Chronic kidney disease (CKD) is a global health issue [1]. The causes of CKD, such as, diabetes mellitus, hypertension, chronic nephritis, acute kidney injury and nephrotoxins, such as arsenic and fluoride, have been discussed. However, in several areas of the world, there is a growing concern of CKD of unknown/uncertain etiology (CKDu), which cannot be attributed to hypertension, diabetes or other known etiologies [2, 3]. For example, in the Dry-zone of Sri Lanka, the epidemic of CKDu has been one of the most serious concerns for public health in the past two decades [4, 5]. The clinical features of CKDu in Sri Lanka are as follows [6, 7]:
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CKDu patients are typically male farmers, 40 to 60 years old, engaged in rice production for more than 10 years at the same area, and usually poor. However, the disease is also observed among women and young children in some parts of the affected regions; and the ratio of male and female is 2.4-3:1.
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The distribution of patients is patchy, i.e. in a village, 2–3 % of population no less than 18 years old are affected by CKDu, but in the neighboring village only a few kilometers away, no patients are found.
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No history of common diseases that cause chronic kidney disease, e.g. chronic nephritis, IgA nephropathy, cystic kidney, idiopathic hypertension and diabetes mellitus.
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Hypertension and edema are rarely found in CKDu patients.
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Typically, their urine is hypotonic with β2-microgloburin; and renal pathological change is mainly in tubules and interstitial tissues.
What is CKDu in Sri Lanka? Many researchers in Sri Lanka and outside have studied this issue; and multidisciplinary approaches have been done to investigate the etiology and clinical course, to search effective biomarkers for early diagnosis and interventions. Diagnostic methods of CKDu, especially in the early stage, is still controversial. To diagnose CKD, serum creatinine, eGFR, and serum Cystatin-C are commonly used as biomarkers. To confirm CKDu, a renal biopsy is the gold standard; however, it is not always performed for all CKD patients [8]. Recent studies reveal that the ability of renal tubule reabsorption begins to decrease at least 10 years preceding the CKDu diagnosis. Minor changes in a urine laboratory test were frequently found in children in the affected areas [9].
As urinary biomarkers of renal tubule activity, albumin and low molecular proteins, which is constantly secreted from glomerulus and absorbed by normal renal tubules have been used, e.g. urine albumin to creatinine ratio (UACR), Kidney injury molecule 1 (KIM-1), Neutrophil gelatinase-associated lipocalin (NGAL), beta 2 microglobulin (B2M), Fibrinogen, Clusterin, Cystatin-C, Osteopontin (OPN), alpha 1 microglobulin (A1M), tissue inhibitor of metalloproteinase 1 (TIMP1), and Retinol binding protein 4 (RBP4) have been examined [10–15].
As the etiology, there are more than 30 factors, such as drinking water contamination of trace metals/metaloids (fluoride, cadmium, arsenic, lead, uranium, and others) [9, 16–27]; hardness of water [28]; mycotoxins [29, 30], snake venoms [31–33], agrochemicals (insecticides, herbicides, and fertilizers) [34–40] and dissolved organic matter (DOM) [41]; infection of Thailand orthohantavirus [42, 43], leptospirosis [44]; dehydration [45–47]; aristolochia [48]; staple diet [49]; and genetic factors [50], have been investigated. Recent retrospective and prospective studies suggested that in addition to the geogenic components, disease manifestation requires (A) prolonged exposure to environmental nephrotoxins and factors, (B) interactions among ions (Ca2+, PO4 − 3, F−, and Mg2+), and (C) vulnerabilities of the person, such as chronic dehydration, antioxidant and micronutrient deficiencies [51–53].
Pesticides are major environmental chemicals that farmers are occupationally exposed to in Sri Lanka, and can be nephrotoxins. Modern conventional agriculture using chemical fertilizers and pesticides achieved an increase in rice production; but not yet enough to export [54]. Although serum dichlorodiphenyltrichloroethane (DDT), p,p'-dichloroethylene (p,p'-DDE), urine 2,4-D, 3,5,6-trichloropyridinol, p-nitrophenol, 1-naphthol, 2-naphthol, glyphosate, AMPA, imidacloprid, N-desmethyl-acetamiprid (DMAP), clothianidin, and thiamethoxam have been detected in the urine of CKDu patients in Sri Lanka [34–40], whether any of these agents have a causative role in the etio-pathogenesis of CKDu has not been established. In 2013, the Sri Lankan government banned four pesticides when the renal toxicity had been reported, i.e. carbaryl, chlorpyrifos, carbofuran, and propanil [55]. They also banned glyphosate import in October 2015 following a campaign over the fears the chemical causes CKD. However, after agricultural organizations pointed out there was no study linking CKD to glyphosate, the import ban was lifted in July 2018; and its use was restricted to tea and rubber plantations [56]. In 2017, 658 tons of insecticides including three neonicotinoids, 1298 tons of herbicides, and 664 tons of fungicides were imported to Sri Lanka as formulations [57]. The active ingredients of three neonicotinoid insecticides, were imidacloprid (6.4 tons), thiamethoxam (2.2 tons), and acetamiprid (3.9 tons), two organophosphates, profenofos (97 tons), and diazinon, (11 tons), one carbamate, BPMC (12 tons), one phenylpyrazole, fipronil (1.3 tons), and one antibiotic, abamectin (1.3 tons).
