Myeloid cells in acute kidney injury

Abstract

Acute kidney injury (AKI) is a serious and common clinical condition characterized by a sudden decline in kidney function. Although kidney function decline is typically reversible, a certain subset of AKI patients eventually develop chronic kidney disease (CKD) and kidney failure. Immune cells are well-known mediators of the injury sequelae. Myeloid cells such as neutrophils, dendritic cells, and macrophages drive the initial inflammatory response following AKI, but can transition their phenotype after resolution of the injury to promote repair. Failure to resolve the initial injury, or improper tubular repair, drives persistent myeloid cell accumulation that can result in development of kidney fibrosis and CKD. In this review, we focus on the role of myeloid cells following AKI including the mechanisms through which they promote injury and repair.

Introduction

Acute kidney injury (AKI) is a clinically significant problem as it occurs in nearly 10% of general hospital admissions and nearly 30% of ICU admissions⁽¹⁾. Etiologies of AKI are broadly defined as pre-renal (hemorrhage, shock, heart failure), intrinsic (nephrotoxins, bacterial infections, medications), or post-renal (renal obstructions, cancer, nephrolithiasis) in nature ⁽²⁾. Clinically, AKI is defined as a rapid, and often reversible, decline in kidney function and is marked by increased serum creatinine (CREA) and blood urea nitrogen (BUN) with decreased urine and glomerular filtration rate (GFR). Glomerular filtration in the kidney is the first functional step in blood plasma filtration and urine production, and it occurs as a result of the pressure differential between the Bowman’s space and the glomerulus ⁽³⁾. Both afferent and efferent renal blood flow can directly affect this pressure gradient, and both can be affected in AKI. Pre-renal AKI typically occurs due to restriction of the renal blood flow, thereby leading to glomerular and tubular injury via hypoperfusion and hypovolemia ⁽³⁾. Intrinsic causes of AKI are typically the result of direct injury to the glomerulus or renal tubules, which leads to the release of vasoconstrictors and subsequently reduces efferent renal blood flow ⁽³⁾. Lastly, post-renal AKI is caused by obstruction of urinary outflow leading to congestion and disruption of kidney filtration; this impacts the glomerular filtration gradients, and leads to renal inflammation and tissue injury⁽³⁾. Regardless of its cause, patients who experience AKI are at a much higher risk of developing chronic kidney disease (CKD) or kidney failure in the future ^{(4, 5)}.

While the pathogenesis of AKI largely depends on the etiology, there are several common pathologies involved regardless of underlying cause: cell damage and death (via necrosis, ferroptosis, and apoptosis), decreased tubular function, oxidative damage, immune cell recruitment, and endothelial dysfunction. The inflammatory response begins upon release of damage associated molecular patterns (DAMPs) by injured renal tubular epithelial cells (TEC). The binding of DAMPs to pattern recognition receptors (PRRs) on resident innate immune cells such as macrophages and dendritic cells begins a cascade of inflammatory signaling, which serves to recruit additional immune cells to the site of injury and drive local inflammation. Immune-cell driven inflammation is a hallmark of the injury response⁽⁶⁾, and the immune sequelae of AKI are known to play a prominent role in the subsequent timeline of injury and repair that follows^(7–9). These cells begin to respond immediately following tissue damage, with kidney resident macrophages and dendritic cells (DCs) responding first, followed by immune populations such as neutrophils and Ly6C^hi monocytes which extravasate to the site of injury after release of chemokines from injured tubular cells and other immune cells ⁽¹⁰⁾.

Currently, there are no available therapies that can prevent the loss of kidney function following AKI and majority of patients are identified after the injury has occurred. Thus, treatment is available only when injury has been detected and primarily consists of palliative therapies such as renal replacement therapy or diuretics ⁽¹¹⁾. As the molecular understanding of AKI pathology deepens, immune-cell focused treatments offer future therapeutic potential. The myeloid cell compartment, while not the only contributor to AKI pathology, plays a diverse and important role in the tissue damage and later repair that occurs and thus is the focus of this review.

