Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

BACKGROUND Development of end-stage renal disease is predominantly attributed to diabetic nephropathy(DN).Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue.Never-theless,the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain.AIM To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism.METHODS Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk.Subsequently,blood and urine indexes were assessed,along with examination of renal tissue pathology.Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin,periodic acid–Schiff,Masson’s trichrome,and Sirius-red.Additionally,high-glucose culturing was conducted on the RAW 264.7 cell line,treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h.In both in vivo and in vitro settings,quantification of inflammation factor levels was conducted using western blotting,real-time qPCR and ELISA.RESULTS In db/db mice,administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis.Notably,we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin,along with a decrease in expressions of inflammatory cytokine-related factors.Furthermore,myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha,interleukin-6,and interluekin-1βinduced by high glucose in RAW 264.7 cells.Additionally,myricetin modulated the M1-type polarization of the RAW 264.7 cells.Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin.The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002.CONCLUSION This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway

Objective Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage.Notably,the knockout of transient receptor potential channel 6(TRPC6)has shown promise in alleviating renal fibrosis.However,the regulatory impact of TRPC6 on renal fibrosis remains unclear.Methods In vivo,TRPC6 knockout(TRPC6−/−)mice and age-matched 129 SvEv(WT)mice underwent unilateral renal ischemia-reperfusion(uIR)injury surgery on the left renal pedicle or sham operation.Kidneys and serum were collected on days 7,14,21,and 28 after euthanasia.In vitro,primary tubular epithelial cells(PTECs)were isolated from TRPC6−/−and WT mice,followed by treatment with transforming growth factorβ1(TGFβ1)for 72 h.The anti-fibrotic effect of TRPC6−/−and the underlying mechanisms were assessed through hematoxylin-eosin staining,Masson staining,immunostaining,qRT-PCR,and Western blotting.Results Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFβ1.TRPC6−/−alleviated renal fibrosis by reducing the expression of fibrotic markers(Col-1,α-SMA,and vimentin),as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro.Additionally,we found that the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/glycogen synthase kinase 3 beta(GSK3β)signaling pathway,a pivotal player in renal fibrosis,was down-regulated following TRPC6 deletion.Conclusion These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3βpathway.Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

Asiaticoside improves diabetic nephropathy by reducing inflammation, oxidative stress, and fibrosis: An in vitro and in vivo study

BACKGROUND Diabetic nephropathy(DN)is a severe microvascular complication of diabetes characterized by inflammation,oxidative stress,and renal fibrosis.Asiaticoside(AC)exhibits anti-inflammatory,antioxidant,and anti-fibrotic properties,suggesting potential therapeutic benefits for DN.This study aimed to investigate the protective effects of AC against DN and elucidate the underlying mechanisms involving the nuclear factor erythroid 2-related factor 2(NRF2)/heme oxygenase-1(HO-1)antioxidant pathway.METHODS The effects of AC on high glucose(HG)-induced proliferation,inflammation,oxidative stress,and fibrosis were evaluated in rat glomerular mesangial cells(HBZY-1)in vitro.A streptozotocin-induced DN rat model was established to assess the in vivo impact of AC on renal injury,inflammation,oxidative stress,and fibrosis.The involvement of the NRF2/HO-1 pathway was examined using pharmacological inhibition studies in the cell model.RESULTS AC inhibited HG-induced HBZY-1 cell proliferation and significantly improved various indicators of DN in rats,including reduced body weight,and elevated blood glucose,serum creatinine,blood urea nitrogen,and 24-h urine protein.Both in vitro and in vivo studies demonstrated that AC decreased inflammation and oxidative stress by reducing interleukin(IL)-6,IL-8,tumor necrosis factor-alpha,reactive oxygen species,and malondialdehyde levels while increasing superoxide dismutase activity.Additionally,AC suppressed the expression of fibrogenic markers such as collagen I,collagen IV,and fibronectin.AC activated NRF2 expression in the nucleus and increased HO-1 and NAD(P)H dehydrogenase(Quinone)1 protein expression in renal tissues and HG-induced HBZY-1 cells.CONCLUSION AC improves DN by reducing inflammation,oxidative stress,and fibrosis through the activation of the NRF2/HO-1 signaling pathway.These findings not only highlight AC as a promising therapeutic candidate for DN but also underscore the potential of targeting the NRF2/HO-1 pathway in developing novel treatments for other chronic kidney diseases characterized by oxidative stress and inflammation.

