Mechanisms of renal interstitial fibrosis: cross-talk between mitophagy and NLRP3 inflammasome

Renal interstitial fibrosis(RIF)is the main pathological basis leading to end-stage renal disease,and is closely related to the prognosis of patients with kidney disease.Increasing evidence as shown that mitophagy and NLRP3 inflammasome play important roles in the pathogenesis of RIF.Studies suggest that inhibiting NLRP3 inflammasome by activating mitophagy can prevent and alleviate RIF.This review summarizes role played by cross-talk between mitophagy and NLRP3 inflammasome in promoting RIF,so as to offer new perspectives on more effective slow the progression of renal diseases and fibrosis prevention.

Exploring the mechanism of Qinghaobiejiatang in treating renal interstitial fibrosis based on network pharmacology

To explore the potential mechanism of the classic ancient prescription Qinghaobiejiatang(QHBJT)in treating renal interstitial fibrosis.Methods:Obtain the active ingredients of Qinghao(Artemisiae annuae herba),Biejia(Trionycis carapax),Dihuang(Rehmanniae radix),Zhimu(Anemarrhenae rhizoma),and Mudanpi(Moutan cortex)through TCMSP and TCMID databases.Collect disease targets through the GENECARDS database.Use Venny2.1.0 platform to draw Venn diagrams to map drugs and disease targets.Import the key targets into Cytoscape 3.7.2 software to draw a network diagram of“drugs-active ingredients-diseases-key targets”.The STRING11.0 database was used to construct the key target protein interaction network diagram.Use R language for Gene ontology function and kyoto encyclopedia of genes and genomes pathway enrichment analysis.Results:A total of 317 active ingredients were obtained through screening,involving 166 targets,and 102 active ingredients related to disease targets,mainly involved in the regulation of key targets such as FOS,IL6,MTOR,MAPK8,RELA,CCND1,TP53,EGFR,and CASP3 signal pathways related to viral infection,tumor-related,apoptosis,signal transduction,fluid shear stress and atherosclerosis play a synergistic role in the treatment of renal fibrosis.Conclusion:The effect mechanism of QHBJT in treating renal interstitial fibrosis is related to inflammation,oxidative stress,hypoxia,apoptosis,pathological activation and damage of renal tubular epithelial cells mediated by the above pathways.

Shen Qi Wan attenuates renal interstitial fibrosis through upregulating AQP1

Renal interstitial fibrosis(RIF)is the crucial pathway in chronic kidney disease(CKD)leading to the end-stage renal failure.However,the underlying mechanism of Shen Qi Wan(SQW)on RIF is not fully understood.In the current study,we investigated the role of Aquaporin 1(AQP1)in SQW on tubular epithelial-to-mesenchymal transition(EMT).A RIF mouse model induced by adenine and a TGF-β1-stimulated HK-2 cell model were etablished to explore the involvement of AQP 1 in the protective effect of SQW on EMT in vitro and in vivo.Subsequently,the molecular mechanism of SQW on EMT was explored in HK-2 cells with AQP1 knockdown.The results indicated that SQW alleviated kidney injury and renal collagen deposition in the kidneys of mice induced by adenine,increased the protein expression of E-cadherin and AQP1 expression,and decreased the expression of vimentin andα-smooth muscle actin(α-SMA).Similarly,treatmement with SQW-containing serum significantly halted EMT process in TGF-β1 stimulated HK-2 cells.The expression of snail and slug was significantly upregulated in HK-2 cells after knockdown of AQP1.AQP1 knockdown also increased the mRNA expression of vimentin andα-SMA,and decreased the expression of E-cadherin.The protein expression of vimentin increased,while the expression of E-cadherin and CK-18 significantly decreased after AQP1 knockdown in HK-2 cells.These results revealed that AQP1 knockdown promoted EMT.Furthermore,AQP1 knockdown abolished the protective effect of SQW-containing serum on EMT in HK-2 cells.In sum,SQW attentuates EMT process in RIF through upregulation of the expression of AQP1.

