Kidney-resident Macrophages Research Articles

Kidney resident macrophages have distinct subsets and multifunctional roles

BackgroundThe kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. MethodsFlow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5−/− (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. ResultsIn wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5−/− (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. ConclusionsWe identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

Interferon-γ and its response are determinants of antibody-mediated rejection and clinical outcomes in patients after renal transplantation.

Interferon-γ (IFN-γ) is an important cytokine in tissue homeostasis and immune response, while studies about it in antibody-mediated rejection (ABMR) are very limited. This study aims to comprehensively elucidate the role of IFN-γ in ABMR after renal transplantation. In six renal transplantation cohorts, the IFN-γ responses (IFNGR) biological process was consistently top up-regulated in ABMR compared to stable renal function or even T cell-mediated rejection in both allografts and peripheral blood. According to single-cell analysis, IFNGR levels were found to be broadly elevated in most cell types in allografts and peripheral blood with ABMR. In allografts with ABMR, M1 macrophages had the highest IFNGR levels and were heavily infiltrated, while kidney resident M2 macrophages were nearly absent. In peripheral blood, CD14+ monocytes had the top IFNGR level and were significantly increased in ABMR. Immunofluorescence assay showed that levels of IFN-γ and M1 macrophages were sharply elevated in allografts with ABMR than non-rejection. Importantly, the IFNGR level in allografts was identified as a strong risk factor for long-term renal graft survival. Together, this study systematically analyzed multi-omics from thirteen independent cohorts and identified IFN-γ and IFNGR as determinants of ABMR and clinical outcomes in patients after renal transplantation.

Modulating inflammation with interleukin 37 treatment ameliorates murine Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of kidney failure and is associated with substantial morbidity and mortality. Interstitial inflammation is attributed to the action of infiltrating macrophages and is a feature thought to aggravate disease progression. Here, we investigated the therapeutic potential of the anti-inflammatory IL37b cytokine as a treatment for ADPKD using genetic mouse models, demonstrating that transgenic expression of human IL37b reduced collecting duct cyst burden in both early and adult-onset ADPKD rodent models. Moreover, injection of recombinant human IL37b could also reduce cyst burden in early onset ADPKD mice, an observation not associated with increased macrophage number at early stages of cyst formation. Interestingly, transgenic IL37b expression also did not alter macrophage numbers in advanced disease. Whole kidney RNA-seq highlighted an IL37b- mediated upregulation of the interferon signaling pathway and single cell RNA-seq established that these changes originate at least partly from kidney resident macrophages. We further found that blocking type I interferon signaling in mice expressing IL37b resulted in increased cyst number, confirming this as an important pathway by which IL37b exerts its beneficial effects. Thus, our studies show that IL37b promotes interferon signaling in kidney resident macrophages which suppresses cyst initiation, identifying this protein as a potential therapy for ADPKD.

Isolation and Flow Cytometry Analysis of Macrophages from the Kidney.

Renal macrophages help maintain homeostasis, participate in tissue injury and repair, and play a vital role in immune surveillance [1-3]. Kidney macrophages can be broken down into two subsets, infiltrating macrophages, which can be further broken down into Ly6Chi and Ly6Clo cells, and kidney resident macrophages. While recent studies have shed light on the differing origins and niches of these cells, a more thorough understanding of kidney macrophage populations and how they may respond to various conditions is needed. This protocol describes how to efficiently isolate murine kidney macrophage populations for flow cytometry analysis.

Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Protecting the kidney in sepsis: resident macrophages to the rescue

Kidney resident macrophages exert pro-inflammatory or reparative effects in experimental acute kidney injury, but their role in sepsis is unclear. In a mouse model of sepsis, Privratsky etal. show that kidney resident F4/80hi macrophages protect against kidney injury by expressing interleukin-1 receptor antagonist, which blocks interleukin-6 production selectively from endothelial cells. Discovery of this novel autocrine loop enhances opportunities for targeted therapies to diminish kidney injury during sepsis.

Defining newly formed and tissue-resident bone marrow-derived macrophages in adult mice based on lysozyme expression.

