Renal Macrophage Infiltration Research Articles

Iguratimod prevents renal fibrosis in unilateral ureteral obstruction model mice by suppressing M2 macrophage infiltration and macrophage–myofibroblast transition

Iguratimod is a novel synthetic, small-molecule immunosuppressive agent used to treat rheumatoid arthritis. Through ongoing exploration of its role and mechanisms of action, iguratimod has been observed to have antifibrotic effects in the lung and skin; however, its effect on renal fibrosis remains unknown. This study aimed to investigate whether iguratimod could affect renal fibrosis progression. Three different concentrations of iguratimod (30 mg/kg/day, 10 mg/kg/day, and 3 mg/kg/day) were used to intervene in unilateral ureteral obstruction (UUO) model mice. Iguratimod at 10 mg/kg/day was observed to be effective in slowing UUO-mediated renal fibrosis. In addition, stimulating bone marrow-derived macrophages with IL-4 and/or iguratimod, or with TGF-β and iguratimod or SRC inhibitors in vitro, suggested that iguratimod mitigates the progression of renal fibrosis in UUO mice, at least in part, by inhibiting the IL-4/STAT6 signaling pathway to attenuate renal M2 macrophage infiltration, as well as by impeding SRC activation to reduce macrophage–myofibroblast transition. These findings reveal the potential of iguratimod as a treatment for renal disease.

Cholinergic Stimulation Exerts Cardioprotective Effects and Alleviates Renal Inflammatory Responses after Acute Myocardial Infarction in Spontaneous Hypertensive Rats (SHRs).

In this investigation, we explored the effects of pharmacological cholinergic stimulation on cardiac function and renal inflammation following acute myocardial infarction (AMI) in spontaneously hypertensive rats (SHRs). Adult male SHRs were randomized into three experimental groups: sham-operated; AMI + Veh (infarcted, treated with vehicle); and AMI + PY (infarcted, treated with the cholinesterase inhibitor, pyridostigmine bromide (PY)-40 mg/kg, once daily for seven days). Rats were euthanized 7 or 30 days post-surgery. The clinical parameters were assessed on the day before euthanasia. Subsequent to euthanasia, blood samples were collected and renal tissues were harvested for histological and gene expression analyses aimed to evaluate inflammation and injury. Seven days post-surgery, the AMI + PY group demonstrated improvements in left ventricular diastolic function and autonomic regulation, and a reduction in renal macrophage infiltration compared to the AMI + Veh group. Furthermore, there was a notable downregulation in pro-inflammatory gene expression and an upregulation in anti-inflammatory gene expression. Analysis 30 days post-surgery showed that PY treatment had a sustained positive effect on renal gene expression, correlated with a decrease in biomarkers, indicative of subclinical kidney injury. Short-term cholinergic stimulation with PY provides both cardiac and renal protection by mitigating the inflammatory response after AMI.

Clonal hematopoiesis of indeterminate potential is associated with acute kidney injury.

Age is a predominant risk factor for acute kidney injury (AKI), yet the biological mechanisms underlying this risk are largely unknown. Clonal hematopoiesis of indeterminate potential (CHIP) confers increased risk for several chronic diseases associated with aging. Here we sought to test whether CHIP increases the risk of AKI. In three population-based epidemiology cohorts, we found that CHIP was associated with a greater risk of incident AKI, which was more pronounced in patients with AKI requiring dialysis and in individuals with somatic mutations in genes other than DNMT3A, including mutations in TET2 and JAK2. Mendelian randomization analyses supported a causal role for CHIP in promoting AKI. Non-DNMT3A-CHIP was also associated with a nonresolving pattern of injury in patients with AKI. To gain mechanistic insight, we evaluated the role of Tet2-CHIP and Jak2V617F-CHIP in two mouse models of AKI. In both models, CHIP was associated with more severe AKI, greater renal proinflammatory macrophage infiltration and greater post-AKI kidney fibrosis. In summary, this work establishes CHIP as a genetic mechanism conferring impaired kidney function recovery after AKI via an aberrant inflammatory response mediated by renal macrophages.

Delayed treatment with erythropoietin attenuates renal fibrosis in mouse model of unilateral ureteral obstruction.

