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.
Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca2+ supersaturation in PT can induce luminal calcium phosphate (CaP) crystal formation. While such CaP crystals are known to act as a nidus for CaP/calcium oxalate (CaOx) mixed stone formation, the regulation of PT luminal Ca2+ concentration ([Ca2+]) under elevated pH and/or high [Ca2+] conditions are unknown. Since we found that transient receptor potential canonical 3 (TRPC3) knockout (KO; -/-) mice could produce mild hypercalciuria with CaP urine crystals, we alkalinized the tubular pH in TRPC3-/- mice by oral acetazolamide (0.08%) to develop mixed urinary crystals akin to clinical signs of calcium nephrolithiasis (CaNL). Our ratiometric (λ340/380) intracellular [Ca2+] measurements reveal that such alkalization not only upsurges Ca2+ influx into PT cells, but the mode of Ca2+ entry switches from receptor-operated to store-operated pathway. Electrophysiological experiments show enhanced bicarbonate related current activity in treated PT cells which may determine the stone-forming phenotypes (CaP or CaP/CaOx). Moreover, such alkalization promotes reactive oxygen species generation, and upregulation of calcification, inflammation, fibrosis, and apoptosis in PT cells, which were exacerbated in absence of TRPC3. Altogether, the pH-induced alteration of the Ca2+ signaling signature in PT cells from TRPC3 ablated mice exacerbated the pathophysiology of mixed urinary stone formation, which may aid in uncovering the downstream mechanism of CaNL.
Melamine induces calcium phosphate (CaP) and calcium oxalate (CaOx) crystal formation; however, the physicochemical mechanism is not clear. Recently, we found that melamine has a discriminatory effect on CaP, CaOx, and CaP + CaOx (Mixed) crystal dissolution. Thus, to delineate the mechanism, we examined crystal interactions through birefringence analysis and found that CaP becomes increasingly birefringent when bound to melamine, while the birefringence of CaOx decreases when it forms CaOx−melamine cocrystals. We also confirmed the feasibility of such melamine− CaP/CaOx co-crystallization at the nanomicromolar range. Interestingly, ammeline, which is a similar triazine, did not accelerate CaP/CaOx/Mixed crystal formation and growth, indicating the specificity of crystal interaction by melamine. Furthermore, melamine stabilizes the CaP/CaOx/Mixed crystals when exposed to a crystal inhibitor (etidronic acid) or dissolution agents (citrate analogues), while it induces crystal growth by increasing crystal retention, suggesting melamine's interference with conventional dissolution remedies. Morphological and elemental analysis of melamine−CaP/CaOx/ Mixed co-crystals using scanning electron microscopy further revealed that melamine harbors such crystals by creating a nucleation site. Finally, we confirmed the physiological relevance of melamine exposure using artificial urine to show the induction, stabilization, and retention of mixed crystals in the presence of crystal-inhibitor/dissolution agent and thus established potential causes of recurrence of kidney stones.
Synthetic vs. biological crystal aggregation in renal tubule. (A) Non-specific recognition by synthetic crystals resulted in larger floppy aggregates. (B) Specific recognition by biogenic crystals with matrix proteins, induced discrete aggregation.
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