Self-maintaining macrophages within the kidney contribute to salt and water balance by modulating kidney sympathetic nerve activity

The kidney is critical in controlling salt and water balance, with the interstitium involved with a variety of components including immune cells in steady state. However, the roles of resident immune cells in kidney physiology are largely unknown. To help unravel some of these unknowns, we employed cell fate mapping, and identified a population of embryo-derived self-maintaining macrophages (SM-MØ) that were independent of the bone marrow in adult mouse kidneys. This kidney-specific SM-MØ population was distinctive from the kidney monocyte-derived macrophages in transcriptome and in their distribution. Specifically, the SM-MØ highly expressed nerve-associated genes; high-resolution confocal microscopy revealed that the SM-MØ in the cortex were in close association with sympathetic nerves and there was a dynamical interaction between macrophages and sympathetic nerves when live kidney sections were monitored. Kidney-specific depletion of the SM-MØ resulted in reduced sympathetic distribution and tone, leading to reduced renin secretion, increased glomerular filtration rate and solute diuresis, which caused salt decompensation and significant weight loss under a low-salt diet challenge. Supplementation of L-3,4-dihydroxyphenylserine which is converted to norepinephrine in vivo rescued the phenotype of SM-MØ-depleted mice. Thus, our findings provide insights in kidney macrophage heterogeneity and address a non-canonical role of macrophages in kidney physiology. In contrast to the well-appreciated way of central regulation, local regulation of sympathetic nerve distribution and activities in the kidney was uncovered. The kidney is critical in controlling salt and water balance, with the interstitium involved with a variety of components including immune cells in steady state. However, the roles of resident immune cells in kidney physiology are largely unknown. To help unravel some of these unknowns, we employed cell fate mapping, and identified a population of embryo-derived self-maintaining macrophages (SM-MØ) that were independent of the bone marrow in adult mouse kidneys. This kidney-specific SM-MØ population was distinctive from the kidney monocyte-derived macrophages in transcriptome and in their distribution. Specifically, the SM-MØ highly expressed nerve-associated genes; high-resolution confocal microscopy revealed that the SM-MØ in the cortex were in close association with sympathetic nerves and there was a dynamical interaction between macrophages and sympathetic nerves when live kidney sections were monitored. Kidney-specific depletion of the SM-MØ resulted in reduced sympathetic distribution and tone, leading to reduced renin secretion, increased glomerular filtration rate and solute diuresis, which caused salt decompensation and significant weight loss under a low-salt diet challenge. Supplementation of L-3,4-dihydroxyphenylserine which is converted to norepinephrine in vivo rescued the phenotype of SM-MØ-depleted mice. Thus, our findings provide insights in kidney macrophage heterogeneity and address a non-canonical role of macrophages in kidney physiology. In contrast to the well-appreciated way of central regulation, local regulation of sympathetic nerve distribution and activities in the kidney was uncovered. Translational StatementSympathetic nerve activities are critical in regulating kidney functions by affecting glomerular filtration rate and the reabsorption of salt via tubular epithelial cells. Given this, sustained overactivation of renal sympathetic nerves will lead to pathogenic outcomes, such as hypertension. This study unveils for the first time that a resident population of macrophages are involved in local regulation of renal sympathetic nerve activities, in contrast to the traditional view that sympathetic tonicity is controlled solely by the central nervous system. Thus, specific manipulation of sympathetic activities in the kidney is now possible via targeting of this subset of macrophages. Sympathetic nerve activities are critical in regulating kidney functions by affecting glomerular filtration rate and