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2086 OXALATE-INDUCED NADPH OXIDASE ACTIVATION IN RENAL EPITHELIAL CELLS: ROLE OF UPSTREAM MEDIATORS
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THE JOURNAL OF UROLOGY姞
2086 OXALATE-INDUCED NADPH OXIDASE ACTIVATION IN RENAL EPITHELIAL CELLS: ROLE OF UPSTREAM MEDIATORS Vijayalakshmi Thamilselvan*, Mani Menon, Sivagnanam Thamilselvan, Detroit, MI INTRODUCTION AND OBJECTIVES: Hyperoxaluria is one of the major risk factors for calcium oxalate nephrolithiasis and several in vitro and in vivo studies have shown that oxalate induced oxidative stress mediated cell injury contributes to renal stone formation. We have recently demonstrated that oxalate induces reactive oxygen species (ROS) via NADPH oxidase activation in renal epithelial cells. However, the upstream signaling mechanisms responsible for NADPH oxidase activation by oxalate are largely unknown. Therefore, we investigated the relevant upstream modulators of NADPH oxidasedependent oxalate-induced cell injury in renal epithelial cells. METHODS: Confluent monolayers of LLC-PK1 cells incubated with or without inhibitors of PKC (PKC␣ : inhibitor peptide, PKC␦: rottlerin), PI3-kinase (LY294002), tyrosine kinase (genistein), c-Src (PP2), p38 MAPK (SB203580), TGF-1 (neutralizing antibody), NADPH oxidase (DPI), RNA synthesis (actinomycin-D), and protein synthesis (cycloheximide) for 30 min were then exposed to 0.5 to 1.0 mM oxalate for different time periods. The ROS production, cell injury (LDH released), and NADPH oxidase activity (lucigenin chemiluminescence) were determined. Phosphorylation of p40phox, activation of Rac1, and membrane translocation of p47phox were determined by Western analysis. RESULTS: Exposure of LLC-PK1 cells to oxalate caused a dose and time dependent increase in ROS production, LDH release, and NADPH oxidase activity. Oxalate induced NADPH oxidase activity was associated with increased p40phox phosphorylation, p47phox membrane translocation, and Rac1 activation. Studies with actinomycin-D and cycloheximide indicated that NADPH oxidase activity was regulated at the transcriptional and translational levels. Inhibitors of PKC, PI3-kinase, tyrosine kinase, c-Src, and TGF-1 significantly attenuated oxalate-induced NADPH oxidase activity, superoxide production, and LDH release. DPI pretreatment completely prevented oxalate-induced NADPH oxidase activity in LLC-PK1 cells, where as p38 MAPK inhibitor did not prevent oxalate-induced ROS, LDH release, and NADPH oxidase activity. CONCLUSIONS: Our data demonstrates that oxalate-induced integrated signaling mechanisms leading to the activation of NADPH oxidase is responsible for sustained increase in ROS generation. The cellular injury as a result of increased ROS generation may lead to calcium oxalate kidney stone formation. Our study suggests that these signaling pathways may represent significant targets for therapeutic intervention in the treatment of recurrent kidney stones.
Vol. 189, No. 4S, Supplement, Wednesday, May 8, 2013
100% ONB water. A mixture comprising half 100% ONB water and half distilled water was defined as 50% ONB water. We divided 60 rats, aged 4 weeks, into 5 groups: (1) control, a water-fed group; (2) 100% ONB, a 100% ONB water-fed group; (3) EG, the EG-treated water-fed group; and (4), and (5) EG⫹50% ONB and EG⫹100% ONB, the 2 EG-treated, ONB water-fed groups. The rats consumed these fluids by free access for 5 weeks, and the EG-treated groups were also administered 1.2 mL of 10% EG and 0.5 g of vitamin D3 via a stomach tube for the last 2 weeks. Six rats in each group were sacrificed after 7 days and 6 after 14 days. The pooled 24-hour urine and blood samples of these rats were collected, and their kidneys were excised. Renal CaOx deposits, urinary excretion of N-acetyl--D-glucosaminidase (NAG), and renal expression of MCP-1 and OPN as inflammation-related proteins, SOD-1 as an oxidative stress biomarker, and the crystal binding molecule hyaluronic acid were compared among the 5 groups. RESULTS: In the EG group, CaOx deposits tended to increase at 14 days compared to 7 days after EG administration. However, CaOx deposits were significantly lower in the EG⫹50% ONB and EG⫹100% ONB groups than in the EG group 14 days after administration (Figure). In the EG⫹50% ONB and EG⫹100% ONB groups, ONB water significantly decreased urinary NAG excretion, and renal MCP-1, OPN, and hyaluronic acid expression, and increased renal SOD-1 expression compared to the EG group. CONCLUSIONS: ONB water exerted substantial effects on kidney stone formation in the rat kidney by protecting renal tubular cell injury. ONB water may be a prophylactic for kidney stones.