Tissue-resident macrophages are derived from different precursor cells and display different phenotypes. Reconstitution of the tissue-resident macrophages of inflamed or damaged tissues in adults can be achieved by bone marrow-derived monocytes/macrophages. Using lysozyme (Lysm)-GFP-reporter mice, we found that alveolar macrophages (AMs), Kupffer cells, red pulp macrophages (RpMacs), and kidney-resident macrophages were Lysm-GFP-, whereas all monocytes in the fetal liver, adult bone marrow, and blood were Lysm-GFP+. Donor-derived Lysm-GFP+ resident macrophages gradually became Lysm-GFP- in recipients and developed gene expression profiles characteristic of tissue-resident macrophages. Thus, Lysm may be used to distinguish newly formed and long-term surviving tissue-resident macrophages that were derived from bone marrow precursor cells in adult mice under pathological conditions. Furthermore, we found that Irf4 might be essential for resident macrophage differentiation in all tissues, while cytokine and receptor pathways, mTOR signaling pathways, and fatty acid metabolic processes predominantly regulated the differentiation of RpMacs, Kupffer cells, and kidney macrophages, respectively. Deficiencies in ST2, mechanistic target of rapamycin (mTOR) and fatty acid-binding protein 5 (FABP5) differentially impaired the differentiation of tissue-resident macrophages from bone marrow-derived monocytes/macrophages in the lungs, liver, and kidneys. These results indicate that a combination of shared and unique signaling pathways coordinately shape tissue-resident macrophage differentiation in various tissues.

Kidney Resident Macrophages Alter MHC II Expression and Location in Response to Acute and Chronic Injury

Kidney Resident Macrophages Alter MHC II Expression and Location in Response to Acute and Chronic Injury

Resident macrophage subpopulations occupy distinct microenvironments in the kidney.

The kidney contains a population of resident macrophages from birth that expands as it grows and forms a contiguous network throughout the tissue. Kidney-resident macrophages (KRMs) are important in homeostasis and the response to acute kidney injury. While the kidney contains many microenvironments, it is unknown whether KRMs are a heterogeneous population differentiated by function and location. We combined single-cell RNA-Seq (scRNA-Seq), spatial transcriptomics, flow cytometry, and immunofluorescence imaging to localize, characterize, and validate KRM populations during quiescence and following 19 minutes of bilateral ischemic kidney injury. scRNA-Seq and spatial transcriptomics revealed 7 distinct KRM subpopulations, which are organized into zones corresponding to regions of the nephron. Each subpopulation was identifiable by a unique transcriptomic signature, suggesting distinct functions. Specific protein markers were identified for 2 clusters, allowing analysis by flow cytometry or immunofluorescence imaging. Following injury, the original localization of each subpopulation was lost, either from changing locations or transcriptomic signatures. The original spatial distribution of KRMs was not fully restored for at least 28 days after injury. The change in KRM localization confirmed a long-hypothesized dysregulation of the local immune system following acute injury and may explain the increased risk for chronic kidney disease.

Blocking CCL8-CCR8-Mediated Early Allograft Inflammation Improves Kidney Transplant Function.

In kidney transplantation, early allograft inflammation impairs long-term allograft function. However, precise mediators of early kidney allograft inflammation are unclear, making it challenging to design therapeutic interventions. We used an allogeneic murine kidney transplant model in which CD45.2 BALB/c kidneys were transplanted to CD45.1 C57BL/6 recipients. Donor kidney resident macrophages within the allograft expanded rapidly in the first 3 days. During this period, they were also induced to express a high level of Ccl8, which, in turn, promoted recipient monocyte graft infiltration, their differentiation to resident macrophages, and subsequent expression of Ccl8. Enhanced graft infiltration of recipient CCR8+ T cells followed, including CD4, CD8, and γδ T cells. Consequently, blocking CCL8-CCR8 or depleting donor kidney resident macrophages significantly inhibits early allograft immune cell infiltration and promotes superior short-term allograft function. Targeting the CCL8-CCR8 axis is a promising measure to reduce early kidney allograft inflammation.

Heat acclimation ameliorated heat stress-induced acute kidney injury and prevented changes in kidney macrophages and fibrosis.

