2020 Volume 45 Issue 9 Pages 589-598
Acute mercury chloride (HgCl2) poisoning may lead to kidney injury, but the underlying mechanism remains largely unknown. Endoplasmic reticulum (ER) stress plays a role in some heavy metal poisoning. Whether it mediates kidney injury in acute HgCl2 poisoning remains unknown. In this study, we examined the kidney injury and the corresponding ER stress in the mouse model of different doses of acute HgCl2 poisoning. To further confirm the role of ER stress, we tested the effects of its chemical chaperone [4-phenylbutyric acid (4-PBA)]. The results revealed that acute HgCl2 poisoning caused more severe kidney injury with dose on and activated ER stress, as indicated by increased expression of GRP78 and CHOP. Inhibition of ER stress restored the functional and morphological changes of kidneys, and partly attenuated renal tubular epithelial cell apoptosis. In summary, ER stress contributes to the acute kidney injury following HgCl2 poisoning, and inhibition of ER stress may alleviate the kidney injury via reducing apoptosis.
Mercury is a toxic heavy metal with special physical and chemical properties, which causes serious biochemical and pathological effects in the human body (Bjørklund et al., 2017). Studies showed that mercury chloride is the most common inorganic mercury which damages many systems, such as the urinary system (Joshi et al., 2014; Elblehi et al., 2019; Yadav et al., 2012), digestive system (Elblehi et al., 2019; Bottino et al., 2016), cardiovascular system (Baiyun et al., 2018), nervous system (Malqui et al., 2018) and reproductive system (Heath et al., 2009). The kidney is the main target organ of inorganic mercury (Zalups, 2000). Inorganic mercury, which enters the body through different pathways, can accumulate in the kidney, damage the kidney, and even lead to acute renal failure (Dhanapriya et al., 2016). However, the molecular mechanism of such injury is still unclear.
The endoplasmic reticulum (ER) is a site for synthesizing, modifying, folding proteins and storing intracellular Ca2+, which plays important roles in signal transduction and maintaining the internal environment stability. When the pathways in the ER are disturbed, accumulated unfolded proteins and (or) misfolded proteins will lead to ER stress, and induce unfolded protein responses (UPR) to maintain the stability of the ER. However, when cells in excessive ER stress are insufficient to resolve the protein-folding defect and restore ER homeostasis through the adaptive UPR pathway, maladaptive UPR will trigger apoptosis (Inagi, 2010). Previous reports suggested that a variety of heavy metals such as lead, cadmium, and mercury can induce ER stress. GRP78 and oxidative stress may be involved in neurotoxicity of C6 rat glioma cells which is caused by lead and mercury (Qian et al., 2000; Qian et al., 2001). Subcytotoxic dose of lead chloride can up-regulate GRP78 in rat renal tubular epithelial cells (NRK-52E) (Stacchiotti et al., 2009). Cadmium chloride induced GRP78 expression in LLC-PK1 renal epithelial cells by phosphorylation of eIF2α and translation of ATF4 (Liu et al., 2006). Meanwhile, a large number of studies have indicated that ER stress is involved in various forms of acute kidney injury (AKI) due to acute nephrotoxicity and ischemia-reperfusion injury (Foufelle and Fromenty, 2016; Gao et al., 2012). Thus, we hypothesized that ER stress is a potential mechanism in acute kidney injury following HgCl2 poisoning.
