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Chronic kidney disease of uncertain aetiology: prevalence and causative factors in a developing country

Chronic kidney disease of uncertain aetiology: prevalence and causative factors in a developing... Background: This study describes chronic kidney disease of uncertain aetiology (CKDu), which cannot be attributed to diabetes, hypertension or other known aetiologies, that has emerged in the North Central region of Sri Lanka. Methods: A cross-sectional study was conducted, to determine the prevalence of and risk factors for CKDu. Arsenic, cadmium, lead, selenium, pesticides and other elements were analysed in biological samples from individuals with CKDu and compared with age- and sex-matched controls in the endemic and non-endemic areas. Food, water, soil and agrochemicals from both areas were analysed for heavy metals. Results: The age-standardised prevalence of CKDu was 12.9% (95% confidence interval [CI] = 11.5% to 14.4%) in males and 16.9% (95% CI = 15.5% to 18.3%) in females. Severe stages of CKDu were more frequent in males (stage 3: males versus females = 23.2% versus 7.4%; stage 4: males versus females = 22.0% versus 7.3%; P < 0.001). The risk was increased in individuals aged >39 years and those who farmed (chena cultivation) (OR [odds ratio] = 1.926, 95% CI = 1.561 to 2.376 and OR = 1.195, 95% CI = 1.007 to 1.418 respectively, P < 0.05). The risk was reduced in individuals who were male or who engaged in paddy cultivation (OR = 0.745, 95% CI = 0.562 to 0.988 and OR = 0.732, 95% CI = 0.542 to 0.988 respectively, P < 0.05). The mean concentration of cadmium in urine was significantly higher in those with CKDu (1.039 μg/g) compared with controls in the endemic and non-endemic areas (0.646 μg/g, P < 0.001 and 0.345 μg/g, P < 0.05) respectively. Urine cadmium sensitivity and specificity were 70% and 68.3% respectively (area under the receiver operating characteristic curve = 0.682, 95% CI = 0.61 to 0.75, cut-off value ≥0.397 μg/g). A significant dose–effect relationship was seen between urine cadmium concentration and CKDu stage (P< 0.05). Urine cadmium and arsenic concentrations in individuals with CKDu were at levels known to cause kidney damage. Food items from the endemic area contained cadmium and lead above reference levels. Serum selenium was <90 μg/l in 63% of those with CKDu and pesticides residues were above reference levels in 31.6% of those with CKDu. Conclusions: These results indicate chronic exposure of people in the endemic area to low levels of cadmium through the food chain and also to pesticides. Significantly higher urinary excretion of cadmium in individuals with CKDu, and the dose–effect relationship between urine cadmium concentration and CKDu stages suggest that cadmium exposure is a risk factor for the pathogensis of CKDu. Deficiency of selenium and genetic susceptibility seen in individuals with CKDu suggest that they may be predisposing factors for the development of CKDu. Keywords: Arsenic, Cadmium, Chronic kidney disease, Kidney disease of uncertain aetiology, Heavy metals, Lead, Pesticides * Correspondence: mendiss@who.int Management of Noncommunicable Diseases, World Health Organization, Geneva, Switzerland Full list of author information is available at the end of the article © 2013 Jayatilake et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 2 of 13 http://www.biomedcentral.com/1471-2369/14/180 Background The objectives of this study were to: determine the The study reported here describes an apparently new prevalence of and identify the risk factors for CKDu; form of chronic kidney disease, which cannot be attrib- compare CKDu cases and controls with regard to expos- uted to diabetes, hypertension or other known aetiol- ure to heavy metals/metalloids and pesticides; and ana- ogies, that has emerged in the North Central region of lyse food, water, soil, fertilizers and weedicides for heavy Sri Lanka [1]. Chronic kidney disease of uncertain aeti- metals, in order to determine whether the levels are ology (CKDu) is slowly progressive, probably starting in above stipulated reference values. the second decade of life, and asymptomatic until ad- A population prevalence study was conducted in three vanced. Peripheral oedema and hypertension are late districts in the endemic area over the period 2010–2012 features. The main histopathological features include (Figures 1 and 2). Arsenic, cadmium, lead and other tubular atrophy, interstitial mononuclear cell infiltration metals, elements and pesticide residues that are potential and interstitial fibrosis [2]. These histological features nephrotoxins [6-9,21-24], were analysed in biological suggest that nephrotoxins play a key role in the aeti- samples. The results from individuals with CKDu were ology of CKDu. compared with those from controls in the endemic area Potential nephrotoxins are widely distributed in the and a non-endemic area (Hambantota where CKDu has environment. Exposure to environmental and other not been reported). The demographic characteristics of nephrotoxins, such as herbal medicines and analgesics, the three groups are shown in Table 1. All individuals are known to play a role in the aetiology of chronic with CKDu (n = 733) had a blood pressure below 160/ kidney disease. Defining their exact role in the aetiology 100 mmHg; 6.8% of these individuals were on treatment of kidney disease is a challenge. Environmental toxins for high blood pressure and had a blood pressure below implicated in kidney damage include heavy metals, such <140/90 mmHg. Three-quarters of those who were on as arsenic, cadmium, lead and uranium; mycotoxins pro- treatment were on angiotensin-converting enzyme inhib- duced by fungi in improperly stored foods; air pollut- itors. Others were on calcium channel blockers, beta- ants, such as tobacco smoke; and pesticides, such as chlorpyriphos, diazinon and propanil [3-9]. Over the last 8 years, several studies have been carried out to determine the prevalence, nature and causes of CKDu in Sri Lanka [10-20]. These studies include hospital-based reviews of case series, in which high-risk areas in North Central Province were identified. The hospital studies gave rise to population-based surveys, which showed that the populations affected by CKDu are scattered in the North Central region of the country [14]. Some studies have reported the point prevalence of CKDu to be about 2–3% among those above 18 years of age [20]. Case–control and cross-sectional studies have provided some insight into associations with the condi- tion. Specific evaluations of exposure to organophosphate and mycotoxins have been conducted. In a cross-sectional study, there was evidence of greater inhibition of acetyl cholinesterase among patients with chronic renal dysfunc- tion in areas of high prevalence of CKDu [17]. Ochratoxin, a naturally occurring mycotoxin with nephrotoxic proper- ties, was not found to be a contaminant of food in the region [13,16]. Some studies have shown high levels of en- vironmental cadmium, lead, aluminium and fluoride in re- gions with high rates of the condition [14,15,17,19]. Recognising the gravity of the public health threat caused by CKDu, in 2010, the Ministry of Health Sri Lanka, in col- laboration with the World Health Organization (WHO), launched a national research project with the aim of Figure 1 Map of Sri Lanka, showing the location of Anuradhapura, investigating the prevalence and aetiology of CKDu in Polonnaruwa and Badulla districts, in the endemic area, and Sri Lanka, with a view to developing appropriate preven- Hambantota district, in the non-endemic area. tive strategies. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 3 of 13 http://www.biomedcentral.com/1471-2369/14/180 Number invited to participate in the study from 220 randomly selected households n=6698 Response rate 74% (n=4957) ACR assay 30 mg/g on two occasions No diabetes, other kidney and satisfy other criteria of CKDu disease, CKDu and snake bite=4044 (n=733) (excluded=180, missing data n=25) Urine arsenic, cadmium and lead n=495 n=132 Urine for other elements n=107 n=0 Serum selenium, strontium etc n=171 n=0 Hair/nails arsenic, cadmium lead n=80 n=48 Urine pesticide residues n=57 n=0 Figure 2 Flow chart of the study conducted in three districts (Anuradhapura, Polonnaruwa and Badulla) in the endemic area. ACR = albumin–creatinine ratio. blockers or diuretics, either alone or in combination  normal glycosylated haemoglobin (HbA ; <6.5%) 1c with angiotensin-converting enzyme inhibitors.  if on treatment for hypertension, blood pressure In both endemic and non-endemic areas, water, food, below <140/90 mmHg; if not on treatment for tobacco, soil and agrochemicals were also analysed for hypertension, blood pressure below <160/ heavy metals and metalloids, to determine whether they 100 mmHg. were within stipulated reference levels. CKDu was graded as follows: Methods Case definition  Stage 1: persistent albuminuria (i.e. ACR ≥30 mg/g The following case definition of CKDu was used. Partici- in initial and repeat urine sample) and estimated pants who had persistent albuminuria, i.e. albumin–cre- glomerular filtration rate (eGFR), using the Chronic atinine ratio (ACR) ≥30 mg/g in an initial urine sample Kidney Disease Epidemiology collaboration (CKD- and at a repeat visit, were considered to have CKDu if EPI) equation [25] >90 ml/min/1.73 m they satisfied the following criteria:  Stage 2: persistent albuminuria and eGFR 60–89 ml/ min/1.73 m no past history of glomerulonephritis,  Stage 3: persistent albuminuria and eGFR 30–59 ml/ pyelonephritis, renal calculi or snake bite min/1.73 m not on treatment for diabetes  Stage 4: persistent albuminuria and eGFR <30 ml/ min/1.73 m . Table 1 Demographic characteristics of CKDu cases in the endemic area, controls from the endemic area and Population prevalence study controls from the non-endemic area Ethical approval for the study was obtained from the Eth- Characteristic Endemic area Non-endemic area ical Review Committee of the Sri Lanka Medical Associ- CKDu cases Controls Controls ation. All participants gave written consent for the study. Total number 733 4044 250 Six divisional secretariat areas (administrative divi- sions) were selected randomly from three districts in the Males, % 37.1 42.5 56 endemic area. Twenty-two villages (Grama Niladari areas) Age (years), mean (SD) 39.1 (14.2) 43.7 (13.9) 35.5 (14.0) were selected randomly from the six divisions. Using the Farmer, % 38 43.9 18.3 electoral lists, 100 households from each village were ran- BMI, kg/m , mean (SD) 22.3 (4.6) 21.1 (4.1) 21.7 (4.4) domly selected for the study. Males and females aged be- ACR ≥30 mg/g 733 0 0 tween 15 and 70 years (n = 6698), with no diagnosed ACR albumin–creatinine ratio, BMI body mass index, SD standard deviation. diabetes, were invited to participate and 74% responded Jayatilake et al. BMC Nephrology 2013, 14:180 Page 4 of 13 http://www.biomedcentral.com/1471-2369/14/180 (Figure 2, flow diagram). Trained interviewers used a Soil, phosphate fertilizer, pesticides and weedicides survey questionnaire to gather information on