LncRNA LOC103694972 promotes fibrosis of NRK-49F cells by regulating STAT3-dependent Smad/CTGF pathway via targeting miR-29c-3p

Background:Renalfibrosis is an important process in the development of chronic kidney disease.Understanding the pathogenesis andfinding effective treatments for renalfibrosis is crucial.This study aims to investigate whether a newly discovered long non-coding RNA(lncRNA)called LOC103694972 could be a potential target for treatingfibrosis of NRK-49F cells.Methods:LncRNA Chip was used to identify differentially expressed lncRNAs between TGF-β1-induced NRK-49F cells and normal cells.The dual-luciferase assay confirmed the binding between miR-29c-3p and signal transducer and activator of transcription(STAT3),as well as between miR-29c-3p and lncRNA LOC103694972.Si-LOC103694972 and miR-29c-3p mimic were then transfected into TGF-β1-induced NRK-49F cells.Results:The study found that LOC103694972 was highly expressed in TGF-β1-induced NRK-49F cells.These cells exhibited increased cell length and activity compared to the control group.The expression levels of Collagen I,α-Smooth muscle actin(α-SMA),and tissue inhibitor of metalloproteinase(TIMP-1)were increased,while matrix Metalloproteinase 2(MMP2)and matrix Metalloproteinase 9(MMP9)expression was decreased.However,transfection with si-LOC103694972 and miR-29c-3p mimics restored cell morphology and reduced cell viability.This led to a decrease in the levels of Collagen I,α-SMA,and TIMP-1,as well as an increase in MMP2 and MMP9 expression.Additionally,TGF-β1-induced NRK-49F cells transfected with miR-29c-3p mimics activated the STAT3-Smad3/CTGF pathway.Conclusion:Based on thesefindings,lncRNA LOC103694972 shows promise as a target for treating renalfibrosis.It negatively regulates miR-29c-3p and activates the STAT3-Smad3/CTGF pathway.

Chronic heat stress induces renal fibrosis and mitochondrial dysfunction in laying hens

Background Heat stress in laying hens negatively affects egg production and shell quality by disrupting the homeo-stasis of plasma calcium and phosphorus levels.Although the kidney plays an important role in calcium and phos-phorus homeostasis,evidence regarding the effect of heat stress on renal injury in laying hens is yet to be elucidated.Therefore,the aim of this study was to evaluate the effects of chronic heat stress on renal damage in hens during laying periods.Methods A total of 16 white-leghorn laying hens(32 weeks old)were randomly assigned to two groups(n=8).One group was exposed to chronic heat stress(33°C for 4 weeks),whereas the other group was maintained at 24°C.Results Chronic heat exposure significantly increased plasma creatinine and decreased plasma albumin levels(P<0.05).Heat exposure also increased renal fibrosis and the transcription levels of fibrosis-related genes(COLA1A1,αSMA,and TGF-β)in the kidney.These results suggest that renal failure and fibrosis were induced by chronic heat exposure in laying hens.In addition,chronic heat exposure decreased ATP levels and mitochondrial DNA copy number(mtDNA-CN)in renal tissue,suggesting that renal mitochondrial dysfunction occurs under conditions of heat stress.Damaged mitochondria leak mtDNAs into the cytosol and mtDNA leakage may activate the cyclic GMP-AMP synthase(cGAS)stimulator of interferon genes(STING)signaling pathway.Our results showed that chronic heat exposure activated the cGAS-STING pathway as indicated by increased expression of MDA5,STING,IRF7,MAVS,and NF-κB levels.Furthermore,the expression of pro-inflammatory cytokines(IL-12)and chemokines(CCL4 and CCL20)was upregulated in heat-stressed hens.Conclusions These results suggest that chronic heat exposure induces renal fibrosis and mitochondrial damage in laying hens.Mitochondrial damage by heat stress may activate the mtDNA-cGAS-STING signaling and cause subse-quent inflammation,which contributes to the progression of renal fibrosis and dysfunction.

