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INTRODUCTION
Kidney stones constitute a common issue encountered in the urology field. Their widespread occurrence, high rates of recurrence, and the financial burden of treatment have significant implications for individuals and society 1. One key factor contributing to kidney stone formation is the harm inflicted on renal tubular epithelial cells due to increased oxalate levels, with hyperoxaluria identified as a significant risk factor for developing urinary stones 2. Exposure to elevated concentrations of oxalic acid over extended periods can trigger oxidative stress in these cells, leading to an overproduction of reactive oxygen species. This process may cause cellular harm, such as cell degeneration, apoptosis, and the exposure of the basement membrane of renal tubular epithelial cells 3, potentially worsening subsequent injuries. Following this, a series of cellular lipid peroxidation and inflammatory responses may occur 4; as a result, antioxidants and anti-inflammatory medications are commonly employed to avert renal injury and the formation of kidney stones.
Lycopene (LYC), a vital carotenoid that falls under the classification of isoprenoid compounds, demonstrates properties such as anti-inflammatory, antioxidant, free radical scavenging, and immune modulation 5,6. Research indicates that lycopene may aid in relieving chronic prostatitis/ chronic pelvic pain syndrome through its ability to diminish inflammation and oxidative stress by engaging the NF-κB, Nrf2, and MAPKs signalling pathways 7. Nonetheless, no prior investigations have directly examined its protective effects against kidney damage caused by calcium oxalate stones. This study intends to explore the role and associated molecular mechanisms of LYC in the damage inflicted on renal tubular epithelial cells by oxalic acid and calcium oxalate crystals in vitro, thereby providing a theoretical foundation for utilizing anti-inflammatory and antioxidant agents, such as LYC, in the prevention and management of kidney stone disorders.
MATERIAL AND METHODS
Cells. HK-2 cells (purchased from BOSTER, catalogue number CX0044) were passed to the ninth passage.
Drugs and Reagents. Lycopene (Shanghai Yuanye Biotechnology Co., Ltd., product number B20378, purity ≥90%), oxalic (Shanghai Macklin Biochemical Technology Co., Ltd., product number O871905), DMEMF-12 (1:1) basic medium (Gibco, USA, product number C11330500BT), Cell Counting Kit-8 (Biosharp, product number BS350B), Reduced glutathione (GSH) assay kit, Lactate dehydrogenase (LDH) assay kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd., product numbers A006-2-1 and A020-2-2), Malondialdehyde (MDA) Colorimetric Assay Kit, Total Superoxide Dismutase (T-SOD) Activity Assay Kit (Wuhan Elabscience Biotechnology Co., Ltd., product numbers E-BC-K028-M and E- BC-K020-M), In this study, we employed the human IL-6 ELISA kit and the human MCP-1 ELISA kit (Quanzhou Ruixin Biotechnology Co., Ltd., product numbers RX106126H and RX106032H), Reactive oxygen species(ROS) detection kit(Shanghai beyotimeBiotechnology Co., Ltd., product numbers S0033S). Additionally, rabbit-derived antibodies include NF-κB p65, Osteopontin (OPN), Bax, Bcl-2, cytochrome C (Cyt C), and active-Caspase3. Secondary antibodies include an anti-mouse antibody from Shanghai Beyotime Biotechnology Co., Ltd. (product numbers: AF5243, AF7662, A0216) and a secondary rabbit antibody from Proteintech Group, Inc. (batch numbers: 50599-2-Ig, 26593-1-AP, SA00001-2). Furthermore, a mouse-derived GAPDH antibody from BOSTER is identified by product numbers: PB9334, BM3937, and BM3876.
Instrumentation
In this research, the equipment used included the Series II Water Jacket CO2 cell culture incubator, the Infinite M1000 Pro full-wavelength microplate reader (Tecan, Switzerland), the Axio Vert A1 inverted fluorescence microscope (Zeiss, Germany), the Mini-Protean 3 Dodeca electrophoresis system, the ChemiDoc XPS+ all-in-one gel imaging system (Bio-Rad Company, USA), and the MoFlo XDP ultra-fast flow cytometer (BD Company, USA).
Method
LYC was dissolved in DMSO, and a blank culture medium was subsequently introduced to formulate a storage solution with a concentration of 1000 μmol/L. The solution was passed through a microporous filter with a pore size of 0.22 μm and kept in a refrigerator at 4°C. Before beginning the experiment, the prepared LYC solution was administered to cultured cells in increasing concentrations (5, 10, 20, 40, 80, 100, 200, 500 μmol/L) to identify LYC’s effective concentration and toxicity range. In a 96-well plate populated with HK-2 cells (1 × 104 cells x well), the effects of different LYC concentrations on HK-2 cytotoxicity were evaluated using the CCK-8 kit, with assessments made 24 hours after administration (refer to Table 1). Ultimately, lycopene concentrations of 5 μM and 10 μM were chosen for further experiments.