age, sex, were analysed for arsenic, cadmium and lead. Soil sam- marital status, education, occupation, smoking, alcohol ples were obtained from paddy fields, other types of consumption, current residence, duration of residence in cultivations, and reservoirs in the endemic (n = 88) and the study area, source of drinking water, storage con- non-endemic (n = 41) areas. tainers for drinking water, exposure to agrochemicals, history of snake bite, glomerulonephritis, pyelonephritis, Specimen handling and analysis renal calculi, use of medications including herbal medi- Samples were collected in uncontaminated collection vials cines, and past medical history. Height was measured to and stored frozen (−20°C) until transfer to the laboratory. the nearest 0.1 cm. Weight was measured to the nearest All analyses were performed in a contract laboratory (La- 0.1 kg, using a calibrated weighing scale. Participants wore boratory of Pathophysiology of the University of Antwerp, light clothes and no shoes. A medical officer verified the Belgium), which has an external quality control scheme medical information gathered and measured the blood for analysis of trace elements. pressure after 15 minutes’ rest, using a mercury sphygmo- Measurements of arsenic, cadmium, lead and other el- manometer. The average of two readings taken 5 minutes ements in urine, water, vegetables, agrochemicals and apart was used. Urine ACR, HbA and serum and urine soil, was performed by inductively coupled plasma mass 1c creatinine concentrations were also measured. spectrometry (MS). Serum analyses were performed by electrothermal atomic Analytical studies absorption spectrometery. Limits of detection for alumin- Arsenic, cadmium and lead in urine, blood, hair and nails ium, strontium, chromium and selenium were 0.1 μg/l, Arsenic, cadmium and lead concentrations were analysed 0.5 μg/l, 0.01 μg/l and 1 μg/l respectively. in urine in a randomly selected subset of CKDu cases (n = 495) and randomly selected matched controls from Determination of pesticide residues in urine the endemic area, as well as from the non-endemic area Samples were shipped in dry ice and stored at −18°C until (n =250). analysis. Analysis used validated liquid chromatography Urine sodium, potassium, calcium, magnesium, cop- with tandem MS (LC-MS/MS), gas chromatography-mass per, zinc and titanium concentrations were analysed in a spectroscopy (GC-MS) and gas chromatography with tan- randomly selected subset of CKDu cases (n = 148). Their dem mass spectrometry (GC-MS/MS) methods. serum was also analysed for selenium, aluminium, stron- Further details of sample preparation and analytical tium and chromium. Hair and nail samples were analysed techniques are provided in the Additional file 1. for cadmium, arsenic and lead in a subset of CKDu cases (n = 80) and controls from the endemic area (n =48). Statistics Urine samples from CKDu cases (n = 57) and controls The normality of data distribution was assessed with histo- from the non-endemic area (n = 39) were analysed for grams. All data on metals had skewed distributions. After pesticide residues (2,4-D, 2,4,5-T, 2,4,5-trichlorophenol, removal of a small number of outliers, log-transformations isopropoxyphenol, pentachlorophenol, 3,5,6-trichloropy- were used to normalise the data, in order to conduct ridinol, p-nitrophenol, 1-naphthol, 2-naphthol, glyphosate, statistical analyses. The mean, median, minimum and aminomethylphosphonic acid (AMPA)). maximum values are reported on original data; t-tests of log-transformed values were used to test differences in Arsenic, cadmium and lead in water, food, tobacco, quantitative variables. The results were also confirmed pasture, weeds, soil, fertilizer, weedicides and pesticides by non-parametric Wilcoxon rank-sum test. Arsenic, cadmium and lead were analysed in samples A multiple logistic regression model was fitted for the (n = 234) obtained from endemic and non-endemic areas. CKDu definition. The model incorporated characteristics They comprised 99 sources of drinking water for indi- of interest, including age, sex, education, smoking, illicit viduals with CKDu (from ground wells, tube wells and alcohol consumption, occupation, type of agriculture, natural springs), 123 other sources of water (from years of agriculture, source of drinking water, drinking ground wells, tube wells, irrigation canals, reservoirs, water from paddy fields, exposure to fertilizer, exposure natural springs) from the endemic area, and 12 from the to weedicides and pesticides, type of water container, non-endemic area. whether using protection against agrochemicals, and Rice; pulses; vegetables, including leafy vegetables, coco- months living in the district. All were entered as categor- nut, yams and roots (e.g. kohila, lotus); freshwater fish; ical variables, except months living in the district. These tobacco; pasture; and weeds obtained from endemic data analyses were performed using Stata 11 and P values (n = 119) and non-endemic (n =32) areas were analysed of less than 0.05 were considered statistically significant. A for arsenic, cadmium and lead. receiver-operating characteristic (ROC) curve was used to Jayatilake et al. BMC Nephrology 2013, 14:180 Page 5 of 13 http://www.biomedcentral.com/1471-2369/14/180 Table 2 Summary results of logistic regression analysis calculate the area under the ROC curve (AUC), to deter- for exposures mine the cut-off values for cadmium and selenium with Exposure 95% CI the best sensitivity and specificity. A multinomial logistic regression was used to assess the dose–effect relationship OR Lower Upper P n value between metal exposure and the outcome CKDu grade. Education The analyses were adjusted for age and sex. No education Reference 174 Results School grades 1–9 0.900 0.612 1.323 0.594 4374 Population prevalence study Higher 1.201 0.588 2.452 0.614 74 The age-standardised prevalence of CKDu was higher in Smoking females 16.9% (95% confidence interval [CI] = 15.5% to Never Reference 3480 18.3%) than in males 12.9% (95% CI = 11.5% to 14.4%; P = Current/former 1.072 0.813 1.415 0.619 1126 0.001). About 37% of those with CKDu were male. The distribution of CKDu stages 1 to 4 in males was 27.0%, Illicit alcohol consumption 27.9%, 23.2% and 22.0% and in females 53.3%, 32.0%, 7.4% Never Reference 3701 and 7.3%, respectively. More severe stages of CKDu were Occasional/frequent/past 1.184 0.905 1.548 0.216 874 seen more frequently in males (stage 3: males versus fe- Occupation males = 23.2% versus 7.4%; stage 4: males versus females = Other Reference 2816 22.0% versus 7.3%; P < 0.001). In both sexes, the preva- Farmer 1.195 1.007 1.418 0.041 1780 lence increased with increasing age (P < 0.001). The preva- lence in the three districts was 15.1% in Anuradhapura, Agriculture type 20.6% in Polonnaruwa and 22.9% in Badulla. Non-paddy Reference 315 There was a family history of kidney disease in parents Paddy 0.732 0.542 0.988 0.042 2620 or siblings in 20% of individuals with CKDu; 2.1% of in- Years working in dividuals with CKDu had a history of ischaemic heart agriculture disease and/or cerebrovascular disease; 0.4% had a his- <10 Reference 660 tory of long-term use of herbal medicines for hyperten- 10–19 0.834 0.603 1.152 0.271 777 sion; 1.8% had a history of long-term use of aspirin; and 20–49 1.092 0.777 1.535 0.611 1182 0.6% had a history of long-term use of analgesics. Being male reduced the risk of CKDu (odds ratio [OR] = 0.745, ≥50 1.322 0.462 3.785 0.602 22 95% CI = 0.562 to 0.988, P < 0.05), and being older than Source of drinking water 39 years increased the risk of CKDu (OR = 1.926, 95% Not well Reference 798 CI = 1.561 to 2.376, P < 0.001). When separate logistic Well 0.971 0.785 1.202 0.793 3819 regressions were run for each potential exposure, only Water storage container occupation type (being a chena cultivation farmer in- Others Reference 1741 creased the OR by 19.5%) and type of agriculture (en- gaging in paddy cultivation compared to cultivation of Aluminium 1.03 0.87 1.22 0.715 2879 vegetables and other crops [chena cultivation] decreased Protection from agrochemicals the OR by 26.8%) were significant (Table 2). Yes Reference 191 Arsenic, cadmium, lead and other elements in urine No 1.011 0.661 1.546 0.959 4271 In CKDu cases, the concentration of cadmium in urine Separate logistic regressions have been run per exposure variable; OR < 1 was significantly higher compared to controls, in both means protective, and OR > 1 means that the exposure increases the odds of CKDu. The total number (n) of observations varies per exposure, owing to the endemic and the non-endemic areas (Table 3). Among missing data. All results are adjusted for sex and age. For all analyses, male sex CKDu cases, the concentration of cadmium in urine was was found to be protective and the risk increased with age. CI confidence interval, OR odds ratio. positively correlated with lead (r = 0.62, P < 0.001) and ar- Being male reduced the risk of CKDu (OR = 0.745, 95% CI = 0.562 to 0.988; P < senic concentrations in urine (r = 0.28, P < 0.001). There 0.05), and being >39 years increased the risk of CKDu (OR = 1.926, 95% CI = was no significant difference in urine arsenic and lead 1.561 to 2.376, P < 0.001). concentrations in CKDu cases compared to controls. The concentration of arsenic in urine were 90% and 23.2% sensitivity and specificity for concentrations of cadmium in respectively (AUC = 0.64, 95% CI = 0.58 to 0.71, cut-off urine were 80% and 53.6% respectively (AUC = 0.682, value ≥88.57 μg/g). The concentration of lead in urine was a 95% CI = 0.61 to 0.75, cut-off value ≥0.23 μg/g; Figure 3). poor predictor of CKDu (AUC = 0.53, 95% CI 0.38 to 0.67). At acut-off valueof ≥0.397 μg/g, sensitivity was 70% and Dose–response analysis showed that cadmium exposure is a specificity 68.3%. The sensitivity and specificity for the Jayatilake et al. BMC Nephrology 2013, 14:180 Page 6 of 13 http://www.biomedcentral.com/1471-2369/14/180 Table 3 Urine concentration of arsenic, cadmium and lead for CKDu cases compared with controls from the endemic and non-endemic areas Mean, median (range) of concentration in urine (μg/g creatinine) Arsenic Cadmium Lead CKDu cases (n = 495) 45.447, 26.3 (0.4 to 616.6) 1.039, 0.695 (0.005 to 8.93) 1.153, 0.95 (0.04 to 8.53) Controls from endemic area (n = 132) 92.443, 6.99 (0.2 to 966.29) 0.646, 0.18, (0.005 to 5.13) 1.254, 0.793 (1.21 to 6.64) Controls from non-endemic area (n = 250) 56.572, 42.025 (5.38 to 350.28) 0.345, 0.265 (0.005 to 2.079) 2.099, 1.434 (0.277 to 20.9) Urine cadmium concentration of cases compared to controls from endemic area P < 0.001. Urine cadmium concentration of cases compared to controls from non-endemic area P < 0.05. risk factor for the development of CKDu: P = 0.019 for (reference range = 54 μg/l to 163 μg/l). A serum selen- stage 3 and P = 0.024 for stage 4. There was no significant ium concentration of 90 μg/l is required to reach the max- dose–effect relationship between the concentration of ar- imum level of glutathione peroxidise [26]. About two-thirds senic, lead or selenium in urine and the stage of CKDu. (63%) of subjects had selenium levels below this cut-off Urine concentrations of sodium, potassium, calcium, value. Serum strontium levels were above normal limits magnesium, copper, zinc, and titanium in CKDu cases (mean = 83.17 μg/l, standard deviation [SD] = 32.15 μg/l; were within normal limits (Additional file 2). reference range = 14 μg/l to 84 μg/l). The sensitivity and specificity for serum selenium were 80% and 60% respect- Serum aluminium, chromium, selenium and strontium in ively (AUC = 0.789, cut = off value ≥94.3 μg/l; Figure 3). CKDu cases Serum aluminium and chromium levels were within nor- Cadmium and arsenic in hair and nails mal limits (Additional file 2). Serum selenium levels in A significantly higher cadmium concentration was also subjects with CKDu ranged from 50.0 μg/l to 121.8 μg/l seen in the nails of CKDu cases (n = 80, mean = 0.017 μg/g, Figure 3 ROC curves generated with urine arsenic, cadmium and lead and serum selenium concentrations. