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.

Hydroxyproline stimulates inflammation and reprograms macrophage signaling in a rat kidney stone model.

Meals rich in oxalate are associated with calcium oxalate (CaOx) kidney stone disease. Hydroxy-L-proline (HLP) is an oxalate precursor found in milk and collagen-containing foods. HLP has been shown to induce CaOx crystal formation in rodents. The purpose of this study was to evaluate the effect of HLP induced oxalate levels on inflammation and renal leukocytes during crystal formation. Male Sprague-Dawley rats (6-8 weeks old) were fed a control diet containing no oxalate for 3 days before being randomized to continue the control diet or 5% HLP for up to 28 days. Blood, 24 h urine, and kidneys were collected on Days 0, 7, 14, or 28. Urinary oxalate levels, crystal deposition, and renal macrophage markers were evaluated using ion chromatography-mass spectrometry, immunohistochemistry, and qRT-PCR. Renal leukocytes were assessed using flow cytometry and RNA-sequencing. HLP feeding increased urinary oxalate levels and renal crystal formation in animals within 7 days. HLP also increased renal macrophage populations on Days 14 and 28. Transcriptome analysis revealed that renal macrophages from animals fed HLP for 7 days were involved in inflammatory response and disease, stress response to LPS, oxidative stress, and immune cell trafficking. Renal macrophages isolated on Day 14 were involved in cell-mediated immunological pathways, ion homeostasis, and inflammatory response. Collectively, these findings suggest that HLP-mediated oxalate levels induce markers of inflammation, leukocyte populations, and reprograms signaling pathways in macrophages in a time-dependent manner. Additional studies investigating the significance of oxalate on renal macrophages could aid in our understanding of kidney stone formation.

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.

Bone marrow Tregs mediate stromal cell function and support hematopoiesis via IL-10.

The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis.

Resident macrophages reprogram toward a developmental state after acute kidney injury.

Acute kidney injury (AKI) is a devastating clinical condition affecting at least two-thirds of critically ill patients, and, among these patients, it is associated with a greater than 60% risk of mortality. Kidney mononuclear phagocytes (MPs) are implicated in pathogenesis and healing in mouse models of AKI and, thus, have been the subject of investigation as potential targets for clinical intervention. We have determined that, after injury, F4/80hi-expressing kidney-resident macrophages (KRMs) are a distinct cellular subpopulation that does not differentiate from nonresident infiltrating MPs. However, if KRMs are depleted using polyinosinic/polycytidylic acid (poly I:C), they can be reconstituted from bone marrow-derived precursors. Further, KRMs lack major histocompatibility complex class II (MHCII) expression before P7 but upregulate it over the next 14 days. This MHCII- KRM phenotype reappears after injury. RNA sequencing shows that injury causes transcriptional reprogramming of KRMs such that they more closely resemble that found at P7. KRMs after injury are also enriched in Wingless-type MMTV integration site family (Wnt) signaling, indicating that a pathway vital for mouse and human kidney development is active. These data indicate that mechanisms involved in kidney development may be functioning after injury in KRMs.

Parabiosis reveals leukocyte dynamics in the kidney.

The immune cellular compartment of the kidney is involved in organ development and homeostasis, as well as in many pathological conditions. Little is known about the mechanisms that drive intrarenal immune responses in the presence of renal tubular and interstitial cell death. However, it is known that tissue-resident leukocytes have the potential to have distinct roles compared with circulating cells. We used a parabiosis model in C57BL/6 CD45 congenic and green fluorescent protein transgenic mice to better understand the dynamics of immune cells in the kidney. We found F4/80Hi intrarenal macrophages exhibit minimal exchange with the peripheral circulation in two models of parabiosis, whether mice were attached for 4 or 16 weeks. Other intrarenal inflammatory cells demonstrate near total exchange with the circulating immune cell pool in healthy kidneys, indicating that innate and adaptive immune cells extensively traffic through the kidney interstitium during normal physiology. Neutrophils, dendritic cells, F4/80Low macrophages, T cells, B cells, and NK cells are renewed from the circulating immune cell pool. However, a fraction of double-negative T (CD4- CD8-) and NKT cells are long-lived or tissue resident. This study provides direct evidence of leukocyte sub-populations that are resident in the renal tissue, cells which demonstrate minimal to no exchange with the peripheral blood. In addition, the data demonstrate continual exchange of other sub-populations through uninflamed tissue.

