An exploratory study of sex differences in thallium-induced nephrotoxicity in rats

Abstract

Sexual dimorphism is recognized in nephrotoxic acute kidney injury, with females being less susceptible than males. Thallium (Tl), a highly toxic heavy metal, induces severe systemic disorders following exposure, including gastrointestinal and neurological disorders and renal failure. However, sex-related differences in Tl-induced nephrotoxicity remain poorly understood. Previous studies have shown that Tl preferentially accumulates in the outer medulla of the kidney of male rats, resulting in mitochondrial dysfunction and medullary thick ascending limb (mTAL) injury, characterized by calcium deposits and impaired renal function. This study investigated the effects of Tl on the kidneys of female rats and the underlying mechanisms. Tl₂SO₄ (30 mg/kg) was administered to rats. Nephrotoxicity was measured 2, 5, and 14 days after Tl-administration using biochemical assays of blood and urine samples, histopathology of the kidney, and transcriptome analysis using microarrays. As in male rats, female Tl-loaded rats developed severe renal dysfunction with calcium deposits in the outer medulla within 5 days after Tl administration. The pathological features were similar to those of male rats; however, the mitochondrial oxidative stress and calcium deposits in the medulla were less extensive in female rats than in male rats. These preliminary findings suggest sex-dependent differences, which might be derived from the differences in sex-hormones, in Tl-induced renal injury and suggest potential involvement of differential oxidative stress handling, mitochondrial responses, and transporter activity. These new insights could assist with the development of therapeutic strategies for treating Tl intoxication in humans.

INTRODUCTION

Thallium (Tl) has been used in medicine and a wide range of industrial applications for many years (Baselt, 2020). Tl(I) salts are readily absorbed after exposure and are highly toxic in both acute and chronic exposure situations, leading to neurological, gastrointestinal, renal, and other severe systemic disorders (Cvjetko et al., 2010). The intracellular toxicity of Tl is primarily attributed to its chemical similarity to potassium (K), which allows it to displace intracellular K and to compete with K in many biological processes (Peter and Viraraghavan, 2005). A previous study in male rats demonstrated that Tl-induced acute nephrotoxicity is characterized by marked calcium (Ca) deposition in the outer medulla of the kidney as a result of preferential Tl reabsorption in the medullary thick ascending limb (mTAL) of the loop of Henle via Na-K-Cl cotransporter 2 (NKCC2) (Unuma et al., 2024). Sexual dimorphism in renal physiology is well-established across species and has been recognized in nephrotoxic acute kidney injury (Ahmed and Dumanski, 2021; Neugarten and Golestaneh, 2022). Multiple human and animal studies suggest that females are less susceptible than males to the development of acute kidney injury (Kher et al., 2005; Rodríguez-Gómez et al., 2012; Swartling et al., 2021). However, the effects of biological sex on Tl-induced nephrotoxicity and the underlying mechanisms are unclear. Although Tl has been reported to accumulate more extensively in females, partly due to the higher body-fat level, leading to persistently elevated urinary Tl concentrations, some cohort studies conducted in human populations have suggested that males are more susceptible than females to nephrotoxicity following environmental Tl exposure (Li et al., 2023; Li et al., 2024; Shelley et al., 2012; Wu et al., 2018). However, evidence from human and animal studies regarding the molecular mechanisms underlying sexual dimorphism in Tl-induced nephrotoxicity is limited. Understanding sexual differences in Tl nephrotoxicity could enhance knowledge of the pathophysiological differences between male and female kidneys in experimental models and inform the development of more effective strategies for treating Tl-induced nephrotoxicity in humans. Thus, this study aimed to compare acute Tl-induced nephrotoxicity in female and male rats and to explore potential underlying mechanisms that could explain sex-related differences in Tl-induced nephrotoxicity.

