Abstract

Interstitial inflammation is an important feature of cystic kidney disease. Renal macrophages are the most well-studied inflammatory cell in the kidney, and their involvement in cyst formation has been reported in different animal models and patients with cystic kidney disease. Originally, it was believed that renal macrophages were maintained from a constant supply of bone marrow–derived circulating monocytes, and could be recruited to the kidney in response to local inflammation. However, this idea has been challenged using fate-mapping methods, by showing that at least two distinct developmental origins of macrophages are present in the adult mouse kidney. The first type, infiltrating macrophages, are recruited from circulating monocytes and gradually develop macrophage properties on entering the kidney. The second, resident macrophages, predominantly originate from embryonic precursors, colonize the kidney during its development, and proliferate in situ to maintain their population throughout adulthood. Infiltrating and resident macrophages work together to maintain homeostasis and properly respond to pathologic conditions, such as AKI, cystic kidney disease, or infection. This review will briefly summarize current knowledge of resident macrophages in cystic kidney disease.

Keywords

resident macrophage, kidney, Cystic Nephropathy, and Cistanche benefits.

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Macrophages in Kidney

Renal macrophages are the largest immune cell population in the kidney and play an important role in homeostasis, monitoring, immune response, tissue injury, and repair in vivo (1-3). Macrophages are a major component of the renal mononuclear phagocyte system and are thought to originate from bone marrow-derived monocytes, which can polarize into distinct phenotypic subpopulations in response to environmental stimuli (4-6). However, they are often confused with dendritic cells due to their co-expression of CD11c and MHCII on the cell surface (7,8). With the advent of new genealogical tracing and single-cell RNA sequencing methods, it is now possible to clearly outline macrophages and dendritic cells in the kidney of mammalian species (9-11). In these studies, the authors demonstrated that renal macrophages are highly heterogeneous and specialized populations derived from two distinct developmental sources (12-14) (Figure 1). One type, infiltrating macrophages, originates from monocyte precursors in the bone marrow and is recruited to the kidney in response to local inflammation (15,16). The other type, resident macrophages, are present in the kidney for a long time, are less mobile, and appear mainly during organogenesis (17). They are derived in a Myb-independent manner from red myeloid progenitor cells that first arise in the fetal yolk sac, colonize the fetal liver, and migrate to the kidney during early development (12,13,17).

The idea that resident macrophages are homogeneous populations in the kidney and are derived exclusively from embryonic precursors has recently been challenged. using a newly generated cre-inducible-hCD59 transgenic line, Liu et al. (18) traced the fate of resident macrophages in the kidney from birth to full maturation and found that some resident macrophages actually originated from peripheral monocytes. The idea that some renal macrophages are monocyte-derived is supported by data from Ms4a3Cre-RosaTdT fate localized mice that faithfully label all adult hematopoietic stem cell-derived monocytes (19). In these studies, the authors found that a large proportion of renal macrophages were derived from adult hematopoietic stem cell-derived monocytes. Liu et al. (18) also demonstrated that resident macrophages of both lineages have a characteristic long-term residence in the kidney, but with functional differences in terms of immune response and metabolic profile under disease conditions. These data suggest that renal macrophages can originate from multiple precursor populations and that their ontological origin may influence their function (6,17,18).

In mice, infiltrating and resident macrophages can be distinguished based on the expression of the surface markers F4/80 and CD11b, with resident macrophages being F4/80high, CD11blow and infiltrating macrophages being F4/80low, CD11bhigh (12,13,20). The exact function of resident macrophages in the kidney is unknown, although emerging evidence suggests that they play an important role in renal development, vascularization, and renal repair (17,21 - 24). Although data on the function of resident macrophages in the kidney are limited, in part because of the nonspecific methods used to study these cells in the past, we may be able to gain insight into their function because of the similarities between m2-like macrophages and resident macrophages. m2 macrophages have anti-inflammatory and pro-fibrotic functions (3,4,25); most resident macrophages also exhibit an M2-like phenotype that with inherent anti-inflammatory properties (3). In addition, renal resident macrophages share CD206 and Arg1 expression with M2 macrophages (20,26), suggesting that M2 macrophages and resident macrophages are similar cell populations. CD2061 M2 macrophages have also been reported to promote tubular regeneration by expressing growth factors during the repair phase after AKI, which is similar to the function of resident macrophages in AKI (27-29). Although these cells have significant functional properties, direct evidence that M2 macrophages and resident macrophages are the same cell type is still lacking.

