These observations claim that endogenous IFN- could regulate the expression of MRP1, a transporter essentially involved in arsenic extrusion

These observations claim that endogenous IFN- could regulate the expression of MRP1, a transporter essentially involved in arsenic extrusion. resistance-associated protein (MRP) 1, a main transporter for NaAs efflux, compared with WT mice. NF-E2-related factor (Nrf) 2 protein, a transcription factor crucial for MRP1 gene expression, was similarly increased in the kidneys of both strains of mice after NaAs treatment. In contrast, the absence of IFN- augmented transforming growth factor–Smad3 signal pathway and eventually enhanced the expression of activating transcription factor 3, which is presumed to repress Nrf2-mediated MRP1 gene expression. Thus, IFN- can protect against NaAs-induced acute renal injury, probably by maintaining Nrf2-mediated intrarenal MRP1 gene expression. Arsenic inhibits the biological functions of various proteins by reacting with their sulfhydryl groups.1 Acute exposure to arsenic can cause profound injury to kidney, H-Ala-Ala-Tyr-OH liver, intestine, and brain,2,3 frequently resulting in acute mortality. Chronic exposure causes dysfunctions in renal and nervous systems.4,5 Moreover, arsenic is a potent carcinogen Igfbp2 to various organs including skin, lung, bladder, liver, and kidney.4,5 Arsenic is H-Ala-Ala-Tyr-OH ubiquitously present in the natural environment in soil, water, and air. Furthermore, groundwater and/or soil are frequently contaminated with a high concentration of arsenic, which is generated during the refinement of various ores such as copper and lead and the consumption of coal. Thus, arsenic intoxication in an acute or a chronic form still remains a serious threat to public health in areas where groundwater and/or soil is contaminated with arsenic. On H-Ala-Ala-Tyr-OH the contrary, accumulating evidence has revealed that As2O3 may be efficacious for acute promyelocytic leukemia without causing bone marrow suppression.6C8 Moreover, As2O3 might be effective also for androgen-independent prostate cancer.9 Its efficaciousness may come from the capacity of As2O3 to induce apoptotic and/or autophagic cell death polymerase (Nippon Gene, Toyama, Japan) using specific sets of primers with an optimal number of cycles at 94C for 1 minute, optimal annealing temperature for 1 minute, and 72C for 1 minute, followed by incubation at 72C for 3 minutes (Table 1). The PCR products H-Ala-Ala-Tyr-OH were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. The band intensities of ethidium bromide fluorescence were measured using NIH Image Analysis Software Version 1.61 (National Institutes of Health, Bethesda, MD). The relative intensities of the bands were determined, and the ratios to -actin were calculated. TABLE 1 Sequences of the Primers Used for RT-PCR hybridization.? Detection of IFN- mRNA in the Kidneys hybridization analyses were performed to detect IFN- mRNA in kidney, as described previously.25 RT-PCR product of IFN- was obtained using the pair of primers with the addition of T7- and Sp6-RNA polymerase promoter to the 5 end of each sense and anti-sense primer of IFN-, respectively (Table 1). Digoxigenin-labeled sense and anti-sense probes were obtained by using DIG RNA labeling kit (Boehringer Mannheim Biochemica, Mannheim, Germany) according to the manufacturers instructions. The sense probe was used as a negative control. Deparaffinized sections were further fixed with 4% paraformaldehyde in PBS for 15 minutes and incubated with 10 g/ml proteinase K in TE buffer (10 mmol/L Tris-HCl and 1 mmol/L ethylenediaminetetraacetic acid) at 37C for 10 minutes. After washing with 5 standard saline citrate at room temperature for 15 minutes, the sections were prehybridized at 55C for 1 hour with a buffer containing 50% deionized formamide, 5 standard saline citrate, and 40 g/ml salmon sperm DNA. After the RNA probes were added to the prehybridization buffer to 400 ng/ml, the slides were incubated under a cover at 55C for 16 hours in a moist chamber. After the section was incubated with anti-digoxigenin Abs for 16 hours, positive signals were visualized with a color-substrate solution containing nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt. Statistical Analysis The means and SEMs were calculated for all parameters determined in this study. Statistical significance was evaluated using analysis of variance or Mann-Whitneys < 0. 05 was accepted as statistically significant. The survival curve by the Kaplan-Meier procedure was analyzed by a log-rank test. Results Intrarenal IFN- Expression in WT Mice after NaAs Challenge We previously observed that NaAs caused severe renal dysfunction.18 Because IFN- can regulate ABC transporter expression,19C21 we determined intrarenal IFN- contents after the subcutaneous administration of NaAs. NaAs challenge increased intrarenal IFN- contents remarkably even at 1 and 2 hours, declining thereafter (Figure 1a). Immunohistochemical analysis demonstrated that most tubular epithelial cells and some interstitial cells expressed IFN- protein even at 1 hour after NaAs challenge (Figure 1b). Consistently, hybridization analyses detected IFN- mRNA exclusively in tubular epithelial cells at 1 hour after NaAs challenge (Figure 1c). Sense probes showed no positive reactions for IFN- mRNA, indicating the specificity of the reaction (Figure 1d). These observations indicated that IFN- might be locally produced by tubular epithelial cells, the main target cells in NaAs-induced renal injury. Open in a separate window FIGURE 1 Analysis.