**1. Introduction**

Cadmium (Cd) and lead (Pb) are environmental toxicants of significant public health concern due to their widespread environmental pollution and persistence, as well as their known adverse impacts on human health, including an enhanced risk of chronic kidney disease (CKD) and various types of cancer [1–5]. The International Agency for Research on Cancer has established Cd as a human carcinogen [6], while the carcinogenicity of chronic Pb exposure in workplace settings has been observed in two large prospective cohort studies [7,8]. Co-exposure to low levels of environmental Cd and Pb has been reported in large population-based studies in the U.S. [9–12], Canada [13], Taiwan [14], and Korea [15]. About half of the participants in the U.S. National Health and Nutrition Examination Surveys (NHANES) 2007–2012, aged ≥6 years, had blood or urinary levels of Cd and Pb above reported median levels [11].

CKD is the cause of significant human morbidity and mortality. Its high worldwide prevalence and escalating treatment costs make developing strategies to prevent CKD of global importance [16–18]. CKD is characterized by albuminuria and/or a decrease of the glomerular filtration rate (GFR) to 60 mL/min/1.73 m<sup>2</sup> that persists for at least three months [19–21]. In theory, the GFR reflects the number of surviving nephrons × the average GFR per nephron [21]. Accordingly, GFR is considered to best indicate nephron function. In practice, the GFR is estimated from equations, including the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations [19–22], and is reported as an estimated GFR (eGFR). Prospective cohort studies in Sweden have linked low-level Pb exposure to decreases in the GFR, CKD onset, and end-stage kidney disease [4,23]. In addition, cross-sectional studies have implicated low-level environmental exposure to Cd as a risk factor for CKD in Spain [24], Korea [25], and the U.S. [26–29].

Elevated dietary Cd intake has been associated with an increased risk of CKD in China [30]. A marked decrease in the GFR has also been observed in residents of an area with Cd pollution in Thailand [2,31–35], as well as in Cd-exposed workers [36,37]. Likewise, marked GFR decreases have been noted in workers and residents of Pb-smelter communities [38]. However, the variable effect of low-level environmental exposure to Cd and Pb on GFR has caused some controversy. Consequently, governments worldwide have not established the necessary regulations to protect their populations. An inverse association has been seen between the GFR and urinary Cd and/or blood Cd [9,28,39,40]. In the opposite direction, other studies have observed a positive association between GFR and urinary Cd [9,12,41,42]. Inverse associations of blood urea nitrogen (BUN) with blood Cd and Pb were noted in a prospective cohort study of premenopausal U.S. women [43]. However, a cross-sectional analysis of data from adolescents (*n* = 2709, aged 12–19 years) who were enrolled in NHANES 2009–2014 reported positive associations of urinary Cd and Pb with an increased eGFR and BUN in models that incorporated urinary creatinine as a covariate [42]. A common practice of normalizing urinary concentrations of Cd to urinary concentrations of creatinine may have caused these disparate findings [33,35,44,45].

The most frequently reported adverse e ffects of chronic Cd exposure in the general population have included tubular injury and reduced tubular re-absorption, as reflected by elevated urinary N-acetyl-β-D-glucosaminidase (NAG) and β2-microglobulin (β2MG) levels, respectively [5]. However, despite numerous reports, the observed Cd-linked tubular dysfunction has not been considered to be clinically relevant [5]. Thus, the present study aimed to clarify the impact on kidney function of long-term environmental exposure to low levels of Cd and Pb with a focus on the GFR, a reliable clinical measure of kidney function and diagnosis of CKD. We used the CKD-EPI equations to derive the eGFR and excretion of Cd (ECd) as indicators of body burden. The associations of the eGFR with ECd and the excretion of Pb (EPb), age, gender, smoking, body iron stores, and BUN (another indicator of kidney e ffect) were evaluated. For a comparative analysis, ECd and EPb were normalized to both the creatinine clearance (Ccr) and the excretion of creatinine (Ecr).

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

### *2.1. Study Population*

We assembled archived data from participants who were drawn from residential areas in the Bangkapi suburb of Bangkok, Thailand, between 2001 and 2003. Our Thai urban population project was undertaken based on the global food monitoring system database, which indicated dietary Cd and Pb may exceed the tolerable intake levels 7 μg Cd/kg body weight/week and 25 μg Pb/kg body weight/week in some countries, Thailand included [46]. The Institutional Ethical Committee, Chulalongkorn Medical Faculty Hospital, Chulalongkorn University, Bangkok, Thailand, approved

the study protocol (Approval No. 142/2544, 5 October 2001). All participants were apparently healthy and had no history of exposure to Cd or Pb in the workplace. Participants took part in the study after giving informed consent. The health status of participants was assessed by a physical examination and was confirmed by routine urinary and blood chemistry analysis. Smoking, diabetes, hypertension, the regular use of medications, educational level, occupation and family health history were obtained by questionnaires. After the exclusion of participants with incomplete datasets, 392 persons (195 men and 197 women) formed the study cohort.