Heatstroke can cause acute kidney injury (AKI), which reportedly progresses to chronic kidney disease. Kidney macrophages may be involved in such injury. Although heat acclimation (HA) provides thermal resilience, its renoprotective effect and mechanism remain unclear. To investigate heat stress-induced kidney injuries in mice and the mitigating effect of HA on them, male C57/BL6J mice were exposed to heat stress (40°C, 1 h) with or without 5-day HA (38°C, 3 h/day) prior to heat stress. Heat stress damaged kidney proximal tubules with an elevation of urinary kidney injury molecule-1. Kidney fibrosis was observed on day 7 and correlated with urinary kidney injury molecule-1 levels on day 3. Kidney resident macrophages decreased on day 1, whereas the number of infiltrating macrophages in the kidney did not change. Both subsets of macrophages polarized to the proinflammatory M1 phenotype on day 1; however, they polarized to the anti-inflammatory M2 phenotype on day 7. HA significantly ameliorated heat stress-induced proximal tubular damage and kidney fibrosis. HA substantially increased heat shock protein 70 expression in the tubules before heat stress and reduced the elevation of cleaved caspase-3 expression after heat stress. HA also induced heat shock protein 70 expression of resident macrophages and prevented heat stress-induced changes in both subsets of kidney macrophages. These results provide pathophysiological data supporting the renoprotective effect of HA. Further studies are needed to confirm that HA can prevent kidney damage due to heat stress in humans.NEW & NOTEWORTHY Heat stress could induce acute kidney injury. Although heat acclimation (HA) reportedly provides thermal tolerance, its effect on heat stress-induced kidney damage remains unclear. This study showed that 5-day HA ameliorates mouse kidney tubular damage and subsequent fibrosis caused by heat stress. It also demonstrated that HA enhances intracellular heat shock protein 70 expression in tubular cells and prevents a decrease in kidney resident macrophages, which explains the renoprotective effect of HA.

Spatial transcriptomics and single cell sequencing identifies a unique injury associated resident macrophage subpopulation and reveals differential response to injury

Abstract Acute kidney injury (AKI) is a growing concern in the U.S. and globally. Despite recovery of kidney function, patients remain at increased risk for developing chronic kidney disease (CKD). The cause of this increased risk is unknown and biomarkers to determine kidney injury are severely lacking. A potential candidate is the network of kidney resident macrophages interwoven throughout the tissue, and specifically a subpopulation of kidney injury associated macrophages (KIA) we first identified to appear after AKI and lack MHC II expression. KIA cells were localized within the tissue using Visium Spatial Transcriptomics and flow cytometry. Comparison of KIA cell kinetics between AKI and CKD indicated a correlation between the presence of KIA cells and the persistence of injury. Additionally, KIA cells have localized to the sight of greatest injury following an ischemic event. As kidney function recovers, KIA cells decrease in number and return to quiescent levels by 14 days post injury. In contrast, in the severe CKD model, KIA cells persist for up to six weeks, composing approximately 20% of the total kidney resident macrophage population. A less severe version of CKD results in fewer KIA cells but similar persistence, indicating appearance of KIA cells directly correlates with injury severity. Targeting KIA cells to either enhance or negate their function is a possible therapeutic target to reduce the risk of developing CKD following AKI. As we have identified KIA cells in multiple models of kidney injury, in the future they should be investigated as a potential injury biomarker. We hypothesize that KIA cells are uniquely involved in the pathogenesis of injury and may be a key to an AKI to CKD transition. Supported by grants from the American Heart Association: (AHA 827257)

Distinct Subpopulations of Mouse and Human Kidney Resident Macrophages in Acute Kidney Injury

Abstract Introduction Kidney resident macrophages (KRMs) are important in renal homeostasis and the response to acute kidney injury. Preliminary data suggests that the KRM population consists of several undescribed subpopulations. Here, we combined single-cell RNA sequencing (scRNAseq) and spatial transcriptomics to identify and localize KRM subpopulations during homeostasis and injury. Methods KRMs were isolated from C57BL/6J mice without treatment and after bilateral ischemia-reperfusion injury (BIRI) as well as from a human donor kidney. ScRNAseq was performed using the 10X Genomics Chromium platform and spatial transcriptomics using the 10X Visium platform. Gene expression data were integrated and analyzed using the R package, Seurat 4.0. Results UMAP plots of integrated data revealed seven major clusters of mouse KRMs with unique transcriptional profiles associated with distinct functions. Spatial transcriptomics revealed that these clusters reside in distinct cellular compartments within the kidney and appear to associate with specific renal structures. Following BIRI, these subpopulations appear in cellular compartments distinct from those occupied in the controls. Several human KRM clusters were correlated with those of the mouse and localized to specific regions of the kidney. Conclusion Transcriptionally distinct subpopulations of mouse KRMs reside within specific kidney microenvironments and change location as a function of injury. Similar subpopulations of KRMs were identified in the human kidney as well. Therefore, further study of the temporal and spatial characteristics and signaling pathways of these subpopulations in the context of homeostasis and injury is warranted. Supported by grants from NIH (R01-DK-59600, T32-AI007051)

