Abstract
Calcium phosphate (CaP) biomineralization is the hallmark of extra-skeletal tissue calcification and renal calcium stones. Although such a multistep process starts with CaP crystal formation, the mechanism is still poorly understood due to the complexity of the in vivo system and the lack of a suitable approach to simulate a truly in vivo-like environment. Although endogenous proteins and lipids are engaged with CaP crystals in such a biological process of stone formation, most in vitro studies use synthetic materials that can display differential bioreactivity and molecular recognition by the cellular component. Here, we used our in vitro microfluidic (MF) tubular structure, which is the first completely cylindrical platform, with renal tubular cellular microenvironments closest to the functional human kidney tubule, to understand the precise role of biological components in this process. We systematically evaluated the contribution of synthetic and biological components in the stone-forming process in the presence of dynamic microenvironmental cues that originated due to cellular pathophysiology, which are critical for the nucleation, aggregation, and growth of CaP crystals. Our results show that crystal aggregation and growth were enhanced by immunoglobulin G (IgG), which was further inhibited by etidronic acid due to the chelation of extracellular Ca2+. Interestingly, biogenic CaP crystals from mice urine, when applied with cell debris and non-specific protein (bovine serum albumin), exhibited a more discrete crystal growth pattern, compared to exposure to synthetic CaP crystals under similar conditions. Furthermore, proteins found on those calcium crystals from mice urine produced discriminatory effects on crystal-protein attachment. Specifically, such biogenic crystals exhibited enhanced affinity to the proteins inherent to those crystals. More importantly, a physiological comparison of crystal induction in renal tubular cells revealed that biogenic crystals are less effective at producing a sustained rise in cytosolic Ca2+ compared to synthetic crystals, suggesting a milder detrimental effect to downstream signaling. Finally, synthetic crystal-internalized cells induced more oxidative stress, inflammation, and cellular damage compared to the biogenic crystal-internalized cells. Together, these results suggest that the intrinsic nature of biogenically derived components are appropriate to generate the molecular recognition needed for spatiotemporal effects and are critical towards understanding the process of kidney stone formation.