Endothelial cell transfusion ameliorates endothelial dysfunction in 5/6 nephrectomized rats.

Endothelial dysfunction is prevalent in chronic kidney disease. This study tested the hypothesis that transfusion of rat aortic endothelial cells (ECs) ameliorates endothelial dysfunction in a rat model of chronic kidney disease. Male Sprague-Dawley rats underwent sham surgery or 5/6 nephrectomy (Nx). Five weeks after Nx, EC (1.5 × 10(6) cells/rat) or vehicle were transfused intravenously. One week later, vascular reactivity of mesenteric artery was assessed on a wire myograph. Sensitivity of endothelium-dependent relaxation to acetylcholine and maximum vasodilation were impaired by Nx and improved by EC transfusion. Using selective pharmacological nitric oxide synthase isoform inhibitors, we demonstrated that the negative effect of Nx on endothelial function and rescue by EC transfusion are, at least in part, endothelial nitric oxide synthase mediated. Plasma asymmetric dimethylarginine was increased by Nx and decreased by EC transfusion, whereas mRNA expression of dimethylarginine dimethylaminohydrolases 1 (DDAH1) was decreased by Nx and restored by EC transfusion. Immunohistochemical staining confirmed that local expression of DDAH1 is decreased by Nx and increased by EC transfusion. In conclusion, EC transfusion attenuates Nx-induced endothelium-dependent vascular dysfunction by regulating DDAH1 expression and enhancing endothelial nitric oxide synthase activity. These results suggest that EC-based therapy could provide a novel therapeutic strategy to improve vascular function in chronic kidney disease.

Endothelial cells overexpressing interleukin-8 receptors reduce inflammatory and neointimal responses to arterial injury.

BACKGROUND: Interleukin-8 (IL8) receptors IL8RA and IL8RB on neutrophil membranes bind to IL8 and direct neutrophil recruitment to sites of inflammation, including acutely injured arteries. This study tested whether administration of IL8RA- and/or IL8RB-transduced rat aortic endothelial cells (ECs) accelerates adhesion of ECs to the injured surface, thus suppressing inflammation and neointima formation in balloon-injured rat carotid arteries. We tested the hypothesis that targeted delivery of ECs by overexpressing IL8RA and IL8RB receptors prevents inflammatory responses and promotes structural recovery of arteries after endoluminal injury. METHODS AND RESULTS: Young adult male rats received balloon injury of the right carotid artery and were transfused intravenously with ECs (total, 1.5×10(6) cells at 1, 3, and 5 hours after injury) transduced with adenoviral vectors carrying IL8RA, IL8RB, and IL8RA/RB (dual transduction) genes, AdNull (empty vector), or vehicle (no EC transfusion). ECs overexpressing IL8Rs inhibited proinflammatory mediators expression significantly (by 60% to 85%) and reduced infiltration of neutrophils and monocytes/macrophages into injured arteries at 1 day after injury, as well as stimulating a 2-fold increase in reendothelialization at 14 days after injury. IL8RA-EC, IL8RB-EC, and IL8RA/RB-EC treatment reduced neointima formation dramatically (by 80%, 74%, and 95%) at 28 days after injury. CONCLUSIONS: ECs with overexpression of IL8RA and/or IL8RB mimic the behavior of neutrophils that target and adhere to injured tissues, preventing inflammation and neointima formation. Targeted delivery of ECs to arteries with endoluminal injury provides a novel strategy for the prevention and treatment of cardiovascular disease.

Curcumin protects dopaminergic neuron against LPS induced neurotoxicity in primary rat neuron/glia culture.