Table 1 Effect of different concentrations of Lycopene on HK-2 cell activity.
| Groups | Concentration /μmol/L | Relative cellular activity/% |
|---|---|---|
|
Control group Lycopene group |
0 5 10 20 40 80 100 200 500 |
100 ± 15.92 98.26 ± 1.09 94.75 ± 0.84 88.13 ± 2.12 86.92 ± 2.75 85.02 ± 2.47 79.82 ± 2.34 69.27 ± 4.76 31.39 ± 2.42 |
Data is expressed as x̄ ± sd, n=3.
“A” Experimental grouping and intervention
The experimental groups were defined as follows: 1) Control group: cultured in basal medium for 24 hours; 2) Model group: cultured in basal medium containing oxalic (2 mmol/L) and COM (100 μg/mL) for 24 hours; 3) LYC I (5 μmol/L) group: treated with 5 μmol/L (LYC) in addition to the model group; 4) LYC II (10 μmol/L) group: treated with 10 μmol/L LYC in addition to the model group. These groups were utilized for subsequent experiments, including cell viability assessments, antioxidant capacity, inflammatory factors, reactive oxygen species, and Western blot analysis. Before the experiments, the original medium in the culture wells was removed, and serum-free medium was added to minimize the influence of proteins present in fetal bovine serum (FBS) on the experimental outcomes.
“B” CCK-8 assay to detect cell activity HK-2 cells were plated in a 96-well plate at a density of 1 × 104 cells x well. After over 18 hours for complete attachment, the cells were allocated into groups for experimental interventions as specified in Section “A”. Once the interventions were completed, the original culture medium was discarded and substituted with serum-free medium in every well. Subsequently, 100 μL of newly prepared culture medium and 10 μL of CCK-8 reagent were added, and the plate was incubated at 37°C for three hours. The absorbance (Ab) at 450 nm was recorded using a microplate reader. Each condition was evaluated in parallel within six replicate wells, and the experiment was conducted three times. The average value of Ab was computed, and cell activity was evaluated using the formula: Ab (experimental group) / Ab (control group) × 100%.
“C” Measurement of indicators related to antioxidant capacity
Cells were plated in a 6-well culture plate at a density of 2 x 105 cells per well until they adhered properly. The experimental groups aligned with those described previously (Experimental grouping). After a 24-hour incubation, cells from each group were collected, and the protein concentration was measured in centrifuge tubes. The instructions of the kit were followed to operate. Finally, the contents of lactate dehydrogenase(LDH), malondialdehyde (MDA), glutathione(GSH), and total superoxide dismutase(T-SOD) were measured in the cells using a Microplate reader at wavelengths of 450, 532, 405, and 450 nm and an ELISA kit was used for detection of IL -6, MCP-1 secretion.
“D” Cells were plated in a 6-well culture plate at a density of 2 x 105 cells per well until they adhered properly. The experimental groups aligned with those described in section “A”. After a 24-hour incubation, the supernatant from each cell group was gathered into centrifuge tubes. Next, 50 μL from each group was transferred to the enzyme plate, following the instructions provided with the kit. Essential procedures included preparing three duplicate wells for every cell group, with the experiment conducted three times. The absorbance (Ab) measurement was taken at a wavelength of 450 nm to assess the levels of the inflammatory cytokines IL-6 and MCP-1.
“E” Observation of cellular ROS
Cells were plated in a 6-well culture dish at a density of 2 x 105 cells per well, adhering to the group allocations outlined before (A). After a 24-hour culture period, a ROS detection kit was utilized to evaluate the levels of intracellular ROS. Specifically, 10 μmol/L DCFH-DA, which was diluted in serum-free culture medium, was introduced in a dark environment. One mL of this fluorescent probe was administered, and the cells were incubated for 20 minutes. After incubation, the cells underwent three washes with 1 mL of serum-free culture medium, after which images were taken using an inverted fluorescence microscope.
“F” Western blot for protein expression in HK-2 cells
Cells were planted in a 6-well plate according to the method described in Section “D”, then collected and denatured at high temperature. Electrophoresis was performed using the SDS gel system, followed by transfer to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours. The primary antibodies (NF-κB p65, OPN, Bax, Bcl-2, Cyt C, active-Caspase3, and GAPDH) were added and incubated at 4°C overnight. Subsequently, they were incubated with a secondary antibody at room temperature for 1.5 hours. The developing agent was added, and images were captured using an automatic gel imager. The ImageJ software was used to measure the gray value of each band and calculate the relative expression of target proteins in each group.
