Role of leukocytes in the pathogenesis of acute kidney injury

Acute kidney injury (AKI) is a significant cause of morbidity and mortality in hospitalized patients, especially those who are critically ill. The mortality rate in patients with severe AKI requiring renal replacement therapy (RRT) can exceed 50 % [1]. Numerous factors contribute to the development of AKI, including reductions in renal blood flow, actions of nephrotoxic drugs, cellular injury/death of proximal tubule epithelial cells, pro-inflammatory responses of renal endothelial cells, influx and activation of inflammatory leukocytes that further reduces renal blood flow through vascular congestion and promotes and extends injury to kidney parenchymal cells [2, 3]. The immune response in AKI involves cells of both the innate and adaptive immune systems. Although numerous studies have demonstrated the detrimental role of many different types of immune cells, recent reports have uncovered a protective and possibly therapeutic role of other immune cells in AKI. Studies in animal models of AKI have revealed that innate immune cells, such as neutrophils, macrophages, dendritic cells, natural killer (NK) cells and natural killer T (NKT) cells, and adaptive CD4+ T cells promote renal injury. Indeed, renal inflammation is a common feature of human AKI [4] and detailed analyses of biopsy samples from patients with AKI demonstrated the presence of mononuclear leukocytes (some CD3+ T cells) and neutrophils [5]. In contrast, CD4+FoxP3+ regulatory T cells (Tregs) can protect the kidney from ischemic and nephrotoxic injury in animal models.


Introduction
Acute kidney injury (AKI) is a signifi cant cause of morbidity and mortality in hospitalized patients, especially those who are critically ill. Th e mortality rate in patients with severe AKI requiring renal replacement therapy (RRT) can exceed 50% [1]. Numerous factors contribute to the development of AKI, including reductions in renal blood fl ow, actions of nephrotoxic drugs, cellular injury/death of proximal tubule epithelial cells, pro-infl ammatory responses of renal endothelial cells, infl ux and activation of infl ammatory leukocytes that further reduces renal blood fl ow through vascular conges tion and promotes and extends injury to kidney parenchymal cells [2,3]. Th e immune response in AKI involves cells of both the innate and adaptive immune systems. Although numerous studies have demonstrated the detrimental role of many diff erent types of immune cells, recent reports have uncovered a protective and possibly therapeutic role of other immune cells in AKI. Studies in animal models of AKI have revealed that innate immune cells, such as neutrophils, macrophages, dendritic cells, natural killer (NK) cells and natural killer T (NKT) cells, and adaptive CD4 + T cells promote renal injury. Indeed, renal infl ammation is a common feature of human AKI [4] and detailed analyses of biopsy samples from patients with AKI demonstrated the presence of mononuclear leukocytes (some CD3 + T cells) and neutrophils [5]. In contrast, CD4 + FoxP3 + regulatory T cells (Tregs) can protect the kidney from ischemic and nephrotoxic injury in animal models. Understanding the immune mechanisms of renal injury and protection should yield new approaches to prevention and treatment of AKI.
Th is review will summarize current knowledge on the role of the immune system in the pathogenesis of AKI.

Neutrophils
Polymorphonuclear cells (PMNs or neutrophils) are critical mediators of innate immunity. Th ey respond rapidly (within minutes) to invading pathogens and to sites of tissue damage. Neutrophils clear invading pathogens by phagocytosis or by releasing toxic granules containing proteases and other enzymes and reactive oxygen species (ROS). For this reason, neutrophil degranu lation can lead to damage of host cells in the infl amed tissue. In several mouse models of AKI (e.g., ischemia/reperfusion injury and cisplatin-induced kidney injury), neutrophil accumulation in the injured kidney is a consistent and early fi nding [6][7][8] and depletion of neutrophils [6] or prevention of neutrophil traffi cking to the kidney [9] reduces kidney injury. In addition to releas ing granules, neutrophils have been shown to produce the pro-infl ammatory cytokines, interferon (IFN)-γ and interleukin (IL)-17, and the chemokine CXCL1, in the injured kidney [7,9]. Th ese fi ndings demonstrate the involvement of neutrophils in the pathogenesis of kidney injury in the commonly used murine model of ischemia/ reperfusion-induced AKI. Studies in some other species have reported a lack of signifi cant neutrophil accumulation and no benefi t after neutrophil depletion; however, these results may be related to diff erences in experimental models and limitations to methods for neutrophil depletion (see [10] and references therein).

