Metabolome evidence of CKDu risks after chronic exposure to simulated Sri Lanka drinking water in zebrafish.

Chronic kidney diseases of uncertain etiology (CKDu) have emerged as significant public health risks, which are linked to various environmental factors such as local water sources, agrochemicals, lifestyle choices, and genetic predisposition (Friedman and Luyckx, 2019, Hettithanthri et al., 2021, Pinto, 2020). Among environmental toxins, the high bioaccumulation of fluoride (higher as 5.47?mg/L), hardness (516?mg/L), heavy metals (60 and 152?µg/L), and cyanobacterial toxins (microcystin-LR, MC-LR at 2.6?µg/L) in drinking water were considered pathogenic factors inducing CKDu in the north dry regions of Sri Lanka country (Chandrajith et al., 2011a, Kulathunga, 2019, McDonough et al., 2020, Wimalawansa, 2016). The environmental factors of fluoride, hardness, and heavy metals in water were highly suspected to be the critical factors in Sri Lanka and similar regions with a high prevalence of CKDu, for their higher levels than WHO standards (Chandrajith, 2011b, Imbulana and Oguma, 2021, Pinto, 2020). Despite previous assessments of various environmental factors in water and food, the full extent of their effects and pathways on the risks of CKDu, particularly regarding metabolite disruptions, remains incompletely understood. Notably, higher concentrations of specific toxic substances, such as nitrate and fluoride, as well as heavy metals like cadmium (Cd) and aluminum (Al), have been reported in local drinking water and daily food intake, posing potential kidney risks as detected in CKD patient samples (Fernando, 2020, Imbulana and Oguma, 2021, Wasana et al., 2016). Until now, most of candidates leading to CKDu were hypothesized but not identified completely by using animal models or checking from the local population, except fluoride and cadmium exposure (Gunasekara et al., 2023, Jayatilake et al., 2013, Priyadarshani, 2023, Sandanayake et al., 2023). Thus, the investigation and in-depth analyses of metabolome and mechanisms by correlation with the omics analyses are still necessary to reveal the impacts of drinking water to avoid the high CKDu incidence around the world.

The identification of causal genes and the mechanism is hindered by few animal models, thus it is highly worth exploring classical animals of mice and rats and leveraging species diversity in zebrafish to develop new models that are more relevant to human kidney disease and therapies (Garrett and Korstanje, 2020). To investigate the toxicity of pollutants in water systems, such as organic chemicals and heavy metals, the zebrafish model can be utilized due to its high similarity to human genomics, renal functions, and multiple disease models (Poureetezadi and Wingert, 2016, Schenk et al., 2017). Furthermore, zebrafish kidney disease models exhibit comparable cell types and shared developmental mechanisms (Drummond and Wingert, 2016, Drummond and Davidson, 2016). More importantly, zebrafish have been developed as models for acute kidney injury, drug screening in genetic kidney diseases, and chronic kidney disease with similar nephrotoxic responses and injury biomarkers similar to those of mammals (Fatma et al., 2021, Gehrig, 2018). Compared to mice, pigs, and salamanders, zebrafish can be made toxin-induce kidney disease models and whole-animal models for nephrotoxicity screening, with advantages of high-throughput, gene-modified, small size, low cost, large quantity, the feasibility of live-time imaging with fluorescent monitoring, and rapid therapeutic discovery before translation (Bauer et al., 2021, Hukriede et al., 2022, Liang and Liu, 2023). In this study, zebrafish were employed to investigate the potential kidney damage and disease risks associated with prolonged exposure to multiple environmental factors.