Nano-Sustained CO-Releasing Molecules Alleviates Cyclosporin-A-Induced Nephrotoxicity and Renal Fibrosis by Inhibiting NLRP3 Inflammasome-Mediated TGF-β/Smad Signaling Pathway

Objective:To investigate the effect nano-sustained CO-releasing molecules on cyclosporin-A(CsA)-induced nephrotoxicity by inhibiting the NLRP3 inflammasome-mediated TGF-β/Smad signaling pathway.Methods:3×105 cell/mL human renal tubular epithelial cells(HK-2)and mouse primary cultured renal tubular epithelial cells(RTECs)were cultured under an inverted microscope and incubated with 10%DMEM and 0.25%β2M in NaCl solution for 3 h.HK-2 and RTECs were divided into 5 complex numbers.MTT assay was used to detect the relative proliferation level of one of the HK-2 cells and calculate the multiplication ratio.Results:The nano-sustained CO-releasing molecules CS-CO had a strong protective effect on the kidney.HK-2 and RTECs cells were treated with siRNA,inhibitors,and NLRP3 knockout mice,and the changes in cell activity and expression of intracellular inflammatory factors were studied.The expression of TGF-β1/Smad signaling pathway related proteins in HK-2 and RTECs was detected by ELISA,western blot,immunofluorescence,and other techniques.Conclusion:SMA/CORM2 alleviates CsA-induced renal fibrosis by inhibiting NLRP3 inflammasome-mediated TGF-β/Smad signaling pathway.

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.

Effect of alprostadil combined with Diammonium glycyrrhizinate on renal interstitial fibrosis in SD rats

Objective:To observe effect of alprostadil combined with Diammonium glycyrrhizinate on renal interstitial fibrosis in SD rate.Methods:A total of 75 SD rate were randomly divided into A,B,C,D,E groups with 15 in each group.Rats in group A served as the control group received just only but tissue separation without modeling operation,while model of unilateral ureteral obstruction(UUO) was established in B,C,D,E groups.Rats in A,B group were given saline lavage placebo treatment,while rats in C,D,E groups were given dianunonium glycyrrhizinate and alprostadil injection.Five rats were sacrificed 1,2,3 weeks after modeling,serum creatinine level of femoral venous blood was determined.Transforming growth factor- β1(TCF- β1) and concentration of connective tissue growth factor(CTGF) were also detected by using ELISA.Line renal interstitial tissue was taken after HE staining,renal interstitial TGF- β1 and CTGF expression were detected by using immunohistochemical method.Results:Serum creatinine levels of B,C,D,E group at different time points in were significantly higher than that of group A(P<0.05);serum creatinine levels in group B were significantly higher than that of C,D,E group at each time point(P<0.05).Serum creatinine level of Croup E was significantly lower than C,D group after 2,3 weeks(P<0.05).Rate in A group at each time point showed no significant changes in TGF- β1 and CREA concentration in serum and kidney tissues(P>0.05);while serum and kidney tissue TGF- β1,concentration of CREA.expression of rats in B,C,D,E groups showed a gradual increasing trend over time.TCF- β1 and CREF of Group B in serum and kidney tissues at each time point were significantly higher than that of the other groups(P<0.05).TCF- β1 and CREF of Group E in serum and kidney tissues at each time point were significantly lower than that of B,C,D group at all time points in serum and kidney tissues(P<0.05).Conclusions:Alprostadil combined with diammonium glycyrrhizinate can significantly lower the expression of TGF- β1 and CTGF in serum and tissues of SD rat with renal interstitial fibrosis,thus inhibit rat renal interstitial fibrosis process.It has synergy protective effect.

