Mechanism of Porphyra Yezoensis Polysaccharides in Inhibiting Hyperoxalate-Induced Renal Injury and Crystal Deposition.

Oxidative damage to the kidneys is a primary factor in the occurrence of kidney stones. This study explores the inhibitory effect of Porphyra yezoensis polysaccharides (PYP) on oxalate-induced renal injury by detecting levels of oxidative damage, expression of adhesion molecules, and damage to intracellular organelles and revealed the molecular mechanism by molecular biology methods. Additionally, we validated the role of PYP in vivo using a crystallization model of hyperoxalate-induced rats. PYP effectively scavenged the overproduction of reactive oxygen species (ROS) in HK-2 cells, inhibited the adhesion of calcium oxalate (CaOx) crystals on the cell surface, unblocked the cell cycle, restored the depolarization of the mitochondrial membrane potential, and inhibited cell death. PYP upregulated the expression of antioxidant proteins, including Nrf2, HO-1, SOD, and CAT, while decreasing the expression of Keap-1, thereby activating the Keap1/Nrf2 signaling pathway. PYP inhibited CaOx deposition in renal tubules in the rat crystallization model, significantly reduced high oxalate-induced renal injury, decreased the levels of the cell surface adhesion proteins, improved renal function in rats, and ultimately inhibited the formation of kidney stones. Therefore, PYP, which has crystallization inhibition and antioxidant properties, may be a therapeutic option for the treatment of kidney stones.

Corn Silk Polysaccharides with Different Carboxyl Contents Reduce the Oxidative Damage of Renal Epithelial Cells by Inhibiting Endocytosis of Nano-calcium Oxalate Crystals.

OBJECTIVE: Renal epithelial cell injury and cell-crystal interaction are closely related to kidney stone formation. METHODS: This study aims to explore the inhibition of endocytosis of nano-sized calcium oxalate monohydrate (nano-COM) crystals and the cell protection of corn silk polysaccharides (CCSPs) with different carboxyl contents (3.92, 7.75, 12.90, and 16.38%). The nano-COM crystals protected or unprotected by CCSPs were co-cultured with human renal proximal tubular epithelial cells (HK-2), and then the changes in the endocytosis of nano-COM and cell biochemical indicators were detected. RESULTS: CCSPs could inhibit the endocytosis of nano-COM by HK-2 cells and reduce the accumulation of nano-COM in the cells. Under the protection of CCSPs, cell morphology is restored, intracellular superoxide dismutase levels are increased, lipid peroxidation product malondialdehyde release is decreased, and mitochondrial membrane potential and lysosomal integrity are increased. The release of Ca2+ ions in the cell, the level of cell autophagy, and the rate of cell apoptosis and necrosis are also reduced. CCSPs with higher carboxyl content have better cell protection abilities. CONCLUSION: CCSPs could inhibit the endocytosis of nano-COM crystals and reduce cell oxidative damage. CCSP3, with the highest carboxyl content, shows the best biological activity.

Cell Protection and Crystal Endocytosis Inhibition by Sulfated Laminaria Polysaccharides Against Nano-COM-Induced Oxidative Damage in Renal Epithelial Cells.

Background: The damage to renal tubular epithelial cells is closely related to the formation of kidney stones. At present, research on drugs that can protect cells from damage remains limited. Methods: This study aims to explore the protective effects of four different sulfate groups (-OSO3 -) of Laminaria polysaccharides (SLPs) on human kidney proximal tubular epithelial (HK-2) cells and determine the difference in the endocytosis of nano-sized calcium oxalate monohydrate (COM) crystals before and after protection. COM with a size of 230 ± 80 nm was used to damage HK-2 cells to establish a damage model. The protection capability of SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO3 - contents of 0.73, 15, 23, and 31%, respectively, against COM crystal damage and the effect of SLPs on the endocytosis of COM crystals were studied. Results: Compared with that of the SLP-unprotected COM-injured group, the cell viability of the SLP-protected group was improved, healing capability was enhanced, cell morphology was restored, production of reactive oxygen species was reduced, mitochondrial membrane potential and lysosome integrity were increased, intracellular Ca2+ level and autophagy were decreased, cell mortality was reduced, and internalized COM crystals were lessened. The capability of SLPs to protect cells from damage and inhibit the endocytosis of crystals in cells enhanced with an increase in the -OSO3 - content of SLPs. Conclusions: SLPs with a high -OSO3 - content may become a potential green drug for preventing the formation of kidney stones.

Seaweed Porphyra yezoensis polysaccharides with different molecular weights inhibit hydroxyapatite damage and osteoblast differentiation of A7R5 cells.

