promising results can be recapitulated in clinical studies, and yet do not represent a great alternative to dialysis or transplantation.

The life-sustaining job of the kidneys comprises filtering and reabsorbing about 180 liters of fluid from the bloodstream every 24 h. As a consequence, it is the organ with the highest metabolic rate as determined by tubular epithelial cell (TEC) metabolism (3). TECs, the most abundant cell type in the kidney, are densely packed with mitochondria. These cells combust fatty acids to generate adenosine triphosphate (ATP) through oxidative phosphorylation (4). Proper mitochondrial function and metabolism of these cells is crucial for the high transport and reabsorption activities. Given the high metabolic demand, the kidneys are sensitive to decreased blood oxygenation and perfusion. Whenever the kidneys experience extremely low oxygen exposure, or metabolic substrates become inadequate, acute kidney injury (AKI) occurs.

AKI is defined as an abrupt reduction in kidney function that results in disturbances in the milieu intérieur and the retention of uremic toxins that especially influence the cardiovascular, immune, and nervous system. With an incidence of 9–15% of hospital admissions and up to 40% in critically ill patients, AKI is a major cause of morbidity, mortality (40–70% for the critically ill needing dialysis), increased health-care costs, and chronic kidney disease (CKD) (5, 6). Although research has made great efforts to understand the pathogenesis of AKI, the only available therapy is still supportive (dialysis) and has not changed for decades. Additionally, AKI patients have a greater risk to develop chronic complications later in life (7). A systematic review and meta-analysis comprising estimates from more than 2,000,000 individuals identified AKI as a risk factor for new or progressive CKD (hazard ratio [HR] = 2.67), ESRD (HR = 4.81), and mortality (HR = 1.80) (7). These important clinical findings have resulted in a shift toward studies that investigate the link between experimental AKI and progressive kidney fibrosis and failure. Pattern recognition receptors (PRRs), the sensors of the innate immune system, are one of the major players determining short-term outcomes after experimental AKI, a topic of many good reviews (8, 9). In this review, we will discuss recent data showing that danger signals and PRRs are involved in cell fate decisions, metabolism, and mitochondrial function in TECs, thereby determining not only whether a pro- or anti-inflammatory phenotype will emerge, but also the success of regeneration and repair. We specifically intend to highlight the link between metabolism and innate immunity in TECs and the altered cell phenotype that occurs during experimental and human ischemia reperfusion injury (IRI) induced AKI.

#### AKI AND EXPERIMENTAL ISCHEMIA-REPERFUSION INJURY

The pathophysiology of AKI is complex. One of the major causes of AKI is IRI (10, 11). IRI is an inevitable event during renal transplantation and is responsible for delayed graft function (DGF), resulting in loss of vital kidney parenchyma and priming of adaptive immune responses that initiate rejection, which altogether lead to graft loss (10, 12). DGF is a clinical syndrome defined as the need for renal replacement therapy in the early phase after transplantation in order to support the function of the newly acquired renal transplant (12). Besides renal transplantation, IRI can also develop in the context of other diseases with low perfusion and/or oxygenation states, including thrombotic diseases, sepsis, trauma, and cardiac surgery (13). Experimental IRI in the mouse is the most widely used preclinical model to mimic human AKI. Despite having many limitations, particularly regarding the immune response (14) it is still the most valuable tool for understanding AKI pathophysiology.

### The Early Phase of Experimental IRI Is Characterized by Programmed Cell Death That Activates a Pro-inflammatory Innate Immune Response

In experimental IRI, following the hypoxic event, TECs, especially the proximal TECs located in the S3 segment of the nephron at the cortico-medullary area, are unable to maintain adequate intracellular ATP levels for the essential processes mentioned earlier. Additionally, restoration of blood Tammaro et al. Innate Immunity in Renal IRI

perfusion, followed by re-oxygenation of the kidney, provokes the production of reactive oxygen species (ROS), eliciting mitochondrial dysfunction. This, together with the ATPdepletion, leads to cell activation and injury, and if severe enough, can lead to programmed cell death (PCD) and secondary necrosis, the hallmark of early IRI (15).

PCD is essential, not only for the maintenance of cellular homeostasis but also in response to irreparable damage caused by injury or disease (15–17). The pathways of cell death execution can present as a diverse morphological pallet in the spectrum from apoptotic to necrotic, with a corresponding degree of danger-associated molecular pattern (DAMP) release and inflammatory potential (18–20). DAMPs are a heterogeneous group of ligands, which are constitutively expressed in different biological compartments of cells hidden from the innate immune system. DAMPs are recognized by a broad spectrum of PRRs, in order to elicit an innate inflammatory response (19).

It has been observed that the severity of the injury determines the path by which cell death is realized, indicating a possible role for PRRs in guiding cell fate decisions by integrating signals from the microenvironment (21). Various currently recognized forms of PCD (18, 22), including necroptosis, pyroptosis, ferroptosis, mitochondrial permeabilization transition (MPT) mediated regulated necrosis, and parthanatos are initiated as a response to hypoxic injury, either directly or indirectly, and blocking their crucial pathways during the early phase of IRI generally leads to reduced necrotic (tubular) damage, reduced inflammation, preservation of renal function, and reduced mortality (15). PRRs are known inducers of PCD, particularly necroptosis, pyroptosis, and apoptosis, which is why these modalities are discussed in more detail below.

## Pattern Recognition Receptors Are Involved in Cell Death Signaling During IRI

In necroptosis, receptor-interacting protein (RIP) kinase-1 and 3 interaction via their RIP–homotypic interacting motif (RHIM) (23) leads to phosphorylation and activation of RIPK3. In turn, RIPK3 catalyzes the phosphorylation and oligomerization of mixed lineage kinase domain–like (MLKL) (24), thereby inducing a molecular switch that leads to plasma membrane rupture and cell death (23–26). Blocking components of the necroptosis pathway in a similar renal phenotype of reduced tubular necrosis, reduced inflammation, better preservation of renal function, and reduced mortality in the first period after reperfusion (27). A recent report by Chen and colleagues showed, in a chimeric bone marrow transplantation model, that in the early phase of IRI, RIPK3 and MLKL in kidney parenchymal cells (including proximal TECs, as shown by in vitro studies) are important for initiation of the vicious inflammatory circle, but that pyroptosis in macrophages is more important in the later stage after reperfusion, suggesting temporal variation in cell death modalities during the course of IRI (28). Pyroptosis is a necrotic form of cell death most often observed in immune cells, such as macrophages and dendritic cells (DCs) (18). During pyroptosis, the presence of DAMPs initiates inflammasome formation, which activates both caspase-1 and caspase-11 (29–31). An effector function of these caspases is to process the inactive precursors of IL-18 and IL-1beta, leading to an intracellular accumulation of pro-inflammatory cytokines (31). These caspases also induce plasma membrane rupture, and essentially cell death, through the cleavage of gasdermin D (GSDMD) (32). The inevitable release of IL-18 and IL-1beta makes this form of cell death highly inflammatory (33). There is some debate as to whether pyroptosis occurs in renal cells as well, however, Yang et al. suggest the occurrence of pyroptosis in TECs based on a significant increase in pyroptosis-related proteins following IRI (34). A recent report by Miao et al. suggests the direct involvement of pyroptosis in IRI and cisplatin toxicity based on Casp11 KO mice (35). In additional experiments they showed that in Gsdmd KO mice, renal tubular damage was less severe, and urinary IL-18 levels were reduced upon cisplatin toxicity (35). Although very suggestive, we do not know whether Gsdmd KO mice have the same phenotype in IRI compared to cisplatin toxicitiy in vivo. Apoptosis is considered a more quiescent form of regulated cell death due to the swift clearance of apoptotic bodies by phagocytes. Apoptosis is initiated through an intrinsic route via mitochondrial outer membrane permeabilization (MOMP) or an extrinsic route via death or dependence receptors (36, 37). Death receptors can initiate apoptosis via cognate ligand binding (e.g., FAS or TNFR1 signaling) whereas dependence receptors initiate apoptosis when there is a lack of ligand binding (i.e., reduced homeostatic survival signaling via e.g., the netrin 1 receptors) (38). After initiation, executioner caspases-3 and−7 are responsible for neat cellular and nuclear fragmentation during apoptosis, releasing "find-me" signals, and flagging apoptotic bodies to be phagocytosed via "eat-me" signals (39). Apoptosis is believed to play a minor role in the early proinflammatory phase after reperfusion. Multiple reports have shown that pharmacologically inhibited apoptosis by a pancaspase inhibitor zVAD or genetic KO of executioner caspase-3 did not reduce but rather exaggerated renal tubular damage and failure (40, 41). In the long-term, Casp3 KO mice appeared to have less peritubular capillary rarefaction, less activated interstitial fibroblasts, less interstitial fibrosis, and evidence of less tubular hypoxia after reperfusion, suggesting a potentially interesting link between late peritubular capillary apoptosis and endothelial-mesenchymal transition and/or pericyte-fibroblast transdifferentiation (41). PRRs can initiate regulated cell death in multiple ways. Toll-like receptor (TLR) signaling via MYD88 results in activation of NFkB, transcriptionally regulating multiple cytokines that can subsequently induce regulated cell death via para- and autocrine signaling to death receptors. However, a more direct route of cell death initiation by TLRs is via Toll/IL-1R domain-containing adaptor-inducing interferon (IFN)-beta (TRIF). TRIF can initiate apoptosis via FADD- and caspase-8-dependent pathways. TRIF also contains a RHIM domain, and could therefore function as a docking site for the RIPK3-MLKL complex during necroptosis initiation (42), as was shown for TLR3 (43). TLR-TRIF-induced active caspase-8 was able to cleave Gasdermin D in macrophages inducing pyroptosis (44), suggesting the bypassing of the inflammasome in these cells.

## (MAL)ADAPTIVE REPAIR RESPONSES AS A MODEL FOR AKI-TO-CKD PROGRESSION

Tubular regeneration and successful renal repair after an episode of AKI can be observed in the majority of surviving patients, especially in cases of mild injury (45–47). Adaptive tubular repair depends on the presence of an appropriate microenvironment, in which inflammation and tubular response to damage are balanced. In the adaptive repair, surviving TECs undergo dedifferentiation and proliferation in order to restore a functional epithelium. However, in case of severe or repetitive injuries or aged kidneys, maladaptive repair of proximal tubules can occur, which can contribute to progressive renal fibrosis (47). Maladaptive repair of kidney tissue after AKI is characterized by rarefaction of peritubular capillaries, interstitial fibrosis and tubular atrophy, glomerulosclerosis, and vascular remodeling, which interfere with repair and eventually lead to a decline in renal function. Therefore, AKI-to-CKD should be regarded as accelerated renal aging (47, 48). As the determinants of renal aging and CKD overlap (49), identifying patients with premature renal aging could be a strategy to identify AKI survivors at risk for CKD.

Among the culprits in the AKI-to-CKD progression, is the persistence of a senescent state in TECs (45). Senescence describes a proliferative arrest with changes in chromatin organization, gene transcription, and protein secretion, which can occur as a response to cell stress and aging (50). Given the high degree of pro-inflammatory molecules released by senescent TECs, it remains elusive whether progressive accumulation of senescent TECs is causally related to an aberrant innate immune response. Recent results from our group and others (51–53) point toward a role for TEC-mediated inflammation, innate immunity, and mitochondrial metabolism in senescence and fibrosis. Given that these mechanisms fall into the new discipline of immunometabolism, and TECs can be regarded as part of the renal innate immune system, further studies are required to characterize the role of immunometabolism in AKIto-CKD progression.

#### Innate Immune Receptors as Gatekeeper of Damage and Repair

The role of the innate immune system was originally to combat infections. However, we now know that its role extends beyond that to include the surveillance of tissue homeostasis, by detecting distinct DAMPs released during tissue injury. Our group pioneered the discovery of TEC-associated innate immune sensors having a crucial role in the initiation of the injury response during IRI, shedding a novel light on the role of TECs as innate immune cells of the kidney (54). Experimental data suggests that the DAMPs released by necrotic cells activate the inflammatory signal initiated through TLRs, and their synergistic receptors, the Nod-like receptors (NLRs) and the NLRP3 inflammasome (55, 56). Initial considerations suggested that pharmaceutical intervention to block TECinduced innate immune cell signaling could lead to novel therapeutics against renal tissue inflammation and injury after IRI (54, 57). However, in the last decade, seminal studies on the role of innate immune sensors and their ligands in renal IRI have provided an additional prospective: the innate immune sensors translate kidney injury into an immune response, essential for shaping adaptive tubular repair and kidney regeneration. Thus, the tubular innate immune response to IRI seems to be a very well-orchestrated phenomenon (58). After the early inflammatory phase, macrophage populations assume a reparative phenotype that is characterized by the production of numerous growth factors, including Platelet-derived growth factor (PDGF), Transforming growth factor beta 1 (TGF-beta1), Insulin-like Growth Factor I (IGF-1), and Vascular endothelial growth factor A (VEGF-A) that promote tubular regeneration (59–61). Long-term sustained inflammation is detrimental, but the absence of the inflammatory response can also predispose to the development and progression of CKD (8). Whether this is dependent on a faulty inflammatory response or the inability of TECs to regenerate due to extensive damage is still not completely understood. Improving our understanding of the role of innate immune receptors, not only in the early pro-inflammatory phase during IRI, but also in (mal)adaptive tubular repair, is therefore crucial for the development of specific therapies or prevention of AKI and its detrimental sequelae.

