Renal Inflammatory Cell Infiltration Research Articles

Dexmedetomidine inhibits ferroptosis and attenuates sepsis-induced acute kidney injury via activating the Nrf2/SLC7A11/FSP1/CoQ10 pathway

ABSTRACT Objectives The molecular mechanism underlying the protective effects of DEX against sepsis-induced acute kidney injury (SAKI) remains to be elucidated. Methods We established S-AKI models in vivo via CLP and in vitro with LPS-induced HK-2 cells. Results The Nrf2/SLC7A11/FSP1/CoQ10 pathway was inhibited in S-AKI both in vitro and in vivo. The overexpression of Nrf2 inhibited LPS-induced ferroptosis by activating the SLC7A11/FSP1/CoQ10 pathway. DEX ameliorated kidney tissue damage, as determined by a decrease in BUN, Cr, and inflammatory factor levels, along with renal tubule vacuolation and inflammatory cell infiltration in S-AKI mice. Additionally, DEX treatment significantly ameliorated ferroptosis in S-AKI in vitro and in vivo, as indicated by an improvement in mitochondrial shrinkage and disruption of cristae, a decrease in iron, ROS, MDA, and 4-HNE levels, and an increase in GSH and GPX4 levels. Mechanistically, DEX treatment restored the reduction of Nrf2 expression and nuclear translocation in S-AKI, as well as, the levels of downstream SLC7A11, FSP1, and CoQ10. Knocking down Nrf2 in vitro and administering brusatol in vivo eliminated the protective effect of DEX against S-AKI. Conclusions DEX mitigated ferroptosis and attenuated S-AKI by activating the Nrf2/SLC7A11/FSP1/CoQ10 pathway. Abbreviation: CLP: Cecal ligation puncture; LPS: Lipopolysaccharide; Nrf2: Nuclear factor-erythroid- 2-related factor 2; SLC7A11: Solute carrier family 7 member 11; FSP1: Ferroptosis suppressor protein 1; CoQ10: Coenzyme Q10; BUN: Blood urea nitrogen; Cr: Serum creatinine; ROS: Reactive oxygen species; MDA: Malondialdehyde; 4-HNE: 4-hydroxynonenal; GSH: Hlutathione; GPX4: Glutathione peroxidase 4.

Protective effect of Huashi Baidu formula against AKI and active ingredients that target SphK1 and PAI-1

BackgroundHuashi Baidu Formula (HBF) is a clinical formula known for its efficacy against coronavirus disease 2019 (COVID-19). HBF may reduce the number of patients with abnormal serum creatinine while improving respiratory symptoms, suggesting that this formula may have potential for treating acute kidney injury (AKI). However, the protective effect of HBF on AKI has not been definitively confirmed, and the mechanism remains unclear. Therefore, the present study explored the renoprotective effects and molecular mechanisms of HBF and screened for its active ingredients to identify new potential applications of renoprotection by HBF.MethodsThe present study first assessed the protective effects of HBF on AKI in a DOX-induced mouse model. Then, RNA-seq and bioinformatics analyses were used to explore the related pathological processes and potential molecular mechanisms, which were subsequently validated using qRT-PCR and Western blotting. Furthermore, candidate compounds with potential binding affinity to two pivotal targets, sphingosine kinase 1 (SphK1) and plasminogen activator inhibitor-1 (PAI-1), were screened from the 29 constituents present in the blood using Microscale Thermophoresis (MST). Finally, to identify the active ingredients, the candidate components were re-screened using the SphK1 kinase activity detection system or the uPA/PAI-1 substrate colorimetric assay system.ResultsIn the DOX-induced AKI mouse model, therapeutic administration of HBF significantly reduced the levels of CRE, BUN, TNF-α, IL-1β, IL-6, and UA in plasma and the levels of MDA, T-CHO, and TG in kidney tissue. Additionally, the levels of TP and Alb in plasma and SOD and CAT in the kidney tissue were significantly increased. Histopathological assessment revealed that HBF reduced tubular vacuolation, renal interstitial inflammatory cell infiltration, tubular atrophy, and positive staining of renal interstitial collagen. RNA-seq and bioinformatics analyses showed that oxidative stress, the immune-inflammatory response, and extracellular matrix (ECM) formation could be the pathological processes that HBF targets to exerts its renoprotective effects. Furthermore, HBF regulated the APJ/SPHK1/NF-κB and APJ/PAI-1/TGFβ signaling axes and reduced the phosphorylation levels of NF-κB p65 and SMAD2 and the expression of cytokines and the ECM downstream of the axis. Finally, six SphK1 inhibitors (paeoniflorin, astragalin, emodin, glycyrrhisoflavone, quercetin, and liquiritigenin) and three PAI-1 inhibitors (glycyrrhisoflavone, licochalcone B, and isoliquiritigenin) were identified as potentially active ingredients in HBF.ConclusionIn brief, our investigation underscores the renoprotective effect of HBF in a DOX-induced AKI model mice, elucidating its mechanisms through distinct pathological processes and identifying key bioactive compounds. These findings offer new insights for broadening the clinical applications of HBF and unravelling its molecular mode of action.

