Expression Of Nephrin、ZO-1and Podoplanin On Podocytes And Intervention Effect Of Sulodexide In Adriamycin Nephropathy Rats

Objective：Nephrotic syndrome (NS) is one of the common clinicalglomerular diseases. Macroalbuminuria (≥3.5g/d),hypoalbuminemia (plasmaalbumin<30g/L),edema and hyperlipidaemia are the typical clinical charactersof NS. Glomerulus will be in the state of high filtration because of Continuousmacoalbuminuria.The changes also lead to irreversible damage of renal, thusaffect the quality of people’s life.Clinically,how to better treat NS and reduceproteinuria is the project which academics pay close attention to.Adriamycin nephropathy (AN) is an animal model that has been studiedextensively,and its clinical characters are similar to NS in human.According toadministration methods and pathological changes,the animal model could bedivided into acute type and chronic type.The model is identified as an acuteAN in the first4weeks,and the pathological changes are similar to minimalchange disease (MCD) in human.From the fifth week to the twelfth week,themodel is identified as a chronic AN,and the pathological changes are similar tofocal segmental glomerulosclerosis (FSGS) in human.The changes about thestructure and relevant protein molecules of podocytes are one of the reasonsthat result in proteinuria.The relevant protein molecules of podocytes includenephrin,ZO-1,podoplanin and so on.And Sulodexide is a new type ofglycosaminoglycan which is extracted from intestinal mucosa ofpigs.Sulodexide contains fast mobility heparin (FMH) and dermatan sulfate(DS),and they play a role together.Many studies have shown that sulodexidehas the effect of reducing proteinuria and protecting kidneys.In this research,the rat models of AN are duplicated by a singleintravenous injection of adriamycin via the tail vein. We observe theexpression of nephrin,ZO-1,podoplanin on the podocytes in adriamycin nephropathy rats through the methods of transmission electron microscope，immunohistochemistry staining and RT-PCR,and discuss the interventioneffect and mechanism of sulodexide.Methods:60healthy and male SD-rats of clean grade,aged12weeks,were fed adaptively for a week.After a week,48rats were randomlychosen to inject adriamycin (6.5mg/kg) via tail vein for establishing theadriamycin nephropathy model.After2weeks,we collected24h urine tomeasure24h urinary protein level,if it was greater than100mg/d,the rat modelwould be considered to be successful.Then,the48successful rat models wererandomly divided into the AN model group (M),the prednison treatment group(P) with prednison (7.5mg·kg-1·d-1),the sulodexide treatment group (S) withsulodexid (10mg·kg-1·d-1),the sulodexide and prednison treatment group (P+S)with prednison (7.5mg·kg-1·d-1) and sulodexid (10mg·kg-1·d-1),and the other12rats were considered as the normal control group (N).At4,8week,we collectedthe24h urine,blood and renal tissue samples to test24h urinary proteinlevel,blood albumin,blood creatinine,blood urea nitrogen,bloodcholesterol,serum triglyceride.And we observed the changes of nephrin,ZO-1，podoplanin on podocytes through the methods of immunohistochemicalstaining and RT-PCR.At8week,we observed the changes in glomerularpathology by transmission electron microscopy.All the data were analyzed bySPSS17.0statistics software,P value<0.05was considered to have statisticalsignificance.Results:1.At4week,the24h urinary protein level,blood cholesterol,serumtriglyceride of group M are higher than group N (P<0.05),but blood albumin islower than group N (P<0.05),and the changes between group P,S,P+S andgroup M are obvious (P<0.05).At8week,the changes of24h urinary proteinlevel,blood cholesterol,serum triglyceride,blood albumin between groupP,S,P+S and group M are obvious (P<0.05).At4week,the changes of bloodcreatinine,blood urea nitrogen between group M,group P,group S,group P+Sand group N are not obvious (P>0.05),thus at8week,the changes between group M and group N are obvious (P<0.05),and the changes between groupP,group S,group P+S and group M are obvious (P<0.05).2.The results of immunohistochemical staining and RT-PCR show thatthe expression of nephrin,ZO-1,podoplanin in group M at4week decreasethan in group N (P<0.05),and the expression of nephrin,ZO-1,podoplanin ingroup P,group S and group P+S increase than in group M (P<0.05).At8week,the expression changes between group P,group S,group P+S and groupM are obvious (P<0.05).3.The results of transmission electron microscopy show that the kidneysof group N are normal,the kidneys of group M exert the feet-fusion,thickeningof glomerular basement membrane and so on at8week.The pathologicalchanges in group P,group S,group P+S are milder than in group M,especiallyin group P+S.Conclusion:1.In the progress of AN,the expression of nephrin,ZO-1,podoplanin aredecreasing gradually. We guess that the changes may be one of pathogenesisabout proteinuria in adriamycin nephropathy rats.2.Sulodexide has the effect of reducing proteinuria and protectingkidneys,and it could increase the expression of nephrin,ZO-1,podoplanin onpodocytes of adriamycin nephropathy rats.So we guess that the regulation maybe one of mechanisms of sulodexide.3.Prednison has the effect of reducing proteinuria and protectingkidneys,and the effect will be enhanced if we use prednison and sulodexidetogether.