Crosstalk between TECs and immune cells in AKI

Kidney injury initiates multiple forms of cell death in the tubular epithelium, including apoptosis and various forms of necrotic cell death. Following the release of DAMPs and chemokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured TECs, the resulting inflammatory response by macrophages and recruited monocytes results in sustained inflammation and tubular apoptosis ⁽¹²⁾. Apoptosis is a caspase mediated form of controlled cell death whereby cells that undergo apoptosis are recognized and removed via phagocytosis in a controlled manner to limit immune activation ^{(13, 14)}. Apoptosis can be triggered via intrinsic signaling, typically as a result of mitochondrial outer membrane permeabilization via Bax/Bak oligomers, or extrinsic signaling via binding of Fas ligand or TNF-α to death receptors on the cell membrane; all signaling mechanisms result in caspase family activation (caspase 8, 9 and/or 12), caspase mediated proteolysis of cellular structures (caspase 3/6/7), and mitochondrial dysfunction ^{(13, 15)}. The plasma membrane integrity is maintained throughout the process, and the removal of dead cells by macrophages and other surrounding cells limits inflammation following apoptotic cell death ⁽¹⁴⁾. The breakdown of tubular epithelium via apoptotic cell death can lead to unregulated diffusion of macromolecules, ions, and water, further contributing to the loss in GFR and aggravating tubular damage ⁽¹⁴⁾. While apoptosis is not typically thought to induce an inflammatory response, it has been reported that apoptotic TECs can induce the activation of plasmacytoid dendritic cells (pDCs), suggesting this process could contribute to their production of IFN-α following kidney injury and thus propagate the inflammatory response ⁽¹⁶⁾. Following AKI, apoptotic TECs are found in both cortical and medullary regions of the kidney and in murine models of ischemic injury, the number of apoptotic TECs cells was found to correlate with the level of renal dysfunction^{(15, 17–19)}.

In contrast to apoptosis, necrosis is a form of cell death accompanied by the breakdown of the cellular membrane and subsequent release of unprocessed cellular components including dsDNA, RNA, and immunogenic proteins such as IL-33, F-actin, and IL-1α ^{(15, 20)}. The release of these DAMPs from necrotic cell death recruits macrophages to phagocytose the cellular debris and induces an inflammatory response ⁽²¹⁾. While originally thought to be unregulated, it is now appreciated there are several regulated pathways of necrotic cell death including necroptosis, pryoptosis, and ferroptosis. Necroptosis in AKI is typically triggered by TNF superfamily receptor activation^{(15, 20, 22)}. This leads to receptor-interacting protein kinase 3 (RIPK3) activation, phosphorylation of mixed lineage kinase domain–like (MLKL), and eventual induction of plasma membrane rupture ^{(15, 20, 22)}. Mice deficient in RIPK3 or treated with Necrostatin-1, an inhibitor of necroptosis, show partially improved kidney function following AKI, increased survival, and decreased tubular injury, demonstrating that necroptosis promotes AKI ^{(23, 24)}.

Pyroptosis is a form of necrotic cell death that is triggered by inflammation and involves the activation of a multi-protein complex called the inflammasome⁽²⁵⁾. The process of pyroptosis is dependent on Caspases (1 and 11) and the Gasdermin D (GSMD) family of proteins; the gasdermin proteins aid in creating small pores in the cell membrane which release both ions and cytokines such as IL-18 and IL-1β, with the cell growing in size until the cell membrane eventually bursts ^{(15, 25)}. The levels of pyroptosis-related proteins, including Caspase-1 and Caspase-11, significantly increase in the first 6 hours following IRI with the highest levels being observed 12 hours post-injury⁽²⁵⁾. Many studies have shown the impact of pyroptosis in AKI; Caspase-11 knockout mice show improved renal function, reduced TEC damage, and reduction in macrophage and neutrophil numbers after IR kidney injury ⁽²⁶⁾. β-hydroxybutyric acid (β-OHB) was found to reduce kidney damage in mice following injury by decreasing expression of IL-18, IL-1β, and Caspase-1 via FOXO3-directed epigenetic regulation ⁽²⁷⁾, and inhibiting Caspase-11 and GSMD proteins via the drug Disulfiram has been shown to reduce pyroptosis and improve renal function after AKI in mice ^{(25, 28, 29)}.