## Mitochondrial Dysfunction and Metabolic Reprogramming

Mitochondrial injury, fragmentation, and ROS generation induce aberrant tubular inflammation and are central mediators of AKI, as well as the AKI-to-CKD progression (62–66). Oxygen tension in TECs is crucial for proper mitochondrial function. When this becomes inadequate, mitochondrial respiration is inhibited and the kidney undergoes a metabolic rewiring toward glycolysis, thereby decreasing ATP production (67). Mitochondrial number and integrity (through fission and fusion processes) (65, 66) are associated with accelerated tubular repair and improved survival after IRI, suggesting that in order to achieve adaptive tubular repair, restoration of mitochondrial homeostasis is an indispensable event (64). Thus, metabolic flexibility seems to be a crucial ability of the tubular epithelium to quickly adapt to the hypoxic environment. However, in order to promote repair, TECs should be able to return to their primary fuel source, and when this flexibility is impaired and metabolic rewiring persists during repair, this leads to a failed re-differentiation and mesenchymal arrest (68).

Hypoxia is likely not the only cause of reduced oxygen availability in the kidney. The widening of interstitial spaces by edema and inflammation and the regression of capillaries during fibrosis could also be involved. Indeed, pathologic hypoxia persists as fibrosis develops and could thus prevent epithelial recovery through feedback effects, ultimately leading to tubular atrophy (69). Consequent to hypoxia, tubular repair after AKI could be impaired by oxidative stress and growth arrest or senescence, which represents the known adverse effects of hypoxia (51, 52, 70, 71). TECs rely mostly on fatty acid oxidation (FAO) for their functions (72), which requires mitochondria and oxygen for efficient ATP generation. All the enzymes required for FAO reside in the mitochondrial matrix, and proper functioning of the mitochondrial cristae is necessary to provide substrates for the respiratory complex. Recently, several groups have reported that mitochondrial function and energy metabolism are involved in the progression from AKI-to-CKD.

Within mitochondria, the coenzyme Nicotinamide adenine dinucleotide (NAD+) carries high-energy electrons from FAO to the electron transport chain. NAD+ is, therefore, a ratelimiting catalyst for FAO (67). In other terms, decreased NAD+ availability results in impaired energy metabolism in these cells (73). Hypoxia and aging (74, 75) have been known to induce NAD+-consuming enzymes, which lowers NAD+ availability. Tran and colleagues recently showed that renal tubular cell NAD+ levels are suppressed in IRI-induced AKI, and this reduction in NAD+ may impede FAO, reduce ATP generation, and elevate susceptibility to AKI stressors (76). They identified a novel function of the mitochondrial biogenesis regulator PPAR-gamma-coactivator-1alpha (PGC1-alpha) to induce the enzymes that sequentially convert the amino acid tryptophan to NAD+, after IRI. Tubular PGC1-alpha expression protects against hypoxia-related stress and enhancing NAD+ could effectively mimic PGC1-alpha's effects in the tubule (76). Interestingly, biopsies of human AKI showed reduced PGC1 alpha expression (77).

The same group, through a metabolomics study, has demonstrated the elevation of urinary quinolinic acid (uQuin) in murine AKI. Quin becomes NAD+ through the action of quinolinate phosphoribosyltransferase (QPRT) and subsequent enzymes. The elevation of uQuin suggested suppression of QPRT during AKI. Genetic targeting of Qprt showed enhanced susceptibility to IRI and recapitulated the majority of the urinary metabolic changes measured in experimental AKI (78). Additionally, few other studies described that either de novo NAD+ biosynthetic pathway activation (79) or replenishment by means of NAD+ precursors (80) are able to protect the kidney from ischemic damage. This is of great importance given that in mammals, only the kidney and the liver exhibit appreciable de novo NAD+ biosynthesis (79, 81). Taken together, these encouraging studies points toward a therapeutic potential of NAD+ enhancers in AKI and its longterm sequelae.

Apart from PGC1-alpha, the AMP-activated protein kinase (AMPK) is a promising component of a signaling cascade that may modulate the severity of ischemic injury (82). AMPK is a ubiquitously expressed serine-threonine kinase that serves as an important intracellular energy sensor. It is activated by conditions that deplete ATP and alter the AMP:ATP ratio, including ischemia and glucose deprivation. AMPK stimulates FAO, glucose uptake, and glycolysis, while downregulating ATPutilizing systems. Other targets of AMPK include pathways modulating inflammation, apoptosis, angiogenesis, blood flow, and maintenance of cell polarity, with or without energetic stress (83). Metformin, a widely used drug for the treatment of type II diabetes mellitus, enhances AMPK activity (84). AMPK preactivation partially ameliorates renal IRI in vivo but also longterm sequelae after IRI (84).

Defective FAO in TECs plays a pivotal role in renal aging (85) and fibrosis (72), but studies directly linking defective FAO to renal IRI are currently missing. The Susztak group showed that the dramatic repression of FAO is induced by TGF-beta signaling (72), which seems to play a role in the early events of renal IRI, therefore, it would be interesting to investigate whether failure to fully return to FAO fuel after IRI might be underlying the AKIto-CKD transition, or whether defective FAO is associated with a senescent tubular phenotype. Although fatty acid accumulation has been shown to be associated with lipid deposition, this is not per se enough to drive fibrosis.

Interestingly, TGF-beta1 stimulates the Warburg-like metabolic reprogramming in kidney cells, which is relevant because it mirrors the metabolic state during AKI (72). Metabolic reprogramming, toward glycolysis, rapidly generates ATP and involves the enzyme Pyruvate Kinase M2 (PKM2), which is involved in the last step of glycolysis (86). Zhou and colleagues found that disabling PKM2 resulted in a significant increase in cell-repair and a concomitant decrease in energy generation, leading to significant protection against kidney injury in mice (87). A key molecule in this process is nitric oxide (NO), which can be transported to different proteins through Co-enzyme A, thereby, switching off their activity. PKM2 is one such protein. Indeed, adding NO to PKM2 activates repair, suggesting it as an important mechanism that can be used to determine whether kidney cells are using their pathways for energy or repair. The same team found that a protein called AKR1A1 could remove NO from PKM2, thereby switching it on and re-activating a robust energy-generating process. Disabling AKR1A1 protected the kidney from disease by stimulating repair (87).

It has been well-established that mitochondrial dysfunction is a principal mediator of AKI through decreased ATP production, oxidative stress, mitochondrial DNA (mtDNA) release, and cell death. Mitochondria also play a crucial role in maintaining organelle function in cells. Organelle stress and crosstalk in the AKI-to-CKD transition has been recently reviewed (64). The ROS generated upon re-oxygenation and the inability to maintain endogenous antioxidant levels, results in mitochondrial oxidative stress and promotes AKI (88). Enhancement of the antioxidant defense via mitochondria-targeted approaches has been successful in ameliorating IRI-induced AKI. These have been summarized in **Table 1**. Additionally, a visual representation of the phenotypic changes occurring in TECs after IRI and the majority of the mechanisms described in this chapter can be found in **Figures 1**, **2**.

## Senescence-Mediated (Mal)adaptive Repair

Kidney regeneration after IRI is accomplished by active tissue repair, in which the role of macrophage phenotype is crucial, as described in recent work (96). A balanced inflammatory response, together with a moderate level of damage, establishes an optimal microenvironment in which surviving TECs fully repair (3, 97). In this scenario of adaptive repair, the kidney regenerates over the course of a few days without scarring. The progression

of AKI-to-CKD is the result of maladaptive tubular repair, which can be mediated by TEC senescence.

The hypoxic injury activates a DNA damage response (DDR) in TECs [Ataxia-Telangiectasia, Mutated(ATM)/ATM and Rad3 related(ATR) pathway] which results in cell cycle arrest (71, 98– 100). This allows TECs to repair the damage and avoid further amplification of the vicious cycle of injury-induced cell death. ATR, an enzyme involved in DDR activation has been shown to protect against maladaptive tubular repair (100), further demonstrating a crucial role for DDR activation in TEC repair.

One of the consequences of irreparable DNA damage and mitochondrial dysfunction, especially in cells with a high energy demand, is a proliferation arrest termed "senescence" (101). Senescence is generally regarded as an irreversible event (102); however, there are reports that describe senescent cells being able to re-enter the cell cycle, suggesting that more research is needed to understand which scenario determines the fate of these cells (103). Senescent TECs, identified by different markers, with Senescence-associated beta-galactosidase (SA-βgal) and the anti-proliferative proteins p16 and p21 being the most common, seem to accumulate acutely following experimental and human IRI (53, 104), as a consequence of oxidative stress, the so-called stress-induced senescence. In vivo studies using the renal IRI model showed that elimination of these cells might hinder regeneration (104, 105). Indeed, senescence also plays a key role in the healing of wounds, tissue repair, and during embryonic development (106). Acute and transient senescence, where damaged cells are eliminated, clearly has beneficial effects for an organism and in the adaptive tubular repair after IRI. Paradoxically, subtle accumulation of senescent cells (chronic senescence), instead, impairs the kidney's regenerative capacity, leading to AKI-to-CKD transition (52, 107, 108).

In this scenario, the use of senolytics (aimed at clearing senescent cells) seems a promising therapeutic strategy (49). However, given the non-specificity of current senescence markers and the existence of different senescence programs, elegantly reviewed by the De Maria group, more research is needed for this novel potential therapeutic field (109). The intervention strategies aimed at senescent cell removal or modulation, which seem to be effective in limiting the progression of IRI, have been reviewed previously (104). Nonetheless, having a specific biomarker for tubular senescence burden could be an effective way to stratify patients that could benefit from the use of senolytics.

Despite being arrested in the cell cycle, senescent cells remain metabolically very active and become fibrogenic due to their innate ability to create a pro-inflammatory environment and secrete pro-fibrotic molecules, components of the senescence-associated secretory phenotype (SASP) (110). Activation of SASP in TECs leads to activation and proliferation of fibroblasts and perivascular pericytes, which in turn induce extracellular matrix production and tubulointerstitial inflammation, with impaired M2 macrophage conversion (47, 71, 96). Perhaps redirecting the metabolism of senescent cells could be an effective way to turn their detrimental phenotype and fate. Recent studies have indeed highlighted the plasticity of these cells, being able to re-enter the cell cycle, posing a new challenge to the postulated theory, that cells can instead be re-programmed to change their cell fate (111).

In addition to the common components of the SASP, senescent cells produce exacerbated levels of mitochondrialderived ROS and might also release mtDNA, which can act as a DAMP, further fueling PRR activation (101, 112). Collectively, they amplify the vicious cycle of inflammation, mitochondrial dysfunction, and senescence, ultimately leading to maladaptive tubular repair. Our group has recently described that the innate immune receptor TREM-1 links mitochondrial dysfunction, tubular senescence and maladaptive repair after AKI. This is the first study linking epithelial immunometabolism to cellular senescence in the context of IRI (52).

However, if persistent, this leads to a pro-inflammatory and profibrotic secretome, ultimately leading to fibrosis.

#### Epithelial Immunometabolism

TECs possess an incredible plasticity. By tightly controlling their metabolism, they are able to anticipate and adapt to constantly changing environments in both health and disease. Activation of the innate immune system and metabolic reprogramming are tightly linked (113). The theory of immunometabolism has been increasingly appreciated to drive effector functions in immune cells. Despite TECs being regarded as innate immune cells of the kidney, research on epithelial immunometabolism is still in its infancy, particularly in the context of renal IRI (113). In the last 2 years, our group has found that the metabolic choice of TECs is driven by either membranebound or intracellular receptors of the innate immune system, proposing the involvement of epithelial immunometabolism in the pathogenesis of IRI and its detrimental consequences (51, 52).

The effect of renal injury on cellular metabolism has been well-established in various AKI models. The induction of AKI with mercuric chloride results in increased glycolytic activity (114), while FAO has been reported to be reduced in folic acid nephropathy (72). The general consensus is that such metabolic shifts are likely necessary to facilitate the initial recovery process; however, their effects may become deleterious when the altered metabolic state persists. Specifically, numerous studies have reported the damaging effects of increased glycolytic activity in TEC repair following IRI. Increased levels of glycolysis and glycolytic enzymes have been observed in TECs that failed to redifferentiate and underwent atrophy (68). As mentioned earlier, Zhou et al. reported the renoprotective effect of PKM2 inhibition by the S-nitroso-CoA reductase system following bilateral IRI.

This obstruction of the glycolytic pathway was accompanied by a shift toward the pentose phosphate pathway (PPP), which generates Nicotinamide adenine dinucleotide phosphate (NADPH) and aids in the supplementation of antioxidant reservoirs, thereby offering protection against ROS-induced injury (87). Similarly, Kim et al. report the renoprotective effect of TP53 Induced Glycolysis Regulatory Phosphatase (TIGAR) activation in IRI, which leads to a metabolic shift away from glycolysis and employs the redox protection of the PPP (115). These studies emphasize the need for 'metabolic fluidity' while simultaneously displaying the need for strict metabolic control during IRI. Results from our group and that of others have shown the effect of TEC-specific expression of PRRs during IRI (8), while our recent reports demonstrate the direct involvement of innate immune signaling (via NLRX1 and TREM1) in regulating TEC metabolism following IRI (51, 52). Altogether these data show the inextricable connection between innate immune signaling and cellular metabolism in TECs, which ultimately defines their fate following an acute insult. It is for these reasons that immunometabolism should be recognized as a crucial mechanism through which TECs respond to, and recover from, IRI.

