Melamine, which induces proximal tubular (PT) cell damage has a greater nephrotoxic effect when combined with cyanuric and uric acids; however, it is unknown whether such effect can stimulate calcium phosphate (CaP)/calcium oxalate (CaOx) stone formation. Here, we show that melamine acts as an inducer of CaP, CaOx and CaP + CaOx (mixed) crystal formations in a time and concentration-dependent manner by stabilizing those crystals and further co-aggregating with melamine. To explore the physiological relevance of such melamine-augmented calcium crystal formation, we used 2-dimensional (2D) and 3D microfluidic (MF) device, embedded with PT cells, which also resembled the effect of melamine-stimulated CaP, CaOx and mixed crystal formation. Significantly, addition of preformed CaP and/or CaOx crystal in the presence of melamine, further potentiated those crystal formations in 3D MFs, which helped the growth and aggregation of mixed crystals. Our data show that the mechanism of such predisposition of stone formation could be largely due to co-crystallization between melamine and CaP/CaOx and pronounced effect on induction of stone-forming pathway activation in 3D MF. Taken together, melamine-induced CaP and/or CaOx crystal formation ex-vivo will help us in understanding the larger role of melamine as an environmental toxicant in producing the pathology in similar cellular microenvironments.

Clinical studies and structural analyses of salivary stones strongly suggest a linkage between higher saliva calcium (Ca2+) and salivary stone formation, sialolithiasis; however, the process and the mechanism leading to Ca2+ overload during sialolithiasis is not well understood. Here, we show that TRPC3 null (−/−) mice presented with a reduction in Ca2+ entry and current in ductal cells with higher saliva [Ca2+] suggesting diminished transepithelial Ca2+ flux across the salivary ductal cells, leaving more Ca2+ in ductal fluid. Significantly, we found that TRPC3 was expressed in mice and human salivary ductal cells, while intraductal stones were detected in both mice (TRPC3−/−) and patient (sialolithiasis) salivary glands. To identify the mechanism, we found that TRPC3 was crucial in preventing the expression of calcification genes (BMP2/6, Runx2) in ductal cells which may be due to higher extracellular Ca2+ in SMG tissues. Similarly, inflammatory (IL6, NLRP3), fibrotic (FN1, TGFβ1) and apoptotic (Bax1/Bcl2) markers were also elevated, suggesting that the loss of TRPC3 induces genetic changes that leads to salivary gland cell death and induction of inflammatory response. Overall, ablation of TRPC3−/− leads to higher saliva [Ca2+], along with elevated detrimental gene expressions, altogether contributing to salivary gland stone formation.