Lastly, ferroptosis is an iron-dependent form of necrotic cell death which involves glutathione metabolism, enhanced lipid reactive oxygen species (ROS) and intracellular iron accumulation ^{(15, 21)}. In homeostasis, glutathione peroxidase 4 (GPX4) uses glutathione (GSH) supplied by X_c-minus, a glu/cys antiporter in the plasma membrane, to continuously reduce peroxidized lipids ^{(21, 30)}. The system therefore indirectly inhibits lipid oxidation reactions (Fenton reactions) between iron substrates and H₂O₂ in the cell ⁽³⁰⁾. In ferroptosis, this system is compromised (decreased GSH, or decreased activity of GPX4) leading to increased intracellular Fe²⁺, increased ROS production through the Fenton reaction, and increased lipid peroxides resulting in cell death ⁽³⁰⁾. The ferroptosis inhibitor ferrostatin-1 improved kidney function and decreased TEC death in folic-acid induced AKI ⁽³¹⁾. Treatment with ferrostatin-1 reduced rolling, adherent, and infiltrated immune cells following IRI, reduced KIM-1 levels, TEC necrosis, and improved kidney function ⁽³²⁾.

Dying TECs are also know to release histones, such as H2A, H2B, H3, H4, and H1; these positively charged proteins can stimulate toll-like receptor (TLR) 2 and 4 on macrophage and dendritic cells, further inducing inflammation through the Myd88 and NF-κb signaling pathway ^{(33, 34)}. High-mobility group box-1 protein (HMGB1), a chromatin-associated protein released by TECs and vascular cells following ischemic injury, further amplifies systemic inflammation⁽³⁵⁾. The renal TECs continue their communication with immune cells by release of pro-inflammatory cytokines such as IL-36α after AKI⁽³⁶⁾. IL-36α is known to activate the NLRP3-inflammasome in macrophages and DCs, stimulating secretion of pro-inflammatory cytokines such as IL-1β that induce CD4 and CD8 T cell activation⁽³⁷⁾. Notably, IL-36R knock-out significantly reduced kidney injury, fibrosis, and pro-inflammatory cytokines in kidney including IL-17A and IL-18 after injury⁽³⁷⁾. In the inflammatory environment following injury, activated T cells secrete CD40L which stimulates CD40 on TECs^{(38, 39)}. This CD40L/CD40 axis induces expression of CCL2 and CXCL8 (also known as IL-8) that functions as a chemoattractant for macrophages and neutrophils, and other immune cells ⁽⁴⁰⁾. CXCL8 also works in an autocrine manner to facilitate TEC expression of ICAM1⁽⁴¹⁾, a molecule that involved in crosstalk with infiltrating neutrophils during inflammation and promotes diapedesis ^{(42, 43)}.

Sphingosine 1-phosphate (S1P), which is known to be a “find-me” signal secreted from dying cells⁽⁴⁴⁾, regulates the helper T cell population in injured kidneys via activation of S1P receptor 3 (S1PR3) on DCs; as S1pr3 KO mice had improved kidney function after IRI, and disrupted DC maturation ⁽⁴⁵⁾. While adoptive transfer of wild-type DC promoted IFNγ and IL-17 levels in ischemic kidney, S1pr3 KO DC induced IL-4 and IL-10 ⁽⁴⁵⁾. This suggests the S1P-S1PR3 axis in DCs dictates the type of helper T cell activation that occurs after injury, with S1PR3 KO DCs driving a Th2 phenotype ⁽⁴⁵⁾. TECs are also reported to produce Fms-related tyrosine kinase 3 ligand (Flt3L) in the early stages of the injury response that promotes the activation of conventional dendritic cells type 1 (cDC1). Intravenous administration of recombinant Flt3L ameliorated kidney injury, suggesting that cDC1 suppress inflammation^{(46, 47)}. It was further found that Flt3L treatment expanded the cDC1 population, decreased conventional dendritic cells type 2 (cDC2) numbers, and promoted Treg responses, characterized by increased Foxp3 and Il10 expression, while simultaneously reducing Th1 responses⁽⁴⁷⁾.