RESULTS
Comparison of cell activity and LDH in each group
Following 24 hours of treatment in the culture medium, the viability of cells in each group was assessed. The findings indicated a notable reduction in cell viability within the model group when juxtaposed with the control group (p<0.001), alongside a significant elevation in LDH levels (p<0.001). Conversely, cell viability in the LYC groups (5 μM, 10 μM) exhibited a marked increase compared to the model group (p<0.05), while LDH levels showed a significant decrease (p<0.05) (Table 2).
Table 2 Comparison of cellular activity and intracellular lactate dehydrogenase content in each group.
| Items | Control group | Model group | LYC (5μM) | LYC (10μM) |
|---|---|---|---|---|
| Cellular activity (%) | 100 | 42±2.98a | 60.37±3.44b | 51.79±1.88b |
| LDH (U/g prot) | 369.9±41.43 | 906±79.97a | 483.7±70.8b | 588.8±33.56b |
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group; as x, x̄ ± sd, n=3. LDH: lactate dehydrogenase; LYC: Lycopene.
Comparison of antioxidant and anti-inflammatory capacities of HK-2 cells
The findings from the biochemical index assessments indicated a notable reduction in GSH levels within the model group when contrasted with the control group (p<0.05). Conversely, MDA levels were found to have increased markedly (p<0.05). MDA levels saw a significant decline due to LYC treatment (p<0.001) (Table 3). Furthermore, when examining the model group, there was a notable surge in inflammatory cytokines IL -6 and MCP-1 compared to the control group (p<0.05). LYC administration at doses of 5 μM and 10 μM demonstrated an inhibitory effect on the secretion of IL -6 and MCP-1 relative to the model group (p<0.05) (Table 4).
Table 3 Comparison of indicators related to intracellular antioxidant capacity in each group.
| Items | Control group | Model group | Lycopene (5μM) | Lycopene (10μM) |
|---|---|---|---|---|
| Glutathione (GSH, umol/g protein) | 327.4±29.98 | 111.8±11.71a | 212.1±21.1b | 201.2±22.19b |
| Total superoxide dismutase (T-S0D, U/mg protein) | 13.54±0.70 | 11.57±1.61 | 15.03±2.56 | 14.59±2.29 |
| Malondialdehyde (MDA, nmol/mg protein) | 2.166±0.3 | 22.89±0.441a | 14.68±1.72b | 15.43±0.85b |
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group. The above data was analyzed using one-way ANOVA. x̄ ± sd, n=3.
Table 4 Effect of Lycopene on Interleukin-6 (IL-6) and Monocytechemotactic protein-1 (MCP-1) released from oxalic acid/calcium oxalate-induced HK-2 cells.
| Groups | IL -6 (pg/mL) | MCP-1(pg/mL) |
|---|---|---|
| Control group | 4.49 ± 0.44 | 9.78 ± 1.38 |
| Model group | 9.25 ± 0.46a | 29.38 ± 3.51a |
| Lycopene (5μM) | 5.42 ± 1.49b | 7.71 ± 6.10b |
| Lycopene (10μM) | 5.28 ± 1.51b | 11.84 ± 4.85b |
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group. The above data was analyzed using one-way ANOVA; (x̄ ± sd, n=3).
Changes in intracellular ROS, mitochondrial membrane potential and apoptosis across different cell groups
In comparison to the control group, the model group exhibited enhanced green fluorescence and a reduced mitochondrial membrane potential. Following intervention with LYC (5 μM, 10 μM), the model group showed an increase in red fluorescence, a decrease in green fluorescence, and an improvement in mitochondrial membrane potential. Moreover, the generation of ROS was increased in the model group compared to the control group. However, after LYC intervention (5 μM, 10 μM), the model group demonstrated a decrease in ROS production (see Fig. 1 and Fig. 2). PI and Hoechst staining indicated that, in contrast to the control group, there was a rise in apoptotic cells within the model group, evidenced by intensified blue fluorescence. In comparison to the model group, treatment with LYC (5 μM, 10 μM) resulted in an improvement and a reduction in cell apoptosis, as shown by diminished blue fluorescence (Fig. 3).
Fig. 1 Effect of LYC on oxalic acid/calcium oxalate crystal-induced intracellular reactive oxygen species ROS in HK-2 cells. A. control group; B. model group; C. LYC (5 μM); D. LYC (10 μM) group (Immunofluorescence, x100).