the reabsorption of salt via tubular epithelial cells. Given this, sustained overactivation of renal sympathetic nerves will lead to pathogenic outcomes, such as hypertension. This study unveils for the first time that a resident population of macrophages are involved in local regulation of renal sympathetic nerve activities, in contrast to the traditional view that sympathetic tonicity is controlled solely by the central nervous system. Thus, specific manipulation of sympathetic activities in the kidney is now possible via targeting of this subset of macrophages. The differentiation and programming of tissue resident macrophages (MØs) are dynamically imprinted by the microenvironment.1Gosselin D. Link V.M. Romanoski C.E. et al.Environment drives selection and function of enhancers controlling tissue-specific macrophage identities.Cell. 2014; 159: 1327-1340Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar,2Lavin Y. Winter D. Blecher-Gonen R. et al.Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.Cell. 2014; 159: 1312-1326Abstract Full Text Full Text PDF PubMed Scopus (1392) Google Scholar Besides playing a role as immune responders, they are critical in maintaining local homeostasis.3Okabe Y. Medzhitov R. Tissue biology perspective on macrophages.Nat Immunol. 2016; 17: 9-17Crossref PubMed Scopus (419) Google Scholar The resident MØs in most tissues have multiple origins. Their entrenchment begins during embryogenesis, when either yolk-sac–derived pro-MØs and/or fetal liver-derived monocytes arrive in the tissues.4Ginhoux F. Guilliams M. Tissue-resident macrophage ontogeny and homeostasis.Immunity. 2016; 44: 439-449Abstract Full Text Full Text PDF PubMed Scopus (1001) Google Scholar These embryo-derived MØs can sustain their number in adulthood by self-renewal. In some organs, including gut, heart, and lung, bone marrow–derived monocytes become a postnatal origin of MØs.4Ginhoux F. Guilliams M. Tissue-resident macrophage ontogeny and homeostasis.Immunity. 2016; 44: 439-449Abstract Full Text Full Text PDF PubMed Scopus (1001) Google Scholar, 5Epelman S. Lavine K.J. Beaudin A.E. et al.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar, 6Gibbings S.L. Goyal R. Desch A.N. et al.Transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages.Blood. 2015; 126: 1357-1366Crossref PubMed Scopus (139) Google Scholar, 7De Schepper S. Verheijden S. Aguilera-Lizarraga J. et al.Self-maintaining gut macrophages are essential for intestinal homeostasis.Cell. 2018; 175: 400-415.e13Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar The embryo-derived self-maintaining MØs (SM-MØs) coexist with their postnatal monocyte-derived counterparts in some tissues in adults but may distribute themselves in disparate niches and possess unique transcriptomes that allow them to meet niche-specific demands.7De Schepper S. Verheijden S. Aguilera-Lizarraga J. et al.Self-maintaining gut macrophages are essential for intestinal homeostasis.Cell. 2018; 175: 400-415.e13Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar,8Zhu Y. Herndon J.M. Sojka D.K. et al.Tissue-resident macrophages in pancreatic ductal adenocarcinoma originate from embryonic hematopoiesis and promote tumor progression.Immunity. 2017; 47: 323-338.e6Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar Thus, an emerging area of interest is identification of the distribution and functions of various subtypes of MØs, which are supposed to fulfill disparate functional demands of the tissues/organs. Kidney is a highly perfused organ that receives intensive efferent sympathetic nerve innervation that targets both nephron and vasculature.9Barajas L. Liu L. Powers K. Anatomy of the renal innervation: intrarenal aspects and ganglia of origin.Can J Physiol Pharmacol. 1992; 70: 735-749Crossref PubMed Scopus (176) Google Scholar The outflow of renal sympathetic nerves is mainly controlled by the autonomic neurons in the brain, where systemic biochemical signals and psychological reactions are integrated. The sympathetic signals from the central nervous system are relayed in the paravertebral ganglia, where postganglionic sympathetic neurons provide axons innervating distal organs, including the kidneys. Sympathetic excitation increases norepinephrine (NE) release, resulting in increased sodium and water reabsorption and reduction in blood flow of the kidney.10Johns E.J. Kopp U.C. DiBona G.F. Neural control of renal function.Compr Physiol. 