| Background: Renal fibrosis is a common result of multiple chronic kidney disease（CKD）development to the end-stage,mainly including glomerular fibrosis,renal vascular fibrosis and renal interstitial fibrosis.In recent years,with the continuous development of society,it has become one of the major public health problems affecting human health.Renal fibrosis present variety of complex clinical manifestations at different stages,including glomerular sclerosis,atrophy and abnormal dilation of renal tubules,reduced capillaries around renal tubules and hyperplasia of tubulointerstitial fibers.Renal fibrosis could be caused by a variety of reasons,such as trauma,infection,inflammation,blood circulation disorders,autoimmune reaction,etc.,the kidney is stimulated after the inherent cell damage inducing a large amount of collagen deposition and accumulation,then further resulting in renal parenchyma hardening,scar formation,until completely loss of organ function.The pathogenesis of renal fibrosis is so complex that there is no unified conclusion of it,limited by the lack of effective treatment drugs and poor prognosis,most of the advanced patients had to rely on renal transplantation to prolong the survival period.D-glucuronyl C5-epimerase is a widely distributed and evolutionarily conserved modified catalytic enzyme encoded by Glce gene.The function of Glce isomerizing glucuronic acid to iduronic acid on Heparin sulfate（HS）chains can enrich the conformation of sugar chain,which is beneficial to molecular signal transduction and contains important biological significance.Glce is a highly conserved single gene enzyme,which is lethal when the whole genome is knocked out in mouse embryos.It is also associated with renal,lung,bone and other organ tissue hypoplasia,suggesting that Glce plays an extremely important role in the process of biological growth and development.Previous studies have found that renal-specific Glce knockout affects the normal development of the kidney,but whether it`s involved in the regulation of the progression of renal fibrosis disease is unknown.Early laboratory studies have shown that plant polysaccharides from Panax notoginseng can inhibit the activation of hepatic stellate cells induced by TGF-β,indicating that it can significantly antagonize fiber formation.However,the protective effect of Panax notoginseng polysaccharides on renal fibrosis disease has not been reported,and further study is needed.Objective: In order to clarify the important role that Glce may play in the normal operation of the kidney and the development of renal fibrosis disease,and to explore whether Panax notoginseng polysaccharide has a certain protective and intervention effect on renal fibrosis,we conducted an in-depth study on its internal molecular regulatory mechanism.This study mainly aims to research D-Glucuronyl C5-epimerase mediated molecular signaling pathway which plays an important part in the formation process of renal fibrosis in vivo and in vitro.On this basis,we discussed the protective effects and the related mechanism of polysaccharide SQD4S2 against abnormal differentiation effect on the renal tubule cells induced by TGF-β and provided strategy for clinical diagnosis and treatment of renal fibrosis.The study also provided experimental basis for the potential application of Panax notoginseng polysaccharide in the improvement and treatment of renal fibrosis.Methods: 1.Some renal tissues of patients with different types of nephropathy were selected,and Masson staining was used to show the profile of collagen expression and deposition in renal tissues.Immunohistochemical experiments were performed to determine the total expression and distribution of Glce protein in human renal tissues,and to observe whether there was any difference in Glce expression between normal kidney and damaged kidney.The model of renal fibrosis in mice was induced by Unilateral ureteral obstruction（UUO）operation and intraperitoneal injection of Folic Acid（FA）.The renal fibrosis model was evaluated by pathological analysis of kidney tissues and immunofluorescence analysis.At the same time,the expression of Glce gene and protein in the renal tissues of normal and renal fibrosis model mice were detected to evaluate its role in renal fibrosis.2.Renal tubule cells Glce-specific knockout mice were constructed by Cre/ loxp gene recombination technology,and the renal tissue organ ratio of mice was measured to evaluate the growth and development status of the kidney.Glce kidney specific knockout(Glce-/-)mice and wild-type mice were treated with UUO surgery to induce renal fibrosis model.The degree of kidney injury and fibrosis development of the animals were determined by animal serum biochemical indexes detection,renal histological analysis,Western blot,and real-time PCR,and on this basis the important role of Glce in the formation of renal fibrosis was evaluated.3.Proteomic analysis techniques were used to extract and compare the differences in protein expression levels in the tissues of Glce kidney specific knockout(Glce-/-)mice and wild-type mice,so as to identify and summarize the specific renal functional areas that may be affected by Glce deletion.4.In Human renal tubular epithelial cells（HK-2 cells）,lentiviral infection was used to knock out or overexpress Glce gene.3-（4,5-dimethylthiazole-2）-2,5-diphenyltetrazolium