Effects of Epigallocatechin-3-Gallate on the Expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in High-Glucose-Stimulated Glomerular Epithelial Cells / 전남의대학술지

Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant polyphenol in green tea. In the present study, we investigated whether EGCG plays a role in the expression of transforming growth factor-beta1 (TGF-beta1), protein kinase C (PKC) alpha/betaII, and nuclear factor-kappaB (NF-kappaB) in glomerular epithelial cells (GECs) against high-glucose injury. Treatment with high glucose (30 mM) increased reactive oxygen species (ROS)/lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 microM EGCG attenuated the increase in ROS/LPO and restored the levels of GSH, whereas ROS, LPO, and GSH levels were not affected by treatment with 30 mM mannitol as an osmotic control. Interestingly, high-glucose treatment affected 3 separate signal transduction pathways in GECs. It increased the expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in GECs, respectively. EGCG (1, 10, 100 microM) pretreatment significantly decreased the expression of TGF-beta1 induced by high glucose in a dose-dependent manner. In addition, EGCG (100 microM) inhibited the phosphorylation of PKC alpha/betaII caused by glucose at 30 mM. Moreover, EGCG (1, 10, 100 microM) pretreatment significantly decreased the transcriptional activity of NF-kappaB induced by high glucose in a dose-dependent manner. These data suggest that EGCG could be a useful factor in modulating the injury to GECs caused by high glucose.

Effects of Epigallocatechin-3-Gallate on the Expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in High-Glucose-Stimulated Glomerular Epithelial Cells / 전남의대학술지

Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant polyphenol in green tea. In the present study, we investigated whether EGCG plays a role in the expression of transforming growth factor-beta1 (TGF-beta1), protein kinase C (PKC) alpha/betaII, and nuclear factor-kappaB (NF-kappaB) in glomerular epithelial cells (GECs) against high-glucose injury. Treatment with high glucose (30 mM) increased reactive oxygen species (ROS)/lipid peroxidation (LPO) and decreased glutathione (GSH) in GECs. Pretreatment with 100 microM EGCG attenuated the increase in ROS/LPO and restored the levels of GSH, whereas ROS, LPO, and GSH levels were not affected by treatment with 30 mM mannitol as an osmotic control. Interestingly, high-glucose treatment affected 3 separate signal transduction pathways in GECs. It increased the expression of TGF-beta1, PKC alpha/betaII, and NF-kappaB in GECs, respectively. EGCG (1, 10, 100 microM) pretreatment significantly decreased the expression of TGF-beta1 induced by high glucose in a dose-dependent manner. In addition, EGCG (100 microM) inhibited the phosphorylation of PKC alpha/betaII caused by glucose at 30 mM. Moreover, EGCG (1, 10, 100 microM) pretreatment significantly decreased the transcriptional activity of NF-kappaB induced by high glucose in a dose-dependent manner. These data suggest that EGCG could be a useful factor in modulating the injury to GECs caused by high glucose.

Angiotensin II Effect on alpha-Actinin in Glomerular Epithelial Cells / 대한신장학회잡지

BACKGROUND: Angiotensin II plays a potential role in renal injury not only by its vasoconstrictive effects but also its biochemical effects. alpha-Actinin, an actin-linked glycoprotein, is expressed in podocytes and known to be rearranged and changed in various glomerular diseases. We investigated the effect of angiotensin II on the alpha-actinin in the glomerular epithelial cells to find out the fact that it could be prevented by losartan, a type 1 angiotensin receptor antagonist. METHODS: Glomerular epithelial cells were treated with various concentrations of angiotensin II in culture media, and then we compared the localization and amount of alpha-actinin by confocal microscopy and Western blot analysis, respectively. We also compared the differences in the localization and protein amount of alpha-actinin by various concentrations of losartan in the presence of angiotensin II. In addition, we tried to observe the mRNA expression of alpha-actinin via RT-PCR. RESULTS: The fluorescent and band intensities of alpha-actinin were decreased by angiotensin II in a dose-dependent manner by confocal microscopy and Western blot analysis, respectively. These changes of alpha-actinin by angiotensin II were reversed by losartan in dose dependent manner. Angiotensin II also changed the distribution of alpha-actinin from peripheral to inner cytoplasm in dose-dependent manner, which was also reversed by losartan. The different expression of alpha-actinin m-RNA by RT-PCR were unremarkable. CONCLUSIONS: Angiotensin II decreases the amount of alpha-actinin protein and and makes cytoskeletal changes in glomerular epithelial cells, which could be reversed by losartan. It suggests that it could be prevented by angiotensin II AT1 receptor blockers.

