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Introduction

Urolithiasis, or kidney stone formation, is one of the most common urological conditions and is characterized by the deposition of crystals or stones within the kidneys (1). Calcium oxalate (CaOX) and/or calcium phosphate are the predominant types of renal stones and their formation is closely associated with increased production of reactive oxygen species (ROS) (2). The key processes underlying stone formation are influenced by multiple factors, including elevated urinary calcium and phosphate levels, as well as the relationship between urinary volume and CaOX crystal formation (3). Renal calculi in urolithiasis may also result from increased glycolic acid oxidase activity induced by sodium oxalate (NaOX), which generates glycolate and oxalate (2). When oxalate crystals deposit and adhere to renal tubular epithelial cells, these cells become injured and impaired (4). In response to renal injury, epithelial cells initiate proliferation and migration to promote regeneration and tissue repair. Collective cell migration is a tightly regulated process essential for normal physiological activities, including tissue repair and wound healing (5). Among the key molecules involved in cell migration and polarization are E-cadherin and vimentin, which play critical roles in regulating these processes. E-cadherin is a key integral protein that mediates lateral cell-to-cell adhesion via adherens junctions, playing a crucial role in maintaining tissue integrity and inhibiting relative cell mobility. By contrast, vimentin functions as a critical regulator of the wound repair process, serving as an intermediate filament that facilitates cell motility and migration (6). Epithelial tight junction proteins, such as zonula occludens (ZOs), occludin, claudins and junctional adhesion molecules, are also essential for regulating paracellular transport in renal tubular epithelial cells (7). These proteins can be altered following renal stone-induced damage. For example, treatment of Madin-Darby canine kidney (MDCK) cells with calcium oxalate monohydrate (COM) crystals has been shown to impair tight junction function by reducing the expression of occludin and ZO-1(8). Similarly, treatment of rat renal tubular epithelial cells (NRK-52E) with COM crystals induces changes in adhesion molecules, including hyaluronic acid, osteopontin and CD44(9). Thus, alterations in cellular signaling and adhesion molecules in renal tubular epithelial cells are critical determinants of oxalate crystal-induced injury and tissue repair (10). Understanding these changes may provide insights into potential therapeutic approaches for diseases related to oxalate crystal deposition.

Currently, there is no specific drug that effectively treats or prevents urolithiasis, despite numerous biological and physical studies aimed at preventing its development (2). However, a number of researchers have turned to natural medicinal agents to evaluate their anti-urolithiasis potential (11,12). The increasing interest in medicinal herbs can be attributed to their wide-spread availability, low cost, long history of traditional use and minimal side effects.

Gracilaria fisheri is a common red seaweed widely found along the coastal areas of Thailand. It is rich in sulfated galactan (SG), which primarily consists of repeating units of D-galactose and 3,6-anhydrogalactose with sulfate residues (13). SG is known for its various biological activities, including immunostimulant (13), antioxidant (14) and wound healing properties (15). Our previous study demonstrated that modifying SG to increase its sulfation, resulting in sulfated galactan from G. fisheri (SGS), inhibited oxalate crystal formation and provided enhanced protection against NaOX-induced human kidney (HK-2) cell death (16). It is considered that certain moieties of the polysulfated chain of SGS can serve as mimetics of natural ligand-protein receptor-glycosaminoglycans, which interact with various proteins, leading to post-translational modifications and modulation of signaling molecules inside the cells (17). These modifications determine cell behavior and responses (18). As previously reported, SG from G. fisheri has been demonstrated to interact with the epidermal growth factor receptor (EGFR), resulting in the regulation of EGFR signaling activity, including p-EGFR and p-Erk levels (19). In addition, studies have also revealed that the expression of adhesion molecules, such as EpCAM, is regulated by EGFR signaling (20,21). Accumulating evidence suggests that SGS may modulate cell signaling pathways and upregulate the expression of cell adhesion molecules, potentially facilitating cell migration and tissue repair. It was hypothesized that SGS promotes cell migration, regulates the expression of adhesion molecules and downstream signaling pathways in HK-2 cells and provides a protective effect against oxalate crystal-induced injury. In addition, the effects of SGS on wound healing and the expression of adhesion molecules in NaOX-induced HK-2 cell injury have not yet been investigated. The present study aimed to evaluate the effects of SGS on cell migration and adhesion molecule expression in NaOX-induced HK-2 cell injury. HK-2 cell migration was assessed using scratch migration and Transwell assays. The mRNA and protein expression levels of adhesion molecules were analyzed using reverse transcription-quantitative (RT-q) PCR, western blotting and immunofluorescence confocal microscopy under NaOX-induced conditions. Additionally, the morphology of HK-2 cells treated with SGS and NaOX was examined using scanning electron microscopy. The present study, for the first time to the best of the authors' knowledge, explored the effects of SGS on cell migration, the expression of cell adhesion molecules and cell signaling pathways under NaOX-induced conditions. These findings are expected to provide evidence supporting the potential use of SGS in the prevention and treatment of urolithiasis and related renal injuries.