Transcriptomic Revealed That Selenium-Rich Lactobacillus plantarum Alleviated Cadmium-Induced Immune Responses in Bulatmai barbel Luciobarbus capito Kidneys

Cadmium (Cd) is a common environmental pollutant that accumulates mainly in the kidneys and thus endangers the physiological health of aquatic animals. Selenium (Se) is a natural antidote to heavy metals that antagonises heavy metal toxicity and enhances the antioxidant capacity of organisms. Lactobacillus plantarum (L. plantarum) can reduce the toxicity of heavy metals through adsorption, reduction and metabolism. Studies have confirmed that the biological synthesis of Se nanoparticles (Bio-SeNPs) using bacterial microorganisms is simple, safe and less toxic than the synthesis of inorganic and organic Se, but the effect on Cd-induced immunosuppression is un-known. One hundred and eighty Bulatmai barbel (Luciobarbus capito: L. capito) plants were randomly divided into control (C), Cd and Cd + Se-enriched L. plantarum groups (S1L1-Cd) and fed for 28 days. The analysis methods included histopathology, test kits, transcriptomics and real-time quantitative PCR. The addition of selenium-enriched L. plantarum significantly attenuated cadmium-induced pathological changes such as glomerular atrophy, detachment of renal tubular epithelial cells, mild swelling, and interstitial inflammatory cell infiltration. Cd stress can lead to significant decreases in RBC, HCT, WBC, LZM, C3, and IgM levels, and the addition of Se-enriched L. plantarum can significantly reverse the changes in these indicators. Transcriptomic analysis revealed 488 DEGs in the Cd groups, 301 of which were upregulated and 187 of which were downregulated. There were 1474 DEGs in the S1L1-Cd group, of which 720 were upregulated and 754 were downregulated. In addition, GO enrichment analysis revealed that the biological regulation of the most differentially expressed genes involved metal ion binding, ATP binding and nucleotide inclusion. KEGG enrichment analysis revealed six of the most enriched pathways: oxidative phosphorylation, Huntington disease, retrograde endocannabinoid signalling, natural killer cell-mediated cyto-toxicity, the IL-17 signalling pathway, and leukocyte transient migration. Moreover, we selected 12 DEGs for qRT-PCR, which showed that the qRT-PCR results were consistent with our RNA-Seq results. Our results suggest that Se-enriched L. plantarum can enhance immunity and alleviate Cd exposure-mediated immunosuppression in L. capito.

Protective effect of olive leaf (Olea europaea L.) extract against chronic exposure of liver and kidney tissues of Wistar rats to aluminum chloride

Introduction: The liver and kidney are the main sites of aluminum (Al) accumulation. Lifetime exposure to significant amounts of Al is inevitable, hence its toxicity on the liver and kidney should be a health concern. Natural antioxidants have been proven to alleviate pathologies in various liver and kidney injuries. However, the effect of olive leaf extract (OLE) on Al-exposed animals is yet to be confirmed. This study aimed to investigate the OLE effect against AlCl3 chronic exposure in rats’ liver and kidneys. Methods:Thirty-two male Wistar rats were divided into four groups (n=8), including the control group, the AlCl3 group treated with 128 mg/kg AlCl3 solution, as well as AlCl3 +OLE50, and AlCl3 +OLE100 groups (Other than AlCl3 they received 50 and 100 mg/kg of OLE, respectively, 2 hours after AlCl3 administration). All treatments were given orally for 12 weeks. All groups were evaluated for liver and kidney histopathological features, then scoring was performed. Results: AlCl3 administrations produced histopathological lesions in the liver and kidney, indicated by increased liver necro-inflammatory grades, ballooning scores, and renal inflammatory cell infiltration (P<0.05). OLE100 mg/kg significantly reduced liver necroinflammatory grade, ballooning score, and kidney inflammatory cell infiltrations. The dose of 50 mg/kg also reduced these parameters (P<0.05), except for the liver necro-inflammatory grade. There was a significant correlation between OLE dose and liver necro-inflammatory grade and ballooning score amelioration. Conclusion: OLE ameliorates liver and kidney histopathological features induced by oral Al chronic exposure in a dose-dependent manner.

Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway

BackgroundPyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI.Methods and resultsThe expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1β production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis.ConclusionOur results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.Graphical We found that the expression of ACSS2 was significantly increased in RTECs in septic AKI. ACSS2-deficient mice displayed resistance to renal damage in this model. ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the KLF5/NF-κB pathway in RTECs. Pharmacological inhibition of KLF5 suppressed NLRP3 activation and caspase-1 cleavage by downregulating of NF-κB. Our results suggest that ACSS2 is a pro-pathogenic mediator of cell pyroptosis of RTECs and renal inflammation in sepsis-induced AKI.

The dysfunction in intestinal microorganisms and enzyme activity as significant contributors to diarrhea with kidney-yang deficiency syndrome.

