Toxicology. 2009 Oct 29;264(3):215-24. Epub 2009 Aug 29.

Stress proteins and oxidative damage in a renal derived cell line exposed to inorganic mercury and lead.

Stacchiotti A, Morandini F, Bettoni F, Schena I, Lavazza A, Grigolato PG, Apostoli P, Rezzani R, Aleo MF.

ABSTRACT:

A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.

PMID: 19720107

INTRODUCTION:

Mercury (Hg) and lead (Pb) are harmful heavy metals that are widely involved in environmental toxicology due to pollution and industrial activities (Zalups, 2000; Lyn Patrick, 2006a). Acute and prolonged exposure to these metals causes severe damage to health in man and animals, where they affect a number of fundamental organs including the brain, blood, liver, bone, reproductive system and kidneys (Clarkson and Magos, 2006). Furthermore, the proximal tubular epithelium is susceptible to toxicants due to the intense filtration of substances from the blood, their transport and the high energy requirement of these functions. In particular, the straight portion or S3 segment in the proximal tubules is often the particular target as reported in vitro and in vivo (Diamond and Zalups, 1998; Clarkson, 2002; Stacchiotti et al., 2003). 

Hg is a widespread environmental pollutant and chronic exposure to low levels is quite common as a result of the contamination of food and drinking water. Among the chemical forms of Hg, the inorganic species are far more  nephrotoxic. Renal proximal tubular cells represent the primary target site, where highly reactive inorganic Hg(II) ions rapidly accumulate and induce acute or chronic kidney injuries (Zalups, 2000). Pb is also a highly toxic and persistent contaminant that is still present in the atmosphere, soil and sediments due to industrial activities and chemical pollution, despite its reduction in petrol and paints since the 1970s.

Inorganic compounds have been classified as possible human carcinogens (IARC, 2005). Pb greatly affects the nervous system in infants and children and contributes to nephrotoxicity in susceptible populations, such as those with hypertension, diabetes and obesity (Logham-Adham, 1997; Ekong et al., 2006). Despite the numerous studies the mechanisms whereby these metals exert their negative effects in the kidney are not fully understood.

Although most of our knowledge of the adverse effects of these metals stems from acute intoxication studies and environmental disasters such as the Minamata Bay accident (Tsuda et al., 2009), the current trend in toxicology is to study the effects of low exposure to a specific metal or a mixture of different metals, which is the situation most commonly encountered by the population in industrialized countries.

One of the main and most common mechanisms behind heavy metal toxicity has been attributed to oxidative stress. Hg(II) induces oxidative stress and ROS production by binding to intracellular thiols (glutathione and proteins) and acting as a catalyst in Fenton-type reactions. This metal also causes direct damage to the mitochondrial and microsomemetabolism, and modifies renal cell membranes and tubular polarity (Lash and Zalups, 1992). Pb(II) ions affect several biochemical activities, such as ion channel function and greatly interfere with calcium signalling in different tissues, mainly in the brain, muscles and heart (Kerper and Hinkle, 1997; White et al., 2007). Exposure to heavy metals is known to activate in all living organisms a universal mechanism of defence called "the stress response", which includes the production of stress proteins to best adapt to adverse conditions (Borkan and Gullans, 2002).

Stress proteins, i.e. heat shock proteins (HSPs), glucoseregulated proteins (GRPs) and metallothioneins (MTs), are highly conserved proteins over-expressed or induced from prokaryotes to mammals to preserve cellular homeostasis, and protect other fundamental proteins or organules damaged by metals (Morimoto and Santoro, 1998). Stress proteins cover several families that are classified according to their molecular weight, with different subcellular locations and cytoprotective functions (Beck et al., 2000). Hsp25 in rodents, alias Hsp27 in mammals, is constitutively expressed in the kidney associated with actin and Na–K-ATPase to regulate proximal tubule polarity and renal flux, and its enhancement is linked to adverse conditions such as ischemia, cancer and drug toxicity (Beck et al., 2000; Arrigo, 2007). Hsp72 is the best known inducible chaperone that is almost undetectable in certain cell lines but over-expressed in the kidney after treatment with nephrotoxic drugs or exposure to environmental toxic metals such as arsenic, cadmium and mercury (Goering et al., 2000; Madden et al., 2002). Grp78/BiP is a chaperone residing in the endoplasmic reticulum but it has also been detected in the external mitochondrial membrane. Recent studies reported that a Grp78 over-expression after exposure to nephrotoxic drugs, such as cisplatin and  cyclosporine, is necessary to counteract overt apoptotic cell death in the kidney (Kimura et al., 2008). Lastly, metallothioneins (MTs) are inducible single-chain proteins with a high proportion of cysteine residues that bind and neutralize metals and are directly involved in the protection of intracellular thiols (Florianczyk, 2007).

The purpose of this study was therefore to relate the incidence of oxidative stress to the expression of specific stress proteins using as an in vitro model a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of Hg(II) and Pb(II). We hypothesized that the up- or down-regulation of particular stress proteins may not only indicate a protective or detrimental role of the chaperone in a specialized organelle or cell compartment, but itmayalso be strictly linked to the specific nephrotoxic mechanism of each metal. We analysed the effects of HgCl2 and PbCl2 on (a) NRK-52E cell viability and ultrastructural features, (b) oxidant/antioxidant cell balance, by determining ROS, RNS, GSH and glutathione-Stransferase (GST) activity, and (c) the expression and subcellular location of Hsp25, Grp78, Hsp72 and MTs. Our experimental data demonstrate that in this renal cell line different stress proteins are over- e expressed early after Hg(II) and Pb(II) exposure and that only Hg(II) induces overt oxidative damage. 

DISCUSSION:

It has been widely demonstrated that the exposure to heavy metals shares several primary mechanisms of toxicity, including oxidative stress, reaction with intracellular thiols and interference with essential metals (Valko et al., 2006; Flora et al., 2008; Wang and Fowler, 2008). If not immediately destroyed by apoptosis or necrosis, cells express a series of early events that favour their survival. The synthesis of antioxidant molecules (GSH and related enzymes) and stress proteins represent the early common mechanisms of cell protection against toxicants (Sabolic, 2006). In this in vitro study we analysed oxidative damage and stress response induced in rat tubular epithelial renal (NRK- 2E) cells by subcytotoxic doses of inorganic mercury [Hg(II)] and lead [Pb(II)]. This cell line is suitable for testing heavy metals (Madden et al., 2002; Giuliani et al., 2005) and has been well characterized for mitochondria content and glutathione status (Lash et al., 2002).

Firstly, our data clearly demonstrate that Hg(II) and Pb(II) have a different influence on oxidant/antioxidant cell balance. Oxidative stress disturbs the equilibrium of pro-oxidant and antioxidant reactions, and occurs in biological systems when there is an overproduction of ROS/RNS and/or a deficiency of enzymatic and non-enzymatic antioxidants.

Of the various mechanisms of defence against free radical-induced oxidative stress, GSH is the major thiol antioxidant of cells (Valko et al., 2007). The tripeptide scavenges hydroxyl radical and singlet oxygen directly, detoxifies hydrogen peroxides and lipid hydroperoxides by the activity of glutathione peroxidase and is able to regenerate other antioxidant molecules (Masella et al., 2005). In this study the quantification of ROS and RNS, as well as total GSH, gave direct evidence confirming the ability of Hg(II) to induce oxidative stress in NRK-52E cells. ROS and RNS levels increased, though at different times, after exposure to 20M, but not to 10M Hg(II). On the contrary, GSH  levels appear to be significantly increased compared to controls after 10M Hg(II) and were slightly enhanced in cells exposed to the highest Hg(II) concentration. This effect really depends on the high Hg(II) affinity to SH-groups that induce a dose-dependent formation of Hg–GSH conjugates, causing depletion of the free GSH cellular amount, thus affecting cell "redox homeostasis".

Although other mechanisms of mercury action on antioxidant defence may be involved (Aleo et al., 2002), its interaction with the main cell redox buffer partially explains the increase in free radicals detected in NRK-52E cells.

Oxidative stress has also been implicated with Pb(II)-associated injury (Hsu and Guo, 2002; Lyn Patrick, 2006b), but  we did not find ROS or RNS generation as a result of Pb(II) exposure in NRK-52E cell line, even at the highest salt concentration. 

Indeed, this heavy metal significantly increased the level of GSH without affecting the concentration of free thiols, thus suggesting that Pb(II) affinity to SH-groups is lower than that of Hg(II) and that the sulphydryl antioxidant reserve may counteract Pb-induced oxidative stress. 

Another important biochemical parameter related to antioxidant response is the activity of glutathione-S-transferases. GSTs are three enzyme families with different subcellular locations that detoxify noxious xenobiotics (including environmental pollutants) and protect against reactive compounds produced during oxidative stress. During GST- mediated reactions, GSH is conjugated with various electrophiles or oxidation end products and the GSH adducts are actively secreted by the cell (Hayes et al., 2005). This GSconjugated efflux can result in the depletion of cellular thiol. In NRK-52E cell line both Hg(II) and Pb(II) enhanced the activity of GSTs, confirming the role of this enzyme in the antioxidant cell response.

Since a close relationship between oxidative damage and stress proteins has been clearly demonstrated (Johnson and Fleshner, 2006), we analysed here the stress response against each metal. Among the major findings reported here we demonstrated that Hg(II) stimulated early MT-I/II transcription at 3 h, and induced Hsp25, Hsp72 and MT protein expression at 24 h; Pb(II) was unable to induce Hsp72, but stimulated Grp78 over-expression and later MT transcription (24–48 h). The different nephrotoxic mechanisms of mercury and lead may explain these differences in the stress response induced inNRK-52Ecell line.Weshowedin fact that Hg(II) and Pb(II) have a different effect on renal cell growth. In particular, 20M HgCl2 was the highest non-cytotoxic concentration, the sensitivity of the model being comparable to that obtained with other renal derived cells (Aleo et al., 2002). At higher Hg(II) concentrations, survival significantly decreased and cells died after 24 h.

This result was corroborated by morphological analysis that highlighted, already at 20M Hg(II), some alterations in cell shape, mitochondrial damage and apoptosis or necrosis. Pb(II) induced cell growth inhibition without affecting cell viability in the NRK- 52E cells, even at the highest concentration. This cytostatic effect may depend on the halting of cell cycle progression induced by the metal in this cell line (Giuliani et al., 2005). Moreover, after Pb(II)- treatment, apoptotic or necrotic events were lacking, although the cells assumed a round shape and decreased their attachment to components of the extracellular matrix. With regard to stress proteins, Hsp25 is known to interact with the actin of the structures mediating cell–cell and cell–substrate attachment in proxima tubular epithelium. After oxidative stress, this chaperone follows F-actin redistribution and the microfilament dynamics playing a role in the regulation and function of these structures (Molitoris et al., 1991; Shelden et al., 2002; Ferns et al., 2006). In Hg(II) treated NRK-52E cells, the redistribution of Hsp25 from the junctional side, where it is bound to actin, to the cytoplasm correlates with actin relocation, thus supporting the role of this chaperone in stabilizing actin cytoskeleton.

This is in agreement with a previous in vivo study, in which we demonstrated that Hsp25 colocalizes with actin on the apical side of renal proximal tubules under control conditions, but moves to a granular cytoplasmic and perinuclear location in rats exposed to inorganic mercury (Stacchiotti et al., 2004).  

Although different studies on rat kidney have reported a nuclear location of Hsp25 in heat-shocked cells or after treatment with nephrotoxic drugs such as cyclosporine (Bryantsev et al., 2007; Pallet et al., 2008), the nuclear translocation of Hsp25 observed here after Pb(II) exposure is unusual. It is known that Pb(II) binds to zinc-binding sites in many proteins, including several DNA-binding proteins, as well as transcription regulators. The displacement of zinc by Pb(II) causes conformational change in these proteins that results in reduced binding toDNAconsensus sequences (Silbergeld, 2003). Hsp25 may well interact with proteins in nuclei to promote refolding or degradation of target damaged proteins. Another important function of Hsp25 is to increase cellular resistance against oxidative damage by different mechanisms, such as preservation of glutathione in its reduced form (Arrigo et al., 2005).

Here, Hsp25 over-expression correlates well with ROS production and with morphological oxidative damage (swollen mitochondria and necrosis) induced by Hg(II). Remarkably, Hsp72 has also been induced by Hg(II) in NRK-52E cells, but not by Pb(II). This result may be explained by the central role of this chaperone in response to oxidative damage; recent studies have reported that this chaperone may ameliorate the cell redox status in transfected MDCK cells by increasing the activity of antioxidant enzymes (Johnson and  Fleshner, 2006; Guo et al., 2007). However, Hsp72 over-expression may represent an early reaction against direct mitochondrial damage, as already reported by Carranza-Rosales et al. (2005), in kidney OK cells or by Park and Park (2007) in BEAS-2B cell line. It is also known that Hsp72 has antiapoptotic effects and modulates the engagement and/or progression of apoptosis by interfering at different points in the apoptotic cascade (Samali and Orrenius, 1998).  In effect Hsp72 has been demonstrated to be the main inducible heat shock protein in renal survival after ATP depletion, as well as in cardiomyocytes after ROS-induced apoptosis (Wang et al., 2002; Jiang et al., 2005).

