Involvement of mitochondrial fission in renal tubular pyroptosis in mice exposed to high and environmental levels of glyphosate combined with hard water.

Chronic interstitial nephritis in agricultural communities (CINAC) has reached epidemic proportions. The combination of glyphosate and hard water has been postulated to play a potent aetiological role in CINAC. Therefore, dynamin-related protein 1 (Drp1)-mediated aberrant mitochondrial fission and subsequent activation of the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (Nlrp3)/caspase1 pathway may be involved in the pathogenesis of nephropathy. In the present study, mice were sub-chronically exposed to high doses and environmental levels of glyphosate (100 mg/kg body weight (mg/kg·bw) glyphosate in Roundup and 0.7 mg/L pure glyphosate, respectively) and hard water (2500 mg/L CaCO3 and 250 mg/L Ca2+, respectively) in drinking water. Moreover, Mdivi-1 (Md-1, 10 mg/kg·bw) was intraperitoneally injected to inhibit Drp1 on the basis of the high-dose experiment. Histopathological examination, biochemical analysis, ELISA, western blotting and fluorescent staining were used to analyse renal structure, renal tubular pyroptosis and mitochondrial fission/fusion alterations. The results showed dramatic proximal tubular injury, particularly in the combined groups. Moreover, significant increases in the protein expression levels of calmodulin (CaM), calmodulin-dependent protein kinase II (CaMKII), Drp1/p-Drp1-Ser616 and the Txnip/Nlrp3/caspase1 signalling pathway, and alterations in oxidative stress were observed in the combined groups, and these effects were attenuated by the Drp1 inhibitor Md-1. Intriguingly, there may be a synergistic effect of glyphosate and hard water on renal injury. Taken together, these results suggest that the combination of glyphosate and hard water, even at environmental exposure levels, enhances pyroptosis and ongoing tubulointerstitial inflammation through excessive Drp1-mediated mitochondrial fission.