## PRRS, THE INFLAMMASOME AND THEIR LIGANDS IN EXPERIMENTAL AKI

### Toll-Like Receptors

In addition to their function as gatekeeper, protecting against micro-organism invasion, TLRs can be activated by a wide range of DAMPs released upon tissue injury. Upon IRI, tubular necrosis occurs together with the release of potential DAMPs, including High mobility group box 1 (HMGB1), histones, heat shock proteins, S100A8/A9, hyaluronic acid, biglycan, mtDNA, ROS, and ATP, which can function as ligands for PRRs. The type of ligands recognized by a specific TLR is partially dependent on their intracellular localization. One group can be found on the cellular surface and includes TLR2 and TLR4 and the other group, including TLR3, TLR7, TLR8, and TLR9, is expressed in intracellular vesicles such as endosomes, lysosomes, and the endoplasmic reticulum. Upon ligand recognition, the intracellular TIR-domain of a TLR functions as a scaffold for the recruitment of specific adaptor proteins via homotypic interactions between their TIR-domains.

Signaling pathways activated downstream of these adaptor molecules promote the expression of pro-inflammatory cytokines, chemokines, and type I and type III IFNs. Although TLRs provide protection against a wide variety of pathogens, inappropriate or unregulated activation of TLR signaling can lead to chronic inflammatory and autoimmune disorders (116, 117).

#### TLR2/4

Tlr2 and Tlr4 mRNA is constitutively expressed by TECs and is upregulated upon IRI in mice (118). Both Tlr2 and Tlr4 overexpression in TECs after IRI induced an exaggerated inflammatory response, resulting in impaired renal function (8, 57, 119). Antisense oligonucleotides targeting Tlr2 protected renal tissue and function after IRI in mice. Compared to the single KO mice, double Tlr2 and Tlr4 KO animals showed no additional protective effects (120). Likewise, mice deficient for Myd88 or Trif were not significantly protected against IRI compared to WT animals (119). This might be the consequence of adaptive mechanisms in these genetically modified animals.

Inhibition of TLRs must take place in the acute phase of injury and cannot be longstanding. Indeed, it has been shown that TLR4 blockade during the recovery phase after IRI slows down the process of tubular repair after IRI in rodents (58). In light of these data and in the context of our review, we believe that inhibiting the endogenous ligands, rather than the receptor itself, might be a safer approach.

#### TLR2/4 Ligands

One such potential ligand is HMGB1. HMGB1 is a nuclear factor that is highly and ubiquitously expressed in nearly all cell types. Upon renal IRI, HMGB1 is overexpressed, and secondary to cell injury, HMGB1 can be leaked into the circulation and activate TLR2, TLR4, and TLR9 (see below). We and others showed that treatment with anti-HMGB1 antibodies reduced renal injury, inflammation, and dysfunction in a murine model of IRI (121, 122). This protective effect of anti-HMGB1 treatment was confirmed in a model of cold and warm renal IRI in miniature swine (123). Another potential agonist for TLR4 is uromodulin. Uromodulin, or Tamm-Horsfall protein, is a highly glycosylated protein, normally secreted by epithelial cells of the thick ascending limb of Henle's loop in the intraluminal compartment. The function of uromodulin remains elusive, but data suggest that uromodulin might prevent the formation of kidney stones and urinary tract infection (124). In vitro, uromodulin has been shown to activate TLR4 signaling in myeloid DCs and bone marrow-derived macrophages (125). Upon kidney injury, uromodulin can leak into the renal interstitial space, potentially leading to the activation of intrarenal resident macrophages and DCs. In contrast with our expectations, uromodulin-deficient mice have markedly fewer resident macrophages. Upon IRI, these uromodulin-deficient mice exhibit aggravated renal injury and impaired polarization of macrophages toward an M2 healing phenotype (126). The discrepancy between in vivo and in vitro data might eventually be due to the different forms of uromodulin, i.e., full-length or truncated, monomeric or aggregating form.

Histones released from dying cells were also found to exacerbate renal tissue injury in AKI in a TLR2/TLR4 dependent manner, and administration of anti-histone antibody suppressed renal inflammation and injury and improved renal function (127).

Among other DAMPs, the calcium binding proteins (S100A8/A9), ligands of TLR4 and the RAGE receptor, are released following renal IRI and seem to play a pivotal role in orchestrating the repair response after hypoxic damage. During the initial injury phase, immune cells, particularly macrophages, are polarized toward an M1-like phenotype and produce pro-inflammatory cytokines, such as TNF-alpha and IL-6, which exacerbate inflammation. However, in a later phase, they switch their phenotype to a more reparative one, the M2-like phenotype, which secretes anti-inflammatory factors, determining the resolution of inflammation and stimulating tubular regeneration (128). Our group showed that S100A8/A9 proteins play a role in this process of macrophage polarization. By controlling excessive M2 polarization, S100A8/A9 fine-tunes the adaptive response of the kidney to IRI-induced AKI (56).

#### TLR3

TLR3 is activated upon the binding of single or double-stranded RNA and induces an antiviral immune response, characterized by the production of type I IFNs. TLR3 is constitutively expressed in mouse and human TECs and is activated earlier (few minutes) than TLR2 and TLR4 after IRI. In a model of bilateral IRI, Tlr3-deficient mice were significantly protected against kidney injury as indicated by less inflammation, less tubular apoptosis and necrosis, and preserved renal function 24 h after reperfusion (129). How TLR3 is involved in IRI remains speculative. Since Tlr3 KO mice had diminished TRIF protein expression, reduction of necroptosis might be one explanation. Also, type 1 IFN-mediated auto- and paracrine activation of IFN receptors might contribute, for instance, to pyroptosis via transactivation of caspase-11 (130, 131). The effects of TLR3 in the later phases of reperfusion were not studied in this paper.

#### TLR3 Ligands

Double-stranded RNA released upon viral replication is the major ligand of TLR3, but TLR3 can also recognize mRNA and mRNA-protein complexes released by necrotic cells (132). Therefore, it is tempting to speculate that both mRNA and mRNA-protein complexes might amplify the pro-inflammatory loop via their interaction with TLR3 upon renal IRI. However, scientific evidences are still lacking.

#### TLR9

TLR9 is a cytosolic DNA sensing receptor and has evolved to detect unmethylated CpG DNA, commonly found in microbial DNA and DNA viruses, and initiate the production of type I IFN and proinflammatory cytokines. TLR9 is highly expressed in professional innate immune cells, such as plasmacytoid DCs and macrophages but also in the kidney. In a model of moderate renal IRI, TLR9 was not involved in renal dysfunction (133). In contrast, in a model of severe IRI, we showed that Tlr9 deficiency resulted in improved survival in mice but not in the improvement of renal function and kidney damage (134). Improved survival was associated with reduced plasma mtDNA

content and a subsequent decrease in hepatic injury. Surprisingly, and somewhat in contrast with our study, a recent study by Han et al. showed that selective intestinal TLR9 deficiency led to increased ischemic AKI and was associated with remote intestinal and hepatic injury. Intestinal Tlr9 deficiency was associated with enlarged Paneth cell granules and increased IL-17A expression (135). We would expect that Tlr9 KO animals would also present with this phenotype, but caution must be applied when interpreting scientific results generated by geneticallymodified animals.

The mitochondrial dysfunction, cellular stress, and cell death involved in renal IRI result in the liberation of mtDNA, both in murine models of IRI and after renal transplantation in humans. Recently, our group reported a correlation between urinary mtDNA levels and the occurrence of DGF following renal transplantation (136). Outside of the mitochondrial matrix, mtDNA acts as a DAMP that can elicit neutrophil-mediated injury through TLR9, and other receptors (137). Indeed, the cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is also able to recognize cytosolic DNA. Cytosolic cGAS binds double-stranded DNA and catalyzes the production of the novel second messenger 2′-3′-cyclic AMP-GMP (2′3′-cGAMP) from ATP and GTP. The binding of cGAMP to the ER-resident protein STING releases DNA-triggered signals and activates the innate immune system (138, 139). Recently, Maekawa et al. showed that cytosolic translocation of mtDNA leads to tubular inflammation via the cGAS-STING pathway, linking mitochondrial dysfunction to enhanced inflammation (112). cGAS-STING is activated in the cortex of animals 24 h after renal IRI and Sting KO animals display mild tubular injury and inflammation. Therefore, we cannot exclude the possibility that the absence of reprotection in Tlr9 KO animals, described in (134) after renal IRI, can be explained by an additional contribution of cGAS-STING. As cGAS-STING is activated in other pathogenic processes such as renal fibrosis (140) and senescence (141), further study into its effect on tubular repair will shed more light on the role of immunometabolism in AKIto-CKD progression. Altogether, these studies strongly suggest that mitochondrial protection and a decrease in oxidative stress can be an elegant way to prevent AKI.

#### TREM-1

Triggering receptor expressed on myeloid cells-1 (TREM-1) is an activating receptor located primarily on cells of the innate immune system and some parenchymal cells (142). TREM-1 signals through its adapter protein, DNAX-activating protein (DAP12), to activate transcription factors capable of inducing the expression of pro-inflammatory cytokines and chemokines. TREM-1 can initiate inflammation, but can also work synergistically with TLRs to enhance an inflammatory response. Although initially studied in the context of infectious diseases, TREM-1 is also active in sterile inflammation (142). TREM-1 seems to be a hypoxia-inducible gene in myeloid DCs and TECs (52, 143) and might be involved in regulated cell death through amplification of inflammatory signals leading to necroptosis and pyroptosis, as has been shown in brain microglia (144). Interestingly, however, TREM-1 was also shown to mediate an inhibitory effect on necroptosis and pyroptosis in neonatal lung tissue, as suggested in previous work by Syed et al. (145). In our hands, in the early phase of renal IRI, TREM-1 modulation did not affect tubular damage or renal function (146). However, we did find that mice lacking TREM-1 displayed maladaptive repair characterized by persistent tubular damage, inflammation, fibrosis, and mitochondrial dysfunction-induced TEC senescence when exposed to renal IRI (52).

## Inflammasomes

#### NOD1/2

Nucleotide-binding oligomerization domain (NOD) 1 and 2 are part of the NOD-like receptor (NLR) family of cytoplasmic PRRs. NOD1/2 can either initiate immune responses to pathogenic invasion through the recognition of PAMPs or sterile inflammation in response to DAMPs released upon cell stress (8). Both NOD1 and NOD2 contain 3 basic structural units: a NOD region, a caspase recruitment domain (CARD), and a ligand-binding domain. Once activated through ligand binding, the resulting oligomerization leads to the binding of signaling molecules to CARD. NOD1/2 activation can lead to apoptotic signaling through RIP-like interacting caspase-like apoptosis-regulatory protein kinase (RIP2) or the release of inflammatory chemokines and cytokines (147). NOD1/2 are expressed on TECs in both mice and humans (148), and due to their structural resemblance to other NLRs and TLRs that are known to play a role in kidney disease, it is plausible that NOD1/2 may play a role in renal injury. Indeed, a study by Shigeoka et al. found that Nod1/2 double KO led to a reduction in apoptosis following IRI (148). Expression of the pro-inflammatory cytokines IL-6, KC, and TNF-alpha were also reduced in mice deficient for NOD1/2, leading to the general suppression of the inflammatory response to IRI. Interestingly, NOD2-deficient mice were better protected against renal injury than NOD1-deficient mice, indicating that although both NOD proteins are involved in renal injury, the underlying mechanisms may be different (148). Endogenous ligands for NOD1/2 remain unknown, therefore, their discovery would certainly aid in the development of a therapeutic intervention.

#### NLRP3

NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) is by far the best-characterized inflammasome-forming protein in the kidney. Once activated, the cytosolic innate immune receptor NLRP3 initiates the assembly of an inflammasome, leading to an inflammatory form of cell death (pyroptosis) and the proteolytic activation of the IL-1beta family of pro-inflammatory cytokines. The NLRP3 inflammasome can trigger inflammation by sensing a wide range of stimuli, but the specific mechanisms are still unclear. Among other factors, K+ efflux, ATP released from damaged mitochondria, and ROS production promote NLRP3 inflammasome activation (149).

Nlrp3 gene expression in murine kidneys increased after IRI and peaked 5 days after reperfusion, corresponding to the repair phase. Although the Nlrp3 gene was primarily expressed by leukocytes, TECs also expressed Nlrp3 after hypoxia or LPS stimulation (150). In a murine model of bilateral IRI, we showed that Nlrp3-deficient animals were protected against mortality, renal dysfunction, and displayed a reduced influx of neutrophils into the kidneys despite similar degrees of tubular necrosis. In this study, activation of NLRP3 was triggered, in part, through ATP produced by mitochondria released from necrotic cells (151).

Uromodulin has also been shown to activate the NLRP3 inflammasome in human peripheral blood mononuclear cells, leading to the secretion of IL-1beta. However, as previously mentioned, uromodulin-deficient mice displayed aggravated renal injury upon IRI. This illustrates the complex role of uromodulin in regulating inflammation.

The role of leukocyte- vs. renal-associated Nlrp3 expression has been studied in chimeric mice. In the early phase following IRI (day 1) only renal Nlrp3 contributed to renal dysfunction based on serum creatinine. In contrast, 5 days after reperfusion (repair phase) both renal- and leukocyte-associated Nlrp3 mediated loss of renal function. Interestingly, Nlrp3-deficient TECs showed increased proliferation and a superior repair response both in vivo and in vitro when compared to wildtype TECs (150).

More recently, it was shown that NLRP3 relocalizes from the cytosol to the mitochondria in TECs during hypoxia. The deletion of NLRP3 in TECs resulted in less mitochondrial ROS production, less mitochondrial damage, and less apoptosis in a model of in vitro hypoxia (152). Understanding the mechanisms of NLRP3 inflammasome activation will boost the development of small-molecule inhibitors for the treatment of NLRP3-related diseases (153).

