Biomarkers for Cadmium

Synonyms

Cadmium: Cd; Cd²⁺

Definitions

Cadmium (Cd): Is an important industrial agent and environment pollutant that is a major cause of kidney disease in many regions of the world.

Nephrotoxicity/proximal tubule: The proximal tubule is the primary target of Cd toxicity in the kidney. Injury of proximal tubule epithelial results in increases in urine volume and excretion of low-molecular-weight proteins, amino acids, glucose, and electrolytes. These effects of Cd may result from even low levels of exposure and are often irreversible.

Biomarkers: The United States National Institutes of Health have broadly defined the term “biomarker” as a “characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” In this entry, the topic of Cd biomarkers will be considered from a more narrow perspective, as “any substance or molecule that can serve as an indicator of the functional state or level of toxic injury in the kidney.”

Biological implications: Biomarkers have proven to be useful tools for evaluating Cd exposure and nephrotoxicity in human populations as well in laboratory studies on animals. However, many fundamental issues regarding the selection of markers and definition of their critical levels have yet to be fully resolved. Recent studies have provided hope that new and even more sensitive biomarkers of Cd-induced kidney injury can be developed.

Cadmium Overview

Cd as an Environmental Health Problem

Cd is an important industrial agent and a widespread environmental pollutant that currently ranks seventh on the United States Environmental Protection Agency’s priority list of hazardous substances. Cd is normally found at low concentrations throughout the lithosphere but has become increasingly concentrated in the biosphere through smelting, mining, agriculture, and industrial activities of humans. As a stable, divalent cation, Cd is not biodegradable and persists in the environment for long periods of time. Despite efforts by many agencies to reduce the usage of Cd, Cd pollution continues to be a major public health problem in many regions of the world (for reviews see ATSDR 2008; Jarup and Akesson 2009).

Workers in smelting industries or industries that utilize Cd and its compounds can be exposed in the workplace by inhaling Cd oxide fumes or Cd-contaminated dust. The general population is more likely to be exposed by the ingestion of Cd-contaminated food or water (ATSDR 2008; Jarup and Akesson 2009). In addition, tobacco contains large amounts of Cd and is a major source of exposure among smokers (ATSDR 2008).

Depending on the dose, route, and duration of exposure, Cd can damage various organs including the lung, liver, kidney, and bone (ATSDR 2008; Jarup and Akesson 2009), and it is carcinogenic (ATSDR 2008). With the chronic, low-level patterns of exposure that are common in human populations, the kidney is the primary target of toxicity, where Cd accumulates in the epithelial cells of the proximal tubule, resulting in a generalized reabsorptive dysfunction that is characterized by polyuria, glucosuria, and low-molecular-weight proteinuria (Jarup and Akesson 2009; Prozialeck and Edwards 2010).

The nephrotoxic actions of Cd are, to a great extent, a consequence of the unique toxicokinetics of Cd in the body (for reviews see ATSDR 2008; Prozialeck and Edwards 2010). Following respiratory exposure, Cd is efficiently absorbed from the lung; up to 40–60% of inhaled Cd reaches the systemic circulation. With oral exposure, the absorption of Cd from the gastrointestinal tract is considerably lower (only 5–10%). However, with long-term exposure, even this low level of absorption from the gastrointestinal tract can lead to systemic accumulation of Cd and subsequent toxicities.

Cd is initially transported to the liver where it is taken up by hepatocytes. In the hepatocytes, Cd induces the synthesis of metallothionein, which binds Cd. However, over time, the Cd-metallothionein complex can be released into the bloodstream. Even though the complex is nontoxic to most organs, it can be filtered at the glomerulus and taken up by the epithelial cells of the proximal tubule. Thus, Cd-metallothionein can have the paradoxical effect of facilitating the delivery of Cd from the liver to the kidney. In addition, Cd in plasma binds to a variety of other proteins as well as low-molecular-weight thiols, such as cysteine and glutathione. The Cd that is associated with the low-molecular-weight compounds is filtered at the glomerulus and be taken up by epithelial cells of the proximal tubule. In addition, there is some evidence for the uptake of ionic Cd²⁺ through metal ion transporters in the proximal tubule. Regardless of the form or speciation of Cd that is present in the bloodstream, Cd eventually accumulates in the epithelial cells of the proximal tubule.