All animal experiments strictly abided by the “Standards of Shandong Provincial Laboratory Animal Management Committee” and “Guidelines for the Use and Care of Experimental Animals in Weifang Medical University”. HgCl2 was obtained from Sigma-Aldrich, St. Louis, MO, USA. 8-week-old healthy male C57BL/6 mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., Jinan, China) were housed at the specific pathogen-free (SPF) animal facility in Weifang Medical University under a 12-hr light-dark cycle with free access to food and water. (a) Mice were randomized into 4 groups: control group (Ctrl group, intraperitoneal injection of saline), 4 mg/kg HgCl2 group, 6 mg/kg HgCl2 group, and 8 mg/kg HgCl2 group, administered by intraperitoneal injection (n = 3-6, 3 mice in the Ctrl group, 6 mice in experimental group). (b) The mice were randomly divided into 3 groups: Ctrl group, 6 mg/kg HgCl2 group, 6 mg/kg HgCl2+4-PBA group (n = 3-6, 3 mice in the Ctrl group, 6 mice in experimental group). 4-PBA was administered before injection of 6 mg/kg HgCl2 (Yang et al., 2020). In the 6 mg/kg HgCl2 + 4-PBA group, 20 mg/kg/day of 4-PBA was injected intraperitoneally for two days of pretreatment, and then 6 mg/kg HgCl2 was injected intraperitoneally. 16 hr after HgCl2 injection, changes of body weight and behaviour were recorded. The mice were sacrificed to harvest blood samples for measuring BUN and Scr, and the kidneys were harvested.
Biochemical analysisLevels of serum creatinine (Scr) and blood serum urea nitrogen (BUN) were measured by automatic biochemistry analyzer.
Hematoxylin-eosin stainingKidney tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. Three-micrometer of renal sections were sectioned and then deparaffinized and rehydrated for the following staining techniques. Hematoxylin-eosin staining was performed to evaluate renal injury according to the protocol of the manufacture.
Periodic Acid-Schiff stainingRehydrated kidney tissues slides were prepared as above and were stained with the PAS Stain Kit according to the manufacturer’s protocol (Solarbio, Beijing, China), staining glycogen-containing components in red-purple.
Immunohistochemistry stainingRehydrated kidney tissues slides were prepared and then subjected to microwave antigen retrieval, followed by blocking with 3% H2O2 and 10% goat serum. The sections were then incubated with primary antibodies against GRP78 (1:300, Cell Signaling Technology, Danvers, MA, USA), CHOP (1:300, Santa-Cruz Biotechnology, CA, USA) at 4°C overnight respectively, washed three times in PBS, incubated with appropriated HRP-conjugated secondary antibody for 1 hr at room temperature and washed with PBS again. The signals were visualized with a DAB kit, followed by counterstaining with hematoxylin and capturing images using an OLYMPUS CX31 microscope.
Western blot analysisRenal cortical and outer medulla tissues were lysed, separated by 10% SDS-PAGE gels, and then transferred onto PVDF membranes. The membranes were blocked with 3% nonfat dried milk + 2% BSA in Tris-buffered saline Tween (TBST) for 2 hr at room temperature, and subsequently incubated with primary antibodies against GRP78 (1:1000, Cell Signaling Technology, 3177s), CHOP (1:1000, Santa-Cruz Biotechnology, sc-7351), Cleaved Caspase-3 (1:500, Cell Signaling Technology, 9661s), GAPDH (1:4000, Cell Signaling Technology) and corresponding secondary antibodies (1:2000, Proteintech Group, Chicago, IL, USA) respectively. The immunoreactive bands were detected by ECL and visualized on Kodak X-ray films. Each band was analyzed using an Image J image analysis system.
Statistical analysisData are presented as (means ± SD). Body weights before and after injection were analyzed by paired-samples t test, and other data were analyzed by Dunnett’s test and independent sample t-test. All data were analyzed with GraphPad Prime 6 software. Values of P < 0.05 were considered significantly different.
The control mice showed no abnormal performance, and were in good active and mental state. Compared with the Ctrl group, the mice of HgCl2 injection groups appeared inactive and emaciated. As the dosage of HgCl2 increased, the exercise capacity and mental state of mice gradually deteriorated and the breathing gradually accelerated. In the Ctrl group and 4, 6 mg/kg HgCl2 dose groups, there were no significant differences in body weights after injection (Table 1). In the 8 mg/kg HgCl2 group, the body weights were significantly lower after HgCl2 injection (P < 0.05) (Table 1).