#### NLRC5

NLR family CARD domain containing 5 (NLRC5) protein is a recently identified member of the NLR family that interferes with the assembly and activity of the NALP3 inflammasome complex by competing with ASC for pro-caspase-1 binding. Although NLRC5 activity leads to caspase-1 activation, induction of pyroptotic cell death dependent on NLRC5 has not yet been shown (154). Nlrc5 is significantly upregulated in the kidney 24 and 48 h after IRI. Nlrc5 deficiency significantly ameliorated renal function, injury, and inflammation 24 and 48 h after IRI. This was associated with less apoptosis in TECs and reduced inflammation in the kidneys (155).

#### NLRX1

In contrast to the hereinabove studied innate immune receptors, NLR family member X1 (NLRX1) exerts inflammasome independent anti-inflammatory effects by interfering with the canonical NF-kB signaling via inhibition of TRAF6 binding to IkB kinase (156). A unique feature of NLRX1 is its localization in the mitochondria. In 2017, we reported that NLRX1 protects against mortality and renal dysfunction after IRI by preventing excessive oxidative stress. We found that NLRX1 may act as an inhibitor of mitochondrial activity and prevents excessive oxidative stress, thereby preventing apoptosis of TECs during IRI (51).

Contrary to the studies by Zhou and Kim, we discovered renoprotective effects of glycolysis in IRI through our studies of NLRX1 (87, 152). Genetic deletion of Nlrx1 potentiates mitochondrial oxidative phosphorylation in TECs, while glycolysis results in enhanced oxidative stress after ischemia and ultimately increases cell death. Using a KO mouse model for acute renal IRI, we found that NLRX1 deficiency enhanced oxidative stress, thereby profoundly enhancing tubular apoptosis, renal dysfunction, and mortality, in the early days after IRI (51). However, the consequences of this metabolic rewiring toward glycolysis on epithelial repair have not yet been investigated. A proper investigation into our hypothesis on the renoprotective effects of NLRX1 in IRI would require NLRX1 activation. In silico studies have postulated polyunsaturated fatty acids as NLRX1 ligands, although further validation is required (157). As these are naturally occurring lipids, we propose that NLRX1 activation through nutritional supplementation may be a possible therapeutic approach to impede AKI-induced oxidative stress.

## INNATE IMMUNITY IN HUMAN IRI AND AKI Pattern Recognition Receptor Expression and Variation During Human IRI

Little is known about the direct in vivo role for PRRs in human AKI and specific data on the spatiotemporal dynamics of activation are lacking. Instead, most of the studies conducted in humans that investigated PRRs tried to identify single nucleotide variants related to certain outcomes as a weak proxy for a human knock-out or knock-in model, depending on the (often estimated) consequence of the genetic variant. We previously conducted a comprehensive screening of TLR single nucleotide variants (TLR1-8 + SIGIRR) that were of interest because of their estimated effect on protein function. In a cohort of over 1,000 matched donor and recipient DNA samples, neither donor (renal) nor recipient (inflammatory cells) genetic variants were associated with DGF and the calculated effect size was low for individual variants, even after stratification for deceased donor type or analyzing the effect of all TLR gene variants in bulk, as compared to a baseline prediction model of cold ischemia time, donor age, and recipient age (158). A smaller study from Germany was able to find a positive association between a TLR3 gene variant and DGF, although for the other TLR genes they were unable to find a relation with DGF (159). Furthermore, in a Brazilian study by Nogueira et al., again, no association between several TLR4 gene variants and DGF were found (160). In the same cohort, with a similar experimental setup, we were also unable to find NLRP3 and TREM1 gene variants associated with DGF (146, 161). These data altogether suggest that the potential impact of these PRR genetic variants do not determine whether a patient will develop acute tubular necrosis and DGF, even though they were estimated to result in a PRR protein anomaly. Redundancy among these relatively common PRR genetic variants might partly explain this lack of association, since complete knockout of the gene of interest is a very rare event and not covered in the variants tested. At the expression

level, polymerase chain reaction on implantation (time = 0) renal transplant biopsies showed a higher expression of TLR4 and MYD88 mRNA in deceased vs. living donor kidneys, but the authors could not find an association with the development of DGF (162). In an interesting study by McGuinness and colleagues from Glasgow, multi-omics analysis on pre- and postperfusion biopies (RNA sequencing, DNA methylation by whole genome bisulphite sequencing, and western blotting) identified a panel of expression markers associated with the development of DGF. They identified a transcriptional panel for DGF that was associated with innate immune signaling, TREM1 signaling, PRRs, and B cell development, with topranked networks related to immune system activation, cell death, and survival and cellular fitness. It seemed that cellular stress and restoration of physiological homeostasis (i.e., regeneration) was exacerbated in patients who developed DGF. At the protein level, DGF and perfusion status were associated with a state of cellular senescence (163). Altogether, the data from this study put PRR signaling, cell death and survival signaling, cellular stress and fitness, an exacerbated regenerative response, and cellular senescence at the center stage in the development of human IRI, and the authors suggest that these events probably occur independently of (minor) genetic differences in PRR. These conceptual findings seem to be in line with animal models.

## The MABSOT Project and the Human TLR2 Opsona OPN-305 Trial

As mentioned before, within the context of the EU FP7 MABSOT project, the humanized anti-TLR2 monoclonal antibody (OPN-305) was tested for the treatment of DGF after transplantation. In 2016, a phase I/II multicenter, randomized, double-blind placebo-controlled trial (NCT01794663) (164) in selected individuals at high risk for DGF showed that a preferred dose of 0.5 mg/kg (lowest dose tested) resulted in a lower percentage of DGF (primary endpoint; 26.5% in the treatment arm vs. 29.4% in the placebo arm) and functional DGF (failure of serum creatinine to decrease at least 10% daily; 38.2 vs. 52.9%, respectively) (165). Doses of 1.5 and 5 mg/kg appeared to be associated with a higher percentage of functional DGF, possibly in part due to the low number of DGF cases in the placebo group, according to the writers of the preliminary report. Data on long-term follow-up of the enrolled patients is pending, and a follow-up randomized controlled trial specifically looking at the clinical impact of the 0.5 mg/kg in recipients of an extended criteria donor kidney is planned according to the preliminary report. What can we learn thus far from this efficacy MABSOT trial as a first proof-of-concept for PRR blockade during human IRI? An interesting finding from the trial is the

efficacy of the lowest antibody dose tested. Mean duration of 100% TLR2 receptor occupancy for this dose was 1 day, whereas for 1.5 mg/kg this was 6 days, and for the highest dose this was even 13 days. The use of OPN-305 in the prevention of IRI is limited by the fact that it does not act specifically on TECs, and the systemic blocking of TLR2 in all cells might result in an increased risk for (severe) infections. It might be the case that TLR blockade in the reparative phase after IRI in humans results in delayed tubular regeneration and even maladaptive repair, increasing the risk of renal fibrosis and infections. Long-term follow-up, with respect to rejection rate and renal function, would be of great interest for further investigation. Efficacy came at the cost of serious infectious and renal side effects in a select number of patients, but these results are the first to suggest the therapeutic potential for early pharmaceutical manipulation of PRRs in the context of human renal IRI.

#### CONCLUDING REMARKS AND FUTURE OUTLOOK

This review described the main phenotypic changes occurring during renal IRI in TECs and the role of innate immunity in dictating inflammation and repair. The central dogma on the role of innate immunity in IRI still remains; excessive inflammation is just as detrimental as a faulty response, as both scenarios predispose to maladaptive repair and chronic progression (see **Figure 3**). We summarized the current evidence that the activation of innate immune receptors, in a very delicate balance (intensity, timing, cell types), is also necessary for adaptive repair. Moreover, here we have provided an additional

#### REFERENCES

and novel perspective about the role of epithelial metabolism and cell fate in IRI, which may be controlled by innate immunity.

As TECs have an incredible phenotypic plasticity and are among the most metabolically active cells, they are crucial for kidney function and homeostasis. Consequently, targeting epithelial immunometabolism holds great potential to alter the initiation of acute renal failure, but also progression towards CKD. As a final consideration, we are in a time where longevity is increasing and research on cellular senescence in the kidney, as a strategy to limit the AKI-to-CKD transition, is a fascinating area to explore for therapeutic potential. Unraveling differences between drivers of acute and chronic senescence will be essential to further investigate whether these cells could be trained to change their cell fate and function, in order to slow down the progression of kidney remodeling. Changes in mitochondrial homeostasis, metabolism, and function of innate immune receptors are known pathological mechanisms associated with aging, and thus could also take place in senescent TECs. Detailed studies on the hallmarks of senescent TECs in the different phases of renal IRI may shed light on novel mechanisms that can be targeted to redirect their phenotype and overcome the issue of repair by elimination. We envision that determining the therapeutic window of opportunity to regulate renal homeostasis by targeting innate immunity, immunometabolism, and cellular senescence should be among the future research goals in the field of nephrology and kidney transplantation.

## AUTHOR CONTRIBUTIONS

AT, JK, and SF designed and directed the project. AS contributed to the study. All authors contributed to the article and approved the submitted version.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Tammaro, Kers, Scantlebery and Florquin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Role of Natural Killer Cells in the Immune Response in Kidney Transplantation

Paola Pontrelli 1 \*, Federica Rascio2 , Giuseppe Castellano2 , Giuseppe Grandaliano3,4 , Loreto Gesualdo1 and Giovanni Stallone2

1 Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari Aldo Moro, Bari, Italy, 2 Nephrology, Dialysis and Transplantation Unit, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy, 3 Nephrology Unit, Department of Translational Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy, 4 Fondazione Policlinico Universitario "A. Gemelli" IRCCS, Rome, Italy

Natural killer cells (NK) represent a population of lymphocytes involved in innate immune response. In addition to their role in anti-viral and anti-tumor defense, they also regulate several aspects of the allo-immune response in kidney transplant recipients. Growing evidence suggests a key role of NK cells in the pathogenesis of immune-mediated graft damage in kidney transplantation. Specific NK cell subsets are associated with operational tolerance in kidney transplant patients. On the other side, allo-reactive NK cells are associated with chronic antibody-mediated rejection and graft loss. Moreover, NK cells can prime the adaptive immune system and promote the migration of other immune cells, such as dendritic cells, into the graft leading to an increased allo-immune response and, eventually, to chronic graft rejection. Finally, activated NK cells can infiltrate the transplanted kidney and cause a direct graft damage. Interestingly, immunosuppression can influence NK cell numbers and function, thus causing an increased risk of post-transplant neoplasia or infection. In this review, we will describe how these cells can influence the innate and the adaptive immune response in kidney transplantation and how immunosuppression can modulate NK behavior.

Keywords: natural killer cells, innate and adaptive immune response, kidney graft rejection, tolerance, immunosuppression

## PHENOTYPE, MATURATION, CYTOTOXIC ACTIVITY, AND DISTRIBUTION OF NATURAL KILLER (NK) CELLS

Natural killer (NK) cells are effector lymphocytes deriving from common lymphoid progenitors and represent 5–10% of circulating lymphocytes. NK cells are natural cytotoxic cells, but, unlike cytotoxic T lymphocytes, they do not require antigen exposure to mediate their effect (1). NK cells represent one of the main cellular components of innate immunity along with mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. They mediate immune responses against intracellular pathogens representing key mediators of the anti-viral and anti-neoplastic defense, but they also play a key role, through the production and release of several cytokines, in many inflammatory diseases, including acute and chronic kidney diseases (2–4). Interestingly, the role of this lymphocyte subset in the progression of kidney injury is starting to be uncovered (5).

#### Edited by:

Jan Fric, International Clinical Research Center (FNUSA-ICRC), Czechia

#### Reviewed by:

Gunnur Deniz, Istanbul University, Turkey Luuk Hilbrands, Radboud University Nijmegen, Netherlands

\*Correspondence: Paola Pontrelli<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Received: 14 March 2020 Accepted: 04 June 2020 Published: 23 July 2020

#### Citation:

Pontrelli P, Rascio F, Castellano G, Grandaliano G, Gesualdo L and Stallone G (2020) The Role of Natural Killer Cells in the Immune Response in Kidney Transplantation. Front. Immunol. 11:1454. doi: 10.3389/fimmu.2020.01454

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The NK cells accomplish their cytolytic effector activity through two main mechanisms of action (6):

In both cases, the lytic function of NK cells depends upon cytolytic molecules, mainly granzyme and perforin, and their activation leads to the production of several inflammatory cytokines (7). Granzyme and perforin are included into cytoplasmic lytic granules, characterized by several lysosomalassociated membrane glycoproteins (LAMPs) into the lipid bilayer. These proteins appear on cell surface after cytotoxic granules exocytosis (8). Among the different LAMPs, CD107a/LAMP-1 has been widely used as a functional marker to identify NK cell activity, since its expression is significantly higher on the surface of NK cells after MHC stimulation and correlates with both cytokine secretion and NK cellmediated lysis of target cells (9, 10). Interestingly, Conehn et al. demonstrated that CD107a/LAMP-1 protects NK cells from self-destruction upon target cell killing, since CD107a/LAMP-1 deficiency, both in human and in mice NK cells, increased NK cell apoptosis after degranulation (11).

