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THE ROLE OF NF-κB ON RENAL STONE FORMATION
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THE JOURNAL OF UROLOGY®
considered significant. The most strongly up and down-regulated genes were grouped based on their gene ontology and function. Genes were also grouped into relevant pathways and changes in functional pathways as a result of oxalate exposure were determined using Ingenuity Pathway Analysis software. Changes in signaling pathways identified by the software were confirmed using western blot analysis. RESULTS: Our study identified a number of genes that are not active in control cells but are highly up-regulated upon oxalate exposure for as little as 4 hours. Expression of many genes that are involved in regulation of gene expression also show changes after oxalate exposure. In addition, using the Ingenuity Pathway Analysis software, we have identified the activation of IL-2 receptor signaling as a result of 4h treatment of HK-2 cells to 1mM oxalate. Changes in expression of the IL-2B receptor and its down stream effectors including STAT5 and JAK1 was confirmed by western blot analysis after treating HK-2 cells with oxalate for various time points. CONCLUSIONS: These exciting observations suggest, oxalate may thus modulate additional, previously unknown intracellular pathways in hyperoxaluria. Further studies into these pathways may provide the key to unlocking novel biochemical targets in stone disease. Source of Funding: NIH-DK-RO1-54084 (Koul H, PI), AEFSOM
1998 THE ROLE OF NF-KB ON RENAL STONE FORMATION Keiichi Tozawa*, Takahiro Yasui, Masahito Hirose, Masayuki Usami, Shuzo Hamamoto, Yasuhiko Hirose, Atsushi Okada, Yasunori Itoh, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVE: Osteopontin (OPN) plays an important role in the development of urolithiasis. Our previous observation revealed that OPN cDNA was encoded in renal calcium oxalate (CaOx) stones protein by molecular sequencing . The transcription factor nuclear factor-KB (NF-KB) is involved in inflammatory and immune responses through the induction of various cytokines and growth factors. Recently, the coordinated action of NF-KB and activator protein-1 (AP-1) was reported in OPN expression. Oxalate, a metabolic end product, is excreted primarily by the kidney and associated with several pathological conditions. This organic dicarboxylate is freely filtered in the glomerulus and is transported bidirectionally into the renal tubules. N-acetyl-l-cysteine (NAC) is not only an antioxidant but also an anti-NF-KB reagent. Previously, we demonstrated the inhibition of NF-KB activation in prostate cancer cells and vascular endothelial cells. In the present study, we demonstrated that oxalate induces OPN expression by activating NF-KB in renal tubular cells. Furthermore, we investigated the inhibitory effect of NAC against NF-KB activation in the human renal tubular cells. METHODS: All the experiments were carried out using human kidney-2 (HK-2) cells, which are human proximal tubular epithelial cells immortalized by transduction with the human papilloma virus 16E6/E7 gene. The time-dependent extraction of total protein was performed after the uptake of 0.5 mM oxalate by the cells. NF-KB activation, OPN expression, and the inhibitory effects of NAC against NF-KB activation were examined by western blotting and immunocytochemistry. RESULTS: As a result of oxalate stimulation, the amount of p65 subunit in the nucleus increased significantly (P < 0.05), and NAC significantly inhibited the translocation of p65 into the nucleus (P < 0.05). The inhibitory effect of NAC on NF-KB activation was demonstrated on a human renal tubular cell line. CONCLUSIONS: NAC is not only an antioxidant but also an anti-NF-KB reagent. Previously, we demonstrated the inhibition of NFKB activation in prostate cancer cells and vascular endothelial cells. Our observations reveal that the inhibition of NF-KB activation and OPN expression using NAC is extremely useful for the prevention of stone formation. These observations indicate that NAC can be used as a drug to prevent stone formation. Source of Funding: None
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
1999 INCREASED PROTEIN INTAKE ON CONTROLLED OXALATE DIETS DOES NOT INCREASE URINARY OXALATE EXCRETION John Knight, Linda Easter, Rebecca Neiberg, Ross P Holmes, Dean G Assimos*, Winston-salem, NC INTRODUCTION AND OBJECTIVE: High protein intake is a risk factor for calcium oxalate stone disease. The effect of dietary protein on the urinary excretion of calcium, acid and citrate is well established. However, its effect on oxalate excretion is unclear, due in part to an inadequate control of dietary oxalate intake in previous studies. This relationship warrants clarification due to the important role of the metabolism of amino acids in endogenous oxalate synthesis. METHODS: Eleven healthy human subjects (6 males and 5 females) of mean age, 32.0 ± 9.4 ± yrs, and mean BMI, 23.5 ± 3.0, completed the study. Subjects consumed controlled, eucaloric diets that contained 0.6, 1.2 or 1.8 g of protein per kg body weight. Dietary oxalate, sodium, calcium and fat consumption was the same for all 3 diets. Individual energy needs were calculated using the Harris-Benedict equation with an added activity factor based on self-reported daily activity. Each diet was consumed for 6 days in random order, with 24 hr urine collections on each of the last 3 days of the diet and a 3 - 5 day washout period between diet periods. RESULTS: The analysis of 24 hr urine collections confirmed that as protein intake increased, urinary calcium increased and urinary pH and citrate decreased. Urinary oxalate excretion did not increase with high protein intake, but urinary glycolate excretion did. The increased glycolate excretion was due in part to an increased hydroxyproline, but not glycolate consumption. CONCLUSIONS: These results indicate that as dietary protein intake increases, the catabolism of diet-derived amino acids is not associated with an increased endogenous oxalate synthesis in normal subjects. Source of Funding: None