The latest research on population indicated the characteristics, prevalence, and risk factors of kidney disease, which supported previous hypotheses of etiology, such as variably linked to environmental exposures, nephrotoxic medications, and/or genetic susceptibility, and the importance of CKD screening (Strasma et al., 2023). The discovery and identification of environmental factors inducing the CKDu epidemic are critical with the screening of potential biomarkers from transcriptomic and metabolomics for early evaluation and kidney disease therapy. For example, environmental fluoride exposure revealed potential pediatric kidney health risks with potential urinary biomarkers (Gunasekara et al., 2023). However, the biomarkers used in the previous studies were known typical ones like urinary kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and albumin-creatinine ratio (ACR), indicating the necessary for more novel and special discoveries (Gunasekara et al., 2023). Among the different profiles, metabolomics, proteomics, and lipidomics (a branch of metabolomics) are powerful approaches to elucidating the exposure of environmental factors in zebrafish with huge potential for more sensitive biomarkers or pathways (Afshinnia et al., 2018, Pereira et al., 2022, Rysz, 2017, Ye and Mao, 2016). The application of metabolomics and multi-omics systems is requested for risk factors, early diagnosis, or to identify therapeutic targets of chronic kidney disease (Cisek et al., 2016, Davies, 2018, Saucedo et al., 2018). Therefore, this study focused on the alternation of metabolomics profile in zebrafish to explore the potential relationships to human kidney health.

As the common complications of metabolic and gastrointestinal outcomes, CKDu is closely influenced by the metabolome and gut microbiota in organisms on metabolism and inflammatory aspects (Mafra et al., 2019, Plata et al., 2019). However, it is still unrevealed whether the CKDu risks will company with the disrupted metabolites, and changed transcriptome and gut microbiota in organisms. In our prior research, we observed that both the local water and simulated high-hardness and fluoride water led to kidney damage in acutely exposed zebrafish larvae (Yang et al., 2022). Importantly, our recent work discovered the basic risks of environmental factors on zebrafish kidney damage and the disruption of transcriptome and gut microbiota after chronic exposure (Jia et al., 2023). Moreover, the pathological process of the pancreas, which possesses both endocrine and exocrine functions and surrounding tissues, was considered to be associated with CKD (Verma and Despa, 2023). For further exploration of pathogenic mechanisms, this study examined the kidney and pancreas tissue damage, and disruption of metabolome profile in chronic treated zebrafish. The critical signaling pathways in metabolomics were elucidated through KEGG pathway analyses, and potential biomarkers were identified by correlating with the disrupted DEGs and bacteria in transcriptome and intestinal microbes of both gender zebrafish. This approach provided deeper insights into the correlations between kidney disease and host regulations from omics analyses. Further, novel candidates screened from comprehensive profiles provide more probability for specific drugs and recognize the development and progression of CKDu.

3. Results

3.1. The histopathological analysis of chronically exposed adult zebrafish

4. Discussion

Previous studies have suggested that CKD is influenced by various factors, particularly drinking water. However, existing evidence and persistent gaps in epidemiological studies conducted in Sri Lanka indicate the need for further research (Pett et al., 2022). Thus, the risks and mechanisms of CKD should be explored by exposure to key factors using the credible models of zebrafish with detection of kidney development and function (Cirio et al., 2015, Jerman and Sun, 2017, Morales and Wingert, 2017, Outtandy et al., 2019). In CKDu patients, the pathological phenomena included renal biopsies, tubular atrophy, glomeruli capsule thickened, glomerular sclerosis, expanded interstitial space, and inflammatory infiltrate (Athuraliya et al., 2011). In this study, the kidney tissue was isolated and detected by histopathological analysis with H&E stain to explore the health risks, according to zebrafish adult (mesonephric) kidney location and the nephron including glomerulus, proximal and distal tubules. The zebrafish kidney had no significant lesions in control, and the glomerulus, proximal and distal tubule, and interstitial stroma appeared normal. Conversely, tubular injuries and micro-villi ruptures, are obvious in the HF and combined exposure groups. The zebrafish have similar proximal tubules to the mammals, which show the brush border and high columnar epithelial cells. These renal structures of the glomerular filtration barrier ensure to reabsorb of the most filtered salts, sugars, and small proteins in the zebrafish. The tubule and brush border injuries were suspected to lead to abnormal renal function and the incidence of kidney diseases under chronic pressure.