Lastly, it is reported that activated TECs can produce CX3CL1 (also called fractalkine) which recruits neutrophils, monocytes and macrophages, and CD1c⁺ cDC2 expressing CX3CR1 ⁽⁴⁸⁾. This CX3CL1-CX3CR1 interaction mediates both the recruitment and retention of these cells within the renal tubulointerstitium, and is reported to promote fibrosis through TGF-β production in cDC2s ^{(48, 49)}. Thus, renal TECs are the first cell type affected by AKI and regulate the downstream recruitment and phenotype of myeloid cells to control AKI outcomes.

Macrophage function during the acute phase of injury

Macrophages rapidly infiltrate injured kidneys and single-cell studies have highlighted the communication networks between tubular cells and macrophages following AKI. For instance, Blazer et al identified a maladaptive PT population following IRI, which express various cytokines and molecules that potentially communicate with myeloid cells ⁽⁵⁰⁾. In a sepsis AKI model, cortical PT cells express Midkine (Mdk), that is associated with macrophage recruitment ⁽⁵¹⁾. Spatial transcriptomic analysis further validated this finding as macrophages were infiltrated into outer cortical part of the kidney ⁽⁵¹⁾. To date, macrophages remain the most well-studied immune cell in AKI. During the course of kidney injury and repair, macrophages display remarkable plasticity in their phenotype, ranging from inflammatory to reparative to profibrotic, depending on the stage and microenvironment ⁽⁶⁾.

In the acute phase of injury, both kidney resident macrophages (KRMs, CD11b^mid F4/80^hi) and recruited infiltrating monocytes (IMs, CD11b^hi, F4/80^lo) play a role in AKI ⁽⁵²⁾. KRMs are tissue resident cells and play a role in tissue homeostasis and immune surveillance; as professional phagocytes they clear both pathogens and cell debris from the kidney milieu and help activate immune responses after infection or injury ⁽⁵³⁾. KRMs are seeded during embryogenesis and are maintained through proliferation and input from bone marrow derived IMs in homeostasis ^{(53, 54)}. In a physiologic homeostatic state, IMs aid in immune surveillance and maintenance of the KRM population, whereas during injury or infection the number of IMs dramatically increases as they infiltrate the tissue in response to local inflammation ^{(53, 55)}. Following kidney injury, both KRMs and IMs adopt a pro-inflammatory phenotype characterized by expression of Ccl3, iNos, and inflammatory cytokines such as IL-6, IL-1β, and TNF- α. These macrophages, often referred to as M1-like, are believed to promote inflammation and tissue damage ^{(9, 56)}, in large part due to their cytokine production. The cytokine IL-6 is known for its effect on a wide variety of cells; IL-6 induces T-cell differentiation and B-cell antibody production, increases serum platelet levels, and induces production of acute phase proteins including C reactive protein and fibrinogen ^{(57, 58)}. IL-1β ligates the IL-1 receptor, which is ubiquitously found on cells in the kidney, and this signaling leads to activation and cytokine release by macrophages and DCs, injury to and chemokine release from surrounding parenchymal cells ⁽⁵⁹⁾. Lastly, TNF- α activates MAPK, caspase, and NF-κB signaling, leading to additional inflammatory cytokine production in a feed forward loop ^{(60, 61)}. While KRM numbers stay relatively stable following kidney injury, IM numbers substantially increase ⁽⁶²⁾. These recruited IMs make up a large portion of the macrophage compartment following injury, and their role in AKI has been shown using Ccr2 deficient mice. In mice lacking Ccr2; Ly6c^hi monocytes are unable to emigrate from the bone marrow thereby preventing IM engraftment in the kidney following AKI ⁽⁶³⁾. Kidney damage was ameliorated in Ccr2^−/− animals after injury and this protection was attributed to the reduction in pro-inflammatory cytokines including IL-6, IL-1β, and TNF- α ^{(64, 65)}. Additionally, Ccl2 expressing macrophages were identified in an atrophic AKI model and were found to be correlated with myofibroblast accumulation and interstitial fibrosis⁽⁶⁶⁾. Mincle receptor⁺(Clec4e⁺) cells have also been identified in the early inflammatory phase of IR AKI; Mincle receptor is a C-type lectin receptor whose activation leads to NF-κB signaling and inflammatory cytokine production ^{(67, 68)}. A spatial analysis showed that Mincle⁺ macrophages were co-localized with fibrotic and inflammatory regions of injured kidney and deletion of Mincle receptor protected against kidney injury and fibrosis after IRI ⁽⁶⁷⁾.

