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.

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.

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.

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.

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.

Effects of Yishenbupi (Tonifying-Kidney and Invigorating-Spleen) prescription on renal fibrosis

Background:This study aims to observe the effects of Yishenbupi(Tonifying-Kidney and Invigorating-Spleen)decoction on renal fibrosis of unilateral ureteral occlusion rats.Methods:Forty-eight sprague-dawley rats were randomly divided into the sham-operated group(sham group),model group,irbesartan group,and Yishenbupi group.Each group was intragastrically administered after the unilateral ureteral occlusion model was established.Rats in the Yishenbupi group were intragastrically administered with Yishenbupi decoction(18 g/kg/d)once every morning.Rats in the irbesartan group were intragastrically administered with 10 mg/kg/d of irbesartan tablets once every morning.Rats in the sham group and model group(unilateral ureteral occlusion group)were intragastrically administered with isovolumetric distilled water twice a day from the day the model was established.All rats were sacrificed 21 days later.Occluded kidney tissues were taken,and pathological sections were prepared.Masson,periodic acid-Schiff,Sirius Red,and immunohistochemical staining were performed to detect the expression of collagen III and fibronectin.Results:The pathological staining of rat kidneys(Masson,periodic acid-Schiff,and Sirius Red)showed that,compared to the unilateral ureteral occlusion model group,the renal interstitial injury was eased and collagen deposition was reduced in the irbesartan and Yishenbupi groups;after immunohistochemical staining,the expression of collagen III and fibronectin positively expressed cells was decreased and decreased more in the Yishenbupi group than in the irbesartan group.Conclusion:Yishenbupi decoction can alleviate the injury to kidney tissues in rats with unilateral ureteral obstruction,reduce the deposition of extracellular matrix,and act against renal fibrosis.

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.

MiR-93-5p in BM-MSCs-derived exosomes amelio­rates renal fibrosis by affecting macrophage polar­ization

MiRNAs and macrophages play important roles in renal fibrosis.The exosomes secreted by bone marrow mesenchymal stem cells(BM-MSCs)can alleviate renal fibrosis.What is not clear,however,is whether a type of miRNAs in the BM-MSCs exosomes can alleviate renal fibrosis by modulating macrophage polarization.First,we take a high-throughput sequencing of miRNAs in exo­somes of BM-MSCs from chronic kidney disease(CKD)and normal people.Then we used the UUO mouse model and injected exosomes into the tail vein.The macrophages were stimulated with lipopolysaccharide(LPS).MSC-Exo or exosomes from BM-MSCs transfected with miR-93-5p inhibitor(Inhi-Exo)were added to the culture medium.The macrophages were transfected with miR-93-5p inhibitor or miR-93-5p mimic alone.The expression of miR-93-5p in exosomes of CKD patients was significantly decreased compared with normal people and in the LPS-stimulated macrophages and UUO mice kidneys.After stimulation with LPS,the macrophages polarized toward M1 subtype.MSC-Exo or miR-93-5p mimic promoted macrophages from M1 to M2 sub­type.Inhi-Exo or miR-93-5p inhibitor blocked the differentiation from M1 to M2 subtype.Significant fibrotic changes occurred in the kidneys of UUO mice,and M1 macrophages were significantly increased.After injecting exosomes into the tail vein of UUO mice,the degree of renal fibrosis was alleviated,the expression of miR-93-5p in the kidney was significantly increased,and the renal macrophages differentiated from M1 to M2 subtype.These results demonstrated that miR-93-5p in the exosomes derived from BM-MSCs can improve renal fibrosis by inducing macrophage differentiation from M1 to M2 subtype.

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.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction(四逆汤)against renal fibrosis