To investigate the pathogenesis of diarrhea with kidney-yang deficiency syndrome by examining characteristic changes in intestinal microorganisms, enzyme activities, oxidative stress, and metabolism indices. Twenty mice were randomly and equally divided into control group (NC) and model group (NM). Mice in NM group received adenine suspension at a dosage of 50 mg/(kg⋅day) by gavage, 0.4 mL/time, once a day for 14 days, and Folium sennae decoction at a dosage of 10 g/(kg⋅day) by gavage, 0.4 mL/time, once a day for 7 days, starting on 8th day. Mice in NC group were administered an equivalent amount of sterile water by gavage once a day for 7 days, and twice a day from the 8th day. After modeling, assessments encompassed microbial culture, organ index calculation, microbial and enzyme activity detection, malondialdehyde (MDA) content determination, superoxide dismutase (SOD) activity, blood biochemical tests, and observation of kidney tissue pathological changes. The results showed that in NM group, a reduction in the number of Lactobacillus and Bifidobacteria was noted, accompanied by an increase in the number of bacteria and E. coli. Xylanase activity in the intestinal contents and mucosa, protease activity in the intestinal mucosa, and intestinal mucosa microbial activity were diminished. Conversely, the activities of amylase, sucrase, and lactase increased in intestinal mucosa. Additionally, there was an elevation in the level of MDA. Renal tubular dilatation and inflammatory cell infiltration were observed in the renal interstitium. These dysfunctions in intestinal microorganisms and enzyme activities suggest potential involvement in diarrhea with kidney-yang deficiency syndrome.

Role of the transcription factor Fli-1 on the CXCL10/CXCR3 Axis.

The transcription factor Fli-1, a member of the ETS family of transcription factors, is implicated in the pathogenesis of lupus disease. Reduced Fli-1 expression in lupus mice leads to decreased renal Cxcl10 mRNA levels and renal infiltrating CXCR3+ T cells that parallels reduced renal inflammatory cell infiltration and renal damage. Inflammatory chemokine CXCL10 is critical for attracting inflammatory cells expressing the chemokine receptor CXCR3. The CXCL10/CXCR3 axis plays a role in the pathogenesis of various inflammatory diseases including lupus. Our data here demonstrate that renal CXCL10 protein levels are significantly lower in Fli-1 heterozygous MRL/lpr mice compared to wild-type MRL/lpr mice. Knockdown of Fli-1 significantly reduced CXCL10 secretion in mouse and human endothelial cells, and human mesangial cells, upon LPS or TNFα stimulation. The Fli-1 inhibitor, Camptothecin, significantly reduced CXCL10 production in human monocyte cells upon interferon stimulation. Four putative Ets binding sites in the Cxcl10 promoter showed significant enrichment for FLI-1; however, FLI-1 did not directly drive transcription from the human or mouse promoters, suggesting FLI-1 may regulate CXCL10 expression indirectly. Our results also suggest that the DNA binding domain of FLI-1 is necessary for regulation of human hCXCR3 promotor activity in human T cells and interactions with co-activators. Together, these results support a role for FLI-1 in modulating the CXCL10-CXCR3 axis by directly or indirectly regulating the expression of both genes to impact lupus disease development. Signaling pathways or drugs that reduce FLI-1 expression may offer novel approaches to lupus treatment.

Dispelling Dampness, Relieving Turbidity and Dredging Collaterals Decoction, Attenuates Potassium Oxonate-Induced Hyperuricemia in Rat Models.

Dispelling dampness, relieving turbidity and dredging collaterals decoction (DED), is a traditional Chinese medicine used in the treatment of hyperuricemia. We aimed to explore the effect and mechanism of DED in the treatment of hyperuricemia. The effects of DED (9.48, 4.74, and 2.37 g/kg/d) on potassium oxonate (750 mg/kg/d)-induced hyperuricemia in rats were evaluated by serum uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), and renal pathological changes. Network pharmacology was used to identify the effective components and targets of DED, and the key targets and signaling pathways for its effects on hyperuricemia were screened. Molecular docking was used to predict the action of DED. H&E, immunohistochemistry, WB, and PCR were used to validate the network pharmacology results. DED can effectively alleviate hyperuricemia, inhibit UA, CRE, BUN, and xanthine oxidase (XOD) activity, and reduce renal inflammatory cell infiltration and glomerular atrophy. The experiment identified 27 potential targets of DED for hyperuricemia, involving 9 components: wogonin, stigmasterol 3-O-beta-D-glucopyranoside, 3β-acetoxyatractylone, beta-sitosterol, stigmasterol, diosgenin, naringenin, astilbin, and quercetin. DED can relieve hyperuricemia mainly by inhibiting RAGE, HMGB1, IL17R, and phospho-TAK1, and by regulating the AGE-RAGE and IL-17 signaling pathways. DED can alleviate hyperuricemia by inhibiting XOD activity and suppressing renal cell apoptosis and inflammation via the AGE-RAGE signaling pathway and IL-17 signaling pathway. This study provides a theoretical basis for the clinical application of DED.

CCN2 Activates RIPK3, NLRP3 Inflammasome, and NRF2/Oxidative Pathways Linked to Kidney Inflammation.