Following injury, activation of pro-inflammatory cytokine production in macrophages is driven through a variety of transcription factors including NF-κB that induce the transcription of nitric oxide, adhesion molecules (ICAM1, VCAM1), chemokines (CXCL1, MCP-1), and cytokines (IL-61, IL-6, TNF- α) that promote inflammation ^(69–72). Inhibition of NF-κB during the acute injury phase decreased inflammation and fibrosis in the kidney following AKI ^{(73, 74)}. Another common signaling pathway in M1 macrophages after injury is JAK/STAT, which results in production of cytokines including IL-6, IL-1β, and TNF- α. Inhibition of JAK/STAT signaling resulted in decreased levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ ), decreased apoptosis, improved renal function, and reduced oxidative stress following AKI in multiple animal models ^(75–78). Further supporting the idea these pro-inflammatory macrophages promote kidney injury, multiple studies have used liposomal clodronate (LC), an encapsulated bisphosphonate that induces apoptosis upon phagocytic uptake^(79–82), to deplete macrophages prior to IR injury and shown significant reduction in damage to the kidney following injury ^{(9, 83–87)}. The reduction in kidney injury observed in these studies was associated with decreased pro-inflammatory cytokine production and signaling. Furthermore, re-introduction of pro-inflammatory macrophages into LC-depleted mice after injury resulted in increased BUN and tubular injury levels, indicating that M1-like macrophages promote injury and inflammation following AKI ⁽⁹⁾. Lastly, a 2014 study reported that prolonged accumulation of the inflammatory M1-like macrophages inhibited kidney repair following injury and led to increased levels of fibrosis ⁽⁸⁸⁾. Xu and colleagues compared immune cells at different days post IRI and found a “Second wave” of the injury. In this study, the macrophages that persisted beyond the repair phase (day 7 post IRI) highly expressed T cell and neutrophil activating molecules including Cxcl16 and Mcp2 (Ccl8), which correlated with worsened injury outcomes⁽⁸⁹⁾. Combined these studies demonstrate that pro-inflammatory macrophages contribute to tissue damage after injury.

Macrophage function during the reparative phase of injury

The production of pro-inflammatory cytokines in the acute phase of injury serves to recruit circulating IMs and neutrophils into the tissue to further perpetuate inflammation and damage. After the initial injury phase, pro-inflammatory macrophages transition to an anti-inflammatory, pro-reparative phenotype ^{(9, 90)}. These macrophages, often referred to as M2-like macrophages, express pro-reparative cytokines such as Il-10 mannose receptor (MR), Arginase, IL-22, and Wnts ⁽⁹¹⁾. Mannose receptor aids in decreasing the inflammatory glycoproteins produced earlier in the immune response, while Arginase, a protein responsible for conversion of the amino acid l-arginine into urea and ornithine, is thought to drive proliferative responses after AKI to facilitate tubular repair ^{(92, 93)}. In support of a pro-reparative role of Arginase 1, data indicate that macrophage specific loss of Arg1 resulted in reduced survival as well as decreased tubular repair and proliferation after AKI ⁽⁹⁴⁾. Intriguingly, recent scRNAseq studies suggest that Ly6c^hi monocytes give rise to Arg1+ macrophages following AKI⁽⁹⁰⁾. The differentiation of Ly6c^hi monocytes into Arg1+ macrophages begins as early as 1 day following injury with Arg1+ macrophages beginning to predominate around day 3. These pro-inflammatory turned reparative macrophages expressed genes related to wound repair, angiogenesis, and phagocytosis⁽⁹⁰⁾. Arg1 expressing macrophages, known for their anti-inflammatory function ⁽⁹⁵⁾, were reported to have enriched TGFβ signaling with renal tubule cells in multiple AKI models⁽⁹⁶⁾