Single Cell RNA Sequencing Identifies a Unique Inflammatory Macrophage Subset as a Druggable Target for Alleviating Acute Kidney Injury

Acute kidney injury (AKI) is a complex clinical disorder associated with poor outcomes. Targeted regulation of the degree of inflammation has been a potential strategy for AKI management. Macrophages are the main effector cells of kidney inflammation. However, macrophage heterogeneity in ischemia reperfusion injury induced AKI (IRI‐AKI) remains unclear. Using single‐cell RNA sequencing of the mononuclear phagocytic system in the murine IRI model, the authors demonstrate the complementary roles of kidney resident macrophages (KRMs) and monocyte‐derived infiltrated macrophages (IMs) in modulating tissue inflammation and promoting tissue repair. A unique population of S100a9hiLy6chi IMs is identified as an early responder to AKI, mediating the initiation and amplification of kidney inflammation. Kidney infiltration of S100A8/A9+ macrophages and the relevance of renal S100A8/A9 to tissue injury is confirmed in human AKI. Targeting the S100a8/a9 signaling with small‐molecule inhibitors exhibits renal protective effects represented by improved renal function and reduced mortality in bilateral IRI model, and decreased inflammatory response, ameliorated kidney injury, and improved long‐term outcome with decreased renal fibrosis in the unilateral IRI model. The findings support S100A8/A9 blockade as a feasible and clinically relevant therapy potentially waiting for translation in human AKI.

Single-Cell and Spatial Transcriptomics Reveal Distinct Subpopulations of Kidney Resident Macrophages in AKI

Single-Cell and Spatial Transcriptomics Reveal Distinct Subpopulations of Kidney Resident Macrophages in AKI

Abstract 46: Vascular Endothelium-derived Endothelin 1 Promotes Salt-induced Kidney Inflammation In Male But Not Female Mice

Endothelin 1 (ET-1), potent vasoactive and pro-inflammatory peptide, plays a criticalrole in hypertension and end-organ damage. The inflammatory response to high saltintake displays sex differences; however, if a sex difference exists in vascular ET-1inducing salt-induced kidney inflammation is unknown. We aimed to assess the role ofendothelium-derived ET-1 in promoting kidney inflammation in male and female micefed high salt. Aged-matched male and female vascular endothelial cell ET-1 knockout(VEET KO) and control floxed ET-1 (VEET fl/fl ) mice were fed 4.0% NaCl chow (HSD).After 3 weeks of HSD, kidney myeloid and lymphoid cell populations and their functionalstatus were examined by flow cytometry. In response to HSD, male VEET fl/fl mice hadsignificantly higher kidney TH17 cell numbers than female VEET fl/fl mice (IL-17A + CD4 + cell absolute count, male vs. female: 229.6±27.7 vs. 84.9±17.2, p=0.0003, n=6-10/group), indicating a substantial sex-dependent difference in kidney TH17 cellabundance. Lack of endothelium-derived ET-1 resulted in 72% reduction in renal TH17cell number in males (VEET fl/fl vs. VEET KO: 229.6±27.7 vs 64.0±64, p=0.0002), but notin females (p>0.05). Frequency of kidney IL-17A + CD4 + cells was not different betweengenotypes in females or between male and female VEET fl/fl mice. However, male VEETKO mice displayed 57% less renal IL-17A + CD4 + cells than male VEET fl/fl (respectively,0.6±0.071 vs. 1.4±0.2, p=0.0055), and 59% reduction compared to female VEET KOmice (1.4±0.2, p=0.0052). Assessment of the effects of vascular ET-1 on the innateimmune response showed that activated kidney resident macrophages(F4/80 hi CD11b + CD64 + ) were increased in male vs. female VEET fl/fl (MFI, respectively,2080±105 vs. 1672±25, p=0.0002). Lack of endothelium-derived ET-1 significantlyreduced CD64 abundance in males (p=0.012), but not females (p>0.05), whencompared to VEET fl/fl . In conclusion, endothelium-derived ET-1 is critical in promotingrenal innate and adaptive inflammatory responses in males in response to high salt, andimportant male-female differences exist in the manner by which vascular ET-1 regulateskidney inflammation in this setting. Funded by NIH F31 HL151264-0 to PAM,P01HL136267 to JSP, and K01HL145324 to CDM.