Effects of Mycophenolate Mofetil on Renal Interstitial Fibrosis after Unilateral Ureteral Obstruction in Rats

To investigate the effects of mycophenolate mofetil (MMF) on the process of renal interstitial fibrosis,unilateral ureteral obstruction (UUO) model was established in rats Twenty Sprague-Dawley rats underwent UUO and received vehicle ( n =10) or MMF (20 mg kg -1 d -1 , by daily gastric gavage, n= 10) during a period of 5 days following surgery, and the additional 10 rats were served as sham-operated group The rats were killed 5 days after surgery Immunohistochemistry was performed on renal tissue for proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and type Ⅰ and Ⅲ collagen (colⅠ, colⅢ) Histological studies were also done by MASSON staining Five days after surgery, proliferating cells in tubules, interstitium as well as interstitial myofibroblast (MyoF) infiltration and interstitial colⅠ, colⅢ deposition were all significantly reduced by MMF treatment MMF also alleviated the histological changes of UUO rats These results suggested that the reduction of interstitial MyoF infiltration may be an important event by which MMF prevents renal injury caused by UUO and MMF could be used to limit the progression of renal fibrosis

Influence of ginsenoside Rg1, a panaxatriol saponin from Panax notoginseng, on renal fibrosis in rats with unilateral ureteral obstruction

Total saponins of Panax notoginseng （PNS） have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg 1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rgl on renal fibrosis in rats with unilateral ureteral obstruction （UUO）. The rats were randomly divided into 3 groups： sham-operation （n=15）, UUO （n=15） and UUO with ginsenoside Rgl treatment （n=15, 50 mg per kg body weight, intraperitoneally （i.p.） injected）. The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rgl significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition, u-smooth muscle actin （α-SMA） and E-cadherin are two markers of tubular epithelial-myofibroblast transition （TEMT）. Interestingly, ginsenoside Rgl notably decreased α-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA （mRNA） of transforming growth factor-β1 （TGF-β1）, a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg 1. Further study showed that ginsenoside Rgl considerably decreased the levels of both active TGF-β1 and phosphorylated Smad2 （pSmad2）. Moreover, ginsenoside Rgl substantially suppressed the expression of thrombospondin-1 （TSP-1）, a cytokine which can promote the transcription of TGF-β1 mRNA and the activation of latent TGF-β1. These results suggest that ginsenoside Rgl inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.

Effects of Losartan on Cell Apoptosis and Expression of Caspase-3 and JNK Proteins in Kidney Tissue in Renal Interstitial Fibrosis Rats

[Objectives]To investigate the effects of losartan on cell apoptosis and the expression of caspase-3 and JNK proteins in kidney tissue in the adenine-induced renal fibrosis rats.[Methods]Thirty Wistar rats were randomly divided into three groups:control group(n=10),model group(n=10)and losartan group(n=10).The rats in the control group received saline,while those in the model group and losartan group both received adenine by gavage,for 21 d.After the renal interstitial fibrosis model was established,the rats in the losartan group were treated with losartan[10 mg/(kg·d)],while the rats in the control group and the model group rats were administered with the same amount of saline.The course of treatment was 30 d.Finally,the renal function,blood urea nitrogen(BUN),serum creatinine(Scr),creatinine clearance rate(Ccr)and the pathological morphology of the rats were detected.The apoptosis of renal tubular epithelial cells was tested by TUNEL.The caspase-3 and JNK protein expression was tested by Western blotting.[Results]After administering adenine for 21 d,the BUN,24 MTP and kidney/body weight in the model group were increased,significantly higher than the control group(P<0.01),and the Ccr was remarkably decreased(P<0.01),signifying that the renal interstitial fibrosis model was successfully built.After treating with losartan for 30 d,the Scr,BUN,and 24 MTP were significantly decreased(P<0.01),and the Ccr was significantly increased in the losartan group(P<0.01).In addition,in comparison to the model group,renal tubular epithelial apoptosis was decreased and caspase-3 and JNK expression was downregulated in the losartan group(P<0.05).[Conclusions]Losartan can reduce the adenine-induced elevation of Scr,BUN and 24 hMPT,increase Ccr,improve general condition of renal interstitial fibrosis in rats and ameliorate the progression of chronic kidney failure(CKD).The effectiveness of losartan is probably due to its ability to regulate caspase-3,JNK protein expression and attenuate renal cell apoptosis.