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 7 of 13 http://www.biomedcentral.com/1471-2369/14/180 median = 0.007 μg/g, minimum = 0.001 μg/g, maximum = taken from a canal and a reservoir, the cadmium con- 0.347 μg/g) compared to controls (n = 48) from the en- centration was 3.46 μg/l in one sample from a reservoir demicarea(mean=0.009 μg/g, median = 0.001 μg/g, mini- and the lead concentration was 12.3 μg/l in one sample mum = 0.001 μg/g, maximum = 0.091 μg/g; P < 0.05). from a reservoir in the endemic area. All other samples Arsenic levels in hair were significantly higher in CKDu from wells, tube wells, irrigation canals, pipe-borne cases (n = 80; mean = 0.144 μg/g, median = 0.139 μg/g, water, reservoirs and natural springs, including those minimum = 0.00 μg/g, maximum = 0.452 μg/g), compared taken from the non-endemic area, had normal arsenic, to controls (n = 48) from the endemic area (mean = cadmium and lead levels. 0.125 μg/g, median = 0.103 μg/g, minimum = 0.006 μg/g, maximum = 1.214 μg/g; P <0.05). Arsenic, cadmium and lead in food, tobacco leaves, pasture and weeds Arsenic, cadmium, lead and uranium in water (endemic Levels of cadmium in rice in both endemic and non- area n = 222, non = endemic area, n = 12) endemic areas were below the allowable limit (0.2 mg/ Levels of cadmium, lead and uranium in sources of kg; Figure 5). The maximum concentration of cadmium drinking water (Figure 4) used by individuals with CKDu in vegetables in the endemic area and in the non- (n = 99) were within normal limits. Arsenic was borderline endemic areas was 0.322 mg/kg and 0.063 mg/kg re- or raised in four samples (9.9 μg/l, 10.2 μg/l, 10.5 μg/l, spectively. Levels of cadmium in certain vegetables such 13.4 μg/l). Repeat analysis (n = 32) from the four sources as lotus root, and in tobacco, were high. Levels of cad- showed normal arsenic levels. mium in lotus and tobacco were higher in endemic than In water samples from other sources, the arsenic con- in non-endemic areas (lotus: mean = 0.413 mg/kg versus centration was 22.2 μg/l and 9.8 μg/l in two samples 0.023 mg/kg, median = 0.066 mg/kg versus 0.023 mg/kg, Figure 4 Concentration of arsenic, cadmium, lead and uranium in water in the endemic area (n = 222) and non-endemic area (n = 12). End canal = endemic area canal; End drink = endemic area drinking water; End reser = endemic area reservoir; End spring = endemic area spring; nonend drink = non-endemic area drinking water. Horizontal lines within the boxes represent the median values. The ends of the solid lines extending on either side of the boxes represent the minimum and the maximum. The dark dots are outliers; defined as being more than 1.5 interquartile ranges away from the box. The interquartile range is the distance between the upper part of the box and the lower part of the box. Reference limits: arsenic <10 μg /l, cadmium <3 μg/l, lead <10 μg/l, uranium <2 μg/l [21]. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 8 of 13 http://www.biomedcentral.com/1471-2369/14/180 Figure 5 (See legend on next page.) Jayatilake et al. BMC Nephrology 2013, 14:180 Page 9 of 13 http://www.biomedcentral.com/1471-2369/14/180 (See figure on previous page.) Figure 5 Content of arsenic, cadmium and lead in food, tobacco leaves, pasture and weeds from the endemic area. Horizontal lines within the boxes represent the median values. The ends of the solid lines extending on either side of the boxes represent the minimum and the maximum. The dark dots are outliers ; defined as being more than 1.5 interquartile ranges away from the box. The interquartile range is the distance between the upper part of the box and the lower part of the box. (The cadmium and lead content in certain food items exceeded the a a maximum stipulated reference value ). The maximum levels of cadmium permitted by the Codex Alimentarius for vegetables is 0.2 mg/kg [22,23] and by the Commission of the European Communities is 0.05 mg/kg [24]. The maximum concentration of cadmium stipulated for certain types of fish by the Commission of the European Communities is 0.05 mg/kg [24]. The maximum concentration of lead stipulated for vegetables by the Commission of the European communities is 0.10 mg/kg [24]. maximum = 1.50 mg/kg versus 0.03 mg/kg; tobacco: socioeconomic groups may have an influence on the ex- mean = 0.351 mg/kg versus 0.316 mg/kg, median = cretion of heavy metals and oxidative stress on the kidney. 0.351 mg/kg versus 0.316 mg/kg, maximum = 0.44 mg/ This would make the kidneys more vulnerable to CKDu, kg versus 0.351 mg/kg in endemic versus non-endemic resulting in a higher prevalence in females. On the other areas respectively). hand, male sex has been reported to be a risk factor for progression to end-stage renal disease [27], and this may Arsenic, cadmium and lead in soil and agrochemicals in partly explain the occurrence of more severe stages of the endemic and non-endemic areas CKDu in men. The level of cadmium in surface soil in the endemic area Previous studies have reported a family history of (n = 94, excluding samples from reservoirs), was 1.16 μg/g chronic kidney disease, ayurvedic treatment, and history compared to 0.49 μg/g in the non-endemic area (n =45, of snake bite as significant predictors for CKDu [10,12,13]. excluding samples from reservoirs) (Additional file 2). In the present study, older age, being female and being a chena cultivation farmer increased the risk of CKDu. Fam- Pesticide residues in urine ily history was positive in one-fifth of those with CKDu, Pesticide residues were detected in the urine from individ- and a history of snake bite was one of the exclusion cri- uals with CKDu (n = 57). The frequency of detection of teria. Long-term use of herbal medicines or analgesics 2,4-D, 3,5,6-trichloropyridinol, p-nitrophenol, 1-naphthol, was reported in only a very small percentage of those 2-naphthol, glyphosate, AMPA was 33%, 70%, 58%, 100%, with CKDu. Fanconi syndrome and other hereditary 100%, 65% and 28% respectively). Isopropoxyphenol, kidney diseases have not been reported in communities 2,4,5-trichlorphenol and pentachlorphenol were below de- in this region. tection limits. The proportions of CKDu cases with pesti- Previous studies have reported divergent information cide levels above reference values are shown in Table 4. on the role of cadmium in the causation of CKDu [14,15,19,20]. In the present study, individuals with Discussion CKDu excreted significantly higher levels of cadmium The prevalence of CKDu found in this study (females compared to those in the control group, in both the en- 16.9%, males 12.9%) was higher than that reported previ- demic and non-endemic areas. Controls in the endemic ously (2% to 3%) [20]. Although the prevalence in females area compared to those in the non-endemic area also was higher, more severe stages of CKDu were seen more had significantly higher urinary excretion of cadmium. often in males. The reason for this discrepancy is not The sensitivity and specificity for urine cadmium were clear. Factors such as low iron stores in females in lower 80% and 53.6% respectively (AUC = 0.682, cut-off value Table 4 Pesticide residues in urine of CKDu cases (n = 57) Parent compound Biomarker Reference limit (μg/l) CKDu cases (μg/l), CKDu cases above reference (minimum, maximum) limit (%) 2,4-D 2,4-D <0.3 0.5, 0.62 3.5 Pentachlorophenol Pentachlorophenol <2 0.3, 2.2 1.7 Chlorpyrifos 3,5,6-trichloropyridinol <11.3 0.5, 34.7 10.5 Parathion p-nitrophenol <25 0.5, 8.88 0 Carbaryl naphthalene 1-naphthol <19.7 0.5, 45.1 10.5 Naphthalene 2-naphthol, <17.1 0.94, 47.88 10.5 Glyphosate Glyphosate <2 0.075, 3.36 3.5 Glyphosate AMPA <0.5 0.075, 2.65 14 Jayatilake et al. BMC Nephrology 2013, 14:180 Page 10 of 13 http://www.biomedcentral.com/1471-2369/14/180 ≥0.23 μg/g). There was a dose–effect relationship be- this to be the case. On the contrary, the cadmium con- tween the concentration of cadmium in urine and the tent in all water samples analysed was within normal stage of CKDu. A significantly higher cadmium concen- limits, except in one sample from a reservoir that had a tration was also seen in the nails of those with CKDu borderline cadmium level (3.45 μg/l). compared to controls from the endemic area. Cadmium Drinking water is a major pathway for entry of inor- is a known nephrotoxin and urinary excretion of ganic arsenic into the human body. The arsenic content cadmium is considered to be a reliable indicator of cu- in 99% of water samples was below the WHO reference mulative long-term exposure to cadmium [6]. The mean value of 10 μg/l [21]. However, it has recently been sug- urine concentration of cadmium in CKDu cases was gested that the concentration of arsenic in drinking above the levels demonstrated in recent studies to cause water should be no more than 5 μg/l [43]. oxidative stress and decreased glomerular filtration rate CKDu occurs in areas where groundwater is the main and creatinine clearance [28-33]. The results of this source of drinking water. Groundwater in this region is study indicate that cadmium exposure is a risk factor known to have a high content of fluoride and calcium. for CKDu. People living in the region for generations have used The mean urine concentration of arsenic in CKDu groundwater for drinking without ill effects. However, cases was also above levels known to cause oxidative hardness of water, the high fluoride content, poor access injury to the kidney [33]. In CKDu cases and controls to drinking water and inadequate intake of water in a from the endemic area, concentrations of arsenic in warm climate may influence the body burden and/or the urine and in fingernails were higher than those reported excretion of heavy metals and oxidative damage to the in people living in low-exposure environments [34,35]. kidneys caused by heavy metals. Urine is a major pathway for excretion of arsenic from The maximum level of cadmium for vegetables permit- the human body, so urine levels reflect exposure. In ted by the Codex Alimentarius is 0.2 mg/kg [22,23] and some studies, markers of oxidative stress have been the level permitted by the Commission of the European demonstrated at urine arsenic concentrations as low as Communities is 0.05 