The Role Of Podocin In Nephrotic Syndrome, Proteinuria Mechanism

Plasma ultrafiltration during primary urine formation is a central function of the kidney, occuring through the glomerular filtration barrier, which is composed of a fenestrated endothelium, the glomerular basement membrane and specialized epithelial cells called podocyte.Defects of this filter lead to proteinuria and potentially to nephrotic syndrome.The podocyte is composed of a cell body and primary processes, which divide into secondary processes, called foot processes, interconnection closely on top of the glomerular basement membrane through the slit diaphragm. Recently, the mutations in several genes encoding novel podocyte proteins were demonstrated to be associated with the development of nephrotic symdrome. Podocin is encoded by the NPHS2 gene, mutations of which are associated with inherited (autosomal recessive) and sporadic steroid-resistant nephrotic syndrome. Podocin-deficient mice develop massive proteinuria after birth, suggesting a crucial role for podocin in the development of proteinuria.In order to understand the role of podocin in the pathomechanisms of proteinuria, two-part study is performed.Part IExpression and Distribution of Podocin in Rats with Minimal Change NephrologyObjective To investigate the expression and distribution of podocin and a-actinin-4 in development of proteinuria in rat puromycin aminonucleoside (PAN) nephropathy and to disclose the possible role of podocin in early podocyte injury by comparing the changes of podocin and a-actinin-4. Methods The nephropathy model was established by asingle injection of PAN. The renal histology was observed under light microscope, electronic microscope. The distribution and mRNA expression of podocin and a-actinin-4 were detected by indirect immunofluorescence staining and semiquantitative reverse transcription PCR-The protein level of podocin and a-actinin-4 expression was detected by western-blot and immunohistochemistry respectively. Results â‘  Podocin staining showed a very fine linear-like pattern along capillary loop in control group. It shifted a discontinuous pattern at day 1, a granular pattern at day 3 ,more coarse granular at day 10 and gradually recovered to line-like pattern, a-actinin-4 showed a dot-line pattern along capillary loop in control group.The staining pattern become more diffusely accompanied by local enhanced intensity. â‘¡ Podocin mRNA slightly elevated at days 1, 3, 10(1.2,1.5,1.4 fold respectively, p<0.01) and recovered to normal level at day 20(P=0.11)compared with the control. The upregulation of a-actinin-4 mRNA started at day 3 and proceeded to increase at day 20. â‘¢ The protein level of podocin expression at day 1 began to decrease and prominently decreased at day 3 and day 10 compared with the control .At day 20, the expression level was still less than the control although it partly retrieved with the decreased proteinuria.The immunohistochemistry showed a-actinin-4 expression upregulated at day 20.â‘£The staining intensity was negatively correlated with proteinuria(r=-0.766, P<0.01).Conclusion A dramatic decrease of podocin expression and abnormal distribution were found in rat PAN nephrology, suggesting involvement of podocin in the development of proteinuria and podocin may serve as a useful early marker of acute podocyte injury.Part IIConstruction of expression vector for podocin and its effects of...