The kidney histopathology of whole-body sections and ultra-structure, as per our previous study, revealed the presence of mesonephric nephrons with eosinophilic deposits, tubular regeneration, tubular epithelial cell death, plasma cell infiltration, vacuolar degeneration, cellular abnormalities, and pyknosis/karyorrhexis (Jia et al., 2023). The histopathological analysis of isolated kidney tissues in this study showed moderate kidney damage in female and male zebrafish exposed to different factors in water. Moreover, the histopathological score of pancreases showed that the tissue damage among the exposed groups especially the combined HFMM exposed zebrafish, including the pancreatic cell arranged irregularly, denatured, necrosis, cell membrane rupture, cytoplasmic release, and the inflammatory cell infiltration at some extent. As an important organ in both the endocrine and the digestive systems, the pancreas is closely connected to other splanchnic organs of the liver, spleen, and intestine with the common blood supply, and also relates to kidney tissues on the kidney-pancreas transplantation and diabetic kidney disease (DKD) aspects (Tang et al., 2020). Here, the histopathological analysis of kidney and pancreas tissues indicated the negative impacts of environmental factors, but it is still difficult to use the pancreas and gut, liver, and gonad to indicate CKD risks in the chronically exposed zebrafish for the incomplete consistent damage scores of these organs. The dysbiosis of the gut-kidney axis contributed to CKD with the link of gut microbiota, pathological barrier loss, and gut derivatives’ metabolic disorders (Feng et al., 2021, Kim and Song, 2020, Mertowska, 2021). Although disruption of gut microbiota was observed, the histopathological analysis of the gut and liver appeared no difference compared to the control. Therefore, it still needs to explore the microstructure abnormal and inner metabolic connection between the kidney and other key organs under the disease risks.

Previously, metabolomics studies revealed that the deviations in metabolic profiles indicated renal pathologies. The progression of CKD was promoted by the disruption of lipid, carbohydrate, amino acid, nucleic acid metabolism, and the TCA cycle, which can provide powerful insights into the pathogenesis and new therapeutic methods (Cargill and Sims-Lucas, 2020, Gagnebin et al., 2018, Hocher and Adamski, 2017, Wang et al., 2019). Except for typical NGAL, BUN, Kim-1, etc, three classifiers of plasma, urinary, and urinary peptide-based metabolites were previously analyzed for the correlation as the novel biomarkers (Rysz et al., 2017). In this study, the metabolome in stimulated water exposed zebrafish was significantly disturbed, especially the following metabolites in the HF group related to renal functions: PS(24:1(15Z)/24:0), germacrenone, ajaconine, montecristin, L-pyridosine, 3-O-p-trans-coumaroylalphitolic acid, NADH, N-hexadecanoylsphinganine-1-phosphocholine, 20-hydroxyeicosatetraenoic acid, 4-hydroxyretinoic acid, 20-hydroxy-LTE4, and 17-hydroxy-E4-neuroprostane. In the HM-exposed zebrafish, the renal-related metabolites were significantly disrupted by the Al and Cd in water, especially the vitamin D, phenol sulfate, nicotine glucuronide, ADP, 6-hydroxypentadecanedioic acid, goshuyic acid, cucurbic acid, buprenorphine glucuronide, tetradecanedioic acid, S-lactoylglutathione, and 5-aminopentanoic acid. In the MC-LR groups, it involved the NADH, PC(18:3(6Z,9Z,12Z)/16:0), (±)?2-propylthiazolidine, bitolterol, 10,20-dihydroxyeicosanoic acid, vitamin D, hexadecanedioic acid, and withanolide B, 20-hydroxyeicosatetraenoic acid, 5b-cyprinol sulfate, threoninyl-tryptophan, D3, isoleucyl-tyrosine, isolimonic acid, and isoleucyl-Tryptophan. In the HFMM exposed zebrafish, significantly changed the 7,8-dihydro-L-biopterin, PS(24:1(15Z)), hexadecanedioic acid, glycyl-Lysine, glutaric acid, hydroxyphenyllactic acid, (±)?2-propylthiazolidine, 20-hydroxy-LTE4, aspartylglycosamine, L-ascorbic acid, threoninyl-tryptophan, N-jasmonoyltyrosine, cortisone acetate, lysoPE and lysoPC, seryllysine, PS(14:1(9Z)), 20-oxo-leukotriene E4, (S)-HDAC-42, and citreoviridin C. Those significantly disrupted metabolites linked to key KEGG pathways, such as lipid, carbohydrate, amino, nucleotide, energy, cofactors and vitamins metabolism, implying the effects of multiple factors on the renal functions. For example, the acylcarnitine, amino acid, lysophospholipid, and carbohydrate metabolism were altered in the CKD progression, and most amino acids catabolized in the kidney and filtered amino acids were almost completely reabsorbed by the proximal tubule into the blood (Kordalewska et al., 2019, Li et al., 2020). Notably, lipid, carbohydrate metabolism, and energy metabolic disruption can be affected by the damaged kidney (Li et al., 2020, Meijers, 2018). Metabolites of arachidonic acid such as 12(S)-hydroxyeicosatetraenoic (12(S)-HETE) were increased in kidney disease and associated with renal function decline (Xu et al., 2022). Glycosphingolipids are highly abundant in the kidney and their accumulation contributes to several common pathologies including end-stage kidney disease, which implicated the role of these lipids in diseases resulting from metabolic syndrome (Mather and Siskind, 2011). Further, vitamin D and its metabolites were considered to relate to chronic kidney disease by regulation of phosphatidylinositol 3 kinase/AKT, MAPK, NF-?B, and Ca²⁺ signaling pathways (Hsu, 2023, Samuel and Sitrin, 2008). Thus, metabolic disruption by environmental factors can indicate damage to kidney development and functions (Gao et al., 2014, Zhang et al., 2015). Therefore, the key metabolites and changed pathways in zebrafish possibly discovered the renal functions or kidney disease risks induced by exposure to multiple environmental factors from drinking water. The interactions between metabolomics and physiology as well as exposure factors will potentially reveal the host functions and CKD pathogenesis (Huang et al., 2019, Tajti et al., 2020, Xu, 2018). In this study, the correlation of dominant metabolites and various factors, histopathological score, and selected DEGs in transcriptome showed obvious links in the chronically exposed zebrafish.