mg/kg [24]. The maximum levels in 3.95 μg/g [36]. The level of total arsenic in urine is asso- certain vegetables grown in the endemic area exceeded ciated with chronic kidney disease in a dose–response these safety levels. The maximum concentration of cad- relationship, especially when the level is greater than mium in fish (0.06 μg/g) also exceeded the European max- 20.74 μg/g [36]. These findings support the contention imum limit of 0.05 mg/kg stipulated for certain types of that chronic exposure to low levels of cadmium may be fish [24]. The maximum level of lead in vegetables permit- a causative factor for CKDu in Sri Lanka. Co-exposure ted by the Commission of the European Communities is to cadmium and arsenic is known to produce additive 0.10 mg/kg [24]. The maximum level of lead in vegetables effects on the kidney that are more pronounced than ex- in the endemic area (0.476 mg/kg) exceeded this cut-off posure to either metal alone [37,38]. value. Levels of cadmium and lead in vegetables and cad- Selenium has been shown to protect the kidney from mium in freshwater fish from the endemic area are above oxidative stress [39]. A selenium concentration of 80– the maximum levels stipulated by certain Food Safety Au- 95 μg/l is needed to maximise the activity of the antioxi- thorities [22-24,44]. dant enzyme glutathione peroxidase and selenoproteins A provisional tolerable weekly intake (PTWI) for in plasma [40,41]. In this context, it is significant that cadmium of 7 μg/kg body weight was established by the serum selenium was below 80 μg/l in 38% and below Joint Food and Agriculture Organization of the United 90 μg/l in 63% of individuals with CKDu. Low selenium Nations (FAO)/WHO Expert Committee on Food Addi- levels may have been a contributory factor increasing tives (JECFA) [45]. In 2011, the JECFA revised the PTWI the vulnerability of the kidneys to oxidative damage for cadmium to 5.8 μg/kg body weight [46]. More re- caused by heavy metals and metalloids. cently, the PTWI for cadmium has been lowered to The association of raised serum strontium levels with 2.52 μg cadmium/kg body weight, in order to ensure a raised serum cadmium levels has been reported previ- high level of protection of all consumers, including ex- ously [42]. Strontium levels were not analysed in food or posed and vulnerable subgroups of the population [44]. water. The most likely explanation is an alteration of Since the cadmium content of certain food items in the strontium handling and excretion, owing to the effect of endemic area is above stipulated levels, the total weekly cadmium on renal tubular function. intake of cadmium in people living in the endemic area Cadmium levels have previously been reported to be could exceed these safe limits, with detrimental effects high in water sources in the domestic environment of on the kidneys, particularly in vulnerable people and people with CKDu, and 10–20 times the maximum stip- those with predisposing factors. ulated level have been found in reservoirs in the en- Reported mean dietary exposure to inorganic arsenic demic area [15]. The results of this study did not show in the United States of America (USA) and various Jayatilake et al. BMC Nephrology 2013, 14:180 Page 11 of 13 http://www.biomedcentral.com/1471-2369/14/180 European and Asian countries ranges from 0.1 to 3.0 μg/ There are several limitations in the study. Other kid- kg body weight per day [45]. Recently, the PTWI for ar- ney disease such as HIV nephropathy could fulfil the senic (0.015 mg/kg body weight per week) was with- case-definition criteria used for CKDu. As HIV is not drawn and environmental authorities are in the process prevalent in Sri Lanka, it was not excluded through of collecting more data for exposure assessment [46]. blood tests. The presence of glomerulonephritis was not The current recommendation is that every effort should excluded by biopsy but was based on past medical re- be made to keep concentrations of arsenic as low as rea- cords and diagnosis cards. The sensitivity and specificity sonably possible. The PTWI for lead is set at 0.025 mg/ of the case definition relative to biopsy-proven CKDu is kg body weight per week [45]. also not known. Stage 1 CKDu is defined by persistent Previous studies have reported high levels of cadmium in microalbuminuria and may overestimate the prevalence fertilizer (mean 47 μg/g) [15]. The maximum cadmium, of CKDu. The case definition required albuminuria. As a lead and arsenic concentrations in phosphate fertilizer result, people with CKDu who have a low eGFR and no from theendemic area in the present studywere30.8 μg/g, albuminuria were excluded from the study. In addition, 823.4 μg/g and 0.19 μg/g respectively. The maximum ac- the CKD-EPI equation used to estimate eGFR [25] has ceptable levels for cadmium, lead and arsenic, in phosphate not been validated in people from South Asia. It is not fertilizer product, at 1% of the nutrient level, are 4 μg/g, known whether the albuminuria of CKDu responds to 20 μg/g and 2 μg/g, respectively [47]. treatment for high blood pressure. If it does, an individ- The mean concentration of cadmium in soil from the ual could then be excluded based on their ACR, despite endemic area was 0.4 μg/g. Surveys of agricultural soils having the disease. in the USA and Sweden have reported lower soil cad- CKDu has been reported in other populations as well mium levels (0.265 mg/kg and 0.23 mg/kg respectively) [54-57]. Lessons learnt from other countries demon- [48,49]. The concentration of cadmium, arsenic and lead strate that sound public health policies to ensure access in soil, and their impact on body burden and excretion, to safe drinking water; regulatory control to ensure ap- is known to be influenced by many environmental factors propriate use of agrochemicals including fertilizer; haz- such as the pH of soil, buffering capacity, content of ardous waste remediation; regulatory control to prevent organic matter and water quality, among others [50-52]. pollution of the environment from discarded batteries Cadmium accumulation by plants, for example, is containing heavy metals; tobacco control; and reduction influenced by the reactive soil cadmium content and of air pollution can reduce exposure to heavy metals pH. It is decreased by high cation exchange capacity of [58,59]. Based on the findings of this study, the Govern- the soil and increased by higher soil temperature ment and the Ministry of Health of Sri Lanka have [49-52]. The hardness and high content of fluoride in already initiated multisectoral collaborative action with water in the endemic area may also influence the dy- the Ministries of Agriculture, Irrigation, Scientific Affairs namics of cadmium in soil, absorption by plants [17] and Social Services, to mitigate the exposure of people and excretion from the kidney. to environmental nephrotoxic substances. Steps are be- Certain pesticide residues were above reference levels ing taken to strengthen the water supply scheme in the in 31.6% of CKDu cases. Residues are demonstrative of endemic area as well as the regulations related to pro- the extent of the environmental distribution of pesticides curement and distribution of fertilizers and pesticides. and certain pesticides are known to be nephrotoxic Further studies are ongoing to investigate the contribu- [4,5,53]. Simultaneous exposure of people to heavy tory role of infections in the pathogenesis of CKDu. metals and nephrotoxic pesticides may be a contributory factor in the pathogenesis and progression of CKDu. Conclusions Compared to previous studies conducted on CKDu, The results of this cross-sectional study indicate that the present study has several strengths: (i) use of a con- multiple agents may play a role in the pathogenesis of sistent case definition for CKDu; (ii) analysis of a range CKDu. Herbal medicines and indiscriminate use of anal- of biological samples from individuals with CKDu; (iii) gesics are unlikely to be causative factors of CKDu. Re- comparison of control groups within and outside the sults show chronic exposure of people in the endemic endemic area; and (iv) use of sensitive analytical tech- area to low levels of cadmium through the food chain niques. Studies conducted hitherto to determine the and also to pesticides. They may also be exposed to lead prevalence and aetiology of CKDu [10,12,13,16,18,20] and arsenic through the food chain. Urine concentra- have relied on dipstick urinalysis to identify kidney dis- tions of cadmium and arsenic in individuals with CKDu ease. The present study is also the first in which heavy were at levels known to cause kidney damage. Significantly metals, metalloids and other elements in environmental higher urinary excretion of cadmium in individuals with and biological samples and pesticide residues in urine CKDu, and the dose–effect relationship between urine have been analysed. cadmium levels and CKDu stages, indicate that cadmium Jayatilake et al. BMC Nephrology 2013, 14:180 Page 12 of 13 http://www.biomedcentral.com/1471-2369/14/180 is a risk factor for the pathogensis of CKDu in Sri Lanka. work to be carried out to seek a sustainable solution for this public health problem is gratefully acknowledged. Deficiency of selenium and genetic susceptibility seen in individuals with CKDu suggest that they may be predis- Author details 1 2 posing factors for the development of CKDu when people Ministry of Health, Colombo, Sri Lanka. Management of Noncommunicable Diseases, World Health Organization, Geneva, Switzerland. World Health are exposed to nephrotoxins. Organization, Colombo, Sri Lanka. Received: 16 March 2013 Accepted: 16 August 2013 Additional files Published: 27 August 2013 Additional file 1: Details of sample preparation and analytical techniques. References Additional file 2: Table S1. Urine concentration of metals (sodium, 1. Gooneratne IK, Ranaweera AK, Liyanarachchi NP, Gunawardane N, Lanerolle potassium, calcium, magnesium, copper, zinc and titanium) in CKDu RD: Epidemiology of chronic kidney disease in a Sri Lankan population. cases. Table S2. Serum concentration of aluminium, chromium, selenium Int J Diabetes Dev Ctries 2008, 28:60–64. and strontium in CKDu cases. Table S3. Concentration of arsenic, 2. Nanayakkara S, Komiya T, Ratnatunga N, Senevirathna ST, Harada KH, Hitomi cadmium and lead in surface soil and in phosphate fertilizer, pesticides T, Gobe G, Muso E, Abeysekera T, Koizumi A: Tubulointerstitial damage as and weedicides, in the endemic area compared with a non-endemic the major pathological lesion in endemic chronic kidney disease among area. Samples of soil from vegetable plots from the endemic area were farmers in North Central Province of Sri Lanka. Environ Health Prev Med obtained from the vicinity of households with CKDu patients. 2012, 17:213–221. 3. Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S, Plattner B, Saran R, Wang AY, Yang CW: Chronic kidney disease: global dimension and perspectives. Abbreviations Lancet 2013, 382(9888):260–272. ACR: Albumin–creatinine ratio; AMPA: Aminomethylphosphonic acid; 4. 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Chronic kidney disease of uncertain aetiology: prevalence and causative factors in a developing country

