The prevalence of CKDu found in this study (females 16.9%, males 12.9%) was higher than that reported previously (2% to 3%) . Although the prevalence in females was higher, more severe stages of CKDu were seen more often in males. The reason for this discrepancy is not clear. Factors such as low iron stores in females in lower socioeconomic groups may have an influence on the excretion of heavy metals and oxidative stress on the kidney. This would make the kidneys more vulnerable to CKDu, resulting in a higher prevalence in females. On the other hand, male sex has been reported to be a risk factor for progression to end-stage renal disease , and this may partly explain the occurrence of more severe stages of CKDu in men.
Previous studies have reported a family history of chronic kidney disease, ayurvedic treatment, and history of snake bite as significant predictors for CKDu [10, 12, 13]. In the present study, older age, being female and being a chena cultivation farmer increased the risk of CKDu. Family history was positive in one-fifth of those with CKDu, and a history of snake bite was one of the exclusion criteria. Long-term use of herbal medicines or analgesics was reported in only a very small percentage of those with CKDu. Fanconi syndrome and other hereditary kidney diseases have not been reported in communities in this region.
Previous studies have reported divergent information on the role of cadmium in the causation of CKDu [14, 15, 19, 20]. In the present study, individuals with CKDu excreted significantly higher levels of cadmium compared to those in the control group, in both the endemic and non-endemic areas. Controls in the endemic area compared to those in the non-endemic area also had significantly higher urinary excretion of cadmium. The sensitivity and specificity for urine cadmium were 80% and 53.6% respectively (AUC = 0.682, cut-off value ≥0.23 μg/g). There was a dose–effect relationship between the concentration of cadmium in urine and the stage of CKDu. A significantly higher cadmium concentration was also seen in the nails of those with CKDu compared to controls from the endemic area. Cadmium is a known nephrotoxin and urinary excretion of cadmium is considered to be a reliable indicator of cumulative long-term exposure to cadmium . The mean urine concentration of cadmium in CKDu cases was above the levels demonstrated in recent studies to cause oxidative stress and decreased glomerular filtration rate and creatinine clearance [28–33]. The results of this study indicate that cadmium exposure is a risk factor for CKDu.
The mean urine concentration of arsenic in CKDu cases was also above levels known to cause oxidative injury to the kidney . In CKDu cases and controls from the endemic area, concentrations of arsenic in urine and in fingernails were higher than those reported in people living in low-exposure environments [34, 35]. Urine is a major pathway for excretion of arsenic from the human body, so urine levels reflect exposure. In some studies, markers of oxidative stress have been demonstrated at urine arsenic concentrations as low as 3.95 μg/g . The level of total arsenic in urine is associated with chronic kidney disease in a dose–response relationship, especially when the level is greater than 20.74 μg/g . These findings support the contention that chronic exposure to low levels of cadmium may be a causative factor for CKDu in Sri Lanka. Co-exposure to cadmium and arsenic is known to produce additive effects on the kidney that are more pronounced than exposure to either metal alone [37, 38].
Selenium has been shown to protect the kidney from oxidative stress . A selenium concentration of 80–95 μg/l is needed to maximise the activity of the antioxidant enzyme glutathione peroxidase and selenoproteins in plasma [40, 41]. In this context, it is significant that serum selenium was below 80 μg/l in 38% and below 90 μg/l in 63% of individuals with CKDu. Low selenium levels may have been a contributory factor increasing the vulnerability of the kidneys to oxidative damage caused by heavy metals and metalloids.
The association of raised serum strontium levels with raised serum cadmium levels has been reported previously . Strontium levels were not analysed in food or water. The most likely explanation is an alteration of strontium handling and excretion, owing to the effect of cadmium on renal tubular function.
Cadmium levels have previously been reported to be high in water sources in the domestic environment of people with CKDu, and 10–20 times the maximum stipulated level have been found in reservoirs in the endemic area . The results of this study did not show this to be the case. On the contrary, the cadmium content in all water samples analysed was within normal limits, except in one sample from a reservoir that had a borderline cadmium level (3.45 μg/l).
Drinking water is a major pathway for entry of inorganic arsenic into the human body. The arsenic content in 99% of water samples was below the WHO reference value of 10 μg/l . However, it has recently been suggested that the concentration of arsenic in drinking water should be no more than 5 μg/l .
CKDu occurs in areas where groundwater is the main source of drinking water. Groundwater in this region is known to have a high content of fluoride and calcium. People living in the region for generations have used groundwater for drinking without ill effects. However, hardness of water, the high fluoride content, poor access to drinking water and inadequate intake of water in a warm climate may influence the body burden and/or the excretion of heavy metals and oxidative damage to the kidneys caused by heavy metals.
The maximum level of cadmium for vegetables permitted by the Codex Alimentarius is 0.2 mg/kg [22, 23] and the level permitted by the Commission of the European Communities is 0.05 mg/kg . The maximum levels in certain vegetables grown in the endemic area exceeded these safety levels. The maximum concentration of cadmium in fish (0.06 μg/g) also exceeded the European maximum limit of 0.05 mg/kg stipulated for certain types of fish . The maximum level of lead in vegetables permitted by the Commission of the European Communities is 0.10 mg/kg . The maximum level of lead in vegetables in the endemic area (0.476 mg/kg) exceeded this cut-off value. Levels of cadmium and lead in vegetables and cadmium in freshwater fish from the endemic area are above the maximum levels stipulated by certain Food Safety Authorities [22–24, 44].