Source of Funding: NIH RO1 DK 056249
2087 OXYGEN NANO-BUBBLE WATER EXERTS INHIBITORY EFFECTS ON KIDNEY STONE FORMATION BY PROTECTING RENAL TUBULAR CELL INJURY Yasuhiko Hirose*, Takahiro Yasui, Kazumi Taguchi, Kazuhiro Niimi, Shuzo Hamamoto, Atsushi Okada, Yasunori Ito, Shoichi Sasaki, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Renal tubular cell injury induced by CaOx crystals plays an important role in kidney stone formation. Water containing oxygen nano-bubbles (ONB: nanometersized bubbles generated from oxygen micro-bubbles) has anti-inflammatory effects. Therefore, we investigated the inhibitory effects of ONB water on kidney stone formation in ethylene glycol (EG)-treated rats. METHODS: ONB water can be produced by rapidly collapsing oxygen micro-bubbles in hard water with a salt concentration of 1.0% by mass. Further, ONB water with a salt concentration of 0% by mass can be prepared by passing the resulting ONB water twice through a reverse osmosis membrane, obtaining ONB water was defined as
Source of Funding: grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT/JSPSKAKENHI Grant Number 22791483)
THE JOURNAL OF UROLOGY姞
2086 OXALATE-INDUCED NADPH OXIDASE ACTIVATION IN RENAL EPITHELIAL CELLS: ROLE OF UPSTREAM MEDIATORS Vijayalakshmi Thamilselvan*, Mani Menon, Sivagnanam Thamilselvan, Detroit, MI INTRODUCTION AND OBJECTIVES: Hyperoxaluria is one of the major risk factors for calcium oxalate nephrolithiasis and several in vitro and in vivo studies have shown that oxalate induced oxidative stress mediated cell injury contributes to renal stone formation. We have recently demonstrated that oxalate induces reactive oxygen species (ROS) via NADPH oxidase activation in renal epithelial cells. However, the upstream signaling mechanisms responsible for NADPH oxidase activation by oxalate are largely unknown. Therefore, we investigated the relevant upstream modulators of NADPH oxidasedependent oxalate-induced cell injury in renal epithelial cells. METHODS: Confluent monolayers of LLC-PK1 cells incubated with or without inhibitors of PKC (PKC␣ : inhibitor peptide, PKC␦: rottlerin), PI3-kinase (LY294002), tyrosine kinase (genistein), c-Src (PP2), p38 MAPK (SB203580), TGF-1 (neutralizing antibody), NADPH oxidase (DPI), RNA synthesis (actinomycin-D), and protein synthesis (cycloheximide) for 30 min were then exposed to 0.5 to 1.0 mM oxalate for different time periods. The ROS production, cell injury (LDH released), and NADPH oxidase activity (lucigenin chemiluminescence) were determined. Phosphorylation of p40phox, activation of Rac1, and membrane translocation of p47phox were determined by Western analysis. RESULTS: Exposure of LLC-PK1 cells to oxalate caused a dose and time dependent increase in ROS production, LDH release, and NADPH oxidase activity. Oxalate induced NADPH oxidase activity was associated with increased p40phox phosphorylation, p47phox membrane translocation, and Rac1 activation. Studies with actinomycin-D and cycloheximide indicated that NADPH oxidase activity was regulated at the transcriptional and translational levels. Inhibitors of PKC, PI3-kinase, tyrosine kinase, c-Src, and TGF-1 significantly attenuated oxalate-induced NADPH oxidase activity, superoxide production, and LDH release. DPI pretreatment completely prevented oxalate-induced NADPH oxidase activity in LLC-PK1 cells, where as p38 MAPK inhibitor did not prevent oxalate-induced ROS, LDH release, and NADPH oxidase activity. CONCLUSIONS: Our data demonstrates that oxalate-induced integrated signaling mechanisms leading to the activation of NADPH oxidase is responsible for sustained increase in ROS generation. The cellular injury as a result of increased ROS generation may lead to calcium oxalate kidney stone formation. Our study suggests that these signaling pathways may represent significant targets for therapeutic intervention in the treatment of recurrent kidney stones.