IL-22 is another cytokine involved in tubular cell repair and proliferation following kidney injury, with macrophages and dendritic cells being the primary producers of this cytokine^(97–100). Increased IL-22 production accelerated epithelial cell growth and reduced serum CREA and BUN in mice compared with controls, while depletion studies have reported that reduced IL-22 impairs epithelial cell repair following AKI^{(100) (98)}. Similarly, macrophage-derived Wnt is known to be necessary for kidney repair as depleting Wnt7b led to a decrease in tubular repair and an increase in fibrosis following AKI⁽¹⁰¹⁾.

Recently TREM2 has been identified as a key mediator of macrophage function following AKI. Loss of TREM2 led to decreased macrophage infiltration, increased kidney fibrosis, and increased inflammation in multiple rodent models of AKI^{(102, 103)}. Zhang et al. also found that loss of TREM2 altered macrophage localization following AKI, suggesting that TREM2 controls kidney macrophage localization ⁽¹⁰³⁾. Transfer of TREM2-expressing macrophages into injured mice significantly reduced immune infiltration, interstitial fibrosis, and glomerular damage further supporting the idea that TREM2 macrophages promote repair following AKI⁽¹⁰³⁾. Lastly, a subset of macrophages expressing both Trem2 and Mmp12, identified in an IRI mouse model, were found to express profibrotic genes (Tgfb1, Tgfbi, Igf1), extracellular matrix components (Fn1, Spp1, Ecm1), and cathepsins (Ctsb, Ctsd, Ctsl), and were predicted to be involved in matrix remodeling and efferocytosis during regeneration following injury ⁽¹⁰⁴⁾. In sum, the function of macrophages in the repair phase of kidney injury is believed to be beneficial for tissue recovery.

Depletion studies have further supported the idea that macrophages promote tubular repair following injury. For example, Lee et al. depleted macrophages 48 and 72 hours after injury ⁽⁹⁾ to study the importance of reparative macrophages after AKI. Importantly, this approach does not alter the initial recruitment of inflammatory cells into the kidney, but instead, directly targets reparative macrophages that appear in the kidney 3-5 days post injury. Data from this study showed that LC treated mice had reduced tubular regeneration when compared to LV injected mice, which was associated with decreased GFR at day 5 and 7 after injury⁽⁹⁾. Supporting these findings, several other groups have reported that macrophage depletion during the reparative phase inhibits tubular repair, decreases kidney function, and leads to an increase in fibrosis^{(9, 105–107)}. Additionally, restoring M2-like macrophages to LC-depleted animals following AKI lowered BUN and reduced tubular injury compared to non-depleted animals, demonstrating a clear anti-inflammatory, pro-reparative phenotype of these macrophages ⁽⁹⁾.

While macrophages can broadly be classified as proinflammatory M1-likeor pro-reparative M2-like, single cell RNA sequencing studies emphasize that macrophages exhibit significant transcriptional heterogeneity following AKI, which is dependent on the microenvironment. Given the plasticity and complexity of these cells, continued studies into the precise function of the various subsets remain a need for the AKI field.

Dendritic cells in acute kidney injury

Dendritic cells are professional antigen presenting cells present in all tissues; in the kidney, their functions in homeostasis include uptake and trafficking of antigens to draining lymph nodes where they interact with naïve T cells and induce tolerance ⁽¹⁰⁸⁾. Dendritic cells are typically classified into three subsets: conventional dendritic cell type 1 (cDC1s) that express CD103⁺ and are CD11b−, conventional dendritic cell type 2 (cDC2s) that express CX₃CR1 and CD11b, and plasmacytoid dendritic cells (pDCs) which express PDCA-1 and B220 ^{(16, 109)}. The vast majority of kidney DCs are cDC2s, with cDC1s comprising approximately 5% of the renal DC population ^{(110, 111)}. Following kidney injury, cDCs become activated and specialized for T cell activation; within 24 hours of AKI these cells are found in the draining lymph nodes where they aid in initiating the adaptive immune response ^(110–112). It is thought cDC2 subsets actively contribute to inflammation following kidney injury; beyond their role in T cell activation cDC2s secrete pro-inflammatory cytokines critical for initiating inflammation post-AKI ^{(113, 114)}. The primary function of pDCs upon activation is type I interferon (IFN) production, and their production of IFN-α is linked to worsened kidney injury following AKI ⁽¹⁶⁾. Using major histocompatibility complex 2 (MHC II) and morphologic assessment, DCs were initially reported to be predominantly expressed in the cortex and outer medulla of rodent kidneys ⁽¹¹⁵⁾. CD1c⁺ CX3CR1⁺ cDC2s have also been suggested to promote fibrosis by secreting TGF-β in the tubulointerstitium ⁽⁴⁹⁾.