MATERIALS AND METHODS

Animals and Tl administration

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Science Tokyo (approval number A2025-057A) and conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Eight-week-old male and female Wistar rats (220–250 g; Oriental Yeast Co. Ltd., Tokyo, Japan) were housed under standard laboratory conditions (25°C, 12-hr light/dark cycles) with ad libitum access to water and food. The rats were randomly assigned to control or Tl groups. Each rat received a single intraperitoneal injection of either double-distilled water or 30 mg/kg Tl₂SO₄ (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) dissolved in double-distilled water. Kidney and blood samples were collected 2, 5, or 14 days after injection, following euthanasia by intraperitoneal injection of sodium pentobarbital (100 mg/kg). Urine samples were collected before euthanasia using a 12-hour metabolic cage. Each analysis was conducted on three or four rats per group. For the 2-day protocol, three female rats were included per group. For the 5-day protocol, the control and Tl groups consisted of four and three female rats, respectively. For the 14-day protocol, the Tl group included three male and three female rats. The 30 mg/kg dose was selected based on a previous study investigating acute Tl toxicity (Leung and Ooi, 2000). To assess reactive oxygen species (ROS) production in the kidneys, a separate group of rats received a single intraperitoneal injection of 120 mg/kg Tl₂SO₄. The kidneys of rats in this group were harvested 4 hr after injection following euthanasia by intraperitoneal injection of sodium pentobarbital (100 mg/kg).

Blood/urine analysis

Blood samples (anticoagulant in the syringe: 1.5 mg/mL K3-EDTA) and urine samples (obtained using a 12-hr metabolic cage at 25°C) were collected from the rats 2, 5, or 14 days after administering Tl (30 mg/kg). The plasma and/or urine levels of albumin (Alb), blood urea nitrogen (BUN), urine urea nitrogen, (U-UN), creatinine (Cr), inorganic phosphorus (IP), uric acid (UA), sodium (Na), K, chlorine (Cl), calcium (Ca), total protein (TP), magnesium (Mg), low-density lipoprotein cholesterol (LDL-C), and N-acetyl-β-D-glucosaminidase (NAG) were measured according to the standard protocols of Oriental Yeast Co. Ltd. (Tokyo, Japan). Urinary proteins were fractionated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with loading amounts normalized to urine specific gravity.

Histological analysis

Central short-axis cross-sections of the rat kidney were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 2.5-µm-thick sections. Histological evaluations were performed using hematoxylin and eosin (H&E), toluidine blue, and von Kossa staining for calcium deposit detection.

Detection of mitochondrial production of ROS

Cryosections (10 μm) of kidneys collected 4 hr after a single intraperitoneal injection of 120 mg/kg Tl₂SO₄ were incubated with hydroxyphenyl fluorescein (HPF, 1:500, Daiichi Chemical Co., Ltd., Tokyo, Japan) at 37°C for 10 min. To detect hydroxyl radicals and peroxynitrites, the sections were observed under a fluorescence microscope (Keyence BZ-9000, Keyence, Osaka, Japan) with excitation and emission wavelengths of 490 and 515 nm, respectively.

DNA microarray

For transcriptome analysis, the cortex and medulla were carefully separated from the kidneys harvested 2 days after injection. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and purified with the RNeasy RNA Purification Kit (Qiagen, Hilden, Germany). RNA integrity was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Hybridization was performed using a Clariom S array (Thermo Fisher Scientific), and data were analyzed using a Transcriptome Analysis Console (Thermo Fisher Scientific).

Statistical analysis

Group comparisons were performed using independent samples t-tests or Dunnett’s post hoc test. Two-tailed P values < 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism (Version 9.0.0, GraphPad Software, San Diego, CA, USA).

RESULTS

Impaired renal function after Tl administration in female rats

Five days after Tl administration (30 mg/kg), female Tl-loaded rats exhibited significantly elevated BUN and Cr levels, and a significant reduction in serum Ca level, compared with those of controls (Fig. 1a). The magnitude of these changes was comparable to those previously reported in male rats 5 days after Tl administration (Unuma et al., 2024). To assess early acute renal effects of Tl exposure, urine samples collected from rats 2 days after Tl administration were analyzed. The urinary Alb level and protein to creatinine ratio were significantly elevated, whereas U-UN, UA, and Ca levels were significantly decreased in Tl-loaded female rats compared with those of controls (Fig. 1b). These results indicate significant impairment of renal function in female rats, characterized by abnormal reabsorption and excretion, at both 2 and 5 days after Tl administration. SDS-PAGE of urine proteins revealed proteinuria of both high-molecular-weight proteins and low-molecular-weight proteins (Fig. 1c). The Alb and IgG proteinuria in female rats was more marked than that previously reported in male rats (Unuma et al., 2024) under the same experimental conditions, suggesting that Tl administration induces more severe glomerular damage in female rats than in male rats. Furthermore, blood and urine analysis results were also compared between male and female rats at 14 days after Tl administration. Serum Alb, BUN, and Cr levels were significantly higher in female rats than in male rats, whereas urinary TP, urinary protein-to-creatinine ratio, K, Cl, IP, and Mg levels were significantly lower in female rats than in male rats (Fig. 1d), suggesting that female rats experience more severe renal dysfunction than that in male rats 14 days after Tl administration. To clearly illustrate the temporal profiles and sex differences in serum Cr, serum Ca, and urinary Ca following Tl exposure, the serial changes in these parameters in both sexes are summarized in Supplementary Table 1, which also includes the reported results from our previous paper (Unuma et al., 2024).