Materials and methods

Materials, chemicals and reagents

SGS was prepared as described by Rudtanatip et al (15). SGS, with a molecular weight of 97.07 kDa and a sulfation level of 26.53±1.09%, consists of a complex structure of alternating 3-linked β-D-galactopyranose and 4-linked 3,6-anhydro-α-L-galactopyranose or α-L-galactopyranose, containing sulfate groups at C-2, C-4 and C-6 of D-galactopyranose, C-2 of L-anhydro-galactopyranose and C-2 and C-6 of L-galactopyranose. The backbone structure of SGS is shown in Fig. 1. The human kidney cell line (HK-2) was purchased from ATCC. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and Anti-Anti (100X) antibiotic-antimycotic were obtained from Thermo Fisher Scientific, Inc. Cystone was procured from the Himalaya Wellness Company. Transwell permeable supports (6.5 mm inserts, 24-well plates) were purchased from Corning Life Sciences. TRIzol® reagent and the RevertAid First Strand cDNA Synthesis Kit were obtained from Thermo Fisher Scientific, Inc. PowerUp SYBR™ Green Master Mix was purchased from Applied Biosystems (Thermo Fisher Scientific, Inc.). The Protease Inhibitor Cocktail (100X) was procured from MedChemExpress. Nitrocellulose membrane was purchased from Global Life Science Operations. Clarity Western ECL substrate was obtained from Bio-Rad Laboratories, Inc. CellMask Deep Red Plasma Membrane Stain was purchased from Thermo Fisher Scientific, Inc. All other chemicals were purchased from Merck KGaA.

Figure 1 The structural features of Gracilaria fisheri SGS. SGS, sulfated galactan with increased sulfation.

HK-2 cell culture and experimental design

HK-2 cells were cultured in DMEM supplemented with 2.2 g/l sodium bicarbonate, 10% FBS and 1% antibiotic-antimycotic in a humidified incubator at 37˚C with 5% CO2. To evaluate the effects of SGS on NaOX-induced injury in HK-2 cells, 1.25 mmol/l of NaOX was used for induction, as described in our previous study (16). The cytotoxicity of NaOX on HK-2 cells was observed at the concentrations ranging from 0.156-5.0 mmol/l, with NaOX showing a dose-dependent reduction in cell proliferation. This concentration (1.25 mmol/l) decreased cell viability by <50% (16). The cells were divided into 5 groups: i) Control-no treatment; ii) NaOX control-treated with 1.25 mmol/l of NaOX; iii) 100-SGS + NaOX-treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; iv) 1000-SGS + NaOX-treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; v) Cystone + NaOX-treated with 100 µg/ml of Cystone, a positive control drug, combined with 1.25 mmol/l of NaOX. Cystone was used as a positive control because it is commonly used to relieve urological problems, including nephrolithiasis (22). It has also been employed as a positive control in numerous experimental studies evaluating its anti-urolithiasis activity, both in vitro and in vivo (23,24). Prior to treatment with NaOX and SGS, the cells were incubated overnight in a serum-free culture medium to achieve synchronization.

Scratch wound healing assay

The rate of HK-2 cell migration was assessed using a scratch wound healing assay. Cells were seeded in 24-well plates at a density of 4.5x104 cells/well in DMEM supplemented with 10% FBS. Once the cells reached 80-100% confluence, they were incubated overnight in a serum-free culture medium and a scratch was created on the bottom of the well using a 200-µl sterile pipette tip. Detached cells were removed by washing twice with 1X PBS. The remaining cells were treated with either the SGS mixed NaOX solution or the Cystone mixed NaOX solution. Cell migration was monitored and imaged at 0, 6, 12 and 24 h post-treatment using a Nikon Eclipse TS100 inverted microscope (Nikon Corporation). The average distance between the edges of the scratch was calculated to determine the wound width.

Transwell cell migration assay

To evaluate cell migration, HK-2 cells were seeded into the upper chamber of a 24-well HTS Transwell Permeable Supports plate at a density of 4.5x104 cells/well in serum-free DMEM. The cells were treated with either the SGS mixed NaOX solution or the Cystone mixed NaOX solution, while DMEM supplemented with 10% FBS was added to the lower chamber as a chemoattractant. After 24 h, the migrated cells in the lower chamber were fixed with 10% neutral buffered formalin at room temperature for 20 min, stained with toluidine blue O at room temperature for 10 min and visualized under an inverted microscope (Leica Biosystems).