2000 DIFFERNTIAL RENAL TISSUE PROTEIN PROFILING IN A MOUSE MODEL OF HYPERCALCIURIA: EFFECT OF HIGH OXALATE DIET Benjamin K Canales*, Leticia Reyes, Patricia A Glenton, Marjorie Chow, Sixue Chen, Saeed R Khan, Gainesville, FL INTRODUCTION AND OBJECTIVE: NPT2a knockout mice have disruptions in Type IIa sodium/phosphate co-transporter and develop hypercalciuria associated with intra-tubular deposits of calcium phosphate crystals. When made moderately hyperoxaluric, these mice express both calcium oxalate and calcium phosphate crystals. We hypothesized that protein differences could be seen between normal and hyperoxaluric groups using a tissue proteomic approach, giving insight into calcium crystal pathogenesis. METHODS: Normal and knockout mice were randomized to control and 3% glyoxylate chow. Food, water consumption, weight, and urine were monitored daily. Mice were sacrificed at 42 days, and renal tissue was obtained for protein, crystal, and immunohistochemical analysis. Crystal distribution was determined by a semiquantitative scoring system. Tissue protein was labeled for two-dimensional difference gel electrophoresis (2D DIGE) and spotted using DeCyder™ version 6.5. Parallel experiments in protein abundance and identity were performed with iTRAQ labeling followed by liquid chromatography and tandem mass spectromety (MS/MS). Statistical analysis was performed using the ANOVA and Student T-test for group to group comparisons on spot average ratios. RESULTS: By the means of 2D-DIGE technique, 80 differential protein spots were detected, where over 600 proteins were identified and quantified by MS. Compared to normals, knockout mice had intratubular calcium phosphate deposits and over 150 differentially expressed proteins, primarily housekeeping and cell membrane. Differential analysis indicates that proteins involved in oxidative stress responses were significantly (p<0.01) increased in hyperoxaluric mice on glyoxylate
THE JOURNAL OF UROLOGY®
considered significant. The most strongly up and down-regulated genes were grouped based on their gene ontology and function. Genes were also grouped into relevant pathways and changes in functional pathways as a result of oxalate exposure were determined using Ingenuity Pathway Analysis software. Changes in signaling pathways identified by the software were confirmed using western blot analysis. RESULTS: Our study identified a number of genes that are not active in control cells but are highly up-regulated upon oxalate exposure for as little as 4 hours. Expression of many genes that are involved in regulation of gene expression also show changes after oxalate exposure. In addition, using the Ingenuity Pathway Analysis software, we have identified the activation of IL-2 receptor signaling as a result of 4h treatment of HK-2 cells to 1mM oxalate. Changes in expression of the IL-2B receptor and its down stream effectors including STAT5 and JAK1 was confirmed by western blot analysis after treating HK-2 cells with oxalate for various time points. CONCLUSIONS: These exciting observations suggest, oxalate may thus modulate additional, previously unknown intracellular pathways in hyperoxaluria. Further studies into these pathways may provide the key to unlocking novel biochemical targets in stone disease. Source of Funding: NIH-DK-RO1-54084 (Koul H, PI), AEFSOM
1998 THE ROLE OF NF-KB ON RENAL STONE FORMATION Keiichi Tozawa*, Takahiro Yasui, Masahito Hirose, Masayuki Usami, Shuzo Hamamoto, Yasuhiko Hirose, Atsushi Okada, Yasunori Itoh, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVE: Osteopontin (OPN) plays an important role in the development of urolithiasis. Our previous observation revealed that OPN cDNA was encoded in renal calcium oxalate (CaOx) stones protein by molecular sequencing . The transcription factor nuclear factor-KB (NF-KB) is involved in inflammatory and immune responses through the induction of various cytokines and growth factors. Recently, the coordinated action of NF-KB and activator protein-1 (AP-1) was reported in OPN expression. Oxalate, a metabolic end product, is excreted primarily by the kidney and associated with several pathological conditions. This organic dicarboxylate is freely filtered in the glomerulus and is transported bidirectionally into the renal tubules. N-acetyl-l-cysteine (NAC) is not only an antioxidant but also an anti-NF-KB reagent. Previously, we demonstrated the inhibition of NF-KB activation in prostate cancer cells and vascular endothelial cells. In the present study, we demonstrated that