2011; 1: 731-767Crossref PubMed Scopus (195) Google Scholar Although central regulation of sympathetic outflow has been investigated extensively, information is limited about local modulation of sympathetic nerve activities in steady state and under stress. Recent studies disclosed the coexistence of embyro-derived SM-MØs and MØs that are continuously replenished by monocytes in the kidney of adult mice,11Liu F. Dai S. Feng D. et al.Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.Nat Commun. 2020; 11: 2280Crossref PubMed Scopus (44) Google Scholar,12Zhu Q. He J. Cao Y. et al.Analysis of mononuclear phagocytes disclosed the establishment processes of two macrophage subsets in the adult murine kidney.Front Immunol. 2022; 13805420Google Scholar but their functions in steady state are unclear. In this study, we performed transcriptomic analysis and high-resolution confocal microscopic examination. These studies revealed a population of SM-MØs that are closely associated with renal sympathetic axon fibers in the cortex. Targeted depletion of renal SM-MØs downregulated distribution and actions of renal sympathetic nerves. Given this, mice could not efficiently preserve salt and water. Thus, we unveiled a nonimmune action of a subset of resident renal MØs that are important to kidney physiology. Cx3cr1CreERT2 mice [020940], Rosa26-stop-TdTomato mice [007914], and iDTR mice [007900] were from The Jackson Laboratory. All the mice are in C57BL/6 background. DbhCre mice were originally provided by Dr. Gunther Schutz (German Cancer Research Center, Heidelberg, Germany) and have been backcrossed to C57BL/6 mice for more than 10 generations.13Xiao Z. Cheng G. Jiao Y. et al.Holo-Seq: single-cell sequencing of holo-transcriptome.Genome Biol. 2018; 19: 163Crossref PubMed Scopus (23) Google Scholar Normal C57BL/6 mice (CD45.2+) were purchased from Shanghai Research Center for Model Organisms. All mice used in this study without specific explanation were 8- to 12-week-old males. Mice were fed with a normal (0.4%) NaCl diet; in some experiments, mice were fed with a low-NaCl (0.1%) diet. Mice were housed in a standard animal facility, with a 12-hour light/dark cycle, in a specific-pathogen-free environment. All animal experiments were approved by the Institutional Animal Care and Use Committee at Zhejiang University (protocol ZJU20190135). Normal healthy mice were recruited for experiments. At the beginning of each experiment, no experimental mice appeared sick or underweight. Kidneys were isolated from mice after perfusion with cold phosphate-buffered saline plus ethylenediamine tetraacetic acid and were cut into small pieces. Each kidney was digested in 6 ml RPMI1640 (GIBICO) with 1.5 mg/ml collagenase IV (Worthington) and 5 μl/ml DNase I (Sigma) for 30 minutes at 37 °C, with gentle shaking. After digestion, cells were passed through a 70-μm strainer (BD) and were then subject to gradient centrifugation by using 72% and 36% Percoll (GE Healthcare). Immune cells were enriched at the 72%/36% interface and were collected for flow cytometry analyses (dead cells were at the bottom so they were not included). Samples were analyzed with a 3-laser flow cytometer (Agilent NovoCyte), and data were processed with FlowJo (version 10.1, FlowJo). Renal MØs were purified by cell sorting with a BD FACS ARIA II. Confocal imaging of live kidney sections ex vivo was developed as a technique for visualizing tissue architecture and cell mobility at close to physiological conditions. Mice were euthanized, and the kidneys were harvested and kept on ice in a typical preoxygenated bath solution (150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose, and 10 mM HEPES). Kidney was sliced into 200-μm sections with a Leica VT1000 S vibrating-blade microtome (Leica Biosystems) at speed 6. Tissue sections were then cultured in complete kidney medium (preoxygenated phenol-red-free Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum and 10 mM HEPES) in a humidified incubator at 