Elucidating environmental factors and their combined effects on CKDu in Sri Lanka using zebrafish.

Introduction

Chronic kidney diseases of uncertain etiology (CKDu) were increasingly reported globally with a high prevalence discovered in Central America, Egypt, India, Sri Lanka, and other regions of Asia (Jayasekara et al., 2013; Weaver et al., 2015). CKDu is closely related to the multiple environmental factors and living habits, posing risks to public health by impacting the quality of drinking water and food sources (Chandrajith et al., 2011b; Hettithanthri et al., 2021; Nayak et al., 2023; Wanigasuriya, 2014). CKDu frequently developed with progressive kidney damage and is evidenced by the structural and gradual decline of functional irreversible abnormalities, which is clinically defined as a sustained loss of renal function and determined as a fall in glomerular filtration rate (GFR) < 60 mL/min or the presence of albuminuria (Levey and Coresh, 2012; Stevens and Levin, 2013). The gradual loss of kidney function and irreversible changes in renal structure, and the progression may cause finally death in severe cases. Previous studies mostly focused on the assessment of environmental factors, agrochemicals, lifestyle, and genetic predisposition on inducing CKDu (Friedman and Luyckx, 2019). However, there are still unsolved problems whether and how those environmental factors or renal toxins in drinking water matter for Sri Lanka and other countries that are suffering from CKDu-related high morbidity and mortality incidences.

Among multiple environmental toxins, heavy metal bioaccumulation (cadmium, Cd, aluminum, Al, etc.), fluoride, hardness of water, cyanobacterial toxins, and disrupted intestinal microbiota were suggested as the possible pathogenic factors leading to CKDu prevalence (Kulathunga et al., 2019; McDonough et al., 2020; Wimalawansa, 2016). For instance, the higher hardness and fluoride levels in drinking water were considered the important parameters to induce kidney diseases in the north-central province (NCP) and dry regions of Sri Lanka (Chandrajith et al., 2011a; Dharma-Wardana, 2018; Fernando et al., 2020). The hardness of water was classified as soft, moderately hard, hard, or very hard, respectively, based on calcium (Ca) and magnesium (Mg) content 0–60, 61–120, 121–180, and >181 mg/L (Water Hardness Classification of the United States Geological Survey, https://water.usgs.gov/owq/hardness-alkalinity). The World Health Organization (WHO) set the levels of hardness water and fluoride in water at 250 mg/L and 1.5 mg/L, respectively, while the detected content in local drinking water samples of Sri Lanka was relatively higher at 516 mg/L and 5.47 mg/L (Rubasinghe et al., 2015; Wickramarathna et al., 2017). Notably, the serum fluoride was the highest at 9.58 mg/L in Sri Lanka CKDu cases, while the urine fluoride was detected to be apex at 6.92 mg/L (Fernando et al., 2020). Moreover, the doses of Al and Cd were also reported in epidemic investigations on the dry region in Sri Lanka to 191.2 and 0.5 ?g/L in drinking or well water, 152.1 and 60 ?g/L in raw or reservoir water, against WHO guidelines at 200 and 3 ?g/L levels, respectively (Dharma-Wardana, 2018; HMA et al., 2018; Wanigasuriya et al., 2011; Wasana et al., 2017). In particular, the leaching of heavy metals and their incorporation into food items, drinking, and freshwater systems with various fluoride concentrations, contribute to major pathways of human exposure and impede kidney development at early stages (Babich et al., 2020). Thus, the evaluation of water quality by acute exposure to aquatic animals was of great significance for human health and criteria, including the toxicity of metals potentially influenced by hardness (expressed as CaCO₃) (Li et al., 2021a; Wu et al., 2013). Additionally, as a serious toxin in the aquatic system, the microcystin-LR (MC-LR) was reported to induce not only the liver but also kidney damage in zebrafish via polluted water (Chen et al., 2018). Overall, the multiple environmental factors including fluoride, hardness, heavy metals, organic matter, agrochemical residues, and bacterial toxins in drinking water were highly suspected critical for Sri Lanka and similar regions with a high prevalence of CKDu or chronic kidney disease of multifactorial origin (CKDmfo) (Imbulana and Oguma, 2021; Pinto et al., 2020; Wimalawansa, 2020). The effects on females and males may be different from the disease incidence rate (Senevirathna et al., 2012). Thus, we suppose that it should be investigated using both genders of zebrafish to simulate the hazardous implications of polluted drinking water in CKDu incidence regions, with the exposure concentrations based on the environmental and detected levels of patients or water samples, and pre-experiments (data unpublished). Although previous studies have focused on the different variables potentially inducing CKD, how those environmental factors cause kidney disorders in the exposed communities from molecular pathways and omics aspects is still unknown.

Zebrafish is considered a credible model for studying CKDu induced by environmental pollutants, with advantages of a small body, transparent embryos, and modeling of genetic kidney disease, acute renal failure (ARF), or acute kidney injury (AKI) (Morales Fenero et al., 2021; Poureetezadi and Wingert, 2016; Schenk et al., 2017). Zebrafish and their special transgenic lines with kidney tissues labeled GFP were cooperatively utilized to build AKI models and other renal impacts to reveal human diseases, because of their high similarity to human genes and kidney functions (Jerman and Sun, 2017; Outtandy et al., 2019). CKDu pathological characteristics were revealed as interstitial fibrosis, interstitial inflammation, glomerulosclerosis, and tubular atrophy in Sri Lanka (Redmon et al., 2014; Wijetunge et al., 2013). Kidney function is mainly achieved by first filtering the blood and then recovering useful ions and small molecules by directed epithelial transport, performed by the renal functional units of nephrons (McKee and Wingert, 2016). The zebrafish model possesses similar cell types and shared developmental mechanisms, which contribute to evaluating the risks of environmental pollution by modeling human kidney organogenesis (Drummond and Wingert, 2016). Therefore, the wild-type and transgenic with GFP-labeled zebrafish lines were applied to explore the potential impacts of various environmental factors with acute and chronic exposure in leading to kidney damage or diseases.

Acute or chronic exposure to environmental factors probably negatively disrupts the critical metabolites of kidney injury molecule (Kim-1), creatinine, creatine kinase (CK), urea nitrogen (BUN), etc (Jayatilake et al., 2013; Rysz et al., 2017). In our previous study, the local water collected from Sri Lanka, as well as the simulated high hardness and fluoride water, were detected to induce kidney damage in zebrafish larvae after acute exposure (Yang et al., 2022). Hence, this study examined the toxicity of acute and chronic exposure to multiple environmental factors by simulated local drinking water, on zebrafish kidney development, tissues damages, response of transcriptome with DEGs and signaling pathways, and regulation of gut microbiota. Furthermore, we explored the in-depth correlation between the major bacteria and environmental factors, developmental indexes, and critical metabolites and DEGs, which can better understand the relationships between kidney disease induced by environmental factors and host response.