Inflammation is a key characteristic of both acute and chronic kidney diseases. Preclinical data suggest the involvement of the NLRP3/Inflammasome, receptor-interacting protein kinase-3 (RIPK3), and NRF2/oxidative pathways in the regulation of kidney inflammation. Cellular communication network factor 2 (CCN2, also called CTGF in the past) is an established fibrotic biomarker and a well-known mediator of kidney damage. CCN2 was shown to be involved in kidney damage through the regulation of proinflammatory and profibrotic responses. However, to date, the potential role of the NLRP3/RIPK3/NRF2 pathways in CCN2 actions has not been evaluated. In experimental acute kidney injury induced with folic acid in mice, CCN2 deficiency diminished renal inflammatory cell infiltration (monocytes/macrophages and T lymphocytes) as well as the upregulation of proinflammatory genes and the activation of NLRP3/Inflammasome-related components and specific cytokine products, such as IL-1β. Moreover, the NRF2/oxidative pathway was deregulated. Systemic administration of CCN2 to C57BL/6 mice induced kidney immune cell infiltration and activated the NLRP3 pathway. RIPK3 deficiency diminished the CCN2-induced renal upregulation of proinflammatory mediators and prevented NLRP3 modulation. These data suggest that CCN2 plays a fundamental role in sterile inflammation and acute kidney injury by modulating the RIKP3/NLRP3/NRF2 inflammatory pathways.

#3581 ACSS2-INDUCED FATTY ACID SYNTHESIS PROMOTES NLRP3 INFLAMMASOME AND PYROPTOSIS OF RENAL TUBULAR CELLS IN SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Cellular fatty acid metabolism was supposed to be tightly associated with immune responses. Pyroptosis is a form of programmed cell death dependent on the NLRP3 inflammasome activation. Previous studies have revealed that acyl-CoA synthetase 2 (ACSS2) promoted fatty acid synthesis under metabolic stress. However, whether ACSS2-mediated fatty acid metabolism can regulate pyroptosis and inflammatory responses of renal tubular cells in AKI remain unclear. Method lipopolysaccharide (LPS) was intraperitoneally injected to establish the sepsis mouse model, and in vitro HK-2 cell culture model was achieved by LPS stimulation. HE and PAS staining were carried out to evaluate pathological injury of kidneys in mice. The renal tissue immunostaining was conducted to detect IL-1β expression and macrophage distribution in kidney tissues of mice. Gene expression of inflammatory factors was evaluated by real-time PCR. Western Blot was conducted to the activation of the NLRP3 pathway and pyroptosis in vivo and in vitro. Results Here, we demonstrated that the expression of ACSS2 was significantly increased in the renal epithelial cells of mice with Lipopolysaccharide (LPS)-induced AKI when compared to wild-type mice. ACSS2 regulates NLRP3-mediated caspase-1 activation and IL-1β production through the stimulation of fatty acid synthase (FASN) in renal epithelial cells. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model. Consistently, ACSS2-deficient mice displayed impaired FASN-mediated lipid synthesis and decreased IL-1β production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and decreased fatty acid synthesis through the downregulation of FASN. The treatment with the chemical inhibitor C75 suppressed NLRP3-mediated caspase-1 activation and pyroptosis of HK-2 cells under LPS treatment in renal tubular cells. Conclusion Our results suggested that ACSS2 regulated the NLRP3 inflammasome activation and pyroptosis by inducing the FASN-mediated fatty acid synthesis pathway in renal epithelial cells. These results identified ACSS2 as a potential therapeutic target in AKI.

Sivelestat protects acute kidney injury by inhibiting the PI3K/AKT pathway in septic rats