Neonicotinoids and renal disease. Neonicotinoids are systemic insecticides with competitive modulator actions on nicotinic acetylcholine receptors (nAChR) in invertebrates and vertebrates including mammals [58–60]. They are lethal to targeted pests and untargeted natural enemies and induce the global collapse of entomophauna and the resurgence of pests [61]. Their half lives in plant and in the environment (soil and water) were much longer than those of organophosphate insecticides, and exert a serious impact on ecosystems in many countries [62, 63]. Preventive use of neonicotinoids and fipronil may disrupt ecosystem service [61, 64].
Neonicotinoids exposure may cause renal dysfunction, which supposed to be caused by human nAChR modulation. The acute toxicity of neonicotinoids for humans had been said to be not as strong as organophosphates; therefore, the use of neonicotinoids is growing rapidly as alternatives to organophosphates. Neonicotinoids formulation containing imidacloprid, acetamiprid and thiacloprid caused acute intoxication by ingestion such as cardiovascular symptoms (tachycardia, bradycardia, arrhythmia, hypertension, hypotension), neurological symptoms (low Glasgow Coma Scale, unconsciousness, sleepiness, dizziness, convulsion, excitation), respiratory symptoms (dyspnea, tachypnea, respiratory arrest, cough, cyanosis), gastrointestinal symptom (nausea, vomiting, diarrhea), secretion symptoms (diaphoresis, anhydrosis, excessive discharge of saliva and bronchial secretion, mouth dryness), pupil symptoms (mydriasis, miosis, abnormal light reflex), abnormal body temperature (fever, low body temperature), skeletal muscle symptoms (muscle weakness, muscle spasm, high creatinine kinase in blood test), metabolic acidosis, and death [65, 66]. Metabolic acidosis is commonly caused by renal tubular disorders. An in vivo study showed that nicotine caused CKD by direct effects on tubular protein reabsorption via α7-nAChR [67]. Another in vitro study showed nicotine induced podocyte apoptosis through reactive oxygen species generation and association [68]. In addition, the pesticide formula contains some additives as surfactants and solvents, which are more toxic than the active substances [69–71]. Common neonicotinoid formulations contain renal toxic additives, such as dimethyl sulfoxide, N-methylpyrrolidone, diethylene glycol, propylene carbonate and mineral oil. Multiple acute kidney injury (AKI) episodes may cause CKD as the final stage of chronic renal pathological conditions [72].
Subacute and chronic neonicotinoids exposure may also cause tubular disorders. We previously reported in our experience that the consecutive intake of tea beverage and/or fruits contaminated with neonicotinoids, may cause similar symptoms as acute intoxication. Some typical symptoms were significantly found in patients with neonicotinoids detection in urine, i.e. general fatigue, headache, chest pains, palpitation, stomachache, muscle pain/ spasm/ weakness, shoulder stiffness, cough, fever (> 37°C), and finger tremor, as well as electrocardiographic abnormalities [73–75]. In those cases, oliguria and the increase of urine Cystatin-C were found [75]. It was assumed oliguria was caused by renal hypoperfusion by nAChR action and increase of urine Cystatin-C was caused by direct or indirect tubule action by neonicotinoids. These patients also complained of dizziness upon standing, skin eruptions, sleeplessness, edema, low urine volume, high urine volume, constipation, diarrhea, skin itching, appetite loss, reduced body weight, and increased body weight. In addition to those symptoms, altered consciousness/dreamy state, recent memory loss with compulsive behaviors, agitation/fear/anger, sudden change of senses of smell, auditory or visual hallucinations, and abnormal behavior [75], which had been reported in a case study of myasthenia gravis patients anti-nAChR antibody were positive [76] were also complained of occasionally. We hypothesized that neonicotinoids caused those symptoms through nAChR action, because all those symptoms were almost always reversible in neonicotinoid intoxication. Chronic occupational exposure of neonicotinoid formulations containing imidacloprid caused renal disorders, such as hematuria and interstitial nephritis, as well as liver dysfunction and leukoclastic vasculitis [77]. In animal study, e.g. 0.2 and 0.4 mg/kg/day thiamethoxam for 15 days to male mice caused renal pathological change in parenchyma [78].
Neonicotinoids absorbed via the intestines and lungs, pass through blood brain barrier and are mainly excreted in the urine [79, 80]. They do not bio-accumulate since they are water soluble; however, their concentrations in tissues may remain at a steady state, or even increase, through continuous exposure [79, 81]. An active neonicotinoid metabolite, N-desmethyl-acetamiprid (DMAP) and dinotefuran pass through placenta and were detected in the urine of newborns [82]. As the route of neonicotinoid exposure in agricultural areas, occupational use and food/beverage intake can be considered.
There is scientific evidence that neonicotinoids and the metabolites are frequently detected in human urine samples from healthy volunteer as well as the patients with neonicotinoid intoxication [74, 75, 79, 81, 82]. DMAP is one of the most frequently detected metabolite of acetamiprid. Spot urine is one of the most popular matrices to use in screening human environmental chemical exposure, such as heavy metals [25], water-soluble pesticides [74, 83], plasticizers [84], parabens [85], perchlorate [86], PAHs [87], phytoestrogens [88, 89], nicotine [90], caffeine [91], and personal care products [10, 92]. Whether urine is an appropriate sample for evaluating neonicotinoid exposure in CKDu patients or not is unknown, because of their low urine concentration ability.
To elucidate the neonicotinoids exposure in people living in CKDu-epidemic area by urine analysis can be the first step of an appropriate pesticide regulation to reduced the patients suffered by CKDu.