Overall, the macrophage ecological niches in the kidney are significantly more diverse than initially understood. To better understand the role of macrophages in the kidney under normal and pathological conditions, the respective functions of these macrophage subpopulations and their underlying molecular mechanisms need to be further investigated.

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Macrophages in Cystic Kidney Disease

Seminal studies in the field of cystic nephropathy have highlighted the association between the number of renal macrophages and the severity of cystic disease (30-33). However, whether macrophages have a pathogenic role in cyst formation or are a secondary consequence of cyst progression and expansion remains controversial. Karihaloo et al. (34) provided the first evidence that macrophages can promote cyst progression in an animal model using a phagocytic toxin, clodronate liposomes (LC), that depletes all macrophages in the kidney. The authors showed that LC treatment of cystic mice not only significantly reduced the number of renal macrophages (by 95%) compared to vector-treated controls, but more importantly, reduced the cystic index and improved renal function (Table 1). Swensen - fields et al.'s follow-up study further supported the conclusion that macrophages can promote cystic nephropathy (35). In these studies, the authors showed that LC treatment also reduced cystic disease in a model of occult cystic nephropathy. These data suggest that macrophages may be involved in promoting cystic progression in multiple forms of cystic kidney disease. However, because these studies removed all phagocytes in the kidney, including infiltrating macrophages, lagging macrophages, and dendritic cells, the contribution of each subpopulation to cyst progression remains unknown.

It has long been assumed that renal macrophages are derived from circulating monocytes and are continuously replenished by them. This led to the idea that targeted recruitment of monocytes to the kidney may be beneficial in the context of cystic kidney disease. This hypothesis is supported by data suggesting that genetic deletion or pharmacological suppression of Ccl2 (also known as monocyte chemotactic agent-1 or MCP-1) and macrophage migration inhibitory factor reduces the severity of cystic kidney disease (36-38). More importantly, the level of cyst reduction with macrophage-targeted therapy is comparable to other inhibitors commonly used in the field of PKD (e.g., tolvaptan or rapamycin) (39,40). Although some studies have shown that inhibition of these macrophage ligands: receptor interactions can inhibit cyst growth, other studies have not reported similar results (41-43). The involvement of infiltrating macrophages in cystic disease remains controversial and has been reviewed previously (44-47). For comparison, we present a table summarizing the results of multiple studies addressing cystic severity macrophages (Table 1).

Resident-Macrophage Involvement in Cystic Kidney Disease

In retrospect, data suggest the presence and involvement of macrophages in cystic kidney disease for decades. For example, several studies have shown that alternatively activated m2-like macrophages expressing CD206 (26,35,48), a newly identified cross-species marker of resident macrophages (11,49), can promote renal cyst formation. microarray analysis by Mrug et al. (50) found that in an autosomal recessive polycystic kidney disease (Cys1^cpk/cpk) non-homozygous model, the genes that are highly expressed are those associated with the innate immune response. Interestingly, several of these innate immune genes were recently found to be specific for macrophages by single-cell RNA sequencing (C1qa, Cxcl16) (11). Furthermore, Viau et al. (36) found that the number of both infiltrating and resident macrophages was increased during cyst progression in the inducible Lkb1 and Pkd1 mouse models. Although the authors in this study emphasized the importance of infiltrating macrophages in controlling cyst progression, they also observed an increase in the number of resident macrophages expressing CCR2 (MCP-1 receptor) in cystic kidneys, suggesting that the MCP1-CCR2 axis may also be important for resident macrophages.

