Colocalization of podocyte-associated molecules in puromycin aminonucleoside nephrotic rats

Objective:To study the expression and distribution of podocyte molecules in nephrotic rats induced by puromycin aminonucleoside (PAN), and to explore the possible interactions among these molecules in the development of proteinuria. Methodology:Forty two Sprague Dawley rats were enrolled in this study, they were randomly divided into experimental group (21 rats) and control group (21 rats). The minimal change nephropathy model was established by a single PAN injection in experimental group. While the control group received only 0.9% sodium chloride solution with the same dose. At the 24th hour, 36th hour, second, 5th, 10th, 15th and 20th day after injection, the rats were sacrificed and the kidneys were acquired. Double immunofluorescence was used to stain the pairs of molecules: CD2AP /nephrin, CD2AP/podocin, CD2AP/α actinin 4, α actinin 4/nephrin and α actinin 4/podocin. A laser scanning confocal microscopy was applied to study the distributions and the relationships of nephrin, podocin, α actinin 4 and CD2AP in the glomerular of the rats. Results:① In PAN nephrosis rats, the distribution of CD2AP was along the glomerular basement membrane with a segmental and linear pattern. Nephrin and podocin showed a similar leaner pattern along the capillary wall but not resembled CD2AP.② In normal rats, there was a partial co localization between nephrin and CD2AP as well as podocin and CD2AP, and their co localizations did not change with the development of the proteinuria. ③ α actinin 4 showed a weaker and granular staining pattern along the glomerular capillary wall in normal rats, but its staining was stronger in 20 days. There was also a little overlapping between the staining of CD2AP and α actinin 4. However, this interaction was stronger with the development of proteinuria, and then the interaction was weaker with the recovery of proteinuria. ④ At the same time, α actinin 4 was also detected to partly co localize with nephrin and podocin, but the degree of the co localization was decreased in the state of proteinuria. However the co localization was recovered at the 20th day. Conclusion:CD2AP maybe a key molecule associated with nephrin, podocin and α actinin 4. The molecular complex of nephrin, podocin and CD2AP may play an important role in the development of proteinuria. In addition, the change of the podocyte molecular behavior may be a common pathway leading to proteinuria in PAN nephrotic rats.