**1. Introduction**

Cadmium (Cd), a divalent metal used for industrial purposes, is an important environmental pollutant in some regions of the world [1–5]. The metal is conveyed to humans in food, air, and tobacco smoke, and subsequently gains access to the circulation through the gu<sup>t</sup> and lungs [5]. Salts of ionized Cd are absorbed in the duodenum; in addition, complexes of Cd with plant metallothioneins (MT) and phytochelatins (PC) may be absorbed in the colon after liberation by bacteria [6]. In the bloodstream, Cd is bound to red blood cells, albumin, glutathione (GSH), sulfur-containing amino acids, MT, and

PC [6–10]. In the liver, hepatocytes take up Cd not bound to MT [10], synthesize MT in response to the metal, and store complexes of CdMT. These complexes are subsequently released from hepatocytes and transported to the kidneys [8,11,12]. Cd in plasma is filterable by glomeruli if it is bound to GSH, amino acids, MT, or PC [8,9], but the fraction of circulating Cd that enters the filtrate is unknown. The proximal tubule reabsorbs and retains most or all of the filtered Cd with an array of channels, solute carriers, and mediators of endocytosis [7,10,13–16]. Basolateral uptake may also add to the cellular content of Cd in the proximal tubule [16–18].

It is currently assumed that the magnitude of a gradually acquired burden determines the toxicity of Cd in tubular cells [19]. The emergence of Cd from lysosomes induces robust intracellular synthesis of MT, which greatly mitigates the injury inflicted by free Cd through complexation of the metal [20]. Nevertheless, a fraction of Cd remains unbound to MT and is presumed to promote autophagy, apoptosis, and necrosis as accumulation of Cd progresses [19,21]. Manifestations of renal toxicity include increased excretion of cellular proteins, impaired reabsorption of filtered substances, histologically demonstrable tissue injury, loss of intact nephrons, and reduction of the glomerular filtration rate (GFR) [5,8,21–27]. GFR may continue to fall for many years after exogenous exposure ceases [22,24], presumably because tra ffic of CdMT from the liver to kidneys persists.

In human studies of Cd-induced nephropathy, the most commonly assayed marker of tubular cell damage is the lysosomal enzyme N-acetyl-β-<sup>d</sup>-glucosaminidase (NAG). Because NAG is too large to be filtered by glomeruli, excessive excretion (ENAG) signifies tubular injury [28]. The most commonly measured indicator of impaired reabsorption is β2-microglobulin (β2MG). This small circulating protein is extremely filterable by glomeruli [29]; ordinarily, more than 99% of filtered β2MG is reabsorbed [30], but that percentage falls early in the course of tubular injury [31].

Although the urinary excretion rate of Cd (ECd) is believed to reflect the body burden of the metal [5], the precise source and pathophysiologic significance of excreted Cd have not been clarified. One possibility is that Cd is excreted because it is filtered and not reabsorbed; an alternate possibility is that excretion reflects liberation of Cd into filtrate from injured or dying tubular cells [32]. This distinction is important because the source of urinary Cd is central to the relationship between Cd accumulation and progression of chronic kidney disease (CKD). Herein, we present evidence that excreted Cd emanates from cells that it has injured. The injury leads to the loss of intact nephrons, reduction of GFR, and impaired reabsorption of filtered β2MG.

#### **2. Materials and Methods**
