| Section Ⅰ IntroductionWith the aging of society,westernization of life style and increasing prevalence of obesity,the prevalence of adult diabetes in China is rising rapidly,which has become a serious public health problem endangering the life and health of the people.Type 2 diabetes mellitus(T2DM)accounts for 90-95% of diabetes cases and is associated with obesity,whereas Type 1 diabetes mellitus(T1DM)and other types of diabetes are rare.Diabetic nephropathy(DN)is considered as the main microvascular complication of diabetes.About 40% of diabetic patients could develop DN,which increases the risk of chronic kidney disease(CKD)and end-stage renal disease(ESRD).The pathogenesis of DN is currently considered to be multifactor,including Advanced Glycation end products(AGEs),hemodynamic changes,oxidative stress,inflammation,hypoxia,autophagy,cellular senescence and epigenetic regulation,and mitochondrial hormesis.Transient receptor potential(TRP)is a superfamily of nonselective cationic channels located in cell membrane,mediating transmembrane transport of various cations.TRPM2,TRPM6 and TRPM7 are unique members of the TRPM supramolecular family,which are structurally composed of a cationic channel coupled with a functional enzyme domain with highly variable carboxyl terminal sequence,also known as channel enzymes.TRPM2 is widely expressed in central nervous system,heart,bone marrow,kidney,lung,liver,pancreas,vascular system and hematopoietic cells.Functional coupling and interaction between channels and kinases,broad expression profiles in vivo,and unique physiological characteristics enable TRPM2 to participate in many physiological and pathological processes.Such as oxidative stress,apoptosis and necrosis,inflammatory response of pulmonary or central nervous system diseases,renal inflammation and fibrosis,Alzheimer’s disease,cardio-cerebral Ischemia reperfusion injury,myocardial fibrosis,diabetes mellitus and tumor.TRPM2 is an intracellular oxidative stress receptor that can be activated by oxidative stress-related diseases such as Alzheimer’s disease,ischemia/reperfusion injury,and diabetes.At present,it is not clear whether TRPM2 plays an important role in the development of DN.With the increasing prevalence of diabetes and the incidence of DN,considering the important role of TRPM2 in cardiovascular diseases and diabetes,we will explore whether TRPM2 protein plays an important role in the molecular pathogenesis of DN,whether it can be used as a potential target for DN intervention,and help formulate the prevention and treatment strategy to improve DN.Section Ⅱ Inflammatory and fibrosis response as well as increased TRPM2 expression in diabetic nephropathy induced by HFD/STZ Objective:In a type 2 diabetic mouse model induced by high-fat diet(HFD)combined with Streptozotocin-induced(STZ),to explore whether inflammation and fibrosis were occurred in kidney tissue,and whether the expression of TRPM2 was elevated.Methods:1)After adaptive feeding for one week,healthy male 7-week-old mice(18 in total)were randomly divided into three groups: Normal Control group(n=6),High fat diet Control group(HFD-Control,n=6)and Diabetes group(n=6).2)Biochemical determination: Fasting blood glucose(FBG)of mice was measured by blood sampling from tail vein.Blood urea nitrogen and creatinine levels were measured by cardiac blood sampling.3)Renal histomorphological observation: The kidney tissue was fixed in 4%neutral paraformaldehyde solution for 2 days and prepared paraffin sections.Hematoxylin-eosin(HE)staining and Periodic acid-Schiff(PAS)staining,Sirius red staining and Masson staining as well as TRPM2 and Antiaquaporin 1(APQ1,a marker of renal tubular epithelial cells)immunofluorescence dual staining were performed.4)The m RNA expression of TRPM2 and related inflammatory factors were detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).5)The Protein expression of TRPM2 and fibrosis factors was detected by Western blot.6)The expression of inflammatory factors in renal homogenate tissue was detected by Enzyme Linked Immuno Sorbent Assay(ELISA).Results:1)Compared to the Control group and the HFD-Control group,HFD/STZ induced diabetic mice had a peak hyperglycemia of around 30 mmol/L accompanied by weight loss,indicating the occurrence of glucose metabolism disorder,suggesting the successful construction of T2 DM mouse model.2)Serum creatinine and urea nitrogen levels were significantly increased in HFD/STZ-induced diabetic mice compared to the Control group and the HFD-Control group,and HE and PAS staining showed glomerulosclerosis,glomerular structural disorder and proximal tubule injury.Masson and Sirius red staining showed glomerulosclerosis,tubular damage,and collagen deposition near several large vessels.3)The m RNA expression level of TRPM2,the m RNA expression levels and tissue homogenate concentrations of inflammatory factors including IL-1β,IL-6,IFN-α,TNF-α and MCP-1 were increased in renal tissue of HFD/STZ induced diabetic mice compared to the Control group and the HFD-Control group.4)The protein expression levels of TRPM2,transforming growth factor-β1(TGF-β1),connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),fibronectin(FN),Collagen I and Collagen III were increased in renal tissue of HFD/STZ induced diabetic mice compared to the Control group and the HFD-Control group.5)The expression of TRPM2 protein in renal tubular epithelial cells was evaluated by using AQP1 antibody specific markers.The abundance of TRPM2 fluorescence expression in renal tubular epithelial cells was enhanced in HFD/STZ-induced diabetic mice compared to the Control group and the HFD-Control group.Conclusion:1)Inflammation and fibrosis was occurred in renal tissue of HFD/STZ induced diabetic mice compared to the Control group and the HFD-Control group.2)The m RNA and protein expression level of TRPM2 as well as the fluorescence abundance within renal tubular epithelial cells were increased in renal tissue of HFD/STZ induced diabetic mice compared to the Control group and the HFD-Control group.Section Ш Knockdown of TRPM2 alleviated renal inflammation and fibrosis in diabetic mice induced by HFD/STZObjective:In the HFD/STZ-induced T2 DM mouse model,the AAV-2 recombinant vector system was injected through the tail vein to target renal tissue infection.After the knockdown of TRPM2 protein,we observe whether renal inflammation and fibrosis were improved in diabetic mice.Methods:1)After adaptive feeding for one week,24 6-week-old male C57BL/6N mice were injected with AAV2/2-U6-Sh TRPM2 recombinant vector or empty vector(~3×1011 gene copy)through tail vein to the knockdown of TRPM2 in kidney,then randomly divided into four groups(6 mice per group): 1)AAV2/2-N.C.;Control group,2)AAV2/2-sh TRPM2;Control group,3)AAV2/2-N.C.;Diabetes group,4)AAV2/2-sh TRPM 2;Diabetes groups.2)Establishment of T2 DM mouse model: After fed with HFD for 4 weeks,8-week-old mouse in the diabetic group were intraperitoneally injected with STZ(the Control group was injected with equal volume of sodium citrate buffer)at the 5th week.After 3 days of injection,blood was collected through tail vein to measure FBG level.The successful construction of T2 DM mouse model was defined as the FBG level of mice was equal or greater than 11.1 mmol/L.3)Biochemical determination: FBG of mice was measured by blood sampling from tail vein.Blood urea nitrogen and creatinine levels were measured by cardiac blood sampling.4)Renal himorphologic observation: Paraffin sections were prepared after fixation with 4% neutral paraformaldehyde solution for 2 days,followed by HE staining,PAS staining,Sirius red and Masson staining as well as TRPM2/APQ1 immunofluorescence double staining.5)The m RNA expression of TRPM2 and related inflammatory factors was detected by RT-PCR.6)The Protein expression of TRPM2 and fibrosis factors as well as signaling pathway molecules were detected by Western blot.7)The expression of inflammatory factors in renal homogenate tissue was detected by ELISA.Results:1)Compared to the Control group,HFD/STZ induced diabetic mice had a peak hyperglycemia of around 20 mmol/L accompanied by weight loss,indicating the occurrence of glucose metabolism disorder,suggesting that the T2 DM mouse model was successfully constructed.However,knockdown of TRPM2 gene did not change the