Analysis of adhesion mRNA transcription by quantitative polymerase chain reaction (qPCR) assay

HK-2 cells were seeded in a 6-well plate at a density of 1.6x105 cells/well in DMEM supplemented with 10% FBS and cultured at 37˚C for 24 h. The cells were then treated with either the SGS mixed NaOX solution or the Cystone mixed NaOX solution and incubated for an additional 24 h. Following treatment, the cells were harvested for RNA extraction. Total RNA was extracted using the TRIzol® reagent (Thermo Fisher Scientific, Inc.) and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.), following the manufacturer's instructions. mRNA expression of CD44, EpCAM, E-cadherin, vimentin, occludin, ZO-1 and GAPDH was analyzed by qPCR using PowerUp SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), as per the manufacturer's protocol. The specific primer sequences used in the experiment are listed in Table I. Each 20-µl PCR reaction mixture contained 10 µl of PowerUp SYBR Green Master Mix, 0.8 µl of 500 nM forward and reverse primers, 7.2 µl of nuclease-free water and 2 µl of cDNA. The cycling conditions were as follows: 50˚C for 2 min, 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec and 72˚C for 30 sec. Amplification and analysis were performed using the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH was used as the housekeeping gene for normalization and relative gene expression was calculated using the 2-∆∆Cq method (25). The experiments were performed in triplicate.

Table I Primer sequences used for qPCR analysis.

Table I

Primer sequences used for qPCR analysis.

Gene	Sequence
CD44-Forward	5'-TGCCGCTTTGCAGGTGTAT-3'
CD44-Reverse	5'-GGCCTCCGTCCGAGAGA-3'
EpCAM-Forward	5'-ATAACCTGCTCTGAGCGAGTG-3'
EpCAM-Reverse	5'-TGAAGTGCAGTCCGCAAACT-3'
E-cadherin-Forward	5'-GAACAGCACGTACACAGCCCT-3'
E-caherin-Reverse	5'-GCAGAAGTGTCCCTGTTCCAG-3'
Vimentin-Forward	5'-AAAACACCCTGCAATCTTTCAGA-3'
Vimentin-Reverse	5'-CACTTTGCGTTCAAGGTCAAGAC-3'
Occludin-Forward	5'-GTCCAATATTTTGTGGGACAAGG-3'
Occludin-Reverse	5'-GGCACGTCCTGTGTGCCT-3'
ZO-1-Forward	5'-AGAAGGATGTTTATCGTCGCATT-3'
ZO-1-Reverse	5'-CCAAGAGCCCAGTTTTCCAT-3'
GAPDH-Forward	5'-GGTGAAGGTCGGTGTGAACG-3'
GAPDH-Reverse	5'-CTCGCTCCTGGAAGATGGTG-3'

Determination of adhesion molecule and signaling protein expression by western blot assay

HK-2 cells were harvested after treatment and used for protein extraction in a protein lysis buffer containing 20 mM Tris-HCl, 100 mM NaCl, 50 mM PMSF and 1X protease inhibitor cocktail. Total protein extracts were quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) to determine protein concentration. The protein samples (50 µg/lane) were then separated on a 12.5% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked with 4% bovine serum albumin (BSA; Merck KGaA) in 1X Tris-buffered saline at room temperature for 2 h and then incubated overnight at 4˚C with primary antibodies specific to CD44, EpCAM, E-cadherin, vimentin, occludin, ZO-1, PI3K, Akt, Erk1/2 and p38 (1:1,000 dilution). Subsequently, the membrane was incubated at room temperature for 1 h with a secondary antibody: HRP-conjugated goat anti-rabbit IgG (1:2,000 dilution) for CD44, EpCAM, occludin, ZO-1, PI3K, Akt, Erk1/2 and p38, or HRP-conjugated goat anti-mouse IgG (1:2,000 dilution) for E-cadherin and vimentin. The antibodies used in the experiment are listed in Table II. The protein signals were developed using a Clarity Western ECL substrate (Bio-Rad Laboratories, Inc.) and band intensities were analyzed relative to the internal control (β-actin) using ImageJ version 1.32j (National Institutes of Health).

Table II List of antibodies used for western blotting and fluorescence analyses.

Table II

List of antibodies used for western blotting and fluorescence analyses.