| Group | Weight | |||||
|---|---|---|---|---|---|---|
| Before injection | After injection | |||||
| Ctrl group | 34.20 | ± | 1.37 | 33.83 | ± | 1.50 |
| 4 mg/kg HgCl2 group | 33.05 | ± | 2.35 | 32.15 | ± | 3.25 |
| 6 mg/kg HgCl2 group | 33.00 | ± | 1.51 | 32.37 | ± | 2.00 |
| 8 mg/kg HgCl2 group | 32.20 | ± | 2.16 | 30.63 | ± | 1.85* |
1Compared with before injection, *P < 0.05, n = 3-6
We examined kidney function by measuring BUN level and found no difference in the 4 mg/kg HgCl2 group compared with Ctrl mice (P > 0.05) (Fig. 1A). Compared with Ctrl mice, there were obvious differences in the 6, 8 mg/kg HgCl2 groups (P < 0.05 and P < 0.01 respectively) (Fig. 1A). Scr level tests showed that Scr increased significantly in the 6, 8 mg/kg HgCl2 groups (P < 0.05 and P < 0.001 respectively) (Fig. 1B). The results indicated that HgCl2 in a dose of 6 mg/kg can damage renal function obviously.
Changes in renal function in mice at different doses of HgCl2. (A) BUN levels in the Ctrl group, 4 mg/kg HgCl2 group, 6 mg/kg HgCl2 group, and 8 mg/kg HgCl2 group for 16 hr; (B) Scr levels in the Ctrl group, 4 mg/kg HgCl2 group, 6 mg/kg HgCl2 group, and 8 mg/kg HgCl2 group for 16 hr; *P < 0.05, **P < 0.01, ***P < 0.001, compared with Ctrl group, n = 3-6.
Next, we examined the kidney morphologic changes. As shown in Fig. 2, there was no obvious pathological change in the Ctrl group. Obvious morphological injuries, which were mainly located in the renal tubule, appeared in all HgCl2 groups. With the dose of HgCl2 on, injuries gradually aggravated. Local renal tubular epithelial cells appeared swollen, with lumen expansion, accompanied by brush border disappearance in the 4 mg/kg HgCl2 group (Fig. 2B, F). More renal tubular epithelial cells experienced local necrosis and more tubular tubes were occluded in the 6 mg/kg HgCl2 group (Fig. 2C, G). Renal tissue displayed flaky necrosis, accompanied with vesicles and protein casts formation in the 8 mg/kg HgCl2 group (Fig. 2D, H).
Changes in morphology of kidney at different dose of HgCl2. (A)-(D) are the results of HE staining in Ctrl group, 4 mg/kg HgCl2 group, 6 mg/kg HgCl2 group and 8 mg/kg HgCl2 group, scale bar, 100 μm; (E)-(H) are the results of PAS staining in Ctrl group, 4 mg/kg HgCl2 group, 6 mg/kg HgCl2 group and 8 mg/kg HgCl2 group, scale bar, 40 μm.
GRP78 and CHOP are the hallmark molecules of ER stress, and their increased expression indicates that ER stress is activated. Immunohistochemical staining showed that GRP78 was almost absent in the tubular cells in the Ctrl group. In kidneys of the HgCl2 groups, high levels of GRP78 were observed in the cytoplasm of the renal tubule. With doses on, the area and intensity of GRP78 became more obvious (Fig. 3A). Similarly, CHOP was nearly negative in the Ctrl kidneys and more obvious in the nucleus of renal tubular epithelial cells after HgCl2 exposure (Fig. 3A). Western blot results showed the same tendency that GRP78 protein was gradually increased in the kidneys of HgCl2. Compared with the Ctrl group, GRP78 was significantly increased in the 6, 8 mg/kg HgCl2 groups (P < 0.01 and P < 0.001 respectively) (Fig. 3B, C). Consistently, western blot showed that CHOP in the 6, 8 mg/kg HgCl2 dose groups was significantly increased than that in the Ctrl group (P < 0.01 and P < 0.001 respectively) (Fig. 3B, D). The above results indicated that 6 mg/kg HgCl2 led to significant changes in the expression of GRP78 and CHOP, and activated ER stress.