Erythropoietin (EPO) exerts tissue-protective effects on various organs including the kidney. However, the effects of EPO on established renal fibrosis remain unclear. In this study, we aimed to examine the therapeutic potential of EPO against established renal fibrosis. Renal fibrosis was induced in mice by unilateral ureteral obstruction (UUO) and the mice were treated with recombinant human EPO (rhEPO) daily during 7 and 13 days after UUO. The degrees of renal fibrosis, myofibroblast accumulation, and macrophage infiltration; the mRNA expression levels of transforming growth factor (TGF)-β1 and α1(I) collagen; and the protein levels of Kelch-like ECH-associated protein 1 (Keap1) and nuclear NF-E2-related factor 2 (Nrf2) in the kidneys were assessed on day 14 after UUO. Treatment with rhEPO significantly decreased fibrosis, myofibroblast accumulation, and α1(I) collagen mRNA expression, but it did not significantly affect TGF-β1 mRNA expression. Although treatment with rhEPO did not significantly affect the total number of interstitial macrophages, it significantly decreased the number of CD86-positive cells (M1 macrophages), while significantly increased the number of CD206-positive cells (M2 macrophages) in the interstitium. Treatment with rhEPO did not affect the Keap1/Nrf2 protein level or the peripheral blood hematocrit value. These results indicate for the first time that EPO exerts antifibrotic effects against the evolution of established renal fibrosis, possibly by influencing the polarization of infiltrating macrophages.

The pathogenic role of succinate-SUCNR1: a critical function that induces renal fibrosis via M2 macrophage

BackgroundRenal fibrosis significantly contributes to the progressive loss of kidney function in chronic kidney disease (CKD), with alternatively activated M2 macrophages playing a crucial role in this progression. The serum succinate level is consistently elevated in individuals with diabetes and obesity, both of which are critical factors contributing to CKD. However, it remains unclear whether elevated succinate levels can mediate M2 polarization of macrophages and contribute to renal interstitial fibrosis.MethodsMale C57/BL6 mice were administered water supplemented with 4% succinate for 12 weeks to assess its impact on renal interstitial fibrosis. Additionally, the significance of macrophages was confirmed in vivo by using clodronate liposomes to deplete them. Furthermore, we employed RAW 264.7 and NRK-49F cells to investigate the underlying molecular mechanisms.ResultsSuccinate caused renal interstitial macrophage infiltration, activation of profibrotic M2 phenotype, upregulation of profibrotic factors, and interstitial fibrosis. Treatment of clodronate liposomes markedly depleted macrophages and prevented the succinate-induced increase in profibrotic factors and fibrosis. Mechanically, succinate promoted CTGF transcription via triggering SUCNR1-p-Akt/p-GSK3β/β-catenin signaling, which was inhibited by SUCNR1 siRNA. The knockdown of succinate receptor (SUCNR1) or pretreatment of anti-CTGF(connective tissue growth factor) antibody suppressed the stimulating effects of succinate on RAW 264.7 and NRK-49F cells.ConclusionsThe causative effects of succinate on renal interstitial fibrosis were mediated by the activation of profibrotic M2 macrophages. Succinate-SUCNR1 played a role in activating p-Akt/p-GSK3β/β-catenin, CTGF expression, and facilitating crosstalk between macrophages and fibroblasts. Our findings suggest a promising strategy to prevent the progression of metabolic CKD by promoting the excretion of succinate in urine and/or using selective antagonists for SUCNR1.

Abstract P188: Hypertensive Stimuli Increase Pro-Inflammatory Bone Marrow Derived Macrophages In Vitro

Renal macrophage infiltration and inflammation is a hallmark of hypertension (HTN). An imbalance in macrophages has been observed in murine salt-sensitive and angiotensin II (AngII)-induced models of HTN. We have identified increased activated macrophages (CD45+ CD11b+ F4/80+ CD38+), pro-inflammatory M1 macrophages (CD45+ CD11b+ F4/80+ CD11c+ CD206-), and activated M1 macrophage (CD45+ CD11b+ F4/80+ CD11c+ CD206- CD38+) in the kidneys of these mice. In this study, we hypothesized that salt and AngII directly induce macrophages to become activated and pro-inflammatory. We exposed bone marrow derived macrophages (BMDMs) grown in GM-CSF to salt (190 μm) and AngII (0.01 μm) and found that there were significant increases in renal activated macrophages (% of CD45 cells: Con: 31 ± 1; AngII: 37 ± 1; Salt: 56 ± 1), pro-inflammatory M1 macrophages (Con: 27 ± 1; AngII: 37 ± 1; Salt: 56 ± 1), and activated M1 macrophages (Con: 22 ± 1; AngII: 28 ± 1; Salt: 43 ± 1) as determined by flow cytometry. However, the cultivation of BMDMs in M-CSF, the more traditional growth factor present in culturing BMDMs, did not produce the same significant results. Next, we will identify renal pro-inflammatory cytokines, chemokines, growth factors, and levels of GM-CSF present in these mouse models of HTN. Currently, these findings suggest that GM-CSF increases macrophage responsiveness to HTN stimuli and induces activated and pro-inflammatory macrophages which may contribute to the development and progression of HTN. Targeting activated renal macrophages or renal GM-CSF may provide a new therapeutic option for the treatment of HTN.