OBJECTIVE:To investigate the mechanism by which Sini decoction(四逆汤,SND)improves renal fibrosis(Rf)in rats based on transforming growth factor β1/Smad(TGF-β1/Smad)signaling pathway.METHODS:Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf.The targets of SND drug components and the targets of Rf were obtained by searching databases,such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCSMP)and GeenCard.The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram.Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-β1/Smad signaling pathway.The Rf rat model was established by unilateral ureteral occlusion(UUO).The expression levels of transforming growth factor,matrix metalloproteinase-9(MMP-9),matrix metal protease-2(MMP-2),connective tissue growth factor(CTGF),and tissue inhibitor of metalloproteinase-1(TIMP-1)were determined by Masson staining of rat renal tissue,and immunohistochemical methods.The expression levels of Smad3,Smad2,and Smad7 in renal tissue were determined by Western blotting(WB).The mechanism of the improving effects of SND on Rf was investigated based on TGF-β1/Smad signaling pathway.RESULTS:A total of 12 drug components of Fuzi(Radix Aconiti Lateralis Preparata),5 drug components of Ganjiang(Rhizoma Zingiber),and 9 drug components of Gancao(Radix Glycy et Rhizoma)were obtained from the database search,and 207 shared targets were found.A total of 1063 Rf targets were found in the database search.According to the Venn diagram,in total,96 intersection targets were found in two database searches.The metabolic pathways involved included TGF-β signaling pathway,phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling(PI3K/Akt)pathway,and hypoxia-inducible factor-1(HIF-1)signaling pathway.Masson staining analysis showed that compared with the model group,the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower(P<0.05).Immunohistochemical analysis,compared with the control group,the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased(P<0.05,P<0.01),and the positive cell area expression levels of CTGF and TGF-β1 were significantly increased(P<0.01).Compared with the model group,the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased(P<0.05,P<0.01),and the positive cell area expression levels of CTGF and TGF-β1 in the kidney tissue were significantly decreased(P<0.05,P<0.01).WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3(P<0.05)and increase the expression of Smad7(P<0.05).

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.

Effects of advanced glycation end products on renal fibrosis and oxidative stress in cultured NRK-49F ceils

Background Advanced glycation end products （AGEs） play a critical role in the development of diabetic nephropathy. Reactive oxygen species （ROS） may play a critical role in AGEs induced growth factor expression. In this study, the effects of AGEs on transforming growth factor β1 （TGF-β1）, connective tissue growth factor （CTGF） and fibronectin （Fn） mRNA expression and oxidative stress in cultured NRK-49F cells were examined. Methods NRK-49F cells were incubated with medium containing different doses of AGEs （50, 100 or 200 μg/ml） for 24 hours, or with AGEs 100 μg/ml for different times （0, 12, 24 or 48 hours）. Cells in the serum-free medium or medium containing 25 mmol/L glucose were controls. Cells were treated with 25 mmol/L glucose and 100 μg/ml AGEs for 24 hours to determine the effects between AGEs and glucose. We clarified the role of antioxidant by pretreating cells with N-acetylcysteine （10 mmol/L）, ginkgo biloba extract （50 or 100 mg/L） for 24 hours and with 100 μg/ml AGEs for further 24 hours. Alamarblue dye assay was used to analyze cell growth; intracellular ROS generation was measured by flow cytometry; intracellular glutathione by fluorescence spectrophotometry; expressions of TGF-β1, CTGF and Fn mRNA by semiquantitative RT-PCR. Results AGEs significantly increased the expressions of TGF-β1, CTGF, Fn mRNA and intracellular ROS generation, and decreased the glutathion level in NRK-49F cells in dose- and time-dependent manners. High glucose and AGEs together significantly increased the expression of TGF-β1, CTGF and Fn mRNA, compared with AGEs and high glucose separately. Preincubation with N-acetylcysteine or ginkgo biloba extract increased GSH level, suppressed AGEs-induced oxidative stress and TGF-β1, CTGF and Fn mRNA overexpression. Conclusions AGEs can significantly increase expression of TGF-β1, CTGF, Fn mRNA in NRK-49F cells through enhancement of oxidative stress. The accumulation of AGEs may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Suppression of AGEs induced TGF-β1, CTGF and Fn mRNA overexpression in renal fibroblasts through inhibition of oxidative stress may be a mechanism underlying effect of ginkgo biloba extract in diabetic nephropathy. In addition, antioxidant therapy may help prevent AGEs accumulation and its induced damage.

Monitoring the Progression of Renal Fibrosis by T2-weighted Signal Intensity and Diffusion Weighted Magnetic Resonance Imaging in Cisplatin induced Rat Models