Macrophages
Macrophages are phagocytic cells that arise from monocytes in the blood. Macrophage numbers increase early in the injured kidney (within 1 hour of reperfusion in an ischemia/reperfusion model [11]), and this infi ltration is mediated by CCR2 and CX3CR1 signaling pathways [12,13]. Th ese macrophages have a distinct 'infl amed' F4/80 low Ly6C high GR-1 + CX3CR1 low phenotype [12]. Depletion of macrophages, using liposomal clodronate, prior to kidney ischemia/reperfusion injury, reduced renal injury and adoptive transfer of macrophages reconstituted AKI [14]. Although macrophage infi ltration is observed in cisplatin-induced experimental AKI, blockade of macrophage traffi cking to the kidney did not prevent renal injury [15]. Analysis of post-ischemic kidney infi ltrating macrophages by fl ow cytometry demonstrated that they are signifi cant producers of many pro-infl amma tory cytokines, including IL-6 and tumor necrosis factor (TNF)-α [12]. Another study identifi ed IL-6 expres sion in renal outer medulla macrophages by in situ hybridization 4 hours after ischemia/reperfusion injury and IL-6 defi cient mice are protected from kidney ischemia/reperfusion injury [16]. Recently, diff erent types of macrophages have been described that either promote or inhibit infl ammation, M1 and M2 macrophages, respect ively [17]. New experimental evidence has demonstrated that once recruited to the post-ischemic kidney, macrophages display an M1 pro-infl ammatory phenotype, but several days later those same macrophages change to an M2 phenotype and are vital in the repair process of the kidney [18].

Natural killer cells
NK cells are similar to lymphocytes of the adaptive immune system but lack a T cell receptor. Th eir activation is governed by signals received from activating and inhibitory receptors on their cell surface. Th e ligands for these NK cell receptors are expressed on target cells and are regulated by cell stress such as viral infection. During experimental AKI, the proximal tubule epithelial cells (TECs) upregulate the expression of an NK cell activating ligand, Rae-1, which promotes TEC killing by activating the NKG2D receptor on NK cells [19]. Th e NK cells utilize perforins to kill the TECs in this model and their numbers in the kidney are elevated as early as 4 h after ischemic insult [19].

Dendritic cells
Dendritic cells classifi ed by the expression of CD11c + are the most abundant leukocyte subset in the normal mouse kidney [20] suggesting an important role in renal immunity and infl ammation. Upon stimulation, dendritic cells transform to a mature phenotype characterized by high levels of class II major histocompatibility complex (MHC class II) and co-stimulatory molecules with low phagocytic capacity. Mature dendritic cells are ideally suited to activate conventional T cells. Dendritic cells migrate to the renal draining lymph nodes after ischemia/ reperfusion injury and induce T cell proliferation suggest ing that kidney dendritic cells are vital to the adaptive immune response to ischemia/reperfusion injury [21]. Dendritic cells are also important in the innate immune response through several mechanisms. Th ese include releasing pro-infl ammatory factors, interacting with NKT cells via the co-stimulatory molecule, CD40, and presenting glycolipid to NKT cells via the CD1d molecule. Dong et al. demonstrated that after ischemia/ reperfusion injury, renal dendritic cells produce the proinfl ammatory cytokines/chemokines, TNF, IL-6, monocyte chemotactic protein (MCP)-1 and RANTES (Regulated on Activation, Normal T Expressed and Secreted), and depletion of dendritic cells prior to ischemia/ reperfusion injury signifi cantly reduced the kidney levels of TNF produced after ischemia/reperfusion injury [22]. IL-12 and IL-23 are mainly produced from activated dendritic cells and their downstream cytokines, IFN-γ and IL-17, promote macrophage activation and neutrophil recruitment and amplify the immune response following ischemic kidney injury [9]. Th e use of a genetically-engineered mouse in which the promoter for the mainly dendritic cell-specifi c surface protein, CD11c, drives expression of the human diphtheria toxin receptor (CD11c-DTR mouse) has facilitated understanding of the role of renal dendritic cells in experimental AKI. Recent studies have demonstrated that dendritic cells can promote or prevent injury to the kidney depending on the stimulus. For example, depletion of dendritic cells prior to ischemia/reperfusion injury reduces subsequent reperfusion injury and renal dysfunction [11]. On the other hand, depletion of dendritic cells prior to cisplatin exposure resulted in worse renal dysfunction and infl ammation [8]. Th ese fi ndings suggest that dendritic cells play an important role in orchestrating the immune response during AKI; additional studies are needed to understand what determines whether dendritic cells promote or inhibit kidney infl ammation as this information may translate into new therapeutic strategies.