CTRP1 Attenuates UUO-induced Renal Fibrosis via AMPK/NOX4 Pathway in Mice

Clq/TNF-related protein 1(CTRP1),a conserved protein of the Clq family,plays a key role in cardiovascular and metabolic diseases.However,the role of CTRP1 in renal injury is unclear.The purpose of this study is to explore the role of CTRP1 in unilateral ureteral obstruction(UUO)-induced renal fibrosis and to elucidate the underlying mechanism.Using gene delivery system,CTRP1 was overexpressed in the kidney,then the mice were operated to induce UUO model after adenovirus transfection.It was found that the expression of CTRP1 in the renal tissue was decreased in mice after UUO.CTRP1 overexpression decreased the kidney function and kidney weight index.Moreover,CTRP1 reduced oxidative stress and renal collagen deposition in vivo.As expected,we found that CTRP1 activated AMP-activated kinase(AMPK)and decreased NOX4 expression,while silencing AMPKal abolished the protective effects of CTRP1 overexpression in mice after UUO.In conclusion,CTRP1 may protect against UUO-induced renal injury via AMPK/NOX4 signaling.Our results indicate that CTRP1 exhibits potential effects to treat renal fibrosis caused by UUO.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Shenkang Ⅶ Recipe Attenuates Unilateral Ureteral Obstruction-induced Renal Fibrosis via TGF-β/Smad, NF-κB and SHH Signaling Pathway

This study aimed to explore the protective effects of the traditional Chinese Medicine formula Shenkang Ⅶ recipe(SK-7)on renal fibrosis and the mechanisms.Renal fibrosis was induced by unilateral ureteral obstruction(UUO)in rats.The rats were then divided into 5 groups:control group(Sham operation),UUO model group,UUO model plus low to high doses of SK-7(0.5,1.0,or 2.0 g/kg/day,for 14 days)groups.The animals were sacrificed on the 7th or 14th day.K idney tissues were collected for histopathological exarminations(hematoxylin and cosin and Masson's trichrome staining).Immunohistochemistry was used to detect the expression of collagen type II(Col II),fibronectin(FN),α-smooth muscle actin(a-SMA),TIMP metallopeptidase inhibitor 2(TIMP2),matrix metallopeptidase 2(MMP2),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and monocyte chemotactic protein-1(MCP-1).The TGF-β1/Smad,NF-κB and Sonic hedgehog signaling proteins were detected by Western blotting.Our results showed that SK-7 prevented UUO-induced renal injury and accumulation of collagen fibrils.Renal fbrosis biomarkers Col Ⅲ,FN,α-SMA and TMP2 were increased in the rats after UUO and decreased by SK-7,while MMP2 was upregulated after treatment.SK-7 also suppressed the levels of TNF-α,IL-1βand MCP-1 in UUO rats.In addition,SK-7 inhibited activation of the TGF-B/Smad,NF-κB and sonic hedgehog signaling(SHH)pathways.Taken together,these findings suggest that SK-7 may regulate the synthesis and degradation of extracellular matrix,reduce inflammation and suppress the proliferation of fibroblasts,by blocking the TGF-β1/Smad,NF-κB and SHH signaling pathways to exert its anti-renal fbrosis effect in UUO rats.

Peroxisome Proliferator-activated Receptor-γ Agonist Pioglitazone Fails to Attenuate Renal Fibrosis Caused by Unilateral Ureteral Obstruction in Mice

Renal tubulointerstitial fibrosis is the common ending of progressive renal disease. It is worth developing new ways to stop the progress of renal fibrosis. Peroxisome proliferator-activated receptor-γ（PPARγ） agonists have been studied to treat diabetic nephropathy, cisplatin-induced acute renal injury, ischemia reperfusion injury and adriamycin nephropathy. In this study, unilateral ureteral obstruction（UUO） was used to establish a different renal fibrosis model. PPARγ agonist pioglitazone was administrated by oral gavage and saline was used as control. At 7th and 14 th day after the operation, mice were sacrificed for fibrosis test and T lymphocytes subsets test. Unexpectedly, through MASSON staining, immunohistochemistry for α-SMA, and Western blotting for α-SMA and PDGFR-β, we found that pioglitazone failed to attenuate renal fibrosis in UUO mice. However, flow cytometry showed that pioglitazone down-regulated Th1 cells, and up-regulated Th2 cells, Th17 cells and Treg cells. But the Th17/Treg ratio had no significant change by pioglitazone. Real-time PCR results showed that TGF-β and MCP-1 had no significant changes, at the same time, CD4＋ T cells associated cytokines were partially regulated by pioglitazone pretreatment. Taken together, pioglitazone failed to suppress renal fibrosis progression caused by UUO.