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Medicine & Public Health; Nephrology; Internal Medicine
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Abstract

Background: This study describes chronic kidney disease of uncertain aetiology (CKDu), which cannot be attributed to diabetes, hypertension or other known aetiologies, that has emerged in the North Central region of Sri Lanka. Methods: A cross-sectional study was conducted, to determine the prevalence of and risk factors for CKDu. Arsenic, cadmium, lead, selenium, pesticides and other elements were analysed in biological samples from individuals with CKDu and compared with age- and sex-matched controls in the endemic and non-endemic areas. Food, water, soil and agrochemicals from both areas were analysed for heavy metals. Results: The age-standardised prevalence of CKDu was 12.9% (95% confidence interval [CI] = 11.5% to 14.4%) in males and 16.9% (95% CI = 15.5% to 18.3%) in females. Severe stages of CKDu were more frequent in males (stage 3: males versus females = 23.2% versus 7.4%; stage 4: males versus females = 22.0% versus 7.3%; P < 0.001). The risk was increased in individuals aged >39 years and those who farmed (chena cultivation) (OR [odds ratio] = 1.926, 95% CI = 1.561 to 2.376 and OR = 1.195, 95% CI = 1.007 to 1.418 respectively, P < 0.05). The risk was reduced in individuals who were male or who engaged in paddy cultivation (OR = 0.745, 95% CI = 0.562 to 0.988 and OR = 0.732, 95% CI = 0.542 to 0.988 respectively, P < 0.05). The mean concentration of cadmium in urine was significantly higher in those with CKDu (1.039 μg/g) compared with controls in the endemic and non-endemic areas (0.646 μg/g, P < 0.001 and 0.345 μg/g, P < 0.05) respectively. Urine cadmium sensitivity and specificity were 70% and 68.3% respectively (area under the receiver operating characteristic curve = 0.682, 95% CI = 0.61 to 0.75, cut-off value ≥0.397 μg/g). A significant dose–effect relationship was seen between urine cadmium concentration and CKDu stage (P< 0.05). Urine cadmium and arsenic concentrations in individuals with CKDu were at levels known to cause kidney damage. Food items from the endemic area contained cadmium and lead above reference levels. Serum selenium was <90 μg/l in 63% of those with CKDu and pesticides residues were above reference levels in 31.6% of those with CKDu. Conclusions: These results indicate chronic exposure of people in the endemic area to low levels of cadmium through the food chain and also to pesticides. Significantly higher urinary excretion of cadmium in individuals with CKDu, and the dose–effect relationship between urine cadmium concentration and CKDu stages suggest that cadmium exposure is a risk factor for the pathogensis of CKDu. Deficiency of selenium and genetic susceptibility seen in individuals with CKDu suggest that they may be predisposing factors for the development of CKDu. Keywords: Arsenic, Cadmium, Chronic kidney disease, Kidney disease of uncertain aetiology, Heavy metals, Lead, Pesticides * Correspondence: mendiss@who.int Management of Noncommunicable Diseases, World Health Organization, Geneva, Switzerland Full list of author information is available at the end of the article © 2013 Jayatilake et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 2 of 13 http://www.biomedcentral.com/1471-2369/14/180 Background The objectives of this study were to: determine the The study reported here describes an apparently new prevalence of and identify the risk factors for CKDu; form of chronic kidney disease, which cannot be attrib- compare CKDu cases and controls with regard to expos- uted to diabetes, hypertension or other known aetiol- ure to heavy metals/metalloids and pesticides; and ana- ogies, that has emerged in the North Central region of lyse food, water, soil, fertilizers and weedicides for heavy Sri Lanka [1]. Chronic kidney disease of uncertain aeti- metals, in order to determine whether the levels are ology (CKDu) is slowly progressive, probably starting in above stipulated reference values. the second decade of life, and asymptomatic until ad- A population prevalence study was conducted in three vanced. Peripheral oedema and hypertension are late districts in the endemic area over the period 2010–2012 features. The main histopathological features include (Figures 1 and 2). Arsenic, cadmium, lead and other tubular atrophy, interstitial mononuclear cell infiltration metals, elements and pesticide residues that are potential and interstitial fibrosis [2]. These histological features nephrotoxins [6-9,21-24], were analysed in biological suggest that nephrotoxins play a key role in the aeti- samples. The results from individuals with CKDu were ology of CKDu. compared with those from controls in the endemic area Potential nephrotoxins are widely distributed in the and a non-endemic area (Hambantota where CKDu has environment. Exposure to environmental and other not been reported). The demographic characteristics of nephrotoxins, such as herbal medicines and analgesics, the three groups are shown in Table 1. All individuals are known to play a role in the aetiology of chronic with CKDu (n = 733) had a blood pressure below 160/ kidney disease. Defining their exact role in the aetiology 100 mmHg; 6.8% of these individuals were on treatment of kidney disease is a challenge. Environmental toxins for high blood pressure and had a blood pressure below implicated in kidney damage include heavy metals, such <140/90 mmHg. Three-quarters of those who were on as arsenic, cadmium, lead and uranium; mycotoxins pro- treatment were on angiotensin-converting enzyme inhib- duced by fungi in improperly stored foods; air pollut- itors. Others were on calcium channel blockers, beta- ants, such as tobacco smoke; and pesticides, such as chlorpyriphos, diazinon and propanil [3-9]. Over the last 8 years, several studies have been carried out to determine the prevalence, nature and causes of CKDu in Sri Lanka [10-20]. These studies include hospital-based reviews of case series, in which high-risk areas in North Central Province were identified. The hospital studies gave rise to population-based surveys, which showed that the populations affected by CKDu are scattered in the North Central region of the country [14]. Some studies have reported the point prevalence of CKDu to be about 2–3% among those above 18 years of age [20]. Case–control and cross-sectional studies have provided some insight into associations with the condi- tion. Specific evaluations of exposure to organophosphate and mycotoxins have been conducted. In a cross-sectional study, there was evidence of greater inhibition of acetyl cholinesterase among patients with chronic renal dysfunc- tion in areas of high prevalence of CKDu [17]. Ochratoxin, a naturally occurring mycotoxin with nephrotoxic proper- ties, was not found to be a contaminant of food in the region [13,16]. Some studies have shown high levels of en- vironmental cadmium, lead, aluminium and fluoride in re- gions with high rates of the condition [14,15,17,19]. Recognising the gravity of the public health threat caused by CKDu, in 2010, the Ministry of Health Sri Lanka, in col- laboration with the World Health Organization (WHO), launched a national research project with the aim of Figure 1 Map of Sri Lanka, showing the location of Anuradhapura, investigating the prevalence and aetiology of CKDu in Polonnaruwa and Badulla districts, in the endemic area, and Sri Lanka, with a view to developing appropriate preven- Hambantota district, in the non-endemic area. tive strategies. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 3 of 13 http://www.biomedcentral.com/1471-2369/14/180 Number invited to participate in the study from 220 randomly selected households n=6698 Response rate 74% (n=4957) ACR assay 30 mg/g on two occasions No diabetes, other kidney and satisfy other criteria of CKDu disease, CKDu and snake bite=4044 (n=733) (excluded=180, missing data n=25) Urine arsenic, cadmium and lead n=495 n=132 Urine for other elements n=107 n=0 Serum selenium, strontium etc n=171 n=0 Hair/nails arsenic, cadmium lead n=80 n=48 Urine pesticide residues n=57 n=0 Figure 2 Flow chart of the study conducted in three districts (Anuradhapura, Polonnaruwa and Badulla) in the endemic area. ACR = albumin–creatinine ratio. blockers or diuretics, either alone or in combination  normal glycosylated haemoglobin (HbA ; <6.5%) 1c with angiotensin-converting enzyme inhibitors.  if on treatment for hypertension, blood pressure In both endemic and non-endemic areas, water, food, below <140/90 mmHg; if not on treatment for tobacco, soil and agrochemicals were also analysed for hypertension, blood pressure below <160/ heavy metals and metalloids, to determine whether they 100 mmHg. were within stipulated reference levels. CKDu was graded as follows: Methods Case definition  Stage 1: persistent albuminuria (i.e. ACR ≥30 mg/g The following case definition of CKDu was used. Partici- in initial and repeat urine sample) and estimated pants who had persistent albuminuria, i.e. albumin–cre- glomerular filtration rate (eGFR), using the Chronic atinine ratio (ACR) ≥30 mg/g in an initial urine sample Kidney Disease Epidemiology collaboration (CKD- and at a repeat visit, were considered to have CKDu if EPI) equation [25] >90 ml/min/1.73 m they satisfied the following criteria:  Stage 2: persistent albuminuria and eGFR 60–89 ml/ min/1.73 m no past history of glomerulonephritis,  Stage 3: persistent albuminuria and eGFR 30–59 ml/ pyelonephritis, renal calculi or snake bite min/1.73 m not on treatment for diabetes  Stage 4: persistent albuminuria and eGFR <30 ml/ min/1.73 m . Table 1 Demographic characteristics of CKDu cases in the endemic area, controls from the endemic area and Population prevalence study controls from the non-endemic area Ethical approval for the study was obtained from the Eth- Characteristic Endemic area Non-endemic area ical Review Committee of the Sri Lanka Medical Associ- CKDu cases Controls Controls ation. All participants gave written consent for the study. Total number 733 4044 250 Six divisional secretariat areas (administrative divi- sions) were selected randomly from three districts in the Males, % 37.1 42.5 56 endemic area. Twenty-two villages (Grama Niladari areas) Age (years), mean (SD) 39.1 (14.2) 43.7 (13.9) 35.5 (14.0) were selected randomly from the six divisions. Using the Farmer, % 38 43.9 18.3 electoral lists, 100 households from each village were ran- BMI, kg/m , mean (SD) 22.3 (4.6) 21.1 (4.1) 21.7 (4.4) domly selected for the study. Males and females aged be- ACR ≥30 mg/g 733 0 0 tween 15 and 70 years (n = 6698), with no diagnosed ACR albumin–creatinine ratio, BMI body mass index, SD standard deviation. diabetes, were invited to participate and 74% responded Jayatilake et al. BMC Nephrology 2013, 14:180 Page 4 of 13 http://www.biomedcentral.com/1471-2369/14/180 (Figure 2, flow diagram). Trained interviewers used a Soil, phosphate fertilizer, pesticides and weedicides survey questionnaire to gather information