Porous Se@SiO₂ nanocomposites play a potential inhibition role in hyperoxaluria associated kidney stone by exerting antioxidant effects

Introduction

Nephrolithiasis is a common urological disease with increasing prevalence worldwide (1,2). If left untreated, kidney stones may lead to different levels of hematuria, urinary infection, and urinary obstruction, which in turn may cause permanent renal function impairment. With the development of endoscopic laser lithotripsy and extracorporeal shock-wave lithotripsy, the treatment of urinary stones has become less invasive and more efficient (3-5). Due to the high recurrence rate (nearly 50% within 5 years of the first occurrence), nephrolithiasis patients suffer from recurrent pain, which leads to increased hospitalization and thus increased medical costs (6-8). It is widely reported that nearly 80% of kidney stones are composed of calcium, especially calcium oxalate (CaOx) (9,10); however, there is still a lack of effective interventions for the clinical prevention of kidney stones.

Hyperoxaluria-induced renal tubular epithelial cell (TEC) injury is an important factor in kidney stone formation (11). Oxidative stress with increasing reactive oxygen species (ROS) is thought to be a significant mechanism underlying the cytotoxicity of high concentrations of oxalate in TECs (12,13). Thus, agents with antioxidant properties might suppress kidney stone formation.

Selenium (Se), which is an essential minor element in the human body with multiple biological functions, is known to have excellent antioxidant properties (14). However, the margin between the safety and toxicity of Se is narrow. The development of cardiac and pulmonary symptoms in cases of severe toxicity can ultimately result in mortality (15). The development of nanotechnology in recent years has promoted the progress of drug research (16). Taking advantage of nanotechnology, the release of Se can be well controlled in vivo. In previous research, we developed Se@SiO₂ nanocomposites (NPs) that have good antioxidant properties and biosafety in the disease process of steroid-induced osteonecrosis and acute lung injury (17,18). We hypothesized that Se@SiO₂ NPs might protect TECs from hyperoxaluria by reducing ROS production.

In this study, we investigated whether Se@SiO₂ NPs ameliorated hyperoxaluria-induced kidney stone formation in mice models. The potential mechanisms underlying the protective effect of Se@SiO₂ NPs on hyperoxaluria-induced renal TEC injury were also explored. The results of this study may provide a new direction for the treatment of kidney stones. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-23-511/rc).

Methods

Synthesis of Se@SiO₂ NPs

We synthesized the Se@SiO₂ NPs as described previously (17,18). First, a mixture of 39.5 mg of Se powder and 5 mL of oleic acid (OA) were stirred continuously for 30 min at 120 ℃ in a nitrogen atmosphere. Second, the Se-OA precursor was formed after the mixture was heated to 220 ℃. Next, a mixture of oleylamine (OAM; 5 mL), OA (5 mL), and cuprous chloride (CuCl) (49.5 mg) was heated to 120 ℃ to remove any moisture. Subsequently, the mixture was heated to 220 ℃, and the above Se-OA precursor was quickly injected into the mixture. The solution was incubated at 220 ℃ for 5 min. For the synthesis, the Cu_2−xSe nanocrystals were washed with ethanol and dispersed in normal hexane. Subsequently, the Cu_2−xSe solution (3 mL), n-hexanol (30 mL), Triton X-100 (3 mL), and deionized water (0.9 mL) were stirred for 30 min, and tetraethyl orthosilicate (0.15 mL) and ammonia (0.20 mL) were then successively added under rapid stirring. The solid Se@SiO₂ NPs were formed by centrifugation after stirring for 24 h. To obtain the porous Se@SiO₂ NPs for the release of Se, the solid Se@SiO₂ NPs were dispersed in 20 mL of polyvinylpyrrolidone (PVP) aqueous solution (10 mg/mL) and heated to 95 ℃ for 2 h under very slow stirring.

Transmission electron microscopy (TEM)

The size and morphology of the Se@SiO₂ NPs were captured by TEM using a JEM-2100F microscope (JEOL, Tokyo, Japan). X-ray diffraction (XRD) was measured by a D/max-2550 PCX-ray diffractometer (Rigaku, Tokyo, Japan).

In-vitro safety of Se@SiO₂ NPs

The mouse kidney TECs (TCMK-1) were plated in 96-well plates (1×10⁴ cells/well) and incubated overnight before undergoing overnight treatments with different concentrations of NPs. After being incubated for 24 h, cell viability was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo, Kyushu Island, Japan) with an ELx800 plate reader (BioTek, Winooski, VT, USA).