The interaction of NK cells with the target cell can occur through distinct inhibitory or stimulatory receptors and, therefore, defines the fate of the target cell (12). Normal cells are protected from NK cell killing since stimulatory receptors signals are balanced by inhibitory receptors signals coming from the interaction with the self-molecules of the MHC class I complex. Neoplastic transformation or cellular infection can induce the expression of stimulatory ligands that overcome the inhibition induced by inhibitory receptors. In this case, an induced-cell recognition occurs (12). In many contexts both missing-self and induced-self recognition are likely to operate simultaneously to provide to NK cells the maximum ability to discriminate normal cells from transformed or infected ones.

NK cells can express on their surface different receptors able to recognize several polymorphic variants of MHC I molecules. Indeed, the human NK cell receptor repertoire is highly complex in each individual (13). In addition, NK cells express specific stimulatory and inhibitory receptors for various other ligands present on the surface of the target cells and the balance of inhibitory and stimulatory signals received by a NK cell determines the outcome of its interactions with target cells (14). These signals involve immunoreceptor tyrosinebased activation motif (ITAM)-bearing molecules and inhibitory receptors, other stimulatory receptors and adhesion molecules,

such as KIR, immunoglobulin-like transcript (LIR), leukocyteassociated immunoglobulin-like receptor (LAIR), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM) (14). Thus, the activation program of NK cells derives from the integration of activator and inhibitor signals, which varies according to the nature of the interacting cells. In addition, NK cells also express cytokines and chemokines receptors that are crucial for the regulation of NK cell functions and Toll like receptors that mediate the production of IFN-g and increase cytotoxicity (15).

NK cell maturation can occur in the medulla of secondary lymphoid organs, lymph nodes, tonsils, and spleen (16). The hematopoietic stem cells Lin–CD34+ can differentiate into multipotent lymphoid progenitors (LMPP) CD45RA+, expressing the stem marker CD34. The expression by multipotent lymphoid progenitors of CD38, CD7, CD10, and the cytokine receptor CD127 (IL-7 receptor-alpha) drives the transition to common lymphoid progenitors that, in turn, may generate progenitors of T cells, B cells, NK cells, and other innate lymphoid cells (ILCs). An ILC restricted progenitor can origin two main ILC lineages, Killer ILC and helper-like ILC (17). Helper-like ILCs express IL-7R-alpha, require GATA-3 for differentiation, and are composed of various cytokine-producing ILC subsets: ILC1, ILC2, and ILC3. ILC1 expresses and requires the transcription factor T-bet for lineage specification and produces large amounts of IFN-g (18). ILC2 produces type 2 cytokines and amphiregulin (19), thus driving type 2 adaptive immune responses through activation of Th2 cells, ILC3 express type 17 cytokines such as IL-22 and IL-17A (20). Similarities between helper-like ILC and T cell subsets led us to propose ILC as the innate counterparts of T cell subsets (21). The expression of CD122 (IL-2 receptor beta) marks the irreversible commitment of common lymphoid progenitors to give rise to conventional NK cells. Conventional NK cells and helperlike ILC1 can be distinguished by the expression of some transcription factors such as the T-box protein in T cells (Tbet) and Eomesodermin (Eomes) since mature NK are Tbet+ Eomes+ while ILC1 are Tbet+ Eomes-; however, the distinction between NK cells and other ILC populations also concerns their cytotoxic properties and other molecules (such as CD200r1, Eomes, CD49b) expressed in different organs and different activation states (22) that can drive different response during homeostasis and viral-induced inflammation (23). Finally, the appearance of the adhesion molecule CD56 (NCAM: neural cell adhesion molecule) designates the final transition of immature NK cells toward a mature phenotype (16).

The NK cell maturation gives rise to a small population of CD56bright (5%) cells, expressing high levels of this adhesion molecule, and a CD56dim population, expressing lower levels of this adhesion molecule, that represent about 90% of mature NK cells (16). Thus, phenotypically, human NK cells are defined as CD3−/CD56+/CD335 (NKp46)+ mononuclear cells that can be further divided into low density CD56dim and high-density CD56bright subsets (24). The NKp46 is a member of the highly conserved family of natural cytotoxicity receptor (NCR), a family of NK-activating receptors, also expressed by a small subset of cytotoxic T lymphocytes (25). The two NK cell subpopulations present specific localization, phenotype, and function. CD56dim NK cells are the dominant subset in peripheral blood and are cytotoxic effector cells, which express high levels of FcγRIII (CD16), an immunoglobulin superfamily member, and CD57 (HNK-1, Leu-7). In addition, they present on their cell surface CXCR1 and the Chemerin receptor that play a role in NK cell recruitment into peripheral inflammatory sites. This subset, however, has a lower ability to produce cytokines in response to activation. On the other side, CD56bright NK cells are present in secondary lymphoid organs and peripheral tissues. They express CCR7 that regulate their homing to lymphonodes, lack perforin, presenting little or no ability to spontaneously kill tumor cell targets, are CD16−, and mediate immune response by secreting pro-inflammatory cytokines, such as interferon-γ and tumor necrosis factor (TNF) alfa (26, 27).

The production of pro-inflammatory or immunosuppressive cytokines is another essential feature of NK cell, distinct from the secretion of cytotoxic granules, and NK cells use diverse activation signals to regulate these two functions in a differential way (16, 28).

As CD4+ T cells, also NK cells can be distinct into Natural killer type-1 (NK1), NK type-2 (NK2) (29), and NK regulatory cells (NKreg) (30). The different functional NK subsets are characterized by different expression of cell surface proteins and cytokines. NK1 cells are NK cells with activating signals, are mainly CD56dim CD11b+ CD27− NK cells, and produce IFN-g. NK2 cells are characterized by inhibitory signals, are mainly CD56bright CD27− CD11b− NK cells, and produce type-2 cytokines, including IL-5 and IL-13; NKreg cells are mainly CD56brightCD27+ NK cells and play their immune regulatory effect by cytokines secretion or cell-to-cell contact (29, 31). The differentiation of NK cell subsets depends on the specific microenvironment in physiological or pathological conditions other than intrinsic regulation by various transcription factors.

In response to tumor ligands or intracellular pathogens, NK cells mainly produce Th1 type cytokines including IFNg, TNF, and GMCSF which facilitate the activation of T cells, dendritic cells, macrophages, and neutrophils (32, 33). NK cells also produce cytokines with chemotactic action, including CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES) which attract effector lymphocytes and myeloid cells toward inflamed tissues (34).

Similarly, different cells can produce inflammatory mediators that act on NK cells influencing their behavior. Dendritic cells play a pivotal role in this setting. These antigen-presenting cells, through the production of critical cytokines such as IL15, 12, 23, 27, and 18 (35–37), can enforce NK cell cytolytic activity. On the other hand, type 1 interferons, IL-12, IL-18, IL-27 released by dendritic cells are powerful activators of NK cell effector functions (38). IL2, produced by T cells, promotes the proliferation, cytotoxicity, and secretion of cytokines by NK cells (16). The NK cell functions can also be regulated by TGF beta released by regulatory T cells (Treg) (39, 40).

NK cells are key regulators of the immune response and of the cross-talk between innate and adaptive immunity, since they not only play a protective role from pathogen infections but also from excessive immune response to these agents (14). In fact, NK stimulation by various soluble factors (such as IL-15, type I IFN, IL-12, IL-18) can increase the maturation and activation of dendritic cells, macrophages, and T cells (41) but simultaneously NK can also present a cytotoxic action on immature dendritic cells, on activated T cells, and on hyperreactive macrophages (42, 43).

All the described NK cell functions play an important role not only in physiological but also in pathological conditions, where NK cells can locally modulate several mechanisms of injury (16). Although many studies on human NK cells focused on peripheral blood, it is now clear that both CD56dim and CD56bright populate also healthy lymphoid and non-lymphoid organs including liver and kidneys. The kidney resident NK cells display a specific surface marker profile in different pathological conditions (44, 45). A significantly increased NK cell number is present in kidney biopsies from patients with chronic kidney disease (CKD). In this setting both NK CD56dim and CD56bright cells were increased in fibrotic renal tissue, but only CD56bright correlated significantly with loss of kidney function (46), thus indicating that these cells, through the production of pro-inflammatory cytokines and IFNg, can play an important role in fibrotic process and in the progression of kidney injury (46).

## NK CELLS IN KIDNEY TRANSPLANTATION

NK cells in the transplanted kidneys are a heterogeneous population of innate lymphocytes with subset-specific functional roles and with complex functions during homeostatic and pathological conditions. In fact, their role in immune reactivity to solid-organ transplant is still controversial. It is well-known that NK cells might promote allograft injury. However, some evidences indicate that NK cells may play a significant role in the priming of allograft tolerance (47). Post-transplantation NK cell subsets can change also at the peripheral level when compared to pre-transplant cells, and these variations affect both the number and the phenotype (48), thus suggesting that NK cell immunoregulatory characteristics can largely influence the graft outcome.

## NK Cell Involvement in Acute and Chronic Allograft Rejection

Kidney graft rejection is classified pathologically into two types: T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) (49–51). Through various interactions with different cell types involved in the immunological response activated by organ transplantation, NK cells can contribute in different ways to the pathogenesis of both acute and chronic T cell-mediated and antibody-mediated rejection (47). Yagisawa et al. recently demonstrated, in a mice model of kidney transplantation, that acute kidney allograft rejection is induced by the presence of both NK cells and donor specific antibodies (DSA), whereas in the absence of NK cell activation the presence of DSA alone cannot induce acute antibody-mediated rejection, although it can still lead to late graft failure (52).

NK cells can also influence maturation of dendritic cells and the subsequent activation of T cells (53). Moreover, NK cells are an early source of IFN-g, which drive a Th1-type immune response. NK cells can interact directly with CD4+ T lymphocytes (54), increasing their reactivity, and these activities can induce acute rejection mechanisms.

Turner et al. (2) proposed a mechanism describing the pathogenic role of NK cells in human antibody-mediated rejection through the expression of CD16. This function can be triggered by anti-HLA antibodies, in particular by DSAs that represent a major risk factor for graft loss. NK cells have been identified in the peri-tubular capillaries of the biopsies of patients ABMR (55) where DSA bind the graft endothelial cells. Once bound to endothelial cells DSA can interact with FcγRIII present on NK cells inducing an ADCC against the graft (2). This model specifically involves the NK CD56dim subset, expressing CD16, and recruited at the graft level. Thus, CD56dim/CD16 NK cells could represent the main NK subset involved in the pathogenesis of antibody-mediated rejection and responsible of ADCC on target cells into the graft (**Figure 1A**). Patients with DSA present, indeed, a reduced number of circulating CD56dim NK cells compared to patients without anti-HLA antibodies or with non-DSA anti-HLA antibodies (56), and this observation might be the result of NK cytotoxic subset homing within the rejecting graft. Sablik et al. recently reported that there are no significant differences in the total percentage and distribution of NK cells, B cells, and T cells between patients with chronic active antibody-mediated rejection and control transplant recipients. However, antibody-mediated graft rejection is characterized by differences in the activation status of circulating monocytes, NK cells, and γδ T cells, mainly regarding the CD16 expression (57).

NK cells may play also a role in the pathogenesis of T cell-mediated rejection. Immunohistochemical characterization of graft infiltrating cells demonstrated that patients with acute T cell-mediated rejection are characterized by a higher number of CD56+ and CD57+ cells within the interstitial compartment, associated with interstitial inflammation and tubulitis, both characteristics of T cell-mediated rejection (58, 59). Authors also established a cut-off of 0.56 cells/mm2 both in the interstitial infiltrate and at the glomerular level, which was significantly associated with a worse graft survival (59). However, the main limits of these studies were the identification of NK cells based only on the expression of a single marker, CD56 or CD16, and the impossibility to identify the NK cell subpopulations involved in this context.

These findings suggest that NK cells need to be carefully evaluated, because variations in NK cell marker expression might be associated with the activation of different immune pathways in graft rejection. A recently published study, where authors isolated lymphomonocytes directly from graft biopsies and used a multi-color flow cytometry to define NK cell subsets involved in different graft rejection, confirmed this hypothesis (60). Biopsies from patients with T cell-mediated rejection showed an increased absolute number of CD56bright NK cells while in the biopsies of patients with antibody-mediated rejection both CD56bright and CD56dim NK cells were increased. Only CD56dim showed the expression of activation markers such as CD69 and high levels of cytotoxic effector molecules (perforin, granzyme A, and granulysin) in supernatants obtained from ABMR biopsies (60). Once again, these data highlight the importance of CD56dim cells activation by the micro-environment featuring ABMR, where they can guide vascular damage. CD56bright NK cells can instead play a specific role in TCMR through the secretion of pro-inflammatory molecules such as IFN-g (**Figure 1A**), which increase the recruitment of alloreactive T cells and up-regulate HLA alloantigens (MHC I and II) on graft target cells, making them more susceptible to cytotoxic killing (60).

Hidalgo et al. analyzed the transcriptional profiles of graft biopsies of patients with antibody-mediated rejection in the presence and absence of DSA (61). They demonstrated that the presence of DSAs was associated with 132 different transcripts, some in common with the T cell mediated rejection. By eliminating these shared transcripts, the authors identified 23 selective associated transcripts. Six of these 23 transcripts showed a high expression in NK cells, while the rest were mainly expressed at the endothelial level (61).