Expression of GRP78 and CHOP in the kidney after exposure to HgCl2 in mice. (A) Immunohistochemical staining for the localization of GRP78 and CHOP in the kidney (scale bar, 100 μm); (B) Western blot assay of GRP78 and CHOP protein level; (C) corresponding statistical analysis of GRP78 protein expression; (D) corresponding statistical analysis of CHOP protein expression; **P < 0.01, ***P < 0.001, compared with Ctrl group, n = 3-6.
Based on the functional and morphological changes of the kidney caused by different doses of HgCl2, we chose 6 mg/kg HgCl2 to continue the subsequent experiments.
4-PBA partially alleviates ER stress caused by HgCl2 and protects kidney4-PBA is an inhibitor of ER stress, which stabilizes protein conformation and enhances ER folding ability, thereby inhibiting ER stress and decreasing expression of ER stress-related factor (Yam et al., 2007). To further determine the role of ER stress in acute HgCl2 poisoning, we examined the effects of 4-PBA on the kidneys in HgCl2 poisoning. 4-PBA was given 2 days before HgCl2 injection. Immunohistochemical staining showed that 4-PBA pretreatment significantly reduced the expression of GRP78 in the renal tubular cytoplasm and inhibited HgCl2-induced CHOP nuclear expression compared with the 6 mg/kg HgCl2 group (Fig. 4A). Consistently, western blot analysis also showed the same results, that 4-PBA pretreatment significantly reduced the expression of GRP78 and CHOP in HgCl2 injection groups (P < 0.05) (Fig. 4B-E). Thus, 4-PBA could efficiently suppress ER stress in acute HgCl2 poisoning.
4-PBA inhibits the expression of GRP78 and CHOP caused by HgCl2. (A) Immunohistochemical staining for the localization of GRP78 and CHOP in the kidney (scale bar, 40 μm); (B) Western blot assay of GRP78 and CHOP protein level; (C) corresponding statistical analysis of GRP78 protein expression; (D) corresponding statistical analysis of CHOP protein expression, *P < 0.05, compared with Ctrl group, #P < 0.05, compared with 6mg/kg HgCl2 group, n = 3-6.
We further determined the effect of 4-PBA on the functional and morphological changes of kidneys in acute HgCl2 poisoning. Functionally, the levels of BUN and Scr in the 6 mg/kg HgCl2 group were significantly higher than those in the Ctrl group, which was consistent with the previous results. Meanwhile, in the group pretreating with 4-PBA, the level of Scr and BUN was significantly reduced (P < 0.05) (Fig. 5). These results indicated that 4-PBA can ameliorate the kidney function of acute HgCl2 poisoning. Morphologically, compared with controls, pretreating with 4-PBA also obviously alleviated the renal tubular injury, and the brush border was relatively intact. The results indicated that inhibition of ER stress can significantly reduce renal functional and morphological damage induced by acute HgCl2 poisoning (Fig. 6).
4-PBA protects against renal function decline induced by HgCl2 injection. (A) BUN levels in the Ctrl group, 6 mg/kg HgCl2 group, and 6 mg/kg HgCl2+4-PBA group; (B) Scr levels in the Ctrl group, 6 mg/kg HgCl2 group, and 6 mg/kg HgCl2+4-PBA group; **P < 0.01, compared with Ctrl group, #P < 0.05, compared with 6 mg/kg HgCl2 group, n = 3-6.
4-PBA protects against morphological changes induced by HgCl2 injection. (A)-(C) was the results of HE staining in Ctrl group, 6 mg/kg HgCl2 group and 6 mg/kg HgCl2+4-PBA group; (D)-(F) was the results of PAS staining in 40 × Ctrl group, 6 mg/kg HgCl2 group and 6 mg/kg HgCl2+4-PBA group, scale bar, 40 μm.
ER stress causes apoptosis after activation of CHOP. And, Cleaved Caspase-3 is an important marker of apoptosis. Immunochemical results showed that the expression of Cleaved Caspase-3 protein in the 6 mg/kg HgCl2 group was dramatically higher than that in the Ctrl group, while treatment with 4-PBA significantly reduced the activation of Caspase-3 (Fig. 7B, C). Consistently, immunoblotting analysis showed the same results (Fig. 7A). Together, these results suggested that 4-PBA can reduce cell apoptosis by inhibiting ER stress in HgCl2 poisoning in mice.
4-PBA alleviates tubular apoptosis caused by HgCl2. (A) Immunohistochemical staining for the localization of Cleaved Caspase-3 in the kidney (scale bar, 200 μm); (B) Western blot assay of Cleaved Caspase-3 protein level; (C) corresponding statistical analysis of Cleaved Caspase-3 protein expression, *P < 0.05, compared with Ctrl group, #P < 0.05, compared with 6 mg/kg HgCl2 group, n = 3-6.
This study showed that acute HgCl2 poisoning, especially the high dose of 8 mg/kg HgCl2, disturbed exercise capacity and mental status of mice and caused a significant weight loss. Except for the significant changes of BUN and Scr, HgCl2 exposure also damaged the morphology of the kidney, particularly the tubular. For mechanism, acute HgCl2 poisoning increased the expression of ER stress chaperone proteins GRP78 and CHOP in a concentration-dependent manner, which suggested that ER stress may be a sensitive pathway for HgCl2. Pretreatment with 4-PBA, a specific blocker of ER stress, significantly reduced GRP78 and CHOP expression and decreased Cleaved Caspase-3 expression induced by HgCl2 exposure. Renal function and morphology also validated renal protection of 4-PBA against HgCl2. These results suggest that ER stress may be the mechanism of renal damage in HgCl2 poisoning.
Mercury poisoning can be seen in active contact cases. In daily life, inadequate occupational protection, unregulated Chinese medicine, and unqualified cosmetics can lead to the accumulation of mercury in the human body. HgCl2 has various complications such as acute renal failure, digestive tract mucosal corrosion, anemia, disseminated intravascular coagulation, and brain abscess (Verma et al., 2010; Murphy et al., 1979), which is particularly severe in the kidney, especially in the proximal tubule (Zalups, 2000). In kidney injury, brush margin detachment, interstitial edema, and intraluminal granule shape may occur, and BUN and Scr may be elevated in a short time (Yadav et al., 2012; Gao et al., 2016), induced by acute HgCl2 exposure. Our findings also showed the same morphological and functional changes. We also found that the effects of HgCl2 on the kidney were significantly correlated with the dose administered. As the dose increased, the degree of kidney damage gradually increased.
Studies have shown that ER stress is involved in glomerular and tubular damage induced by a variety of causes, which plays an important role in multiple pathogeneses of AKI (Gao et al., 2012; Xu et al., 2016). However, whether it is involved in kidney damage caused by HgCl2 is not fully understood. GRP78, glucose-regulated protein 78, also known as immunoglobulin-binding protein (Bip), is expressed in the cytoplasm. It is responsible for guiding protein folding and assembly, and targeting unfolded or misfolded proteins for degradation (Zhu and Lee, 2015). GRP78 is a marker of the occurrence and presence of ER stress and its expression reflects the ability of ER stress to initiate and regulate ER homeostasis (Zhu and Lee, 2015). Heavy metals such as cadmium (Liu et al., 2006) and lead (Wang et al., 2019) induced GRP78 expression in nephrogenic cell lines in vitro. GRP78 is the target of heavy metal exposure and ER is an intracellular sensor. The subcellular cytotoxic dose of mercury chloride did not significantly activate GRP78 in NRK-52E cells (Stacchiotti et al., 2009). Our results showed that the up-regulation of GRP78 in the low-level mercury chloride group (4 mg/kg HgCl2 group) was not obvious, and that as the dose increased, the expression of GRP78 began to increase significantly. This indicated that the obvious ER stress activation appeared to be related to the dose of HgCl2. ER stress initially ensures cell survival by activating ER stress chaperones such as GRP78 and GRP94. If the ER stress is too severe or requires prolonged repair, it will trigger apoptosis by regulating the expression of CHOP (Sun et al., 2016). UPR is mediated by three ER-localized proteins: IRE1, PERK, and ATF6, all of which induce CHOP transcription during ER stress (Schröder and Kaufman, 2005). CHOP is a member of the transcription factor family C/EBP, which is mainly expressed in the nucleus and is extremely low under normal physiological conditions (Ron and Habener, 1992). When ER stress occurs, CHOP increases significantly and regulates apoptosis-related genes in downstream cells (Hu et al., 2019). Pb induces apoptosis and autophagy inhibition by activating the PERK-eIF2α-ATF4-CHOP pathway in primary rat proximal tubule (rPT) cells (Hu et al., 2019), indicating that excessive ER stress activates CHOP and induces apoptosis. Our results showed that except for the 4 mg/kg HgCl2 group, CHOP was significantly up-regulated as the HgCl2 dose increased. Therefore, there is a link between kidney damage and ER stress induced by acute HgCl2 exposure. Kidney damage is positively correlated with the expression of GRP78 and CHOP. In order to confirm this, we inhibited the ER stress before HgCl2 exposure to observe the damage of kidney.