#4544 THE ASSOCIATION OF TAMOXIFEN TO THE CONSERVATIVE CKD TREATMENT PROMOTED ADDITIONAL ANTIFIBROTIC EFFECTS IN A MODEL OF HYPERTENSIVE NEPHROSCLEROSIS

Abstract Background and Aims Chronic kidney disease (CKD) is an insidious, progressive and highly debilitating condition, which leads to the loss of renal function and to the need of life-sustaining renal replacement therapy. The conservative treatment of CKD is mainly based in the blockade of the renin-angiotensin-aldosterone system (RAAS), which can be associated to immunosuppressant drugs, according to the etiology of the renal injury. However, this pharmacological approach is not able to prevent CKD progression completely. Until the present moment, medical community still lack of a specific drug to effectively stop the progression of renal fibrosis associated to CKD, which once triggered became irreversible. Tamoxifen (TAM) is an estrogen receptor antagonist, widely employed for the clinical treatment of breast cancer, and responsible for saving a number of lives worldwide, in the past decades. This well-tolerated and cost-effective drug have been reported to exert antifibrotic effects in both experimental and human peritoneal fibrosis. Moreover, our research group already demonstrated that TAM efficiently prevented albuminuria, glomerulosclerosis, and interstitial fibrosis in a model of CKD. In the present study, we investigated if the association of TAM to the classic conservative treatment of CKD, here obtained by the association of Losartan (LOS) and Micofenolate Mofetil (MMF), could promote further renoprotection in an experimental model of hypertensive nephrosclerosis, induced by the chronic nitric oxide synthase blockade, obtained by the oral administration of L-NAME (NAME), associated to a high sodium (HS) diet, in rats. Method The experimental protocol was approved by the local Research Ethics Committee (CAPPesq) and was developed in strict conformity with the international standards for care and manipulation of laboratory animals. Thirty male Wistar rats were kept with a 3.2% HS diet for 15 days of adaptation, before the protocol start. After this period, the animals continued to receive the HS diet and were divided among 5 groups. CONT: receiving no further drugs or treatments, NAME: treated with 70 mg/kg/d of L-NAME, diluted in drinking water, LOS: NAME rats treated with 50mg/Kg/d of LOS, also diluted in drinking water, MMF: NAME rats treated with 10 mg/Kg/d of MMF given by gavage, TAM: NAME rats treated with 10 mg/Kg/d also by gavage, and LOS+MMF+TAM: NAME rats treated with all the drugs simultaneously. Systolic blood pressure (SBP), urinary albumin excretion (24 h uAE), glomerulosclerosis (GS), glomerular ischemia (GI), interstitial fibrosis (INT), tubulointerstitial infiltration of macrophages (CD68) and T-cells (CD3), as well as renal cortical interstitial collagen I (COLL1) and fibronectin (FIBRO) accumulation were evaluated after 30 days of treatment. Results The association of TAM to the classic treatment of CKD improved the renoprotection obtained by LOS+MMF. The triple combined treatment significantly reduced hypertension, albuminuria, glomerular structural damage, renal macrophage and T-cell infiltration in NAME rats, compared to the animals treated with the respective monotherapies. Moreover, both TAM alone or the association, were equally effective in reducing interstitial collagen and fibronectin accumulation, in NAME rats. Data are presented as Mean ± SE. For One-way ANOVA statistical analysis, we considered: p<0.05 vs.*CONT, #NAME, †LOS, §MMF, &TAM. Conclusion Our preliminary results shown TAM to be effective in reducing renal inflammation and fibrosis in the chronic nitric oxide synthase blockade model and to exert additional renoprotective effects when associated to LOS and MMF in all the analyzed parameters. Although further studies with different nephropathy models are still required in order to confirm our findings, here we suggest TAM to be a potential adjuvant in the conservative management of CKD.