on age, sex, were analysed for arsenic, cadmium and lead. Soil sam- marital status, education, occupation, smoking, alcohol ples were obtained from paddy fields, other types of consumption, current residence, duration of residence in cultivations, and reservoirs in the endemic (n = 88) and the study area, source of drinking water, storage con- non-endemic (n = 41) areas. tainers for drinking water, exposure to agrochemicals, history of snake bite, glomerulonephritis, pyelonephritis, Specimen handling and analysis renal calculi, use of medications including herbal medi- Samples were collected in uncontaminated collection vials cines, and past medical history. Height was measured to and stored frozen (−20°C) until transfer to the laboratory. the nearest 0.1 cm. Weight was measured to the nearest All analyses were performed in a contract laboratory (La- 0.1 kg, using a calibrated weighing scale. Participants wore boratory of Pathophysiology of the University of Antwerp, light clothes and no shoes. A medical officer verified the Belgium), which has an external quality control scheme medical information gathered and measured the blood for analysis of trace elements. pressure after 15 minutes’ rest, using a mercury sphygmo- Measurements of arsenic, cadmium, lead and other el- manometer. The average of two readings taken 5 minutes ements in urine, water, vegetables, agrochemicals and apart was used. Urine ACR, HbA and serum and urine soil, was performed by inductively coupled plasma mass 1c creatinine concentrations were also measured. spectrometry (MS). Serum analyses were performed by electrothermal atomic Analytical studies absorption spectrometery. Limits of detection for alumin- Arsenic, cadmium and lead in urine, blood, hair and nails ium, strontium, chromium and selenium were 0.1 μg/l, Arsenic, cadmium and lead concentrations were analysed 0.5 μg/l, 0.01 μg/l and 1 μg/l respectively. in urine in a randomly selected subset of CKDu cases (n = 495) and randomly selected matched controls from Determination of pesticide residues in urine the endemic area, as well as from the non-endemic area Samples were shipped in dry ice and stored at −18°C until (n =250). analysis. Analysis used validated liquid chromatography Urine sodium, potassium, calcium, magnesium, cop- with tandem MS (LC-MS/MS), gas chromatography-mass per, zinc and titanium concentrations were analysed in a spectroscopy (GC-MS) and gas chromatography with tan- randomly selected subset of CKDu cases (n = 148). Their dem mass spectrometry (GC-MS/MS) methods. serum was also analysed for selenium, aluminium, stron- Further details of sample preparation and analytical tium and chromium. Hair and nail samples were analysed techniques are provided in the Additional file 1. for cadmium, arsenic and lead in a subset of CKDu cases (n = 80) and controls from the endemic area (n =48). Statistics Urine samples from CKDu cases (n = 57) and controls The normality of data distribution was assessed with histo- from the non-endemic area (n = 39) were analysed for grams. All data on metals had skewed distributions. After pesticide residues (2,4-D, 2,4,5-T, 2,4,5-trichlorophenol, removal of a small number of outliers, log-transformations isopropoxyphenol, pentachlorophenol, 3,5,6-trichloropy- were used to normalise the data, in order to conduct ridinol, p-nitrophenol, 1-naphthol, 2-naphthol, glyphosate, statistical analyses. The mean, median, minimum and aminomethylphosphonic acid (AMPA)). maximum values are reported on original data; t-tests of log-transformed values were used to test differences in Arsenic, cadmium and lead in water, food, tobacco, quantitative variables. The results were also confirmed pasture, weeds, soil, fertilizer, weedicides and pesticides by non-parametric Wilcoxon rank-sum test. Arsenic, cadmium and lead were analysed in samples A multiple logistic regression model was fitted for the (n = 234) obtained from endemic and non-endemic areas. CKDu definition. The model incorporated characteristics They comprised 99 sources of drinking water for indi- of interest, including age, sex, education, smoking, illicit viduals with CKDu (from ground wells, tube wells and alcohol consumption, occupation, type of agriculture, natural springs), 123 other sources of water (from years of agriculture, source of drinking water, drinking ground wells, tube wells, irrigation canals, reservoirs, water from paddy fields, exposure to fertilizer, exposure natural springs) from the endemic area, and 12 from the to weedicides and pesticides, type of water container, non-endemic area. whether using protection against agrochemicals, and Rice; pulses; vegetables, including leafy vegetables, coco- months living in the district. All were entered as categor- nut, yams and roots (e.g. kohila, lotus); freshwater fish; ical variables, except months living in the district. These tobacco; pasture; and weeds obtained from endemic data analyses were performed using Stata 11 and P values (n = 119) and non-endemic (n =32) areas were analysed of less than 0.05 were considered statistically significant. A for arsenic, cadmium and lead. receiver-operating characteristic (ROC) curve was used to Jayatilake et al. BMC Nephrology 2013, 14:180 Page 5 of 13 http://www.biomedcentral.com/1471-2369/14/180 Table 2 Summary results of logistic regression analysis calculate the area under the ROC curve (AUC), to deter- for exposures mine the cut-off values for cadmium and selenium with Exposure 95% CI the best sensitivity and specificity. A multinomial logistic regression was used to assess the dose–effect relationship OR Lower Upper P n value between metal exposure and the outcome CKDu grade. Education The analyses were adjusted for age and sex. No education Reference 174 Results School grades 1–9 0.900 0.612 1.323 0.594 4374 Population prevalence study Higher 1.201 0.588 2.452 0.614 74 The age-standardised prevalence of CKDu was higher in Smoking females 16.9% (95% confidence interval [CI] = 15.5% to Never Reference 3480 18.3%) than in males 12.9% (95% CI = 11.5% to 14.4%; P = Current/former 1.072 0.813 1.415 0.619 1126 0.001). About 37% of those with CKDu were male. The distribution of CKDu stages 1 to 4 in males was 27.0%, Illicit alcohol consumption 27.9%, 23.2% and 22.0% and in females 53.3%, 32.0%, 7.4% Never Reference 3701 and 7.3%, respectively. More severe stages of CKDu were Occasional/frequent/past 1.184 0.905 1.548 0.216 874 seen more frequently in males (stage 3: males versus fe- Occupation males = 23.2% versus 7.4%; stage 4: males versus females = Other Reference 2816 22.0% versus 7.3%; P < 0.001). In both sexes, the preva- Farmer 1.195 1.007 1.418 0.041 1780 lence increased with increasing age (P < 0.001). The preva- lence in the three districts was 15.1% in Anuradhapura, Agriculture type 20.6% in Polonnaruwa and 22.9% in Badulla. Non-paddy Reference 315 There was a family history of kidney disease in parents Paddy 0.732 0.542 0.988 0.042 2620 or siblings in 20% of individuals with CKDu; 2.1% of in- Years working in dividuals with CKDu had a history of ischaemic heart agriculture disease and/or cerebrovascular disease; 0.4% had a his- <10 Reference 660 tory of long-term use of herbal medicines for hyperten- 10–19 0.834 0.603 1.152 0.271 777 sion; 1.8% had a history of long-term use of aspirin; and 20–49 1.092 0.777 1.535 0.611 1182 0.6% had a history of long-term use of analgesics. Being male reduced the risk of CKDu (odds ratio [OR] = 0.745, ≥50 1.322 0.462 3.785 0.602 22 95% CI = 0.562 to 0.988, P < 0.05), and being older than Source of drinking water 39 years increased the risk of CKDu (OR = 1.926, 95% Not well Reference 798 CI = 1.561 to 2.376, P < 0.001). When separate logistic Well 0.971 0.785 1.202 0.793 3819 regressions were run for each potential exposure, only Water storage container occupation type (being a chena cultivation farmer in- Others Reference 1741 creased the OR by 19.5%) and type of agriculture (en- gaging in paddy cultivation compared to cultivation of Aluminium 1.03 0.87 1.22 0.715 2879 vegetables and other crops [chena cultivation] decreased Protection from agrochemicals the OR by 26.8%) were significant (Table 2). Yes Reference 191 Arsenic, cadmium, lead and other elements in urine No 1.011 0.661 1.546 0.959 4271 In CKDu cases, the concentration of cadmium in urine Separate logistic regressions have been run per exposure variable; OR < 1 was significantly higher compared to controls, in both means protective, and OR > 1 means that the exposure increases the odds of CKDu. The total number (n) of observations varies per exposure, owing to the endemic and the non-endemic areas (Table 3). Among missing data. All results are adjusted for sex and age. For all analyses, male sex CKDu cases, the concentration of cadmium in urine was was found to be protective and the risk increased with age. CI confidence interval, OR odds ratio. positively correlated with lead (r = 0.62, P < 0.001) and ar- Being male reduced the risk of CKDu (OR = 0.745, 95% CI = 0.562 to 0.988; P < senic concentrations in urine (r = 0.28, P < 0.001). There 0.05), and being >39 years increased the risk of CKDu (OR = 1.926, 95% CI = was no significant difference in urine arsenic and lead 1.561 to 2.376, P < 0.001). concentrations in CKDu cases compared to controls. The concentration of arsenic in urine were 90% and 23.2% sensitivity and specificity for concentrations of cadmium in respectively (AUC = 0.64, 95% CI = 0.58 to 0.71, cut-off urine were 80% and 53.6% respectively (AUC = 0.682, value ≥88.57 μg/g). The concentration of lead in urine was a 95% CI = 0.61 to 0.75, cut-off value ≥0.23 μg/g; Figure 3). poor predictor of CKDu (AUC = 0.53, 95% CI 0.38 to 0.67). At acut-off valueof ≥0.397 μg/g, sensitivity was 70% and Dose–response analysis showed that cadmium exposure is a specificity 68.3%. The sensitivity and specificity for the Jayatilake et al. BMC Nephrology 2013, 14:180 Page 6 of 13 http://www.biomedcentral.com/1471-2369/14/180 Table 3 Urine concentration of arsenic, cadmium and lead for CKDu cases compared with controls from the endemic and non-endemic areas Mean, median (range) of concentration in urine (μg/g creatinine) Arsenic Cadmium Lead CKDu cases (n = 495) 45.447, 26.3 (0.4 to 616.6) 1.039, 0.695 (0.005 to 8.93) 1.153, 0.95 (0.04 to 8.53) Controls from endemic area (n = 132) 92.443, 6.99 (0.2 to 966.29) 0.646, 0.18, (0.005 to 5.13) 1.254, 0.793 (1.21 to 6.64) Controls from non-endemic area (n = 250) 56.572, 42.025 (5.38 to 350.28) 0.345, 0.265 (0.005 to 2.079) 2.099, 1.434 (0.277 to 20.9) Urine cadmium concentration of cases compared to controls from endemic area P < 0.001. Urine cadmium concentration of cases compared to controls from non-endemic area P < 0.05. risk factor for the development of CKDu: P = 0.019 for (reference range = 54 μg/l to 163 μg/l). A serum selen- stage 3 and P = 0.024 for stage 4. There was no significant ium concentration of 90 μg/l is required to reach the max- dose–effect relationship between the concentration of ar- imum level of glutathione peroxidise [26]. About two-thirds senic, lead or selenium in urine and the stage of CKDu. (63%) of subjects had selenium levels below this cut-off Urine concentrations of sodium, potassium, calcium, value. Serum strontium levels were above normal limits magnesium, copper, zinc, and titanium in CKDu cases (mean = 83.17 μg/l, standard deviation [SD] = 32.15 μg/l; were within normal limits (Additional file 2). reference range = 14 μg/l to 84 μg/l). The sensitivity and specificity for serum selenium were 80% and 60% respect- Serum aluminium, chromium, selenium and strontium in ively (AUC = 0.789, cut = off value ≥94.3 μg/l; Figure 3). CKDu cases Serum aluminium and chromium levels were within nor- Cadmium and arsenic in hair and nails mal limits (Additional file 2). Serum selenium levels in A significantly higher cadmium concentration was also subjects with CKDu ranged from 50.0 μg/l to 121.8 μg/l seen in the nails of CKDu cases (n = 80, mean = 0.017 μg/g, Figure 3 ROC curves generated with urine arsenic, cadmium and lead and serum selenium concentrations. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 7 of 13 http://www.biomedcentral.com/1471-2369/14/180 median = 0.007 μg/g, minimum = 0.001 μg/g, maximum = taken from a canal and a reservoir, the cadmium con- 0.347 μg/g) compared to controls (n = 48) from the en- centration was 3.46 μg/l in one sample from a reservoir demicarea(mean=0.009 μg/g, median = 0.001 μg/g, mini- and the lead concentration was 12.3 μg/l in one sample mum = 0.001 μg/g, maximum = 0.091 μg/g; P < 0.05). from a reservoir in the endemic area. All other samples Arsenic levels in hair were significantly higher in CKDu from wells, tube wells, irrigation canals, pipe-borne cases (n = 80; mean = 0.144 μg/g, median = 0.139 μg/g, water, reservoirs and natural springs, including those minimum = 0.00 μg/g, maximum = 0.452 μg/g), compared taken from the non-endemic area, had normal arsenic, to controls (n = 48) from the endemic area (mean = cadmium and lead levels. 0.125 μg/g, median = 0.103 μg/g, minimum = 0.006 μg/g, maximum = 1.214 μg/g; P <0.05). Arsenic, cadmium and lead in food, tobacco leaves, pasture and weeds Arsenic, cadmium, lead and uranium in water (endemic Levels of cadmium in rice in both endemic and non- area n = 222, non = endemic area, n = 12) endemic areas were below the allowable limit (0.2 mg/ Levels of cadmium, lead and uranium in sources of kg; Figure 5). The maximum concentration of cadmium drinking water (Figure 4) used by individuals with CKDu in vegetables in the endemic area and in the non- (n = 99) were within normal limits. Arsenic was borderline endemic areas was 0.322 mg/kg and 0.063 mg/kg re- or raised in four samples (9.9 μg/l, 10.2 μg/l, 10.5 μg/l, spectively. Levels of cadmium in certain vegetables such 13.4 μg/l). Repeat analysis (n = 32) from the four sources as lotus root, and in tobacco, were high. Levels of cad- showed normal arsenic levels. mium in lotus and tobacco were higher in endemic than In water samples from other sources, the arsenic con- in non-endemic areas (lotus: mean = 0.413 mg/kg versus centration was 22.2 μg/l and 9.8 μg/l in two samples 0.023 mg/kg, median = 0.066 mg/kg versus 0.023 mg/kg, Figure 4 Concentration of arsenic, cadmium, lead and uranium in water in the endemic area (n = 222) and non-endemic area (n = 12). End canal = endemic area canal; End drink = endemic area drinking water; End reser = endemic area reservoir; End spring = endemic area spring; nonend drink = non-endemic area drinking water. Horizontal lines within the boxes represent the median values. The ends of the solid lines extending on either side of the boxes represent the minimum and the maximum. The dark dots are outliers; defined as being more than 1.5 interquartile ranges away from the box. The interquartile range is the distance between the upper part of the box and the lower part of the box. Reference limits: arsenic <10 μg /l, cadmium <3 μg/l, lead <10 μg/l, uranium <2 μg/l [21]. Jayatilake et al. BMC Nephrology 2013, 14:180 Page 8 of 13 http://www.biomedcentral.com/1471-2369/14/180 Figure 5 (See legend on next page.) Jayatilake et al. BMC Nephrology 2013, 14:180 Page 9 of 13 http://www.biomedcentral.com/1471-2369/14/180 (See figure on previous page.) Figure 5 Content of arsenic, cadmium and lead in food, tobacco leaves, pasture and weeds from the endemic area. Horizontal lines within the boxes represent the median values. The ends of the solid lines extending on either side of the boxes represent the minimum and the maximum. The dark dots are outliers ; defined as being more than 1.5 interquartile ranges away from the box. The interquartile range is the distance between the upper part of the box and the lower part of the box. (The cadmium and lead content in certain food items exceeded the a a maximum stipulated reference value ). The maximum levels of cadmium permitted by the Codex Alimentarius for vegetables is 0.2 mg/kg [22,23] and by the Commission of the European Communities is 0.05 mg/kg [24]. The maximum concentration of cadmium stipulated for certain types of fish by the Commission of the European Communities is 0.05 mg/kg [24]. The maximum concentration of lead stipulated for vegetables by the Commission of the European communities is 0.10 mg/kg [24]. maximum = 1.50 mg/kg versus 0.03 mg/kg; tobacco: socioeconomic groups may have an influence on the ex- mean = 0.351 mg/kg versus 0.316 mg/kg, median = cretion of heavy metals and oxidative stress on the kidney. 0.351 mg/kg versus 0.316 mg/kg, maximum = 0.44 mg/ This would make the kidneys more vulnerable to CKDu, kg versus 0.351 mg/kg in endemic versus non-endemic resulting in a higher prevalence in females. On the other areas respectively). hand, male sex has been reported to be a risk factor for progression to end-stage renal disease [27], and this may Arsenic, cadmium and lead in soil and agrochemicals in partly explain the occurrence of more severe stages of the endemic and non-endemic areas CKDu in men. The level of cadmium in surface soil in the endemic area Previous studies have reported a family history of (n = 94, excluding samples from reservoirs), was 1.16 μg/g chronic kidney disease, ayurvedic treatment, and history compared to 0.49 μg/g in the non-endemic area (n =45, of snake bite as significant predictors for CKDu [10,12,13]. excluding samples from reservoirs) (Additional file 2). In the present study, older age, being female and being a chena cultivation farmer increased the risk of CKDu. Fam- Pesticide residues in urine ily history was positive in one-fifth of those with CKDu, Pesticide residues were detected in the urine from individ- and a history of snake bite was one of the exclusion cri- uals with CKDu (n = 57). The frequency of detection of teria. Long-term use of herbal medicines or analgesics 2,4-D, 3,5,6-trichloropyridinol, p-nitrophenol, 1-naphthol, was reported in only a very small percentage of those 2-naphthol, glyphosate, AMPA was 33%, 70%, 58%, 100%, with CKDu. Fanconi syndrome and other hereditary 100%, 65% and 28% respectively). Isopropoxyphenol, kidney diseases have not been reported in communities 2,4,5-trichlorphenol and pentachlorphenol were below de- in this region. tection limits. The proportions of CKDu cases with pesti- Previous studies have reported divergent information cide levels above reference values are shown in Table 4. on the role of cadmium in the causation of CKDu [14,15,19,20]. In the present study, individuals with Discussion CKDu excreted significantly higher levels of cadmium The prevalence of CKDu found in this study (females compared to those in the control group, in both the en- 16.9%, males 12.9%) was higher than that reported previ- demic and non-endemic areas. Controls in the endemic ously (2% to 3%) [20]. Although the prevalence in females area compared to those in the non-endemic area also was higher, more severe stages of CKDu were seen more had significantly higher urinary excretion of cadmium. often in males. The reason for this discrepancy is not The sensitivity and specificity for urine cadmium were clear. Factors such as low iron stores in females in lower 80% and 53.6% respectively (AUC = 0.682, cut-off value Table 4 Pesticide residues in urine of CKDu cases (n = 57) Parent compound Biomarker Reference limit (μg/l) CKDu cases (μg/l), CKDu cases above reference (minimum, maximum) limit (%) 2,4-D 2,4-D <0.3 0.5, 0.62 3.5 Pentachlorophenol Pentachlorophenol <2 0.3, 2.2 1.7 Chlorpyrifos 3,5,6-trichloropyridinol <11.3 0.5, 34.7 10.5 Parathion p-nitrophenol <25 0.5, 8.88 0 Carbaryl naphthalene 1-naphthol <19.7 0.5, 45.1 10.5 Naphthalene 2-naphthol, <17.1 0.94, 47.88 10.5 Glyphosate Glyphosate <2 0.075, 3.36 3.5 Glyphosate AMPA <0.5 0.075, 2.65 14 Jayatilake et al. BMC Nephrology 2013, 14:180 Page 10 of 13 http://www.biomedcentral.com/1471-2369/14/180 ≥0.23 μg/g). There was a dose–effect relationship be- this to be the case. On the contrary, the cadmium con- tween the concentration of cadmium in urine and the tent in all water samples analysed was within normal stage of CKDu. A significantly higher cadmium concen- limits, except in one sample from a reservoir that had a tration was also seen in the nails of those with CKDu borderline cadmium level (3.45 μg/l). compared to controls from the endemic area. Cadmium Drinking water is a major pathway for entry of inor- is a known nephrotoxin and urinary excretion of ganic arsenic into the human body. The arsenic content cadmium is considered to be a reliable indicator of cu- in 99% of water samples was below the WHO reference mulative long-term exposure to cadmium [6]. The mean value of 10 μg/l [21]. However, it has recently been sug- urine concentration of cadmium in CKDu cases was gested that the concentration of arsenic in drinking above the levels demonstrated in recent studies to cause water should be no more than 5 μg/l [43]. oxidative stress and decreased glomerular filtration rate CKDu occurs in areas where groundwater is the main and creatinine clearance [28-33]. The results of this source of drinking water. Groundwater in this region is study indicate that cadmium exposure is a risk factor known to have a high content of fluoride and calcium. for CKDu. People living in the region for generations have used The mean urine concentration of arsenic in CKDu groundwater for drinking without ill effects. However, cases was also above levels known to cause oxidative hardness of water, the high fluoride content, poor access injury to the kidney [33]. In CKDu cases and controls to drinking water and inadequate intake of water in a from the endemic area, concentrations of arsenic in warm climate may influence the body burden and/or the urine and in fingernails were higher than those reported excretion of heavy metals and oxidative damage to the in people living in low-exposure environments [34,35]. kidneys caused by heavy metals. Urine is a major pathway for excretion of arsenic from The maximum level of cadmium for vegetables permit- the human body, so urine levels reflect exposure. In ted by the Codex Alimentarius is 0.2 mg/kg [22,23] and some studies, markers of oxidative stress have been the level permitted by the Commission of the European demonstrated at urine arsenic concentrations as low as Communities