*3.2. Cadmium*

Cadmium (Cd) naturally exists in zinc, lead, and copper ores as a divalent cation in the Earth's crust and marine environments at low concentrations and accumulates in air water and soil through volcanic activity and erosion [124]. While trace amounts of Cd in the environment are byproducts of these processes, the majority of environmental Cd is the result of industrial and agricultural use [124]. Soluble Cd ions from phosphate fertilizers can contaminate water and soil and subsequently accumulate in aquatic organisms or plants such as tobacco, grains, and root vegetables [125,126]. Because of accumulation in tobacco plants, individuals who smoke are exposed to significant levels of Cd through the inhalation of cigarette smoke. Cigarettes may contain 1.8–2.5 μg/g Cd [127], and data from the National Health and Nutrition Examination Study (NHANES) reported that smokers had average blood and urine Cd levels of 0.376 μg/L and 0.232 μg/L, respectively [128].

Following exposure, Cd is absorbed readily by epithelial cells in the gastrointestinal tract and lungs. Once ingested, ionized Cd2+ binds to albumin and the resulting complexes are then transported to target organs, including the kidney, bone, liver, and lung. Cd is taken up into hepatocytes via Ca2+ channels and membrane transporters [129]. Within the cell, Cd has been shown to impair electron transport chain complexes II and III, which impedes electron flow and generates ROS [112]. The generation of ROS promotes binding of the metal-regulated transcription factor 1 (MTF-1) to metal response elements (MRE), which subsequently activates transcription of metallothionein (MT) [110]. Intracellularly, MT binds to Cd to create a MT–Cd complex, and a fraction of the complex is exported into the circulation [130]. MT–Cd complexes are filtered freely by the glomerulus and are reabsorbed by proximal tubular epithelial cells via multiple mechanisms, including

megalin and cubilin receptor-mediated endocytosis, ZIP8 and ZIP14, and the divalent metal transporter 1 (DMT1) [131,132].

Environmental and occupational exposure to low levels of Cd has been shown to cause renal tubular injury [133]. Owing to its toxic renal effects, chronic exposure to Cd increases the risk of developing CKD from 10% in the average population to 25% in exposed individuals [134]. Analyses of NHANES data showed that individuals with blood Cd levels over one mcg/L had a significantly higher association with CKD and albuminuria [135]. It is clear that exposure to Cd reduces GFR and impairs overall renal function. Here, we summarize the molecular mechanisms that underlie these pathologies and contribute to the development and/or progression of CKD (Figure 4).

**Figure 4.** Schematic to summarize major factors that contribute to progression of CKD following exposure to cadmium (Cd). Additional factors not included here also contribute to Cd-induced progression of CKD.

As discussed in the previous sections, diabetes is a major risk factor for developing CKD. Exposure to Cd appears to increase the risk of developing diabetes in some individuals [136]. Diabetic mice (db/db) exposed to Cd were found to be hyperglycemic and exhibited an increase in white adipose tissue and weight gain [137]. Interestingly, exposure of these mice to Cd also decreased serum leptin levels, which may enhance appetite and lead to weight gain. In contrast to these findings, a recent study using streptozotocininduced diabetic mice (C57BL/6) exposed to Cd showed that body weight decreased after exposure [138]. This variation could be due to differences in the frequency and dose of Cd exposure. This study, however, confirmed findings that exposure of hyperglycemic animals to Cd enhances the risk of renal injury [138]. Specifically, in vitro studies using cultured podocytes indicate that exposure to Cd under hyperglycemic conditions leads to mitochondrial dysfunction and ROS. Podocyte viability is reduced, leading to apoptosis, fibrosis, and decreased renal function [138]. These studies suggest that exposure of diabetic individuals to Cd may exacerbate renal injury and lead to CKD or enhance the progression of CKD by causing additional injury.