Fig. 2 Effect of LYC on mitochondrial membrane potential induced by oxalic acid/calcium oxalate crystals in HK-2 cells. A. control group; B. model group; C. LYC (5 μM); D. LYC (10 μM) group (Immunofluorescence, x100).
Expression of inflammation and apoptosis-related proteins
The levels of NF-κB p65 and OPN in the model group were significantly elevated (p<0.05) compared to the control group, whereas LYC (5 μM, 10 μM) led to a decrease relative to the model group (Fig. 4). The expression levels of Bax, CytC, and active caspase3 were markedly increased (p<0.05) in the model group when compared to the control group, while Bcl-2 expression was significantly decreased (p<0.05). The LYC (5 μM, 10 μM) treatment group exhibited a downregulation in Bax, CytC, and active caspase3 expression, along with an upregulation in Bcl-2 expression compared to the model group (Table 5, Fig. 5).
Fig. 4 LYC ameliorates oxalic acid/calcium oxalate crystal-induced changes in inflammation-associated protein levels in HK-2 cell injury.
Table 5 Relative expression of proteins related to inflammation and mitochondrial damage in cells of each group.
| Items | Control group | Model group | Lycopene (5μM) | Lycopene(10μM) |
|---|---|---|---|---|
| P65 | 0.708±0.077 | 1.014±0.053a | 0.807±0.163 | 0.782±0.229 |
| Osteopontin (OPN) | 0.546±0.70 | 0.864±0.006a | 0.746±0.260 | 0.642±0.127b |
| BAX | 0.427±0.062 | 1.109±0.205a | 0.734±0.221 | 0.542±0.203b |
| Bcl-2 | 1.13±0.301 | 0.466±0.228a | 0.536±0.261 | 0.544±0.245 |
| Cytochrome C (Cyt C) | 0.539±0.066 | 2.242±0.428a | 0.930±0.264b | 0.775±0.490b |
| Active caspase3 | 0.355±0.155 | 1.16±0.231a | 0.585±0.212b | 0.771±0.165 |
Note: a is p<0.001 compared with the control group, and b is p<0.05 compared with the model group. The above data was analyzed using one-way ANOVA.
DISCUSSION
The main goals in treating kidney stones include removing the stones, protecting kidney function, and tackling the root causes to reduce the likelihood of recurrence 8. Thus, it is vital to identify specific pharmacological agents that target the condition’s etiology for preventing and treating stones. The formation of kidney stones is a complicated process that entails the supersaturation of factors contributing to urolithiasis, harm to renal tubular epithelial cells, and the mechanisms of crystal adhesion, aggregation, nucleation, and growth 9. A significant contributor to the development of kidney stones is oxalic acid. Elevated concentrations of oxalic acid may result in oxidative damage and initiate an inflammatory reaction in renal tubular epithelial cells 10.
An expanding array of studies has demonstrated a relationship between inflammation, oxidative stress, and kidney stone formation 4,11. Hence, investigating effective anti-inflammatory and antioxidant mechanisms is crucial for alleviating kidney injury associated with calcium oxalate stones.
Malondialdehyde (MDA) is the end product generated from the peroxidation of cellular lipids. The levels of MDA offer valuable information regarding the degree of lipid peroxidation in the body, thereby acting as an indirect indicator of cellular damage.
Although free radicals can inflict considerable harm, human cells also harbor substances that neutralize these free radicals. Among these protective agents, super-oxide dismutase (SOD) stands out as a key antioxidant enzyme that aids in reducing the damage inflicted by oxygen-derived free radicals 12. A reduction in SOD activity indicates a lower ability of the organism to combat free radical-induced damage, implying that the organism may be undergoing oxidative stress. For example, continuous exposure to elevated levels of oxalic acid can promote the production of free radicals, which initiate lipid peroxidation within biological membranes.
This chain of events may result in changes to the ultrastructural integrity of cell membranes, enable cellular penetration, inflict harm on mitochondria and DNA, and ultimately lead to cell necrosis and apoptosis13. These experimental findings reveal that the oxidative damage to HK-2 cells induced by oxalic acid/calcium oxalate is significant, as evidenced by compromised cell proliferation, decreased cell viability, heightened release of lipid peroxidation byproducts like MDA and LDH, along with lower levels of antioxidant enzymes such as SOD and GSH. Following treatment with LYC, we noted an enhancement in cell viability, reduced oxidative injury, and increased antioxidant activity.