Zimmerman et al. (20) provided evidence of macrophage involvement in cystic disease using conditional Ift88 cilia mutant mice. These mutants develop a cystic kidney disease phenotype, the severity of which is largely influenced by the age at which cilia loss is induced (51). Induction of Ift88 deletion in the early years (before postnatal day 14) leads to rapid and severe cyst formation. In contrast, induction in adults leads to slow progression of cysts in focal areas of the kidney. Following ischemia-reperfusion injury, adult-induced mutants have a substantially higher and more extensive rate of cyst formation. In correlation with these cystogenesis rates, the authors show that macrophages in the kidney of wild-type mice undergo a phenotypic switch (from CD11clow to CD11chigh) after birth. CD11clow resident macrophages are enriched in juveniles, negligible in adult mice, and reappear after ischemia-reperfusion injury in the kidney of adult ciliated mutant mice. More importantly, the number of CD11clow resident macrophages was increased before and during cyst formation, suggesting that these cells may be responsible for or at least contribute to cyst formation. Confocal image analysis revealed that most resident macrophages co-expressed F4/80, Ki67, and CD206 in the cyst area, suggesting that there is mutual communication between resident macrophages and cyst wall epithelial cells (Figure 2). To understand the mechanism of resident macrophage accumulation, the authors performed heterozygous symbiosis experiments by combining circulating CD45.2 control or ciliated mutant mice with homozygous CD45.1 wild-type mice and found that macrophage accumulation in ciliated mutant kidneys was independent of peripheral blood input.Ki67 cell proliferation analysis showed that macrophage accumulation in injured ciliated mutant mice was mainly driven by self-proliferation The accumulation of macrophages in damaged ciliated mutant mice was mainly driven by their own proliferation. To identify the cell types driving the proliferation of resident macrophages, the authors performed flow classification of epithelial cells and showed significantly increased expression of membrane-bound colony-stimulating factor 1 in proximal tubule cells of damaged ciliated mutant kidneys compared to injured controls (Figure 2). Inhibition of the CSF1R kinase signaling pathway using GW2580 (52), which reduces the proliferation of resident macrophages, prevented the accumulation of cd11cllow macrophages and reduced the severity of cystic disease in both the injury-conditioned Ift88 model and the faster-progressing CPK mouse model. Interestingly, GW2580 treatment had no effect on the number of infiltrating macrophages, suggesting that the effect of GW2580 is resident macrophage-specific.

Follow-up studies examining the involvement of macrophage subpopulations in a direct mouse model of autosomal dominant polycystic kidney disease (ADPKD) support the data that resident macrophages can promote cystic disease (53). using unilateral nephrectomized conditional Pkd1 mice, Zimmerman and colleagues found that compared with sham-operated mice, unilateral nephrectomized conditional Pkd1 mice had infiltrating macrophages, and resident macrophage numbers were increased and the increase occurred prior to severe cystogenesis. In addition, the authors found that IFN regulatory factor 5 (Irf5), a transcription factor known to induce the production of inflammatory cytokines by macrophages (54), was increased in infiltrating and resident macrophages in cystic kidneys. To determine the function of IRF5 in macrophages and its importance in cyst formation, the authors used antisense oligonucleotide (ASO) treatment to inhibit IRF5 expression and found that inhibition of IRF5 reduced the number of renal macrophages, decreased inflammatory gene expression, and reduced cyst growth. More careful characterization of infiltrating and resident macrophages showed that IRF5 ASO treatment specifically reduced the expression of IRF5 and Il6 in resident macrophages but did not affect their expression in infiltrating macrophages. More importantly, the authors found that IRF5 ASO-treated mice reduced STAT3 phosphorylation and p-STAT3 target gene expression compared to vector-treated mice, suggesting that IRF5-expressing macrophages release inflammatory factors (IL-6) that stimulate STAT3 phosphorylation in epithelial cells, thereby promoting cyst growth in Pkd1-deficient mice.

The data presented in this review suggest the involvement of macrophages in cystic kidney disease. However, the exact mechanisms by which macrophages affect cyst growth are largely unknown. Although Zimmerman and colleagues provided evidence that cytokines such as il - 6 may affect cyst growth through a STAT-dependent mechanism, other direct or indirect mechanisms may be involved in regulating cyst formation and disease progression (55) (Figure 2). For example, macrophages control the renal injury and repair process by promoting the proliferation and dedifferentiation of renal tubular epithelial cells, a hallmark of renal cyst formation (55). RNA sequencing of resident macrophages after AKI revealed transcriptional reprogramming of resident macrophages, including upregulation of several Wnt genes (Wnt4, Wnt7b, Wnt10a, and Wnt10b) (21). wnt-induced b-catenin signaling pathway prevents epithelial cell apoptosis and promotes proliferative repair (27,56,57). In addition, Wnt signaling can also drive the activation and proliferation of mesenchymal myofibroblasts, leading to increased stromal protein deposition and renal fibrosis (56,58). These data suggest that macrophage-derived wnt promotes the proliferation of cystic epithelial cells and drives interstitial fibrosis during cystic disease progression.

It is also possible that macrophages act as "first responders" in the kidney and control the accumulation and effector functions of other immune cells (e.g., neutrophils, infiltrating macrophages, and T cells), which have been observed in patients with cystic kidney disease and in mouse models (47,59 - 62). Indeed, resident macrophages are well suited for this role because they are able to maintain a persistent residence in the adult kidney through self-proliferation and are located directly adjacent to the tubular epithelium (63). Thus, they may act as sentinels in the kidney to regulate the accumulation of other immune cells that influence cyst growth and progression. Indeed, due to their resident advantage, resident macrophages can act faster than neutrophils, which have been considered the first responders to renal injury. Uderhardt et al. (24), using a combination of live imaging and confocal multi-microscopy, observed that macrophages exert "stealth " behavior, which would prevent injury-induced neutrophil activation and neutrophil-driven inflammation. Thus, resident macrophages may play a similar role in cystic disease.