Hypertension is another major risk factor for the development of CKD. A meta-analysis study of published literature found a positive association between hypertension and Cd levels in blood and hair [139]. Indeed, Cd has been shown to decrease plasma levels of atrial natriuretic peptide (ANP) [140], an important regulator of blood pressure. Cd appears to reduce the affinity of the ANP receptor for ANP and also decreased the number of binding sites available [141]. Reduced levels of ANP and reduced sensitivity of the receptor may decrease the ability to regulate blood pressure, which may lead to hypertension and subse-

quent renal injury. The development of hypertension is characterized by low ANP plasma concentrations [142] and its suppressed ability to regulate blood pressure via inhibiting the renin–angiotensin–aldosterone system. In addition to causing vasodilation of the afferent arterioles, ANP binds to natriuretic peptide receptor-A, catalyzing the conversion of GTP to cGMP. cGMP phosphorylates and allosterically binds to basolateral sodium-potassium ATPase channels and apical cyclic nucleotide-gated, heterometric channels of transient receptor potential V4 and P2 [143]. While the mechanism remains entirely unclear, damage to the kidney's response to ANP may potentially be mediated by Cd-induced oxidative damage. In one study, increased production of thiobarbituric acid reactive substance after exposure to Cd was accompanied by decreased glomerular filtration rate and increased creatinine levels; upon bolus injection of ANP in compromised rats, high blood pressure and low glomerular filtration rate remained remarkably uncorrected. Without the counteraction of salt and fluid retention from ANP due to compromised receptor response, Cd may play a role in exacerbating hypertensive conditions precipitating kidney injury and eventual CKD [143].

In addition to the association with diabetes and hypertension, exposure to Cd has been shown to cause generalized cellular injury in renal epithelial cells. A major consequence of Cd exposure is intracellular oxidative stress. Studies using male Sprague Dawley rats found swelling, deformation, and vacuolation in mitochondria of renal tubular epithelial cells. In addition, expression of superoxide dismutase 2 (SOD2), found in mitochondria, decreased, indicating an inability to counteract the production of ROS. Indeed, increased cellular content of ROS was accompanied by increased expression of cytoplasmic superoxide dismutase 1 (SOD1). Interestingly, expression of catalase was reduced, which would prevent cells from responding appropriately to oxidative stress [144]. Other studies reveal the association of Cd exposure to substantial activity reduction in antioxidant enzymes, including superoxide dismutase, catalase, and glutathione reductase, that may amplify the progression of chronic kidney disease from oxidative species overwhelming antioxidants [145].

In addition to oxidative stress, Cd has been shown to induce ER stress and autophagy (via BNIP3) in HK2 cells and SD rats [146]. Studies in cultured rat pheochromocytoma cells (PC-12) showed that exposure to Cd enhanced autophagy [147]. In contrast, studies in mice exposed to Cd showed that protein components of autophagosomes (e.g., p62, Sirt6, and LC3-II) accumulated in the cytoplasm of renal tubular cells rather than participate in the formation of autophagosomes. This resulted in the inhibition of autophagy and the initiation of apoptosis [148]. Similarly, a study in cultured proximal tubular cells reported that treatment with Cd led to the accumulation of p62 in the cytoplasm and the inhibition of autophagy. Furthermore, it was reported that elevated levels of p62 led to increased nuclear translocation of Nrf2 [149]. Persistent activation of Nrf2 can lead to lysosomal dysfunction, which prevents fusion of lysosomes and autophagosomes [150]. Cd may also induce apoptosis in renal epithelial cells through p-53 mediated, DNA damage autophagy modulator (DRAM) and BAX signaling [151]. Cd has also been shown to activate inflammatory cytokines, such as NF-κb, TNF-α, and iNOS, and induce necroptosis [152,153].

Exposure to Cd has also been shown to disrupt cadherin-dependent cell adhesion in proximal tubular cells. Alterations in cellular adhesion have been shown to alter the membrane localization of the Na+K+-ATPase, which can lead to alterations in transport [154]. Similarly, other studies in proximal tubules showed that exposure to Cd decreased expression of SGLT1 and SGLT2. This decrease was attributed to the replacement of Zn in the Sp1 DNA binding domain, which reduced activation of SGLT1 and SGLT2 promoters [155].

Cd is a toxic metal to which humans continue to be exposed throughout their lives. Continued studies related to Cd-induced cellular injury in renal tubular epithelial cells are necessary to understand how exposure to this metal affects patients with renal disease.
