Volume 73, Issue 5, Pages 578-587 (March 2008) Prostacyclin protects renal tubular cells from gentamicin-induced apoptosis via a PPARα-dependent pathway  Y.-H. Hsu, C.-H. Chen, C.-C. Hou, Y.-M. Sue, C.-Y. Cheng, T.-H. Cheng, H. Lin, W.-L. Tsai, P. Chan, T.-H. Chen  Kidney International  Volume 73, Issue 5, Pages 578-587 (March 2008) DOI: 10.1038/sj.ki.5002704 Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 1 Gentamicin-induced apoptosis in NRK-52E cells. (a) Time-dependent apoptosis induced by gentamicin as revealed by TUNEL assays. NRK-52E cells were treated with 3 mM of gentamicin from 12 to 48 h, harvested, stained with TUNEL assay, and examined by fluorescence microscopy. The level of apoptosis was presented as the percentage of TUNEL-positive cells for each treatment. (b) Dose-dependent apoptosis induced by gentamicin as revealed by TUNEL assays. NRK-52E cells were treated with gentamicin at 1–5 mM for 24 h. Results are the mean±s.d. (n=6). *P<0.05 compared with the group without gentamicin treatment. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 2 The influence of Ad-COX-1/PGIS transfection on NRK-52E cells. (a) The levels of 6-keto-PGF1α in transfected NRK-52E cells. NRK-52E cells were transfected with Ad-COX-1/PGIS (Ad-C/P) at different MOI for 2 days. Ad-HPGK transfection was included as a control. Results are the mean±s.d. (n=6). *P<0.05 compared with the expression level of 6-keto-PGF1α in Ad-HPGK transfected cells. (b) The protective effect of Ad-COX-1/PGIS transfection against the gentamicin-induced apoptotic injury in NRK-52E cells. NRK-52E cells were transfected with Ad-COX-1/PGIS (Ad-C/P) or Ad-HPGK at different MOI for 2 days, and then treated with 3 mM of gentamicin for 24 h. The level of apoptosis was presented as the percentage of TUNEL-positive cells for each treatment. Results are the mean±s.d. (n=6). *P<0.05 compared with the gentamicin alone group; C represents untreated control. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 3 Effects of Ad-COX-1/PGIS transfection on gentamicin-induced apoptotic signaling. (a) The concentrations of cytosol gentamicin in NRK-52E cells. The NRK-52E cells were treated with 3 mM gentamicin for different time periods with Ad-COX-1/PGIS (solid bars) or Ad-HPGK transfection (blank bars) at 40 MOI. The concentration of gentamicin in each sample was detected as described in Materials and Methods. Data are expressed as the mean±s.d. (n=6). (b) The inhibitory effect of Ad-COX-1/PGIS transfection on gentamicin-induced ROS generation in NRK-52E cells. NRK-52E cells were transfected with Ad-COX-1/PGIS or Ad-HPGK at 40 MOI for 2 days, and then treated with 3 mM of gentamicin for 24 h. Gentamicin-induced increases in intracellular ROS were revealed by fluorescent intensities of DCF. Fluorescence intensities of cells are shown as the relative intensity of experimental groups compared with untreated control cells. Results are the mean±s.d. (n=6). *P<0.05 compared with the Ad-HPGK-transfected group with gentamicin treatment. (c, d) The influence of Ad-COX-1/PGIS transfection on the activity of SOD and catalase in NRK-52E cells. The transfected cells were harvested and the activity of SOD and catalase was analyzed using ELISA assay. Results are the mean±s.d. (n=6). *P<0.05 compared with the Ad-HPGK-transfected group with gentamicin treatment. (e) Effects of Ad-COX-1/PGIS transfection on caspases, cytochrome c, and Bcl-xL in gentamicin-treated NRK-52E cells. Transfected cells were treated with 3 mM of gentamicin for 24 h. Western blotting was carried out with the specific antibody against cleaved caspase-3 and caspase-9, cytochrome c, and Bcl-xL. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. C/P, Ad-COX-1/PGIS transfection; H, Ad-HPGK transfection; C, untreated control. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 4 Effects of Ad-COX-1/PGIS transfection on PPARα expression in NRK-52E cells. (a) The relative mRNA levels of PPARα in NRK-52E cells. Gentamicin decreased PPARα mRNA levels more than 50% compared with controls as revealed by real-time reverse transcriptase-polymerase chain reaction (*P<0.05). NRK-52E cells were also transfected with 40 MOI of Ad-COX-1/PGIS or Ad-HPGK for 2 days, and then treated with or without gentamicin. There was no significant influence of the transfection on the gentamicin-induced decrease of PPARα mRNA levels. A bar is mean±s.d. of three experiments. (b) The effect of Ad-COX-1/PGIS transfection on the quantity of PPARα in NRK-52E cells. NRK-52E cells were transfected with Ad-COX-1/PGIS or Ad-HPGK at 40 MOI for different periods. Total protein and cell nuclei in each sample were purified. PPARα of each sample was immunoprecipitated and immunoblotted with anti-PPARα antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. C/P, Ad-COX-1/PGIS transfection; H, Ad-HPGK transfection; C, untreated control. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 5 Effects of PPARα overexpression on gentamicin-induced apoptotic signaling. (a) The PPARα expression level in NRK-52E cells with Flag-PPARα construct transformation. The level of Flag-PPARα fusion protein in each sample was determined by western blot analysis with anti-Flag and anti-PPARα antibodies. (b) Protein levels of cleaved caspase-3 in transformed NRK-52E cells treated with gentamicin. Transformed NRK-52E cells were treated with 3 mM of gentamicin for 24 h, and then analyzed using western blotting with the specific antibody against cleaved caspase-3. (c) The protective effect of Flag-PPARα transformation against the gentamicin-induced apoptosis in NRK-52E cells. Transformed NRK-52E cells were treated with 3 mM of gentamicin for 24 h, and then stained by TUNEL assay. Results are the mean±s.d. (n=6). *P<0.05 compared with the blank vector transformants with gentamicin treatment. C, untransformed controls; V, blank vector transformants; P, Flag-PPARα construct transformants. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 6 The influence of Flag-PPARα transformation on gentamicin-induced ROS generation in NRK-52E cells. (a) The inhibition effect of Flag-PPARα transformation on gentamicin-induced ROS generation. NRK-52E cells were transformed with the Flag-PPARα construct and then treated with 3 mM of gentamicin for 24 h. Gentamicin-induced increases in intracellular ROS were revealed by fluorescent intensities of DCF. Fluorescence intensities of cells are shown as the relative intensity of experimental groups compared with untreated control cells. Results are the mean±s.d. (n=6). *P<0.05 compared with the blank vector transformants with gentamicin treatment. (b, c) The influence of Flag-PPARα transformation on the activity of SOD and catalase in NRK-52E cells. The transformed cells were harvested and the activity of SOD and catalase was analyzed using ELISA assay. Results are the mean±s.d. (n=6). *P<0.05 compared with the blank vector transformants group with gentamicin treatment. C, untransformed controls; V, blank vector transformants; P, Flag-PPARα construct transformants. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 7 Effects of the blocking of PPARα expression on the protective effect of Ad-COX-1/PGIS transfection in NRK-52E cells. (a) The PPARα mRNA level in NRK-52E cells with PPARα siRNA transformation. Total RNA in each sample was extracted and applied in real-time polymerase chain reaction assays. (b) The influence of PPARα siRNA transformation on the protective effect of Ad-COX-1/PGIS in NRK-52E cells. NRK-52E cells transformed with PPARα siRNA or mock control siRNA were transfected with or without Ad-COX-1/PGIS at 40 MOI for 2 days, treated with 3 mM of gentamicin for 24 h, and then stained by TUNEL assays. Results are the mean±s.d. (n=6). (c) Protein levels of cleaved caspase-3 in siRNA-transformed NRK-52E cells treated with gentamicin. Each sample was analyzed using western blotting with the specific antibody against cleaved caspase-3. C, untransformed controls; M, mock controls; sP, PPARα siRNA transformants. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 8 The protective effect of DHA against gentamicin-induced renal injury in vivo. Normal and PPARα-deficient mice were injected with saline, gentamicin, or gentamicin and DHA. Concentrations of blood urea nitrogen and serum creatinine were measured at the end of the treatment. (a) The concentration of blood urea nitrogen in treated mice. (c) The concentration of serum creatinine in treated mice. Results are the mean±s.d. (n=6). *P<0.05 compared with the gentamicin alone group. (b) Accumulation of gentamicin in mouse kidney. The gentamicin quantity in the kidneys of normal and PPAR-α-deficient mice was measured at 120 min after the intraperitoneal injection of gentamicin without or with DHA. Results are the mean±s.d. (n=3).(d) Representative photomicrographs of hematoxylin and eosin staining. Bar=50 μm (n=5). (e) Apoptotic cells in the renal cortex of the gentamicin-treated mice. (f) Apoptotic cells in the renal cortex of the PPARα-deficient mice with gentamicin treatment. TUNEL-labeled nuclei were revealed as bright spots in cortex sections from untreated and treated mice. The identical fields stained for TUNEL were also stained using 4′,6-diamidino-2-phenyindole to reveal the positions of cell nuclei. Bar=100 μm. Kidney International 2008 73, 578-587DOI: (10.1038/sj.ki.5002704) Copyright © 2008 International Society of Nephrology Terms and Conditions