Chronic interstitial nephritis in agricultural communities (CINAC) is a severe and widespread disease that has been associated with environmental and occupational exposure to glyphosate and hard water. However, the potential underlying mechanisms remain incompletely understood. Melatonin is reported to exert protective effects on the kidney, but whether melatonin can attenuate renal tubular injury in mice exposed to glyphosate combined with hard water is unclear. Here, mice were treated with high doses and environmentally relevant doses of glyphosate (100 mg/kg·bw and 0.7 mg/L, respectively) and/or hard water (2500 mg/L CaCO₃ and 250 mg/L Ca²⁺, respectively) via their drinking water for 12 weeks. We found that high-dose glyphosate or hard water treatment significantly increased the levels of biomarkers of renal damage, including β₂-microglobulin, neutrophil gelatinase-associated lipid carrier protein, and/or albumin, in the urine; these increased biomarker levels were correlated with obvious morphological changes, and all of these changes were also observed in animals exposed to environmentally relevant doses of glyphosate and/or high Ca²⁺ water. Melatonin (10 mg/kg·bw, intraperitoneal injection, daily for 12 weeks) administered concomitantly with high doses of glyphosate and hard water inhibited the glyphosate- and hard water-induced increases in the levels of kidney injury biomarkers and changes in morphology; this result was intriguing. Additionally, glyphosate combined with hard water at both high and environmentally relevant doses significantly upregulated the expression of the endoplasmic reticulum (ER) stress marker proteins Bip, ATF6, and PERK as well as the pyroptosis-related proteins (NLRP3 and caspase 1 signaling proteins) in renal tissues. Similarly, melatonin significantly attenuated the increased ER stress and pyroptosis induced by high doses of glyphosate and hard water. In summary, we conclude that exposure to glyphosate and hard water at both high doses and environmentally relevant doses causes renal dysfunction in mice, and this dysfunction can be attenuated by melatonin, possibly through the inhibition of ER stress and pyroptosis. Our results support the notion that melatonin may have therapeutic potential for the treatment of chronic kidney diseases.

The combination of glyphosate (Gly) and hard water (Hwt) is a suspected risk factor for chronic interstitial nephritis in agricultural communities (CINAC). Accumulated mitochondrial damage and proximal tubular epithelial (PTE) cell senescence have been implicated in CINAC pathogenesis. Melatonin (Mel) has potential mitochondrial function and renoprotective properties, but its role and mechanism in CINAC are unknown. Here, we detected PTE cell senescence and PTEN-induced putative protein kinase 1 (PINK1)-parkin RBR E3 ubiquitin protein ligase (Parkin)-dependent mitophagy in mice orally administered with different doses of Gly combined with Hwt (Gly: 100 mg/kg·bw and 0.7 mg/L; Hwt: 2,500 mg/L CaCO₃ and 250 mg/L Ca²⁺) for different durations (12 and 36 w) using histological examination, transmission electron microscopy (TEM), immunofluorescence (IF) analysis, and immunohistochemistry (IHC), immunoblotting, ELISA and biochemical assays with kits. The same assays were performed after combination treatment with Mdivi-1 (an inhibitor of mitophagy, i.p. 10 mg/kg·bw, twice a week for 12 w) or Mel (i.p. 10 mg/kg·bw, once a day for 12 w) under high-level exposure. Gly combined with Hwt (Gly-Hwt) significantly increased P16-P21-dependent PTE cell senescence, mitochondrial fission and oxidative stress, and activated PINK1-Parkin-mediated mitophagy, accompanied by defective autophagic flux at high doses but unaltered autophagic flux at low doses. Improved senescence occurred after Mdivi-1 administration, suggesting that mitophagy is involved in cellular senescence. Mel significantly decreased senescence induced by Gly-Hwt. Furthermore, PINK1-Parkin-dependent mitophagy and autophagic flux were markedly enhanced, and mitochondrial function was improved, as evidenced by reductions in mitochondrial fission and subsequent oxidative damage. Thus, Gly and Hwt synergistically promote PTE cell senescence through PINK1-Parkin-mediated mitophagy, and Mel exerts renoprotective effects by modulating mitophagy, suggesting therapeutic applications in ageing-related CINAC.

Background: The combined effects of glyphosate and hard water on chronic kidney disease of unknown etiology (CDKu) have attracted much interest, but the mechanisms remain unknown. Cytoplasmic phospholipase A₂ (cPLA₂) plays a key role in the acute and chronic inflammatory reactions. This study explored the effect of glyphosate combined with hard water on renal tubules and the possible targets and mechanisms involved. Methods: In vivo experiments were conducted to investigate the synergistic effects and potential mechanisms of glyphosate and hard water on renal tubular injury in mice. Results: Administration of glyphosate in mice resulted in elevated levels of β2-microglobulin (β₂-MG), albumin (ALB), and serum creatinine (SCr) compared to control mice. This increase was more pronounce when glyphosate was combined with hard water. In the glyphosate-treated mice, small areas of the kidney revealed fibroblast proliferation and vacuolar degeneration, particularly at the higher dose of 400 mg/kg glyphosate. However, the combination of glyphosate and hard water induced an even greater degree of pathological changes in the kidney. Immunofluorescence and western blot analyses showed that glyphosate and hard water had a coordinated effect on calcium ions (Ca²⁺)-activated phospholipase A₂ and the activation may play a key role in inflammation and renal tubular injury. Exposure to glyphosate alone or glyphosate plus hard water increased the levels of oxidative stress markers and inflammatory biomarkers, namely, thromboxane A₂ (TX-A₂), leukotriene B₄ (LTB₄), prostaglandin E₂ (PGE₂), nitric oxide synthase (NOS), and nitric oxide (NO). Parameters of oxidative stress, including the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were decreased. Further analysis showed that the levels of these biomarkers were significantly different between the mice treated with glyphosate plus hard water and the mice treated with glyphosate alone. Conclusions: These findings suggested that hard water combined with glyphosate can induce renal tubular injury in mice, and this may involve mitogen-activated protein kinases (MAPK)/cytosolic phospholipase A₂ (cPLA₂)/arachidonic acid (AA) and its downstream factors.