Name	Host	Cat. no.	Supplier
CD44	Rabbit	Ab157107	Abcam
EpCAM	Rabbit	Ab71916	Abcam
E-cadherin	Mouse	M3612	Dako (Agilent Technologies, Inc.)
Vimentin	Mouse	M0725	Dako (Agilent Technologies, Inc.)
Occludin	Rabbit	71-1500	Thermo Fisher Scientific, Inc.
ZO-1	Rabbit	40-2200	Thermo Fisher Scientific, Inc.
PI3K	Rabbit	4255	Cell Signaling Technology, Inc.
Akt	Rabbit	4058	Cell Signaling Technology, Inc.
Erk1/2	Rabbit	4695	Cell Signaling Technology, Inc.
p38	Rabbit	MA5-15177	Thermo Fisher Scientific, Inc.
β-actin	Rabbit	AF7018	Affinity biosciences
HRP-conjugated goat anti-rabbit		31466	Thermo Fisher Scientific, Inc.
HRP-conjugated goat anti-mouse		31430	Thermo Fisher Scientific, Inc.
FITC-conjugated goat anti-rabbit		12-507	Merck KGaA
FITC-conjugated goat anti-mouse		12-506	Merck KGaA

Immunofluorescence confocal microscopy

HK-2 cells were seeded onto round glass coverslips in a 24-well plate at a density of 4.5x104 cells/well in DMEM supplemented with 10% FBS and cultured at 37˚C for 24 h. The cells were then treated with either the SGS mixed NaOX solution or the Cystone mixed NaOX solution and allowed to grow for an additional 24 h before being fixed with 10% formalin at room temperature for 20 min. The fixed cells were washed three times with 1X PBS and incubated overnight with primary antibodies specific to CD44, E-cadherin, vimentin, EpCAM, occludin and ZO-1 (1:250 dilution), as shown in Table II. They were then incubated at room temperature for 1 h with FITC-conjugated goat anti-rabbit IgG (1:500 dilution) for CD44, EpCAM, occludin and ZO-1, or FITC-conjugated goat anti-mouse IgG (1:500 dilution) for E-cadherin and vimentin. Finally, the cells were stained at room temperature for 20 min with DAPI for nuclear visualization and CellMask Deep Red Plasma Membrane Stain to label the cytoplasm and plasma membranes, following the manufacturer's protocols. Images were captured using a Zeiss LSM800 inverted confocal laser scanning microscope (Carl Zeiss AG) equipped with a 63x Plan-Apochromat 1.4 NA oil immersion objective lens (magnification, x630). Laser wavelengths and pinhole size were set as follows: 488 nm with a 43 µm pinhole for FITC staining, 405 nm with a 42 µm pinhole for DAPI staining and 561 nm with a 53 µm pinhole for CellMask Deep Red Plasma Membrane staining.

Observation of cell morphology by scanning electron microscopy

HK-2 cells were seeded onto round glass coverslips in a 24-well plate at a density of 4.5x104 cells/well in DMEM supplemented with 10% FBS and cultured at 37˚C for 24 h. The cells were then treated with either the SGS mixed NaOX solution or the Cystone mixed NaOX solution and allowed to grow for an additional 24 h. Following incubation, the cells were fixed with 2.5% glutaraldehyde at room temperature for 30 min. The coverslips were then washed with 1X PBS, dehydrated and air-dried overnight. The dried coverslips were mounted onto aluminum stubs and coated with gold particles. Cell morphology was subsequently examined using a JSM-IT200 InTouchScope scanning electron microscope (JEOL, Ltd.).

Statistical analysis

All data were expressed as mean ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison test using GraphPad Prism version 9 (Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Results

Gracilaria fisheri sulfated galactan with increased sulfation (SGS) enhances HK-2 cell migration, counteracting NaOX-induced inhibition of cell migration

The present study investigated the effect of SGS on cell migration using a scratch wound assay. The initial wound edges were marked to measure cell migration by tracking the decrease in wound width over time. The scratch distances between wound edges were compared across time points (0, 6, 12 and 24 h). The results showed no significant differences in wound gape among the groups at 0 and 6 h after scratching. However, at 12 and 24 h after scratching, the wound gape in the NaOX group was markedly larger compared with the normal control group. Notably, a significant reduction in wound gape was observed at 12 h in NaOX-induced HK-2 cells treated with SGS (1,000 µg/ml) compared with the NaOX group. By 24 h, NaOX-induced HK-2 cells treated with SGS (100 and 1,000 µg/ml) and Cystone showed a significant decrease in wound gape compared with the NaOX group (Fig. 2A and B). To further confirm the cell migration promoting ability of SGS, a Transwell plate assay was performed. As shown in Fig. 3, NaOX treatment reduced the number of migratory HK-2 cells compared with the control. However, treatment with SGS (100 and 1,000 µg/ml) and Cystone markedly increased the number of migratory cells in NaOX-induced HK-2 cells. These results suggest that SGS markedly enhances HK-2 cell migration at the wound edge and promotes cell migration in NaOX-induced HK-2 cells.

