Abstract
To evaluate cell morphology, crystal adhesion, cell damage, Calcium sensitive receptor (CaSR), and Claudin protein‐14 (Claudin‐14) expression at different time intervals and explore the role of nanobacteria in the formation of urinary calculi. In this experiment, HK‐2 cells were cocultured with nanobacteria (NB) in the absence or presence of tetracycline (Tet). Cells treated with calcium oxalate monohydrate (COM) crystals were used as a positive control of urinary stone‐induced cell damage. After which, cell morphology was evaluated by hematoxylin‐eosin staining in comparison to untreated HK‐2 cells (negative control). Use different methods to assess cell damage, crystal adhesion, and protein expression. (The degree of cell damage, crystal adhesion, and protein expression were evaluated by various methods). It was found that the degree of cell damage observed in Tet + NB‐treated cells was significantly lower than that in NB‐treated cells. Lactate dehydrogenase (LDH) leakage was higher in COM‐exposed than in control cells (P < 0.05). However, LDH release from both NB‐ and Tet + NB‐treated cells was significantly lower than from COM‐treated cells (P < 0.05). The relative expression of CaSR and Claudin‐14 proteins was higher in NB, COM, and TET + NB cells than in control cells (P < 0.001) and was lower in Tet + NB than in NB cells (P < 0.01). And P < 0.05 means that the difference was statistically significant, and P < 0.001 means that there was a significant difference between the both things. From the cell morphology, the cell damage in the COM group was greater than that in the NB group, and the cell damage markers in the COM group and the NB group were elevated. NB caused damage to HK‐2 cells by inducing lipid peroxidation, and the degree of damage was increased in processing time. The adhesion of HK‐2 cells to COM crystals increased after injury and was proportional to the duration of NB coculture. NB upregulated the expression of CaSR and Claudin‐14 in HK‐2 cells.
Highlights
To evaluate cell morphology, crystal adhesion, cell damage, Calcium sensitive receptor (CaSR), and Claudin protein-14 (Claudin-14) expression at different time intervals and explore the role of nanobacteria in the formation of urinary calculi
Cells treated with calcium oxalate monohydrate (COM) crystals were used as a positive control of urinary stone-induced cell damage
It is reported that approximately 33% of patients with kidney stones develop urinary calcium reabsorption disorders. e activation of calcium-sensing receptor plays an important role in this process by upregulating, in the thick ascending limb (TAL), the downstream molecule Claudin-14, which in turn inhibits the resorption of calcium
Summary
Urine samples (2 ml) were collected from patients with nephrolithiasis and diluted five times with saline under aseptic conditions. E filtered sample was diluted 5 times with saline and centrifuged at 4°C for 45 min (14,000 × g). Slides in 12-well plates were removed, rinsed 3 times with PBS, and fixed with 95% alcohol for 20 min. After washing with PBS 3 times, the cells were stained with Harris hematoxylin for 1 min and rinsed with tap water for 2 min. After fixation with 4% paraformaldehyde for 20 min and incubation with phalloidin-FITC for 20 min, the coverslips were examined with a laser scanning confocal microscope with 488 nm and 633 nm excitation light to evaluate the crystal retention by cells. E Detection of CaSR and Claudin-14 mRNA Expression in HK-2 Cells by Quantitative Real-Time PCR. After four 5-min washes with TBST, the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase for 2 h at 20°C. e signals were captured using an ECL advanced system. e bands were quantified through Quantity One software and normalized to GAPDH
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