Elsevier

Toxicology

Volume 339, 2 January 2016, Pages 9-18
Toxicology

Exaggerated arsenic nephrotoxicity in female mice through estrogen-dependent impairments in the autophagic flux

https://doi.org/10.1016/j.tox.2015.11.005Get rights and content

Abstract

Gender is one of the essential factors in the development of various diseases and poisoning. Therefore, we herein examined gender differences in sodium arsenite (NaAsO2)-induced acute renal dysfunction. When male and female BALB/c mice were subcutaneously injected with NaAsO2 (12.5 mg/kg), serum and urinary markers for proximal tubular injury were significantly higher in female mice than in male ones. NaAsO2-induced histopathological alterations were consistently more evident in females than in males. Ovariectomy, but not orchiectomy significantly attenuated NaAsO2-induced renal injury. These results imply that the hypersusceptibility of female mice is attributed to estrogen signals. NaAsO2 suppressed the autophagic flux in tubular cells through the activation of ERK. Enhancements in the activation of ERK were significantly greater in females than in males, with the eventual accumulation of LC3-II and P62 in the kidneys, implying that the autophagic flux is impaired in females. The IL-6/STAT3 signaling pathway had protective roles in NaAsO2-induced nephrotoxicity through the suppression of ERK activation. Despite the absence of differences in intrarenal IL-6 expression between male and female mice, STAT3 was less activated with enhanced SOCS3 expression in females than in males. An in vitro study using mProx24 cells revealed that the estrogen treatment induced SOCS3 expression, and eventually suppressed the autophagic flux, as evidenced by greater increases in the accumulation of LC3-II and p62 with ERK activation, which was canceled by the knockdown of Socs3. Collectively, these results indicate that estrogen has a negative impact on the development of NaAsO2 nephrotoxicity through its suppression of the autophagic flux.

Introduction

The ubiquitous distribution of arsenic causes environmental pollution, which has led to serious health issues in several developing countries (Oremland and Stolz, 2003). Chronic exposure to arsenic (such as not acutely toxic dose of arsenic in drinking water) results in the dysfunction of renal and nervous systems (Snow, 1992, Thompson, 1993) and eventually acts as a carcinogen in the skin, lung, bladder, liver, and kidney (Goering et al., 1999, Abernathy et al., 1999). Higher doses of arsenic have been shown to cause acute severe injuries to various organs including the kidney, liver, intestine, and brain, resulting in high mortality and morbidity. Renal tubular necrosis is a hallmark of renal dysfunction caused by acute arsenic exposure (Kimura et al., 2005, Kimura et al., 2006).

Gender differences in susceptibilities to various stresses have been linked to sexually dimorphic diseases. Gender differences are presumed to be induced by sex hormones and structural and functional differences in organs (Sabolic et al., 2007). The female hormone, estrogen, is widely recognized as a suppressor of cellular immunity and enhancer of humoral immune responses, indicating that it plays a protective role in acute organ injury (Nadkarni and McArthur, 2013). Previous studies demonstrated that the female gender was more resistant to ischemia-reperfusion insults in the brain (Goering et al., 1999) and kidney (Hu et al., 2009). Moreover, the male gender exhibited greater sensitivity to mercury-induced renal injury (Hazelhoff et al., 2012). Since As2O3 has recently been shown to be effective for acute promyelocytic leukemia without causing bone marrow suppression (Soignet et al., 1998, Huang et al., 2014, Niu et al., 1999, Matthews et al., 2005, Douer and Tallman, 2005), its anticancer efficiency is being extended to several types of solid tumors (Dilda and Hogg, 2007). Thus, it is important to minimize the adverse effects of As2O3 in its clinical use. However, gender differences in susceptibility to arsenic have not yet been examined in detail.

We previously reported that interleukin-6 (IL-6) played a protective role in sodium arsenite (NaAsO2)-induced acute renal injury (Kimura et al., 2010). In NaAsO2-induced acute renal injury, autophagy was augmented in renal tubular cells and was closely related to the loss of renal function. IL-6-dependent signal transducer and activator of transcription-3 (STAT3) activation is involved reduces autophagic cell death because it inhibits the activation of extracellular signal-regulated kinase (ERK). These findings imply that the modulation of IL-6/STAT3 signaling pathways may be used to determine the magnitude of NaAsO2-induced renal dysfunction. Moreover, estrogen has been suggested to regulate IL-6/STAT3 signaling pathways (Matthews et al., 2005, Leong et al., 2004).

Thus, we examined gender differences in NaAsO2-induced acute nephrotoxicity in mice with a subcutaneous injection of a high dose of NaAsO2. Although no significant difference was observed in intrarenal IL-6 expression between the genders, female mice were more sensitive to NaAsO2-induced nephrotoxicity with attenuated STAT3 activation and the reciprocally enhanced expression of suppressor of cytokine signaling-3 (SOCS3). Moreover, the results of an in vitro study revealed that estrogen up-regulated the expression of SOCS3, which ultimately resulted in an impaired autophagic flux in NaAsO2-treated mProx24 cells. Collectively, these results demonstrate that estrogen has a negative impact on the development of NaAsO2-induced nephrotoxicity through its induction of SOCS3.

Section snippets

Reagents and antibodies (Abs)

NaAsO2 (reagent grade >90% purity) and flutamide, a non-steroidal antiandrogen drug, were purchased from Wako Chemical Industries (Osaka, Japan). The following monoclonal Abs (mAbs) or polyclonal Abs (pAbs) were used in the present study: anti-LC3A/B mAb, anti-STAT3 mAb, anti-phospho-STAT3 mAb (Tyr705), anti-ERK1/2 mAb, anti-phosphor-ERK1/2 mAb (Thr202/Tyr204), anti-SOCS3 mAb (Cell Signaling Technology, Tokyo, Japan), anti-p62/A170/SQSTM1 (p62) pAbs (Wako Chemical Industries), and β-actin pAbs

Gender differences in susceptibility to NaAsO2-induced renal injury

Under the physiological conditions used in the present study, no significant differences were observed in serum BUN or CRE levels, urine NGAL, the urine protein/CRE ratio, or renal morphology between male and female BALB/c mice (Fig. 1a–e). The subcutaneous administration of NaAsO2 (12.5 mg/kg) induced renal dysfunction in both genders of BALB/c mice, as evidenced by elevations in serum BUN and CRE levels (Fig. 1a and b), urine NGAL, an early marker of renal injury (Mishra et al., 2003), and the

Discussion

Previous studies demonstrated gender differences in various kinds of disease models (Hu et al., 2009, Park et al., 2006, Hodeify et al., 2013, Pezeshki et al., 2013). Female mice were found to be significantly more resistant to nephrotoxicity induced by ischemia-reperfusion, cisplatin, or ER stress than male mice (Hu et al., 2009, Hodeify et al., 2013, Pezeshki et al., 2013). Moreover, the male gender is generally more sensitive to chronic arsenic exposure (Watanabe et al., 2001, Ahsan et al.,

Funding

This work was supported in part by Grants-in-Aids from the Ministry of Education, Culture, Science, and Technology of the Japanese Government (Toshikazu K. and A.K.), and Research Grant from the Uehara Memorial Foundation (Toshikazu K.).

Acknowledgment

We thank Dr. Tamotsu Yoshimori (Osaka University, Japan) for his gift of the EGFP-LC3 plasmid.

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