Urine samples were collected from patients and controls in Padavi-Sripura and from Colombo. Padavi-Sripura is a divisional secretariat (sub-division) of Trincomalee district of the Eastern province bordering to NCP with a population of 11,858. This area has been identified as having one of the highest prevalence of SAN. Cases (group 1; N = 10) were recruited from the patients who attended a medical clinic at Padavi-Sripura divisional hospital in Sri Lanka. These patients were diagnosed by a qualified Nephrologist according to the Ministry of health (Sri Lanka) criteria for the diagnosis of CKDu (for further details on diagnosis see reference 24). Stated briefly, criteria for diagnosis are, no history of diabetes mellitus, hypertension, urological disease or glomerulonephritis, history of snake bite, blood pressure < 160/100 mmHg untreated or < 140/90 mmHg when treated with two or less anti hypertensive agents and with a HbA1c < 6.5. Ultra sound evidence of contracted kidneys and serum creatinine > 1.3 mg/dL was used to diagnose CKDu.

There were two groups of controls. The first group of control participants (N = 10) was selected from the same Padavi-Sripura area as the cases (group 2 - endemic controls). Their kidney functions were normal. They had negative albuminuria (albumin creatinine ratio less than 30 mg/g) and serum creatinine less than 1.3 mg/dL. The participants in this endemic control group shared the same physical environment as the patients. The other group of control participants (N = 10) was recruited from Malabe, a semi-urban area located in the Colombo district about 20 km away from city of Colombo (group 3 - non endemic controls). Participants’ in-group 1 and 2 are farmers but the participants in-group 3 were not. Both groups of controls did not have any history of kidney disease any other chronic disease.

Non-fasting spot urine samples were collected in the morning from all the cases and controls into empty polypropylene bottles. All samples were immediately sealed and stored at 4⁰ C and then transferred to -20⁰ C within 6 hours of collection. Sample were shipped to Institute for Integrated Research in Materials, Environments and Society (IIRMES) lab, California State University, Long Beach (CSULB), USA and stored at -40⁰ C until analysis. Urine creatinine level was measured by a modified Jaffe test. (Aluminum (Al), Antimony (Sb), Arsenic (As), Barium (Ba), Cadmium (Cd), Chromium (Cr), Cobalt (Co), Copper (Cu), Iron (Fe), Lead (Pb), Manganese (Mn), Molybdenum (Mo), Nickel (Ni), Selenium (Se), Strontium (Sr), Tin (Sn), Titanium (Ti), Vanadium (V) and Zinc (Zn) levels were measured in the urine using an Inductively Coupled Plasma Mass Spectrometer (ICP-MS; HP 4500, Agilent Technologies, Palo Alto, CA) equipped with a quadrupole analyzer and octopole collision/reaction cell.

Heavy metal and glyphosate concentrations of urine samples of the SAN patients (group 1) were compared with two control groups, one from the endemic area (group 2) and the other from a non-endemic area (group 3) using Kruskal-Wallis test. Non-parametric tests were used for the analysis because of the non-normality of data and small sample size. Mann-Whitney U test was used to analyze the difference between each group. Because of multiple testing, alpha level of 0.05 was divided by three for a more stringent alpha level (Bonferroni adjustment). Effect size was calculated by dividing the Z values by square root of the total number of participants in the two groups [18]. All the statistical analysis was performed using SPSS 22.0.

Except for Ba, Mo, Sn, and Ti creatinine unadjusted median value of all other heavy metals and glyphosate in the urine is higher in patients (group 1) and endemic controls (group 2) when compared to the controls in the non-endemic area (group 3). Creatinine unadjusted heavy metals and glyphosate concentrations were lower in cases (group 1) than in controls from endemic Padavi-Sripura area (group 2).