Monitoring of Human Populations

The monitoring of human populations for early signs of Cd exposure and toxicity has posed a major challenge (Bernard 2004; Prozialeck and Edwards 2010). Intuitively, it might seem that the most direct way to monitor levels of Cd exposure would be to simply measure blood or urinary levels of Cd. However, this issue is greatly complicated by the tendency of Cd to be sequestered in organs such as liver and kidney. While blood levels of Cd can yield information regarding recent exposures, they often do not provide information regarding the total body burden of Cd or the severity of injury in specific target organs. Likewise, the monitoring and interpretation of data on urinary levels of Cd are not as straightforward as one might expect. With low, or even moderate, levels of exposure, any Cd that is filtered at the glomerulus is almost completely reabsorbed by epithelial cells of the proximal tubule; little or no Cd is excreted in the urine. It is only when the body burden of Cd is fairly large and/or kidney injury begins to appear that urinary excretion of Cd increases significantly. As a result of these limitations in interpreting data on blood and urinary levels of Cd, investigators have utilized various biomarkers to assess levels of Cd exposure and toxicity. As a result of Cds tendency to accumulate in epithelial cells of the proximal tubule, the kidney is, in effect, a sentinel of Cd exposure. Consequently, the most useful biomarkers of Cd exposure and toxicity have been markers of the various effects of Cd in the kidney.

This entry will highlight some of the urinary biomarkers that have proven to be most useful in monitoring Cd exposure and toxicity in human populations and in experimental animals. In addition, novel markers that appear to offer increased sensitivity will be described. All of the biomarkers that will be described represent different events in the pathophysiology of Cd-induced kidney injury. With continuous exposure, the levels of Cd in the proximal tubule cells continue to increase until a critical threshold concentration of about 150–200 μg/g of tissue is reached. The classic view is that as this threshold concentration is approached, the cells undergo oxidative stress that leads to injury and either necrotic or apoptotic cell death (reviewed by Prozialeck and Edwards 2010). The cellular injury causes alterations in proximal tubule function as well as the shedding of injured cells and cytosolic contents into the urine. The shedding of dead or injured cells triggers a repair process in which neighboring noninjured cells dedifferentiate in a process known as epithelial-mesenchymal transformation. The dedifferentiated cells migrate to the denuded area of the basement membrane and replace the injured cells.

Biomarkers of Cd Nephrotoxicity

As noted previously, the traditional urinary biomarkers that have been used to monitor Cd toxicity reflect various steps in this sequence of pathologic events. These urinary markers can be classified into four broad categories: (1) Cd-binding proteins such as metallothionein; (2) low-molecular-weight proteins; (3) proteins and enzymes derived from the brush border, intracellular organelles, or the cytosol of proximal tubule epithelial cells; and (4) proteins expressed in response to cellular injury. Figure 1 shows the typical patterns for the urinary excretion of each of these classes of markers along with a timeline describing specific pathophysiologic events in the proximal tubule. Reference will be made to this figure as each of the classes of markers is described below.

Biomarkers for Cadmium, Fig. 1

Patterns of urinary excretion of representative markers of Cd exposure and toxicity. The graphs show the general patterns for the urinary excretion of various classes of biomarkers of Cd-induced kidney injury. These are not the results of single experiments but rather depict the general results of a wide variety of studies (reviewed in Prozialeck and Edwards 2010 and Shaikh and Smith 1986). The y-axis shows the relative urinary excretion of the various markers (0 to maximum), and the x-axis indicates the relative duration of Cd exposure. The actual time frame for the various events depends on the level of Cd exposure. In humans who are exposed to low levels of Cd, the time frame can involve years of exposure. With higher levels of exposure that are used in experimental animals, the same events can occur in weeks. The bracketed comments at the bottom describe the stages of exposure and proximal tubular injury. The downward arrows (↓) indicate the point at which excretion of each marker usually becomes significantly elevated

Cd and Metallothionein

The urinary excretion of Cd and metallothionein can serve as markers of both Cd exposure and of Cd-induced proximal tubule injury (Bernard 2004; Prozialeck and Edwards 2010; Shaikh and Smith 1986). During early stages of exposure, circulating Cd which is bound to low-molecular-weight molecules such as metallothionein, cysteine, or glutathione in the plasma is filtered at the glomerulus and efficiently taken up by the epithelial cells of the proximal tubule. Only extremely small amounts are excreted in the urine. During this stage of exposure (labeled as “Early exposure” in the timeline in the figure), the presence of Cd or metallothionein in the urine most likely results from the normal turnover and shedding of epithelial cells and is a reflection of the level of Cd exposure and the body burden of Cd (Prozialeck and Edwards 2010). However, over time, the concentration of Cd in the epithelial cells increases to the point that Cd injures the cell and/or disrupts tubular reabsorptive processes. At this stage (labeled “Intermediate exposure” in the figure), the excretion of Cd and metallothionein begins to increase in a linear manner. However, as the intracellular levels of Cd increase further, more of the epithelial cells begin to die and slough off. At this point, the urinary excretion of Cd and metallothionein increases markedly (Prozialeck and Edwards 2010; Shaikh and Smith 1986). This surge in the urinary excretion of Cd and metallothionein coincides with the onset of polyuria and proteinuria. Thus, the early, linear phases of Cd and metallothionein excretion are a reflection of Cd exposure, whereas the later increases in excretion are a reflection of Cd-induced tubular injury.