Depletion of CUL4B in macrophages ameliorates diabetic kidney disease via miR-194-5p/ITGA9 axis

Diabetic kidney disease (DKD) is the most prevalent chronic kidney disease. Macrophage infiltration in the kidney is critical for the progression of DKD. However, the underlying mechanism is far from clear. Cullin 4B (CUL4B) is the scaffold protein in CUL4B-RING E3 ligase complexes. Previous studies have shown that depletion of CUL4B in macrophages aggravates lipopolysaccharide-induced peritonitis and septic shock. In this study, using two mouse models for DKD, we demonstrate that myeloid deficiency of CUL4B alleviates diabetes-induced renal injury and fibrosis. Invivo and invitro analyses reveal that loss of CUL4B suppresses migration, adhesion, and renal infiltration of macrophages. Mechanistically, we show that high glucose upregulates CUL4B in macrophages. CUL4B represses expression of miR-194-5p, which leads to elevated integrin α9 (ITGA9), promoting migration and adhesion. Our study suggests the CUL4B/miR-194-5p/ITGA9 axis as an important regulator for macrophage infiltration in diabetic kidneys.

Chronic treatment with IL-25 increases renal M2 macrophages and reduces renal injury in obese Dahl salt-sensitive rats during the prepubescent stage.

Recently, we have reported that the early progression of proteinuria in the obese Dahl salt-sensitive (SS) leptin receptor mutant (SSLepRmutant) strain was associated with increased renal macrophage infiltration before puberty. Macrophages can be divided into two distinct phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory). Moreover, previous studies have demonstrated that interleukin (IL)-25 converts resting macrophages and M1 into M2. Therefore, the present study examined whether treatment with IL-25 would reduce the early progression of renal injury in SSLepRmutant rats by increasing renal M2. We also investigated the impact of IL-25 on M2 subtypes: M2a (wound healing/anti-inflammatory), M2b (immune mediated/proinflammatory), M2c (regulatory/anti-inflammatory), and M2d (tumor associated/proangiogenic). Four-wk-old SS and SSLepRmutant rats were treated with either control (IgG) or IL-25 (1 µg/day ip every other day) for 4 wk. The kidneys from SSLepRmutant rats displayed progressive proteinuria and renal histopathology versus SS rats. IL-25 treatment had no effect on these parameters in SS rats. However, in the SSLepRmutant strain, proteinuria was markedly reduced after IL-25 treatment. Chronic treatment with IL-25 significantly decreased glomerular and tubular injury and renal fibrosis in the SSLepRmutant strain. Although the administration of IL-25 did not change total renal macrophage infiltration in both SS and SSLepRmutant rats, IL-25 increased M2a by >50% and reduced M1 by 60% in the kidneys of SSLepRmutant rats. Overall, these data indicate that IL-25 reduces the early progression of renal injury in SSLepRmutant rats by inducing M2a and suppressing M1 and suggest that IL-25 may be a therapeutic target for renal disease associated with obesity. NEW & NOTEWORTHY For the past few decades, immune cells and inflammatory cytokines have been demonstrated to play an important role in the development of renal disease. The present study provides strong evidence that interleukin-25 slows the early progression of renal injury in obese Dahl salt-sensitive rats before puberty by increasing systemic anti-inflammatory cytokines and renal M2a macrophages.

Periodontitis deteriorates renal fibrosis and macrophage infiltration in rats with chronic kidney disease.

The objective of this study was to examine the effect of periodontitis on renal function and morphology in rats with or without nephrectomy (Nx)-induced chronic kidney disease (CKD). Rats were divided into sham surgery (Sham), Sham with tooth ligation (ShamL), Nx, and NxL groups. Periodontitis was induced by tooth ligation at 16-week olds. Creatinine, alveolar bone area, and renal histopathology were analyzed at 20-week olds. Creatinine did not differ between the Sham and ShamL groups or between the Nx and NxL groups. The ShamL and NxL groups (both p=0.002) had less alveolar bone area than the Sham group. The NxL group had fewer glomeruli than the Nx group (p < 0.000). The periodontitis groups demonstrated more tubulointerstitial fibrosis (Sham vs. ShamL p=0.002, Nx vs. NxL p < 0.000) and macrophage infiltration (Sham vs. ShamL p=0.002, Nx vs. NxL p=0.006) than the groups without periodontitis. Only the NxL group had greater renal TNFα expression than the Sham group (p < 0.003). These suggest that periodontitis increases renal fibrosis and inflammation in the presence or absence of CKD but does not affect renal function. Periodontitis also increases TNFα expression in the presence of CKD.

