The Roles Of Nephrin, α3/β1-integrin And α-actinin In The Development Of Proteinuria In Adriamycin Nephrosis

The roles of nephrin,a3/pl-integrin and a-actinin in the development of proteinuria in adriamycin nephrosisObjectiveGlomerular podocyte and slit diaphragm between podocyte foot processes constitute the important part of glomerular filtration membrane . Recently , several podocyte molecules and slit diaphragm proteins such as a3/pl-integrin, nephrin, podocin,a-actinin-4 were found and confirmed that they play critical role in maintaining normal slit diaphragm stucture and filtration barrier function.Podocytes consist of three different segments: cell body, major processes and foot processes. In general, cell bodies and major processes are not directly connected to the glomerular basement membrane(GBM) but hang freely in the urinary space, fixed to the underlying capillaries only via attachment of their foot processes to the GBM. a3/B1-integral is the matrix receptor of podocytes and binds to extracellular matrix proteins in the GBM, so a3/B1-integrin play an important role in attachment of podocyte cell membrane to the GBM. Nephrin is a member of the immunoglobulin(Ig) superfamily of cell adhesion molecules, and belongs to transmembrane protein with a large extracellular domain, a single membrane spanning domain ,and a cytoplasmic portion.In addition to its function in the slit diaphragm structure, nephrin plays an essential role in mediating cell signaling, a-actinin is a member of cytoskeletal proteins, to date, four isoforms of human a-actinin have been identified: non-muscle actinin-1, muscle actinin-2, muscle actinin-3,and recently indentified nonmuscle actinin-4. Muscle a-actinins form part of the contractile machinery by anchoring actin filaments at the Z lines in striated muscle and dense bodies insmooth muscle. By contrast, nonmuscle a-actinins not only cross-link actin filaments but also interact with other cytoskeletal and membrane-associated proteins. Nonmuscle a-actinins are therefore considered to be involved in the organization of the cortical cytoskeleton adjacent to adherent junctions. Kaplan et al. generated anti-peptide antibodies specific for human actinin-1 and -4.They examined expression of the proteins in the human kidney by Western blot analysis and immunofluorescence studies using the antibodies. Interestingly, their results showed expression of actinin-4 in the glomeruli in an epithelial cell patern, but did not show expression of actinin-1 in the kidney. a-actinin-4 is considered to have a central role for linking the actin cytoskeleton to the slit diaphragm components as well as to the glomerular basement membrane via interaction with integrins.Minimal change disease(MCD) model is used in developing glomerular proteinuria.lt was well known that rat adriamycin nephrosis model is one of MCD models. To explore the effects ofa3/pl-integrin, nephrin and a-actinin-4 on proteinuria in adriamycin nephrosis, we observed urine protein, serum biochemical parameters and renal tissue pathological changes at day3,day7, day14,day21 and day28 after venous injection of adriamycin 6.5mg/kg, and examined the expression levels of a3/B1-integrin, nephrin,a-actinin-4 mRNA and protein,as well as the protein localization in immunofluorescence(IF) staining in glomeruli of adriamycin nephrosis rats .Materials and methodsAnimal experimentsYoung male rats of Sprague-Dawley strain(n=45)weighting 200 to 250g were divided into experiment group(n=40) and control group(n=5). Rats received a single tail-vein injection of adriamycin(6.5mg/kg,Shenzhen pharmacy company,China) in experiment group and 0.9% saline in equal amounts in control group.Otherwise, the animals were fed with standard rat chow and had free access to tap water.Sample collectionUrine was collected using metabolic cages at days 0,3,7,14,21,and 28. five rats were killed after 3,7,14,21,28 days in experiment group and after 28 days in control group, kidney specimens were removed through a flank incision under amobarbital anesthesia to define morphologic changes, cortical kidney tissue were sectioned and snap frozen in l...