The different role of NK cells in T cell-and B cell-mediated rejections was also confirmed by the analysis of transcriptomic profiles of 403 kidney graft biopsies (62). Gene expression profiling of human kidney allografts identified high levels of NK cell transcripts in early T cell-mediated rejection, thus suggesting a distinct role for NK cells in this tubule-interstitial disease, while late biopsies showed increased number of NK cell transcripts in patients with antibody-mediated rejection, microvascular inflammation, and DSA. These data support the different role of NK cells in ABMR compared to the TCMR. Shin et al. also reported a positive correlation between the number of CD56+ cells and the severity of T cell-mediated rejection (63). In addition, it has been recently demonstrated that transcripts from activated NK cells are the only among those from leukocyte types that differentiate antibody- and T cell-mediated rejections and correlate with transplant outcome (55).

Several immune cells subtypes such as CD4+ and CD8+ T lymphocytes, monocytes/macrophages, dendritic cells, and NK cells infiltrate the kidney during graft rejection; however, kidney biopsies from ABMR patients are specifically characterized by a significant enrichment of NK cell transcripts, and activated NK cell infiltration can discriminate ABMR from TCMR and can predict graft failure after kidney transplantation (55). NK-cell depletion in mice models can, indeed, significantly attenuate the frequency and severity of antibody-mediated chronic rejection and the presence of NK cells is important in the pathogenesis of antibody-mediated graft lesions (64).

Although different cell types and different cell subsets are involved in ABMR and TCMR, respectively, NK cells and CD8 T cells, shared transcripts are expressed in the graft in both antibody- and T cell-mediated rejections, such as CD160, XCL1, TNFRSF9, and IFN-g, thus indicating possible similar effector systems, important in rejection mechanisms (65).

Our group has recently identified specific transcripts from NK cells also on peripheral blood mononuclear cells isolated from patients with antibody-mediated rejection compared to control transplant recipients with normal graft function and histology (Pontrelli P, personal communication). In particular, we observed

that patients with antibody-mediated rejection are characterized by the increased presence of specific NK cell receptors that are essential for NK cell behavior. The key mediators of NK cell alloreactivity, in fact, are multiple receptors, KIRs, that predominantly recognize HLA Class I molecules (66). The different KIR expression on NK cells allows the ability of these cells to evaluate minute changes in MHC class I expression (67). Inhibitory KIRs expressed by NK cells of solid organ transplant recipients with donor mismatched for HLA KIR ligands may not recognize HLA class I molecules of donor and may dramatically induce NK cell alloreactivity against the graft (68, 69). Indeed, Littera et al. demonstrated, in a retrospective study including 174 donor/recipient pairs, a significantly higher risk of chronic rejection when recipient and donor pairs completely lacked the two KIR-HLA ligand combinations rKIR2DL1/dHLA-C2

immunosuppressive regiments is important to control and predict the onset of infections and neoplasia.

and rKIR3DL1/dHLA-Bw4 corresponding to a low level of NK cell inhibition (70). Van Bergen et al. in a retrospective cohort study of 397 HLA-DR-compatible kidney transplantations demonstrated that KIR-ligand mismatching contributes to the rejection of human solid allografts as an independent risk factor in HLA-A-B-DR-compatible transplantations, indicating that suppression of NK-cell activity can improve the kidney graft survival (71).

NK (mis)matching between KIR receptors and HLA molecules can largely influence a transplant outcome and many studies confirmed the importance of NK KIR mismatching in graft-vs.-host disease (72). Recently, it has been demonstrated that in hematopoietic stem cell transplantation, activation of donor NK cells, in the absence of appropriate inhibitory ligands, can largely influence the outcome of transplantation. The specific analysis on KIRs genotype and HLA-A/B genotypes on a cohort of 100 patients with acute leukemia who received hematopoietic stem cell transplantation from their HLA-matched siblings suggested that an appropriate selection based on donor-recipient KIR genotypes and recipient HLA class I molecules can modulate the risk of host's disease and the efficacy of transplantation (73).

#### How Can NK Cells Influence Transplant Tolerance?

Despite their essential role in allograft rejection, NK cells might also promote allograft tolerance (74). Immunological tolerance to a set of antigens is the absence of an immune response against those antigens, while normal responses to other antigens are preserved. Therefore, tolerance is an active antigen-specific process, is achieved under conditions that suppress the immune reaction, and is not just the absence of an immune response (75).

In specific settings, NK cells have potent immunoregulatory properties that promote tolerance induction. In a skin transplant model in mice it has been demonstrated that recipient's NK cells can contribute to the induction of graft tolerance by killing allogeneic antigen presenting cells (76). Donor antigenpresenting cells, in the absence of host NK cells, can survive and directly induce the activation of alloreactive T cells that are resistant to co-stimulatory blockade treatment (76). Thus, in those models in which NK cells have an altered function or are reduced, it will be difficult to obtain tolerance toward a MHC mismatched graft. After transplantation, in fact, both antigen presenting cells and T cells may represent potential targets of NK cell regulation (77). In this scenario, NK cells can be activated through different mechanisms: detection of the missing MHC I-self on the target cells, recognition of the Fc portion of the IgG, recognition of altered molecules on cells under stress conditions, inflammatory environment mediated by the cytokines produced by dendritic cells and T cells (78, 79). Activated NK cells can kill donor-derived dendritic cells through direct lysis (77), thus dampening the immune response and promoting a tolerogenic environment (**Figure 1B**). It is not yet clear which factors move NK cells toward immature donorderived dendritic cells. Moreover, NK cells regulation could also affect recipient dendritic cells, thus influencing allograft antigen presentation (77). It is conceivable that NK cells might be able to integrate stimulating and inhibiting signals influenced also by T cell behavior, thus defining the final immune response (47).

The maintenance of transplant tolerance could be also associated with the production of IL-10 by NK cells (**Figure 1B**). Upon stimulation with glycolipids, such as galactosyl ceramide, NK cells produce high levels of IL-10 that can promote the development of regulatory dendritic cells (80). Moreover, NK cells from tolerant mice show high IL10 levels and can influence the immune response mediating heart transplant tolerance (81).

Lozano et al. described the presence of genes transcripts associated with NK cell-mediated cytotoxicity in the expression profiles of peripheral lymphocytes obtained from tolerant recipients, especially in liver transplant recipients (82). Even in kidney transplantation, the NK cell signature characterizes a tolerogenic action in recipients and kidney transplant patients with spontaneous operational tolerance are characterized by specific transcriptional profiles (82, 83). Dugast et al. showed that, although the frequency of circulating NK cells was normal in these spontaneously tolerant patients, these cells showed a reduced activation profile with a reduced expression of activating KIR2DS5 gene, NKp46, and CD16 with a subsequent reduction in the effector functions of these cells including cytotoxicity and the release of cytokines such as IFN-g (84).

In the induction of tolerance by suppressing the immune response, Tregs play a leading role. Tregs are typically CD4+CD25+ and express the foxp3 transcription factor, which is the main inducer and regulator of Treg development and functions (85). CD4+CD25+T cells suppress the proliferation of CD4+ and CD8+ T lymphocytes. Thus, their major role is to shut down an immune reaction mediated by T cells and to suppress auto-reactive T lymphocytes that escaped the negative selection in the thymus (86). Tregs can influence the NK cell function in different ways, and this interaction can be positive in physiological conditions, such as pregnancy, or negative in some pathological conditions, such as autoimmune diseases or neoplasms, where Tregs suppress NK cells and inhibit their effector functions (87). On the other hand, NK cells maintain a complex crosstalk with different cells of the immune system (monocytes, B and T cells) (88–92) through direct contact or secretion of cytokines including TGF-beta. In correlation with higher TGF-beta level in inflammatory response, NK cells are able to induce Tregs (87, 93). However, how NK cells and Treg cells can influence each other in physiological and pathological conditions is still largely unknown.

A direct correlation between NK cells and Tregs in inducing tolerance is currently controversial (94). To date, most published evidencessupport the possibility of a mutual antagonism between NK cells and Tregs (94). An alternative proposal is that the reactivity of NK cells and Tregs are temporally distinct during the induction of tolerance (47). NK cells would induce tolerance in the first 3 weeks after transplantation by blocking dendritic cells and/or T cells that could start rejecting the graft, while Tregs, by maturing later, would maintain the long-term tolerance toward the graft (74). It is therefore possible that NK cells per se do not induce tolerance but simply allow the survival of the graft while the recipient develop a regulatory response (47) (**Figure 1B**).

#### HOW DOES IMMUNOSUPPRESSION INFLUENCE NK CELL BEHAVIOR?

Information regarding the influence of immunosuppressive drugs on the activity of NK cells in transplant recipients is rather limited compared to T cells, which represent the main target of immunosuppressive therapies.

It has been demonstrated that certain KIR genotypes and their specific HLA class I ligands could affect kidney transplantation outcome by interfering with the efficacy of immunosuppressive drugs (70). The interference of KIR with therapy effectiveness has been already explored in allogenic transplantation of hematopoietic stem cells in chronic myeloid leukemia (95–97).

Immunosuppressive drugs might modulate the phenotype of NK cells after kidney transplantation, thus suggesting that NK cells can serve as sensors for immunosuppression and can be considered for personalized immunosuppression therapy adjustment (98). In fact, among kidney transplant recipients with a reduced expression of CD16 and CD56 on NK cells compared to healthy controls, patients in immunosuppressive therapy with tacrolimus showed more significant phenotypic changes on the expression of these markers than patients treated with cyclosporine or tacrolimus in combination with mTOR inhibitors (98). In addition, the presence of mTOR inhibitors in vitro also had functional consequences regarding de-granulation and IFN-g production (98) (**Figure 1C**). However, it is unclear whether these phenotypic changes of NK cells, induced by immunosuppressive drugs, may represent an activation signal of NK cells rather than functional exhaustion.

Hoffmann et al. demonstrated that NK cells of kidney transplant recipients under immunosuppression retain their ability to respond to stimulation since they produce equal amounts of IFN-g, perforin, and granzyme compared to NK cells from healthy individuals in response to strong, non-specific stimulation by PMA/Ionomycin (3). Thus, the inability of current immunosuppressive regimens to down-regulate the function of NK cells represents an opportunity from a therapeutic point of view, and new treatments targeted to activated NK cells and/or their effector functions should be explored.

However, immunosuppression may influence the number of NK cells over time. In patients treated with cyclosporine compared to patients treated with tacrolimus, the number of NK cells as well as the ratio CD56dim/CD56bright is lower and the cytotoxic activity is reduced 1 year after transplantation (99). It will be useful in the future to routinely monitor and evaluate NK cell function in the context of specific algorithms to personalize immunosuppressive regimens (**Figure 1C**).

Monitoring NK cell number and especially NK cell function in transplanted patients is also important to control and predict the onset of infections and neoplasia (100). NK cells, indeed, play an important role in cancer defense, and the incidence of cancer is deeply increased after transplantation (101). Peraldi et al., in a cross-sectional multi-center case control study, demonstrated that kidney transplant recipients with cancer had a lower

#### REFERENCES

frequency of the cytokine-enriched CD56bright NK cell subset compared to normal kidney graft recipients. The percentage of NKp46+NK cells in these patients was significantly reduced (45 vs. 53%, P = 0.001) along with a significant reduction in the ability of NK cells to degranulate CD107a+ cytolytic vesicles and to secrete IFN-g (102). In addition, Dendle et al. recently demonstrated that NK cell cytotoxic functions predict the appearance of severe infections in kidney graft recipients 2 years after transplantation better than NK number (103). Activated KIR genes have been associated with the protection from human Cytomegalovirus infection in renal transplantation (85), an infection associated with graft loss and reduced survival. In particular the presence after Cytomegalovirus infection of a specific subset of mature NK cells expressing the CD94/NKG2Cactivating receptor can control the viral infection in kidney transplant recipients (104, 105).

## CONCLUDING REMARKS

The phenotype of NK cells in peripheral blood of kidney transplant recipients might be informative of the immune status after transplantation in terms of rejection vs. tolerance induced by immunosuppressive drugs.

A more careful evaluation of the number and function of these cells will allow us to balance the activation of mechanisms underlying graft rejection, favoring the immunological tolerance of the graft. This will achieve an equilibrium condition that allows the best survival of the graft and a reduction in the risk of developing malignancies or infections.

#### AUTHOR CONTRIBUTIONS

PP, GG, LG, and GS designed, wrote, and critically revised the review. FR and GC analyzed NK cell involvement in acute and chronic allograft rejection. All authors contributed to the article and approved the submitted version.