Alpha‐kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

BackgroundAlpha‐kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1β in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1β and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined.MethodsImmunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1β, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1β and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD.ResultsThe heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1β in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1β, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA.ConclusionsOur data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Astragaloside IV Targets Macrophages to Alleviate Renal Ischemia-Reperfusion Injury via the Crosstalk between Hif-1α and NF-κB (p65)/Smad7 Pathways.

(1) Background: Astragaloside IV (AS-IV) is derived from Astragalus membranous (AM), which is used to treat kidney disease. Macrophages significantly affect the whole process of renal ischemia-reperfusion (I/R). The regulation of macrophage polarization in kidneys by AS-IV was the focus. (2) Methods: Renal tubular injury and fibrosis in mice were detected by Hematoxylin and Eosin staining and Masson Trichrome Staining, separately. An ELISA and quantitative real-time polymerase chain reaction were used to explore the cytokine and mRNA expression. Western blot was used to determine protein expression and siRNA technology was used to reveal the crosstalk of signal pathways in RAW 264.7 under hypoxia. (3) Results: In the early stages of I/R injury, AS-IV reduced renal damage and macrophage infiltration. M1-associated markers were decreased, while M2 biomarkers were increased. The NF-κB (p65)/Hif-1α pathway was suppressed by AS-IV in M1. Moreover, p65 dominated the expression of Hif-1α. In the late stages of I/R injury, renal fibrosis was alleviated, and M2 infiltration also decreased after AS-IV treatment. Hif-1α expression was reduced by AS-IV, while Smad7 expression was enhanced. Hif-1α interferes with the expression of Smad7 in M2. (4) Conclusions: AS-IV promoted the differentiation of M1 to M2, relieving the proinflammatory response to alleviate the kidney injury during the early stages. AS-IV attenuated M2 macrophage infiltration to prevent kidney fibrosis during the later stages.

Immunosuppressive therapies attenuate paraquat-induced renal dysfunction by suppressing inflammatory responses and lipid peroxidation.

Although paraquat (PQ) induces oxidative damage and inflammatory responses in the lungs, the mechanism underlying PQ-induced acute kidney injury in patients is unclear. Immunosuppressive therapy with glucocorticoids and the immunosuppressant cyclophosphamide (CP) has been employed to treat patients with PQ poisoning. This study examined whether PQ could concurrently cause renal injury, inflammatory responses, and oxidative damage in the kidneys, and whether CP and dexamethasone (DEX) could suppress PQ-induced alterations. Mice were assigned to eight groups: Control, PQ, DEX, PQ plus DEX, CP, PQ plus CP, DEX plus CP, and PQ plus DEX with CP. DEX, CP, and DEX plus CP reversed PQ-induced renal injury, as indicated by urinary albumin-to-creatinine ratios and urea nitrogen levels in serum. The treatments also attenuated PQ-induced renal infiltration of leukocytes and macrophages and induction of the Il6, Tnf, Icam, Cxcl2, Tlr4, and Tlr9 genes encoding the inflammatory mediators in the kidneys. However, DEX only partially suppressed the macrophage infiltration, whereas DEX plus CP provided stronger protection than DEX or CP alone for the induction of Il6 and Cxcl2. Moreover, through the detection of F2-isoprostanes (F2-IsoPs) and isofurans in the kidneys and lungs and F2-IsoPs in the plasma and urine, the therapies were found to suppress PQ-induced lipid peroxidation, although DEX was less effective. Finally, PQ decreased ubiquinol-9:ubiquinone-9 ratios in the kidneys. This effect of PQ was not found under CP treatment, but the ratio was lower than that of the control group. Our findings suggest that the suppression of PQ-induced inflammatory responses by DEX and CP in the kidneys can mitigate oxidative damage and acute kidney injury.

Clopidogrel delays and can reverse diabetic nephropathy pathogenesis in type 2 diabetic db/db mice.

