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.

High glucose stimulates the expression of erythropoietin in rat glomerular epithelial cells / 한국실험동물학회지

It has been reported that the levels of erythropoietin are associated with diabetes mellitus. Glomerular epithelial cells, located in the renal cortex, play an important role in the regulation of kidney function and hyperglycemia-induced cell loss of glomerular epithelial cells is implicated in the onset of diabetic nephropathy. This study investigated the effect of high glucose on erythropoietin and erythropoietin receptor expression in rat glomerular epithelial cells. We found that 25 mM D-glucose, but not mannitol or L-glucose, stimulated erythropoietin mRNA and protein expression in a time dependent manner (>4 h) in rat glomerular epithelial cells. In addition, 25 mM glucose, but not mannitol or L-glucose, also increased the phosphorylation of erythropoietin receptor, suggesting a role for erythropoietin receptor phosphorylation in erythropoietin synthesis. We conclude that high glucose stimulates erythropoietin production and erythropoietin receptor phosphorylation in rat glomerular epithelial cells.

High Glucose and Advanced Glycosylation Endproducts (AGE) Modulate the CD2AP Expression in Glomerular Epithelial Cells (GEpC) / 대한신장학회잡지

BACKGROUND: Regardless of the underlying diagnosis, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the foot processes and componental changes in slit diaphragm. We examined the molecular basis for this alteration of the podocyte phenotype, involving quantitative and distributional changes especially on CD2AP as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and AGE induce podocyte cytoskeletal changes, we cultured rat GEpC under normal (5 mM) or high glucose (HG, 30 mM) and AGE- or BSA-added conditions and examined the distribution of CD2AP by confocal microscope and measured the change of CD2AP expression by Western blotting and RT-PCR. RESULTS: We found that CD2AP moved from peripheral to inner cytoplasm in the HG condition by confocal microscopy. In Western blotting, administration of high glucose or AGE decreased the CD2AP productions by 36.9% (p<0.05) and 16.0% (p< 0.05), respectively. Furthermore, both high glucose and AGE decreased the amount of CD2AP more significantly by 64.6% compared to those of control (p<0.01). Such changes was not seen in osmotic control. In RT-PCR, administration of high glucose, AGE or both high glucose and AGE decreased the expression of CD2AP mRNA by 44.9%, 27.9%, and 29.3% (p<0.05), respectively, compared to that of control. CONCLUSION: We could find that HG induce the inward translocation of CD2AP molecule and HG and AGE suppress the production of CD2AP at transcriptional and partly translational level. We suggest that these changes may explain the structural and functional changes of podocytes in diabetic conditions.

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.

Effect of Tumor Necrosis Factor-Alpha on Glomerular Epithelial Cells in Glomerular Permeability

PURPOSE: Minimal Change Disease (MCD) is the most common primary nephrotic syndrome in children. Some suggested that tumor necrosis factor-alpha (TNF-alpha) are involved in the pathogenesis of MCD. METHODS: This study was done to see the changes of plasma and urinary TNF-alpha, and its effect on the determination of permeability of the glomerular basement membrane (BM) contributed by heparan sulfate proteoglycan (HSPG). Study patients consisted of 19 biopsy-proven MCD children aged 2-15 years old. Both plasma and urinary TNF-alpha were measured. Employing the Millicell system, TNF-alpha was screened for the permeability factors. We examined whether TNF-alpha regulated BM HSPG gene expression and HS synthesis in the glomerular epithelial cells (GECs). RESULTS: Urinary TNF-alpha during relapse was significantly increased when compared with that of during remission or controls (364.4+/-51.2 vs 155.3+/-20.8, 36.0+/-4.5 ng/mg cr) (P< 0.05). However, negative results were obtained in the permeability assay using the Millicell system. No difference was seen in the BM HSPG gene expression and HS synthesis in the GECs. CONCLUSION: It seems that TNF-alpha may not play a disease-specific role in the pathogenesis of MCD.

Effects of High Glucose and Advanced Glycosylation Endproducts (AGE) on the alpha-actinin-4 Expressed by Glomerular Epithelial Cells (GEpC) / 대한신장학회잡지

BACKGROUND: Regardless of the underlying diagnosis, the proteinuric condition demonstrates ultrastructural changes in GEpC with retraction and effacement of the highly specialized interdigitating podocyte foot processes. I examined the molecular basis for this alteration of the podocyte phenotype, involving cytoskeletal changes especially on alpha-actinin-4 as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration and regulation of the podocyte actin cytoskeleton. METHODS: To investigate whether high glucose and AGE induce podocyte cytoskeletal changes, we cultured rat GEpC under normal (5 mM) or high glucose (30 mM) and AGE- or BSA-added conditions and examined the distribution of alpha-actinin-4 by confocal microscope and measured the change of alpha-actinin-4 production by Western blotting and RT-PCR. RESULTS: I found that alpha-actinin-4 moved from peripheral cytoplasm to inner actin filaments complexes in the condition of AGE and high glucose by confocal microscopy. In Western blotting, administration of high glucose or AGE decreased the alpha-actinin-4 productions by 22.3% (p>0.05) and 28.1% (p<0.05), respectively. Furthermore, both high glucose and AGE decreased the amount of alpha-actinin-4 more significantly by 53.6% compared to those of control (p<0.01). S uch changes could not be seen by osmotic control. The expression of mRNA for alpha- actinin-4 were not changed in condition of high glucose or AGE-coated surface, however, both high glucose and AGE significantly decreased the expression of alpha-actinin-4 mRNA by 15.7% compared to those of control. CONCLUSION: I could suggest that both high glucose and AGE induce the cytoplasmic translocation and suppress the production of alpha-actinin-4 at transcriptional level and these changes may explain the cytoskeletal changes of GEpC in diabetic conditions.