Using primary rat mesencephalic neuron-glia cultures as an in vitro model of Parkinson's disease (PD), we tested the effect of curcumin, a natural dietary pigment with well-known anti-inflammation effects, on dopaminergic (DA) degeneration. Curcumin pretreatment mitigated LPS-induced DA neurotoxicity in a concentration-dependent manner and curcumin post-treatment also showed protective effect. Microglia depletion abolished this protective effect of curcumin, indicating that microglia play an important role in this effect. Supportively, observation by immunocytochemistry staining using OX-42 antibody showed that curcumin treatment inhibited LPS-induced morphological change of microglia. Besides, LPS-induced production of many proinflammatory factors and their gene expressions decreased dramatically after curcumin treatment. Results also revealed that curcumin treatment decreased LPS-induced activation of two transcription factors--nuclear factors kappaB (NF-kappaB) and activator protein-1 (AP-1). Taken together, our study implicated that curcumin might be a potential preventive and therapeutic strategy for inflammation-related neurodegenerative diseases.

Protective effects and mechanism of total coptis alkaloids on a beta 25-35 induced learning and memory dysfunction in rats.

OBJECTIVE: To observe the effect of total coptis alkaloids (TCA) on beta -amyloid peptide (A beta 25-35) induced learning and memory dysfunction in rats, and to explore its mechanism. METHODS: Forty male Wistar rats were randomly divided into four groups: the control group, the model group, the TCA low dose (60 mg/kg) group and the TCA high dose (120 mg/kg) group, 10 in each. A beta 25-35 (5microl, 2 microg/microl) was injected into bilateral hippocampi of each rat to induce learning and memory dysfunction. TCA were administered through intragavage for consecutive 15 days. Morris Water Maze test was used to assess the impairment of learning and memory; concentration of malondialdehyde (MDA) in cerebral cortex was determined by thiobarbituric acid reactive substance to indicate the level of lipid peroxidation in brain tissues; activity of manganese-superoxide dismutase (Mn-SOD) in cerebral cortex was determined by xanthine-oxidase to indicate the activity of the enzyme; and NF- kappa B protein expression in cerebral cortex was measured by SP immunohistochemistry. RESULTS: (1) Morris Water Maze test showed that, during the 4 consecutive days of acquisition trials, the rats in the model group took longer latency and searching distance than those in the control group (P<0.01), which could be shortened by high dose TCA (P<0.05); during the spatial probe trial on the fifth day, the rats in the model group took shorter searching time and distance on the previous flat area than those in the control group (P<0.01), which could be prolonged after TCA treatment (for low dose group, P<0.05; for high dose group, P<0.01). (2) Analysis of cerebral cortical tissues showed that, compared with the control group, MDA level got significantly increased and Mn-SOD activity decreased in the model group (both P<0.01). After having been treated with TCA, the MDA level got significantly decreased (P<0.05 and P<0.01 respectively for low and high dose group), while relative increase of Mn-SOD activity only appeared in high dose group (P<0.05). (3) Immunohistochemistry analysis showed the protein expression of NF- kappa B got significantly increased after modeling, while high dose TCA can significantly inhibit it. CONCLUSION: TCA could improve A beta 25-35 induced dysfunction of learning and memory in rats, and its protective mechanism is associated with its actions in decreasing MDA level, increasing Mn-SOD activity and inhibiting the expression of NF-kappa B in cerebral cortex.

Cadmium-induced toxicity in rat primary mid-brain neuroglia cultures: role of oxidative stress from microglia.

This study examined the role of oxidative stress in neurotoxic effects of cadmium chloride (Cd) in rat primary mid-brain neuron-glia cultures. Cd accumulated in neuron-glia cultures and produced cytotoxicity in a dose-dependent manner, with IC(50) of 2.5microM 24 h after exposure. (3)H-dopamine uptake into neuron-glia cultures was decreased 7 days after Cd exposure, with IC(50) of 0.9microM, indicative of the sensitivity of dopaminergic neurons to Cd toxicity. To investigate the role of microglia in Cd-induced toxicity to neurons, microglia-enriched cultures were prepared. Cd significantly increased intracellular reactive oxygen species production in microglia-enriched cultures, as evidenced by threefold increases in 2',7'-dichlorofluorescein signals. Using 5,5-dimethyl-1-pyrroline N-oxide as a spin-trapping agent, Cd increased electron spin resonance signals by 3.5-fold in microglia-enriched cultures. Cd-induced oxidative stress to microglia-enriched cultures was further evidenced by activation of redox-sensitive transcription factor nuclear factor kappa B and activator protein-1 (AP-1), and the increased expression of oxidative stress-related genes, such as metallothionein, heme oxygenase-1, glutathione S-transferase pi, and metal transport protein-1, as determined by gel-shift assays and real-time reverse transcription-PCR, respectively, in microglia-enriched cultures. In conclusion, Cd is toxic to neuron-glia cultures, and the oxidative stress from microglia may play important roles in Cd-induced damage to dopaminergic neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP4-6 are neuroprotective through inhibition of NADPH oxidase: potent regulators of microglia-mediated oxidative stress.