4-PBA can inhibit the ER stress signaling pathway, so we evaluated its function on HgCl2-induced acute kidney injury in mice. The results showed that 4-PBA down-regulated the protein expression of GRP78 and CHOP. CHOP is a major regulator of ER stress-induced apoptosis (Zinszner et al., 1998), which is capable of mediating ER stress-specific apoptotic pathways (Tabas and Ron, 2011). The currently main mechanism for CHOP-induced apoptosis is inhibiting the expression of the apoptosis inhibitory protein Bcl-2. Bcl-2 can form a homodimer with the other member of the Bcl-2 family in the cytoplasm, Bax (pro-apoptotic factor), and then move to the mitochondrial membrane. It plays a role in anti-apoptosis by regulating mitochondrial membrane potential and permeability transition channels, blocking the release of cytochrome c from the mitochondrial membrane compartment and blocking the initiation of the downstream caspase-3 apoptosis signaling cascade (Tao et al., 2017). In addition, ER stress activates Caspase-12, initiates the Caspase cascade, and then induces apoptosis by activating Caspase-3 (Hitomi et al., 2004). Therefore, Cleaved Caspase-3 can be used as a basis for cell apoptosis, and can reflect the degree of apoptosis to some extent. Our results showed that Cleaved Caspase-3 in high-dose HgCl2 group was significantly higher than that in the Ctrl group. Studies have shown that the expression of CHOP is up-regulated within 24 hr, while the expression of Cleaved Caspase-3 is down-regulated in a model of kidney injury caused by acute HgCl2 poisoning (Rojas-Franco et al., 2019). However, CHOP mediates the endoplasmic reticulum stress-specific apoptosis pathway, which can cause up-regulation of Cleaved Caspase-3 expression in a short time (Yang et al., 2020). The reason for the result is not clear, and further studies are needed. Moreover, the specific ER stress blocker, 4-PBA, showed that it significantly reduced the expression of Cleaved Caspase-3 induced by HgCl2. This indicated that the increase of apoptosis level induced by a high dose of HgCl2 is also effectively inhibited. At the same time, renal function results showed that 4-PBA can reduce the level of BUN caused by HgCl2 effectively. And, the morphological results showed that after the ER stress was inhibited, the necrosis of renal tissues, especially the renal tubules, was significantly improved. These results suggest that inhibition of GRP78 and CHOP expression alleviates kidney damage induced by acute HgCl2 poisoning, and this effect may be done by the inhibition of apoptosis.
In summary, ER stress involved the renal damage caused by acute HgCl2 poisoning, and inhibition of ER stress can reduce cell apoptosis to protect HgCl2-induced AKI. This study provides a new research direction for clarifying the mechanism of renal damage induced by acute HgCl2 poisoning. In this experiment, we proved that ER stress is one of the mechanisms of renal damage caused by acute HgCl2 poisoning. The role of ER stress in the damage of other organs is unknown, which is also the next step of our research.
This study was supported by a grant from the National Nature Science Foundation of China (NFSC: 81802474), Nature Science Foundation of Shandong Province, China (ZR2018MH040, ZR2018MC012).
Conflict of interestThe authors declare that there is no conflict of interest.
