MRP-1 expression levels determine strain-specific susceptibility to sodium arsenic-induced renal injury between C57BL/6 and BALB/c mice

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Abstract

To clarify the pathophysiological mechanism underlying acute renal injury caused by acute exposure to arsenic, we subcutaneously injected both BALB/c and C57BL/6 mice with sodium arsenite (NaAs; 13.5 mg/kg). BALB/c mice exhibited exaggerated elevation of serum blood urea nitrogen (BUN) and creatinine (CRE) levels, compared with C57BL/6 mice. Moreover, half of BALB/c mice died by 24 h, whereas all C57BL/6 mice survived. Histopathological examination on kidney revealed severe hemorrhages, acute tubular necrosis, neutrophil infiltration, cast formation, and disappearance of PAS-positive brush borders in BALB/c mice, later than 10 h. These pathological changes were remarkably attenuated in C57BL/6 mice, accompanied with lower intrarenal arsenic concentrations, compared with BALB/c mice. Among heavy metal inducible proteins including multidrug resistance-associated protein (MRP)-1, multidrug resistance gene (MDR)-1, metallothionein (MT)-1, and arsenite inducible, cysteine- and histidine-rich RNA-associated protein (AIRAP), intrarenal MDR-1, MT-1, and AIRAP gene expression was enhanced to a similar extent in both strains, whereas NaAs challenge augmented intrarenal MRP-1 mRNA and protein expression levels in C57BL/6 but not BALB/c mice. Moreover, the administration of a specific inhibitor of MRP-1, MK-571, significantly exaggerated acute renal injury in C57BL/6 mice. Thus, MRP-1 is crucially involved in arsenic efflux and eventually prevention of acute renal injury upon acute exposure to NaAs.

Introduction

Arsenic is ubiquitously present in the natural environment such as soil, water, and air (National Research Council, 1999). Moreover, arsenic is generated as a by-product during the refinement of various ores such as copper and lead, and coal consumption in various types of workplaces. Furthermore, arsenic has been frequently used as a toxicant for suicide or homicide. Thus, arsenic intoxication still remains a serious health problem. Arsenic reacts with sulfhydryl groups of various proteins and eventually inhibits their functions (Goering et al., 1999). As a result, acute exposure induces toxicity of the liver, kidney, intestine, and brain (Klaassen, 1996, Liu et al., 2000), while chronic exposure causes dysfunctions in nervous and renal systems, and cancers in various organs including skin, lung, bladder, liver, and kidney (Snow, 1992, Thompson, 1993).

In contrast, a trivalent arsenical salt has been used for many decades in traditional Chinese medicine. A low dose of As2O3 can modulate an anti-apoptotic molecule, Bcl-2 (Mahieux et al., 2001) and glutathione redox system (Dai et al., 1999), and activate caspases (Kitamura et al., 2000), thereby inducing apoptosis in acute promyelocytic leukemia (APL) cell lines (Zhu et al., 1997). Probably due to these activities, 10 mg As2O3 (10 ml 0.1% solution) is efficacious for APL without causing severe side effects such as bone marrow suppression (Chen et al., 1997, Shen et al., 1997, Soignet et al., 1998). Moreover, As2O3 can also induce apoptotic or autophagic cell death in malignant lymphoid cells (Zhu et al., 1999) and solid tumor cells (Akao et al., 1998, Park et al., 2001, Zhang et al., 1999). This reflected the clinical experience that As2O3 is effective also for androgen-independent prostate cancer (Maeda et al., 2001). These observations suggest that As2O3 can be a promising agent for non-hematopoietic as well as hematopoietic malignancies.

The clinical application of As2O3 necessitates the development of the measures to suppress or minimize its side effects. Because heavy metals and metalloid cannot undergo catabolic conversion, active transport for extrusion is the most common mechanism by which cells can reduce stresses arising from exposure to heavy metals. Active transport systems are well conserved in various species from bacteria to mammals. Accumulating evidence indicates that ATP-binding cassette (ABC) transporter proteins have a central role in active transport of heavy metals and that among ABC transporter proteins, multidrug resistance gene protein (MDR)/P-glycoprotein (Borst et al., 1993, Gottesman and Pastan, 1993) and multidrug resistance-associated protein (MRP)-1 (Zaman et al., 1994) can transport arsenic in mammalian cells ( Chen et al., 1998, Liu et al., 2001, Vernhet et al., 2000). However, it still remains elusive which ABC transporter protein(s) is essential for arsenic transport in body.

Moreover, accumulating evidence indicates that mercury, one of the major heavy metals, induces autoimmunity by augmenting T helper type 2 (Th2) response (Kono et al., 1998). Thus, we hypothesized that Th1 or Th2 immune responses might also be involved in arsenic-induced renal injury. To prove this hypothesis, we examined the susceptibility to arsenic in C57BL/6 and BALB/c mice, since C57BL/6 and BALB/c mice tend to exhibit predominantly Th1 and Th2 immune responses after antigen challenge, respectively. Here, we demonstrated that BALB/c but not C57BL/6, exhibited acute lethality to subcutaneously administered sodium arsenite. However, the differences in sensitivities arose mainly from arsenite-induced intrarenal MRP-1 expression levels, which resulted in differences in intrarenal arsenic concentrations between these two strains.

Section snippets

Sodium arsenite-induced renal injury

Pathogen-free 8-week-old male BALB/c and C57BL/6 mice were obtained from Sankyo Laboratories (Tokyo, Japan). Sodium arsenite (NaAs) was purchased from Wako Chemical Industries (Osaka, Japan) and dissolved in phosphate-buffered saline (PBS, pH 7.4) at a concentration of 2.5 mg/ml. In our preliminary experiments, the mice of both strains were injected subcutaneously with various doses of NaAs from 12.0 to 15.0 mg/kg. All doses caused significantly higher levels of serum blood urea nitrogen (BUN)

Lethality and renal injury due to NaAs administration

There was no significant difference in serum BUN and CRE levels between untreated BALB/c and C57BL/6 mice (BUN, 22.4 ± 1.5 mg/dl versus 21.0 ± 1.1 mg/dl; CRE, 0.40 ± 0.01 mg/dl versus 0.39 ± 0.01 mg/dl). A half of BALB/c mice (5 death/10 mice) died by 24 h after NaAs challenge, whereas all of C57BL/6 mice survived until 48 h (Fig. 1a). Because acute NaAs intoxication can cause acute renal failure, we determined serum BUN and CRE levels. Both levels began to increase in BALB/c mice later than 6

Discussion

Here, we investigated pathophysiological changes induced by NaAs treatment in two different mouse strains, BALB/c and C57BL/6. BALB/c mice exhibited enhanced susceptibility as evidenced by severe acute renal failure and lethality, accompanied with an exaggerated increase in arsenic accumulation in kidney, compared with C57BL/6 mice. These findings suggest that between these two strains, there are differences in active extrusion, which is commonly used in various organisms from bacteria to

Acknowledgments

The work is supported by Grants-in-Aids from the Ministry of Education, Culture, Science, and Technology of the Japanese Government.

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