The World Health Organization, United States Environmental Protection Agency and other agencies have established guidelines for the monitoring of populations for Cd exposure and for Cd exposure limits (ATSDR 2008; Huang 2004; World Health Organization (WHO) 2000). Even though there are variations among the standards from these different agencies, some generalizations can be made. The blood levels of Cd in nonexposed populations are typically less than 0.5 μg/L. Blood levels higher than 1.0 μg/L are generally indicative of Cd exposure; levels higher than 5 μg/L are considered hazardous. Urinary levels of Cd in nonexposed populations are usually below 0.5 μg/g creatinine; values above 1–2 μg/g are indicative of exposure or elevated body burden. The critical urinary Cd concentration that is associated with the onset of renal injury is usually about 2–10 μg/g creatinine, which corresponds to a renal cortical Cd concentration of about 150–200 μg/g tissue (Prozialeck and Edwards 2010). It should be emphasized that these generalizations are derived from consensus-based standards from various regulatory agencies. However, there is evidence that even lower urinary levels of Cd may be associated with adverse effects (for review see Prozialeck and Edwards 2010). With regard to metallothionein, the critical urinary level that is associated with the onset of overt kidney injury is about approximately 300 μg/g creatinine (Chen et al. 2006; Shaikh and Smith 1986).

It should be noted that the majority of studies on which these standards/recommendations are based involved the measurement of total urinary metallothionein; they did not differentiate/identify specific metallothionein isoforms. There are currently four known isoforms of metallothionein. Although the relationships between urinary excretion of Cd and these different metallothionein isoforms have not been established, there are a few reports indicating possible differential effects of Cd on the expression of the different isoforms. The application of using specific isoforms of metallothionein as biomarkers of Cd exposure remains to be fully explored.

Low-Molecular-Weight Proteins

The second category of Cd urinary biomarkers includes a variety of low-molecular-weight proteins such as β₂-microglobulin, Clara cell protein (CC-16), α₁-microglobulin, retinol-binding protein, and vitamin D–binding protein. These low-molecular-weight proteins are present in plasma and are small enough to be easily filtered at the glomerulus. Under normal circumstances, these filtered proteins are efficiently reabsorbed by the proximal tubule and are not excreted to any great extent in the urine (Bernard 2004; Prozialeck and Edwards 2010). However, as Cd accumulates in the proximal tubule, absorption of these proteins becomes impaired, and the proteins begin to appear in the urine. Of these proteins, β₂-microglobulin has been most widely employed as a standard marker for monitoring the early stages of Cd exposure and toxicity in humans. Urinary levels of β₂-microglobulin of 1,000 μg/g creatinine (or greater) are considered to indicate specific renal injury. This level is typically associated with urinary Cd of greater than 5 μg/g creatinine. For population monitoring, a cutoff value of 300 μg/g β₂-microgobulin/creatinine has been used (Huang 2004). However, other investigators have recommended lower critical exposure levels (Uno et al. 2005). Even though β₂-microglobulin has proven to be a very useful biomarker, its lack of stability in acidic urine can be problematic.

As with β₂-microglobulin, increased levels of retinol-binding protein are suggestive of impairment of tubular reabsorptive function. Unlike β₂-microglobulin, however, retinol-binding protein is stable in acidic urine, and no special preservative or alkaline treatment is required (for review see Prozialeck and Edwards 2010).

Proximal Tubule–Derived Enzymes

Some of the most extensively used markers of Cd-induced proximal tubule injury have been enzymes that are expressed in proximal tubule epithelial cells. A variety of enzymes including: N-acetyl-β-d-glucosaminidase (NAG), lactate dehydrogenase (LDH), alkaline phosphatase, and more recently, alpha-glutathione-S-transferase (α-GST) have been studied in this context. The appearance of these enzymes in urine is classically thought to result from the leakage of intracellular contents when necrotic proximal tubule epithelial cells lose their membrane integrity and/or slough off into the urine (Vaidya et al. 2008).

NAG has proven to be especially useful in the monitoring of human populations. NAG is a lysosomal enzyme that exists as multiple isoforms. Both forms A and B are expressed in kidney. However, the B form, which is more abundant in the proximal tubule, is regarded as the more sensitive and reliable marker of Cd-induced injury. However, assays that do not differentiate between the two isoforms can also yield useful results. Several epidemiologic studies have shown that NAG outperforms other traditional markers (Jin et al. 1999; Moriguchi et al. 2009; Noonan et al. 2002; Suwazono et al. 2006). However, it is also noteworthy that it does not perform as well in animal (rat) models of Cd nephrotoxicity (Prozialeck and Edwards 2010). One major advantage of NAG for large-scale population studies is that it is relatively stable in nonpreserved urine.