Except for Sn creatinine adjusted median value of all other heavy metals and glyphosate in the urine is higher in patients (group 1) and endemic controls (group 2) when compared to the controls in the non-endemic area (group 3) (Table 2). When comparing group 1 and 2 except for Ba, Fe, Sr and Ti adjusted mean urinary concentrations of all other heavy metals and glyphosate is higher among patients with SAN (group 1).

Creatinine adjusted urinary heavy metal excretion (with the exception of Sn) is higher in subjects living in the endemic area as compared to the inhabitants of the non-endemic region (group 3 from Colombo district). In endemic areas urine heavy metal excretion (except for Ba, Fe, Sr and Ti) is higher among patients (group 1) than in controls (group 2). Apart from a few exceptions there seems to be graded excretion of heavy metals in the urine with highest values recorded among patients in the endemic area and lowest among non-endemic controls (group 3) and the values among endemic controls (group 2) placed in between. The glyphosate excretion is very high in endemic controls (39 times more) and patients (46 times more) compared to non-endemic controls. The reason for high excretion of Ti and V by patients and participants from both control groups is another intriguing finding.

We have demonstrated that urinary excretion of heavy metals and glyphosate is markedly high in people living in endemic areas when compared to those living in non-endemic areas. These data supports toxicological origin of SAN that is confined to specific geographical areas [13]. Previous research has given clues about the origin of these heavy metals and glyphosate. All of the implicated heavy metals are present in the fertilizer samples (Triple super phosphate) commonly used in the paddy cultivation in SAN endemic area [19, 20]. Further, we have shown that the total As content (range 52.4-540.4 μg/Kg) of rice cultivated in the endemic region (Padavi-Sripura) is high [21]. The amount of Cd (range 5-800 μg/Kg) and Pb (range 3-93 μg/Kg) in market samples of rice obtained from Sri Lanka is also high [22, 23]. Analysis of Cd, As and Pb content in tobacco and vegetables grown in the endemic area has shown to be high [15]. A case-control study showed that farmers from the endemic area, who spray glyphosate, drink well water, and had a history of drinking from an abandoned well are at a higher risk of developing SAN [24]. In addition, there was a significantly higher amount of glyphosate in the well water from the endemic region when compared to the non-endemic area (Colombo district) [24]. Rice, vegetables, tobacco, and drinking water are possible sources of ingestion of heavy metals and glyphosate by the inhabitants living in the endemic area. In addition, pesticides or residues may be absorbed through skin and by inhalation [25].

The limited attention directed to the synergistic effect of multiple metals or chemical compounds is one of the main drawbacks of the previous toxicological studies carried out on SAN epidemic. Low concentrations of heavy metals in biological samples of the patients with SAN have prompted investigators to overlook the effect of these elements without taking into account their synergistic effects. Possible role of As and Cd in epidemic of SAN in Sri Lanka was the subject of several studies [8, 14, 15]. Such studies have revealed high As and Cd levels in hair and nail samples of SAN patients with correspondingly lower levels of excretion of the same metals in urine. Once the renal functions are compromised, SAN patients lose their ability to excrete the heavy metals resulting in their accumulation in the body tissues over the time and reduced excretion in urine. However, it should be noted that the total biochemical, clinical, histo-pathological picture of SAN does not match coherently with classical As or Cd poisoning.

All heavy metals excessively present in the urine samples of SAN patients cause oxidative damage to kidneys in animal studies. Nephrotoxicity of Cd, As, Cr, Ni, Pb and V on humans and animals have previously been discussed [26–32]. There are few isolated animal studies describing exposure to combined heavy metals [33], however we found no studies related to the concurrent exposure to heavy metals and pesticides. Studies have shown Cr and V undergo redox cycling, while Cd, Pb and Ni deplete glutathione and protein-bound sulfhydryl groups, resulting in the production of reactive oxygen species as superoxide ion, hydrogen peroxide, and hydroxyl radicals. Consequently, enhanced lipid peroxidation, DNA damage, and altered calcium and sulfhydryl homeostasis could occur [34].

