Epidemiology Of Membranous Nephropathy And Exploration Of New Biomarkers In Idiopathic Membranous Nephropathy

Membranous nephropathy?MN?is a common pathological type of nephrotic syndrome in adults.The typical pathological features of MN are diffuse thickening of glomerular basement membrane and diffuse immune complex deposition under basement membrane epithelial cells.According to the etiology,MN can be divided into idiopathic membranous nephropathy?IMN?and secondary membranous nephropathy?SMN?.About 25%of membranous nephropathy caused by secondary factors,such as systemic lupus erythematosus and other autoimmune diseases,hepatitis B virus infection,malignancies,drugs and toxic substances and so on.Studies have shown that IMN accounted for 9.89%of the primary glomerular disease in China,and there is an upward trend.High incidence occurs in the middle-aged and elderly people.MN easily relapses and is chronic persistent.Clinical outcome is variable and unpredictable,only 1/3 of MN patients may have spontaneous remission,while about 30%to 40%of patients have insufficient therapeutic response and bad prognosis,those will develop end-stage kidney disease?ESRD?in 5 to 15 years.In recent years,due to the clinical popularization and upgrading of renal biopsy technique,and regional nephrotic syndrome registration system,the epidemiological characteristics of MN gradually get the attention of kidney disease medical staff.A large number of epidemiological research data at home and abroad shows that the incidence of MN is increasing year by year.A national multicenter study has shown that the incidence was growing at a rate of 13%per year over the past 11 years.At present,the exact pathogenesis of IMN is not clear.However,in recent years,with the understanding of IMN and the development of basic research technology,domestic and foreign research on the pathogenesis of IMN has made significant progress.NEP,M-type phospholipase A2 receptor?PLA2R?,aldose reductase?AR?,superoxide dismutase?SOD2?,?-enolase and cationized bovine serum albumin and other target antigens were disovered after the success of Heymann nephritis rat model.A large number of studies have shown that pathogenic target antigen,circulating antibodies and complement activation play an important role in the pathogenesis of IMN.Research during the recent years has suggested that oxidative stress has emerged as being a key pathophysiological mechanism in renal injury in diabetic nephropathy,hypertensive kidney damage,acute or chronic interstitial nephritis,but there is less research related to IMN.This study is divided into the following three parts:?1?Epidemiological progress of membranous nephropathy;?2?Explore the new biomarkers of IMN,and detect the abnormal expression of LncRNA in renal tissues and blood in IMN patients;?3?Verify the difference expression of LncRNA from the clinical control perspective and verify the pathogenesis of the downstream oxidative stress effect in IMN.The aim of the study is to reveal the epidemiological characteristics of MN,to screen MN new biomarkers,and to explore the pathogenesis of MN,and then provide theoretical basis and data support for reducing the occurrence of MN,delaying the progress and reducing mortality of MN.Part I Epidemiology progress of membranousnephropathyBackgroundMembranous nephropathy?MN?is a common pathological type of nephrotic syndrome in adults.The typical pathological features of MN are diffuse thickening of glomerular basement membrane and diffuse immune complex deposition under basement membrane epithelial cells.High incidence occurs in the middle-aged and elderly people.MN easily relapses and is chronic persistent.Clinical outcome is variable and unpredictable,only 1/3 of MN patients may have spontaneous remission,while about 30%to 40%of patients have insufficient therapeutic response and bad prognosis,those will develop end-stage kidney disease?ESRD?in 5 to 15 years.In recent years,due to the clinical popularization and upgrading of renal biopsy technique,and regional nephrotic syndrome registration system,the epidemiological characteristics of MN gradually get the attention of kidney disease medical staff.ObjectiveThis study was to understand the progress of MN epidemiology by summarizing the recent high-quality literatures of MN epidemiology at home and abroad.The epidemiological status and progress of MN were reviewed and summarized to reveal MN epidemiological characteristics,and