Vol. 189, No. 4S, Supplement, Wednesday, May 8, 2013
100% ONB water. A mixture comprising half 100% ONB water and half distilled water was defined as 50% ONB water. We divided 60 rats, aged 4 weeks, into 5 groups: (1) control, a water-fed group; (2) 100% ONB, a 100% ONB water-fed group; (3) EG, the EG-treated water-fed group; and (4), and (5) EG⫹50% ONB and EG⫹100% ONB, the 2 EG-treated, ONB water-fed groups. The rats consumed these fluids by free access for 5 weeks, and the EG-treated groups were also administered 1.2 mL of 10% EG and 0.5 g of vitamin D3 via a stomach tube for the last 2 weeks. Six rats in each group were sacrificed after 7 days and 6 after 14 days. The pooled 24-hour urine and blood samples of these rats were collected, and their kidneys were excised. Renal CaOx deposits, urinary excretion of N-acetyl--D-glucosaminidase (NAG), and renal expression of MCP-1 and OPN as inflammation-related proteins, SOD-1 as an oxidative stress biomarker, and the crystal binding molecule hyaluronic acid were compared among the 5 groups. RESULTS: In the EG group, CaOx deposits tended to increase at 14 days compared to 7 days after EG administration. However, CaOx deposits were significantly lower in the EG⫹50% ONB and EG⫹100% ONB groups than in the EG group 14 days after administration (Figure). In the EG⫹50% ONB and EG⫹100% ONB groups, ONB water significantly decreased urinary NAG excretion, and renal MCP-1, OPN, and hyaluronic acid expression, and increased renal SOD-1 expression compared to the EG group. CONCLUSIONS: ONB water exerted substantial effects on kidney stone formation in the rat kidney by protecting renal tubular cell injury. ONB water may be a prophylactic for kidney stones.
Source of Funding: NIH RO1 DK 056249
2087 OXYGEN NANO-BUBBLE WATER EXERTS INHIBITORY EFFECTS ON KIDNEY STONE FORMATION BY PROTECTING RENAL TUBULAR CELL INJURY Yasuhiko Hirose*, Takahiro Yasui, Kazumi Taguchi, Kazuhiro Niimi, Shuzo Hamamoto, Atsushi Okada, Yasunori Ito, Shoichi Sasaki, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Renal tubular cell injury induced by CaOx crystals plays an important role in kidney stone formation. Water containing oxygen nano-bubbles (ONB: nanometersized bubbles generated from oxygen micro-bubbles) has anti-inflammatory effects. Therefore, we investigated the inhibitory effects of ONB water on kidney stone formation in ethylene glycol (EG)-treated rats. METHODS: ONB water can be produced by rapidly collapsing oxygen micro-bubbles in hard water with a salt concentration of 1.0% by mass. Further, ONB water with a salt concentration of 0% by mass can be prepared by passing the resulting ONB water twice through a reverse osmosis membrane, obtaining ONB water was defined as
Source of Funding: grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT/JSPSKAKENHI Grant Number 22791483)