Lymphocytes
T and B cells are the major eff ector cells of the adaptive immune system. Recognition of antigens, presented by antigen-presenting cells, in the presence of suffi cient costimulation, causes expansion and activation of T cells with a T-cell receptor specifi c for that antigen. B cells recognize soluble antigens through cell surface immunoglobulin type receptors. T cell contribution to the pathogenesis of kidney ischemia/reperfusion injury has been established in diff erent mouse models lacking certain types of lymphocytes. In mice which lack CD4 and CD8 T cells (nu/nu mice), kidney injury and dysfunction were signifi cantly reduced compared to wild-type controls after ischemia/reperfusion injury and cisplatin induced injury [23,24]. Reconstitution of nu/nu mice with CD4+ T cells alone but not with CD8+ T cells alone restored kidney injury after ischemia/reperfusion injury [23] and mice lacking CD8 or CD4 T cells alone suff ered less kidney dysfunction after cisplatin administration [24]. To investigate the requirement of antigendependent CD4 T cell activation in ischemia/reperfusion injury, Satpute et al. reconstituted nu/nu mice with either polyclonal CD4 T cells (with a diverse array of T-cell receptor specifi cities) or CD4 T cells from the DO11.10 mouse which nearly all express the same T-cell receptor, specifi c for a chicken ovalbumin peptide [25]. Whereas polyclonal T cells reconstituted injury in the T cell defi cient mice, DO11.10 T cells did not, unless their cognate antigen was co-administered with the T cells [25]. Th ese results suggest that antigen specifi c activation of CD4 T cells is important for early (within 24 h) AKI.
Th e results of B cell defi ciency in mouse models of AKI are not consistent. Mice lacking T and B cells (RAG-1 KO) were protected in some studies [26,27] and in others had similar injury to wild-type mice [28,29]. Defi ciency of B cells alone in mu MT mice conferred protection against ischemia/reperfusion injury in one study [30] but resulted in more severe renal dysfunction after ischemia/ reperfusion injury in another [31]. Th erefore, more studies are needed to determine the role of B cells in AKI.

Natural killer T cells
NKT cells are a unique subset of T lymphocytes with surface receptors and functional properties shared with conventional T cells and NK cells. Invariant or Type I NKT cells express a conserved T-cell receptor (Vα14/ Jα18 and Vβ8.2,Vβ2 or Vβ7) together with the NK cell marker, NK1.1. In contrast to conventional T cells, this invariant NKT cell T-cell receptor does not recognize pep tide antigens presented by MHC-class I or II; it recognizes glycolipids in the context of the class I-like molecule, CD1d. One of the most important functions of NKT cells is their ability to rapidly produce large amounts of cytokines, including Th 1-type (IFN-γ, TNF) and Th 2-type (IL-4, IL-13) at the same time. Th e rapid response by NKT cells following activation can amplify and regulate the function of dendritic cells, Tregs, NK and B cells, as well as conventional T cells. Th e number of IFN-γ producing type I NKT cells in the kidney is signifi cantly increased by 3 hours of reperfusion after ischemic insult [7] and NKT cells were observed in kidneys of patients with acute tubular necrosis (ATN) [32]. Blockade of NKT cell activation with the anti-CD1d mAb, NKT cell depletion with an anti-NK1.1 monoclonal antibody in wild-type mice, or use of type I NKT celldefi cient mice (Jα18 -/-) inhibited the accumulation of IFN-γ producing neutrophils after IRI and prevented AKI [7]. Type II NKT cells are not restricted to an invariant T-cell receptor but still recognize glycolipids, such as sulfatide. In a recent study by Yang et al. sulfatideactivated Type II NKT cells migrated to the injured kidney and protected mice from ischemia/reperfusion injury [32]. Taken together these studies suggest that diff er ent types of NKT cells may play divergent roles in AKI.