37° C for 2 hours. Sections were held down with tissue anchors in 22-mm glass-bottom dishes and imaged using an IX83-FV3000-Olympus Super Resolution inverted high-resolution confocal microscope (Olympus) equipped with an environmental chamber and a motorized stage. Microscope configuration was set up for 4-dimensional analysis (x, y, z, t). Four fields of sympathetic nerve–associated MØs were simultaneously recorded with a ×20 objective lens every 5 minutes for 4 hours with 1.5-μm z-axis increments and 512 × 512–pixel resolution. The range of the Z stack of images was 25–30 μm. Images and videos were processed using Imaris software (Bitplane). Eight-week-old Cx3cr1CreER/+: Rosa26-iDTR mice were i.p. injected with tamoxifen (75 mg/kg) for 5 consecutive days. After 1 day or 3 months, the mice were anesthetized and kept on a homeothermic pad to maintain body temperature during the surgery. Dorsal incision was made to expose kidney pedicles, and a 30-gauge needle was inserted into the subcapsular space through the kidney pedicle. For each kidney, 25-μl DT in phosphate-buffered saline (10 ng/g body weight) was injected through the needle. Both kidneys of a mouse were subjected to the same procedure. The kidney was then gently returned into the original positions in the abdominal cavity, and the skin was sutured. Statistical analysis was performed with Prism 9.0 (GraphPad). Data are presented as mean ± SEM. One-way analysis of variance with Tukey’s multiple-comparisons testing was used to compare multiple groups. Paired and unpaired Student t tests were used for 2-group comparisons. All statistical tests were 2-tailed, and P values of <0.05 were considered significant. Detailed methods are described in the Supplementary Methods. In a previous study, we showed that SM-MØs and their monocyte-derived counterparts could be distinguished by employing Cx3cr1CreER/+: R26Td mice.12Zhu Q. He J. Cao Y. et al.Analysis of mononuclear phagocytes disclosed the establishment processes of two macrophage subsets in the adult murine kidney.Front Immunol. 2022; 13805420Google Scholar In the Cx3cr1CreER/+: R26Td mice, renal MØs can be labeled with fluorescent tdTomato upon tamoxifen treatment. Afterwards, SM-MØs and their progeny keep expressing tdTomato, whereas the monocyte-derived MØ population gradually decreases tdTomato expression due to replacement. Thus, only SM-MØs retain tdTomato labelling 3 months after tamoxifen treatment. To define the functional properties of renal MØ subtypes in steady state, we performed whole-transcriptome (RNA-sequencing) analysis on tdTomato+ SM-MØs and tdTomato–monocyte-derived MØs derived from Cx3cr1CreER/+: R26Td mice 4 months after tamoxifen injection. The 2 MØ subtypes were sorted according to a gating strategy we developed (Supplementary Figure S1),12Zhu Q. He J. Cao Y. et al.Analysis of mononuclear phagocytes disclosed the establishment processes of two macrophage subsets in the adult murine kidney.Front Immunol. 2022; 13805420Google Scholar in which the SM-MØs were F4/80hitdTomato+ and the monocyte-derived MØs were F4/80hi tdTomato–. Both populations highly expressed genes important to MØ ontology and functions, including Cebpa, Csf1r, Fcgr2b, and Ctsb (Figure 1a). Hierarchical clustering analysis showed that the replicates of each cell subset were clustered together (Figure 1b). A distinction in the transcriptomes between SM-MØs and monocyte-derived MØs was unveiled, as over 1000 genes were differentially expressed (fold change >2; false discovery rate [FDR] ≤ 0.05; P < 0.05). The level of expression of Ccr2 and Ly6c2, 2 characteristic genes of classical monocytes, was higher in tdTomato–cells than in tdTomato+ cells, supporting the conclusion that the tdTomato–cells are monocyte-derived (Figure 1c; Supplementary Figure S2). Surprisingly, gene ontology (GO) biological process enrichment analysis showed that the self-maintaining tdTomato+ MØs were characterized by enriched expression of transcripts involved in neuronal differentiation and excitation (Figure 1d). The characteristic genes included Ppp1r9a, Sema6d, Pmp22, and Gria1 (Figure 1c and e). Kidney is heavily innervated by sympathetic nerves. Consistent with this, SM-MØs highly expressed