To explore the protective effect of sivelestat (SV) against sepsis-induced acute kidney injury (AKI) and its molecular mechanism. According to the random number table method, 64 male Wistar rats were divided into sham operation group (Sham group), sepsis due to cecal ligation and puncture group (CLP group), low dose of SV treatment group (SL group, 50 mg/kg SV was injected into the tail vein at 12 hours and 24 hours after CLP), and high dose of SV treatment group (SH group, 100 mg/kg SV was injected into the tail vein at 12 hours and 24 hours after CLP), with 16 rats in each group. 48 hours after CLP, the 48-hour survival of rats were recorded, all rats were sacrificed and samples were harvested. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of kidney injury molecule-1 (KIM-1), interleukins (IL-1β, IL-6), tumor necrosis factor-α (TNF-α) and neutrophil elastase (NE). Hematoxylin-eosin (HE) staining was used to observe histopathological changes and assess renal tubule injury score. Masson staining was used to detect the collagen volume fraction (CVF) of kidney tissue. Western blotting was used to detect the protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphorylation PI3K (p-PI3K), protein kinase B (AKT), phosphorylation AKT (p-AKT), nuclear factor-κB p65 (NF-κB p65) and NE. The protein expressions of p-PI3K, p-AKT, NF-κB p65 were detected by immunohistochemistry. Compared with Sham group, the 48-hour survival rate of CLP group was significantly reduced. Histopathological results showed that large tubular epithelial cells and brush margins were shed, tubular casts were formed, some tubular atrophy, glomerular hyperemia, renal interstitial inflammatory cell infiltration and increased renal tubular injury score. Renal interstitial fibrosis was obvious and CVF increased. The levels of KIM-1, IL-1β, IL-6, TNF-α and NE in serum were significantly elevated in the CLP group. The proteins expression of inflammatory pathway-related p-PI3K/PI3K, p-AKT/AKT, NF-κB p65 and NE were significantly increased in kidney tissue. It suggested that septic rats had renal injury and the PI3K/AKT inflammatory pathway was activated. Compared with CLP group, there was no significant difference in 48-hour survival in SL group and SH group (68.75%, 75.00% vs. 56.25%, both P > 0.05), but kidney injury was significantly relieved. Specifically: renal tubular injury score and CVF significantly decreased [tubular injury score: 2 (1, 2), 1 (1, 1) vs. 2 (2, 3); CVF: (22.36±0.86)%, (18.74±1.05)% vs. (58.38±0.79)%, all P < 0.05]; the serum levels of KIM-1, IL-1β, IL-6, TNF-α and NE also decreased significantly [KIM-1 (ng/L): 145.03±8.88, 117.58±7.02 vs. 158.22±12.00; IL-1β (ng/L): 108.32±9.00, 92.98±8.06 vs. 133.78±8.48; IL-6 (ng/L): 124.33±10.11, 115.42±8.17 vs. 165.19±5.70; TNF-α (ng/L): 321.56±19.29, 289.68±21.57 vs. 424.88±22.76, NE (mol/L): 93.84±9.14, 75.01±10.56 vs. 113.45±6.39, all P < 0.05]; the proteins expression of inflammatory pathway-related p-PI3K/PI3K, p-AKT/AKT, NF-κB p65 and NE were significantly decreased (p-PI3K/PI3K: 0.93±0.06, 0.67±0.04 vs. 1.27±0.08; p-AKT/AKT: 0.78±0.09, 0.47±0.05 vs. 0.96±0.12; NF-κB p65/GAPDH: 1.43±0.13, 0.85±0.08 vs. 1.88±0.17; NE/GAPDH: 1.45±0.06, 0.91±0.04 vs. 1.71±0.08, all P < 0.05), the positive expressions of p-PI3K, p-AKT and NF-κB p65 in kidney tissue were decreased [p-PI3K positive expression area: (13.36±1.84)%, (8.03±1.12)% vs. (21.56±1.20)%; p-AKT positive expression area: (21.57±0.91)%, (15.21±2.76)% vs. (30.81±2.12)%; NF-κB p65 positive expression area: (25.17±1.38)%, (17.07±2.11)% vs. (37.85±2.50)%, all P < 0.05]. Serum inflammatory factor level, and PI3K/AKT pathway related protein, NF-κB p65, NE protein expression level and p-PI3K, p-AKT, NF-κB p65 positive area and other indicators in renal tissue in SH group were further lower than those in SL group (all P < 0.05). SV can ameliorate sepsis-induced AKI. The mechanism may be related to the inhibition of PI3K/AKT pathway, and high dose of SV has better efficacy.

Macrophage promotes fibroblast activation and kidney fibrosis by assembling a vitronectin-enriched microenvironment.

Background: Renal infiltration of inflammatory cells including macrophages is a crucial event in kidney fibrogenesis. However, how macrophage regulates fibroblast activation in the fibrotic kidney remains elusive. In this study, we show that macrophages promoted fibroblast activation by assembling a vitronectin (Vtn)-enriched, extracellular microenvironment. Methods: We prepared decellularized kidney tissue scaffold (KTS) from normal and fibrotic kidney after unilateral ischemia-reperfusion injury (UIRI) and carried out an unbiased quantitative proteomics analysis. NRK-49F cells were seeded on macrophage-derived extracellular matrix (ECM) scaffold. Genetic Vtn knockout (Vtn-/-) mice and chronic kidney disease (CKD) model with overexpression of Vtn were used to corroborate a role of Vtn/integrin αvβ5/Src in kidney fibrosis. Results: Vtn was identified as one of the most upregulated proteins in the decellularized kidney tissue scaffold from fibrotic kidney by mass spectrometry. Furthermore, Vtn was upregulated in the kidney of mouse models of CKD and primarily expressed and secreted by activated macrophages. Urinary Vtn levels were elevated in CKD patients and inversely correlated with kidney function. Genetic ablation or knockdown of Vtn protected mice from developing kidney fibrosis after injury. Conversely, overexpression of Vtn exacerbated renal fibrotic lesions and aggravated renal insufficiency. We found that macrophage-derived, Vtn-enriched extracellular matrix scaffold promoted fibroblast activation and proliferation. In vitro, Vtn triggered fibroblast activation by stimulating integrin αvβ5 and Src kinase signaling. Either blockade of αvβ5 with neutralizing antibody or pharmacological inhibition of Src by Saracatinib abolished Vtn-induced fibroblast activation. Moreover, Saracatinib dose-dependently ameliorated Vtn-induced kidney fibrosis in vivo. These results demonstrate that macrophage induces fibroblast activation by assembling a Vtn-enriched extracellular microenvironment, which triggers integrin αvβ5 and Src kinase signaling. Conclusion: Our findings uncover a novel mechanism by which macrophages contribute to kidney fibrosis via assembling a Vtn-enriched extracellular niche and suggest that disrupting fibrogenic microenvironment could be a therapeutic strategy for fibrotic CKD.