BACKGROUNDDiabetic nephropathy (DN) is the principal cause of end-stage renal disease. Previous studies have shown that clopidogrel can prevent the early progression of renal injury.AIMTo elucidate whether clopidogrel is beneficial against DN by using a db/db mouse model.METHODS db/db mice with a higher urinary albumin/creatinine ratio (ACR) relative to age- and sex-matched wild-type control mice were randomly allocated to clopidogrel and vehicle treatment groups. Clopidogrel was administered at doses of 5, 10, and 20 mg/kg by gavage for 12 wk. Body mass, blood glucose level, and urinary creatinine and albumin concentrations in each group were measured before and after the intervention. Renal fibrosis was evaluated using periodic acid-Schiff and Masson’s trichrome staining. The renal protein expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and F4/80 was assessed using immunohistochemistry. Urinary TNF-α, monocyte chemoattractant protein-1 (MCP-1), and IL-6 levels were analyzed using enzyme-linked immunosorbent assay; TNF-α and IL-1β mRNA expression was measured using real-time quantitative polymerase chain reaction. The protein expression of fibronectin (FN) and collagen I was assessed using immunohistochemistry.RESULTSClopidogrel treatment did not affect the body mass or blood glucose level of the db/db mice; however, it increased bleeding time and reduced urinary ACR in a dose-dependent manner. Immunohistochemical staining revealed an amelioration of renal fibrosis, significantly lower deposition of FN and collagen I, and significantly lower expression of the proinflammatory cytokines TNF-α and IL-1β and lower levels of urinary TNF-α and MCP-1 in the clopidogrel-treated db/db mice (P < 0.05). Furthermore, clopidogrel significantly reduced macrophage infiltration into the glomeruli of the db/db mice.CONCLUSIONClopidogrel significantly reduced renal collagen deposition and fibrosis and prevented renal dysfunction in db/db mice, most likely through inhibition of renal macrophage infiltration and the associated inflammation.

LNA-anti-miR-150 alleviates renal interstitial fibrosis by reducing pro-inflammatory M1/M2 macrophage polarization.

Renal interstitial fibrosis (RIF) is a common pathological feature contributing to chronic injury and maladaptive repair following acute kidney injury. Currently, there is no effective therapy for RIF. We have reported that locked nuclear acid (LNA)-anti-miR-150 antagonizes pro-fibrotic pathways in human renal tubular cells by regulating the suppressor of cytokine signal 1 (SOCS1)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. In the present study, we aimed to clarify whether LNA-anti-miR-150 attenuates folic acid-induced RIF mice by regulating this pathway and by reducing pro-inflammatory M1/M2 macrophage polarization. We found that renal miR-150 was upregulated in folic acid-induced RIF mice at day 30 after injection. LNA-anti-miR-150 alleviated the degree of RIF, as shown by periodic acid–Schiff and Masson staining and by the expression of pro-fibrotic proteins, including alpha-smooth muscle actin and fibronectin. In RIF mice, SOCS1 was downregulated, and p-JAK1 and p-STAT1 were upregulated. LNA-anti-miR-150 reversed the changes in renal SOCS1, p-JAK1, and p-STAT1 expression. In addition, renal infiltration of total macrophages, pro-inflammatory M1 and M2 macrophages as well as their secreted cytokines were increased in RIF mice compared to control mice. Importantly, in folic acid-induced RIF mice, LNA-anti-miR-150 attenuated the renal infiltration of total macrophages and pro-inflammatory subsets, including M1 macrophages expressing CD11c and M2 macrophages expressing CD206. We conclude that the anti-renal fibrotic role of LNA-anti-miR-150 in folic acid-induced RIF mice may be mediated by reducing pro-inflammatory M1 and M2 macrophage polarization via the SOCS1/JAK1/STAT1 pathway.

Effects of Suramin on Polycystic Kidney Disease in a Mouse Model of Polycystin-1 Deficiency.