changes of FBG level and body weight in mice,suggesting that TRPM2 does not affect DN by changing blood glucose level and body weight.2)Compared to AAV2/2-N.C.;Control group,the levels of serum urea nitrogen and creatinine in AAV2/2-N.C.;Diabetes group were significantly increased.Compared to AAV2/2-N.C.;Diabetes group;the levels of serum urea nitrogen and creatinine were decreased in AAV2/2-sh TRPM2;Diabetes group.These results suggested knockdown of TRPM2 gene could partially improve renal dysfunction in HFD/STZ-induced T2 DM mice.3)Compared to AAV2/2-N.C.;Control group,the protein expression level and fluorescence abundance within renal tubular epithelial cells of TRPM2 in renal tissues of diabetic mice was increased.However,the expression of TRPM2 protein was effectively down-regulated by tail-vein injection of recombinant AAV2/2-sh TRPM2 vector.4)Compared to AAV2/2-N.C.;Control group,HE and PAS staining showed glomerular structural disorder and proximal tubule injury in AAV2/2-N.C.;Diabetes group mice.Masson and Sirius red staining showed glomerular sclerosis,tubule injury and interstitial fibrosis in diabetic mice.Knockdown of TRPM2 could ameliorate the above pathological damage in diabetic mouse kidney tissue.5)Compared to AAV2/2-N.C.;Control group,the m RNA expression levels and tissue homogenate concentrations of inflammatory factors including IL-1β,IL-6,IFN-α,TNF-α and MCP-1 were increased in renal tissue of AAV2/2-N.C.;Diabetes group mice,while knockdown of TRPM2 could improve the expression levels of inflammatory factors in diabetic mice.6)Compared to AAV2/2-N.C.;Control group,the protein expression levels of renal fibrosis markers(TGF-β1,CTGF,α-SMA,FN,Collagen I and III)were increased in AAV2/2-N.C.;Diabetes group,while knockdown of TRPM2 could improve the protein expression levels of renal fibrosis factors in diabetic mice.7)Compared to AAV2/2-N.C.;Control group,the phosphorylation levels of JNK1,IKKα and NF-κB were increased in renal tissue of AAV2/2-N.C.;Diabetes group,while knockdown of TRPM2 could down-regulate the phosphorylation levels of JNK1,IKKα and NF-κB in diabetic mice.Conclusion:In HFD/STZ-induced T2 DM mice,knockdown of TRPM2 protein improved renal inflammation and fibrosis,and was associated with TGF-β1/JNK1/NF-κB signaling pathway.Section Ⅳ Knockdown of TRPM2 improved the inflammatory and fibrosis response of HK-2 cells stimulated by high glucose by blocking the activation of TGF-β1/JNK1 pathwayObjective:In vitro cell experiments,human proximal tubular epithelial cell line(HK-2) was stimulate by high glucose(HG)to simulate diabetic kidney injury,and knockdown of TRPM2 was realized by Small interfering RNA(si RNA)to observe whether it could improve the inflammatory and fibrosis response induced by HG.Methods:1)HK-2 was cultured in DMEM medium contained with 5.5 m M D-glucose.In the treatment with HG,HK-2 cells were stimulated with 33 m M D-glucose for 24 hours.2)Si RNA transfection and cell grouping: HK-2 cells were transfected with Si-TRPM2 or Si-NC for 48 hours,and stimulated with normal or HG for 24 hours.In another set of parallel experiments,HK-2 cells were pre-incubated with 10μM JNK inhibitor SP600125 for 1 hour and then stimulated with normal or HG for 24 hours.3)The expression levels of TRPM2 and related proteins in HK-2 cells were detected by Western blot.4)The m RNA expression levels of TRPM2 and related genes in HK-2 cells were detected by RT-PCR.Results:1)HG treatment significantly increased the protein expression levels of TRPM2,and si-TRPM2 could effectively down-regulate the protein expression level of TRPM2 in HK-2 cells.Compared with the control group,HG treatment significantly increased the expression levels of TGF-β1 and phosphorylated JNK1,while TRPM2 knockdown by si-TRPM2 down-regulated the expression levels of TGF-β1 and phosphorylated JNK1 in HK-2 cells.2)HG treatment significantly increased the m RNA expression levels of inflammatory cytokines including IL-1β,IL-6,IFN-α,TNF-α and MCP-1 in HK-2 cells.The increased m RNA expression levels of IL-1β,IL-6,IFN-α,TNF-α and MCP-1 in HK-2 cells stimulated by HG were partly down-regulated by si-TRPM2.In addition,the