Microglial activation is implicated in the progressive nature of numerous neurodegenerative diseases, including Parkinson's disease. Using primary rat mesencephalic neuron-glia cultures, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) 38, PACAP27, and its internal peptide, Gly-Ile-Phe (GIF; PACAP4-6), are neuroprotective at 10(-13) M against lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity, as determined by [(3)H]DA uptake and the number of tyrosine hydroxylase-immunoreactive neurons. PACAP38 and GIF also protected against 1-methyl-4-phenylpyridinium(+)-induced neurotoxicity but only in cultures containing microglia. PACAP38 and GIF ameliorated the production of microglia-derived reactive oxygen species (ROS), where both LPS- and phorbol 12-myristate 13-acetate-induced superoxide and intracellular ROS were inhibited. The critical role of NADPH oxidase for GIF and PACAP38 neuroprotection against LPS-induced DA neurotoxicity was demonstrated using neuron-glia cultures from mice deficient in NADPH oxidase (PHOX(-/-)), where PACAP38 and GIF reduced tumor necrosis factor alpha production and were neuroprotective only in PHOX(+/+) cultures and not in PHOX(-/-) cultures. Pretreatment with PACAP6-38 (3 microM; PACAP-specific receptor antagonist) was unable to attenuate PACAP38, PACAP27, or GIF (10(-13) M) neuroprotection. PACAP38 and GIF (10(-13) M) failed to induce cAMP in neuronglia cultures, supporting that the neuroprotective effect was independent of traditional high-affinity PACAP receptors. Pharmacophore analysis revealed that GIF shares common chemical properties (hydrogen bond acceptor, positive ionizable, and hydrophobic regions) with other subpicomolar-acting compounds known to inhibit NADPH oxidase: naloxone, dextromethorphan, and Gly-Gly-Phe. These results indicate a common high-affinity site of action across numerous diverse peptides and compounds, revealing a basic neuropeptide regulatory mechanism that inhibits microglia-derived oxidative stress and promotes neuron survival.

Protective effect of ecdysterone on PC12 cells cytotoxicity induced by beta-amyloid25-35.

OBJECTIVE: To examine the protective effect of ecdysterone (ECR) against beta-amyloid peptide fragment(25-35) (Abeta(25-35))-induced PC12 cells cytotoxicity, and to further explore its mechanism. METHODS: Experimental PC12 cells were divided into the Abeta group (treated by Abeta(25-35) 100 micromol/L), the blank group (untreated), the positive control group (treated by Vit E 100 micromol/L after induction) and the ECR treated groups (treated by ECR with different concentrations of 1, 50 and 100 micromol/L). The damaged and survival condition of PC12 cells in various groups was monitored by lactate dehydrogenase (LDH) release and MTT assay. The content of malondialdehyde (MDA) was measured by fluorometric assay to indicate the lipid peroxidation. And the antioxidant enzymes activities in PC12 cells, including superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), were detected respectively. RESULTS: After PC12 cells were treated with Abeta(25-35) (100 micromol/L) for 24 hrs, they revealed a great decrease in MTT absorbance and activity of antioxidant enzymes, including SOD, CAT and GSH-Px as well as a significant increase of LDH activity and MDA content in PC12 cells (P < 0.01). When the cells was pretreated with 1-100 micromol/L ECR for 24 hrs before Abeta(25-35) treatment, the above-mentioned cytotoxic effect of Abeta(25-35) could be significantly attenuated dose-dependently, for ECR 50 micromol/L, P < 0.05 and for ECR 100 micromol/L, P < 0.01. Moreover, ECR also showed significant inhibition on the Abeta(25-35) induced decrease of SOD and GSH-Px activity, but not on that of CAT. CONCLUSION: ECR could protect PC12 cells from cytotoxicity of Abeta(25-35), and the protective mechanism might be related to the increase of SOD and GSH-Px activities and the decrease of MDA resulting from the ECR-pretreatment.