The aberrant activation of the purinergic signaling pathway has been shown to promote cyst growth and fluid secretion in autosomal dominant polycystic kidney disease (ADPKD). Suramin is an anti-parasitic drug that has strong anti-purinergic properties. Whether suramin could have a therapeutic effect on ADPKD has not been fully investigated. We examined the effect of suramin on cyst progression in a Pkd1 microRNAs transgenic mouse model that presented stable Pkd1 knockdown and moderate disease progression. The Pkd1-deficient mice were treated with suramin (60 mg/kg) by intraperitoneal injection twice a week from postnatal days 35 to 90. Kidney-to-body weight ratios, cyst indices, and blood urea nitrogen (BUN) levels were measured. Cell proliferation and macrophage infiltration were determined by immunohistochemistry. The suramin-treated group had significantly lower renal cyst densities, cell proliferation, and macrophage infiltration compared with saline-treated controls. Suramin significantly inhibited ERK phosphorylation and the expression of Il1b, Il6, Nlrp3, Tgfb, Fn1, P2rx7, and P2ry2 mRNAs in the kidneys. However, BUN levels remained high despite the reduction in cyst growth. Furthermore, plasma cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels were significantly higher in the suramin-treated group compared with the control group. Periodic acid-Schiff staining revealed degenerative changes and epithelial cell vacuolation in the non-cystic renal tubules, which indicated phospholipidosis following suramin treatment. These results suggest that suramin may reduce renal cyst growth and inflammation, but the associated tubular cell injuries could limit its therapeutic potential. Other purinergic receptor antagonists with less nephrotoxicity may deserve further investigation for the treatment of ADPKD.

TGF-β1 peptide-based inhibitor P144 ameliorates renal fibrosis after ischemia-reperfusion injury by modulating alternatively activated macrophages.

ObjectivesIschemia–reperfusion injury (IRI) is a major cause of chronic renal fibrosis. Currently, numerous therapies have shown a minimal effect on the blockade of fibrosis progression. Here, the therapeutic potential of peptide‐based TGF‐β1 inhibitor P144 in IRI‐induced renal fibrosis and the underlying mechanism were analyzed.Materials and MethodsThe unilateral ischemia–reperfusion injury with the contralateral nephrectomy model was established, and the P144 was administered intravenously 1d/14d after the onset of IRI. The histopathology and immunofluorescence staining were used to detect renal fibrosis and macrophage infiltration. The in vivo fluorescence imaging was used to measure the bio‐distribution of P144. The transwell assays were used to observe the migration of macrophages. RT‐qPCR and western blot were used to analyze TGF‐β1 signaling.ResultsP144 ameliorated the accumulation of extracellular matrix in the kidney and improved the renal function in the unilateral ischemia–reperfusion injury plus contralateral nephrectomy model. Mechanistically, P144 downregulated the TGF‐β1‐Smad3 signaling at both the transcriptional and translational levels and further reduced the TGF‐β1‐dependent infiltration of macrophages to the injured kidney. Additionally, P144 blocked the polarization of macrophages to an M2‐like phenotype induced by TGF‐β1 in vitro, but showed no effect on their proliferation.ConclusionsOur study showed that the TGF‐β1 peptide‐based inhibitor P144 decreased renal fibrosis through the blockade of the TGF‐β1–Smad3 signaling pathway and the modulation of macrophage polarization, suggesting its potential therapeutic use in IRI‐induced renal fibrosis.

Modified chitosan for effective renal delivery of siRNA to treat acute kidney injury

Acute kidney injury (AKI) is characterized by a sudden decrease in renal function and impacts growing number of people worldwide. RNA interference (RNAi) showed potential to treat diseases with no or limited conventional therapies, including AKI. Suitable carriers are needed to protect and selectively deliver RNAi to target cells to fully explore this therapeutic modality. Here, we report on the synthesis of chitosan modified with α-cyclam-p-toluic acid (C-CS) as a novel siRNA carrier for targeted delivery to injured kidneys. We demonstrate that conjugation of the α-cyclam-p-toluic acid to chitosan imparts the C-CS polymer with targeting and antagonistic properties to cells overexpressing chemokine receptor CXCR4. In contrast, the parent α-cyclam-p-toluic acid showed no such properties. Self-assembled C-CS/siRNA nanoparticles rapidly accumulate in the injured kidneys and show long retention in renal tubules. Apoptosis and metabolic and inflammatory pathways induced by p53 are important pathological mechanisms in the development of AKI. Nanoparticles with siRNA against p53 (sip53) were formulated and intravenously injected for attenuation of IRI-AKI. Due to the favorable accumulation in injured kidneys, the treatment with C-CS/sip53 decreased renal injury, extent of renal apoptosis, macrophage and neutrophil infiltration, and improved renal function. Overall, our study suggests that C-CS/siRNA nanoparticles have the potential to effectively accumulate and deliver therapeutic siRNAs to injured kidneys through CXCR4 binding, providing a novel way for AKI therapy.