Animal experimental design

The study was approved by the Animal Care and Use Committee of Shanghai Changhai Hospital (hospital project license, No. 2020QH08). All the animals were kept under specific pathogen-free conditions, in compliance with Guide for the Care and Use of Laboratory Animals (Eighth Edition). A protocol was prepared before the study without registration. In total, 24 C57BL/6 male mice (aged 8 weeks old and weighing 20±1 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The mice were randomly divided into the following 4 groups: (I) the control group (n=6), which was intraperitoneally injected with normal saline/day; (II) the Se@SiO₂ group (n=6), which was intraperitoneally injected with Se@SiO₂ (1 mg/kg/day); (III) the glyoxylic acid monohydrate (GAM) group (i.e., the nephrolithiasis group), which was intraperitoneally injected with GAM (100 mg/kg/day) for 7 days consecutively; and (IV) the GAM + Se@SiO₂ group, which was intraperitoneally injected with GAM (100 mg/kg/day) after 8 h of Se@SiO₂ (1 mg/kg/day) pre-treatment. At the end of the experimental procedures, all the mice were euthanized. Serum samples and urine samples were collected and stored at −80 ℃. Left renal samples were collected and fixed with 4% polyformaldehyde (PFA). Right renal samples were collected and stored in liquid nitrogen.

Basic body characteristics and urinary oxalate and Se quantitative analysis

The body weight of the mice was measured. Urinary oxalate excretion levels were determined using an oxalate assay kit (Sigma, MAK179; St. Louis, MO, USA). The standard curves and samples were prepared and measured according to the instructions. After the treatment of the part renal tissues with aqua regia, the mixed solutions were filtered. The concentration of Se was measured by inductively coupled plasma atomic emission spectroscopy as previously described (17,18).

Histology examination analysis

The left renal samples were fixed in 4% PFA, embedded with paraffin, and serially sectioned at 6-µm thickness for staining. Hematoxylin and eosin (H&E) staining was performed to conduct the histology examination and assess the renal injury level. We then evaluated 10 non-continuous fields from the cortex of each section (400× and 200× magnification).

TUNEL analysis

Apoptosis was detected using a TUNEL apoptosis assay kit (Beyotime, Shanghai, China). The whole process was performed in accordance with the manufacturer’s instructions, and the samples were observed under a fluorescence microscope before anti-fluorescence quenching.

ROS activity analysis

The ROS levels of the kidney tissues were measured using the dihydroethidium in accordance with the manufacturer’s instructions. The tissues were incubated with dihydroethidium staining solution for 30 min at 37 ℃. The tissues were then washed with phosphate buffered saline (PBS) in the dark and incubated with 4',6-diamidino-2-phenylindole staining solution for 10 min. The tissues were then washed with PBS in the dark. Fluorescence images were captured using a Pannoramic MIDI (3DHISTECH), and ROS production was measured using ImageJ software.

The intracellular ROS levels were measured using the Carboxy-H2DFFDA kit in accordance with the manufacturer’s protocol. The TCMK-1 cells were plated in a 6-well plate at 10⁶ cells/well with an oxalate (0.5 mM) or Se@SiO₂ (90 µg/mL) treatment for 3 h, then washed with PBS and incubated with carboxy-H2DCFDA staining solution for 1.5 h at 37 ℃. The cells were subsequently rinsed with PBS under low light conditions. Fluorescent images were acquired utilizing a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek), and quantification of ROS generation was performed using ImageJ software.

Quantitative real-time polymerase chain reaction (qRT-PCR)

The RNA was isolated from the tissues using the RNeasy Mini kit (Qiagen, Dusseldorf, Germany) following the manufacturer’s instructions. Complementary DNA synthesis was performed using a PrimeScript RT Master Mix Kit (Takara Bio, Dalian, China). QRT-PCR analysis was conducted on an ABI PRISM^® 7500 system (Thermo Fisher Scientific, Waltham, MA, USA) using SYBR Premix Ex TaqTM II, specific forward and reverse primers, and double distilled water. The cycling conditions consisted of an initial denaturation step at 95 ℃ for 5 min followed by 42 cycles of denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 20 s, extension at 72 ℃ for 20 s, and a final extension step at 72 ℃ for 20 s. The primers were as follows: bone morphogenetic protein 2 (BMP-2; Forward: 5'-CCTATATGCTCGACCTGTACCG-3'; Reverse: 5'-CTGGCTGTGGCAGGCTTTAT-3'), osteopontin (OPN; Forward: 5'-CTCGAACGACTCTGATGATGT-3'; Reverse: 5'-TGTCAGGTCTGCGAAACTTCT-3'), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Forward: 5'-ACCCAGAAGACTGTGGATGG-3'; Reverse: 5'-CACATTGGGGGTAGGAACAC-3'). The expression of the target genes was normalized to GAPDH levels. All the assays were repeated 3 times. Gene expression was calculated with 2^−∆∆CT.