is 0.05 mg/kg [24]. The maximum levels in 3.95 μg/g [36]. The level of total arsenic in urine is asso- certain vegetables grown in the endemic area exceeded ciated with chronic kidney disease in a dose–response these safety levels. The maximum concentration of cad- relationship, especially when the level is greater than mium in fish (0.06 μg/g) also exceeded the European max- 20.74 μg/g [36]. These findings support the contention imum limit of 0.05 mg/kg stipulated for certain types of that chronic exposure to low levels of cadmium may be fish [24]. The maximum level of lead in vegetables permit- a causative factor for CKDu in Sri Lanka. Co-exposure ted by the Commission of the European Communities is to cadmium and arsenic is known to produce additive 0.10 mg/kg [24]. The maximum level of lead in vegetables effects on the kidney that are more pronounced than ex- in the endemic area (0.476 mg/kg) exceeded this cut-off posure to either metal alone [37,38]. value. Levels of cadmium and lead in vegetables and cad- Selenium has been shown to protect the kidney from mium in freshwater fish from the endemic area are above oxidative stress [39]. A selenium concentration of 80– the maximum levels stipulated by certain Food Safety Au- 95 μg/l is needed to maximise the activity of the antioxi- thorities [22-24,44]. dant enzyme glutathione peroxidase and selenoproteins A provisional tolerable weekly intake (PTWI) for in plasma [40,41]. In this context, it is significant that cadmium of 7 μg/kg body weight was established by the serum selenium was below 80 μg/l in 38% and below Joint Food and Agriculture Organization of the United 90 μg/l in 63% of individuals with CKDu. Low selenium Nations (FAO)/WHO Expert Committee on Food Addi- levels may have been a contributory factor increasing tives (JECFA) [45]. In 2011, the JECFA revised the PTWI the vulnerability of the kidneys to oxidative damage for cadmium to 5.8 μg/kg body weight [46]. More re- caused by heavy metals and metalloids. cently, the PTWI for cadmium has been lowered to The association of raised serum strontium levels with 2.52 μg cadmium/kg body weight, in order to ensure a raised serum cadmium levels has been reported previ- high level of protection of all consumers, including ex- ously [42]. Strontium levels were not analysed in food or posed and vulnerable subgroups of the population [44]. water. The most likely explanation is an alteration of Since the cadmium content of certain food items in the strontium handling and excretion, owing to the effect of endemic area is above stipulated levels, the total weekly cadmium on renal tubular function. intake of cadmium in people living in the endemic area Cadmium levels have previously been reported to be could exceed these safe limits, with detrimental effects high in water sources in the domestic environment of on the kidneys, particularly in vulnerable people and people with CKDu, and 10–20 times the maximum stip- those with predisposing factors. ulated level have been found in reservoirs in the en- Reported mean dietary exposure to inorganic arsenic demic area [15]. The results of this study did not show in the United States of America (USA) and various Jayatilake et al. BMC Nephrology 2013, 14:180 Page 11 of 13 http://www.biomedcentral.com/1471-2369/14/180 European and Asian countries ranges from 0.1 to 3.0 μg/ There are several limitations in the study. Other kid- kg body weight per day [45]. Recently, the PTWI for ar- ney disease such as HIV nephropathy could fulfil the senic (0.015 mg/kg body weight per week) was with- case-definition criteria used for CKDu. As HIV is not drawn and environmental authorities are in the process prevalent in Sri Lanka, it was not excluded through of collecting more data for exposure assessment [46]. blood tests. The presence of glomerulonephritis was not The current recommendation is that every effort should excluded by biopsy but was based on past medical re- be made to keep concentrations of arsenic as low as rea- cords and diagnosis cards. The sensitivity and specificity sonably possible. The PTWI for lead is set at 0.025 mg/ of the case definition relative to biopsy-proven CKDu is kg body weight per week [45]. also not known. Stage 1 CKDu is defined by persistent Previous studies have reported high levels of cadmium in microalbuminuria and may overestimate the prevalence fertilizer (mean 47 μg/g) [15]. The maximum cadmium, of CKDu. The case definition required albuminuria. As a lead and arsenic concentrations in phosphate fertilizer result, people with CKDu who have a low eGFR and no from theendemic area in the present studywere30.8 μg/g, albuminuria were excluded from the study. In addition, 823.4 μg/g and 0.19 μg/g respectively. The maximum ac- the CKD-EPI equation used to estimate eGFR [25] has ceptable levels for cadmium, lead and arsenic, in phosphate not been validated in people from South Asia. It is not fertilizer product, at 1% of the nutrient level, are 4 μg/g, known whether the albuminuria of CKDu responds to 20 μg/g and 2 μg/g, respectively [47]. treatment for high blood pressure. If it does, an individ- The mean concentration of cadmium in soil from the ual could then be excluded based on their ACR, despite endemic area was 0.4 μg/g. Surveys of agricultural soils having the disease. in the USA and Sweden have reported lower soil cad- CKDu has been reported in other populations as well mium levels (0.265 mg/kg and 0.23 mg/kg respectively) [54-57]. Lessons learnt from other countries demon- [48,49]. The concentration of cadmium, arsenic and lead strate that sound public health policies to ensure access in soil, and their impact on body burden and excretion, to safe drinking water; regulatory control to ensure ap- is known to be influenced by many environmental factors propriate use of agrochemicals including fertilizer; haz- such as the pH of soil, buffering capacity, content of ardous waste remediation; regulatory control to prevent organic matter and water quality, among others [50-52]. pollution of the environment from discarded batteries Cadmium accumulation by plants, for example, is containing heavy metals; tobacco control; and reduction influenced by the reactive soil cadmium content and of air pollution can reduce exposure to heavy metals pH. It is decreased by high cation exchange capacity of [58,59]. Based on the findings of this study, the Govern- the soil and increased by higher soil temperature ment and the Ministry of Health of Sri Lanka have [49-52]. The hardness and high content of fluoride in already initiated multisectoral collaborative action with water in the endemic area may also influence the dy- the Ministries of Agriculture, Irrigation, Scientific Affairs namics of cadmium in soil, absorption by plants [17] and Social Services, to mitigate the exposure of people and excretion from the kidney. to environmental nephrotoxic substances. Steps are be- Certain pesticide residues were above reference levels ing taken to strengthen the water supply scheme in the in 31.6% of CKDu cases. Residues are demonstrative of endemic area as well as the regulations related to pro- the extent of the environmental distribution of pesticides curement and distribution of fertilizers and pesticides. and certain pesticides are known to be nephrotoxic Further studies are ongoing to investigate the contribu- [4,5,53]. Simultaneous exposure of people to heavy tory role of infections in the pathogenesis of CKDu. metals and nephrotoxic pesticides may be a contributory factor in the pathogenesis and progression of CKDu. Conclusions Compared to previous studies conducted on CKDu, The results of this cross-sectional study indicate that the present study has several strengths: (i) use of a con- multiple agents may play a role in the pathogenesis of sistent case definition for CKDu; (ii) analysis of a range CKDu. Herbal medicines and indiscriminate use of anal- of biological samples from individuals with CKDu; (iii) gesics are unlikely to be causative factors of CKDu. Re- comparison of control groups within and outside the sults show chronic exposure of people in the endemic endemic area; and (iv) use of sensitive analytical tech- area to low levels of cadmium through the food chain niques. Studies conducted hitherto to determine the and also to pesticides. They may also be exposed to lead prevalence and aetiology of CKDu [10,12,13,16,18,20] and arsenic through the food chain. Urine concentra- have relied on dipstick urinalysis to identify kidney dis- tions of cadmium and arsenic in individuals with CKDu ease. The present study is also the first in which heavy were at levels known to cause kidney damage. Significantly metals, metalloids and other elements in environmental higher urinary excretion of cadmium in individuals with and biological samples and pesticide residues in urine CKDu, and the dose–effect relationship between urine have been analysed. cadmium levels and CKDu stages, indicate that cadmium Jayatilake et al. BMC Nephrology 2013, 14:180 Page 12 of 13 http://www.biomedcentral.com/1471-2369/14/180 is a risk factor for the pathogensis of CKDu in Sri Lanka. work to be carried out to seek a sustainable solution for this public health problem is gratefully acknowledged. Deficiency of selenium and genetic susceptibility seen in individuals with CKDu suggest that they may be predis- Author details 1 2 posing factors for the development of CKDu when people Ministry of Health, Colombo, Sri Lanka. Management of Noncommunicable Diseases, World Health Organization, Geneva, Switzerland. World Health are exposed to nephrotoxins. Organization, Colombo, Sri Lanka. Received: 16 March 2013 Accepted: 16 August 2013 Additional files Published: 27 August 2013 Additional file 1: Details of sample preparation and analytical techniques. References Additional file 2: Table S1. Urine concentration of metals (sodium, 1. Gooneratne IK, Ranaweera AK, Liyanarachchi NP, Gunawardane N, Lanerolle potassium, calcium, magnesium, copper, zinc and titanium) in CKDu RD: Epidemiology of chronic kidney disease in a Sri Lankan population. cases. Table S2. Serum concentration of aluminium, chromium, selenium Int J Diabetes Dev Ctries 2008, 28:60–64. and strontium in CKDu cases. Table S3. Concentration of arsenic, 2. Nanayakkara S, Komiya T, Ratnatunga N, Senevirathna ST, Harada KH, Hitomi cadmium and lead in surface soil and in phosphate fertilizer, pesticides T, Gobe G, Muso E, Abeysekera T, Koizumi A: Tubulointerstitial damage as and weedicides, in the endemic area compared with a non-endemic the major pathological lesion in endemic chronic kidney disease among area. Samples of soil from vegetable plots from the endemic area were farmers in North Central Province of Sri Lanka. Environ Health Prev Med obtained from the vicinity of households with CKDu patients. 2012, 17:213–221. 3. Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S, Plattner B, Saran R, Wang AY, Yang CW: Chronic kidney disease: global dimension and perspectives. Abbreviations Lancet 2013, 382(9888):260–272. ACR: Albumin–creatinine ratio; AMPA: Aminomethylphosphonic acid; 4. 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