A provisional tolerable weekly intake (PTWI) for cadmium of 7 μg/kg body weight was established by the Joint Food and Agriculture Organization of the United Nations (FAO)/WHO Expert Committee on Food Additives (JECFA) . In 2011, the JECFA revised the PTWI for cadmium to 5.8 μg/kg body weight . More recently, the PTWI for cadmium has been lowered to 2.52 μg cadmium/kg body weight, in order to ensure a high level of protection of all consumers, including exposed and vulnerable subgroups of the population . Since the cadmium content of certain food items in the endemic area is above stipulated levels, the total weekly intake of cadmium in people living in the endemic area could exceed these safe limits, with detrimental effects on the kidneys, particularly in vulnerable people and those with predisposing factors.
Reported mean dietary exposure to inorganic arsenic in the United States of America (USA) and various European and Asian countries ranges from 0.1 to 3.0 μg/kg body weight per day . Recently, the PTWI for arsenic (0.015 mg/kg body weight per week) was withdrawn and environmental authorities are in the process of collecting more data for exposure assessment . The current recommendation is that every effort should be made to keep concentrations of arsenic as low as reasonably possible. The PTWI for lead is set at 0.025 mg/kg body weight per week .
Previous studies have reported high levels of cadmium in fertilizer (mean 47 μg/g) . The maximum cadmium, lead and arsenic concentrations in phosphate fertilizer from the endemic area in the present study were 30.8 μg/g, 823.4 μg/g and 0.19 μg/g respectively. The maximum acceptable levels for cadmium, lead and arsenic, in phosphate fertilizer product, at 1% of the nutrient level, are 4 μg/g, 20 μg/g and 2 μg/g, respectively .
The mean concentration of cadmium in soil from the endemic area was 0.4 μg/g. Surveys of agricultural soils in the USA and Sweden have reported lower soil cadmium levels (0.265 mg/kg and 0.23 mg/kg respectively) [48, 49]. The concentration of cadmium, arsenic and lead in soil, and their impact on body burden and excretion, is known to be influenced by many environmental factors such as the pH of soil, buffering capacity, content of organic matter and water quality, among others [50–52]. Cadmium accumulation by plants, for example, is influenced by the reactive soil cadmium content and pH. It is decreased by high cation exchange capacity of the soil and increased by higher soil temperature [49–52]. The hardness and high content of fluoride in water in the endemic area may also influence the dynamics of cadmium in soil, absorption by plants  and excretion from the kidney.
Certain pesticide residues were above reference levels in 31.6% of CKDu cases. Residues are demonstrative of the extent of the environmental distribution of pesticides and certain pesticides are known to be nephrotoxic [4, 5, 53]. Simultaneous exposure of people to heavy metals and nephrotoxic pesticides may be a contributory factor in the pathogenesis and progression of CKDu.
Compared to previous studies conducted on CKDu, the present study has several strengths: (i) use of a consistent case definition for CKDu; (ii) analysis of a range of biological samples from individuals with CKDu; (iii) comparison of control groups within and outside the endemic area; and (iv) use of sensitive analytical techniques. Studies conducted hitherto to determine the prevalence and aetiology of CKDu [10, 12, 13, 16, 18, 20] have relied on dipstick urinalysis to identify kidney disease. The present study is also the first in which heavy metals, metalloids and other elements in environmental and biological samples and pesticide residues in urine have been analysed.
There are several limitations in the study. Other kidney disease such as HIV nephropathy could fulfil the case-definition criteria used for CKDu. As HIV is not prevalent in Sri Lanka, it was not excluded through blood tests. The presence of glomerulonephritis was not excluded by biopsy but was based on past medical records and diagnosis cards. The sensitivity and specificity of the case definition relative to biopsy-proven CKDu is also not known. Stage 1 CKDu is defined by persistent microalbuminuria and may overestimate the prevalence of CKDu. The case definition required albuminuria. As a result, people with CKDu who have a low eGFR and no albuminuria were excluded from the study. In addition, the CKD-EPI equation used to estimate eGFR  has not been validated in people from South Asia. It is not known whether the albuminuria of CKDu responds to treatment for high blood pressure. If it does, an individual could then be excluded based on their ACR, despite having the disease.
CKDu has been reported in other populations as well [54–57]. Lessons learnt from other countries demonstrate that sound public health policies to ensure access to safe drinking water; regulatory control to ensure appropriate use of agrochemicals including fertilizer; hazardous waste remediation; regulatory control to prevent pollution of the environment from discarded batteries containing heavy metals; tobacco control; and reduction of air pollution can reduce exposure to heavy metals [58, 59]. Based on the findings of this study, the Government and the Ministry of Health of Sri Lanka have already initiated multisectoral collaborative action with the Ministries of Agriculture, Irrigation, Scientific Affairs and Social Services, to mitigate the exposure of people to environmental nephrotoxic substances. Steps are being taken to strengthen the water supply scheme in the endemic area as well as the regulations related to procurement and distribution of fertilizers and pesticides. Further studies are ongoing to investigate the contributory role of infections in the pathogenesis of CKDu.