Acteoside Alleviates Renal Fibrosis by Inhibiting β-Catenin/CTGF Signaling Pathway in UUO Rats

Objective: Acteoside (ACT) has been reported to regulate the inflammation and immune response. The study aims to explore the effect of ACT on renal fibrosis in unilateral ureteral obstruction (UUO) rats. Methods: Eighteen Sprague-Dawley rats were randomly divided into 3 groups: sham group, opened the abdominal cavity and sutured abdominal; UUO group, performed UUO surgery; and ACT + UUO group, ACT (40 mg/kg) was given by gavage every day after UUO surgery. After 2 weeks of rat model construction, urine and blood samples were collected for biochemical analysis, while kidney tissues were harvested for hematoxylin and eosin (H&amp;E), Masson's trichrome, and immunohistochemistry staining. The expression of connective tissue growth factor (CTGF), alpha smooth muscle actin (α-SMA), collagen III, heat shock protein 47 (HSP47), and β-catenin in the renal tissue was detected and the correlation between these proteins was analyzed. Results: ACT improved the parameters of renal function in UUO rats, including decreased creatinine and urea nitrogen, and declined urinary protein. Pathological analysis suggested that ACT improved the conditions of renal tubule lesion (including structure destruction, atrophy and lumen obstruction), renal interstitial fibrosis and inflammatory cell infiltration in UUO rats. It also down-regulated the expressions of fibrin-related proteins β-catenin, CTGF, α-SMA, collagen III, and HSP47. Correlation analysis found that β-catenin and CTGF were correlated with the expressions of α-SMA, collagen III, and HSP47. Conclusions: ACT could alleviate renal fibrosis in UUO rats probably via inhibiting β-catenin/CTGF signaling pathway.

In Vivo Inhibition of TRPC6 by SH045 Attenuates Renal Fibrosis in a New Zealand Obese (NZO) Mouse Model of Metabolic Syndrome.

Metabolic syndrome is a significant worldwide public health challenge and is inextricably linked to adverse renal and cardiovascular outcomes. The inhibition of the transient receptor potential cation channel subfamily C member 6 (TRPC6) has been found to ameliorate renal outcomes in the unilateral ureteral obstruction (UUO) of accelerated renal fibrosis. Therefore, the pharmacological inhibition of TPRC6 could be a promising therapeutic intervention in the progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome. In the present study, we hypothesized that the novel selective TRPC6 inhibitor SH045 (larixyl N-methylcarbamate) ameliorates UUO-accelerated renal fibrosis in a New Zealand obese (NZO) mouse model, which is a polygenic model of metabolic syndrome. The in vivo inhibition of TRPC6 by SH045 markedly decreased the mRNA expression of pro-fibrotic markers (Col1α1, Col3α1, Col4α1, Acta2, Ccn2, Fn1) and chemokines (Cxcl1, Ccl5, Ccr2) in UUO kidneys of NZO mice compared to kidneys of vehicle-treated animals. Renal expressions of intercellular adhesion molecule 1 (ICAM-1) and α-smooth muscle actin (α-SMA) were diminished in SH045- versus vehicle-treated UUO mice. Furthermore, renal inflammatory cell infiltration (F4/80+ and CD4+) and tubulointerstitial fibrosis (Sirius red and fibronectin staining) were ameliorated in SH045-treated NZO mice. We conclude that the pharmacological inhibition of TRPC6 might be a promising antifibrotic therapeutic method to treat progressive tubulo-interstitial fibrosis in hypertension and metabolic syndrome.

Sirtuin 7 Deficiency Reduces Inflammation and Tubular Damage Induced by an Episode of Acute Kidney Injury.

Acute kidney injury (AKI) is a public health problem worldwide. Sirtuins are a family of seven NAD+-dependent deacylases, Overexpression of Sirtuin 1, 3, and 5 protect against AKI. However, the role of Sirtuin 7 (Sirt7) in AKI is not known. Here, we analyzed how Sirt7 deficient mice (KO-Sirt7) were affected by AKI. As expected, wild-type and Sirt7 heterozygotes mice that underwent renal ischemia/reperfusion (IR) exhibited the characteristic hallmarks of AKI: renal dysfunction, tubular damage, albuminuria, increased oxidative stress, and renal inflammation. In contrast, the KO-Sirt7+IR mice were protected from AKI, exhibiting lesser albuminuria and reduction in urinary biomarkers of tubular damage, despite similar renal dysfunction. The renoprotection in the Sirt7-KO+IR group was associated with reduced kidney weight, minor expression of inflammatory cytokines and less renal infiltration of inflammatory cells. This anti-inflammatory effect was related to diminished p65 expression and in its active phosphorylation, as well as by a reduction in p65 nuclear translocation. Sirt7 deficient mice are protected from AKI, suggesting that this histone deacetylase promotes tubular damage and renal inflammation. Therefore, our findings indicate that Sirt7 inhibitors may be an attractive therapeutic target to reduce NFκB signaling.

