Effect of Puromycin Aminonucleoside on Podocyte P-Cadherin

PURPOSE: To test whether the expression of P-cadherin, a component of slit diaphragms between podocyte foot processes, would be altered by puromycin aminonucleoside (PAN) in a cultured podocyte in vitro. METHODS: Rat glomerular epithelial cells (GEpC) were cultured with various concentrations of PAN. The distribution of P-cadherin was examined with a confocal microscope. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to measure the change in P-cadherin expression. RESULTS: This study found that P-cadherin was concentrated in the inner and peripheral cytoplasm with high concentrations of PAN under immunofluorescence views. Western blotting of GEpC revealed that PAN induced a decrease of P-cadherin in dose- and time-dependent manners. A high dose (50 microg/mL) of PAN decreased P-cadherin expression by 21.9% at 24 h (P<0.05) and 31.9% at 48 h (P<0.01) compared to those without PAN. In RT-PCR, high concentrations (50 microg/mL) of PAN also decreased P-cadherin mRNA expression, similar to protein suppression, by 23.5% at 48 h (P<0.05). CONCLUSION: Podocytes exposed to PAN in vitro concentrated P-cadherin internally, and reduced P-cadherin mRNA and protein expression. This could explain the development of proteinuria in experimental PAN-induced nephropathy.

The Change of Podocyte beta-Catenin by Puromycin Aminonucleoside

PURPOSE: To test whether the expression of beta-catenin, a component of podocyte as a filtration molecule, would be altered by puromycin aminonucleoside (PAN) in the cultured podocyte in vitro. METHODS: We cultured rat glomerular epithelial cells (GEpC) with various concentrations of PAN and examined the distribution of beta-catenin by confocal microscope and measured the change of beta-catenin expression by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We found that beta-catenin relocalized from peripheral cytoplasm to inner cytoplasm, therefore, intercellular separations were seen in confluently cultured cells by high concentrations of PAN in immunofluorescence views. In Western blotting of GEpC, PAN (50 microg/mL) decreased beta-catenin expression by 34.9% at 24 hrs and 34.3% at 48 hrs, compared to those in without PAN condition (P<0.05). In RT-PCR, high concentrations (50 microg/mL) of PAN also decreased beta-catenin mRNA expression similar to protein suppression by 25.4% at 24 hrs and 51.8% at 48 hrs (P<0.05). CONCLUSION: Exposure of podocytes to PAN in vitro relocates beta-catenin internally and reduces beta-catenin mRNA and protein expression, which could explain the development of proteinuria in experimental PAN-induced nephropathy.