Kidney resident macrophages in the rat have minimal turnover and replacement by blood monocytes.

Kidney resident macrophages (KRMs) are involved in maintaining renal homeostasis and in controlling the pathological outcome of acute kidney injury and cystic kidney disease in mice. In adult mice, KRMs maintain their population through self-renewal with little or no input from the peripheral blood. Despite recent data suggesting that a transcriptionally similar population of KRM-like cells is present across species, the idea that they are self-renewing and minimally dependent on peripheral blood input in other species has yet to be proven due to the lack of an appropriate model and cross-species expression markers. In this study, we used our recently identified cross-species KRM cell surface markers and parabiosis surgery in inbred Lewis rats to determine if rat KRMs are maintained independent of peripheral blood input, similar to their mouse counterparts. Flow cytometry analysis indicated that parabiosis surgery in the rat results in the establishment of chimerism of T/B cells, neutrophils, and monocyte-derived infiltrating macrophages in the blood, spleen, and kidney 3 wk after parabiosis surgery. Analysis of KRMs using the cell surface markers CD81 and C1q indicated that these cells have minimal chimerism and, therefore, receive little input from the peripheral blood. These data indicate that KRM properties are conserved in at least two different species.NEW & NOTEWORTHY In this report, we performed parabiosis surgery on inbred Lewis rats and showed that rat kidney resident macrophages (KRMs), identified using our novel cross-species markers, are minimally dependent on peripheral blood input. Thus, for the first time, to our knowledge, we confirm that a hallmark of mouse KRMs is also present in KRMs isolated from another species.

Macrophage Heterogeneity in Kidney Injury and Fibrosis.

Kidney macrophages are central in kidney disease pathogenesis and have therapeutic potential in preventing tissue injury and fibrosis. Recent studies highlighted that kidney macrophages are notably heterogeneous immune cells that fulfill opposing functions such as clearing deposited pathogens, maintaining immune tolerance, initiating and regulating inflammatory responses, promoting kidney fibrosis, and degrading the extracellular matrix. Macrophage origins can partially explain macrophage heterogeneity in the kidneys. Circulating Ly6C+ monocytes are recruited to inflammatory sites by chemokines, while self-renewed kidney resident macrophages contribute to kidney repair and fibrosis. The proliferation of resident macrophages or infiltrating monocytes provides an alternative explanation of macrophage accumulation after kidney injury. In addition, dynamic Ly6C expression on infiltrating monocytes accompanies functional changes in handling kidney inflammation and fibrosis. Mechanisms underlying kidney macrophage heterogeneity, either by recruiting monocyte subpopulations, regulating macrophage polarization, or impacting distinctive macrophage functions, may help develop macrophage-targeted therapies for kidney diseases.

Single-Cell Transcriptomics Reveal Immune Mechanisms of the Onset and Progression of IgA Nephropathy

IgA nephropathy (IgAN) is the leading cause of kidney failure due to an incomplete understanding of its pathogenesis. We perform single-cell RNA sequencing (RNA-seq) on kidneys and CD14+ peripheral blood mononuclear cells (PBMCs) collected from IgAN and normal samples. In IgAN, upregulation of JCHAIN in mesangial cells provides insight into the trigger mechanism for the dimerization and deposition of IgA1 in situ. The pathological mesangium also demonstrates a prominent inflammatory signature and increased cell-cell communication with other renal parenchymal cells and immune cells, suggesting disease progress from the mesangium to the entire kidney. Specific gene expression of kidney-resident macrophages and CD8+ Tcells further indicates abnormal regulation associated with proliferation and inflammation. A transitional cell type among intercalated cells with fibrosis signatures is identified, suggesting an adverse outcome of interstitial fibrosis. Altogether, we systematically analyze the molecular events in the onset and progression of IgAN, providing a promising landscape for disease treatment.

Research Highlights

Research Highlights