[Effect of ecdysterone on the expression of c-fos in the brain of rats induced by microinjection beta-AP25-35 into the hippocampus].

AIM: To observe the behavior in learning and memory and the expression of c-fos gene from the brain of rats induced by beta-AP25-35, and the intervention of ecdysterone, in order to explore the protective mechanism of ecdysterone on the dysfunction of learning and memory of the rat induced by beta-AP25-35. METHODS: Microinjection of beta-AP25-35 into hippocampus induced learning and memory dysfunction of rats. The learning and memory of rats were observed by Morris Water Maze. The expression of c-fos gene in the brain was detected by immunohistochemistry. RESULTS: The results of Morris Water Maze showed that after rats were microinjected beta-AP25-35 into hippocampus, the rats in model group took longer latency and searching distance compared with the ones in control group (P < 0.01), and the rats in treated group (ECR 4 mg x kg(-1), ECR 8 mg x kg(-1) and nimodipine 7.2 mg x kg(-1)) took shorter latency and searching distance, especially the ECR 8 mg kg(-1) group (P < 0.01). At the same time, after the 5 days training, there was a higher expression of c-fos in hippocampus and cortex from the rats in control group than that in model group (P < 0.01), but in the treated group, there was a relatively higher expression of c-fos, especially the ECR 8 mg x kg(-1) group (P < 0.01). CONCLUSION: Microinjection of beta-AP25-35 into the rat hippocampus resulted in dysfunction of learning and memory. Ecdysterone was shown to improve the learning and memory of the rats and increase the expression of c-fos. Increasing the expression of c-fos is probably one of the most molecular mechanism of its protection.

[The effects of genistein on epidermal growth factor receptor mediated signal transduction pathway in human ovarian carcinoma cells lines SKOV3 and its xenograft in nude mice].

OBJECTIVE: It was reported previously that genistein could inhibit proliferation of human ovarian carcinoma cell line SKOV(3), but mechanism was not clear. There is a close relationship between EGFR mediated signal transduction pathway and the development of ovarian tumor. This study aimed to investigate the effects of genistein on the EGFR mediated signal transduction pathway and to clarify its mechanisms of proliferation inhibition on human ovarian carcinoma cell line SKOV(3) and its xenograft in nude mice. METHODS: The expression of c-erbB-2 protein was determined using immunocytochemistry. The expressions of c-jun and c-fos protein were determined using Western blotting. The expression of c-erbB-2, c-raf-1, c-jun and c-fos mRNA were tested by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of c-erbB-2, c-raf-1 and its downstream gene c-jun and c-fos were decreased at mRNA level in the 20 micromol/L genistein group. The expression of c-erbB-2 protein were decreased, its average absorbency (A) were decreased after treatment of SKOV(3) with 20 micromol/L genistein for 48 h, reached at 0.42 +/- 0.02 (P < 0.05). Western blotting demonstrated that the expressions of c-jun and c-fos protein were decreased gradually after being treated with 20 micromol/L genistein for 12 - 72 h. CONCLUSIONS: Genistein could down-regulate the expression of two key genes, c-erbB-2 and c-raf-1 at mRNA and protein level in the EGFR mediated signal transduction pathway, and down-regulate the expression of its downstream nuclear transcription factors c-jun and c-fos at mRNA and protein level. It is suggested that interfering the expressions of some key signal molecules in EGFR mediated signal transduction system by genistein may account for its molecular foundation of proliferation inhibition in ovarian carcinoma.