Correlation between the Expression of TGF-β1,Rhoa,SOX9 and Renal Interstitial Fibrosis in Rats with Chronic Kidney Disease

Objective:To study the correlation between the expression of transforming growth factor-β1(TGF-β1),Rho A,SOX9 and renal interstitial fibrosis in rats with chronic kidney disease.Methods:Forty specific pathogen-free(SPF)male SD rats were randomly divided into study group and control group,with 20 cases in each group.The study group was given adenine suspension by gavage,while the control group was given the same amount of saline by gavage.Blood,urine and renal tissue specimens were collected from all rats at 3rd and 6th weeks after modeling.The kidney weight,kidney weight/body weight,renal function indexes,the expression of TGF-β1,Rho A and SOX9 m RNA in renal tissues,Masson staining and renal interstitial fibrosis score were compared between the two groups.Pearson correlation was used to analyze the relationship between the renal interstitial fibrosis score and the expression of TGF-β1,Rho A and SOX9 m RNA in renal tissues of rats with chronic kidney disease.Results:The kidney weight and kidney weight/body weight of rats in the study group were higher than those in the control group at 3rd and 6th weeks after modeling(P<0.05).The quantitative levels of creatinine,urea nitrogen and 24-hour urinary protein in the study group were higher than those in the control group at 3rd and 6th weeks after modeling(P<0.05).The expression levels of TGF-β1,Rho A and SOX9 m RNA in renal tissues of rats in the study group were higher than those in the control group at 3rd and 6th weeks after modeling(P<0.05).The renal interstitial fibrosis score in the study group was higher than that in the control group at 3rd and 6th weeks after modeling(P<0.05).Pearson correlation analysis confirmed that the renal interstitial fibrosis score in rats with chronic kidney disease was positively correlated with the expression of TGF-β1,Rho A and SOX9 m RNA in renal tissues(P<0.05).Conclusion:The expression of TGF-β1,Rho A and SOX9 was abnormally high in rats with chronic kidney disease and was closely related to renal interstitial fibrosis,which may play a promoting role in the process of renal interstitial fibrosis.

Fuzheng Huayu recipe, a traditional Chinese compound herbal medicine, attenuates renal interstitial fibrosis via targeting the miR-21/PTEN/AKT axis

Objective: MicroRNAs(miRNAs) may be viable targets for treating renal interstitial fibrosis(RIF). Fuzheng Huayu recipe(FZHY), a traditional Chinese compound herbal medicine, is often used in China to treat fibrosis. This study sought to assess the mechanisms through which FZHY influences miRNAs to treat RIF.Methods: RIF was induced in rats by mercury chloride and treated with FZHY. Hydroxyproline content,Masson’s staining and type I collagen expression were used to evaluate renal collagen deposition.Renal miRNA profiles were evaluated using a miRNA microarray. Those miRNAs that were differentially expressed following FZHY treatment were identified and subjected to bioinformatic analyses. The miR-21 target gene phosphatase and tensin homolog(PTEN) expression and AKT phosphorylation in kidney tissues were assessed via Western blotting. In addition, HK-2 human proximal tubule epithelial cells were treated using angiotensin II(Ang-II) to induce epithelial-to-mesenchymal transition(EMT), followed by FZHY exposure. miR-21 and PTEN expressions were evaluated via quantitative reverse transcriptionpolymerase chain reaction(qRT-PCR), while E-cadherin and a-smooth muscle actin(a-SMA) expressions were assessed by immunofluorescent staining and qRT-PCR. Western blotting was used to assess PTEN and AKT phosphorylation.Results: FZHY significantly decreased kidney collagen deposition, hydroxyproline content and type I collagen level. The miRNA microarray identified 20 miRNAs that were differentially expressed in response to FZHY treatment. Subsequent bioinformatic analyses found that miR-21 was the key fibrosis-related miRNA regulated by FZHY. FZHY also decreased PTEN expression and AKT phosphorylation in fibrotic kidneys. Results from in vitro tests also suggested that FZHY promoted E-cadherin upregulation and inhibited a-SMA expression in Ang-II-treated HK-2 cells, effectively reversing Ang-II-mediated EMT. We also determined that FZHY reduced miR-21 expression, increased PTEN expression and decreased AKT phosphorylation in these cells.Conclusion: miR-21 is the key fibrosis-related miRNA regulated by FZHY. The ability of FZHY to modulate miR-21/PTEN/AKT signaling may be a viable approach for treating RIF.

Chinese herbal decoction astragalus and angelica exerts its therapeutic effect on renal interstitial fibrosis through the inhibition of MAPK,PI3K-Akt and TNF signaling pathways

Astragalus and Angelica decoction(A&A)has been clinically used as a classical traditional Chinese medicine(TCM)formula in China for many years for the treatment of kidney diseases,especially renal interstitial fibrosis(RIF).However,the mechanisms underlying the therapeutic effects of A&A on RIF remains poorly understood.In the present study,systematic network pharmacology and effective experimental verification were utilized for the first time to elucidate the pharmacological efficacy and potential mechanism.The outcomes indicated that 22 active components and 87 target genes of A&A were identified and cross-referenced with RIF-associated genes,contributing to confirmation of 74 target genes of A&A for RIF.Pathway and functional enrichment analyses revealed that A&A had substantial effects on MAPK,PI3K-Akt and TNF signaling pathways.In addition,seven core targets with relatively higher betweenness and degree were identified in the constructed Chinese medicine material-chemical component-target-signal pathway network.Moreover,we verified the potential therapeutic effect of A&A in vivo(using a mouse model of RIF),confirming that A&A could effectively protect the kidney by regulating these target genes.The therapeutic effect of A&A on RIF could be attributed to its role in regulating the cell cycle,limiting the apoptosis,and inhibiting the inflammation.

