Effects of alternative splicing events and transcriptome changes on kidney stone formation

During the development of urinary stone disease, the formation of tiny crystals that adhere to the renal tubular epithelium induces epithelial cell damage. This damage and repair of the epithelium is associated with the establishment of more crystal adhesion sites, which in turn stimulates further crystal adhesion and, eventually, stone formation. Deposited crystals typically cause changes in epithelial cell gene expression, such as transcriptome changes and alternative splicing events. Although considered important for regulating gene expression, alternative splicing has not been reported in studies related to kidney stones. To date, whether alternative splicing events are involved in the regulation of stone formation and whether crystallographic cell interactions are regulated by alternative splicing at the transcriptional level have remained unknown. Therefore, we conducted RNA sequencing and alternative splicing-related bioassays by modeling the in vitro stone environment. Many alternative splicing events were associated with crystallographic cell interactions. Moreover, these events regulated transcription and significantly affected the capacity of crystals to adhere to renal tubular epithelial cells and regulate apoptosis.

Rosiglitazone Suppresses Renal Crystal Deposition by Ameliorating Tubular Injury Resulted from Oxidative Stress and Inflammatory Response via Promoting the Nrf2/HO-1 Pathway and Shifting Macrophage Polarization

Oxidative stress and inflammatory response are closely related to nephrolithiasis. This study is aimed at exploring whether rosiglitazone (ROSI), a regulator of macrophage (Mp) polarization, could reduce renal calcium oxalate (CaOx) deposition by ameliorating oxidative stress and inflammatory response. Male C57 mice were equally and randomly divided into 7 groups. Kidney sections were collected on day 5 or day 8 after treatment. Pizzolato staining and polarized light optical microscopy were used to detect crystal deposition. PAS staining and TUNEL assay were performed to assess the tubular injury and cell apoptosis, respectively. Gene expression was assessed by immunohistochemistry, immunofluorescence, ELISA, qRT-PCR, and Western blot. The reactive oxygen species (ROS) level was assessed using a fluorescence microplate and fluorescence microscope. Hydrogen peroxide (H2O2), malonaldehyde (MDA), and glutathione (GSH) were evaluated to determine oxidative stress. Lactic dehydrogenase (LDH) activity was examined to detect cell injury. Adhesion of CaOx monohydrate (COM) crystals to HK-2 cells was detected by crystal adhesion assay. HK-2 cell death or renal macrophage polarization was assessed by flow cytometry. In vivo, renal crystal deposition, tubular injury, crystal adhesion, cell apoptosis, oxidative stress, and inflammatory response were significantly increased in the 7-day glyoxylic acid- (Gly-) treated group but were decreased in the ROSI-treated groups, especially in the groups pretreated with ROSI. Moreover, ROSI significantly reduced renal Mp aggregation and M1Mp polarization but significantly enhanced renal M2Mp polarization. In vitro, ROSI significantly suppressed renal injury, apoptosis, and crystal adhesion of HK-2 cells and markedly shifted COM-stimulated M1Mps to M2Mps, presenting an anti-inflammatory effect. Furthermore, ROSI significantly suppressed oxidative stress by promoting the Nrf2/HO-1 pathway in HK-2 cells. These findings indicate that ROSI could ameliorate renal tubular injury that resulted from oxidative stress and inflammatory response by suppressing M1Mp polarization and promoting M2Mp polarization. Therefore, ROSI is a potential therapeutic and preventive drug for CaOx nephrolithiasis.

Preliminary Evaluation of Nanobacteria on Crystal Retention, CaSR, and Claudin-14 Expression in HK-2 Cells

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.

Adaptive Response of HK-2 Cells to High Levels of COM Crystals Highlights CaSR as an Enhancer of Crystal Adhesion