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Furthermore, epithelial apoptosis was detected during cyst formation (64), and macrophages are known to be specialized phagocytic cells. Thus, resident macrophages may control cyst growth by phagocytosis of damaged epithelial cells and subsequent activation of inflammatory signaling pathways. There are data suggesting that macrophages can detect and remove immune complexes or foreign debris from the mesenchyme and upregulate several inflammatory signaling pathways including NF-kB and JAK/STAT, both of which are associated with cystic disease progression (22,65).

In addition, macrophages may also promote cyst progression through other mechanisms, including regulation of vascular abnormalities through their proposed pro-angiogenic function (23,66,67). It is also possible that resident macrophages regulate fluid secretion directly or indirectly, as data suggest that macrophage-derived cytokines mediate the localization and activity of multiple ion channels in the kidney and other tissues (68-71). All of these processes have been reported to be associated with cystic kidney disease (72,73).

Targeting Resident Macrophages as a Potential Therapeutic Intervention

In multiple preclinical models, inhibition or reduction of resident macrophage numbers has had beneficial effects on both cyst burden and disease progression (20,53). However, targeting these cells in patients with cystic kidney disease has significant limitations because the mechanisms by which macrophages are involved in cyst growth are unclear and methods for identifying these cells across species are difficult. Despite these limitations, it should be noted that in animal models, inhibition of two proinflammatory signaling pathways present in macrophages, namely NF-kB and JAK/STAT pathways, significantly improves the degree of cyclicity (44,46). For example, the STAT3 inhibitor S3I-201 significantly inhibited cyst formation and growth in a neonatal PKD mouse model (74). In addition, rebaudioside has well-known anti-inflammatory effects through the inhibition of NF-kB transactivation, and its beneficial effects on cystogenesis in a mouse model of ADPKD have been reported for decades (75,76). The results of phase 3 clinical trial (NCT02115659) of rationing in ADPKD are highly anticipated.

Another caveat is the difficulty in translating macrophage-centric animal studies to humans. This difficulty is due to the fact that the marker used to profile mouse macrophages (i.e., F4/80) is not expressed in human macrophages, which makes it challenging to identify similar populations between species. In addition, there is no way to distinguish infiltrating macrophages of monocyte origin from resident macrophages of embryonic seeds due to the lack of appropriate methods. Using recently developed single-cell RNA sequencing technology, Zimmerman et al. (11) identified a cross-species renal macrophage-specific gene expression signature by sequencing CD451 cells isolated from mouse, rat, pig, and human kidney tissues. As part of this marker, the authors identified C1QC, CD81, and CD74 as the novel, cross-species resident macrophage markers. The authors go on to show that these markers are expressed at the protein level in mouse resident macrophages and co-expressed at the protein level in CD451 cell populations isolated from rats and humans. Thus, it is likely that resident macrophages are present in other species and that C1q, CD81, and CD74 can be used to identify these cells.

The identification of macrophages in the human kidney will greatly facilitate clinically relevant translational studies from mouse models to human patients. Macrophage targeting as a potential therapeutic intervention has yielded promising results in preclinical models of inflammatory diseases and cancer (77). However, specifically targeting macrophages in the kidney of patients is extremely challenging due to the lack of precise methods to deplete macrophages in the kidney from other tissues. Thus, any resident macrophage inhibitor would deplete all tissues of resident macrophages that are essential for basic biological functions such as synaptic pruning, cardiac conduction, and infection prevention (78,79). Therefore, it is critical to develop kidney-specific approaches to selectively deplete macrophages.

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Conclusions and Future Perspectives

In conclusion, studies have shown that renal macrophages are involved in cyst progression and that targeting macrophages using genetic deletion or pharmacological inhibition is a promising therapeutic target for reducing cyst growth. Understanding the function of macrophages under physiological and pathological conditions is important to reveal the mechanisms of their role in cystic kidney disease and to translate these novel mechanisms into benefits for patients with cystic kidney disease.

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Zhang Li¹ ; Kurt A. Zimmerman² ; and Bradley K. Yoder¹

1 Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama.

2 Division of Nephrology, Department of Internal Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma.