*3.3. Mercury*

Mercury (Hg) is a toxic metal found in various environmental and occupational settings. It may exist in an elemental (metallic), inorganic, and/or organic form. Elemental

mercury (Hg0) is particularly unique because it exists as a liquid at room temperature. Inorganic mercury is usually found as mercurous (Hg1+) or mercuric (Hg2+) ions salts. Organic forms of mercury include phenylmercury, dimethylmercury, and monomethylmercury (MeHg), which is the most common form encountered by humans. The majority of human exposure is due to the ingestion of contaminated food. Upon ingestion, MeHg is absorbed readily by enterocytes along the gastrointestinal tract [1], after which they can enter systemic circulation and be delivered to target organs. Within biological systems, a fraction of MeHg is slowly transformed to Hg2+ [156–159].

Exposure to all forms of Hg has been shown to have significant renal effects (Figure 5). Experimental models (uninephrectomy) of early-stage CKD suggest that acute renal injury is more pronounced in uninephrectomized rats exposed to a nephrotoxic dose of HgCl2 than in corresponding sham rats [160–162]. It was found that mercury-induced proximal tubular necrosis was more extensive in 50% nephrectomized animals than in sham animals. Additionally, the urinary excretion of cellular enzymes and plasma proteins, including lactate dehydrogenase, γ-glutamyltransferase, and albumin, was greater in uninephrectomized animals than in sham animals [162,163]. Interestingly, when 75% nephrectomized rats were used as models of late-stage CKD, it was found that the accumulation of mercury per g kidney is significantly greater in 75% nephrectomized rats than in sham rats, suggesting that cellular accumulation of Hg may be greater in the remnant renal mass from 75% nephrectomized animals than in kidneys of sham animals [164].

**Figure 5.** Flowchart outlining major mechanisms involved in mercury (Hg)-induced progression of CKD. Other factors not specified here also play a role in the progression of CKD induced by exposure to Hg.

In humans, chronic exposure to mercury has been associated with glomerulonephritis, particularly membranous nephropathy [165,166]. Membranous nephropathy is characterized by tissue damage due to activation of membrane attack complexes (MAC) by antigen– antibody complexes deposited on the glomerular basement membrane (GBM) [167]. This damage results in podocyte damage and disruption of the anionic charge barrier, leading to massive proteinuria [167]. Analyses of patient biopsies found that patients with Hg-induced membranous nephropathy exhibited more mesangial deposits and smaller podocyte foot processes than patients with idiopathic membranous nephropathy. Interestingly, podocyte effacement was less severe in Hg-induced cases than in idiopathic cases [168]. In vitro

studies have shown that exposure to Hg leads to autoimmune disease characterized by anti-GBM antibodies, glomerular deposits of immunoglobin G (IgG), proteinuria, and acute tubulointerstitial nephritis [169–171]. Studies in Brown Norway rats have shown that exposure to HgCl2 leads to a T-cell dependent autoimmune syndrome that leads to the production of anti-laminin antibodies that interact with the GBM [172,173]. Exposure to HgCl2 leads to the appearance of non-antigen-specific CD8+ T-cells [174]. Additional studies in Brown Norway rats showed that RT6+ T cells decreased, which inversely corresponded with the autoimmune response to the GBM [175]. Escudero et al. showed that the HUTS-21 epitope of the beta-1 integrin on lymphocytes appears to be involved in Hg-induced nephritis by promoting lymphocyte infiltration into renal interstitium and deposition of anti-GBM antibodies [176].

Exposure to Hg may also play a role in the development of hypertension. A study of non-Hispanic Asians using NHANES data found that higher blood Hg levels were associated with hypertension [177]. Studies using spontaneously hypertensive rats (SHR) found that exposure to Hg accelerated the development of hypertension by increasing the production of nitric oxide and other ROS [178,179]. However, it appears that Hg also induces vasoprotective mechanisms such as increased plasma levels of nitric oxide and hydrogen peroxide to counteract other vasoconstrictive effects [179]. In addition, plasma levels of angiotensin-converting enzyme (ACE) were found to be increased in SHR rats following exposure to Hg [180], which can lead to vasoconstriction and hypertension. In a recent review, Habeeb et al. outlined the molecular mechanisms by which Hg exposure leads to hypertension [181]. Hg has been shown to increase atherosclerosis as well as stimulate the proliferation of vascular smooth muscle cells, which would further increase the risk of hypertension.