Figure 2 The effect of SGS on HK-2 cell migration, evaluated using a scratch wound healing assay. (A) Phase contrast images show the wound distance in HK-2 cells treated with NaOX, SGS + NaOX and Cystone + NaOX at 0, 6, 12 and 24 h after scratching. (B) Bar graphs represent the wound gape at 0, 6, 12 and 24 h after scratching. Data are presented as mean ± SEM (n=3) from three independent experiments. *P<0.05 vs. normal control; #P<0.05 vs. NaOX control. Scale bars, 100 µm. SGS, sulfated galactan with increased sulfation; NaOX, sodium oxalate; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

Figure 3 Photomicrographs show the effect of SGS on the Transwell migration of HK-2 cells, stained with toluidine blue O at 24 h, with accompanying graphs quantifying the number of migrated cells. Data are presented as mean ± SEM (n=3) from three independent experiments. *P<0.05 vs. normal control; #P<0.05 vs. NaOX control. Scale bar, 500 µm. SGS, sulfated galactan with increased sulfation; NaOX, sodium oxalate; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

SGS positively regulates the expression of adhesion molecules in HK-2 cells induced by NaOX

Cell adhesion molecules play a critical role in maintaining cell polarity, integrity and function (7). To evaluate the effects of SGS on the expression of adhesion molecules in NaOX-induced HK-2 cell damage, qPCR and western blot analyses were conducted to detect the expression levels of CD44, EpCAM, E-cadherin, vimentin, occludin and ZO-1. The qPCR results showed significant alterations in mRNA expression in HK-2 cells treated with NaOX, SGS + NaOX and Cystone + NaOX. Specifically, the expression levels of EpCAM, E-cadherin, occludin and ZO-1 were markedly downregulated following NaOX exposure, while CD44 and vimentin were markedly upregulated. Treatment with SGS (1,000 µg/ml) markedly upregulated the expression levels of CD44, EpCAM, E-cadherin and occludin and markedly downregulated the expression of vimentin, compared with the NaOX control. However, no significant changes in ZO-1 expression were observed. Similarly, the expression levels of adhesion molecule mRNA in NaOX-induced HK-2 cells treated with Cystone (positive control) were consistent with those in SGS-treated cells (Fig. 4). Western blotting results (Fig. 5) further corroborated the qPCR findings. In NaOX-induced HK-2 cell damage, the protein expression levels of adhesion molecules followed a similar trend. Following treatment with SGS, the protein expression levels of CD44 and vimentin were markedly downregulated, while the expression levels of EpCAM, E-cadherin, occludin and ZO-1 were markedly upregulated, compared with the NaOX control. These results suggested that SGS can modulate the expression of adhesion molecules at both the mRNA and protein levels, providing protective effects against NaOX-induced damage in HK-2 cells.

Figure 4 mRNA expression levels of adhesion molecules in HK-2 cells treated with NaOX, SGS + NaOX and Cystone + NaOX, assessed by qPCR analysis. The transcription levels of CD44, EpCAM, E-cadherin, vimentin, occludin and ZO-1 are shown. Data are presented as mean ± SEM (n=3) from three independent experiments. *P<0.05 vs. normal control; #P<0.05 vs. NaOX control. NaOX, sodium oxalate; SGS, sulfated galactan with increased sulfation; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

Figure 5 Expression levels of adhesion molecules in NaOX-induced HK-2 cells following SGS treatment. (A) Protein expression levels of adhesion molecules in HK-2 cells treated with NaOX, SGS + NaOX and Cystone + NaOX, analyzed by western blotting. (B) Quantitative data of CD44, EpCAM, E-cadherin, vimentin, occludin and ZO-1 normalized to β-actin, with fold changes relative to the control group. Data are presented as mean ± SEM (n=3) from three independent experiments. *P<0.05 vs. normal control; #P<0.05 vs. NaOX control. NaOX, sodium oxalate; SGS, sulfated galactan with increased sulfation; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

Additionally, confocal immunofluorescence staining was performed to confirm the distribution of adhesion molecules in HK-2 cells. The results showed that NaOX-induced HK-2 cells exhibited increased expression of CD44 and vimentin, while the expression of EpCAM, E-cadherin, occludin and ZO-1 was decreased (Fig. 6). By contrast, HK-2 cells treated with SGS + NaOX showed decreased expression of CD44 and vimentin, alongside increased expression of EpCAM, E-cadherin, occludin and ZO-1. These findings indicate that NaOX alters the expression patterns of adhesion molecules, while SGS mitigates NaOX-induced toxicity and restores adhesion molecule expression to a more normal state.