oxalate induces OPN expression by activating NF-KB in renal tubular cells. Furthermore, we investigated the inhibitory effect of NAC against NF-KB activation in the human renal tubular cells. METHODS: All the experiments were carried out using human kidney-2 (HK-2) cells, which are human proximal tubular epithelial cells immortalized by transduction with the human papilloma virus 16E6/E7 gene. The time-dependent extraction of total protein was performed after the uptake of 0.5 mM oxalate by the cells. NF-KB activation, OPN expression, and the inhibitory effects of NAC against NF-KB activation were examined by western blotting and immunocytochemistry. RESULTS: As a result of oxalate stimulation, the amount of p65 subunit in the nucleus increased significantly (P < 0.05), and NAC significantly inhibited the translocation of p65 into the nucleus (P < 0.05). The inhibitory effect of NAC on NF-KB activation was demonstrated on a human renal tubular cell line. CONCLUSIONS: NAC is not only an antioxidant but also an anti-NF-KB reagent. Previously, we demonstrated the inhibition of NFKB activation in prostate cancer cells and vascular endothelial cells. Our observations reveal that the inhibition of NF-KB activation and OPN expression using NAC is extremely useful for the prevention of stone formation. These observations indicate that NAC can be used as a drug to prevent stone formation. Source of Funding: None
Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009
1999 INCREASED PROTEIN INTAKE ON CONTROLLED OXALATE DIETS DOES NOT INCREASE URINARY OXALATE EXCRETION John Knight, Linda Easter, Rebecca Neiberg, Ross P Holmes, Dean G Assimos*, Winston-salem, NC INTRODUCTION AND OBJECTIVE: High protein intake is a risk factor for calcium oxalate stone disease. The effect of dietary protein on the urinary excretion of calcium, acid and citrate is well established. However, its effect on oxalate excretion is unclear, due in part to an inadequate control of dietary oxalate intake in previous studies. This relationship warrants clarification due to the important role of the metabolism of amino acids in endogenous oxalate synthesis. METHODS: Eleven healthy human subjects (6 males and 5 females) of mean age, 32.0 ± 9.4 ± yrs, and mean BMI, 23.5 ± 3.0, completed the study. Subjects consumed controlled, eucaloric diets that contained 0.6, 1.2 or 1.8 g of protein per kg body weight. Dietary oxalate, sodium, calcium and fat consumption was the same for all 3 diets. Individual energy needs were calculated using the Harris-Benedict equation with an added activity factor based on self-reported daily activity. Each diet was consumed for 6 days in random order, with 24 hr urine collections on each of the last 3 days of the diet and a 3 - 5 day washout period between diet periods. RESULTS: The analysis of 24 hr urine collections confirmed that as protein intake increased, urinary calcium increased and urinary pH and citrate decreased. Urinary oxalate excretion did not increase with high protein intake, but urinary glycolate excretion did. The increased glycolate excretion was due in part to an increased hydroxyproline, but not glycolate consumption. CONCLUSIONS: These results indicate that as dietary protein intake increases, the catabolism of diet-derived amino acids is not associated with an increased endogenous oxalate synthesis in normal subjects. Source of Funding: None
2000 DIFFERNTIAL RENAL TISSUE PROTEIN PROFILING IN A MOUSE MODEL OF HYPERCALCIURIA: EFFECT OF HIGH OXALATE DIET Benjamin K Canales*, Leticia Reyes, Patricia A Glenton, Marjorie Chow, Sixue Chen, Saeed R Khan, Gainesville, FL INTRODUCTION AND OBJECTIVE: NPT2a knockout mice have disruptions in Type IIa sodium/phosphate co-transporter and develop hypercalciuria associated with intra-tubular deposits of calcium phosphate crystals. When made moderately hyperoxaluric, these mice express both calcium oxalate and calcium phosphate crystals. We hypothesized that protein differences could be seen between normal and hyperoxaluric groups using a tissue proteomic approach, giving insight into calcium crystal pathogenesis. METHODS: Normal and knockout mice were randomized to control and 3% glyoxylate chow. Food, water consumption, weight, and urine were monitored daily. Mice were sacrificed at 42 days, and renal tissue was obtained for protein, crystal, and immunohistochemical analysis. Crystal distribution was determined by a semiquantitative scoring system. Tissue protein was labeled for two-dimensional difference gel electrophoresis (2D DIGE) and spotted using DeCyder™ version 6.5. Parallel experiments in protein abundance and identity were performed with iTRAQ labeling followed by liquid chromatography and tandem mass spectromety (MS/MS). Statistical analysis was performed using the ANOVA and Student T-test for group to group comparisons on spot average ratios. RESULTS: By the means of 2D-DIGE technique, 80 differential protein spots were detected, where over 600 proteins were identified and quantified by MS. Compared to normals, knockout mice had intratubular calcium phosphate deposits and over 150 differentially expressed proteins, primarily housekeeping and cell membrane. Differential analysis indicates that proteins involved in oxidative stress responses were significantly (p<0.01) increased in hyperoxaluric mice on glyoxylate