#### *2.2. Specimen Collection and Analysis*

Blood samples were collected within 1 h after drinking 300 mL of water following an overnight fast. Urine samples were collected within 3 h of blood sampling. Urine and blood samples were transported on ice to the Department of Laboratory Medicine, Chulalongkorn University Hospital, where plasma samples were prepared for routine chemistry by using an automated system. The assay for plasma and urinary creatinine concentrations was based on the Ja ffe reaction, while the plasma ferritin assay was based on an electrochemiluminescence immunoassay (Boehringer Mannheim Elecsys 1010, Roche Diagnostics GmbH, Mannheim, Germany). Aliquots of urine, with 5 mL per aliquot, were shipped on dry ice and kept frozen throughout shipment period. They were delivered to the National Research Centre for Environmental Toxicology, Australia, where they were stored at −80 ◦C for later analysis. Urinary concentrations of Cd and Pb were determined with inductively-coupled plasma/mass spectrometry (ICP/MS, Agilent 7500, Agilent Technologies, Santa Clara, CA, USA), which had been calibrated with multi-element standards (EM Science, EM Industries, Inc., NJ, USA). Quality assurance and control were conducted with simultaneous analyses of samples of the reference urine Lyphochek ® (Bio-Rad, Gladesville, New South Wales, Australia), which contained low- and high-range Cd and Pb levels. A coe fficient of variation value of 2.5% was obtained for Cd and Pb in the reference urine. The low limit of detection (LOD) was 0.05 μg/<sup>L</sup> for urinary Cd and 0.03 μg/<sup>L</sup> for urinary Pb. The urine samples containing Cd and Pb levels below the LOD were assigned as the LOD divided by the square root of 2. Fifty-eight subjects (14.8%) had urinary Cd levels below the LOD, while 26 subjects (6.6%) had urinary Pb levels below the LOD.

#### *2.3. Estimation of Excretion Rates*

The procedures for the simultaneous collection of blood and urine samples enabled the normalization of the excretion rates of metals to creatinine clearance (Ccr) by using the following equation: Ex/Ccr = [x]u[cr]p/[cr]u, where Ex/Ccr = excretion of x per volume of filtrate; [x]u = urine concentration of x (mass/volume); [cr]p = plasma creatinine concentration (mg/dL); and [cr]u = urine creatinine concentration (mg/dL) [33,35,47]. The normalization of Ex to Ccr circumvents the e ffect of muscle mass on Ex/Ecr and [x]u/[cr]u while nullifying urine volume (Vu) as a confounder on concentration ([x]u).

As is typical, the excretion of Cd (ECd) was normalized to the excretion of creatinine (Ecr) as [Cd]u/[cr]u, where [Cd]u = urine concentration of Cd (μg/L), and [cr]u = urine creatinine concentration (mg/dL). The ratio [Cd]u/[cr]u was expressed as μg/g of creatinine. This allows for the correction of the urine flow rate (Vu) on concentration ([x]u). However, it introduces another confounder or bias given that Ecr is a ffected by muscle mass and many other factors unrelated to nephron function [48].

#### *2.4. Estimated Glomerular Filtration Rates*

The eGFR was calculated by using the CKD-EPI equations [19,21]. The male eGFR = 141 × [serum creatinine / 0.9]<sup>Y</sup> × 0.993age, where Y = −0.411 if serum creatinine ≤ 0.9 mg/dL, Y = −1.209 if serum creatinine > 0.9 mg/dL. The female eGFR = 144 × [serum creatinine / 0.7]<sup>Y</sup> × 0.993age, where Y = −0.329 if serum creatinine ≤ 0.7 mg/dL, Y = −1.209 if serum creatinine > 0.7 mg/dL. CKD is defined as an eGFR <60 mL/min/1.73 m<sup>2</sup> for three months or more [19,22]. CKD stages 1, 2, 3, 4, and 5 corresponded to an eGFR of 90–119, 60–89, 30–59, 15–29, and <15 mL/min/1.73 m2, respectively [19,21].