Figure 6 Distribution of adhesion molecules in NaOX-induced HK-2 cells following SGS treatment. (A) Confocal fluorescence micrographs show the expression of adhesion molecules (CD44, EpCAM, E-cadherin, vimentin, occludin and ZO-1) in HK-2 cells treated with NaOX, SGS + NaOX and Cystone + NaOX. Scale bars, 10 µm. (B) Quantitative analysis of fluorescence intensities for each adhesion molecule. Data are presented as mean ± SEM (n=3) from three independent experiments. *P<0.05 vs. normal control; #P<0.05 vs. NaOX control. NaOX, sodium oxalate; SGS, sulfated galactan with increased sulfation; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

NaOX-induces morphological changes of HK-2 cells were reversed by SGS

The effect of SGS on the morphological changes of NaOX-induced HK-2 cells was examined using scanning electron microscopy, as shown in Fig. 7. Normal HK-2 cells (control) exhibited thick, long, slender and regular cytoplasmic processes that were tightly interconnected with the cytoplasmic processes of neighboring cells. By contrast, NaOX-induced HK-2 cell damage resulted in significant morphological alterations, including cell volume retraction and the presence of very thin, long, slender and irregular cytoplasmic processes. However, NaOX-induced HK-2 cells treated with SGS displayed thin, long, slender and regular cytoplasmic processes, resembling those observed in the control and Cystone-treated cells. These findings suggested that SGS exerts a cell recovery effect, mitigating the morphological damage caused by NaOX in HK-2 cells.

Figure 7 Scanning electron microscopy images show morphological changes in HK-2 cells from various treatment groups, including normal control, NaOX control, 100-SGS + NaOX, 1000-SGS + NaOX and Cystone + NaOX. Scale bar, 10 µm. NaOX, sodium oxalate; SGS, sulfated galactan with increased sulfation; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

SGS alters the expression of PI3K, Akt, Erk1/2 and p38 signaling proteins in NaOX-induced HK-2 cell damage

The PI3K/Akt and MAPK signaling pathways play a critical role in regulating various cellular processes, including the cell cycle, survival and death. These pathways are closely associated with the pathological mechanisms of kidney diseases (26). To evaluate the effects of SGS on signaling pathways, we investigated the expression of key signaling proteins, including PI3K, Akt, Erk1/2 and p38, in NaOX-induced HK-2 cell damage. As shown in Fig. 8, NaOX exposure markedly upregulated the expression levels of Akt and p38, while downregulating the expression levels of PI3K and Erk1/2. However, treatment of NaOX-induced HK-2 cells with SGS markedly reversed these changes. Specifically, SGS treatment (1,000 µg/ml) markedly upregulated the expression levels of PI3K and Erk1/2 while downregulating the expression levels of Akt and p38, compared with the NaOX control. These effects were consistent with those observed in Cystone-treated NaOX-induced HK-2 cells.

Figure 8 Expression levels of PI3K/Akt and MAPK signaling proteins in NaOX-induced HK-2 cells following SGS treatment. (A) Protein expression levels of PI3K, Akt, Erk1/2 and p38 in HK-2 cells treated with NaOX, SGS + NaOX and Cystone + NaOX, assessed by western blotting. (B) Quantitative data of PI3K, Akt, Erk1/2 and p38 normalized to β-actin, with fold changes relative to the control group. Data are presented as mean ± SEM (n=3) from three independent experiments. *P<0.05 vs. normal control; #P<0.05 vs. NaOX control. NaOX, sodium oxalate; SGS, sulfated galactan with increased sulfation; Control, no treatment; NaOX control, treated with 1.25 mmol/l of NaOX; 100-SGS + NaOX, treated with 100 µg/ml of SGS combined with 1.25 mmol/l of NaOX; 1000-SGS + NaOX, treated with 1,000 µg/ml of SGS combined with 1.25 mmol/l of NaOX; Cystone + NaOX, treated with 100 µg/ml of Cystone combined with 1.25 mmol/l of NaOX.

Discussion

Biological activities of sulfated polysaccharides are strongly influenced by their molecular structures, including molecular weight and degree of sulfation (27). The present study demonstrated that SGS enhanced cell migration ability and wound healing activity in NaOX-induced HK-2 cells. Kidney stone (oxalate crystal) formation has been linked to increased glycolic acid oxidase activity induced by NaOX, leading to injury and impairment of renal tubular epithelial cells (2,28). In response to injury, renal tubular epithelial cells compensate through proliferation and migration to promote tissue repair (5). However, NaOX inhibits renal epithelial cell migration, potentially exacerbating kidney disease progression, as observed in MDCK cells exposed to NaOX (29). In the present study, SGS markedly enhanced wound healing activity in HK-2 cells, which aligns with previous findings showing that SGS promoted wound closure in an L929 fibroblast scratch model (15). This suggests that increased sulfation of polysaccharides is closely associated with enhanced wound healing properties (15).