The renal damage and the HF factors were more related to the metabolite changes than the pancreas tissue analysis and HM and MC-LR factors, respectively. The critical “kidney” and “renal” related DEGs of ptpn11b, akt1, jun, ca2, egln1b, pkhd1l1, egln3, gls2b, FO704813.1, and slc2a1a checked in our previous work were deeply related to the changed metabolites in this study. The enhanced DEGs related to “pancreas” and “liver” in zebrafish were screened and analyzed to be positively and negatively related to the changed metabolites in the individual group, indicating the potential connection between organs in kidney disease. Moreover, the KEGG analysis of metabolomics, reveals the significantly enriched pathways of oxidative phosphorylation, calcium signaling pathway, folate biosynthesis, metabolism of thiamine, ascorbate and aldarate, arachidonic acid, pyruvate, purine, phenylalanine, sphingolipid, FoxO signaling pathway, lysosome, AGE-RAGE signaling pathway in diabetic complications, adipocytokine signaling pathway, necroptosis, citrate cycle (TCA cycle), vascular smooth muscle contraction, lysine degradation, and fatty acid degradation. Notably, the disrupted crucial pathways of calcium signaling pathway and necroptosis were the shared significantly changed pathway of metabolome and transcriptome in exposed zebrafish. The dysregulation of Ca²⁺ signaling and Calpains, aerobic glycolysis, fatty acid metabolism, utilization of tryptophan, TCA cycle, and the downstream metabolites may contribute as biomarkers to the renal abnormality in terms of autophagy, injury, epithelial cell growth, renal cell carcinoma, and tubular morphogenesis and function (Kaushal, 2020, Lemos and Ehrlich, 2018, Lucarelli et al., 2019). Metabolomics plays an important role in assessing environmental toxicity and health risks, providing altered metabolites as response biomarkers in patient samples, laboratory animals, or wildlife (Gu et al., 2023, He et al., 2022, Jadhav et al., 2019). Thus, the different changes in zebrafish metabolome and the correlation between environmental factors indicated potential CKDu risks by various regulating pathways after chronic exposure.