Fig. 1

Impaired renal function in female rats after thallium (Tl) administration. Analysis of blood (a) and urine (b) samples of female rats collected 5 days and 2 days after Tl administration (30 mg/kg, n = 4 for control and n = 3 for Tl group), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of urine (c) samples of female rats collected 5 days after Tl administration (30 mg/kg, n = 3). Analysis of blood and urine (d) samples of female rats collected 14 days after Tl administration (30 mg/kg, n = 3). Each bar represents the mean and standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ALB, albumin; BUN, blood urea nitrogen; Ca, calcium; Cl, chloride; Cr, creatinine; IP, inorganic phosphorus; K, potassium; LDL-C, low-density lipoprotein cholesterol; Mg, magnesium; Na, sodium; NAG, N-acetyl-β-D-glucosaminidase; Tf, transferrin; THP, Tamm-Horsfall glycoprotein; TP, total protein; U-, urinary level of; UA, uric acid; UN, urea nitrogen; U-P/C ratio, urinary protein to creatinine ratio

Ca deposits and mTAL damage in the outer medulla of female rats after Tl administration

A previous study in male rats demonstrated that Tl-induced nephrotoxicity is characterized by prominent Ca depositions in the outer medulla, especially associated with mTAL, resulting in tubular obstruction and structural damage (Unuma et al., 2024). Von Kossa staining of short-axis kidney cross-sections of female rats obtained 5 days after Tl administration, revealed similar pathological findings to those in male rats, with Ca deposits localized to the outer medulla (Fig. 2a). Toluidine blue staining of the kidney sections from Tl-loaded female rats revealed vacuolar degeneration and desquamation of mTAL cells in the outer medulla with disrupted organelles, whereas the other nephron segments retained normal morphology (Fig. 2b). H&E staining confirmed the presence of basophilic Ca deposits in both female and male Tl-loaded rats, although the extent of the deposits appeared less in female rats (Fig. 2c). The similar pathological manifestations between sexes after Tl administration suggest a shared underlying mechanism, despite the histopathological changes being less severe in female rats than in male rats.

Fig. 2

Calcium deposits and medullary thick ascending limb damage in the outer medulla of rats after Tl administration. (a)Von Kossa staining of kidney sections of control and Tl-loaded female rats 5 days after Tl administration (30 mg/kg). (b) Toluidine blue staining of kidney sections from Tl-loaded female rats 2 days after Tl administration (30 mg/kg). (c) Hematoxylin and eosin staining of kidney sections from female and male Tl-loaded rats 5 days after Tl administration (30 mg/kg). CCD, cortical collecting tubule; Cx, cortex; DCT, distal convoluted tubule; DL, descending limb of the loop of Henle; IM, inner medulla; IMCD, inner medulla collecting duct; OM, outer medulla; OMCD, outer medulla collecting duct; S1, proximal tubule segment 1; S3, proximal tubule segment 3; TAL, thick ascending limb of the loop of Henle; VR, vasa recta. Scale bars = 100 μm

Mitochondrial dysfunction and sexual differences in oxidative stress responses in female and male rats after Tl administration

