*2.1. Study Subjects*

The Institutional Ethical Committees of Chulalongkorn University, Chiang Mai University and the Mae Sot Hospital approved the study protocol (Approval No. 142/2544, 5 October 2001) [33]. All participants gave informed consent before participation. Subjects were recruited from urban communities in Bangkok in 2001/2002 and from subsistence farming areas in Mae Sot District, Tak Province, Thailand in 2004/2005 [33]. They had lived at their current addresses for at least 30 years. Exclusion criteria were pregnancy, breast-feeding, a history of metal work, and a hospital record or physician's diagnosis of an advanced chronic disease. Because occupational exposure was an exclusion criterion, we presumed that all participants had acquired Cd from the environment.

Cd exposure was low in Bangkok and moderate or high in Mae Sot [33,34]. Determination of exposure was based on reported levels of Cd in rice grains grown in the Cd-a ffected areas of the Mae Sot District [35,36]. After exclusion of subjects with incomplete datasets, we studied 172, 310, and 222 persons from the low, moderate, and high exposure areas, respectively.

#### *2.2. Collection of Biological Specimens and Laboratory Analyses*

Second morning-void urine samples were collected after an overnight fast. Within three hours after urine sampling, specimens of whole blood were obtained and serum samples were prepared. Aliquots

of urine, whole blood, and serum were transported on ice from a mobile clinic to a laboratory and stored at −20 ◦C or −80 ◦C for later analysis. The assay for urine and serum creatinine concentrations ([cr]u, [cr]p]) was based on the Jaffe reaction. The urine NAG assay was based on colorimetry (NAG test kit, Shionogi Pharmaceuticals, Sapporo, Japan). The urine β2MG assay was based on the latex immunoagglutination method (LX test, Eiken 2MGII; Eiken and Shionogi Co., Tokyo, Japan). When the urine concentration of β2MG ([β2MG]u) was below the limit of detection (LOD), 0.5 μg/L, the value assigned to [β2MG]u was LOD/(square root of 2).

For the Bangkok group, [Cd]u was determined by inductively-coupled plasma mass spectrometry (ICP/MS, Agilent 7500, Agilent Technologies), because it had the high sensitivity required to measure Cd concentrations below the detectable limit of atomic absorption spectrophotometry. Multi-element standards (EM Science, EM Industries, Inc., Newark, NJ, USA) were used to calibrate Cd analyses. The accuracy and precision of those analyses were evaluated with reference urine (Lyphochek®, Bio-Rad, Sydney, Australia). When [Cd]u was less than the detection limit of 0.05 μg/L, the concentration assigned was the detection limit divided by the square root of 2.

For the Mae Sot groups, [Cd]u was determined by atomic absorption spectrophotometry (Shimadzu Model AA-6300, Kyoto, Japan). Urine standard reference material No. 2670 (National Institute of Standards, Washington, DC, USA) was used for quality assurance and control purposes. None of the urine samples from the Mae Sot groups were found to have [Cd]u below the detection limit.

#### *2.3. Estimated Glomerular Filtration Rate (eGFR)*

The glomerular filtration rate was estimated with equations from the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) [37,38]. CKD stages 1, 2, 3, 4, and 5 corresponded to eGFR of 90–119, 60–89, 30–59, 15–29, and <15 mL/min/1.73 m2, respectively. For dichotomous comparisons, CKD was defined as eGFR < 60 mL/min/1.73 m2.

#### *2.4. Normalization of Excretion Rates to Creatinine Clearance (Ccr)*

Excretion rates of Cd, NAG, and β2MG were normalized to Ccr to yield the ratios ECd/Ccr, ENAG/Ccr, and <sup>E</sup>β2MG/Ccr in units of mass (amount excreted) per volume of filtrate. For *x* = Cd, NAG, or β2MG, E*x*/Ccr was calculated as [*x*]u[cr]p/[cr]u ([39]; Supplementary Materials).
