A major limiting factor in the use of cyclosporine A (CsA) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of CsA tubulotoxicity, we examined mechanisms (DNA synthesis, necrosis and apoptosis) of cellular injury induced by CsA in cultured LLC-PK1 renal tubular cell line. The possible role of Fas antigen in the mediation of CsA-induced cell death and the hypothesis that CsA-mediated injury activates the glucose transporter GLUT1, a stress response gene in renal tubular cells were also investigated. CsA treatment for 24 hours in LLC-PK1 cells showed significantly decreased 3H-thymidine uptake in a dose dependent manner (0.1 microgram/ml to 1 mg/ml), indicating that DNA damage is a sensitive indicator of CsA induced nephrotoxicity. A dose of 10 microgramml CsA caused a significant increase in LDH release (M+/-S.D., 11.0+/-3.0% vs 27.0+/-9.8, p<0.05). On flow cytometric analysis, 9.9 4.2% of control cells appeared in a region of decreased forward light scatter and increased side scatter, respectively. Both indices representing characteristics of apoptotic cell death. Compared to control, treatment with 10 ng/ml of CsA for 24 hours significantly increased the proportion of cells in apoptotic region to 38.9 13.5%. This finding was supported by electrophoretic analysis of the DNA extracted from CsA-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. CsA (0.1 microgram/ml) treatment for 24 hours was seen to cause a significant elevation in the expression of the 45 kD Fas protein by Western blot analysis. In addition, the exposure to CsA was also associated with an increase of GLUT1 protein levels up to 2.2 fold (mean) on Western blot analysis. In conclusion, CsA is directly toxic to tubular cells with inhibiting DNA synthesis and inducing cell death in the form of necrosis or apoptosis. Fas antigen-ligand system and glucose transporter GLUT1 may play roles in mediating CsA induced tubular cytotoxicity.
Animals ; Antigens, CD95 ; Apoptosis* ; Base Pairing ; Blotting, Western ; Cell Death ; Cell Line ; Cyclosporine* ; DNA ; DNA Damage ; Epithelial Cells* ; Glucose Transport Proteins, Facilitative ; Glucose Transporter Type 1 ; LLC-PK1 Cells ; Necrosis ; Negotiating ; Swine

A major limiting factor in the use of cyclosporine A (CsA) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of CsA tubulotoxicity, we examined mechanisms (DNA synthesis, necrosis and apoptosis) of cellular injury induced by CsA in cultured LLC-PK1 renal tubular cell line. The possible role of Fas antigen in the mediation of CsA-induced cell death and the hypothesis that CsA-mediated injury activates the glucose transporter GLUT1, a stress response gene in renal tubular cells were also investigated. CsA treatment for 24 hours in LLC-PK1 cells showed significantly decreased 3H-thymidine uptake in a dose dependent manner (0.1 microgram/ml to 1 mg/ml), indicating that DNA damage is a sensitive indicator of CsA induced nephrotoxicity. A dose of 10 microgramml CsA caused a significant increase in LDH release (M+/-S.D., 11.0+/-3.0% vs 27.0+/-9.8, p<0.05). On flow cytometric analysis, 9.9 4.2% of control cells appeared in a region of decreased forward light scatter and increased side scatter, respectively. Both indices representing characteristics of apoptotic cell death. Compared to control, treatment with 10 ng/ml of CsA for 24 hours significantly increased the proportion of cells in apoptotic region to 38.9 13.5%. This finding was supported by electrophoretic analysis of the DNA extracted from CsA-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. CsA (0.1 microgram/ml) treatment for 24 hours was seen to cause a significant elevation in the expression of the 45 kD Fas protein by Western blot analysis. In addition, the exposure to CsA was also associated with an increase of GLUT1 protein levels up to 2.2 fold (mean) on Western blot analysis. In conclusion, CsA is directly toxic to tubular cells with inhibiting DNA synthesis and inducing cell death in the form of necrosis or apoptosis. Fas antigen-ligand system and glucose transporter GLUT1 may play roles in mediating CsA induced tubular cytotoxicity.
Animals ; Antigens, CD95 ; Apoptosis* ; Base Pairing ; Blotting, Western ; Cell Death ; Cell Line ; Cyclosporine* ; DNA ; DNA Damage ; Epithelial Cells* ; Glucose Transport Proteins, Facilitative ; Glucose Transporter Type 1 ; LLC-PK1 Cells ; Necrosis ; Negotiating ; Swine