bromide(3-（4,5-dimethyl-thiazol-2-yl）2,5-diphenyltetrazolium bromide（MTT）assay was used to detect the effect of Glce on the growth viability of normal HK-2 cells.The effects of Glce deletion or overexpression on TGF-β signaling pathway activation and EMT mediated by epithelial-mesenchymal transition（EMT）in animal kidney tissues and cultured renal tubular Epithelial cells were observed by western blot and real-time PCR.5.Renal fibrosis was induced in Glce kidney specific knockout(Glce-/-)mice and wild-type mice by UUO surgery,and the TGF-β1 receptor inhibitor SB431542 was intraperitoneally injected to prevent the activation of TGF-β signaling pathway.After the materials were collected,biochemical indexes of animal serum were detected as well as the pathological section staining of kidney tissue.In addition,renal immunohistochemical labeling,Western blot,and real-time PCR were performed to investigate whether the effects of Glce on the development of kidney injury and fibrosis in animals were mainly through the regulation of TGF-β signaling pathway.6.The Glce gene was knockdown or overexpressed by lentivirus infection in human normal renal tubular epithelial cells（HK-2 cells）,and the effects of Glce deletion or overexpression on EGFR expression and activation of downstream signaling pathway were detected by Western blot and Real-time PCR.The expression and distribution of Glce and EGFR in HK-2 cells were observed by immunofluorescence technique,and the interaction between Glce and EGFR in HK-2 cells was explored by coimmunoprecipitation（CO-IP）and surface plasma-resonance（SPR）experiments.7.Glce kidney specific knockout(Glce-/-)mice and wild-type mice were induced by UUO surgery to form renal fibrosis models,and EGFR signaling pathway activation was inhibited by the EGFR tyrosine kinase receptor inhibitor Erlotinib orally.Serum biochemical indexes,pathological sections of renal tissues and renal morphology were detected.Then Western blot and Real-time PCR experiments were used to explore whether Glce could mediate activation of EGFR signaling pathway to regulate renal injury and fibrosis formation and whether inhibition of EGFR signaling pathway activation has any effect on TGF-β expression and pathway activation.8.At the age of 6 weeks,the adeno-associated virus（AAV）vector with Glce gene fragment and GFP fragment was introduced into the renal pelvis of Glce-/-mice by in situ injection.6 weeks later,UUO surgery was performed to induce renal fibrosis.The successful introduction and expression of AAV vectors were observed by two-photon confocal microscopy and immunofluorescence technique.The degree of kidney injury was evaluated by animal serum biochemical index detection,renal tissue pathological analysis,renal tissue morphology analysis and fibrosis related protein expression detection.Thereby the important role of Glce in regulating TGF-β and EGFR signaling pathway in the formation of renal fibrosis was verified by Western blot,Real-time PCR and Co-IP methods to further observe whether Glce protein supplementation could significantly improve renal injury.9.In the stable HK-2 cell lines with Glce knockdown,the plasmid of site-directed mutated Glce gene（The mutation sites were Y500 F,Y560F and Y578F）was introduced by lentiviral infection technology to re-express Glce protein that had lost its enzyme activity function,then the activation degree of EGFR signaling pathway and the expression and localization of Glce protein in cells were observed.At the age of 6 weeks,Glce kidney specific knockout(Glce-/-)mice were injected in situ into the renal pelvis with a vector carrying GFP fragment adeno-associated virus（AAV）,and the Glce gene fragment with site-directed mutagenesis was introduced to cause the loss of enzyme activity.6 weeks later,UUO model was made to induce renal fibrosis.Two-photon confocal microscope immunofluorescence technique was used to observe the express condition of AAV virus carrier.The degree of kidney injury was evaluated by serum biochemical index detection,renal tissue pathology analysis,renal tissue morphology analysis and fibrosis related index protein expression detection.Moreover,Western blot and real-time PCR were used to investigate whether the effect of Glce enzyme activity on TGF-β and EGFR signaling pathway activation interfered with the formation and development of renal fibrosis.10.MTT assay was used to detect the effects of different concentrations of Panax notoginseng polysaccharide SQD4S2 on the growth activity of human normal renal tubular epithelial cells（HK-2 cells）;After TGF-β-induced cell transdifferentiation,the effect of SQD4S2 on the morphology of HK-2 cells was observed under microscope,and the molecular mechanism of SQD4S2’s improvement of TGF-β-induced HK-2 cell fiber proliferation was further explored by Western blot and Real-time PCR experiments.Results: 1.Compared with normal human tissues,Masson staining showed a large amount of collagen deposition and renal unit destruction in renal tissues of patients with nephropathy,and Glce protein labeled by immunohistochemical staining was significantly reduced.The mouse renal fibrosis model was successfully induced by UUO operation or intraperitoneal injection of folic acid.In the renal tissue of the affected mice,there were atrophy,death and abnormal dilation of renal tubules,thickened glomerular basement membrane,and