provide theoretical basis and data support for clinical prevention and treatment of MN.Results1.MN accounted for 25.6%of total renal biopsy cases,annual cases and percentage are 591?19.6%?cases,938?27.9%?cases,1047?29.7%?and 1254?32.6%?,respectively,from 2010 to 2015 in henan province.The trend test shows that the trend is increasing?P<0.05?.2.From 2010 to 2015,the proportion of IMN in henan province also increased year by year?P<0.05?.The proportion of IMN in PGD accounting for 209?14.5%?,336?16.8%?,486?22.6%?,666?29.4%?,867?34.4%?,and 1115?40.4%?from 2010 to 2015,3.System analysis shows that the proportion of IMN in PGD approximately account for 8.6%?23.9%in China,and its incidence has increased in recent years.The incidence also increase oversea.4.There are regional differences in MN epidemiology,and there is a large difference in MN prevalence in different parts of the world,and the prevalence of MN in developing countries is generally higher than that in developed countries.Conclusion1.The incidence of MN in henan province,especially the IMN,has been increasing year by year and has been getting younger.2.The prevalence of MN in the worldwide is generally high,account for about 6.0%to 28.8%of primary glomerular disease;Part II The difference expression of LncRNA in patients with idiopathic membranous nephropathy BackgroundLong chain noncoding RNA?Long non coding RNA,LncRNA?is a kind of more than 200 nt,noncoding protein RNA,which can be combined with DNA,RNA and proteins and control gene expression level in the form of RNA on multiple levels?epigenetic regulation,transcriptional regulation and transcription regulation,etc.?.At present,IncRNA is considered to have the following functions:?1?interfere with the expression of downstream genes by transcribing in the upstream promoter region of protein-coding genes;?2?affect the expression of downstream genes by inhibiting RNA polymerase ? or mediated chromatin reconstruction and histone modification;?3?through the transcription of the protein coding gene,it forms a complementary double strand,which interferes with the shear of mRNA,resulting in different shear forms.?4?through the transcription of protein-coding genes,a complementary double strand was formed,which further produced endogenous siRNA under Dicer enzyme,regulating the expression level of genes;?5?by binding to specific proteins,LncRNA transcriptional instinct regulates the activity of corresponding proteins;?6?nucleic acid protein complexes formed as structural components and proteins;?7?change the cytoplasmic localization of the protein by binding to specific proteins:?8?as small molecule RNA,such as microRNA,piRNA precursor molecular transcription,etc.Therefore,LncRNA and target genes have formed complex regulatory networks to regulate cell proliferation,differentiation and apoptosis,and participate in many life activities.In recent years,the research on LncRNA has been progressing rapidly and the related new databases have been kept emerging.However,the number of LncRNAS that are now known is extremely limited,and the function of most LncRNAs is still not clear.Research on LncRNA and disease has been carried out in various fields.Tumor,cardiovascular system,immune system and other diseases are all related to the regulation of LncRNA.More scholars have suggested that LncRNA can be used as biomarkers and therapeutic targets for disease.The study and correction of LncRNA anomalies have become a new way to explore the pathogenesis and therapeutic targets of diseases.In the study of diabetic complications and kidney diseases,the researchers also gradually discovered the important biological functions of LncRNA and began to explore them.In 2015,it was reported that LncRNA MIR210HG,linc-atp13a4-8,and linc-kiaa 1737-2 were involved in the injury of renal tubular epithelial cells caused by inflammation and hypoxia.In the study of membranous nephropathy,LncRNA Xist was involved in foot cell injury induced by inflammatory and hypoxia,and the expression of increased Xist in the patient's urine may be considered as biomarker for membranous nephropathy.These indicate that LncRNA is closely related to the occurrence of membranous nephropathy,but the related research is rarely reported,menawhile whether LncRNA expression changes are related to oxidative stress is unknown.ObjectiveTo analyze the long non-coding-RNA?LncRNA?in patient with Idiopathic membranous nephropathy?IMN?expression in renal tissues