Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells M.E.I. Y.I. Huang, Lakshmi S. Chaturvedi, Sweaty.

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Oxalate stimulates IL-6 production in HK-2 cells, a line of human renal proximal tubular epithelial cells M.E.I. Y.I. Huang, Lakshmi S. Chaturvedi, Sweaty Koul, Hari K. Koul Kidney International Volume 68, Issue 2, Pages 497-503 (August 2005) DOI: 10.1111/j.1523-1755.2005.00427.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Oxalate and lipopolysaccharide (LPS) stimulate interleukin-6 (IL-6) production from HK-2 cells. Confluent, growth-arrested, and serum-starved HK-2 cells were exposed to oxalate (1mmol/L) or LPS (0.1, and 10 μg/mL) for 24 hours. The conditioned mediums were collected and assayed for IL-6 by ELISA, as described inMethods. The IL-6 levels were expressed as pg/mL, and each value represents the mean ± SD of at least 3 to 5 experiments, each performed in duplicate. The star indicates a significant difference (P < 0.001, Studentt test). Kidney International 2005 68, 497-503DOI: (10.1111/j.1523-1755.2005.00427.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Oxalate stimulates dose-dependent increases in interleukin-6 (IL-6) production from HK-2 cells. Confluent, growth-arrested, and serum-starved HK-2 cells were exposed to oxalate (0.2 to 2.0) for 24 hours. The conditioned mediums were collected and assayed for IL-6 by ELISA, as described inMethods. The IL-6 levels were expressed as pg/mL, and each value represents the mean ± SD of at least 3 to 5 experiments, each performed in duplicate. The star indicates a significant difference (P < 0.001, Studentt test). Kidney International 2005 68, 497-503DOI: (10.1111/j.1523-1755.2005.00427.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Oxalate exposure increases time-dependent increase in interleukin-6 (IL-6) mRNA in HK-2 cells. Confluent, growth-arrested, and serum-starved HK-2 cells were exposed to oxalate (1.0mmol/L) for various time points (4, 6, and 24 h). Total RNA was isolated according to the guanidium thiocyanate-phenol-chloroform extraction method, and the human IL-6 gene-specific relative quantitative RT-PCR was carried out as described inMethods. (A) Representative relative quantitative RT-PCR results for IL-6 and 18s ribosomal mRNA. (B) Relative IL-6 mRNA levels of HK-2 cells treated with oxalate (1mmol/L) for various time points. The values were obtained by dividing the densitometric reading of the IL-6 band by that of the 18s band. Kidney International 2005 68, 497-503DOI: (10.1111/j.1523-1755.2005.00427.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Oxalate exposure increases dose-dependent increase in interleukin-6 (IL-6) RNA in HK-2 cells. Confluent, growth-arrested, and serum-starved HK-2 cells were exposed to various concentrations of oxalate (1.0 to 2.0mmol/L) for 4 hours. Total RNA was isolated according to the guanidium thiocyanate-phenol-chloroform extraction method, and human IL-6 gene specific relative quantitative RT-PCR was carried out as described inMethods. (A) Representative relative quantitative RT-PCR results for IL-6 and 18s ribosomal mRNA. (B) Relative IL-6 mRNA levels of HK-2 cells treated with various concentrations of oxalate (1.0 to 2.0mmol/L) for 4 hours. The values were obtained by dividing the densitometric reading of the IL-6 band by that of the 18s band. *Indicates a significant difference compared to control (P < 0.001, Studentt test). Kidney International 2005 68, 497-503DOI: (10.1111/j.1523-1755.2005.00427.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Oxalate-induced IL-6 expression is mediated in part by free radical production. Effect of pretreatment with SOD on free-radical production: For these experiments, confluent monolayers of HK-2 cells were exposed to DMEM containing 25 μg/mL NBT plus varying oxalate (0 or 1mmol/L total), alone or in the presence of SOD. *Indicates a significant difference compared to control (P < 0.005, Studentt test).a, Indicates a significant difference compared to oxalate treated group (P < 0.005, Studentt test). Kidney International 2005 68, 497-503DOI: (10.1111/j.1523-1755.2005.00427.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Effect of SOD on IL-6 expression. Confluent, growth-arrested, and serum-starved HK-2 cells were exposed to oxalate (0 to 2.0) alone or in combination with SOD for 24 hours. The conditioned mediums were collected and assayed for IL-6 by ELISA, as described inMethods. The IL-6 levels were expressed as pg/mL, and each value represents the mean ± SD of at least 3 experiments, each performed in duplicate.a, Indicates a significant difference compared to control (P < 0.001, Studentt test). **Indicates a significant difference compared to oxalate treated group (P < 0.001, Studentt test). Kidney International 2005 68, 497-503DOI: (10.1111/j.1523-1755.2005.00427.x) Copyright © 2005 International Society of Nephrology Terms and Conditions