Effect of electroacupuncture on renal function and expression of autophagy-related proteins in kidney of rats with diabetic nephropathy

To observe the effect of electroacupuncture(EA)on renal function and autophagy-related protein expression in diabetic nephropathy (DN) rats，so as to explore its mechanisms underlying improvement of DN. Male Wistar rats were randomly divided into normal control，DN model and EA groups (n=8 rats in each group). The DN model was established by high-fat diet (for 4 weeks) + streptozotocin (STZ，35 mg/kg，i.p.). EA (2 Hz/15 Hz，1 mA) was applied to bilateral "Shenshu"(BL23)and "Zusanli"(ST36)for 20 min，5 times a week for 6 consecutive weeks. The urine volume in 24 h (24 h-UV) was recorded，the levels of urine protein in 24 h (24 h-UP)，blood urea nitrogen (BUN) and serum creatinine (Scr) were detected by using an automatic biochemical analyzer，and the fasting blood glucose (FBG) was detected using rapid blood glucose paper test. The histopathological changes of glomerulus of the left kidney were observed after periodic acid-Schiff stain (PAS) and the ultra-microstructural changes，the numbers of autophagosomes and autophagic vacuoles of the right kidney were observed by transmission electronic microscope (TEM). The expression and localization of podocyte marker protein Nephrin was detected by using immunofluorescence assay，and the expression levels of autophagy-related proteins microtubule associated protein 1 (LC3)，Beclin-1，p62 and Nephrin in the kidney tissue measured by Western blot. Before treatment,compared with the normal control group，the levels of 24 h-UV，24 h-UP and FBG were significantly increased (P<0.01) in the model and EA groups. After treatment, compared with the normal group，the levels of 24 h-UV，24 h-UP，FBG，BUN，Scr and p62 protein expression were signi-ficantly increased (P<0.01)，and the expression levels of LC3Ⅱ，Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰ were significantly decreased (P<0.01) in the model group. Compared with the model group，the levels of 24 h-UV，24 h-UP，FBG，BUN and p62 were apparently decreased (P<0.01，P<0.05)，while the expression levels of LC3Ⅱ，Beclin-1 and Nephrin proteins and ratio of LC3Ⅱ/Ⅰ were obviously up-regulated (P<0.01，P<0.05) in the EA group. No significant changes were found in the level of Scr after EA (P>0.05) and in the expression levels of LC3 Ⅰ after modeling and EA (P>0.05). Outcomes of PAS showed blurred structure of the glomerulus with obvious proliferation of glomerular mesangial cells，increase of cell matrix，and thickening of basement membrane of the glomerulus and renal tubule，atrophy of epithelia cells of renal tubules and infiltration of inflammatory cells in the renal interstitum; and TEM revealed fusion and exfoliation of some foot processes，bareness or thickening of some basement membrane，and reduction of number of autophagosomes or autophagobubbles in podocytes in the model group，which was relatively milder in the EA group. EA can effectively alleviate kidney damage in DN rats，which may be related to its function in facilitating autophagy in the kidney.

Cxcl10 deficiency attenuates renal interstitial fibrosis through regulating epithelial-to-mesenchymal transition

IFN-γ-inducible protein 10 (IP-10, CXCL10) has been widely demonstrated to be involved in multiple kidney pathological processes. However, the role of CXCL10 in renal fibrosis remains unclear. In this study, Cxcl10-deficient (Cxcl10−/−) mice were used to generate the unilateral ureteral obstruction (UUO) model. The level of renal fibrosis and inflammatory cell infiltration was examined in vivo and the effects of CXCL10 on EMT process of HK-2 cells was investigated in vitro. We observed that the injury degree of renal tissue and the collagen deposition levels were lighter and the expression of α-SMA, collagen I and fibronectin was significantly reduced in Cxcl10−/− mice, while the expression of E-cadherin was increased. However, interstitial F4/80-positive macrophages and CD4-positive T lymphocytes were unaffected by knockout of Cxcl10. Furthermore, IFN-γ or CXCL10 stimulation could obviously promote the expression of α-SMA, collagen I, fibronectin and reduce the expression of E-cadherin in HK-2 cells, which could be inhibited by transfection of Cxcl10-siRNA. Our findings suggested Cxcl10 knockout could reduce renal dysfunction and inhibit renal fibrosis through regulating EMT process of renal tubular epithelial cells in murine UUO model. These results may provide a novel insight into the mechanism and a potential therapy target of renal fibrosis.

FGF21 prevents low-protein diet-induced renal inflammation in aged mice.