Nephrotoxicity is a major limiting factor in the use of aminoglycoside antibiotics, the mechanisms for which are still speculative. To clarify the mechanisms of renal tubular cell death induced by aminoglycosides, we examined the renal proximal tubule-like cell line, LLC-PK1, after inducing apoptosis through a chronic treatment with gentamicin (GM). Changes in the expression of the Fas were also investigated. On flow cytometric analysis, 5.7 +/- 3.3% of the control cells appeared in a region of decreased forward light scatter and increased side light scatter, where both indices represent the characteristics of apoptotic cell death. Compared to the control, treatment with 10 mM of GM for 15 days significantly increased the proportion of cells in the apoptotic region to 23.9 +/- 8.5%. This finding was supported by electrophoretic analysis of the DNA extracted from the GM-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. However, the 15-day GM treatment did not cause a significant elevation in the expression of the 45 kD Fas protein, the cell surface molecule that stimulates apoptosis, by Western blot analysis. In conclusion, long-term exposure to GM induces apoptosis of the renal tubular epithelial cells, and this process may contribute to some of the aminoglycoside nephrotoxicities. Further studies are needed on the mechanism(s) of apoptosis induced by GM.
Animal ; Antibiotics, Aminoglycoside/toxicity* ; Antigens, CD95/analysis ; Apoptosis/drug effects* ; Cell Line ; Gentamicins/toxicity* ; Kidney Tubules, Proximal/pathology ; Kidney Tubules, Proximal/drug effects* ; Swine

Thickening of tubular basement membrane and progressive tubulointerstitial fibrosis has been reported as important components of diabetic nephropathy, In order to investigate the mechanisms of tubulinterstitial changes in diabetic nephropathy, we evaluated the effects of a high concentration of glucose(25mM; 450mg/dL) on glucose transporter GLUT1 level, fibronectin production and tissue inhibitors of metalloproteinases (TIMP)-1 concentration in renal tubular(LLC-PK1) cells. As the effect of high glucose-induced alteration in LLC-PK1 cells, the expression of facilitative glueose transporter, GLUT1 was decreased after longer than 24-hours exposure to 25mM glucose, compared to control(5.6mM). The administration of protein kinase C (PKC) inhibitor GF109203X(10 microM) did not show significant effect on high glucose-induced decrease of GLUT1 level. On western blot analysis of fibronectin production, The exposure of LLC-PK cells to 25mM glucose for 48 hours significantly increasc4 fibro- nectin production, dose-dependently. The addition of GF102903X at the concentration of 10pM induced the significant increase of fibronectin level in LLC-PK1cells under glucose-free condition, whereas there was no significant effect on the high glucose-induced increase of fibronectin production. The addition of anti-TGF-beta antibody at 30 microgram/mL partly inhibited the high glucose-induced increase of fibronectin production. Concerning the changes of tissue inhibitor of metallo-proteinase(TIMP)-1 levels in the presence of high glucose, the exposure to high glucose for 24 and 43 hours increased TIMP-1 levels in culture supernatant of LLC-PK1 cells, dose-dependently. The TIMP-1 levels of 48-hour exposure to 15 and 25mM glucose were also significantly higher than those of 24-hourexposure. The treatment with 10 microM GF102903X or 30 microgram/mL anti-TGF-Beta antibody had no significant effects on TIMP-1 levels measured under the high glucose culture condition. In conclusion, the expression of facilitative glucose transporter, GLUT1 is inhibited and the production of fibronectin is increased in renal tubular cells cultured in the presence of high concentration of glucose, which is partly mediated by TGF-beta. The TIMP-1 level is also increased under high glucose culture condition. The enhanced productions of fibronectin and TIMP-1 of renal tubular cells under high glucose concentration may contribute to tubulointerstitial fibrosis that occurs in diabetic nephropathy.
Animals ; Basement Membrane ; Blotting, Western ; Diabetic Nephropathies ; Epithelial Cells* ; Extracellular Matrix* ; Fibronectins ; Fibrosis ; Glucose Transport Proteins, Facilitative ; Glucose* ; LLC-PK1 Cells ; Metalloproteases ; Protein Kinase C ; Swine ; Tissue Inhibitor of Metalloproteinase-1 ; Transforming Growth Factor beta