Vascular calcification (VC) is a common pathological manifestation in patients with cardiovascular diseases, leading to high mortality in patients with chronic kidney diseases. The deposition of hydroxyapatite (HAP) crystals on vascular smooth muscle cells leads to cell damage, which promotes osteogenic transformation. In this study, four different molecular weights (MWs ) of Porphyra yezoensis polysaccharides (PYP1, PYP2, PYP3, and PYP4 with MWs of 576, 49.5, 12.6, and 4.02 kDa, respectively) were used to coat HAP, and the differences in toxicity and calcification of HAP on A7R5 cells before and after coating were studied. The results showed that PYPs could effectively reduce HAP damage to the A7R5 cells. Under the protection of PYPs, cell viability increased and lactate dehydrogenase release, active oxygen level, and cell necrosis rate decreased; also, the amount of the HAP crystals adhering to cell surfaces and entering cells decreased. PYPs with low molecular weights presented better protective effects than high-molecular-weight PYPs. PYPs also inhibited the osteogenic transformation of the A7R5 cells induced by HAP and decreased alkaline phosphatase (ALP) activity and expressions of bone/chondrocyte phenotype genes (runt-related factor 2, ALP, osteopontin, and osteocalcin). In the adenine-induced chronic renal failure (CRF) mouse VC model, PYP4 was found to obviously inhibit the aortic calcium level, and it also inhibited the serum creatinine, serum phosphorus and serum BUN levels. PYP4 (least molecular weight) showed the best inhibitory effect on calcification and may be considered as a candidate drug with therapeutic potential for inhibiting cellular damage and osteoblast differentiation induced by the HAP crystals.

Shape-dependent adhesion and endocytosis of hydroxyapatite nanoparticles on A7R5 aortic smooth muscle cells.

The interaction between nanohydroxyapatite (HAP) and smooth muscle cells is an important step in vascular calcification. However, the effect of the shape of HAP on adhesion and endocytosis to aortic smooth muscle cells has been rarely reported. Four different morphological HAP crystals (H-Rod, H-Needle, H-Sphere, and H-Plate) were selected to interact with rat aortic smooth muscle cells (A7R5). Fluorescence-labeled HAP was used to detect crystal adhesion and endocytosis and then pretreated with different endocytic inhibitors to explore the pathway of endocytotic crystals. The distribution of crystals inside and outside the cells and the crystal localization in lysosomes was observed through laser confocal microscopy. The effect of crystal on the cell cycle and the changes in the expression of phosphatidylserine, osteopontin, α-actin, core binding factor alpha 1, and osterix on the surface of A7R5 cells were detected. The adhesion and endocytosis of HAP on A7R5 cells were closely related to crystal shapes and ranked as follows: H-Plate > H-Sphere > H-Needle > H-Rod. H-Sphere and H-Needle were internalized into the cells mainly via the clathrin-mediated pathway, whereas H-Plate and H-Rod were internalized into the cells mainly via macropinocytosis. The endocytosed nano-HAP was mainly distributed in the cell lysosome. The adhesion and endocytosis of HAP to A7R5 cells were positively correlated with the specific surface area, and contact area of HAP and negatively correlated with the absolute value of Zeta and contact angle of HAP. This study provided insights into the effect of crystal morphology on vascular calcification and its mechanism.

High-phosphorus environment promotes calcification of A7R5 cells induced by hydroxyapatite nanoparticles.

This study simulated the high-phosphorus (Pi) environment in patients with chronic kidney disease. Nano-hydroxyapatite (HAP) crystals were used to damage rat aortic smooth muscle cells (A7R5) pre-damaged with different concentrations of Pi solution to compare the differences in HAP-induced calcification in A7R5 cells before and after injury by high-Pi condition. After the A7R5 cells were damaged by high-Pi environment, the following were observed. HAP resulted in declined cell viability and lysosomal integrity, release of lactate dehydrogenase, and increased reactive oxygen species production. The ability of high-Pi damaged cells to internalize HAP crystals declined; crystal adhesion and calcium deposition on the cell surface and alkaline phosphatase activities increased. Osteopontin expression and level of Runt-related transcription factor 2 were increased, and HAP-induced osteogenic transformation was enhanced. High-Pi condition promoted the adhesion of A7R5 cells to nano-HAP crystals and inhibited HAP endocytosis, increasing the risk of calcification.

Shape-dependent toxicity and mineralization of hydroxyapatite nanoparticles in A7R5 aortic smooth muscle cells.

Vascular smooth muscle cell damage is a key step in inducing vascular calcification that yields hydroxyapatite (HAP) as a major product. The effect of the shape of HAP on the damage to vascular smooth muscle cells has yet to be investigated. In this study, we compared the differences in toxicity of four various morphological nano-HAP crystals, namely, H-Rod, H-Needle, H-Sphere, and H-Plate, in rat aortic smooth muscle cells (A7R5). The sizes of these crystals were 39 nm × 115 nm, 41 nm ×189 nm, 56 nm × 56 nm, and 91 nm × 192 nm, respectively. Results showed that all HAPs decreased cell viability, disorganized cell morphology, disrupted cell membranes, increased intracellular reactive oxygen species concentration, decreased mitochondrial membrane potential, decreased lysosome integrity, increased alkaline phosphatase activity, and increased intracellular calcium concentration, resulting in cell necrosis. The cytotoxicity of the four kinds of HAP was ranked as follows: H-Plate > H-Sphere > H-Needle > H-Rod. The cytotoxicity of each crystal was positively correlated with the following factors: large specific surface area, high electrical conductivity and low surface charge. HAP accelerated calcium deposits on the A7R5 cell surface and induced the expression of osteogenic proteins, such as BMP-2, Runx2, OCN, and ALP. The crystals with high cytotoxicity caused more calcium deposits on the cell surface, higher expression levels of osteogenic protein, and stronger osteogenic transformation abilities. These findings elucidated the relationship between crystal shape and cytotoxicity and provided theoretical references for decreasing the risks of vascular calcification.