Reactive oxygen species (ROS) are recognized as primary regulators of oxidative stress and identified as significant contributors to the damaging effects of pathological stone formation 14. The role of the NF-κB signaling pathway is critical in facilitating intrarenal inflammation mediated by oxidative stress 15. The significant production of ROS caused by oxidative stress can additionally activate various signalling pathways associated with inflammation, creating diverse inflammatory mediators and promoting the buildup of inflammatory cells, thus triggering and enhancing the inflammatory response. Studies show that heightened oxalic acid levels can provoke the activation of the NF-κB intracellular signalling pathway, which leads to an increased expression of inflammatory factors like OPN and MCP-1/ IL -6, ultimately resulting in the infiltration of inflammatory cells and causing interstitial damage 4. Monocyte chemotactic protein-1 is an essential inflammatory mediator contributing to the inflammatory reactions linked to calcium oxalate kidney stones. Under standard physiological conditions, renal tissue cells only produce a minimal amount of MCP-1. However, when oxalic acid or calcium oxalate crystals precipitate in urine due to supersaturation, these substances can damage and stimulate these cells, leading to a substantial increase in MCP-1 production, subsequently attracting monocytes into the inflamed tissue 16. Boonla et al. 17 compared MCP-1 and IL -6 mRNA expression levels in kidney tissues adjacent to stones and those in normal kidneys. Their findings indicated more severe tubular damage in the tissues surrounding the stones and significantly reduced expression levels of MCP-1 and IL -6 compared to normal kidney tissues. This observation implies that MCP-1 and IL -6 may be involved in advancing kidney stone disease.
Furthermore, during cellular damage, various negatively charged molecules-including osteopontin (OPN), hyaluronic acid (HA), and CD44 18-are showcased on the cell surface. These molecules can bind to Ca2+ ions and attach to positively charged calcium oxalate crystals. The adhering crystals can trigger cellular production of free radicals, further harming the renal epithelium via lipid peroxidation, thus heightening the possibility of kidney stone formation. The study utilized ELISA experiments to show that MCP-1 and IL -6 release were elevated in the model group, while LYC was found to have a protective effect. Western blot analyses indicated that NF-κB and OPN levels were upregulated in the model group relative to the control group; however, their expression was reduced after LYC treatment. In addition, a combined analysis of reactive oxygen species levels in cells revealed that LYC could ameliorate the intrarenal ROS levels triggered by oxalic acid/calcium oxalate crystals in HK-2 cells, effectively inhibiting the NF-κB signalling pathway and thereby diminishing the intrarenal inflammatory response.
The deposition of calcium oxalate crystals may also cause damage to mitochondria by increasing cellular ceramide levels. Mitochondrial abnormalities or oxidative stress can trigger the initiation of cell apoptosis programs. The family of Bcl-2 proteins is crucial in governing mitochondrial permeability to various proteins and the permeabilization of the outer mitochondrial membrane, playing an essential role in the intrinsic apoptosis pathway. Bax and Bcl-2 are pro-apoptotic and anti-apoptotic agents, respectively 19. While Bcl-2 shields cells from mitochondrial injury and suppresses apoptosis, Bax enhances the permeability of the mitochondrial membrane, facilitating the release of cytochrome C (Cyt C) 20. This sequence of events leads to an increase in hydrogen peroxide production, a decrease in glutathione peptide levels, and a drop in mitochondrial membrane potential, coupled with the liberation of apoptotic factors into the cytosol, which ultimately activates caspase-3 and induces cell death 21. The expression levels of proteins associated with the mitochondrial pathway were evaluated in this study.
Results indicate that treatment with oxalic acid/calcium oxalate crystals led to an upregulation of Bax, Cyt C, and active-caspase3, while a downregulation of Bcl-2 was observed. Following the intervention with LYC, improvements in cell apoptosis were noted. These findings imply that LYC may mitigate HK-2 cell apoptosis by inhibiting the Bax/caspase3 signalling pathway.In conclusion, LYC demonstrates a significant ability to reduce oxidative stress and inflammatory responses in HK-2 cells, enhances cellular health, and may operate through the modulation of the ROS/NF-κB inflammasome pathway while also mitigating mitochondrial damage by inhibiting the Bax/caspase3 signalling pathway associated with mitochondria. This research offers an initial insight into the potential mechanisms by which LYC could aid in the clinical prevention and treatment of kidney stones, thereby opening new avenues and concepts for addressing calcium oxalate kidney stones and the clinical utilization of LYC and analogous medications. Nonetheless, as the investigation primarily focuses on cellular models, it may not wholly replicate the mechanisms involved in the human body, indicating that the study has inherent limitations, warranting further exploration and validation.
