Melamine promotes calcium crystal formation in three-dimensional microfluidic device

Abstract

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.

Abrogation of store-operated Ca2+ entry protects against crystal-induced ER stress in human proximal tubular cells

“…CaP, CaOx, and mixed crystals were prepared as described previously 6 . Briefly, CaP crystals were prepared by mixing solutions of 2.4 mM CaCl 2 , 0.9 mM Na 2 HPO 4 , and 5.8 mM NaH 2 PO 4 .…”

“…Alizarin red (3,4-Dihydroxy-9, 10-dioxo-2-anthracenesulfonic acid sodium) staining was performed as previously described to detect the presence of CaP and/or CaOx crystals 6 . Briefly, following crystal induction, cells were rinsed with HBSS without phenol red, Ca 2+ , and Mg 2+ , fixed with 3% paraformaldehyde for 10 min at RT, then washed with HBSS three times.…”

“…HK2 cells were seeded and grown to 80% confluency. Cells were induced with crystals as described previously 6 . Following crystal induction, cells were trypsinized and stained with toluidine blue stain.…”

“…Interestingly, several studies have proposed that the onset of nephrolithiasis lies within the nephron, moreso, renal biopsies of some stone-formers presented with crystals inside renal tubular cells 4,5 . Thereafter, many in vitro studies have shown that calcium crystals, such as CaOx monohydrate, are taken up by renal tubular cells and subsequently influence the process of kidney-stone formation 6 . The proximal tubular (PT) cells are more prone to crystal internalization than distal tubular (DT) cells, due to their greater crystal binding affinity 7 .…”

“…However, the effect of mixed (CaP + CaOx) crystals, which are usually the most abundant in calcium nephrolithiasis, has not yet been studied. Further, this crystal-induced cytotoxicity as a downstream mechanism toward cell death has been extensively studied 6,11–13 , which could be linked to the upstream mechanisms involving endoplasmic reticular (ER) and plasma membrane (PM) signaling in crystal-induced condition.…”