Recent studies suggest that α-GST may also be especially useful early marker of Cd-induced kidney injury. Garcon et al. (2007) reported that α-GST was a sensitive indicator of kidney injury in workers who had been exposed to Pb and Cd. Results of studies from our laboratories showed that α-GST was a more sensitive marker of kidney injury than NAG in a rat model of Cd-induced kidney injury (Prozialeck and Edwards 2010).

Miscellaneous Markers of Proximal Tubule Dysfunction, Amino Acids, Glucose, Na⁺, K⁺, and Ca²⁺

In addition to its effects on these protein biomarkers, Cd causes a generalized proximal tubule dysfunction that results in an increase in the urinary excretion of amino acids, Na⁺, K⁺, PO ⁻₄ , and Ca²⁺ (Shaikh and Smith 1986). Even though these effects are characteristic of Cd nephrotoxicity, the urinary excretion of these substances can be influenced by many factors other than Cd exposure (Shaikh and Smith 1986). In general, these substances have not been widely applied in the monitoring of human populations for early signs of Cd exposure. One notable exception, however, is Ca²⁺, which has been shown to be a reliable indicator of Cd-induced proximal dysfunction in exposed human subjects (Wu et al. 2001).

Injury Response Proteins

Most of the traditional biomarkers of Cd nephrotoxicity are based on the assumption that Cd causes necrotic or apoptotic cell death of proximal tubule epithelial cells. However, an increasing volume of evidence indicates that the early stages of Cd toxicity involve changes in proximal tubule cell adhesion and function that occur before the onset of cell death (Prozialeck et al. 2009a; Prozialeck and Edwards 2010). In addition, several recent studies indicate that the onset of Cd-induced kidney injury may be preceded by changes in specific markers of metallothionein expression, immune function, and glucose metabolism (for review see Prozialeck and Edwards 2010). Together, these recent findings raise the possibility of identifying more specific and earlier biomarkers of Cd exposure and toxicity. One of the more promising urinary markers that have been described recently is kidney injury molecule-1 (Kim-1).

Kim-1 is a transmembrane protein that is not detectable in normal kidney but is expressed at high levels in the proximal tubule after ischemic or toxic injury (Vaidya et al. 2008). Kim-1 acts as a regulator of cell adhesion and endocytosis in regenerating cells of the injured tubule as they reform a functional epithelial barrier. This process is associated with the proteolytic cleavage of the ectodomain of Kim-1 into the urine. The ectodomain is stable in urine and has been shown to be a sensitive marker of renal injury induced by a variety of agents (Vaidya et al. 2008).

In studies utilizing a rat model of Cd-induced kidney injury, Kim-1 outperformed traditional urinary markers (Prozialeck et al. 2007; Prozialeck et al. 2009a, 2009b). Kim-1 was detected in the urine 4–5 weeks before the onset of proteinuria and 2–5 weeks before the appearance of other markers such as metallothionein and CC-16. Other studies showed that the Cd-induced increase in Kim-1 expression occurred at a time when there was little or no evidence of either necrosis or apoptosis of proximal tubule epithelial cells (Prozialeck et al. 2009b). The fact that Kim-1 can be detected at a time before lethal injury to proximal tubule epithelial cells has occurred may be especially significant. Perhaps, with earlier detection via Kim-1, it may be possible to reverse, or at least more effectively treat, Cd-induced kidney injury. In light of this possibility, studies on the utility of Kim-1 as marker of Cd toxicity in humans are certainly warranted.

Markers of Glomerular Injury

While the proximal tubule is the primary target of Cd-induced kidney injury, there is evidence that Cd can also affect the glomeruli. Changes in classic markers of glomerular dysfunction such as BUN and serum or urinary creatinine are generally not seen during the early or mild stages of Cd-induced kidney injury (Prozialeck and Edwards 2010). However, other investigators have reported associations between Cd exposure and alterations (in glomerular function). For example, Weaver et al. (2011) have recently reported significant increases in creatinine clearance in subjects exposed to Cd and Pb. At present, the relative contributions and relationship of glomerular injury and proximal tubule injury to these finding remain unclear.

Summary and Perspective

Biomarkers have proven to be useful tools for evaluating Cd exposure and nephrotoxicity in human populations as well in laboratory studies on animals. While many fundamental issues regarding the selection of markers and definition of their critical levels have yet to be fully resolved, recent studies have provided hope that new and even more sensitive Cd biomarkers can be developed.

Cross-References

Cadmium Absorption

Cadmium and Metallothionein

Cadmium and Oxidative Stress

Cadmium and Stress Response

Cadmium Exposure, Cellular and Molecular Adaptations

Cadmium, Effect on Transport Across Cell Membranes

Cadmium Transport

Cadmium, Physical and Chemical Properties