increased m RNA expression levels of IL-1β,IL-6,IFN-α,TNF-α and MCP-1 stimulated by HG were partly down-regulated in HK-2 cells after pre-incubation with 10μM JNK inhibitor SP600125.3)HG treatment significantly increased the expression levels of TGF-β1,CTGF,α-SMA,Collagen I and III proteins in HK-2 cells.The increased expression levels of TGF-β1,CTGF,α-SMA,Collagen I and III proteins in HK-2 cells stimulated by HG were partly down-regulated by si-TRPM2.In addition,the increased expression levels of TGF-β1,CTGF,α-SMA,Collagen I and III proteins stimulated by HG were partly down-regulated in HK-2 cells after pre-incubation with 10μM JNK inhibitor SP600125.4)HG treatment significantly increased the expression levels of TGF-β1,phosphorylated IKKα and NF-κB in HK-2 cells,while TRPM2 knockdown by si-TRPM2 down-regulated the expression levels of TGF-β1,phosphorylated IKKα and NF-κB in HK-2 cells.In addition,the increased expression levels of TGF-β1,phosphorylated IKKα and NF-κB stimulated by HG were partly down-regulated in HK-2 cells after pre-incubation with 10μM JNK inhibitor SP600125.5)HG treatment significantly increased the expression levels of nucleoprotein NF-κB in HK-2 cells,while TRPM2 knockdown by Si-TRPM2 down-regulated the expression levels of nucleoprotein NF-κB in HK-2 cells.In addition,the increased expression levels of nucleoprotein NF-κB stimulated by HG were partly down-regulated in HK-2 cells after pre-incubation with 10μM JNK inhibitor SP600125.Conclusion:1)HG stimulation significantly increased inflammation and fibrosis in HK-2 cells in vitro,and activated the TGF-β1/JNK1/NF-κB signaling pathway.2)TRPM2 knockdown by si-TRPM2 could down-regulate the inflammation and fibrosis of HK-2 cells stimulated by HG,and inhibit the activation of TGF-β1/JNK1/NF-κB signaling pathway.3)After pre-incubated with JNK inhibitor SP600125,the inflammation and fibrosis was alleviated in HK-2 cells stimulated by HG,and could inhibit the activation of NF-κB inflammatory signaling pathway.Section V SummaryAt present,it is not clear whether TRPM2 plays an important role in the development of DN.In this study,we found that in HFD/STZ-induced T2 DM mouse model,inflammation and fibrosis occurred in renal tissues,and the expression levels of TRPM2 m RNA and protein were increased,and the abundance of TRPM2 fluorescence expression was enhanced in renal tubular epithelial cells.These results suggest that TRPM2 might play an important role in the development of DN.Next,to explore whether TRPM2 knockdown could alleviate renal damage in HFD/STZ-induced T2 DM mouse,the expression of TRPM2 was knocked down by a single tail vein injection of AAV2/2-U6-Sh TRPM2 recombinant vector(~3×1011 gene copy)at the age of 8 weeks.Knockdown of TRPM2 protein was found to improve renal inflammation and fibrosis in diabetic mice,and was associated with TGF-β1/JNK1/NF-κB signaling pathway.The phosphorylation levels of JNK1,IKKα and NF-κB in renal tissues of diabetic mice were increased in diabetes group,and the phosphorylation levels of JNK1,IKKα and NF-κB in renal tissues of diabetic mice were partly down-regulated by TRPM2 knockdown.In vitro cell experiments,HK-2 was stimulated by HG to simulate diabetic kidney injury.HG stimulation significantly increased the inflammation and fibrosis of HK-2 cells,and activated the TGF-β1/JNK1/NF-κB signaling pathway.However,knockdown of TRPM2 by si-TRPM2 could down-regulate the inflammation and fibrosis of HK-2 cells stimulated by HG and inhibit the activation of TGF-β1/JNK1/NF-κB signaling pathway.After pre-incubation with 10μM JNK inhibitor SP600125,the inflammation and fibrosis of HK-2 cells stimulated by HG were partly relieved,and the activation of NF-κB inflammatory signaling pathway were partly down-regulated.These results demonstrated for the first time that knockdown of TRPM2 expression improveed inflammation and fibrosis in DN by inhibiting TGF-β1/JNK1 pathway activation in HFD/STZ-induced T2 DM mice.This study might provide a new therapeutic target for the diagnosis and treatment of DN and contribute to the formulation of prevention and treatment strategies to improve DN. |
PDF Full Text Request