Decreasing Insulin Resistance Reduces Early Progressive Proteinuria by Decreasing Renal Hyperfiltration and Inflammation in Obese Dahl Salt‐Sensitive Rats

Prepubertal/childhood obesity (PPO) is quickly becoming an epidemic and is considered a major risk factor for proteinuria. Previous studies have demonstrated that insulin resistance alters renal hemodynamics leading to inflammation and renal injury in obese adults. However, studies examining the relationship between insulin resistance and renal hyperfiltration (RH) and inflammation in obese children are lacking. Recently, we reported that insulin resistance in the obese Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) rat is associated with progressive proteinuria and RH prior to puberty. Therefore, the aim of the present study was to examine whether decreasing insulin resistance with metformin will reduce proteinuria in SSLepRmutant rats. Four‐week‐old SS and SSLepRmutant rats were separated into 4 groups (n=5/group) and treated with either vehicle (PBS) or metformin (300 mg/kg/day, ip) for 4 weeks. Blood glucose levels were similar among the groups throughout the study (≤ 120 mg/dL). However, at the end of study, plasma insulin levels were significantly elevated in SSLepRmutant rats vs SS rats (9±2 vs 1±0.2 ng/ml respectively; p&lt;0.05), and glucose tolerance was significantly impaired in SSLepRmutant rats compared to values measured in SS rats. Chronic treatment with metformin markedly reduced plasma insulin (2±1 ng/ml; p&lt;0.05) and improved glucose tolerance in SSLepRmutant rats. At baseline, proteinuria was significantly higher in SSLepRmutant rats vs SS rats and remained elevated throughout the study (449±123 vs 25±7 mg/day, respectively; p&lt;0.05). Metformin treatment significantly decreased proteinuria in SSLepRmutant rats (177±80 mg/day; p&lt;0.05) with no effect in SS rats (36±13 mg/day). We observed a significant increase in creatinine clearance (CCr) in SSLepRmutant rats vs SS rats indicating RH (3.3±0.4 ml/min vs 1.9±0.1 ml/min, respectively; p&lt;0.05). Chronic treatment with metformin significantly decreased CCr in SSLepRmutant rats (1.3±0.2 ml/min; p&lt;0.05) while not affecting SS rats. Renal histology revealed marked glomerular injury in vehicle‐treated SSLepRmutant compared to SS rats, and treatment with metformin reduced glomerular injury in SSLepRmutant rats. We observed a significant increase in the renal infiltration of stimulatory dendritic cells and macrophages in vehicle‐treated SSLepRmutant rats compared to their SS counterparts, and metformin reduced the infiltration of these immune cells only in SSLepRmutant rats. MIP3α (pro‐inflammatory cytokine) was significantly increased and IL‐4 (anti‐inflammatory cytokine) was markedly decreased in the kidneys of SSLepRmutant rats compared to their SS counterparts (23±3 vs 5±1 ng/mg and 11±2 vs 54±5 ng/mg, respectively; p&lt;0.05). Treatment with metformin decreased renal MIP3α (10±2 ng/mg; p&lt;0.05), while increasing renal IL‐4 (26±5 ng/mg) in SSLepRmutant rats. These data suggest that decreasing insulin resistance reduces RH, proteinuria, and inflammation and may be therapeutically useful in managing renal injury during PPO.

Epigenetic activation of RBM15 promotes clear cell renal cell carcinoma growth, metastasis and macrophage infiltration by regulating the m6A modification of CXCL11

Clear cell renal cell carcinoma (ccRCC) is a common kidney malignancy that is characterized by poor prognosis. RNA-binding motif protein 15 (RBM15) has been identified as an oncogene in multiple tumors. Nevertheless, the function and mechanism of RBM15 in ccRCC are not clear. In this study, RBM15 was found to be upregulated in ccRCC cells and tissues. RBM15 enhanced the proliferation, clone formation, migration, invasion and epithelial-interstitial transition of ccRCC cells. Enhanced RBM15 was caused by the abundant histone 3 acetylation modification of the RBM15 promoter induced by EP300/CBP. RBM15 enhanced the stability of CXCL11 mRNA in an m6A-dependent manner. Moreover, RBM15 was found to promote macrophage infiltration and M2 polarization by promoting the secretion of CXCL11 in ccRCC cells in vitro and in vivo. Our findings highlight the function of RBM15 in ccRCC and reveal a novel identified EP300/CBP-RBM15-CXCL11 signaling axis, which promotes ccRCC progression and provides new insight into ccRCC therapy.