As for mechanisms of CKDu progression, the investigation should focus on transcriptome and metabolome pattern, but also pay attention to the relationship of gut microbiota (gut-kidney axis) in the organism, which always altered and increased risks in CKD patients to sensitively warn at early stages (Al Khodor and Shatat, 2017, Chen et al., 2019, Hobby et al., 2019, Wang et al., 2020). Our previous study showed that the disrupted dominant phyla and significantly changed genera including Pseudomonas, Rhodobacter, Rhizobiales_Incertae_Sedis, and ZOR0006 were selected to explore the environmental factors’ effects on the kidneys of females and males, according to the Spearman correlation outcomes (Jia et al., 2023). The richness of Firmicutes and Bacteroidetes, Proteobacteria were notably linked to host aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and p-cresol/p-cresyl sulfate metabolism, which were the products of gut bacterial metabolism, to regulate the CKD progression (Cigarran Guldris, 2017, Kulathunga, 2020, Lau et al., 2018, Meijers et al., 2019, Wu et al., 2020). In this study, the changed metabolites of 12S-HHT, G2 2-glyceryl Ester, L-Pyridosine, 7,8-Dihydroneopterin, 20-hydroxy-LTE4, 6-O-Acetylaustroinulin, 20-Hydroxyeicosatetraenoic acid, D-Glucuronic acid, 4-Hydroxyretinoic acid, 12-HEPE 24,25-Dihydroxyvitamin D, phenol sulfate, lysoPE(0:0/22:4(7Z,10Z,13Z,16Z)), 2-Methoxy-3-methyl-9?H-carbazole, 3-Deoxyarabinohexonic acid, cis-Piceid, N-Palmitoylsphingosine, PG(18:0/18:1(11Z)), SM(d18:1/22:1(13Z)), 20-Oxo-leukotriene E4, avenestergenin B2, antibiotic CP 412065, vinaginsenoside R12, citreoviridin C, PS(14:1(9Z)/18:3(9Z,12Z,15Z)), and lysoPE(22:4(7Z,10Z,13Z,16Z)/0:0), were significantly positively and negatively related to major genera of g_Cetobacterium, g_Nocardiag_Paracoccus, g_Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, g_ZOR0006, g_Shewanella. g_Tsukamurella, g_Brevibacillus, g_Pseudomonas, g_Aneurinibacillus, g_Clostridium_sensu_stricto_1, and g_Rhodobacter. Thus, these results provided the significantly changed metabolites and their inner correlation between major genera, which were beneficial to further discovering the biomarkers in CKD animals. However, the function of a single gut bacteria strain in association with chronic kidney disease still needs further investigation by the isolation and infection of the animal models.

The notable genes involving various pathways, show relationships with the biologically active metabolites influencing renal functions, and reveal in-depth mechanisms (Hsu and Tain, 2020; Jia et al., 2023). In this study, the potential novel biomarkers were revealed by omics analysis for early diagnosis in CKDu, with the regulating signaling pathways of lipids, carbohydrates, energy, nucleotide, metabolism of cofactors and vitamins, and amino acids. Notably, the comprehensive analysis explored the metabolome and its correlation analyses with transcriptome and gut microbiota, environmental factors, histopathological score, and then the network for multiple relationships. It discovered that the gut bacteria of g_ZOR0006, g_Pseudomonas, and DEGs of egln3, ca2, jun, slc2a1b, gls2b, were markedly closely related to metabolites of 20-hydroxy-LTE4, PS(18:0/22:2(13Z,16Z)), neuromedin N, 20-Oxo-leukotriene E4, and phenol sulfate, etc, in chronically exposed zebrafish, and the shared significantly enriched the calcium signaling pathways and necroptosis. Among these changed metabolites, 20-hydroxy-LTE4 as a primary urinary metabolite, PS(18:0/22:2(13Z,16Z)), LysoPC products, and gut microbiome-derived phenyl sulfate have been proven as key biomarkers in Cd-induced and diabetic kidney diseases by influencing the lipid, albuminuria, and Arachidonic acid (AA) metabolism (Gong et al., 2017, Kikuchi, 2019).