Cell adhesion molecules play a crucial role in the processes of tissue injury and repair (30). Dysregulation of these molecules is also critical in triggering signaling cascades that can lead to apoptotic or necrotic cell death. The detachment of injured cells is linked to changes in adhesion molecules such as cadherins, integrins and occludin junction proteins, which are essential for maintaining cell-cell and cell-extracellular matrix adhesions (31). Oxalate crystals interact with renal epithelial cells, altering tight junctions by decreasing the expression levels of key proteins such as ZO-1, occludin and claudin (32). Additionally, oxalate crystals promote renal epithelial cell damage by upregulating adhesion proteins, including hyaluronic acid, osteopontin, CD44 and annexin A2, on the cell surface (9). Conversely, oxalate crystal deposition in the kidneys of hyperoxaluric rats is reduced when osteopontin expression is inhibited, highlighting its role in crystal formation and retention (33). Additionally, E-cadherin and vimentin are key proteins involved in regulating cell migration and polarization. E-cadherin, an integral membrane protein, mediates lateral cell-to-cell adhesion through adherens junctions, playing a critical role in maintaining tissue integrity and inhibiting cell mobility. By contrast, vimentin serves as an intermediate filament that regulates the wound repair process by promoting cell motility and migration (6). The inhibition of MDCK cell migration by oxalate crystals, as reported by Ji et al (29), may be linked to the dysregulation of E-cadherin and vimentin. In the present study, NaOX exposure altered the expression of CD44, EpCAM, E-cadherin, vimentin, occludin and ZO-1 in HK-2 cells, suggesting damage to barrier function, loss of cell adhesion and impaired cell migration. Additionally, increased expression of CD44 has been shown to upregulate cytoskeleton function through ankyrin, activating the actomyosin contractile complex to mediate cell migration (34) and may directly affect wound healing (35). The increased expression of CD44 induced by NaOX in the present study may reflect a compensatory cellular response by enhancing contraction, migration and wound healing. Another possible reason for the increased CD44 expression under NaOX-induced conditions is an imbalance between cellular oxidants and antioxidants, leading to the expression of inflammatory molecules (36).

However, SGS treatment alleviated the effects of NaOX by restoring the expression levels of these molecules to near-normal levels. Several studies further support the protective effects of compounds on the expression of proteins and RNA involved in cell adhesion and migration, which mitigate kidney injury. For example, downregulation of osteopontin by caffeic acid inhibited kidney stone formation (37). In a glyoxylate-induced nephrolithiasis mouse model, curcumin administration reduced calcium oxalate crystal deposition and renal tissue damage by decreasing the expression of osteopontin and CD44(38). Similarly, treatment of MDCK cells with the plant alkaloid trigonelline attenuated oxalate-induced epithelial-to-mesenchymal transition by increasing the expression of epithelial markers E-cadherin and ZO-1(39).

Oxalate crystals not only alter the transcription and translation of adhesion molecules but also markedly alter cell morphology. Renal cells exposed to oxalate crystals exhibit distinct morphological changes, including structural disorganization, cell edema, chromatin condensation, translocation of phosphatidylserine to the cell surface and displacement of membrane proteins (40,23). These morphological changes are closely linked to and functionally integrated with the actin-based cytoskeleton (41). In the present study, NaOX exposure induced marked morphological changes in HK-2 cells, as observed under scanning electron microscopy. However, treatment with SGS restored the morphology of oxalate-injured cells, which gradually returned to a normal appearance. These findings align with a previous study demonstrating the cytoprotective effects of D. pedicellata ethanolic extract and Cystone in oxalate crystal-induced cell damage, where restoration of cell morphology was also observed (23).

The mechanisms underlying kidney injury caused by oxalate crystals are multifactorial and complex, with a number of aspects that remain to be elucidated. The PI3K/Akt and MAPK signaling pathways are closely linked to the pathological processes of kidney diseases and play crucial roles in cellular functions, including the cell cycle, survival and tissue repair (26,42). The findings of the present study revealed that NaOX exposure increased the expression of Akt and p38 while decreasing the expression of PI3K and Erk1/2. Conversely, SGS treatment reversed these effects, increasing the expression of PI3K and Erk1/2 and reducing the expression of Akt and p38, highlighting the possible protective effects of SGS against NaOX-induced damage. The potential mechanisms by which SGS modulates Akt, p38, PI3K and Erk1/2 in NaOX-induced cell death may involve its high sulfate ester content, which imparts a strong negative charge, enabling binding to cell surface receptors and regulation of the PI3K/Akt/MAPK pathways (43,44). Additionally, SGS interferes with crystal formation, reducing crystal-cell binding and subsequently inhibiting reactive oxygen species (ROS) production, which contributes to tight junction alteration via regulation of the ROS/Akt/p38 MAPK signaling pathway (8). The antioxidant properties of SGS may also neutralize NaOX-induced free radicals, thereby suppressing oxidative stress and modulating downstream signaling molecules (16). However, the protective effect of SGS against NaOX-induced damage in HK-2 cells requires further confirmation using PI3K/Akt and MAPK signaling inhibitors.