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.

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.

The Effect of Tumor Necrosis Factor-Alpha to Glomerular Epithelial Cells in Glomerular Permeability / 대한해부학회지

Minimal Change Disease (MCD) is the most common primary nephrotic syndrome in children. Some suggested that tumor necrosis factor-alpha (TNF-alpha) are involved in the pathogenesis of MCD. This study was done to see changes of plasma and urinary TNF-alpha, and its effect on determination of permeability of glomerular basement membrane (BM) contributed by heparan sulfate proteoglycan (HSPG). Study patients consisted of 19 biopsy-proven MCD children aged 2-15 years old. Both plasma and urinary TNF-alpha were measured. Employing the Millicell system, TNF-alpha was screened for the permeability factors. We examined whether TNF-alpha regulated BM HSPG gene expression and HS synthesis in the glomerular epithelial cells (GECs). Urinary TNF-alpha during relapse was also significantly increased (364.4+/-51.2 vs 155.3+/-20.8, 36.0+/-4.5 ng/mg.cr) (p<0.05). However, the negative results were obtained in the permeability assay using the Millicell system. No difference was seen in BM HSPG gene expression and HS synthesis in the GECs. Therefore, it seems that TNF-alpha may not play a disease-specific role in the pathogenesis of MCD.

Effects of Advanced Glycation Endproducts (AGE) on Rat Glomerular Epithelial Cells (GEC): Roles of Reactive Oxygen Species (ROS) / 대한신장학회잡지

BACKGROUND: AGE-induced oxidative stress is implicated in the development and progression of diabetic nephropathy. AGE also affect the GEC to increase their permeability, therefore, we investigate the possibility that AGE may induce oxidative stress and subsequent injury to GEC. METHODS: We cultured rat GEC on the AGE- or BSA-coated plate with high glucose (HG) to produce more pathophysiologic conditions similar to prolonged diabetic environment in vivo and measured the change of ROS and their anti-oxidants systems. We also evaluated the effects of probucol as an antioxidant on this system. RESULTS: The amount of superoxide anion slightly decreased on AGE condition without significance. However, the production of hydrogen peroxide was significantly enhanced by 10% on AGE-coated and HG condition compared to control (BSA-coated and 5 mM glucose) (p< 0.05) and hydroxyl radical have also showed similar increase on AGE-coated and HG condition by 10% above control (p< 0.01), and both increases were attenuated by probucol (both, p< 0.05). The activity of superoxide dismutase (SOD) was decreased by 10% on AGE-coated and HG condition (p< 0.05) and recovered by probucol partially. However, there were no significant changes on the activity of other anti-oxidant enzymes including catalase, glutathione peroxidase, and glutathione reductase. Therefore, glomerular epithelial injury presenting proteinuria may be provoked by hydrogen peroxide and subsequently increased hydroxyl radical induced by AGE and high glucose. CONCLUSION: We might assume that superoxide had been converted to hydrogen peroxide by consumptive SOD in the presence of AGE, and subsequently produced hydroxyl radical, which could be reversed by anti-oxidant, may induce diabetic glomerular epithelial injury and eventually proteinuria.

Colocalization of ANG II and mRNA for the Renin-Angiotensin System Components in Cultured Rat Glomerular Epithelial and Mesangial Cells / 대한해부학회지

Mesangial cells are found to have renin and angiotensin II-AT1 receptors, but the presence of other components of the renin-angiotensin system and production of angiotensin II within the cell have not been demonstrated. The presence of the renin-angiotensin system components in the glomerular epithelial cell has not been previously reported. We studied expression of each component of the renin-angiotensin system in primary cultured rat glomerular epithelial cells and mesangial cells. We assessed mRNA expression by RT-PCR and the presence of angiotensin II by immunocytochemistry. Both cultured glomerular epithelial cells and mesangian cells expressed mRNA for components of the renin-angiotensin system such as renin, angiotensinogen and angiotensin II type 1A and 1B receptor subtypes. Immunocytochemical studies with specific antibody for angiotensin II demonstrated significant immunoreactivity in both glomerular epithelial cells and mesangian cells. These results, for the first time, provide direct evidence that both the glomerular epithelial cells and mesangian cells contain a complete renin-angiotensin system and generate angiotensin II with intracellular mechanisms. Further studies are required to define the subcellular localization of angiotensin II with electron microscopy and to elucidate the physiological importance of the intracellular reninangiotensin system.