Tl aggregation and Tl-induced Ca deposition in the outer medulla have been shown to lead to mitochondrial ROS generation and severe mitochondrial dysfunction in male rats (Unuma et al., 2024). Given the observed sex differences in pathological severity, we evaluated mitochondrial dysfunction and measured mitochondrial ROS in female rats and compared them with those in male rats. A fluorometric mitochondria superoxide assay detected ROS accumulation in the outer medulla of female rats 4 hr after Tl administration (120 mg/kg) but the fluorescence intensity was lower in female rats than that observed in male rats (Supplemental Fig. 1), suggesting milder Tl-induced oxidative stress in female rats. DNA microarray analysis of kidney cortex and medulla tissue collected 2 days after Tl exposure was conducted to explore potential sex differences in oxidative stress responses. The top 10 upregulated and downregulated genes in the cortex and medulla are listed in Tables 1 and 2, respectively. Marked upregulation of tubular injury markers such as kidney injury molecule-1 (KIM1) (Ichimura et al., 2008) and neutrophil gelatinase-associated lipocalin (lcn2) (Paragas et al., 2011) was observed in both sexes. Notably, KIM1 expression in the medulla of male rats was approximately double that in female rats (Table 2). Genes associated with calcium homeostasis, including voltage-dependent calcium channel γ5 subunit (Cacng5), calcium-sensing receptor (Casr), and regucalcin (Rgn), were downregulated in the medulla of both male and female rats, which may have contributed to the formation of Ca deposits in the outer medulla. Microarray analysis of antioxidant defense-related genes, including pathway genes involved in ROS detoxification and glutathione metabolism, showed smaller fold changes in female rats than in male rats, as reflected by the ratio of female-to-male fold changes (Table 3). These findings suggest that the ROS load in the medulla following Tl exposure was lower in female rats than in male rats (Supplemental Fig. 1). Furthermore, because sexual dimorphism in the expression and abundance of renal transport is well-established (Ransick et al., 2019; Veiras et al., 2017; Veiras et al., 2020), we analyzed the expression profiles of major renal transporter genes in the medulla of Tl-loaded rats (Table 4). Genes associated with water transport (Aqp6, Aqp2, Aqp3) and electrolyte reabsorption (Kcnq1, Kcnma1, Slc5a2, Slc20a2, Scnn1a) were upregulated in female Tl-loaded rats compared with male Tl-loaded rats, suggesting that female rats have better-preserved reabsorption activity in the medulla following Tl administration. Expression of Slc12a1 (encoding NKCC2) was higher in female rats than in male rats, suggesting that this co-transporter was less suppressed in female rats under conditions of Tl exposure.

Table 1. Top 10 upregulated or downregulated genes in the cortex of female and male Tl-loaded rats (30 mg/kg, 2d) in order of fold change.

Table 2. Top 10 upregulated or downregulated genes in the medulla of female and male Tl-loaded rats (30 mg/kg, 2d) in order of fold change.

Table 3. Expression of pathway genes related to defense against oxidative stress in the medulla of Tl-loaded rats (30 mg/kg, 2d).

Table 4. Expression of major kidney transporter and channel genes in the medulla of Tl-loaded rats (30 mg/kg, 2d).

DISCUSSION

This study investigated sex differences in Tl-induced nephrotoxicity in female and male rats. In the early phase, 2 and 5 days after Tl administration, female rats exhibited significant renal dysfunction, characterized by abnormal reabsorption and excretion, and Ca deposits preferentially located in the outer medulla. Although the overall pattern and severity of renal injury resembled that previously reported in male rats (Unuma et al., 2024), in contrast to the focal and concentrated outer medullar Ca deposits and tubular lesions in male rats, female rats developed more pronounced proteinuria of high-molecular-weight proteins such as IgG, exhibited fewer Ca deposits, and showed a milder mitochondrial oxidative stress response in the outer medulla.

These findings suggest that although Tl-induced nephrotoxicity involves similar underlying mechanisms in both sexes, susceptibility to Tl-induced nephrotoxicity differs according to sex. Specifically, male rats are more vulnerable to medullar tubular injury following Tl exposure. whereas female rats appear more prone to impairment of glomerular function and more resistant to outer medullary damage induced by Tl exposure. Moreover, blood and urine analyses at a later time point (14 days after administration) revealed more severe renal dysfunction in female rats than in male rats, showing that glomerular functional impairment is critical to subsequent renal dysfunction. It should be noted renal function analyses were made in only three time point (2d, 4d, 14d) applying relatively small sample size without any time-dependent observation. This may limit our interpretation on the sex-dependent renal dysfunction by Tl exposure. Further studies with larger sample size and temporal scale are needed to clarify the mechanisms underlying long-term and time-dependent sex differences in Tl-induced nephrotoxicity.