#### ACKNOWLEDGMENTS

Authors thank Dr. Rossana Franzin for her support in the revision of the English language and of the figure.

killer lymphocyte reconstitution one year after kidney transplant by reference to cyclosporine/azathioprine. Transplantation. (2006) 82:558–66. doi: 10.1097/01.tp.0000229390.01369.4a

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Pontrelli, Rascio, Castellano, Grandaliano, Gesualdo and Stallone. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Ischemia Reperfusion Injury Triggers CXCL13 Release and B-Cell Recruitment After Allogenic Kidney Transplantation

Kirill Kreimann1†, Mi-Sun Jang1†, Song Rong1 , Robert Greite1 , Sibylle von Vietinghoff 1 , Roland Schmitt 1 , Jan Hinrich Bräsen2 , Lena Schiffer 1 , Jessica Gerstenberg1 , Vijith Vijayan3 , Oliver Dittrich-Breiholz 4 , Li Wang1 , Christian M. Karsten5 , Wilfried Gwinner 1 , Hermann Haller 1 , Stephan Immenschuh3 \* † and Faikah Gueler 1†‡

#### Edited by:

Cees Van Kooten, Leiden University, Netherlands

#### Reviewed by:

Matthew S. Macauley, University of Alberta, Canada Guseppe Grandaliano, University of Foggia, Italy

#### \*Correspondence:

Stephan Immenschuh immenschuh.stephan @mh-hannover.de

†These authors have contributed equally to this work

‡Deceased March 20, 2020

#### Specialty section:

This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology

Received: 30 September 2019 Accepted: 14 May 2020 Published: 06 August 2020

#### Citation:

Kreimann K, Jang M-S, Rong S, Greite R, von Vietinghoff S, Schmitt R, Bräsen JH, Schiffer L, Gerstenberg J, Vijayan V, Dittrich-Breiholz O, Wang L, Karsten CM, Gwinner W, Haller H, Immenschuh S and Gueler F (2020) Ischemia Reperfusion Injury Triggers CXCL13 Release and B-Cell Recruitment After Allogenic Kidney Transplantation. Front. Immunol. 11:1204. doi: 10.3389/fimmu.2020.01204 1 Department of Nephrology, Hannover Medical School (MHH), Hannover, Germany, 2 Nephropathology Unit, Institute of Pathology, Hannover Medical School (MHH), Hannover, Germany, 3 Institute for Transfusion Medicine, Hannover Medical School (MHH), Hannover, Germany, 4 Research Core Unit Genomics, Hannover Medical School, Hannover, Germany, 5 Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany

Ischemia reperfusion injury (IRI) is linked with inflammation in kidney transplantation (ktx). The chemokine CXCL13, also known as B lymphocyte chemoattractant, mediates recruitment of B cells within follicles of lymphoid tissues and has recently been identified as a biomarker for acute kidney allograft rejection. The goal of this study was to explore whether IRI contributes to the up-regulation of CXCL13 levels in ktx. It is demonstrated that systemic levels of CXCL13 were increased in mouse models of uni- and bilateral renal IRI, which correlated with the duration of IRI. Moreover, in unilateral renal IRI CXCL13 expression in ischemic kidneys was up-regulated. Immunohistochemical studies revealed infiltration of CD22+ B-cells and, single-cell RNA sequencing analysis a higher number of cells expressing the CXCL13 receptor CXCR5, in ischemic kidneys 7 days post IRI, respectively. The potential relevance of these findings was also evaluated in a mouse model of ktx. Increased levels of serum CXCL13 correlated with the lengths of cold ischemia times and were further enhanced in allogenic compared to isogenic kidney transplants. Taken together, these findings indicate that IRI is associated with increased systemic levels of CXCL13 in renal IRI and ktx.

Keywords: ischemia reperfusion injury, delayed graft function, kidney transplantation, B-cell activation, CXCL13

## INTRODUCTION

Ischemia reperfusion injury (IRI) in kidney transplantation (ktx) is linked with inflammation and leukocyte recruitment (1). The extent of IRI depends on donor-related factors and duration of the ischemia time. Short ischemia times (2–3 h) that are encountered in living donor ktx have been associated with better long-term graft survival as compared to deceased donor ktx. In deceased donor renal transplants, median cold ischemia time (CIT) in Germany can be up to 14 to 16 h. Extended CITs (longer than 25 h) have been reported in other countries (2). Prolonged CIT and old donor age increase the risk of developing delayed graft function (DGF) (3). Immunosuppressive agents—typically, a combination of prednisolone, mycophenolatmofetil (MMF), and calcineurin inhibitors (CNI)—are used to inhibit rejection. B-cell inhibitors are only used for pretreatment of

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recipients in ABO-incompatible living donor ktx to reduce or eliminate blood group antibodies prior to transplantation. Despite these interventions, acute rejection occurs shortly after ktx in 6–12% of deceased allograft recipients and some of these patients have signs of antibody-mediated rejection although donor-specific antibodies are not detectable. Thus, activation of a humoral response may be triggered by IRI itself and immediate production of the chemokine CXCL13 after surgery could be an important mediator of B-cell activation and subsequent antibody- mediated transplant rejection. Recently, we showed that CXCL13 is a biomarker for acute mixed allograft rejection in kidney transplant recipients (4). The major function of CXCL13 is recruitment of CXCR5+ cells (mainly naïve B cells) into lymphoid follicles (5, 6). Moreover, CXCL13 causes resident kidney cells to produce pro-inflammatory cytokines and chemokines (4, 7–9). In liver transplantation, increased numbers of circulating CXCR5+CXCR3-CD4+ T-cells have been shown to correlate with acute transplant rejection (10) and CXCR5+ follicular T-helper cells (Tfh) have been linked to humoral immunity (11). Furthermore, it has been shown that CXCR5+CD8+ T-cells localize to B-cell follicles, express costimulatory proteins, and promote B-cell differentiation along with antibody isotype class switching (12). Here, we demonstrate that renal IRI causes increased levels of systemic CXCL13 with subsequent infiltration of CXCR5+ leukocytes in kidneys not only in models of IRI, but also in that of allogenic ktx.

## MATERIALS AND METHODS

#### Animals

Male C57BL/6 and BalbC mice (11–15 weeks of age, 22–28 g in weight) were housed in a 14/10 h light/dark cycle under conventional conditions. Mice had free access to food (Altromin 1324) and tap water, and were monitored daily for behavior and well-being. All experiments were approved by the local authorities (Lower Saxony State office for Consumer Protection and Food Safety, LAVES: 33.12-42502-04-14/1569 and 33.9- 42502-04-07/1396; 09/1637).

#### Models of Uni- and Bilateral Kidney IRI

Mice were anesthetized with isoflurane (3–5% induction and 1.5% maintenance). Prior to surgery butorphanol 1 mg/kg s.c was injected for analgesia. Depending on the model, bi- or unilateral renal IRI was induced after their abdomens were opened by renal pedicle clamping using a micro-aneurysm clip for 15–45 min to induce subclinical, transient or progressive acute kidney injury (AKI) (13, 14). Sham surgery was performed in the same manner, but the renal pedicle was not clamped. After surgery mice received metamizol (p.o. 200 mg/kg) in drinking water over 3 consecutive days for postoperative pain control. The animals were monitored daily. At the designated endpoints they were deeply anesthetized and euthanized by total body perfusion with ice cold PBS via the left ventricle. Sham kidneys as well as contralateral kidneys from the unilateral experiments served as control tissues.

### Kidney Transplantation Model

C57BL/6 (H2b ) mice served as donors and fully mismatched BalbC (H2d ) mice as recipients for allogenic ktx. For isogenic ktx C57BL/6 mice served as donors as well as recipients. Ktx was performed by a vascular surgeon with >20 year experience in small animal microsurgery. Surgeries were performed after the animals were anesthetized with isoflurane inhalation (35% induction 1.5–2% maintenance) following i.p. injection of butorphanol (2 mg/kg) for analgesia. After surgery mice received metamizol (p.o. 200 mg/kg) in drinking water over 3 consecutive days for postoperative pain control. For graft retrieval the donor's left kidney including the renal vein, renal artery, and ureter were removed en bloc. The recipient was prepared for transplantation by removal of its left kidney. The kidney graft was then transplanted into the lower abdomen of the recipient by end-to-side anastomosis of the renal artery to the aorta and the renal vein to the inferior vena cava (15). The ureter was implanted into the bladder dome (16). Normal renal function was ensured by the remaining native, unaffected right kidney of the recipient. After surgery mice were monitored daily for general health, activity, and well-being. Reasons for study termination included behavioral changes (reduced activity, no food intake) or surgical complications (graft thrombosis, hind limb paralysis, bleeding, urinary leakage, or significant urinary congestion).

## Single Cell RNA sequencing (sc-RNAseq) in Control and IRI kidneys

Kidneys from three male C57BL/6 control mice (no surgery) and three male C57BL/6 mice 7 days after unilateral IRI for 27 min were processed for sc-RNAseq to determine whether CXCR5+ cells were infiltrating the kidney. After kidney retrieval, tissue was sliced and digested by collagenase as described (17). Following red blood cell lysis and dead cell removal using a Miltenyi Dead Cell Removal Kit according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany) 10,000 cells per sample were subjected to single cell mRNA-Seq analysis (Chromium Single Cell 3 Reagent Kits v3 User Guide, Document Number CG000183, Rev A; 10x Genomics). Equimolar amounts of libraries were pooled, denatured with NaOH, and finally diluted to 1.8 pM according to the Denature and Dilute Libraries Guide (Document # 15048776 v02; Illumina). 1.3 ml of the denatured pool was sequenced on an Illumina NextSeq 550 sequencer using one third of a High Output Flowcell for 75 cycles per sample (#20024906; Illumina). The proprietary 10x Genomics CellRanger pipeline (v3.0.2) was employed with default parameters. CellRanger was used to build a "pre-mRNA" reference package from reference genome provided by 10x Genomics (Mouse reference dataset 3.0.0; November 19, 2018; mm 10) as described in https://support.10xgenomics.com/single-cell-gene-expression/ software/pipelines/latest/advanced/references. Read data were then aligned to the "premRNA" reference package with CellRanger using the aligner STAR to count aligned reads per gene and calculate clustering and summary statistics. Finally, the Loupe Cell Browser from 10x was used to view

FIGURE 1 | Cytokine levels in serum samples and kidney tissue. Serum levels of pro-inflammatory cytokines were measured in models of uni- and bilateral renal IRI. CXCL13 levels significantly increased within 24 h in 30 min bilateral IRI as compared to 15 min IRI or baseline (BL) levels prior to IRI (A). In unilateral IRI for 35 and 45 min a time-dependent increase of serum CXCL13 levels were observed 24 h after IRI (B). To longitudinally determine the kinetics of CXCL13 release in 45 min unilateral IRI blood samples were taken at the indicated times. A maximum was measured 24 h after IRI (C). MCP-1 (D) and IL-6 (E) were measured in comparison to sham surgery prior to IRI and 24 h after bilateral IRI. Both markers were significantly increased in comparison to baseline. 24 h after unilateral IRI a significant increase of CXCL13 (F) and of IL-6 (G) mRNA expression in renal tissue was observed in the 45 min unilateral IRI model (n = 6–10 mice per group, one-way ANOVA, \*p < 0.05; \*\*p < 0.01, \*\*\*p < 0.001). BL, baseline.

and revise annotated clusters, based on the implemented tSNE algorithm.

#### RNA Extraction and qPCR

Gene-specific primers for CXCL13 (Primer-sequence: fwd-TCT GGA CCA AGA rev-TGA AGA AAG TT), monocyte chemoattractant protein-1 (MCP-1; Mm\_Ccl2\_1\_SG QuantiTect Primer Assay QT00167832), IL\_6 (Mm\_IL6\_1\_SG QuantiTect Primer Assay QT00098875), and TNF-α (Mm\_TNF\_1\_SG QuantiTect Primer Assay QT00104006) were used. qPCR was performed as described previously (18). After fixation of renal tissue sections in RNAlater (Ambion) overnight, total RNA was isolated using RNeasy Mini Kit (Qiagen). For quantitative real time PCR (qPCR), 1 µg of DNase-treated total RNA was reverse transcribed using PrimeScript Reverse Transcriptase reagent Kit (Takara) and qPCR was performed with a LightCycler 96 (Roche Diagnostics). For each reaction 10 µl TB Green premix Ex Taq II (Takara), 3 µl DEPCtreated water (Ambion), and 10 pmol of forward and reverse primer were used in each well of a 96-well plate. The PCR reaction was initiated at 95◦C (30 s), then 40 cycles followed: 5 s 95◦C and 1 min at 60◦C. HPRT (Mm\_HPRT\_1\_SG QuantiTect Primer Assay QT00166768) was used as house keeping gene for normalization.

#### Cytokine Assays

CXCL13 serum levels were analyzed by ELISA (R&D Systems, MCX130) as described previously (19). Color development was measured by a Tecan spectra ELISA reader (Tecan, Crailsheim, Germany). In addition, a CBA bead assay was used to measure proinflammatory cytokines (IL-6, TNF-a, MCP-1) (BD Biosciences) in blood samples.

#### Histology of Renal Tissue After IRI and ktx

Paraffin-embedded renal tissue was serially sectioned (2µm) and stained with periodic acid Schiff (PAS) reaction. Signs of AKI varied from loss of brush border, an early lesion, to cell detachment from the basement membrane and accumulation of cell debris in the tubular lumen. AKI was characterized using a semi-quantitative grading system: 0 = no acute tubular injury (ATI), 1 = focal ATI with <10% of tubuli in the cortex affected, 2 = moderate ATI with 10–25% of tubuli affected, 3 = severe ATI with 25–50% of tubuli affected, 4 = very severe with >50% of the tubuli affected. Scores for interstitial inflammation and leukocyte infiltration ranged from

0 to 4 correlating with mild, moderate, marked, and severe cell infiltration. Kidney grafts were graded according to the updated Banff classification (20). Analysis was performed without knowledge of the animal group identity by a nephropathologist with >20 years of experience. For further characterization of infiltrating leukocytes, immunostaining was performed with antibodies against Gr-1 (neutrophils) (Biorad, MCA 771G), F4/80 (monocytes/macrophages) (Acris Antibodies, BM4007), CD22 (Southern Biotech, 1580-01), and CD45R (B cells) (eBioscience, 14-0452-82), CD3 (T cells) (Dako, A0452), respectively.