and blood,and screened with the pathogenesis of 1MN is related to LncRNA,to investigate the role of LncRNA in the diagnosis of IMN.MethodsFresh kidney tissues and whole blood samples of 3 patients with IMN were collected from January 2017 to March 2017.The total RNA was extracted by Trizol reagent,and RNA was detected by denatured agarose gel electrophoresis and NanoDrop ND-1000 UV-Vis spectrophotometer.Screening of differential expression of LncRNA in renal tissues of patients with IMN and whole blood samples with normal kidney tissue and whole blood samples by Arraystar Human LncRNA Microarray chip,and to analyze its expression,then the blood and kidney tissues at the same time increase or down-regulation of LncRNA in cross analysis.Results1.compared with normal kidney tissues,there were 1771 differentially expressed LncRNA more than 2 times in renal tissues of patients with IMN,712 of which were up-regulated and 1059 were down regulated.2.Compared with normal human whole blood specimens,there were 509 differences in LncRNA expression in renal tissues of patients with IMN,242 of which were up-regulated and 267 were down-regulated.3.Cross analysis showed that up-regulated in IMN patients and the difference expressed between the samples of whole blood and renal tissue more than 2 times have 9 LncRNAs,respectively:ENST00000510094,NR₀33412,ENST00000433035,ENST00000602973,T297967,ENST00000426412,ENST00000433505,NR₁10236,NR₀40073.There were 6 LncRNAs down-regulated in the whole blood specimens and kidney tissues of IMN patients,which were expressed more than 2 times.respectively:T074169 and T302890,NR₀46174,T307836,GSE61474_TCONS₀0281832 and ENST00000606186.ConclusionENST00000510094 and other LncRNA is different expressed in renal tissue and blood of patients with IMN.These LncRNA may be closely related to the occurrence and development of IMN,and may be a new target for molecular diagnosis and gene therapy of IMN.Part ? Mechanism of oxidative stress in IdiopathicMembranous NephropathyBackgroundMembranous nephropathy?MN?is a common nephrotic syndrome,named after its pathologic diagnosis,is a common pathological type of adult nephrotic syndrome,The typical pathological features of MN are diffuse thickening of glomerular basement membrane and diffuse immune complex deposition under basement membrane epithelial cells,immunofluorescence showed C3 and IgG along the glomerular capillary wall was fine granular deposition.High incidence in the elderly population.In 2013,a retrospective study from abroad shows that IMN accounted for 27.5%of primary glomerular disease,has become the second major pathological type of the disease.Recently,a multicenter study from the China showed that MN accounted for 23.41%of 71151 patients renal biopsy from 2004 to 2014 and became the second major pathological type after IgA nephropathy,predicted the growing rate is 13%per year.In the second part,we screen differentially expressed LncRNA in the blood and urine specimen with IMN patients.Among them,there are nine LncRNAs which are more than twice as high as the difference expression and up-regulated in the whole blood specimens and kidney tissues,respectively:ENST00000510094,NR₀33412,ENST00000433035,ENST00000602973,T297967,ENST00000426412,ENST00000433505,NR₁10236,NR₀40073.By further retrieving gene pool,we find ENST00000433035 downstream is closely associated with oxidative stress,and in this section we intends to explore the mechanism of oxidative stress involved in IMN through controlled clinical research.In recent years,many domestic and foreign research data have put forward the key role of oxidative stress?OS?in kidney disease.OS refers to the body in a variety of external stimuli,activity of reactive oxygen species in the body increase,oxidation and antioxidant system is in the imbalance condition,the body is in a state of stress.Superoxide dismutase?SOD?and heme oxygenase?HO-1?are important biomarkers of antioxidants,which are generally contribution in animals and plants and so on.It is divided into three types according to the distribution position:SOD2?mainly distributed in the mitochondria of prokaryotic cells and eukaryotic cells,and also play an important part of the antioxidant protection system?,SOD2?mainly distributed in the cytoplasm?,SOD3?mainly distributed in prokaryotic cells?.They can transfer the toxic superoxide anion into hydrogen peroxide?H202?and diatomic oxygen?02?,play an important role in the body OS