Huangqi decoction(黄芪汤) attenuates renal interstitial fibrosis via transforming growth factor-β1/mitogen-activated protein kinase signaling pathways in 5/6 nephrectomy mice

OBJECTIVE: To investigate the effect of Huangqi decoction( 黄芪汤) on renal interstitial fibrosis and its association with the transforming growth factor-β1(TGF-β1)/mitogen-activated protein kinase(MAPK) signaling pathway. METHODS: 120 C57/BL mice were randomly divided into six groups: sham group, Enalapril(20 mg/kg) group, 5/6 nephrectomy model group, and 5/6 nephrectomy model plus Huangqicoction(0.12, 0.36 and 1.08 g/kg respectively) groups. Detecting 24hours urinary protein, blood pressure, serum creatinine, urea nitrogen content changes. Periodic Acid-Schiff stain(PAS) and Masson’s trichrome staining was used to observe the renal tissue pathological changes. Protein expression of TGF-β1, Phosphorylated P38 mitogen activated protein kinases(P-P38), Phosphorylated c-jun N-terminal kinase(P-JNK), Phosphorylated extracellular regulated proteinhnase(PERK), Fibroblast-specific protein-1(FSP-1), Alpha smooth muscle actin(α-SMA), Type Ⅲ collagen(Collagen Ⅲ), Connective tissue growth factor(CTGF),Bcl-2 Assaciated X protein(Bax) and B cell lymphoma 2(Bcl-2) were measured with western blot and immunohistochemical. RESULTS: Both Huangqi decoction and Enalapril improved the kidney function, 24 h urinary protein and the fibrosis in 5/6 nephrectomy mice, Huangqi decoction downregulated the expressions of TGF-β1, FSP-1, α-SMA, Collagen Ⅲ and CTGF in a dose-dependent manner, and it has a significant difference(P < 0.01) compared with model group.Huangqi decoction downregulated the expressions of P-P38, P-JNK, P-ERK and Bcl-2 in a dose-dependent manner, while upregulated the expression of Bax. CONCLUSIONS: The protective effect of Huangqi decoction for renal interstitial fibrosis in 5/6 nephrectomized mice via the inhibition of EpithelialMesenchymal Transitions and downregulating the TGF-β1/MAPK signaling pathway.

Z-Guggulsterone alleviated renal fibrosis and G_(2)/M cycle arrest through Klotho/P53 signaling

OBJECTIVE Chronic kidney disease(CKD)has become a global public health problem with 10%-15%incidence rate,and inhibiting the renal interstitial fibrosis is considered to be a potential strategy to delay the progression of CKD.Z-Guggulsterone(Z-GS),an active compound from derived from Commiphora mukul,has been proved to be effective in various diseases.The present study aimes to determine the protective effect and the molecular mechanism of Z-GS on renal fibrosis.METHODS Unilateral ureteral obstruction(UUO)mice and hypoxia-induced HK-2 cells were used to simulate renal fibrosis in vitro and in vivo,respectively.The mice and cells were treated with different doses of Z-GS to observe the pharmacological action.Renal function,including Scr,BUN,and UA,were detected by commercial kits.H&E and Masson staining were performed to observe histopathological changes of kidney.Cell viability and LDH release of HK-2 cells were detected by commercial kits.Cell cycle distribution and apoptosis rate were analyzed by flow cytometry.Fibrosis markers were detected by immunohistochemistry and immunofluorescence analysis.Cell cycle related proteins and Klotho/p53 signaling were analyzed by Western blotting.RESULTS The results showed that Z-GS decreased the rise of Scr,BUN,and UA and lightened renal histopathological injury,which were induced by UUO.Besides,Z-GS administration alleviated renal fibrosis in mice by inhibiting the expressions ofα-SMA,TGF-βand collagenⅣ,and delayed G2/M cell cycle arrest by promoting the expressions of CDK1 and cyclinD1/B1 rate.Experiments in vitro indicated that Z-GS treatment significantly increased the cell viability while decreased the LDH release in hypoxia-induced HK-2 cells.In addition,hypoxia induced fibrosis and G2/M cycle arrest in HK-2 cells were retarded by Z-GS.The study of its possible mechanism exhibited that Z-GS treatment increased the level of Klotho and inhibited P53 level.Nevertheless,the effect of Z-GS on Klotho/P53 signaling was reversed by siRNA-Klotho.Moreover,siRNA-Klotho treatment eliminated the effects of Z-GS on G2/M cell cycle arrest and fibrosis.CONCLUSION This study clarified that Z-GS alleviated renal fibrosis and G2/M cycle arrest through Klotho/P53 signaling pathway.People who have suffered CKD may potentially benefit from treatment with Z-GS.