collagen hyperplasia in the inter tubular space.The expression of Glce gene and protein in fibrosis model mice decreased significantly with the aggravation of the disease and the protein level decreased in a time dependent manner.At the same time,alpha-smooth muscle actin（α-SMA）increased obviously.These results indicated that UUO surgery or FA administration successfully induced renal fibrosis in mice,and Glce played an important role in the process of renal fibrosis.2.Compared with wild-type mice,Glce-specific renal tubule knockout mice showed significantly lower organ coefficient ratio and delayed renal development.After UUO operation,the kidney injury and collagen deposition of the knockout mice were more serious,the Serum creatinine（Scr）,Blood Nitrogen（BUN）,and the levels of α-SMA、Fn、Col1、Col3、tgfb1 genes and proteins related to fibrosis were higher than those of the wild model mice.Taken together,these results suggest that Glce deficiency significantly aggravates the progression of renal injury and fibrosis.3.Significant differences in proteomics Protein cluster analysis results showed that specific renal Glce knockout could affect the expression of extracellular space related proteins,hemorheological procoagulation-related proteins,and extracellular transmembrane related proteins,suggesting that the biological function of Glce in the kidney may be related to such proteins.4.MTT assay results showed that Glce knockdown or overexpression did not affect the cell viability of human renal tubular epithelial cells.Glce knockdown significantly upregulated the expression levels of TGFBR1 and Smad3 genes and downregulated the expression levels of Smad2 genes,while overexpression of Glce was the opposite.After UUO modeling of Glce-/-mice,the phosphorylation degree of Smad2/3 protein in kidney tissue of wild-type mice was significantly higher than that of EMT-related Snail,Slug,and N-cadherin genes and proteins.TGF-β stimulated HK-2 cells significantly decreased Glce gene and protein levels,and Glce knockdown significantly upregulated TGFBR1 and Smad2/3 phosphorylation levels,as well as TGF-β-regulated downstream EMT-related proteins Snail,Slug,N-cadherin levels.SIS3,a Smad2/3 phosphorylation inhibitor,could not eliminate the TGF-β-induced difference in EMT-related protein expression between the Glce-knockdown group and the control group,while the TGFBR1 phosphorylation activation inhibitor SB431542 could suppress it.These results suggested that Glce deficiency promoted the phosphorylation and activation of TGFBR1,which mediated the activation of TGF-β signaling pathway and accelerated the EMT process.5.Compared with wild-type mice,intraperitoneal injection of TGFBR1 phosphorylation inhibitor SB431542 after UUO model in Glce-/-mice significantly inhibited the abnormal increase of serum creatinine and urea nitrogen,reduced the expression of fibrosis related proteins COL1,Fibronectin and α-SMA,and improved renal damage and collagen deposition.Also,TGF-β signaling pathway activation was inhibited and the expression levels of EMT-related gene or protein were abnormally increased.But in the operation group,SB431532 could only inhibit the activation of TGF-β signaling pathway and the expression of EMTrelated molecular genes or proteins,and could not relieve kidney injury and fibrosis in mice.These results suggest that inhibition of TGFBR1 receptor activation can improve the aggravation of Glce deficiency-induced fibrosis,suggesting that Glcemediated activation of TGF-β signaling pathway plays an important role in the development of renal fibrosis.6.Glce knockdown in human renal tubule HK-2 cells promoted EGFR gene expression,while overexpression of Glce had no significant effect on EGFR gene expression.Also,Glce knockdown promoted EGFR Y1068 tyrosine site and downstream MEK and ERK1/2 protein phosphorylation,while EGFR kinase inhibitor Erlotinib significantly inhibited abnormal activation of EGFR signaling pathway in Sh Glce group,while overexpression of Glce had no significant effect on EGFR signaling pathway.Immunofluorescence showed that the expression regions of EGFR protein and Glce protein coincided to a certain extent.Co-IP assay showed that there was an interaction between Glce and EGFR in HK-2 cells,and Glce deficiency induced increased activation of EGFR phosphorylation.SPR assay further showed that Glce protein and EGFR could bind to the intracellular domain of EGFR protein,and there was a direct interaction(Kd =1.243×10-8)and interact directly with each other.These results suggest that Glce can directly bind to EGFR to play a regulatory role,and Glce deficiency induces abnormal phosphorylation and activation of EGFR,which may have important significance in the development of renal fibrosis.7.Wild type mice with Glce-/-mice oral lavage EGFR tyrosine kinase receptor inhibitor Erlotinib can significantly inhibit abnormally elevated serum creatinine and urea nitrogen caused by UUO surgery.Erlotinib could also inhibit the expression of protein involved in inhibiting fibrosis formation such as COL1、Vimentin、α-SMA、TGF-β,improve renal cell injury and collagen deposition as well as the activation of EGFR and TGF-β signaling pathways,and abnormal increase of EMT related gene protein expression.These results indicate that inhibition of EGFR signaling pathway activation can affect TGF-β signaling pathway