Protective actions of regulatory T cells
Tregs make up an indispensible counter-balance to the pro-infl ammatory cells of the immune system. Th is aspect is illustrated in patients with immunodysregu lation, polyendocrinopathy, enteropathy, x-linked (IPEX) syndrome and scurfy mice which both have devastating autoimmunity caused by a lack of functional Tregs [33]. Numerous types of Tregs have been described in the literature, but the most abundant are CD4 + T cells, which express CD25, and the Treg-specifi c transcription factor, FoxP3. FoxP3 suppresses the expression of pro-infl ammatory genes and promotes the anti-infl ammatory phenotype of Tregs [34]. Th e main mechanisms of suppression employed by Tregs include production of anti-infl am matory cytokines, such as IL-10 and transforming-growth factor (TGF)-β, generation of extracellular adenosine, direct contact-mediated inhibition of dendtritic cells through cell surface molecules such as lymphocyte activation gene (LAG)-3 and cytotoxic T-lymphocyte antigen (CTLA)-4 and several others [35,36]. Based on the important contribution of the pro-infl ammatory immune cells to AKI discussed above we hypothesized that Tregs would serve to protect the kidney from infl ammation and injury. To test this hypothesis, Tregs were partially depleted from naïve wild-type mice prior to a short ischemic insult that was not severe enough to induce kidney dysfunction in control mice [37]. Reduction in numbers of Tregs predisposed mice to tubular necrosis, renal infl ammation and loss of function [37]. In other studies, Treg depletion prior to more severe ischemia also worsened renal injury measured at 72 h of reper fusion [38]. Th ese results suggest that enhancement of Treg numbers or function may be an eff ective preventative therapy for AKI. Indeed, adoptive transfer of isolated Tregs, by i.v. injection prior to ischemia/reperfusion injury [37,39] or cisplatin administration [40] protected mice from kidney injury. Th is protection was associated with a reduction in infi ltrating innate immune cells and pro-infl ammatory gene expression in the kidney [37,39,40]. In the ischemia/reperfusion injury model, IL-10-defi cient Tregs were unable to off er any protection [37], suggesting that IL-10 production is an important mechanism for Treg-mediated protection from kidney injury. Two other methods of protecting the kidney, ischemic preconditioning [39,41] and use of FTY720 [42], have been shown to be partially dependent on the presence of Tregs.
In addition to prevention of injury, Tregs also promote the recovery of kidney function after ischemia/reperfusion injury [43]. Th is eff ect was demonstrated by depleting Tregs after ischemic injury, which caused reduced renal repair and increased mortality; on the other hand, adoptive transfer of Tregs 24 h after ischemic insult accelerated the recovery of renal function in mice [43]. Th e eff ects on kidney recovery did not appear to be mediated through eff ects on infi ltrating innate immune cells, but were associated with increased tubular epithelial cell proliferation and reduced cytokine production from infi ltrating T cells [43]. Th is study is important since it is diffi cult to detect renal injury in a timely manner and this suggests that manipulations to enhance Treg numbers or function can have benefi cial eff ects after injury has occurred.
Recently, a clinical trial was conducted to determine the safety and feasibility of ex vivo expansion of human Tregs for adoptive transfer into patients [44]. Th e authors demonstrated that it is possible to expand Tregs in vitro and then infuse up to 3 million Tregs/kg to patients, with no adverse eff ects observed [44]. Although this trial was conducted in the study of a diff erent disease model, it demonstrates that Treg cells themselves may be a novel therapeutic strategy in humans for many diseases, including AKI.

Conclusion
Th e immune response to kidney damage during AKI is an important contributor to the prolonged lack of renal function and progression of kidney injury. Th is response is complex, involving numerous pro-infl ammatory leukocytes which employ diverse eff ector mechanisms inside the kidney (Figure 1). Th e response also involves an antiinfl ammatory arm that protects the kidney from subthreshold insults, which is mediated by Tregs and possibly dendritic cells in some conditions ( Figure 1). In the future, it may be possible to prevent AKI or treat existing AKI by inhibiting the pro-infl ammatory immune response to kidney injury and/or promoting the antiinfl ammatory response through drugs or maneuvers that enhance Treg action or by direct administration of Tregs themselves.

Competing interests
The authors declare there are no competing interests.

Figure 1. Toxic or ischemic insults initiate infl ammation in the kidney which consists of interferon (IFN)-γ-and interleukin (IL)-17producing neutrophils (PMN), IL-6-producing macrophages (MØ), dendritic cells (DC) which produce tumor necrosis factor (TNF)-α, IL-12 and IL-23, IFN-γ-producing CD4 + T cells, invariant type I natural killer T (NKT) cells and natural killer (NK) cells which kill renal tubule epithelial cells in a perforin-dependent manner.
On the other hand toxic and ischemic injuries are prevented by regulatory T cells (Tregs) which produce IL-10, and in cisplatin-induced nephrotoxicity DCs protect the kidney by an unknown mechanism.