Background：Diffusion weighted imaging （DWI）,with the applying of intravoxel incoherent motion model,has showed promising results in obtaining additional information about microperfusion and tubular flow associated with morphologic changes in chronic kidney diseases.The study aims to evaluate the potential of T2-weighted signal intensity （SI） and DWI with mono-and bi-exponential models to reflect the serial changes on cisplatin （CP） induced rat renal fibrosis models.Methods：Magnetic resonance exams were performed prior to and 2nd day,4th day,6th day,8th day,2nd week,3rd week and 4th week after CP injection at a 3.0T with an animal coil.Besides T2-weighted images （T2WI）,DWI of 13 b values from 0 to 1500 s/mm^2 was acquired.Apparent diffusion coefficient （ADC）,fluid fraction f,pure diffusivity D and pseudodiffusivity D＊ values were calculated.The regions of interest were placed on cortex （CO）,outer stripe of the outer medulla （OM） and inner stripe of the outer medulla （OM）,parameters were measured and compared among different time points.Five rats were scarified at each time point for pathological examination.Results：OM revealed remarkable hyperintense and broadened before it became an obscure thread,while CO demonstrated moderate hyperintense and IM didn＇t show significant change on T2WI.On all three stripes,ADC values decreased firstly then kept increasing since the 4th day;f values decreased on all stripes; D values had a tendency to increase with fluctuations but the changes didn＇t achieve statistical significance; D＊ values increased at the 2nd day then tended to be steady thereafter.Pathological findings revealed tubules epitheliums swelling followed by inflammation cells infiltration,interstitial fibrosis was observed since the 2nd week.Conclusions：All of T2-weighted SI,ADC,and biexponential models parameters vary during fibrotic process; biexponential model is superior to monoexponential model in separating changes of microperfusion together with tubular flow from pure diffusion.

An injectable micelle-hydrogel hybrid for localized and prolonged drug delivery in the management of renal fibrosis

Localized delivery,comparing to systemic drug administration,offers a unique alternative to enhance efficacy,lower dosage,and minimize systemic tissue toxicity by releasing therapeutics locally and specifically to the site of interests.Herein,a localized drug delivery platform ("plum?pudding"structure) with controlled release and long-acting features is developed through an injectable hydrogel ("pudding") crosslinked via self-assembled triblock polymeric micelles ("plum") to help reduce renal interstitial fibrosis.This strategy achieves controlled and prolonged release of model therapeutics in the kidney for up to three weeks in mice.Following a single injection,local treatments containing either anti-inflammatory small molecule celastrol or anti-TGFb antibody effectively minimize inflammation while alleviating fibrosis via inhibiting NF-k B signaling pathway or neutralizing TGF-b1 locally.Importantly,the micelle-hydrogel hybrid based localized therapy shows enhanced efficacy without local or systemic toxicity,which may represent a clinically relevant delivery platform in the management of renal interstitial fibrosis.

Role of reactive oxygen species in the renal fibrosis

Renal fibrosis is a common pathway of progressive renal diseases leading to end-stage renal disease regardless of the etiology. Accumulating evidence indicates that oxidative stress, resulting in generation of reactive oxygen species （ROS）, plays a critical role in the initiation and progression of fibrotic diseases. Nicotinamide adenine dinucleotide phosphate （NADPH） oxidase is the predominant enzyme source for ROS generation and is now recognized as a key mediator of cell proliferation and matrix accumulation in renal disease. Multiple stimuli and agonists, such as transforming growth factor , tumor necrosis factor, platelet derived growth factor, angiotensin II, hyperglycemia, oxidized low-density lipoprotein and albumin have been shown to alter the activity or expression of the NADPH oxidase and ultimately increase ROS production. ROS directly incites damage to biologically important macromolecules and leads to generation of the so-called advanced oxidation protein products （AOPPs） and advanced glycation end products, which are not only markers of oxidative stress but also cause renal injury. Targeting NADPH oxidase and/or reducing AOPPs production miaht be a novel strateav for the theraoeutic intervention of varietv of fibrotic kidney disorders.

Efficacy of Danggui Buxue decoction(当归补血汤)on diabetic nephropathy-induced renal fibrosis in rats and possible mechanism

OBJECTIVE:To observe the efficacy of Danggui Buxue decoction(当归补血汤,DBD) on diabetic nephropathyinduced renal fibrosis in rats,and to study the possible mechanism.METHODS:Sixty male Goto Kakizaki(GK) rats were randomly assigned to the model group,gliquidone group,astragaloside Ⅳ group,and high-,medium-and lowdoses DBD groups.After 8 weeks,changes in body weight,blood glucose,serum creatinine,serum urea nitrogen,and total cholesterol were observed.Changes in transforming growth factor-β1(TGF-β1),Smad3,and Smad5 pathways and the expression of the fibrosisrelated proteins collagen Ⅳ(col Ⅳ),α-smooth muscle actin(α-SMA),and vimentin were assessed.The degree of renal fibrosis was observed by immunohistochemistry and Mason staining.The expression of interleukin 6(IL-6),interleukin 10(IL-10),tumor necrosis factor(TNF-α),and C-reactive protein(CRP) in the kidneys was assessed using enzyme linked immunosorbent assay.RESULTS:Our experiments showed that DBD effectively reduced blood glucose,blood urea nitrogen,and creatinine levels after 8 weeks of administration,improved renal function in diabetic rats,alleviated renal fibrosis,and reduced the renal tissue levels of IL-6,IL-10,TNF-α,and CRP.Furthermore,DBD decreased the expression of TGF-β1,Smad3,col IV,α-SMA,and vimentin in renal tissues and increased the expression of Smad5.CONCLUSIONS:DBD ameliorates diabetic renal interstitial fibrosis by modulating the TGF-β1/Smads pathway.