Background: Calcium oxalate monohydrate (COM) is an aetiologic factor for urolithiasis. However, how Human Kidney-2 (HK-2) cells respond to a high COM has not yet been completely elucidated.Materials and methods: A gel-based proteomics approach was applied to investigate COM-induced cellular proteomic changes. The COM-induced upregulation of calcium-sensing receptor (CaSR) in HK-2 cells was studied. Surface phospholipids (PS), which play a role in urolithiasis formation by mediating adhesion of HK-2 cells, were labelled in the inner or outer leaflet of the plasma membrane of HK-2 cells with fluorescent nitrobenzoxadiazole (NBD) to form NBD-PS to detect transmembrane movements of PS. After labelling, HK-2 cells were exposed to COM in the presence of the CaSR-specific agonist gadolinium chloride (GdCl3) or the CaSR-specific antagonist NPS2390. Inward and outward transmembrane movements of PS were tracked with a fluorescence quenching assay. Surface-expressed PS was detected by an annexin V binding assay. Changes in aminophospholipid translocase (APLT), oxidative stress (OS), levels of apoptosis-related proteins in HK-2 cells and crystal adhesion were also assessed.Results: COM increased CaSR and surface-expressed PS levels, decreased APLT activity, impaired inward transport of PS, and enhanced outward transport of PS. However, pretreatment with GdCl3 further effectively inhibited the inward movement of PS and APLT activity and increased surface-expressed PS levels compared with COM treatment alone. In contrast, NPS2390 promoted the inward movement of PS and APLT activity and decreased surface-expressed PS levels compared with COM treatment alone. COM increased OS, apoptosis of HK-2 cells and crystal adhesion onto cells, and this increase was further enhanced by GdCl3 pretreatment but attenuated by NPS2390 treatment.Conclusions: These results strongly suggest that COM-induced CaSR generation may affect crystal adhesion by regulating PS externalization, apoptosis and OS in HK-2 cells.

Preprotection of Tea Polysaccharides with Different Molecular Weights Can Reduce the Adhesion between Renal Epithelial Cells and Nano-Calcium Oxalate Crystals

Crystal adhesion is an important link in the formation of kidney stones. This study investigated and compared the adhesion differences between nano-calcium oxalate monohydrate (COM) and human renal proximal tubule epithelial (HK-2) cells before and after treatment with tea polysaccharides (TPSs) TPS0, TPS1, TPS2, and TPS3 with molecular weights of 10.88, 8.16, 4.82, and 2.31 kDa, respectively. TPS treatment effectively reduced the damage of COM to HK-2 cells, thereby resulting in increased cell activity, decreased release of lactate dehydrogenase, cell morphology recovery, decreased level of reactive oxygen species, increased mitochondrial membrane potential, increased lysosomal integrity, decreased expression of adhesion molecule osteopontin and eversion of phosphatidylserine, and decreased crystal adhesion. Among the TPSs, TPS2 with moderate molecular weight had the best protective effect on cells and the strongest effect on the inhibition of crystal adhesion. Thus, TPS2 may be a potential anticalculus drug.

Repair activity and crystal adhesion inhibition of polysaccharides with different molecular weights from red algae Porphyra yezoensis against oxalate-induced oxidative damage in renal epithelial cells

Porphyra yezoensis polysaccharide repaired oxalate-injured renal epithelial cells and decreased COM crystal adhesion on the cell surface.

Downregulated Expression of Solute Carrier Family 26 Member 6 in NRK-52E Cells Attenuates Oxalate-Induced Intracellular Oxidative Stress

Solute carrier family 26 member 6 (Slc26a6), which is mainly expressed in the intestines and kidneys, is a multifunctional anion transporter that is crucial in the transport of oxalate anions. This study is aimed at investigating the effect of Slc26a6 expression on oxalate-induced cell oxidation and crystal formation. Lentivirus transfection was used to upregulate or downregulate Slc26a6 expression in NRK cells. Cell viability and apoptosis, reactive oxygen species (ROS) and malondialdehyde (MDA) generation, and superoxide dismutase (SOD) activity were measured. Crystal adhesion and the cell ultrastructure were observed using light and transmission electron microscopy (TEM). Three groups of rats, normal control, lentivirus-vector, and lentivirus-small interfering RNA (lv-siRNA) groups, were used, and after lentivirus transfection, they were fed 1% ethylene glycol (EG) and 0.5% ammonium chloride (NH4Cl) for 2 weeks. Dihydroethidium (DHE), terminal deoxynucleotidyl transferase (TdT) deoxyuridine dUTP nick-end labeling (TUNEL), and von Kossa staining were performed, and nuclear factor κB (NFκB) and osteopontin (OPN) expression were measured. In the vitro study, compared to the control group, downregulated Slc26a6 NRK cells showed alleviation of the cell viability decrease, cell apoptosis rate, ROS generation, and SOD activity decrease after oxalate treatment. Crystal adhesion and vesicles were significantly less after oxalate exposure than in the untreated controls. Rats infected with lentivirus-siRNA exhibited attenuated SOD generation, cell apoptosis, and crystal formation in the kidneys. Increased phosphorylation of NFκB and OPN was involved in the pathological process. In conclusion, the results of the present study indicate that reducing the expression of Slc26a6 in the kidney may be a potential strategy for preventing stone formation.