sympathetic nerve–relevant genes Maoa, Npy1r, and Slc6a2, relative to monocyte-derived MØs (Figure 1c). In contrast, genes involved in metabolic processes and molecule transportation were underrepresented in SM-MØs (Figure 1d and e). To identify potential distinctive surface markers of SM-MØs, we focused on the genes of membrane proteins with a high abundance of transcripts in the tdTomato+ subset and a low P value when comparing these 2 MØ subsets. Cd4 and Vcam1 are among the top 10 upregulated genes in the tdTomato+ subset (Figure 1e). Flow cytometry analysis showed that CD4+ or Vcam1+ MØs were exclusively tdTomato+, although not all of tdTomato+ cells were CD4+ or Vcam1+ (Figure 1f). In conclusion, by combining a fate-mapping approach with bulk RNA-sequencing, we were able to capture the heterogeneity of renal MØs. The transcriptome disparity between SM-MØs and their monocyte-derived counterparts suggests that the SM-MØs may serve at specific niches surrounding nerves in the kidney. Kidneys are innervated by efferent sympathetic nerves and afferent sensory nerves.9Barajas L. Liu L. Powers K. Anatomy of the renal innervation: intrarenal aspects and ganglia of origin.Can J Physiol Pharmacol. 1992; 70: 735-749Crossref PubMed Scopus (176) Google Scholar By immunohistostaining for typical markers of sympathetic nerves (tyrosine hydroxylase [TH]) and sensory nerves (calcitonin gene-related peptide [CGRP]), we were able to examine the microanatomy of sympathetic and sensory nerves. Sympathetic nerves were seen around the afferent and efferent glomerular arterioles and extended along tubules in the kidney (Supplementary Figure S3A). Employing Dbhcre: R26Td reporter mice, which have tdTomato expression in sympathetic neurons,14Parlato R. Otto C. Begus Y. et al.Specific ablation of the transcription factor CREB in sympathetic neurons surprisingly protects against developmentally regulated apoptosis.Development. 2007; 134: 1663-1670Crossref PubMed Scopus (51) Google Scholar we confirmed this distribution pattern of sympathetic nerves (Supplementary Figure S3B). Contrary to the wide distribution of the sympathetic nerves, the majority of the sensory nerves were located in the pelvic area, with few distributed in the kidney interstitium (Supplementary Figure S3A), a pattern consistent with previous reports.9Barajas L. Liu L. Powers K. Anatomy of the renal innervation: intrarenal aspects and ganglia of origin.Can J Physiol Pharmacol. 1992; 70: 735-749Crossref PubMed Scopus (176) Google Scholar,15Kopp U.C. Role of renal sensory nerves in physiological and pathophysiological conditions.Am J Physiol Regul Integr Comp Physiol. 2015; 308: R79-R95Crossref PubMed Scopus (110) Google Scholar When examining the kidneys of Cx3cr1CreER/+:R26Td mice that were aged for 4 months after tamoxifen injection with confocal microscopy, we found a divergence in the spatial association of SM-MØs between nerves, with many more SM-MØs being in close contact with sympathetic fibers than sensory fibers (“close contact” was defined by the attachment of MØ soma to the nerve fiber, as verified by Z-stacked images; Figure 2a). Not all SM-MØs were nerve-associated, as some tdTomato+ cells also were scattered in the stroma where nerve components were absent. Considering that F4/80 was still the most faithful marker of renal MØs,11Liu F. Dai S. Feng D. et al.Distinct fate, dynamics and niches of renal macrophages of bone marrow or embryonic origins.Nat Commun. 2020; 11: 2280Crossref PubMed Scopus (44) Google Scholar,12Zhu Q. He J. Cao Y. et al.Analysis of mononuclear phagocytes disclosed the establishment processes of two macrophage subsets in the adult murine kidney.Front Immunol. 2022; 13805420Google Scholar,16Zimmerman K.A. Bentley M.R. Lever J.M. et al.Single-cell RNA sequencing identifies candidate renal resident macrophage gene expression signatures across species.J Am Soc Nephrol. 