Arterially transplanted mesenchymal stem cells in a mouse reversible unilateral ureteral obstruction model： in vivo bioluminescence imaging and effects on renal fibrosis

Background Chronic kidney disease （CDK） is a worldwide health problem, but there is currently no effective treatment that can completely cure this disease. Recently, studies with mesenchymal stem cells （MSCs） on treating various renal diseases have shown breakthroughs. This study is to observe the homing features of MSCs transplanted via kidney artery and effects on renal fibrosis in a reversible unilateral ureteral obstruction （R-UUO） model. Methods Thirty-six Balb/c mice were divided into UUO group, UUO-MSC group, and sham group randomly, with 12 mice in each group. The MSCs had been infected by a lentiviral vector to express stably the luciferase reporter gene and green fluorescence protein genes （Luc-GFP-MSC）. Homing of MSCs was tracked using in vivo imaging system （IVIS） 1, 3, 14, and 28 days after transplantation. Imaging results were verified by detecting GFP expression in frozen section under a fluorescence microscope. E-cadherin, α-SMA, TGF-β1, and TNF-α mRNA expression in all groups at 1 and 4 weeks after transplantation were analyzed by quantitative PCR. Results Transplanted Luc-GFP-MSCs showed increased Luciferase expression 3 days after transplantation. The expression decreased from 7 days, weakened thereafter and could not be detected 14 days after transplantation. Quantitative PCR results showed that all gene expressions in UUO group and UUO-MSC group at 1 week had no statistical difference, while at 4 weeks, except TGF-β expression （P〉0.05）, the expression of E-cadherin, α-SMA, and TNF-α in the above two groups have statistical difference （P〈0.01）. Conclusion IVIS enables fast, noninvasive, and intuitive tracking of MSC homing in vivo. MSCs can be taken home to kidney tissues of the diseased side in R-UUO model, and renal interstitial fibrosis can be improved as well.

Human dendritic cell-specific ICAM-3-grabbing non-integrin downstream signaling alleviates renal fibrosis via Raf-1 activation in systemic candidiasis

We generated a human dendritic cell-specific ICAM-3-grabbing non-integrin(DC-SIGN)transgenic mouse in which renal tubular epithelial cells expressed DC-SIGN.The transgenic mice were infected with Candida albicans intravenously to study how DC-SIGN expression affected the pathogenesis of systemic candidiasis.We discovered that,while C.albicans infection induced renal fibrosis in both transgenic and littermate control mice,the transgenic mice had significantly lower levels of Acta2,Col1a2,Col3a1,and Col4a1 mRNA transcripts compared to the controls.KIM-1,an emerging biomarker for kidney injury,along with Tnf,Il6,and Tgfb1 transcripts,were lower in infected transgenic mice,and yet,the levels of Il10 remained comparable to the controls.While renal CD45+infiltrating cells were the source of Tnf,Il6,and Il10,LTL+renal proximal tubular epithelial cells were TGF-β1 producers in both infected transgenic and littermate controls.DC-SIGN-expressing tubular epithelial cells produced less TGF-β1 in response to C.albicans infection.In vivo experiments demonstrated that renal proximal tubular epithelial cell production of TGF-β1 was key to C.albicans-induced renal fibrosis and injury.Infection of transgenic mice induced a marked increase of phosphorylated Raf-1 and p38 in the kidney.However,ERK1/2 and JNK phosphorylation was more pronounced in the infected-littermate controls.Interestingly,treating the infected transgenic mice with a Raf-1 inhibitor increased the levels of the Tgfb1,Kim1,and Acta2 transcripts.These results indicate that DC-SIGN signaling,through activation of Raf-1 and p38 and suppression of JNK and ERK1/2 phosphorylation,reduces TGF-β1 production and C.albicans-induced renal fibrosis.Our study reveals for the first time the effect of DC-SIGN expression on C.albicans-induced renal fibrosis.