The effects of Hg on renal tubule epithelial cells can be detrimental to total renal function. Studies in cultured proximal tubular cells have demonstrated that exposure to Hg induces significant cellular alterations [182,183]. Specifically, the most profound modifications were noted as increased oxidative stress, cytoskeletal rearrangements, increased intracellular calcium, and reduced cellular viability.

In the mitochondria, mercury-induced oxidative stress has been shown to disrupt the overall structure, leading to swelling, destruction of mitochondrial membrane potential, altered membrane, and increased release of Cytochrome C [184]. Exposure of human embryonic kidney epithelial (HEK-293T) cells to HgCl2 revealed a decrease in cell viability due to a downregulation in the expression of the silent information regulator (Sirt1) and PGC-1α signaling pathway, a key mechanism in mitochondrial homeostasis [185].

Cytoskeletal alterations have been detected following exposure of normal rat kidney cells (NRK-52E) to MeHg [186]. These alterations are a result of epigenetic modulation of matrix metalloproteinase 9 (MMP9) via demethylation of its regulatory site. The subsequent increased expression of MMP9 led to loss of cell-to-cell adhesion and disturbances in cytoskeletal proteins such as F-actin, vimentin, and fibronectin [186]. Similarly, exposure of NRK-52E cells to HgCl2 also led to loss of cytoskeleton integrity [182].

The ER is another cellular target in acute HgCl2 toxicity. Experiments in NRK-52E cells showed that ER stress, as indicated by expression of GRP78 (78-kDa glucose regulated protein) and CHOP (C/EBP homologous protein), is a marker of renal cell injury [187]. GRP78 is an ER chaperone, which is upregulated upon stress; however, if the ER experiences prolonged stress, CHOP, a transcription factor specializing in regulation of apoptosis-related genes, will also be upregulated. Both of these proteins are positively correlated with renal damage. In addition, HgCl2 has been shown to enhance the activity of Caspase 3 and the expression of IRE1a (inositol-requiring enzyme 1), GADD-153 (growth arrested and DNA damage-inducible gene 153), and Caspase 12, resulting in the death of tubular and glomerular cells [188].

Exposure to Hg also affects the activity of various transporters, which may lead to tubular injury and renal disease. Studies in Wistar rats exposed to a low dose of HgCl2 showed that Hg inhibited the Na+/H+ exchanger (NHE3) in proximal tubular cells. It was suggested that Hg enhanced phosphorylation of NHE3 and thereby reduced its activity [189]. NHE3 is the main isoform of the Na+/H+ exchanger in the proximal tubule and it plays a major role in the reabsorption of sodium from the lumen. Alterations in the activity of NHE3 could indirectly affect reabsorption and secretion of important molecules and fluid. In addition, HgCl2 has been shown to inhibit Na-K-ATPase [190], which would alter solute gradients necessary for water reabsorption and lead to increased urinary output, a common sign of renal injury. Mercury has also been shown to bind to cysteine residues in aquaporin 1 (AQP1) [191], located in the proximal tubule and thin limbs in the loop of Henle [192], and inhibit its activity. AQP1 facilitates reabsorption of 70% of water from the ultrafiltrate entering the proximal tubule [193]. Therefore, the inhibition of AQP1 is also a likely cause of increased urinary output following Hg intoxication.

Collectively, the results of these studies suggest that kidneys of animals with reduced renal mass are more susceptible to the toxic effects of Hg. Similarly, individuals who have reduced renal function, due to CKD or other disease processes, may be more susceptible to renal injury following exposure to a nephrotoxicant such as Hg.
