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

Chronic kidney disease with uncertain etiology (CKDu) in Sri Lanka has attracted much attention as a global health issue. However, how environmental factors in local drinking water induce kidney damage in organisms is still elusive. We investigated multiple environmental factors including water hardness and fluoride (HF), heavy metals (HM), microcystin-LR (MC-LR), and their combined exposure (HFMM) to elucidate their toxic effects on CKDu risk in zebrafish. Acute exposure affected renal development and inhibited the fluorescence of Na, K-ATPase alpha1A4:GFP zebrafish kidney. Chronic exposure influenced the body weight of both genders of adult fish and induced kidney damage by histopathological analyses. Furthermore, the exposure significantly disturbed differential expression genes (DEGs), diversity and richness of gut microbiota, and critical metabolites related to renal functions. The transcriptomic analysis revealed that kidney-related DEGs were linked with renal cell carcinoma, proximal tubule bicarbonate reclamation, calcium signaling pathway, and HIF-1 signaling pathway. The significantly disrupted intestinal microbiota was closely related to the environmental factors and H&E score, which demonstrated the mechanisms of kidney risks. Notably, the Spearman correlation analysis indicated that the changed bacteria such as Pseudomonas, Paracoccus, and ZOR0006, etc were significantly connected to the DEGs and metabolites. Therefore, the assessment of multiple environmental factors provided new insights on "bio-markers" as potential therapies of the target signaling pathways, metabolites, and gut bacteria to monitor or protect residents from CKDu.