Exposure to Sri Lanka’s local groundwater in a CKDu prevalent area causes kidney damage in zebrafish.

Since the mid-1970s, chronic kidney disease of unknown etiology (CKDu) attracted global attention (Dharma-Wardana et al., 2015; JH. et al., 2014), which etiology excludes commonly known risk factors, such as diabetes mellitus, hypertension, and glomerular nephritis (Woo et al., 2012). CKDu was frequently reported with tubular atrophy, interstitial fibrosis (Abraham et al., 2019; Nanayakkara et al., 2012a), and acute kidney injury (Liano et al., 2010; S. et al., 2014). CKDu cases were also found in Asia, Central America, and the Balkan Peninsula (Pearce et al., 2019), and became a significant burden on public health and health care systems (Hettithanthri et al., 2021). In Sri Lanka, CKDu was first reported in 1994 (Jayasumana et al., 2015b; Wimalawansa, 2016), and higher incidences were mainly found in Anuradhapura and Polonnaruwa of the North Central Province (NCP) (Senevirathna et al., 2012). The age of most of CKDu patients ranged from 40 to 60 years old, and the majority were male farmers (Carlos et al., 2011; Ranasinghe et al., 2019). Because of the slow progressive loss of renal functions, CKDu patients often get worst slowly (Gunatilake et al., 2014). However, the etiological cause of CKDu is still elusive. Previous studies implied that the development CKDu may be related to many environmental factors, such as high fluoride (Chandrajith et al., 2011a; Nanayakkara et al., 2020), hard water (Gobalarajah et al., 2020), heavy metals (cadmium, plumbum, and arsenic) (Chandrajith et al., 2020; Fernando et al., 2020a; Jayasumana et al., 2013; Navarathna et al., 2021), the organic carbon in shallow groundwater (Makehelwala et al., 2019), heat stress and dehydration (Jayasekara et al., 2019; Nerbass et al., 2017), pesticides (Jayasinghe et al., 2019; Jayatilake et al., 2013), as well as genetic background (Nanayakkara et al., 2014).

In Sri Lanka, particularly in rural communities of dry zones, over 87% of the population drink groundwater directly extracted from shallow wells (10 m depth) or deeper wells (about 60 m) (Dissanayake and Chandrajith, 2018). Previous studies revealed that the local water possesses high hardness and high fluoride in the CKDu prevalent area (Chandrajith et al., 2011a, 2011b, 2012; Dissanayake and Chandrajith, 2019; Jayasumana et al., 2014; Wickramarathna et al., 2017), which is also confirmed by the overlap distribution of hardness, fluoride, and CKDu prevalence (Fig. 1). Chronic exposure to high levels of fluoride was reported to affect the functions of the liver, kidney, heart, lungs, brain, nervous system, and reproductive systems (Kumari and Kumar, 2011; Nakamoto and Rawls, 2018; Zuo et al., 2018). Wang et al. found that long-term drinking of high fluoride (>25 mg/L F^–) water can hurt kidney structure and inhibit renal cell proliferation (Wang et al., 2020). The damaged kidneys can accumulate more fluoride, resulting in further injuries in the kidney, bone, and other organs (Nanayakkara et al., 2020). A previous study showed that significantly higher fluoride in serum and urine was found in CKDu patients, compared to endemic healthy control (Fernando et al., 2020b). The accumulation of fluoride in the body can further aggravate renal tissue damage along with the progression of CKDu (Nanayakkara et al., 2020). Moreover, water hardness is significantly higher in the CKDu regions, compared to non-CKDu regions (Dissanayake and Chandrajith, 2019; Liyanage et al., 2022).

Zebrafish are widely used in toxicological studies because of their rapid organogenesis, sensitivity to harmful chemicals, and high homology with the human genome (Dai et al., 2014). In this study, we aim to investigate whether Sri Lanka’s local groundwater in a CKDu prevalent area can cause kidney damage using the zebrafish model, which may provide valuable evidence for the pathogenesis of CKDu. Thus, we assessed the renal toxicity after exposure to drinking groundwater collected from CKDu regions in Sri Lanka, which was detected to be high hardness and fluoride. Further, human 293T cells were used to validate the disturbing signaling pathway after exposure to local groundwater. To our knowledge, this is the first report of exposure to original local groundwater in a CKDu prevalent area using the zebrafish model, and our results implied that high water hardness and high fluorine in Sri Lanka’s local groundwater is likely to be potential reasons for the etiological of CKDu.