There are no comprehensive studies reporting nephrotoxic properties of glyphosate in humans. However, several animal studies provide some evidence for nephrotoxic properties of glyphosate [35–40]. At the same time, glyphosate is capable of inducing oxidative stress in animals at low dose exposures [41]. Kidney is particularly susceptible to oxidative stress and it is one of the leading pathological mechanisms contributing totubulo-interstitial nephritis. The predominant histopathological presentation of SAN is a tubulo-interstitial nephritis [9].

Toxicity of individual metals in isolation was the main consideration when calculating renal threshold levels for the heavy metals [18]. These values do not hold true for multi-elemental exposure or when presented to the human tissue in combination with glyphosate. Animal studies have already shown that nephrotoxicity of multi-metalic compositions are more toxic than the additive effects of its components per se [42]. Hence, assessments of individual toxicity as well as synergistic effects are needed in order to understand the holistic picture of nephrotoxicity caused by a mixture of heavy metals. Furthermore, the potential additive effects of a simultaneous exposure to more than one pesticide compound was shown earlier [43]. The WHO study group demonstrated the presence of excessive amount of multiple pesticides and pesticide residues in the urine samples of individuals from SAN in endemic area [15]. Synergism of toxic heavy metals and pesticides may cause damage to microstructure of the filtering system of the kidney that ultimately results in kidney damage and low glomerular filtration rate. The possibility of formation and/or presence of toxic organo-metallic structures should also be taken in to consideration. Many toxic metals including As, Cd, Pb, Cr, Sn are capable of forming covalent bonds with carbon resulting in organo-metalic compounds [44]. Such a transformation by methylation or alkylation influences their mobility, accumulation and toxicity [45].

Another study done in Medawachchiya a neighboring area to Padavi-Sripura showed consumption of less than three liters of water per day (P < 0.04) is a risk factor for SAN [46]. Cyclical dehydration may leads to reabsorption of heavy metals and pesticide residues in the renal tubules. In addition, the dehydration will promote thirst that is quenched by drinking well water that is often contaminated with heavy metals and pesticides [24]. In a mouse model, it was shown that recurrent volume depletion caused by repeated heat stress, water deprivation could induce proximal tubular injury, early renal fibrosis and increase in serum creatinine through hyperactivation of the aldose reductase pathway in the renal cortex [47]. Almost all the people in the endemic area who participated to the study excrete heavy metals and glyphosate but only some develop disease. Apart from dehydration, other causes such as hereditary factors, infections may also contribute to the development of SAN. However, heat stress and cyclical dehydration could not be considered as the major factor responsible for SAN as similar kidney disease epidemic or even isolated outbreaks were not reported from Northern Province of Sri Lanka, a cultivating area adjacent to the SAN endemic area in spite of having similar or even harsher climatic factors. Northern Province of Sri Lanka had not been using or minimally using imported agrochemicals due to prohibition imposed by the government due to the potential of these agrochemicals being used in the production of Improvised Explosive Devices by the terrorist groups [13].

A major limitation of this study is the small sample size (10 each in three groups) due to logistical and financial constraints. We also did not measure serum creatinine in non-endemic controls (group 3).

Agrochemicals and heavy metals ingested and absorbed from various routes can accumulate in the body of inhabitants living in the endemic area and are excessively excreted in their urine. This finding is in favor of the toxicological origin of SAN in specific geographical areas. Research has failed to localize a single nephrotoxin responsible for SAN. Multiple heavy metals and agrochemicals such as glyphosate and their residues acting synergistically may play a role in the pathogenesis of SAN. Hypothesized pathophysiological mechanisms leading to renal damage by these toxicants were previously described [11]. Currently, this epidemic of kidney disease has escalated to become the most important health issue in the paddy farming areas in the dry zone of Sri Lanka. More research is needed to investigate the mechanism of renal damage caused by chronic low dose exposure to multiple nephrotoxins.