Angiotensin II Effect on alpha-Actinin in Glomerular Epithelial Cells / 대한신장학회잡지

BACKGROUND: Angiotensin II plays a potential role in renal injury not only by its vasoconstrictive effects but also its biochemical effects. alpha-Actinin, an actin-linked glycoprotein, is expressed in podocytes and known to be rearranged and changed in various glomerular diseases. We investigated the effect of angiotensin II on the alpha-actinin in the glomerular epithelial cells to find out the fact that it could be prevented by losartan, a type 1 angiotensin receptor antagonist. METHODS: Glomerular epithelial cells were treated with various concentrations of angiotensin II in culture media, and then we compared the localization and amount of alpha-actinin by confocal microscopy and Western blot analysis, respectively. We also compared the differences in the localization and protein amount of alpha-actinin by various concentrations of losartan in the presence of angiotensin II. In addition, we tried to observe the mRNA expression of alpha-actinin via RT-PCR. RESULTS: The fluorescent and band intensities of alpha-actinin were decreased by angiotensin II in a dose-dependent manner by confocal microscopy and Western blot analysis, respectively. These changes of alpha-actinin by angiotensin II were reversed by losartan in dose dependent manner. Angiotensin II also changed the distribution of alpha-actinin from peripheral to inner cytoplasm in dose-dependent manner, which was also reversed by losartan. The different expression of alpha-actinin m-RNA by RT-PCR were unremarkable. CONCLUSIONS: Angiotensin II decreases the amount of alpha-actinin protein and and makes cytoskeletal changes in glomerular epithelial cells, which could be reversed by losartan. It suggests that it could be prevented by angiotensin II AT1 receptor blockers.

Radiation Induced Changes in the Expression of Fibronectin, Pai-1, MMP in Rat Glomerular Epithelial Cell / 대한방사선종양학회지

PURPOSE: Renal irradiation can lead to the development of radiation nephropathy, and this is characterized by the accumulation of extracellular matrix and final fibrosis. To determine the possible role of the glomerular epithelial cell, the radiation-induced changes in the expression of its genes associated with the extracellular matrix were analyzed. MATERIALS AND METHODS: Rat glomerular epithelial cells (GEpC) were irradiated with a single dose of 0, 2, 5, 10 and 20 Gy with using 6 MV LINAC (Siemens, USA), and the samples were collected 6, 24, 48 and 72 hours post-irradiation, respectively. Northern blotting, western blotting and zymography were used to measure the expression level of fibronectin (Fn), plasminogen activator inhibitor-1 (Pai-1), matrix metalloproteinases-2, 9 (MMP-2, 9), tissue inhibitor of metalloproteinase-2 (TIMP-2), tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). RESULTS: Irradiation with a single dose of 10 Gy resulted in a significant increase in Fn mRNA since 24 hours post-irradiation, and a single dose of 5 and 10 Gy significantly increased the Fn immunoreactive protein measured 48 hours post-irradiation. An increase in Pai-1 mRNA and protein was also observed and especially, a single dose of 10 Gy significantly increased the mRNA measured 24 and 48 hours post-irradiation. The active MMP-2 measured 24 hours post-irradiation slightly increased in a dose dependent manner, but this increase did not reach statistical significance. The levels of MMP-9, TIMP-2, t-PA and u-PA appeared unaltered after irradiation. CONCLUSION: Irradiation of the glomerular epithelial cells altered the expression of genes associated with the extracellular matrix, implying that the glomerular epithelial cell may be involved in the development of radiation nephropathy.

Effect of epidermal growth factor on glomerular epithelial cells cytoskeleton / 中华肾脏病杂志