Another remarkable result of this study is that Pb(II) stimulates Grp78 expression in NRK-52E cells, whereas Hg(II) appears to be ineffective. Different in vitro studies reported an increase in Grp78 in cell lines of different organs exposed to Pb(II) (Tully et al., 2000; Qian et al., 2001; Xu et al., 2009). We hypothesize that Grp78 overexpression in our in vitro system may be related to a protective  function against Pb(II) and to a request for synthesis/translocation of new proteins, as corroborated by the morphological findings, i.e. the RER assembly in the perinuclear area and nucleolar segregation. Besides mitochondria, endoplasmic reticulum is also involved in cell death mechanisms (Inagi, 2009) and when its ability to fold polypeptides properly is affected, the unfold protein response (UPR) is activated. A major UPR-regulated target is Grp78 (Li et al., 2008).

It is also worth noting that Pb(II) affects intracellular Ca2+ metabolism by mimicking or inhibiting its physiological action (Godwin, 2001; Li et al., 2008). Since ER plays a vital role controlling both the level and the release of the calcium stores (Hendershot, 2004), the Pb-induced Grp78 expression may be functional in preserving calcium homeostasis. Interestingly, Qian et al. (2005) demonstrated in astrocytoma cells that Grp78 interacts directly with Pb(II), thus protecting cells from the free metal ion to maintain ROS homeostasis.

As previously found in this cell line, another intracellular target of Pb(II) is microtubular protein tubulin (Giuliani et al., 2005). Grp78 immunostaining in the mitotic spindle may indicate this chaperone's requirement to maintain microtubule integrity for chromosomal segregation and cell division (Bonacker et al., 2005). Lastly, MTs isoforms can be considered the first line of defence against metals, but they also act as endogenous antioxidants by reacting with free radicals and reactive oxygen species (Coyle et al., 2002). It is noteworthy that Hg(II) stimulates MT expression in renal tubular cells (Zalups and Koropatnik, 2000; Aleo et al., 2005). Here in NRK-52E cells, MT mRNA transcription and protein expression happen within 24 h of Hg(II) exposure, as well as expected by a stress response, and then decline. On the contrary, the delay in MT induction after Pb(II) could be explained by the metal's lower sulfhydryl- binding affinity for these proteins (Wang et al., 2009) or the lesser degree of nephrotoxicity induced in this in vitro model.

However, a remarkable characteristic of Pb poisoning is the production of a protein–Pb complex, which appears as nuclear inclusion bodies in renal cells of chronically exposed humans or animals that mitigate toxicity. Qu et al. (2002) demonstrated in MT-null mice that the inability to express the major isoforms of the MT gene increased the sensitivity to Pb(II) toxicity and formed no inclusion bodies in the kidney. So in our renal cell line, MT I/II may prevent cell damage by Pb(II) accumulation even if a longer incubation time may be needed to induce inclusion bodies. 

Taken together, all data demonstrated that the pattern of stress protein expression in NRK-52E renal cells is strongly influenced by upstream biochemical changes, such as oxidative damage or thiol modification, induced by different pollutants such as mercury or lead. Also, stress response is clearly toxicant-dependent. On this basis, the over expression of specific SPs induced by exposure to Hg(II) or Pb(II) should not be considered as the result of a generalized reaction to stress condition, but conversely as a specific antioxidant response activated by NRK-52E cells to counteract the action of each metal on different cellular targets (organelle or cellular compartment).

The relationship between stress response and pollutant metals of this study provided significant information on the multiple mechanisms of their nephrotoxicity, and might be useful to identify early biomarkers of metal environmental injury. Moreover, because the lack of a stress protein response acts on cell viability leading to an irreversible damage, the possibility to modulate specific stress proteins by low doses of metals might offer new opportunities in therapeutic approaches.