Oxalate crystal induction is associated with increased cell death and dysregulation of the PI3K/Akt and MAPK signaling pathways. Consistent with this, treating MDCK cells with oxalate crystals has been shown to reduce the expression of junctional proteins, such as ZO-1 and occludin, while increasing the expression of Akt/ASK1/p38 MAPK signaling proteins (8). Transcriptomic analysis of renal tubular epithelial cells and mouse kidneys exposed to oxalate crystals has revealed that kidney stone formation is associated with activation of the PI3K/Akt signaling pathway (45). Additionally, p38 MAPK has been implicated in oxalate crystal-induced tight junction alteration in rat renal tubular epithelial (NRK-52E) cells. The mRNA and protein expression levels of p38 MAPK-related molecules [such as phosphorylated (p-)p38] and adhesion molecules (such as osteopontin, hyaluronic acid and CD44) were markedly increased in NRK-52E cells treated with oxalate crystals (9,32). These findings are consistent with the present study, which showed that NaOX increased the expression of Akt and p38 while decreasing the expression of PI3K and Erk1/2. This response may reflect cellular compensation and repair mechanisms in response to oxalate crystal-induced ROS generation and cell injury (46,47). In addition, the decreased expression of Erk1/2 by NaOX may be attributed to increased ROS generation through the inhibition of the AMPK/mTOR/ERK signaling axis (48).

SGS treatment demonstrated opposing effects on the expression levels of PI3K, Akt, p38 and Erk1/2, effectively alleviating the effect of NaOX by restoring these signaling molecules to near-normal levels. Inhibition of p38 MAPK has been shown to reduce oxalate crystal-induced cell adhesion and injury. For example, treatment with SB239063, a p38 MAPK inhibitor, decreases p38 expression and inhibits oxalate crystal adhesion and cell damage in NRK-52E cells (9). Similarly, quercetin, a molecule with potent antioxidant and anti-inflammatory effects, suppresses p38 MAPK activation, mitigating oxidative stress and reducing oxalate crystal-induced injury in HK-2 cells (49). These findings are consistent with the results of the present study, in which SGS decreased the expression of p38 in NaOX-induced HK-2 cell injury. By contrast, Puerarin, an isoflavone derived from the root of Pueraria lobata, has been reported to activate the SIRT1/Akt/p38 signaling pathways, thereby reducing oxalate crystal-induced oxidative stress and autophagy, suggesting its potential to directly inhibit autophagy activation (50). Similarly, it can be implied that SGS suppressed Akt and p38 activation, mitigating ROS generation and reducing HK-2 cell death induced by NaOX. The observed increase in PI3K and Erk1/2 expression following SGS treatment may be related to the migration of non-injured epithelial cells adjacent to the site of injury, promoting wound healing activity (46). PI3K/Akt activation mediates VEGF-driven cell survival (47), while Erk1/2 signaling regulates cell migration and proliferation during wound healing. Specifically, inhibition of Erk1/2 delays wound healing, whereas its activation stimulates cell migration and proliferation (51). Two effects of SGS on NaOX-induced cell death can be proposed: i) SGS inhibits NaOX-induced cell death by suppressing ROS production mediated by Akt and p38 and ii) SGS promotes cell migration by activating PI3K and Erk1/2, thereby enhancing wound healing. These findings suggested that SGS may contribute to the prevention and treatment of urolithiasis by modulating the expression of molecules involved in cell migration, adhesion and related signaling pathways in response to oxalate crystal-induced injury. However, a limitation of the present study is that the modulation of the PI3K/Akt and MAPK (p38 and Erk1/2) signaling pathways, through the examination of phospho-protein levels, was not assessed. Future studies should incorporate western blot or ELISA to assess phosphorylated proteins (p-Akt, p-p38, p-ERK1/2) to provide direct evidence of signaling pathway regulation. This would strengthen the mechanistic understanding of how SGS modulates the expression of molecules in response to oxalate crystal-induced injury.

In conclusions, the present study demonstrated that NaOX exposure altered the expression of cell adhesion molecules and impaired the migration ability of HK-2 cells. Notably, SGS treatment alleviated these effects by restoring cell adhesion molecule expression to near-normal levels and enhancing cell migration. These protective effects are likely mediated through the regulation of the PI3K/Akt and MAPK (p38 and Erk1/2) signaling pathways. Furthermore, the findings highlighted the potential of SGS in the prevention and treatment of urolithiasis, providing valuable new insights into therapeutic strategies for this disease.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the Faculty of Medicine, Khon Kaen University, Thailand (grant no. IN63318), Research and Graduate Studies, Khon Kaen University (grant no. RP65-2-002) and Postgraduate Study Support Grant of the Faculty of Medicine, Khon Kaen University (grant no. 647070001-4).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

TR, JP and WS conceived and designed the experiments. TR, JP, SS and WS performed the experiments. TR, JP, SS, JEA and WS were responsible for the analysis of data. TR and JP participated in the drafting of the manuscript. KW, JK and TR edited and finalized the final version of the manuscript. KW and JK supervised the study. WS provided funding and project administration. TR, JP and WS confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References