Objective To study the changes of adriamycin-induced glomerular epithelial cells (GECs) permeability and cytoskeleton, and to explore the role and possible mechanism of epithelia growth factor (EGF) on adriamycin-induced glomerular epithelial barrier function. Methods Rat GECs on Milicell-PCF Inserts were exposed to adriamycin (0.5 ?mmol/L) 24 hours in the absence or presence of EGF. The paracelluar permeability to BSA and cell viability were evaluated. The expression of F-actin and alpha-actinin were analyzed by immunofluorescence and Leica laser scan confocal microscopy. Results After induced by adriamycin for 24 hours, paracelluar permeability to BSA and F-actin reorganization rate increased. Disassembling of cortical cytoskeleton architecture was observed. Stress fiber in cytoplasm disappeared. Alpha-actinin staining showed perinuclear enhancement, which is different from normal cytosolic pattern. EGF significantly reduced adriamycin induced effect. Higher level of BSA passed through GEC monolayer in a dose-dependent manner. EGF prevented adriamycin-induced cytoskeletal reorganization. Disassembled cortical cytoskeleton was recovered, and stress fiber appeared again. Alpha-actinin staining mainly exhibited cytosolic uniform distribution as control GEC. In addition, administration of either AG1478, a specific inhibitor of EGF-receptor tyrosine kinase, or U73122, a specific inhibitor of PLC? before EGF treatment attenuated the EGF-mediated effect, whereas neither AG1478 nor U73122 exerted influence in the absence of EGF. Conclusions Adriamycin increases GEC paracellular permeability to BSA as a result of adriamycin-induced cytoskeleton disassembling and disruption. EGF may prevent adriamycininduced cytoskeleton reorganization and maintain glomerular epithelial barrier function through EGF mediated EGF-EGFR-PLC? signal pathway.

The Effect of Cyclic AMP on Gene Regulation of Glomerular Basement Membrane Heparan Sulfate Proteoglycan in Rat Glomerular Epithelial Cells / 대한신장학회잡지

It is well known that the glomerular basement membrane heparan sulfate proteoglycan(GBM HSPG) synthesized by glomerular epithelial cell(GEC) has an important role in the permeability of glomerular basement membrane and cyclic AMP(cAMP) is involved in regulation of a wide variety of genes maybe including GBM HSPG gene. The direct effect of cAMP on GBM HSPG gene expression and metabolism was not evaluated as yet. Proteinuria represents an impairment of permselectivity function of glomerular basement membrane regulated by GBM HSPG and could be associated with increased glomerular level of cAMP in nephrotic syndrome of diverse causes. RPD-I(rat GBM HSPG core protein domain-I) detected a >9.5kb transcript of GBM HSPG in RNA of rat GEC. Emp1oying a riboprobe synthesized from RPD-I in RNase protection assay, we examined whether cAMP regulated perlecan expression in the GEC. At l, 6, 24 and 48 hrs of incubation, l mM cAMP caused 43%, 32%, 47% and 40% reduction in mRNA expression of perlecan, respectively. Immunoprecipitation showed a corresponding reduction of 51%, 70% and 68% in the synthesis of 35SO4 labeled GBM HSPG by the GEC fol1owing l2, 24 and 48 hrs of incubation with cAMP. Our results show that decrease in GBM HSPG gene expression and synthesis by cAMP may be of relevance to proteinuric states characterized by activation of these mediators.

Experimental Study of the Progressive Glomerulosclerosis Induced by Long-term Administration of Puromycin Aminonucleoside in Rats

Pathogenetic mechanisms of progressive glomerulosclerosis are not clear. We studied the long-term(10 weeks) effects of puromycin aminonucleoside(PAN) in Sprague-Dawley rats with or without uninephrectomy(UN). Compared to rats with PAN injections only, rats with uninephrectomy and PAN injections showed significantly higher serum levels of urea nitrogen(153 +/- 155 mg/dl vs. 16 +/- 4 mg/dl, p<0.01), ceatinine(2.96 +/- 1.21 mg/dl vs. 0.92 +/- 0.36 mg/dl, p<0.01), cholesterol(466 +/- 125 mg/dl vs. 94 +/- 27 mg/dl, p<0.01), and triglyceride(337 +/- 237 mg/dl vs. 111 +/- 36 mg/dl, p<0.05) as well as increased amounts of proteinuria(428 +/- 90 mg/day vs. 136 +/- 130 mg/day, p<0.01). Lesions of focal segmental glomerulosclerosis(FSGS) were more frequently observed in rats with UN and PAN injections than rats with PAN infections only(39.5 +/- 17.2% vs. 4.3 +/- 4.7%, p<0.01). Ultrastructural examination of the glomeruli from rats with UN and PAN injections revealed severe epithelial cell changes including foot process effacement, vaculoar change or pseudocyst formation and focal detachment of epithelial cells from the underlying basement membrane. The results suggest that chronic nephrosis induced by PAN showed functional and morphologic features similar to those of human FSGS. Cytotoxic effect of PAN on the glomerular epithelial cells may be an initiating factor for the development of FSGS. which may be aggravated by some hemodynamic changes induced by uninephrectomy.