A previous study in male rats demonstrated that Tl-induced Ca deposits in the outer medulla arises from NKCC2-dependent Tl accumulation in mTAL, resulting in mitochondrial dysfunction and mTAL epithelial cell damage (Unuma et al., 2024). Because the mTAL plays a critical role in Ca reabsorption (Greger, 1985; Mount, 2014), its disruption leads to impaired Ca homeostasis, intratubular Ca crystal formation, nephron obstruction, and consequent renal injury (Appenroth et al., 1995; Danilewicz et al., 1980; Unuma et al., 2024). Tl exerts cell toxicity by depleting antioxidant defenses (e.g., glutathione) and inducing oxidative damage via lipid peroxidation and endoplasmic reticulum stress (Anaya-Ramos et al., 2021; Morel Gómez et al., 2023; Sugahara et al., 2024). Mitochondria from females are reported to possess superior bioenergetic profiles, higher antioxidant capacity, and lower ROS production than those from males (Gaignard et al., 2017; Khalifa et al., 2017; Ventura-Clapier et al., 2017). The milder Ca deposition and ROS accumulation observed in the outer medulla of Tl-loaded female rats may thus reflect stronger mitochondrial resilience. However, the relatively modest changes in oxidative stress-related gene expression in the female medulla are more consistent with less pronounced oxidative stress burden rather than enhanced antioxidant activation. This aligns with the upregulated gene expressions of water transport and electrolyte reabsorptions in the medulla of female Tl-loaded rats (Table 4), suggesting a better-preserved reabsorption of K, P, and Cl, as well as potentially intact paracellular Ca²⁺ reabsorption. Previous studies have reported higher NKCC2 abundance in the medullary nephron segments of the female rats compared with those of males (Harris et al., 2018; Musselman et al., 2010; Veiras et al., 2017), as well as greater medulla filtered volume (Li et al., 2020). These findings indicate that female rats may possess stronger renal concentrating ability and more diluted medulla perfusion than that of males (Musselman et al., 2010). Collectively, these effects could contribute to less aggregation of Ca deposits in outer medulla in female rats than in male rats, and reduced susceptibility to Tl-induced mTAL injury. Nevertheless, further investigation is required to clarify why female rats were more susceptible to the glomerular effects of Tl exposure. It should be noted that the ratio of female-to-male fold changes was used to evaluate sex-dependent differences. Although this approach provides an initial indication of differential responsiveness between sexes, the potential influence of baseline expression differences cannot be fully excluded. Therefore, fold changes relative to the control group in each sex and comparisons of basal levels between the two sex groups have been included. Nevertheless, these comparisons should be interpreted cautiously, and further validation using larger sample sizes and absolute expression levels will be necessary to confirm the present findings. The milder oxidative stress in female medulla may result from relatively limited Tl reabsorption in this region, the protective effect of sex hormones, or a combination of these two factors. Sex differences have been reported in renal transport of xenobiotics and heavy metals (Morris et al., 2003; Pamphlett et al., 1997). Similar mechanisms may underlie the observed sexual dimorphism in Tl nephrotoxicity. However, the mechanisms require further investigation.

Sex hormones regulate kidney structures and functions and protect against ROS generation in the kidney (Harris et al., 2018; Koenig et al., 1980; Lima-Posada et al., 2017; Veiras et al., 2017). However, this study did not investigate hormone levels or signaling pathways, limiting our ability to interpret their contribution to the observed sex differences. Further research is needed to clarify the roles of estrogen, androgen, and other sex-related factors in modulating the renal response to Tl. The main limitation of this study is its small sample size. However, our findings are consistent with those of previous studies (Sugahara et al., 2024; Unuma et al., 2024).

In conclusion, the study demonstrates that Tl-induced nephrotoxicity shares common features and underlying mechanisms in female and male rats, with oxidative stress generation, Ca deposits in the medulla, and severely impaired kidney function. However, compared with male rats, female rats appear to be less susceptible to Tl-induced outer medullary damage, yet more susceptible to glomerular injury. These findings indicate sex-dependent differences in Tl-induced renal injury and suggest potential involvement of differential ROS handling, mitochondrial responses and transporter activity, which may be derived from different sex hormone expressions between female and male rats. The current findings should contribute to the understanding of Tl nephrotoxicity and the development of more targeted strategies for managing Tl poisoning in humans.

Funding

Funding was provided by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 23 K19799 (to S.W.) and 22 K10606 (to K. U.). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

Data availability

The authors confirm that the data supporting the findings of this study are available in the article and its supplementary materials.

Author contributions

S.W. prepared the initial draft. A.T. and K.U. conceived and designed the experiments. S.W., K.U., and A.T. conducted the experiments. A.T. and K.U. analyzed and interpreted the results. T.A., A.T., and K.U. critically revised the manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Science Tokyo (approval number A2025-057A) and conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Consent to participate was not applicable as there were no human subjects.

Patient consent for publication

Not applicable.

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