High Glucose and Advanced Glycosylation Endproducts(AGE) Modulate the P-cadherin Expression in Glomerular Epithelial Cells(GEpC)

PURPOSE: Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the integrity of slit diaphragm(SD) proteins including nephrin, P-cadherin, and others. Diabetic proteinuric condition demonstrates defects in SD molecules as well as ultrastructural changes in podocytes. We examined the molecular basis for this alteration of SD molecules especially on P-cadherin as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and AGE induce changes in SD, we cultured rat GEpC under normal(5 mM) or high glucose(30 mM) and AGE- or BSA-added conditions and measured the change of P-cadherin expression by Western blotting and RT- PCR. RESULTS: We found that administration of high glucose decreased the P-cadherin production significantly in the presence or absence of AGE by Western blotting. In RT-PCR high glucose with or without AGE also significantly decreased the expression of P-cadherin mRNA compared to those of controls. Such changes were not seen in the osmotic control. CONCLUSION: We suggest that high glucose with or without AGE suppresses the production of P-cadherin at the transcriptional level and that these changes may explain the functional changes of SD in diabetic conditions.

Effects of Angiotensin II on Glomerular Epithelial Cells Permeability Model; Role of Oxidative Stress / 대한신장학회잡지

BACKGROUND: Glomerular injury induced by angiotensin II (Ang II) may arise from its hemodynamic or non-hemodynamic actions including oxidative stress, or from such effects of Ang II acting in concert. The release of reactive oxygen species from podocytes may play a role in the pathogenesis of glomerular damage and proteinuria. METHODS: To investigate whether Ang II induces oxidative stress in vitro in glomerular epithelial cells (GEpC) and whether such oxidant stress may increase in vitro glomerular permeability model using cultured GEpC, we studied GEpC culture exposed to Ang II and antioxidant, probucol. For oxidative system assay, we measured the production of superoxide anion and hydrogen peroxide and the activity of superoxide dismutase (SOD). Scanning electron microscopy was performed on cells grown for one week on chamber slides. RESULTS: We found that in vitro permeability, which was prevented from probucol, increased significantly in media with 10-4 and 10-5 M of Ang II by 15.9% and 13%, respectively. Administration of the 10-5 M of Ang II significantly increased the superoxide anion productions by 39%, 61% and 30% at 1, 2 and 6 hours exposure time, respectively, compared to those of control and suppressed by probucol to control levels. At high concentration (10-5 M) Ang II suppressed the activity of SOD without affecting the production of hydrogen peroxide on the other hand, at low concentration (less than 10-5 M) Ang II showed reverse results. On ultrastructural examination, we could see the shortened and fused microvilli on GEpC surface by 10-5 M of Ang II, which change could be prevented by probucol. CONCLUSION: We could suggest that Ang II induces the generation of superoxide anion and the suppression of the activity of SOD, and subsequent oxidative stress leading to increase glomerular permeability by disruption of glomerular filtration barrier.

Effects of High Glucose and Advanced Glycosylation Endproducts (AGE) on ZO-1 Expression in cultured Glomerular Epithelial Cells (GEpC)

PURPOSE: Regardless of the underlying diseases, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. We examined the molecular basis for this alteration of the podocyte phenotypes, including quantitative and distributional changes of ZO-1 protein as a candidate contributing to the pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and advanced glycosylation endproduct(AGE) induce podocyte cytoskeletal changes, we cultured rat GEpC under 1) normal glucose(5 mM, =control) or 2) high glucose(30 mM) or 3) AGE-added or 4) high glucose plus AGE-added conditions. The distribution of ZO-1 was observed by confocal microscope and the change of ZO-1 expression was measured by Western blotting and RT-PCR. RESULTS: By confocal microscopy, we observed that ZO-1 moves from peripheral cytoplasm to inner actin filaments complexes in both AGE-added and high glucose condition. In Western blotting, high glucose or AGE-added condition decreased the ZO-1 protein expression by 11.1%(P>0.05) and 2.3%(P>0.05), respectively compared to the normal glucose condition. High glucose plus AGE-added condition further decreased ZO-1 protein expression to statistically significant level(12%, P<0.05). No significant change was seen in the osmotic control. In RT-PCR, high glucose plus AGE-added condition significantly decreased the expression of ZO-1 mRNA by 12% compared to normal glucose condition. CONCLUSION: We suggest that both high glucose and AGE-added condition induce the cytoplasmic translocation and suppresses the production of ZO-1 at transcriptional level and these changes may explain the functional changes of podocytes in diabetic conditions.