activation and improve the aggravation of fibrosis induced by Glce deficiency,suggesting that Glce-mediated activation of EGFR signaling pathway plays an important role in the development of renal fibrosis.8.Immunofluorescence results show that the GFP spontaneous green fluorescent recombinant protein can be observed under the condition of 488 nm excitation wavelength protein,in addition,red fluorescent protein markers Glce was observed under the condition of 647 nm excitation wavelength in AAV-m Glce control group mice kidney tissues.While only green fluorescence of GFP can be observed in the negative control AAV-Zs G1 group and it show that mice in AAV-m Glce control group kidney successfully re-express Glce protein.Compared with Glce-/-mice in the AAV-ZSG1 control group,the re-expression of Glce protein in the AAV-m Glce group significantly improved the increase of serum creatinine and urea nitrogen induced by UUO operation model,reduced the area of renal tissue fibrosis,and alleviated the damage of renal cells and collagen deposition.Furthermore,the abnormally elevated levels of COL1,Vimentin,α-SMA,Fibronectin genes and proteins associated with fibrosis were significantly down-regulated,the activation of EGFR and TGF-β signaling pathways and the abnormally increased expression of EMT-related genes were also significantly inhibited.Co-IP assay showed that the activation of EGFR protein phosphorylation in kidney tissues of mice after UUO operation was increased and the binding with Glce protein was significantly decreased compared with normal mice,but the level of EGFR phosphorylation was significantly down-regulated after the re-expression of Glce protein.These results suggest that renal supplementation Glce inhibits the activation of EGFR and TGF-β signaling pathways,thereby inhibiting renal injury and the progression of renal fibrosis.9.The EGFR signaling pathway was not abnormally activated compared with the Sh Glce control group after the mutation of Glce-catalyzed active sites Y500,Y560 and Y578 gene sequences,and Glce protein expressed both in Golgi and cytoplasm in normal HK-2 cells,while the mutagen-induced enzyme function loss had no effect on the expression or distribution of Glce in HK-2 cells.GFP green fluorescence could be observed in Glce-/-mice after introducing the negative AAV vector,at this time as well both GFP green fluorescence and the red fluorescence of the labeled Glce protein could be observed simultaneously after introducing the mutant AAV-mut Glce plasmid without enzyme activity function,suggesting the successful expression of Glce protein without enzyme activity function.After UUO operation,the serum creatinine and urea nitrogen of Glce-/-mice in AAV-mut Glce group were significantly reduced,kidney injury and fibrosis development were slowed down,fibrosis related protein expression was decreased,EGFR and TGF-β signaling pathway activation and EMT-related gene protein levels were significantly down-regulated,compared with negative control group.The above results showed that renal regeneration of Glce protein without enzyme activity could still inhibit the activation of EGFR and TGF-β signaling pathways,and thus inhibit the development of renal injury and fibrosis,suggesting that the regulatory and protective role of Glce in the development of renal fibrosis disease may not depend on its catalytic enzyme activity.10.MTT test showed that Panax notoginseng polysaccharide SQD4S2 had no effect on the growth activity of HK-2 cells,which proved that it had certain safety.1mg/ m L SQD4S2 can significantly inhibit the TGF-β-induced morphological changes of HK-2 cells,suggesting that SQD4S2 may regulate the epithelial mesenchymal transformation of renal tubular cells.After administration of SQD4S2 cells,TGF-β-induced decreased Glce gene and protein expression was significantly improved,and fibrogenic factors COL1A1,COL3A1,ACTA2,FN1 gene expression and α-SMA,Vimentin,Fibronectin protein expression were inhibited.At the same time,SQD4S2 significantly promoted the expression of characteristic marker protein Ecadherin in epithelial cells,and inhibited the genes and proteins expression of EMTrelated regulatory molecules including N-cadherin,Snail,and Slug.These results suggested that SQD4S2 may inhibit the fibrous proliferation and interstitial transformation of HK-2 cells by promoting Glce expression..Conclusion: 1.Glce expression levels in renal tissues of nephrotic patients and animal models decreased significantly during the formation and development of renal fibrosis,and Glce deficiency aggravated renal injury and renal fibrosis.2.Glce can bind to the intracellular region of EGFR protein to inhibit the abnormal activation of the EGFR signaling pathway.The deletion of Glce leads to the activation of the EGFR pathway and promotes the expression of TGF-β1 protein.At the same time,it induces the increased phosphorylation and activation of TGF-β1 receptor,aggravates the activation of the TGF-β signaling pathway and the process of EMT,and further induces the development of renal fibrosis.3.Renal supplementation of Glce protein can inhibit the activation of EFGR and TGF-β signaling pathways,block the EMT process and improve renal fibrosis,and this regulatory and protective mechanism has nothing to do with its enzyme activity. |
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