Following kidney injury, CD11c⁺ DCs were found to be highly enriched and localized in the tubulointerstitium but not within inflamed glomeruli ⁽¹¹⁶⁾. A similar accumulation of DCs was reported in an IRI model of injury ⁽¹¹⁷⁾. FITC-dextran uptake experiments have shown that kidney CD11c⁺ DCs exhibit greater phagocytic activity than their splenic counterparts, suggesting a heightened capacity for antigen surveillance within the renal microenvironment ⁽¹¹³⁾. Mechanistically, DCs are a primary source of tumor necrosis factor (TNF) 24 hours after ischemic injury ⁽⁴⁶⁾. Renal DCs also produce inflammatory cytokines including IL-6 and MCP-1 following AKI, suggesting they contribute to the initial injury and inflammatory response ⁽⁴⁶⁾. It should also be noted that DCs and macrophages share overlapping functions as well as molecular and genetic markers, making it challenging to clearly distinguish between the two populations. Traditional identification of DCs has relied on markers such as CX3CR1, CD11c, and F4/80. However, these are also expressed by various macrophage subsets, albeit at differing levels ⁽¹¹¹⁾. Consequently, advanced approaches like single-cell transcriptomic analysis are essential for validating previous findings and enabling a more precise characterization of DC function in the context of AKI.

Neutrophils in Acute Kidney Injury

Neutrophils are one of the first cells to be recruited to the kidney following injury, often accumulating within 24 hours of tissue damage⁽¹⁰⁾. In the vasculature, E-selectin mediated signaling to circulating neutrophils leads to slow-rolling and eventual tethering of the neutrophils along the vascular endothelium; tethering of the neutrophils occurs via integrin signaling including ICAM1, ICAM2, and VCAM ^{(43, 118, 119)}. Chemokines produced by the injured tubular epithelial cells such as CXCL1 and CXCL2 help enhance the ligand-receptor affinity, ensuring firm adhesion and neutrophil arrest so that transendothelial migration can occur ⁽¹²⁰⁾. Downregulation or silencing of CXCL1 or CXCL2 expression inhibited neutrophil infiltration following kidney injury and was renoprotective ^{(118, 121)}. Following adhesion, neutrophils become polarized and flattened while they scan the endothelium for an extravasation site; these changes are driven in part by PI3K/PIP3, Rac1/2, and PIP2/RhoA signaling⁽¹²²⁾. Slit2 protein, which blocks Rho-family activation, was found to inhibit neutrophil infiltration and led to decreased serum creatinine and tubular necrosis in a murine injury model⁽¹²³⁾. Once transmigration begins, neutrophils follow chemotactic gradients as they traverse the endothelial barrier and enter the tissue. A recent spatial transcriptomics study found that activated neutrophils primarily migrate to outer medulla following kidney injury⁽⁵¹⁾. It is thought S3 proximal tubule (PT) cells may secrete a chemoattractant for neutrophils, as neutrophils have been reported to be colocalized with Atf3+ PT cells⁽⁵¹⁾.