基于糖原合成酶激酶-3β及内质网应激探讨Renalase抗肾脏纤维化的作用机制

目的 探讨糖原合成酶激酶-3β(GSK-3β)在Renalase抗肾脏纤维化过程中的作用及其可能的机制。方法 选用C57BL/6小鼠,予以下分组:(1)Sham+Ad-β-gal组;(2)Sham+Ad-Renalase组;(3)UUO+Ad-β-gal组;(4)UUO+Ad-Renalas组。观察GSK-3β表达的变化,其中UUO组为单侧输尿管结扎致肾脏纤维化模型组,Sham组为假手术对照组,Ad-Renalase为过表达Renalase腺病毒,Ad-β-gal为对照腺病毒。然后应用病毒转染技术上调(UUO+Ad-Renalase+AAV-GSK-3β组)及下调(UUO+Ad-Renalase+Ad-GSK-3β-RNAi组)GSK-3β后观察Renalase抗纤维化作用的变化。结果 与假手术(Sham)组相比,UUO组GSK-3β表达增加;与UUO+Ad-β-gal组相比,UUO+Ad-Renalase组纤维组织沉积减少、纤维化标志蛋白Col-Ⅰ和FN表达下降的同时GSK-3β表达下调;当上调GSK-3β后,Renalase抗纤维化作用被抵消,而当下调GSK-3β后,与UUO+Ad-Renalase组相比,UUO+Ad-Renalase+Ad-GSK-3β-RNAi组纤维化没有进一步改善。同时观察到与Sham组相比,UUO组内质网应激激活明显,过表达Renalase后,内质网应激被抑制。当加入内质网应激激动剂TM后,与UUO+Ad-Renalase组相比,UUO+Ad-Renalase+TM组纤维化加重,GSK-3β表达升高;但无论GSK-3β升高或者降低,内质网应激均无明显变化。结论 本研究证实在单侧输尿管结扎致肾脏纤维化过程中,Renalase通过抑制内质网应激下调GSK-3β表达从而延缓肾脏纤维化。

Ginkgo biloba extract inhibits Akt/mTOR signaling mediated renal fibrosis in diabetic nephropathy

Aim To investigate the effects of Ginkgo biloba extract （GbE） on renal fibrosis in STZ induced diabetic rats and high glucose （HG） cultured proximal tubular epithelial cells （NRK-52E）. Methods In vivo, rats were randomized into six groups termed normal control, diabetes mellitus , low dose of GbE （50 mg · kg^-1 · d^-1） , in- termediate dose of GbE （100 mg · kg^-1·d^-1）, high dose of GbE （200 mg · kg^-1 · d^-1） and rapamycin （1 mg·kg^-1·d^-l）. After 12 weeks, the rats were sacrificed and then fasting blood glucose, creatinine （Cr） , blood u- rea nitrogen （BUN） , urine protein, kidney index, glycogen and collagen accumulation, and collagen IV and lami- NRK-52E cells were divided into six groups： normal nin expression were measured by different methods. In vitro, glucose （5.56 mmol · L^-1）, high glucose （60 mmol · L^-1）, low dose of GbE （10 mg · L^-1）, intermediate dose of GbE （20 mg· L^-1）, high dose of GbE （40 mg· L^-1） and rapamycin （20 nmol · L^-l）. The morphological changes of cells were observed by microscopy after culturing for 48 h. Akt, roTOR and p70S6K, were examined by western blotting both in the renal cortex of rats and NRK-52E cells. Results Compared with diabetic rats, the lev- els of Cr, BUN, urine protein, kidney index, accumulation of glycogen and collagen, and expression of collagen IV and laminin in the renal cortex were all decreased in GbE treated rats. Furthermore, GbE ameliorated the morpho- logical changes of NRK-52E cells caused by HG. In addition, GbE reduced the expression of E-cadherin, oL-SMA, snail and phosphorylation of Akt, roTOR and p70S6K in diabetic renal cortexes and NRK-52E cells exposed to HG. Conclusion GbE was a satisfactory agent to prevent renal fibrosis in diabetic nephropathy, and this effect might be associated with the inhibition of the Akt/mTOR signaling pathway.

