Carrying Cd2ap And Its Role In The Mechanism Of Nephrotic Syndrome, Proteinuria

The glomerulus is a specialized filtration unit of the kidney. Its function is to provide a molecular sieve for blood plasma so that low molecular waste products are excreted into the urine, while essential macromoleculesare retained in the blood. The glomerular filtration barrier present in the capillary walls is comprised of a fenestrated endothelium, the glomerular basement membrane (GBM), and highly specialized visceral epithelial called podocytes, the foot processes of which cover the outer surface of the GBM. Each actin-rich foot process is attached to its neighbor along its length by slit diaphragm (SD), which is located just above the GBM.The recent discovery of several novel podocyte proteins and the description of their physical and functional interactions highlight the critical role of the SD complex and of the actin cytoskeleton in the maintenance of the glomerular filtration barrier, which will result in better understanding of proteinuria and nephrotic syndrome. CD2-associated protein (CD2AP) is a newly found cytoplasmic protein. In the glomeruli, CD2AP is expressed in podocytes. Mice completely lacking CD2AP die of massive proteinuria shortly after birth, suggesting an important role for CD2AP in the filtration barrier of the kidney.In order to understand the role of CD2AP in the pathomechanisms of proteinuria, two-part study is carried out.Part one: CD2AP expression in rats with puromycin aminonucleoside nephrosisObjective: To investigate the role of CD2AP in the pathogensis of puromycin aminonucleoside nephrosis in rats.Methods: Puromycin aminonucleoside nephrosis was induced by single intraperitoneal injection of puromycin aminonucleoside(PAN). Renal tissues were studied at 3, 7, 10 and 20 days after PAN injection by meansof immunohistochemistry, RT-PCR ,Western blot and fluorescence.Results: At day 3, CD2AP expression in podocyte began to decrease, and significantly decreased at day 7 and 10. These occurred in association with an increase in 24h urinary protein content. At day 10, a remarkable decrease of F- actin was found in the glomeruli of PAN rats.Conclusion: CD2AP expression may be a useful marker of acute podocyte injury. CD2AP is also likely to be involved in the pathomechanisms of proteinuria.Part two: The influence of CD2AP antisense oligonucleotide on HEK293 cell cytoskeletonObjective: To investigate the influence of CD2AP antisense oligonucleotide on the expression of CD2AP mRNA and protein, and its influence on the cytoskeleton of HEK293 cell.Methods: 24h after the transfection of CD2AP antisense oligonucleotide, the expression levels of CD2AP mRNA and protein in HEK293 cells were studied by means of RT-PCR and Western blot, and the cytoskeleton changes were observed by cytofluorescence.Results: CD2AP antisense oligonucleotide could inhibit the expression of CD2AP mRNA and protein, which also resulted in the decrease of F-actin fluorescent intensity.Conclusion: CD2AP may be useful to the stability of F-actin.