Once in the tissue, neutrophils contribute to tissue damage through production of reactive oxygen species (ROS) via NADPH oxidase (NOX2), reactive nitrogen species (RNS), and neutrophil extracellular traps (NETs)^(124–126). NETs are comprised of decondensed DNA in a complex with histone and neutrophil granule proteins and promote inflammation and tissue injury ⁽¹²⁴⁾. Inhibiting the formation of NETs has been shown to be protective in multiple models of AKI^(127–130). The histones released during NET formation are cytotoxic and inhibition of free circulating histones alone was sufficient to reduce tubular injury in IR injury ⁽¹³⁰⁾. The number of Chi3l1 expressing neutrophils has also been reported to correlate with kidney fibrosis after injury ⁽⁶⁶⁾. In the liver, neutrophils play a role in angiogenesis and cell debris clearance and their absence negatively affects liver repair after injury ⁽¹³¹⁾. Despite the obvious role neutrophils play in tissue damage and inflammation, neutrophil depletion has shown minimal effects in kidney injury studies ^{(132, 133)}. Recently, epidermal growth factor receptor (EGFR) deletion in neutrophils has been found to shorten their lifespan and limit kidney fibrosis following injury ⁽¹³⁴⁾. In these studies loss of EGFR did not impact neutrophil infiltration but was instead found to promote neutrophil apoptosis, as EGFR^−/− neutrophils showed minimal expression of antiapoptotic protein Mcl-1 when compared with their EGFR^WT counterparts ⁽¹³⁴⁾. These results suggest EGFR expression and activation functions to extend neutrophil lifespan after AKI by upregulating Mcl-1 expression in neutrophils ⁽¹³⁴⁾.

Conclusions

Immune-cell driven inflammation is a hallmark of AKI, and specifically targeting these cells is a promising therapeutic avenue. Myeloid cells have diverse functions in AKI and affect both the injury and repair process, thus are a favorable target. The spectrum of phenotypes observed across macrophages, dendritic cells, and neutrophils is heavily influenced by the kidney microenvironment, specifically the chemokines and cytokines present therein, and there is a need for a deeper understanding of the molecular mechanisms behind AKI pathology. In this review, we examined the current understanding of tubular epithelial cell, macrophage, dendritic cell, and neutrophil response to acute kidney injury, and their phenotypes and function in the kidney following injury.

Figure 1. Crosstalk between tubular epithelial cells (TECs) and immune cells during acute kidney injury (AKI).

Initial injury induces cell death in TECs, releasing damage-associated molecular patterns (DAMPs), which activate pattern recognition receptors (PRRs) on immune cells, triggering downstream signaling to induce inflammation and fibrosis. TEC-derived cytokines and immune stimulatory molecules, further amplify inflammation by activating macrophage and DC.

Figure 2: Macrophage phenotypes in AKI.

Following AKI, the M1-like population rapidly expands, and is comprised of subsets reported to express iNos and ccl3; these cells produce cytokines such as IL-6, IL-1β, and TNF- α. The M2-like population gradually begins to predominate following the initial stage of injury. These cells are characterized by Arginase and mannose receptor 1 expression, and produce cytokines including IL-10, IL-22, and TGF-β.

Figure 3. Functional roles of dendritic cells (DCs) in kidney injury.

DCs reside predominantly in the cortex and outer medulla of normal kidneys, with significant expansion following acute kidney injury (AKI). Mature DCs express high MHC II and exhibit enhanced antigen uptake and phagocytic activity. Conventional DCs (cDCs) are subdivided into two major subsets: inflammation-suppressing cDC1 and inflammation-promoting cDC2. While cDC1 subsets recruit regulatory T cells (Tregs) to facilitate tissue repair, cDC2 subsets exacerbate inflammation and fibrosis via secretion of pro-inflammatory cytokines. Plasmacytoid dendritic cells (pDCs) are the primary producers of type I interferons (IFN-α and IFN-β).

Figure 4: Neutrophils in AKI.

Circulating neutrophils are recruited into the renal interstitium via expression of selectins and integrins on the endothelium, including CXCL8, ICAM1, ICAM2, and VCAM1. Neutrophils undergo transendothelial migration to move into the interstitium; here they begin to mediate their inflammatory effects via production of reactive oxygen and nitrogen species (ROS and RNS), NADPH oxidase, and matrix metalloproteases. Formation of extracellular NETs is another known contributor to neutrophil mediated renal inflammation following AKI.