Previously, renal function monitoring and potential genetic biomarkers were researched to explain the appearance and development of CKDu in the local population, which was significant for guaranteeing the safety of drinking water and protecting from renal toxins (Floris et al., 2021, Xiao and Meierhofer, 2019). As the metabolites and products of gut microbiota, the uremic toxins activate important cellular processes that contribute to oxidative states, inflammation, proliferation, fibrosis development, and apoptosis, which usually appear in the intestinal and renal organs (Chen et al., 2019, Plata et al., 2019). Interestingly, calcium metabolism plays a key role in CKD patients through the gut-kidney axis from homeostasis, oxalate balance, and renal pathophysiology aspects, during which intestinal absorption and renal excretion take place, and can be supplemented by vitamin D (Peacock, 2010, Ticinesi, 2020). Notably, the CKD and germ-free (GF) mice models were found to have worse kidney damage than the models with commensal microbes, and the carboxyl-containing metabolites including bile acids and fatty acids, might contribute to disease risk by circulating microbiota-derived metabolites (Kanemitsu et al., 2019). Here, the changed metabolites and microbiota, suspected to be related to kidney diseases with the disrupted KEGG pathways, including primary immunodeficiency, renal cell carcinoma, proximal tubule bicarbonate reclamation, pyruvate metabolism, purine metabolism, etc., can be the biomarkers combined with the DEGs and environmental factors to detect the abnormal functions and warn the renal risks.

In humans, various pathways were discovered with abnormal changes in the metabolome, transcriptome, and gut microbiota, which also highlight that age, gender, occupation, and environmental factors should be taken into consideration to avoid CKDu risks (Abdissa, 2020, Evenepoel et al., 2017, Mahmoodpoor et al., 2017). The omics changes and correlation analysis that revealed from animal models probably be applied to human disease by providing accurate, safe, and non-invasive biomarkers capable of diagnosing and staging CKD. Therefore, the biomarkers and signaling pathways from deep omics analyses, screened by the serious impacts of high hardness, fluoride, and heavy metals (Al, Cd), among these potential toxic factors, on zebrafish kidneys based on the profile of transcriptome, metabolome, and intestinal microbes, are significant for populations who are suffering from the threat of kidney disease. These findings indicated that the control of diverse exposure factors in drinking water quality, regulation of gut microbiota, transplanted microbiota from patients to germ-free animals, probiotics and derivates supplementation will benefit CKDu from a public health aspect. A recent study identified dozens of metabolite markers most associated with kidney function by analyzing 53 participants’ serum glomerular filtration rate (GFR) index (Peng et al., 2022). However, the limitations of microbiota and kidney-related metabolites still exist with the underlying molecular causes of the disease or affected cellular pathways. After all, the potential biomarkers should be further identified on animal models to ensure the effectiveness of monitoring kidney risks and combined with the detection outcomes of cohort research.

Oxo-leukotriene E4, and phenol sulfate. These metabolites, pathways, and bacteria were suspected as promising detection candidates. The next challenge is to use them for screening and treating CKDu, which would prevent the disease caused by renal toxic factors in drinking water.

5. Conclusion

The chronic exposure to toxic factors, such as higher hardness and fluoride, heavy metals, and combined solutions, via water system, was conducted to investigate the CKDu risks to human health based on the fish renal damage outcomes. The metabolome profile of exposed zebrafish and correlation analyses with transcriptome and intestinal microbes revealed that the significantly changed metabolites, closely related to the critical DEGs and shifted intestinal bacterial strains, indicated potential biomarkers for CKDu. Notably, the enriched signaling pathways in zebrafish were: oxidative phosphorylation, folate biosynthesis, arachidonic acid metabolism, FoxO signaling pathway, lysosome, pyruvate metabolism, and purine metabolism. The gut bacteria of g_ZOR0006 (Zoogloea ramigera), g_Pseudomonas (Pseudomonas spp.), and DEGs of egln3, ca2, jun, slc2a1b, gls2b, were closely related to major critical renal metabolites, including 20-hydroxy-LTE4, PS(18:0/22:2(13Z,16Z)), neuromedin N, 20-sease caused by renal toxic factors in drinking water.

CRediT authorship contribution statement

Pan-Pan Jia: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing – original draft. Yan Li: Methodology, Investigation. Lan-Chen Zhang: Resources, Validation. Ming-Fei Wu: Software, Visualization. Tian-Yun Li: Resources, Project administratio. Desheng Pei: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.