2019; 30: 767-781Crossref PubMed Scopus (84) Google Scholar monocyte-derived MØs were identified as F4/80+tdTomato–in immunohistologic assessment. Examining sympathetic nerve–associated MØs unveiled that tdTomato+ SM-MØs were dominant over tdTomato– monocyte-derived MØs (Figure 2b). Some SM-MØs were tightly wrapped around the nonmyelinated sympathetic nerve fibers with their dendritiform processes; some MØs were soma-bound to the axons; and some had protruding pseudopodia along the nerve bundles over a large area, the details of which were revealed by high optical magnification and 3D reconstruction (Figure 2c). We next set out to observe possible dynamic interaction between MØs and sympathetic nerves. The penetration ability of 2-photon excitation microscopy makes it difficult to locate sympathetic nerves, which are relatively scarcely distributed under the surface of the kidney. We thus resorted to monitoring live kidney sections. To do this, we first labeled MØs using i.v. infusion of fluorophore-conjugated anti-F4/80 antibody into Dbhcre: R26Td mice. In a preliminary experiment, we verified that almost all tdTomato+ SM-MØs in the kidney were labeled by an anti-F4/80 antibody 1 hour after i.v. administration of this antibody to Cx3cr1CreER/+:R26Td mice (Supplementary Figure S4). Thus, we collected the kidneys of Dbhcre: R26Td mice 1-hour post-labeling, and then we sectioned them by vibrotome in ice-cold buffer to preserve the microarchitecture of the kidney and the viability of cells. After warming the sections to 37 °C to restore cell mobility, we tracked the cells by confocal microscope for up to 4 hours. While remaining sessile and fixed in position with respect to the main cell body in this timeframe, the nerve-bound MØs displayed dynamic extensions and retractions of pseudopodia over time, suggesting axonal scanning and surveillance (Figure 2d; Supplementary Movies S1 and S2). To investigate whether the nerve-interactive pattern of MØ varies in response to physiological challenge, we monitored the SM-MØs under conditions of water deprivation that could stimulate sympathetic outflow.17Brooks V.L. Qi Y. O'Donaughy T.L. Increased osmolality of conscious water-deprived rats supports arterial pressure and sympathetic activity via a brain action.Am J Physiol Regul Integr Comp Physiol. 2005; 288: R1248-R1255Crossref PubMed Scopus (38) Google Scholar,18Stocker S.D. Hunwick K.J. Toney G.M. Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water-deprived rats.J Physiol. 2005; 563: 249-263Crossref PubMed Scopus (64) Google Scholar We verified that water deprivation for 24 hours significantly raised both osmolality and the NE level in blood plasma (Supplementary Figures S5A and B). Water deprivation increased the movement of MØ pseudopodia along the nerve bundles (Figure 2e). Also, the density of sympathetic nerve–bound SM-MØs was increased (Figure 2f). Based on these findings, we concluded that the renal sympathetic nerves are closely associated with a distinct SM-MØ population. Renal sympathetic innervation is important in regulating salt and water balance. In particular, sympathetic axons release NE, which stimulates a reduction of blood flow and an increase of sodium and water reabsorption in the kidney, ultimately leading to reduced excretion of salt and water.10Johns E.J. Kopp U.C. DiBona G.F. Neural control of renal function.Compr Physiol. 2011; 1: 731-767Crossref PubMed Scopus (195) Google Scholar Enlightened by the observation above, we sought to determine whether SM-MØs play a role in regulating sympathetic nerve activities. To this end, we adopted a depletion strategy by crossing Cx3cr1CreERT2 to Rosa26-iDTR mice. After injection of tamoxifen at 8 weeks of age, Cx3cr1CreER/+:Rosa26-iDTR mice were aged for 3 months before receiving DT treatment (Figure 3a). This strategy was used so that the renal SM-MØ pool would be deleted specifically, sparing monocyte-derived MØs. Indeed, upon DT administration 3 months post–tamoxifen treatment, a complete loss of CD4+ renal MØs was noticed, indicative of loss of SM-MØs (Supplementary Figure S6A). In line with this finding, renal MØs were reduced by ∼37% after DT application (Supplementary Figure S6B), close to the estimated ratio of SM-MØs.12Zhu Q. He J. Cao Y. et al.Analysis of mononuclear phagocytes disclosed the establishment processes of two macrophage subsets in the adult murine kidney.Front Immunol. 