reaction process,which is known as "oxygen free radical scavenger".In the OS reaction process,there are a variety of body damage markers,those have different function and performance,including malondialdehyde?MDA?.MDA is as the final product of peroxidation,is a lipid peroxidation of unsaturated fatty acids when the body is stimulated by the outside world,which is manifested as oxidative damage of lipid;8-Hydroxy-2'-deoxyguanosine?8-OHdG?,as a sensitive indicator of DNA damage,is a large amount of ROS released in the body when stimulated by the outside world,resulting in accumulation in the body and oxidation and antioxidation system imbalance,which expresses as oxidative damage of nucleic acids.Previous studies have found that membrane attack complexes can lead to activation of glomerular podocytes,resulting in OS response,and specific expression of superoxide dismutase?SOD2?IgG4 antibody have been found in MN patients,immunofluorescence detection found that it also expresses in the glomerular.Research during the recent years has suggested that oxidative stress has emerged as being a key pathophysiological mechanism in renal injury in hypertensive kidney damage,diabetic nephropathy,interstitial nephritis and acute renal injury,but there is less research related to IMN.Objective1.Verification:whether SOD,HO-1,MDA and 8-OHdG as important markers of OS in renal tissue and serum and urine of IMN patients highly expresses or not2.To investigate the relationship between oxidative stress-related factors SOD,HO-1,MDA,8-OhdG and pathogenesis of idiopathic membranous nephropathy.MethodsAll of the selected subjects were from patients admitted to the Department of Nephrology,the XX Hospital from June 20 to June 2017.Among them,50 patients with idiopathic membranous nephropathy,30 patients with secondary membranous nephropathy,30 Cases of patients with minimal change disease,all patients were diagnosed by the first renal biopsy.10 cases of normal renal tissue from our hospital kidney resection and 30 cases of healthy adult blood and urine specimens for the blank control.The levels of SOD,HO-1,MDA and 8-OHdG in serum and urine were measured by ELISA.The expression area and distribution of SOD,HO-1,8-OHdG in renal tissue were detected by immunohistochemistry..Results1.ENST00000433035 expression levels in serum of patients with IMN is higher than that in secondary membranous nephropathy group and MCD group.2.The levels of SOD,HO-1,MDA and 8-OHdG oxidative stress-related factors in serum samples were analyzed by ELISA.The results showed that the levels of SOD,HO-1,MDA and 8-OHdG in IMN group were significantly higher than MCD and NC group?P<0.05?,slightly higher than SMN group?P>0.05?.The levels of SOD,HO-1,MDA and 8-OHdG in SMN group were significantly higher than those in NC group?P<0.05?.3.The levels of oxidative stress-related factors SOD,HO-1,MDA and 8-OHdG in urine samples were analyzed by ELISA.The results showed that the levels of SOD,HO-1,MDA and 8-OHdG in IMN group were significantly higher than MCD and NC group?P<0.05?,slightly higher than SMN grou?P>0.05?.The levels of SOD,HO-1,MDA and 8-OHdG in SMN group were significantly higher than those in NC group?P<0.05?.The levels of SOD,HO-1,MDA and 8-OHdG in MCD group were significantly higher than those in NC group?P<0.05?4.The levels of SOD,HO-1,MDA and 8-OHdG oxidative stress-related factors in renal tissue were quantitatively analyzed by immunohistochemistry.The results show that SOD mainly distribute in glomeruli and renal tubules,the distribution in tubules is significantly more widespread than in the glomeruli.HO-1 distributes in the glomeruli and renal tubules,8-OHdG mainly distributes in the nucleus,the cytoplasm is less.The expression of SOD,HO-1,8-OHdG in MCD and NC group are significantly lower than that in IMN group?P<0.05?,slightly higher than SMN group?P>0.05?.The expression of SOD,HO-1,8-OHdG in SMN group was significantly higher than that in NC group?P<0.05?,which was slightly higher than that of MCD group.Except for SOD?P<0.05?.The expression area of SOD,HO-1,8-OHdG in MCD group was slightly bigger than NC group,but the difference was not significant?P>0.05?.Conclusion1.The levels of SOD,HO-1,MDA and 8-OHdG in serum,urine and renal tissue of IMN patients were significantly increased,which confirmed the important role of oxidative stress in IMN pathogenesis?2.IMN and SMN pathogenesis is in the presence of DNA damage and lipid peroxidation damage.