Porphyra yezoensis polysaccharide and potassium citrate synergistically inhibit calcium oxalate crystallization induced by renal epithelial cells and cytotoxicity of the formed crystals.

Mineralization crystallization is considered to be the initial stage of stone formation. However, the formation of crystals and subsequent cell damage have rarely been investigated. An oxidatively damaged cell model was established using oxalic acid to injure human proximal tubular epithelial cells (HK-2). Subsequently, CaOx crystallization was induced by adding 2.0 mmol/L sodium oxalate solution. We compared the synergistic effects of PYPs with molecular weights of 49.54 kDa (PYP1) and 4.02 kDa (PYP2) and K3Cit on the inhibition of CaOx crystallization and studied the nucleation, growth, and retention process of CaOx crystals on the cell surface and the subsequent damage of the formed crystals to the cells. Normal HK-2 cells mainly induced the formation of CaOx dihydrate (COD), whereas the damaged cells mainly induced the formation of CaOx monohydrate (COM) crystals. Under the protection of PYPs, the state of cells was improved, and the proportion of COD crystals in the formed crystals increased. Small-molecular-weight PYP2 exhibited better abilities of inhibiting CaOx crystallization and improving cell state compared with PYP1. Under the synergistic effects of PYPs and K3Cit, the number of formed crystals was obviously reduced, and the size was obviously decreased. PYPs can repair damaged cells and inhibit the conversion of COD phase to COM phase. K3Cit can obviously inhibit the nucleation of CaOx crystal and reduce the amount of crystal formation. The repair of damaged cells by PYPs and the synergistic inhibition of CaOx crystallization by PYPs and K3Cit reduce cell damage and crystal formation on the cell surface. By simultaneously repairing damaged cells and inhibiting crystallization, this strategy is expected to exert a desirable effect in preventing the formation and recurrence of stones.

Regulatory Effects of Damaged Renal Epithelial Cells After Repair by Porphyra yezoensis Polysaccharides with Different Sulfation Degree on the Calcium Oxalate Crystal-Cell Interaction.

BACKGROUND: The interaction between urinary microcrystals and renal epithelial cells is closely related to kidney stone formation. However, the mechanism of cell state changes that affect crystal-cell interaction remains unclear. METHODS: This study investigated the relationship between the sulfate group (-OSO3 -) content in Porphyra yezoensis polysaccharide (PYP) and the ability to repair damaged cells, as well as the changes in cell adhesion and endocytosis of nano-calcium oxalate monohydrate (COM) crystals before and after PYP repair of damaged renal tubular epithelial cells. The sulfur trioxide-pyridine method was used to sulfate PYP (-OSO3 - content of 14.14%), and two kinds of sulfated PYPs with -OSO3 - content of 20.28% (SPYP1) and 27.14% (SPYP2) were obtained. The above three PYPs were used to repair oxalate-damaged human proximal tubular epithelial cells (HK-2), and the changes in the biochemical indicators of the cells before and after the repair and the changes in cell adhesion and endocytosis of nano-COM crystals were detected. RESULTS: After repair by PYPs, the cell viability increased, the number of reactive oxygen species decreased, and the reduction of mitochondrial membrane potential and the release of intracellular Ca2+ were suppressed. The cells repaired by PYPs inhibited the adhesion of nano-COM crystals while promoting the endocytosis of the adhered crystals. The endocytosed crystals mainly accumulated in the lysosome. The ability of PYPs to repair cell damage, inhibit crystal adhesion, and promote crystal endocytosis was enhanced when the -OSO3 - content increased. Among them, SPYP2 with the highest -OSO3 - content showed the best biological activity. CONCLUSION: SPYP2 showed the best ability to repair damaged cells, followed by SPYP1 and PYP. SPYP2 may become a potential green drug that inhibits the formation and recurrence of calcium oxalate stones.

Effects of Porphyra yezoensis Polysaccharide with Different Molecular Weights on the Adhesion and Endocytosis of Nanocalcium Oxalate Monohydrate in Repairing Damaged HK-2 Cells.

The adhesion and endocytosis of renal epithelial cells to urinary microcrystals are closely related to kidney stone formation; however, the mechanism of cell state changes that affect adhesion and endocytosis remains unclear. In this study, a damaged cell model was established using oxalate to impair human kidney proximal tubular epithelial cells (HK-2). Then, we used four degraded Porphyra yezoensis polysaccharides (PYPs), namely, PYP1, PYP2, PYP3, and PYP4, with molecular weight of 576.2, 49.5, 12.6, and 4.02 kDa, respectively, to repair the damaged cells. After repairing the damaged HK-2 cells by PYPs, the cell morphology gradually recovered to near normal; the cell migration speed increased; the phosphatidylserine eversion ratio and osteopontin expression decreased; and the intracellular adenosine triphosphate was promoted. The adhesion and endocytosis of calcium oxalate monohydrate (COM) crystals were closely related to cell state. Normal cells could endocytose crystals more than the damaged cells, whereas damaged cells could adhere to crystals more than the normal cells. The cells repaired by PYPs inhibited the adhesion of nano-COM crystals and promoted the endocytosis of the adherent crystals, thereby reducing the adhesion of crystals on the cell surface under both effects. As the molecular weight of PYP decreased, the ability of PYP to repair cells, inhibit crystal adhesion, and promote endocytosis of cells was enhanced; that is, PYP4, which had the lowest molecular weight, exhibited the best biological activity. Therefore, PYPs, especially PYP4, may become an optional green drug to inhibit the formation and recurrence of calcium oxalate stones.