Low-protein (LP) diets extend lifespan through a comprehensive improvement in metabolic health across multiple tissues and organs. Many of these metabolic responses to protein restriction are secondary to transcriptional activation and release of FGF21 from the liver. However, the effects of an LP diet on the kidney in the context of aging has not been examined. Therefore, the goal of the current study was to investigate the impact of chronic consumption of an LP diet on the kidney in aging mice lacking FGF21. Wild-type (WT; C57BL/6J) and FGF21 knockout (KO) mice were fed a normal protein diet (20% casein) or an LP (5% casein) diet ad libitum from 3 to 22 mo of age. The LP diet led to a decrease in kidney weight and urinary albumin-to-creatinine ratio in both WT and FGF21 KO mice. Although the LP diet produced only mild fibrosis and infiltration of leukocytes in WT kidneys, the effects were significantly exacerbated by the absence of FGF21. Accordingly, transcriptomic analysis showed that inflammation-related pathways were significantly enriched and upregulated in response to LP diet in FGF21 KO mice but not WT mice. Collectively, these data demonstrate that the LP diet negatively affected the kidney during aging, but in the absence of FGF21, the LP diet-induced renal damage and inflammation were significantly worse, indicating a protective role of FGF21 in the kidney.NEW & NOTEWORTHY Long-term protein restriction is not advantageous for an otherwise healthy, aging kidney, as it facilitates the development of renal tubular injury and inflammatory cell infiltration. We provide evidence using FGF21 knockout animals that FGF21 is essential to counteract the renal injury and inflammation during aging on a low-protein diet.

Nephroprotective effect of umbelliferone against cisplatin-induced kidney damage is mediated by regulation of NRF2, cytoglobin, SIRT1/FOXO-3, and NF- kB-p65 signaling pathways.

Cisplatin (Cis) is one of the most potent and effective broad-spectrum antitumor drugs, but its use is limited due to nephrotoxicity. The current study investigated the renoprotective effect of umbelliferone (UMB) on Cis-induced nephrotoxicity in rats. Renal injury was induced by a single injection of Cis (7 mg/kg, ip). Our results exhibited that the injection of Cis significantly disrupted renal function biomarkers as well as KIM-1 expression. The expressions of TNF-α, IL-1β, NF-kB-p65, and IKKβ were elevated along with downregulation of IkBα expression. Also, Cis disrupted cellular oxidant/antioxidant balance through the reduction of glutathione (GSH), glutathione-S-transferase (GST), and superoxide dismutase (SOD) levels and elevation of malondialdehyde (MDA) content. On the contrary, the levels of renal function biomarkers, cytokines, NF-kB-p65, IkBα, IKKβ, and oxidant/antioxidant status have been improved after UMB treatment. Mechanistically, rats administered Cis only exhibited a significant decrease in NRF2 and cytoglobin expressions as well as the CREB, SIRT1, FOXO-3, and PPAR-γ genes. Treatment with UMB significantly upregulated NRF2 and cytoglobin proteins, as well as effectively increased the expression of CREB, SIRT1, FOXO-3, PPAR-γ, and NRF2 genes. Histopathological findings strongly supported our biochemical results, as evidenced by attenuation of renal hemorrhage, cast diffusion, and inflammatory cell infiltration. Interestingly, UMB significantly enhanced Cis cytotoxicity in both HL-60 and HeLa cells in a dose-dependent manner. Together, our results demonstrated that UMB can protect against Cis-induced nephrotoxicity in normal rats along with the enhancement of its in vitro antitumor activity. These findings suggested that UMB could be used as a potential adjuvant therapy in Cis chemotherapeutic protocols.

Preliminary study on effect of Phellinus igniarius ethanol extract on serum uric acid metabolism and gut microbiome in rats

The aim of this paper was to investigate the effect of ethanol extract of Phellinus igniarius in lowering uric acid and changing the gut microbiome in hyperuricemia rats. A total of 36 SD rats were randomly divided into normal control group, model control group, positive drug control group, and high-dose, middle-dose and low-dose P. igniarius ethanol extract groups, with 6 rats in each group. Hyperuricemia rats were established by D-fructose combined with oteracil potassium(OAPS). One week later, the positive control group was given allopurinol 50 mg·kg~(-1) intragastrically, and P. igniarius ethanol extract groups were treated with 30, 60 and 90 mg·kg~(-1) drugs for 14 consecutive days. Body weight, blood glucose and serum uric acid(SUA) were monitored every week. After the model rats were administered with the ethanol extracts of P. igniarius by gavage for two weeks, the activities of creatinine, BUN, xanthine oxidase(XOD) and adenosine deaminase(ADA) were detected. The right kidney was taken to analyze the histological and morphological changes and the degree of damage to main organs of the extract of P. igniarius. The 16 S rDNA gene sequence technique was used to analyze the guts microbiota composition in feces. The results indicated that ethanol extract of P. igniarius could significantly lower the SUA level(P&lt;0.01), while inhibiting the activities of XOD and ADA(P&lt;0.05, P&lt;0.01). Histological examination showed that the allopurine group showed slight renal tubular dilation and inflammatory cell infiltration compared with the normal group, with no significant difference between the P. igniarius ethanol extract groups and the normal group. The 16 S sequencing results showed that the composition of gut microbiota has changed in each group. Therefore, ethanol extracts of P. igniarius may reduce the level of SUA in rats by inhibiting the activities of XOD and ADA, with a certain effect on the composition of gut microbiota.