2022; 13805420Google Scholar Depletion of SM-MØs did not lead to overt kidney inflammation (Supplementary Figure S6C), which was also reflected by the normal counts of intrarenal neutrophils and classical monocytes (Supplementary Figure S6B) and unaltered expression of proinflammatory cytokines (Supplementary Figure S6D). Based on the observation that renal MØs would not be fully repopulated until 18 days post-depletion by liposome clodronate,12Zhu Q. He J. Cao Y. et al.Analysis of mononuclear phagocytes disclosed the establishment processes of two macrophage subsets in the adult murine kidney.Front Immunol. 2022; 13805420Google Scholar we chose a 14-day window to observe the effects of SM-MØ depletion. Time-course analysis revealed that depletion of SM-MØs caused an overall decrease of the kidney NE level, as measured by mass spectrometry, suggesting reduced sympathetic tonicity (Figure 3b). In alignment with this finding, the levels of L-DOPA and dopamine—the NE precursors generated in sympathetic nerves—in the kidneys were reduced by ∼20%–25% in the SM-MØ–depleted mice, compared to the levels in the SM-MØ–intact littermates (Figure 3c). Moreover, a pronounced reduction of sympathetic nerve distribution in the renal cortex of SM-MØ-depleted mice was revealed by immunofluorescence staining of either TH or synaptophysin, a presynaptic marker (Figure 3d and e), which was accompanied by reduced renal sympathetic nerve activity at baseline (Figure 3f). Collectively, these data provide supporting evidence that SM-MØs play a role in maintaining renal sympathetic innervation. Actions of renal sympathetic nerves include stimulation of renin release from juxtaglomerular cells, limitation of blood flow through glomeruli, and enhancement of water/salt (sodium, potassium, chloride) reabsorption by segments of tubular epithelial cells that express adrenergic receptors. Indeed, depletion of SM-MØs led to a decline in plasma renin concentration and an increase in glomerular filtration rate (Figure 3g and h). Consistent with weakened sympathetic modulation, loss of SM-MØs led to a significantly increased urine output (Figure 3i). Moreover, SM-MØ–depleted mice had approximately twice the urine osmolality as that of control mice (Figure 3j); their 24-hour urinary excretion of sodium, potassium, and chloride were all significantly increased by a similar magnitude (Figure 3k). These data strongly indicate a solute diuresis rather than a water diuresis in the SM-MØ–depleted mice, consolidating a reduced sympathetic innervation. To more specifically determine the roles of renal SM-MØs, we administrated DT into the subcapsular space of the kidneys of tamoxifen-treated Cx3cr1CreER/+:Rosa26-iDTR mice. This procdeure can specifically affect kidney while sparing other organs. Indeed, subcapsular delivery of DT specifically removed renal MØs but had minimal effects on brain microglia and intestinal MØs, which also homogenously express CX3CR1 (Supplementary Figure S7A–C). Again, this led to a reduction of sympathetic nerve distribution in the cortex, a decrease of kidney NE level, and a significant increase of water output and of Na+, K+, Cl– excretion (Supplementary Figure S7D–G), underscoring that renal SM-MØs play a role in modulating local sympathetic nerves. In the kidney, renal afferent sensory nerves collect chemical and mechanical signals and exert negative feedback control of sympathetic outflow via the renorenal reflex.15Kopp U.C. Role of renal sensory nerves in physiological and pathophysiological conditions.Am J Physiol Regul Integr Comp Physiol. 2015; 308: R79-R95Crossref PubMed Scopus (110) Google Scholar Thus, excitement of the inhibitory renorenal reflex would contribute to salt and water excretion. To exclude the possibility that the reduction of sympathetic tone after deprivation of SM-MØs was mediated indirectly through an altered microenvironment sensed by sensory nerves, we selectively ablated afferent sensory nerves by periaxonal application of capsaicin (Figure 4a).19Foss J.D. Wainford R.D. Engeland W.C. et al.A novel method of selective ablation of afferent renal nerves by periaxonal application of capsaicin.Am J Physiol Regul Integr Comp Physiol. 2015; 308: R112-R122Crossref PubMed Scopus (79) Google Scholar Capsaicin treatment led to a dramatic reduction of sensory nerve distribution, as demonstrated