#### RESULTS

#### IRI Causes Increased Serum Levels and Renal Expression of CXCL13

Severity of AKI is dependent on duration of renal ischemia times. Here, we compared systemic serum levels of CXCL13 in various models of renal IRI 24 h after surgery. A 15 min ischemia time causes subclinical IRI without elevation of creatinine and blood urea nitrogen (BUN), whereas a 30 min IRI induced these clinical parameters of AKI (**Supplementary Figure 1**). A pronounced increase of CXCL13 levels in serum was observed in animals that received 30 min compared to 15 min of bilateral IRI (**Figure 1A**). For comparison, the pro-inflammatory mediators MCP-1 and IL-6 were also significantly elevated after 24 h in the 30 min bilateral IRI model (**Figures 1D,E**). To allow for longitudinal follow-up without mortality after IRI, a model of unilateral IRI was used, in which only one kidney is clipped with unaltered overall renal function (21). In the unilateral model, 35 min of IRI caused a minor increase, but 45 min of IRI caused a marked increase in the levels of CXCL13 24 h after surgery (**Figure 1B**). Further time course studies in the 45 min unilateral IRI model revealed that the maximum increase in CXCL13 occurred at day 1 (24 h after IRI), which rapidly returned to basal levels at day 2 and did not increase further

pooled IRI kidneys 7 days after IRI. Expression of CXCR5 was increased after IRI (orange) compared to control (blue). Clusters of CXCR5 positive cells and scattered single cells were identified (B, enlarged view of A). The highest proportion of CXCR5+ cells were identified amongst CD79+ B-cells, followed by Thy1+ T-cells and C1qc+ macrophages (C).

until day 7 (**Figure 1C**). In addition, qPCR analysis of renal samples showed an increase in CXCL13 mRNA expression in ischemic kidneys at day 1 after IRI, but not at day 7 in comparison to renal samples from animals with sham surgery (**Figure 1F**). Although, a similar time-dependent up-regulation and decline was noted for the pro-inflammatory cytokine IL-6 in ischemic kidneys, the expression levels were still significantly elevated in comparison to sham controls at day 7 after IRI (**Figure 1G**).

In summary, renal IRI leads to an early increase in systemic serum CXCL13 levels and CXCL13 mRNA expression in ischemic kidneys.

#### IRI Causes AKI, Inflammation and Renal Infiltration of B-cells

Histomorphological changes were determined in animals after sham surgery or after 35 min and 45 min of unilateral IRI. In both groups of IRI typical alterations of severe AKI including the presence of apoptotic and necrotic cells at day 1 after surgery were observed and were still detectable at day 7 after IRI. Leukocyte infiltration increased from day 1 to day 7 after 35 and 45 min IRI (**Figures 2A–C**). As determined by immunohistochemistry the majority of infiltrating cells was GR-1 positive at day 1 (**Figures 3A,B**), whereas the more prominent cell type was F4/80+ myeloid cells at day 7 (**Figures 3A,C**). More importantly, at day 7 after IRI an infiltration of CD22+ B-cells was detected in ischemic kidneys, which was almost absent in animals that received sham surgery (**Figures 4A,B**). In addition, CD3+ T lymphocytes were found in the outer medulla at day 7 (**Figures 4A,C**). Since CXCL13 recruits cells expressing CXCR5, we performed sc-RNA seq analysis with three pooled control kidneys in comparison to three pooled IRI kidneys to identify CXCR5+ cells (**Figure 5**). In tSNE analysis IRI kidneys showed a cluster of CXCR5 expressing cells and some scattered single cells (**Figures 5A,B**). The majority of CXCR5+ cells were of B-cell origin (CD79+) followed by a subset of macrophages (C1qc+) and some T-cells (Thy1+). In control kidneys only few CXCR5+ cells were detected (**Figure 5C**).

Taken together, the data indicate that renal IRI causes recruitment of B cells to the inflamed tissue.

FIGURE 6 | Serum CXCL13 levels in mouse ktx. Post ktx levels of serum CXCL13 at day 1 were significantly increased compared to baseline. A higher increase was observed in longer cold ischemia time (30 vs. 60 min cold ischemia time). Isogenic ktx with prolonged cold ischemia time of 60 min had significantly lower CXCL13 levels compared to allogenic ktx (A). PAS stain at day 7 revealed enhanced cell infiltration in allogenic compared to isogenic ktx (B). Double staining for CD3+ T-lymphocytes (green) and CD45R+ B-cells (red) was performed at day 7. More interstitial CD3+ T-lymphocytes were observed in allografts compared to isografts. Allografts exhibited scattered B-cells in interstitial tissue, but also clusters of CD45R+ cells. Isografts showed only few B cells in the interstitium at day 7 (B) (bar: 100µm, n = 6 per group, one-way ANOVA \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001). BL, baseline.

#### CXCL13 Release Depends on Duration of Ischemia Time After Kidney Transplantation

In the following, allogenic ktx [C57BL/6 (H2b ) on BalbC (H2d )] or isogenic ktx was performed with either 30 or 60 min of CIT. Serum levels of CXCL13 were significantly higher in animals that received kidneys with longer CITs. In addition, the increase in levels of systemic serum CXCL13 was amplified in the allogenic as compared to the isogenic transplant setting (**Figure 6A**). Histopathology revealed inflammation (i.e., Banff 1a rejection) in allografts with enhanced CD45R+ B-cell and CD3+ T lymphocyte infiltration 7 days after transplantation (**Figure 6B**). To summarize, increased levels of serum CXCL13 levels after ktx are dependent on the duration of CIT. Moreover, systemic levels of CXCL13 and B-cell infiltration into renal grafts post transplantation were higher in the allogenic setting.

## DISCUSSION

This study provides evidence that the chemokine CXCL13, also termed B-lymphocyte chemoattractant, is up-regulated rapidly after renal IRI. Duration of IRI correlated with increased systemic serum CXCL13 levels in both models of uni- and bilateral clamping. Increased levels of CXCL13 in response to IRI were transient and reached a maximum after 24 h of IRI (**Figure 1**). B-cell infiltrates were detected in the kidney 1 week after IRI (**Figure 4**). The majority of cells expressing CXCR5 appeared to be of B-cell origin, but some T-cells and macrophages were also CXCR5 positive (**Figure 5**). Furthermore, in a mouse model of allogenic ktx systemic CXCL13 serum levels correlated with the length of graft ischemia time duration (**Figure 6**).

It is noteworthy that allogenic ktx caused higher levels of serum CXCL13 levels than isogenic ktx (**Figure 6**). This finding raises the question of whether the host inflammatory response, which is markedly higher in allogenic ktx may contribute to CXCL13 secretion. A limitation of the current study is that the source of CXCL13 was not identified. A very early event in IRI is rapid complement activation orchestrating the inflammation that follows. Complement activation might also influence the secretion of immune cell-mediated CXCL13 release. Accordingly, recent experimental evidences indicates that activated peritoneal macrophages release CXCL13 via a mechanism that involves the complement anaphylatoxin C5a. Macrophages that are deficient for the C5a receptor C5aR1 showed markedly reduced release of CXCL13. Alternatively, CXCL13 release may also be regulated via Toll-like receptor (TLR)2- and IL-10-dependent mechanisms (22). Since myeloid cells are early drivers of inflammation and the extent of macrophage infiltration is directly linked to the duration of ischemia times and the severity of IRI (21) it is conceivable that infiltrating macrophages after IRI may contribute to CXCL13 production and release. The observation that the mRNA levels of CXCL13 in ischemic kidneys were induced after 24 h also indicates that the source of CXCL13 production might be the damaged kidney. Although Tfhs are considered to be the major source of CXCL13, it has previously been shown that stromal cells can also produce CXCL13 in response to IL-17 in inflamed lung tissue in mice (23). Moreover, peripheral T helper cells, macrophages and damaged tubuli are potential candidate cells for CXCL13 production in kidney. Further studies are warranted to elucidate the underlying mechanisms of how CXCL13 expression is up-regulated in IRI and transplantation.

sc-RNAseq has emerged as a powerful technique for determination of different cell types (24, 25) and availability of new transcriptomic data expands the understanding of molecular mechanisms in various disease states. Accordingly, immune cell landscapes in particular renal disorders have been demonstrated (i.e., diabetic nephropathy, lupus nephritis) (26, 27). In the current study sc-RNAseq analysis was applied to identify cells that express the CXCL13 receptor CXCR5. It is shown that IRI alone caused substantial B-cell infiltration into the kidney within 7 days of IRI and a subset of B-cells was positive for CXCR5. The expression of CXCR5 was not limited to B-cells as a subset of renal T-cells and macrophages were also identified to be positive for CXCR5. Several lines of existing evidence points to a detrimental role for CXCR5+ cells in transplant rejection. Notably, in a recent study, the presence of CXCR5+ CD4+ cells correlated with acute rejection after liver transplantation (10). In ABMR, CXCR5 containing exosomes from Tfhs were significantly higher compared to controls and in co-culture experiments these Tfh derived exosomes were able to promote Bcell proliferation and maturation (28). Tfhs play a critical role in germinal center reactions and development of tertiary lymphoid structures which have been reported in chronic allograft rejection of kidney (29, 30), lung (31), and heart transplants (32). These germinal centers are the primary sites of B-cell expansion and maturation directing the production of antibodies (33). However, it needs to be pointed out that the findings in this study are correlational with regard to CXCL13 and CXCR5. Further studies using strategies to block CXCL13 are required to validate the hypothesis that infiltration of CXCR5 positive cells in ischemic kidneys is a direct result of CXCL13 secretion. Alternatively, it is feasible that the recruitment of these cells is due to ongoing renal inflammation, independent of CXCL13 secretion. Due to necrotic cell death in ischemic tissue a variety of damageassociated molecular patterns (DAMPs) are released which can interact with TLR2 and−4 expressed on myeloid, dendritic, tubular epithelial and endothelial cells. TLR activation mediates downstream production of TNF-α, IL-1ß, and IL-6 promoting activation of the adaptive immune response (34, 35). Based on the findings we propose that systemic expression of CXCL13 after IRI and DGF might be a potential clinical candidate for early detection of interstitial inflammation and B-cell activation.

Further clinical studies are needed to determine the role of CXCL13 as a predictive biomarker for AKI, DGF and rejection in ktx.

#### DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

#### REFERENCES

#### ETHICS STATEMENT

The animal study was reviewed and approved by Lower Saxony State Office for Consumer Protection and Food Safety.

#### AUTHOR CONTRIBUTIONS

FG designed and supervised the experimental studies. FG and SI drafted the manuscript. KK, M-SJ, SR, VV, and LW conducted the experimental studies. SR performed IRI and ktx surgeries. FG and JB analyzed the histology and immunohistochemistry. WG, CK, LS, and HH discussed the results and edited the manuscript. SV, RS, and OD-B performed single cell sequencing experiments. All authors participated in the interpretation of data, editing, and approval of the manuscript.

#### FUNDING

This study was supported by the Deutsche Forschungsgemeinschaft (DFG) grant GU 613/1-1 to FG and grant IM 20/4-1 to SI.

## ACKNOWLEDGMENTS

We thank Herle Chlebusch, Heike Lührs, and Michaela Beese for excellent technical support. We thank Dr. Alexandra Helmke and Dr. Payel Sen for great experimental support in single-cell RNA sequencing. We thank Dr. Michael Brownstein for editing the manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01204/full#supplementary-material

Supplementary Figure 1 | Clinical parameters in bilateral ischemia model. Fifteen minutes bilateral IRI did not cause relevant increase of s-creatinine (A) or BUN (B). Bilateral IRI for 30 min caused significant deterioration of renal function with highly significant s-creatinine and BUN elevation (n = 5 sham mice, n = 6 mice per IRI group, one-way ANOVA, ∗∗∗p < 0.001). BL, baseline.

acute T cell mediated kidney allograft rejection. Int J Mol Sci. (2019) 20:2552. doi: 10.3390/ijms20102552

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling editor declared a past co-authorship with one of the authors FG.

Copyright © 2020 Kreimann, Jang, Rong, Greite, von Vietinghoff, Schmitt, Bräsen, Schiffer, Gerstenberg, Vijayan, Dittrich-Breiholz, Wang, Karsten, Gwinner, Haller, Immenschuh and Gueler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Coagulation and Fibrinolysis in Kidney Graft Rejection

Giovanni Stallone1 , Paola Pontrelli 2 \*, Federica Rascio1 , Giuseppe Castellano1 , Loreto Gesualdo2 and Giuseppe Grandaliano3,4

1 Nephrology, Dialysis and Transplantation Unit, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy, 2 Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari Aldo Moro, Bari, Italy, 3 Nephrology Unit, Department of Translational Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy, 4 Department of Medical and Surgical Sciences, Fondazione Policlinico Universitario "A. Gemelli" IRCCS, Rome, Italy

Coagulation system is currently considered an integrated part of innate immunity. Clotting activation in response to bacterial surface along with complement cascade priming represents the first line of defense against pathogens. In the last three decades, we learned that several coagulation factors, including factor II or thrombin and factor X, can interact with specific cell surface receptors activated by an unusual proteolytic mechanism and belonging to a novel class of G-protein-coupled receptors known as protease-activated receptors (PARs). PARs are expressed by a variety of cells, including monocytes, dendritic cells, and endothelial cells and may play a key role in the modulation of innate immunity and in the regulation of its interaction with the adaptive branch of the immune system. Also, the fibrinolytic system, in which activation is controlled by coagulation, can interact with innate immunity, and it is a key modulator of extracellular matrix deposition eventually leading to scarring and fibrosis. In the setting of kidney transplantation, coagulation and fibrinolytic systems have been shown to play key roles in the ischemia/reperfusion injury featuring delayed graft function and in the pathogenesis of tissue damage following acute and chronic rejection. In the present review, we aim to describe the mechanisms leading to coagulation and fibrinolysis activation in this setting and their interaction with the priming of the innate immune response and their role in kidney graft rejection.

Keywords: coagulation, fibrinolysis, protease-activated receptors, innate immunity, graft function

## INTRODUCTION