Effects of Yishenbupi (tonifying-kidney and invigorating-spleen) prescription on the expression of renal fibrosis-associated proteins in unilateral ureteral occlusion rats

Objective:To evaluate the effects and mechanism of the Yishenbupi(tonifying-kidney and invigorating-spleen)prescription on the expression of renal fibrosis-associated vimentin,α-SMA,and fibronectin in unilateral ureteral occlusion rats.Methods:A total of 48 SD(Sprague-Dawley)rats were randomly divided into the model,sham-operated(sham),irbesartan,and Yishenbupi groups,with 12 rats in each group.After the unilateral ureteral occlusion model was established,rats in the model and sham groups were administered normal saline,whereas rats in the Yishenbupi group were administered Yishenbupi prescription(18 g/kg/d)intragastrically and those in the irbesartan group were administered irbesartan(10 mg/kg/d)intragastrically.All rats were sacrificed 21 days later.Pathological changes in rat renal tissue were evaluated by H&E staining.The expression of vimentin,α-SMA,and fibronectin in renal tissues was detected by western blotting.Results:Compared with the sham group the model group had renal tubular epithelial cell atrophy,inflammatory cell infiltration accompanied with the proliferation of interstitial collagen fibers,fewer glomeruli,or glomerulosclerosis.Compared with the model group,significantly less renal tubular and glomerular damages,inflammatory cell infiltration,and collagen fibers were observed in different intervention groups,especially in the Yishenbupi group.Compared with the sham group,significantly higher expressions of fibrosis markers,including vimentin,α-SMA,and fibronectin,were observed in the model group.Compared with the model group,the expression of anti-fibrosis markers,including vimentin,α-SMA and fibronectin,was significantly decreased in both the irbesartan and Yishenbupi groups(P<0.01);however,the Yishenbupi group showed higher efficacy than the irbesartan group(P<0.05).Conclusion:The Yishenbupi prescription may improve renal fibrosis by reducing the expression of fibrosis-associated vimentin,α-SMA,and fibronectin.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.

Involvement of hydrogen sulfide in the progression of renal fibrosis

Objective:Renal fibrosis is the most common manifestation of chronic kidney disease(CKD).Noting that existing treatments of renal fibrosis only slow disease progression but do not cure it,there is an urgent need to identify novel therapies.Hydrogen sulfide(H2S)is a newly discovered endogenous small gas signaling molecule exerting a wide range of biologic actions in our body.This review illustrates recent experimental findings on the mechanisms underlying the therapeutic effects of H2S against renal fibrosis and highlights its potential in future clinical application.Data sources:Literature was collected from PubMed until February 2019,using the search terms including"Hydrogen sulfide,""Chronic kidney disease"MRenal interstitial fibrosis,"Kidney disease,""Inflammation factor,""Oxidative stress,""Epithelialto-mesenchymal transition,""H2S donor,""Hypertensive kidney dysfunction,""Myofibroblasts,""Vascular remodeling,""transforming growth factor(TGF)-beta/Smads signaling,"and"Sulfate potassium channels."Study selection:Literature was mainly derived from English articles or articles that could be obtained with English abstracts.Article type was not limited.References were also identified from the bibliographies of identified articles and the authors5 files.Results:The experimental data confirmed that H2S is widely involved in various renal pathologies by suppressing inflammation and oxidative stress,inhibiting the activation of fibrosis-related cells and their cytokine expression,ameliorating vascular remodeling and high blood pressure,stimulating tubular cell regeneration,as well as reducing apoptosis,autophagy,and hypertrophy.Therefore,H2S represents an alternative or additional therapeutic approach for renal fibrosis.Conclusions:We postulate that H2S may delay the occurrence and progress of renal fibrosis,thus protecting renal function.Further experiments are required to explore the precise role of H2S in renal fibrosis and its application in clinical treatment.