Kidney tissue targeted metabolic profiling of renal interstitial fibrosis rats induced by unilateral ureteral obstruction using NMR

Aim Renal interstitial fibrosis （RIF） is a common final pathological process in the progression of kid- hey disease. To investigate the pathogenesis of RIF and offer invaluable instructions for diagnosis and therapy treat- 1 ment of RIF. Method： H NMR based-metabolomics study on targeted kidney tissue of RIF rats induced by uni- lateral ureteral obstruction was conducted combined with multivariate data analysis to characterize the alteration of endogenous metabolites and elucidate the molecular mechanism of RIF. Results The combination of a variety of statistical methods was used to screen out 14 potential significantly changed metabolites, including increased levels of lactate, methionine, aspartate, allantoin, uracil, 3-HB and decreased levels of TMAO, leucine, valine, lysine, adenosine, adenine, tyrosine and phenylalanine in the left kidney of UUO rats, compared with SO rats. To gain ad- ditional insight about the relationship between metabolites, they were mapped to KEGG IDs and built compound network by Metscape reflecting the complex pathology and providing evidence for the involvement of such processes as altered amino acid metabolism, adenine metabolism, energy metabolism, osmolyte change and induced oxidative stress. In addition, we have explored the morphology and size, calculated the degree of fibrosis based on altered differential metabolites, and speculated the probable causes of moderate RIF of contralateral kidneys to help to un- derstand the disease, which was also supported by serum biochemistry and kidney histopathology results. In addi- tion, the correlation analysis of the pathological parameters （ clinical chemistry, histological and immunohistochem- istry results） with the significantly changed differential metabolites responsible for the cluster （different groups） was also performed. Conclusion Our work shows that target tissue metabolomics analysis can be used as a power-ful tool to gain a better understanding of the mechanism of the disease and provide a novel insight in the pathogene- sis of RIF.

Effects of Manshenkangning Prescription on Adenine-induced Renal Interstitial Fibrosis in Rats

[Objectives]To study the protective effects of Manshenkangning Prescription on adenine-induced renal interstitial fibrosis in rats,and explore the possible mechanism.[Methods]Sixty Wistar male rats were divided into normal group,model group,control group(administered with 10 mg/(kg·d)losartan)and high,medium and low dose experimental groups(30,15,7.5 mg/(kg·d)Manshenkangning).The rat models of renal interstitial fibrosis were induced by intragastric administration of adenine(250 mg/(kg·d)).After 2 h,the above drugs were administered intragastrically for 21 consecutive days and the administration time was 30 consecutive days.Serum creatinine(SCr),blood urea nitrogen(BUN),24 h urinary protein(24 h MTP)and glomerular filtration rate(eGFR)were measured by biochemical method;renal histopathological changes were observed by hematoxylin-eosin(HE)staining.Renal collagen deposition in rats was observed by Masson staining.[Results]The SCr in model group and the high,medium and low dose experimental groups were(340.00±22.99),(176.80±18.60),(234.75±13.59),(266.11±14.78)μmol/L,and BUN were(23.74±2.51),(14.53±2.25),(18.78±0.88),(18.90±2.14)mmol/L;24 h MTP were(675.86±74.58),(323.81±41.83),(438.84±34.69),(493.76±37.04)mg/d;eGFR were(19.30±2.48),(49.96±10.95),(32.61±10.75),(27.18±5.98)mL/min,and the difference was statistically significant compared with the normal group(all P<0.05).HE staining and Masson staining showed that compared with normal group,the renal interstitial lesions in model group were severe and the renal interstitial collagen material was deposited in a large amount.The renal interstitial tubule injury was relieved and the renal interstitial collagen deposition was reduced in experimental groups.And the difference was statistically significant(all P<0.01).[Conclusions]Manshenkangning can significantly protect the kidney against the progress of interstitial fibrosis in rats.Its possible mechanism is to regulate the activity of SIRT1 and inhibit the expression of COX-2 in order to resist the inflammatory reaction of kidney and improve the ability of anti-oxidative stress of kidney,thus delaying the occurrence and development of chronic renal failure.

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.