Sulfated Laminarin Polysaccharides Reduce the Adhesion of Nano-COM Crystals to Renal Epithelial Cells by Inhibiting Oxidative and Endoplasmic Reticulum Stress

Abstract

Purpose: Adhesion between calcium oxalate crystals and renal tubular epithelial cells is a vital cause of renal stone formation; however, the drugs that inhibit crystal adhesion and the mechanism of inhibition have yet to be explored. Methods: The cell injury model was constructed using nano-COM crystals, and changes in oxidative stress levels, endoplasmic reticulum (ER) stress levels, downstream p38 MAPK protein expression, apoptosis, adhesion protein osteopontin expression, and cell-crystal adhesion were examined in the presence of Laminarin polysaccharide (DLP) and sulfated DLP (SDLP) under protected and unprotected conditions. Results: Both DLP and SDLP inhibited nano-COM damage to human kidney proximal tubular epithelial cell (HK-2), increased cell viability, decreased ROS levels, reduced the opening of mitochondrial membrane permeability transition pore, markedly reduced ER Ca²⁺ ion concentration and adhesion molecule OPN expression, down-regulated the expression of ER stress signature proteins including CHOP, Caspase 12, and p38 MAPK, and decreased the apoptosis rate of cells. SDLP has a better protective effect on cells than DLP. Conclusions: SDLP protects HK-2 cells from nano-COM crystal-induced apoptosis by reducing oxidative and ER stress levels and their downstream factors, thereby reducing crystal-cell adhesion interactions and the risks of kidney stone formation.

Keywords: apoptosis; crystal adhesion; endoplasmic reticulum stress; sulfated polysaccharide.

Figure 1

Characterization and fluorescence labeling of Nano-COM crystals. (A) XRD spectrum; (B) FT-IR spectrum; (C) SEM image; (D) average COM diameter from the analysis of (C) by Nano Measurer 1.2 software; (E,F) Nano-COM microscopy images before and after FITC labeling; scale bar: 100 μm; (G,H) fluorescence of Nano-COM before and after FITC labeling detected by flow cytometry.

Figure 2

Cytotoxic (A) and protective effect on HK-2 cells (B) of different concentrations of DLP and SDLP determined by CCK-8 method. NC: normal control group; DC: nano-COM crystal control group, crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, * p < 0.05, ** p < 0.01.

Figure 3

Changes in ROS levels in HK-2 cells before and after protection by different concentrations of DLP and SDLP. (A) Fluorescence microscopy; (B) columnar quantitative graph. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, ** p < 0.01.

Figure 4

Laser confocal microscopy observation of DLP and SDLP inhibiting nano-COM damage to HK-2 cells to reduce mPTP opening. (A) Laser confocal micrograph; (B) columnar quantitative graph. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, * p < 0.05, ** p < 0.01 Scale bar: 20 µm.

Figure 5

Changes in ERS before and after protection of HK-2 cells by different concentrations of DLP and SDLP. (A) Fluorescence micrograph; (B) column quantitative graph. The blue fluorescence indicates the nucleus, red fluorescence indicates the endoplasmic reticulum (ER), and green fluorescence indicates the calcium ion concentration in the endoplasmic reticulum (ER Ca²⁺). NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, * p < 0.05, ** p < 0.01. Scale bar: 20 µm.

Figure 6

Changes in CHOP expression levels before and after protection of HK-2 cells by different concentrations of DLP and SDLP. (A) Fluorescence micrographs; (B) quantitative bar graphs. Blue fluorescence indicates nucleus and red fluorescence indicates CHOP. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Scale bar: 20 µm. Compared with DC group, ** p < 0.01.

Figure 7

Changes in Caspase 12 expression levels before and after protection of HK-2 cells by different concentrations of DLP and SDLP. (A) Qualitative fluorescence plot; (B) quantitative bar graph. The blue fluorescence indicates the nucleus and the green fluorescence indicates Caspase 12. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Scale bar: 20 µm. Compared with DC group, ** p < 0.01.

Figure 8

Changes in P-p38 expression levels before and after protection of HK-2 cells by different concentrations of DLP and SDLP. (A) Fluorescence micrographs; (B) quantitative bar graphs. The blue fluorescence indicates the nucleus and the green fluorescence indicates P-p38. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Scale bar: 20 µm. Compared with DC group, * p < 0.05. ** p < 0.01.

Figure 9

Changes in apoptosis/necrosis before and after protection of HK-2 cells by different concentrations of DLP and SDLP. (A) Qualitative assay; (B) flow-through quantitative histogram; (C) flow-through quantitative histogram. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, ** p < 0.01.

Figure 10

Changes in OPN expression levels before and after protection of HK-2 cells by different concentrations of DLP and SDLP. (A) Qualitative fluorescence plot; (B) quantitative bar graph. The blue fluorescence indicates the nucleus and the green fluorescence indicates OPN. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, * p < 0.05, ** p < 0.01. Scale bar: 20 µm.

Figure 11

Changes in crystal adhesion on the cell surface before and after polysaccharide protection by laser confocal microscopy. The blue fluorescence is the DAPI-stained nucleus; the green fluorescence is the FITC-labeled nano-COM crystals; the red fluorescence is the DiI-stained cell membrane. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Scale bar: 20 µm.

Figure 12

Changes in crystal adhesion on the surface of HK-2 cells before and after protection by different concentrations of DLP and SDLP. (A) Flow histogram; (B) quantitative bar graph. NC: normal control group; DC: nano-COM crystal damage group. DLP-10 and DLP-60 are polysaccharide protection groups, indicating polysaccharide concentrations of 10 μg/mL and 60 μg/mL, respectively. Crystal concentration: 200 μg/mL. Protection time: 24 h. Compared with DC group, * p < 0.05, ** p < 0.01.