Maleic dialdehyde (MDA) and superoxide dismutase (SOD) assays

We took part of the kidney tissue, rinsed it in normal saline, sucked it dry with filter paper, weighed it, added normal saline 9 times the weight of the kidney tissue, homogenized it for 5 min, centrifuged it for 10 min at 3,000 r/min, and sucked it up with a micropipette. The absorbance values of SOD and MDA were detected at 550 and 532 nm by ultraviolet-visible (UV-Vis) spectrophotometry according to the operation requirements of the SOD and MDA kit.

Cell apoptosis assay

The cell apoptosis analysis kit was utilized to assess cell apoptosis. TCMK-1 cells were trypsinized, PBS-washed, and then resuspended in 1 × Annexin-binding buffer. Early apoptosis was identified by staining with Alexa Fluor^® 488 Annexin V (Thermo Fisher Scientific) and propidium iodide (PI). The stained cells were analyzed using a CytoFLEX flow cytometer from Beckman Coulter Inc. (Brea, CA, USA). Data acquisition was performed using Cell Quest software (version 3.0).

Statistical analysis

The data analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). The data are expressed as the mean ± standard deviation. The unpaired Student’s t-test and a one-way analysis of variance were used for the comparisons. P values <0.05 were considered significant.

Results

The characterization of porous Se@SiO₂ NPs

We prepared the porous Se@SiO₂ NPs as previously described (17,18). The XRD pattern showed an increase in the low angle region of the solid Se@SiO₂ NPs due to the silica coating (Figure 1A). According to the standard Se phase (Joint Committee on Powder Diffraction Standards, JCPDS card No. 65-1876), the distinctive peaks of the solid Se@SiO₂ NPs had a hexagonal shape (Figure 1A). The morphology and size of the Se@SiO₂ NPs were also observed by TEM (Figure 1B-1D). The diameters of the solid Se@SiO₂ NPs were about 55 nm. Many small (size <5 nm) and irregular Se quantum dots were interspersed from the center to the surface of the NPs. After the hot PVP solution treatment, the Se@SiO₂ NPs showed a porous structure (Figure 1D), which is facile for the release of Se. The CCK-8 assays showed that the Se@SiO₂ NPs had almost no obvious cytotoxicity in the TCMK-1 (Figure 1E).

Figure 1 Characterization of the porous Se@SiO₂ nanocomposites. (A) XRD pattern of the porous Se@SiO₂ nanocomposites and standard Se phase (JCPDS and No. 65-1876). (B) TEM of the porous Se@SiO₂ nanocomposites. (C,D) Low-magnified and medium-magnified images of the porous Se@SiO₂ nanocomposites. (E) The cell viability of the TCMK-1 cells treated with different concentrations of the porous Se@SiO₂ nanocomposites for 24 h. XRD, X-ray diffraction; TEM, transmission electron microscopy; ppm, parts per million.

General results of the hyperoxaluria mouse model following the porous Se@SiO₂ nanocomposite treatment

The schematic protocol for the animal experiment in our study is shown in Figure 2A. There were no significant differences in the body weight of the mice in the control, Se@SiO₂, GAM, and GAM + Se@SiO₂ groups (Figure 2B). The urinary oxalate excretion levels differ significantly among the GAM and GAM + Se@SiO₂ groups, (Figure 2C). The mice kidney Se concentration levels of the Se@SiO₂ and GAM + Se@SiO₂ groups were significantly higher than those of the control and GAM groups (Figure 2D).

Figure 2 General results of the hyperoxaluria mouse model treated with porous Se@SiO₂ nanocomposites. (A) The schematic protocol of the animal experiment. (B) The body weight curves of the mice. (C) The urinary oxalate excretion levels of the mice. (D) The mice kidney Se concentration levels. **, P<0.01; ***, P<0.001. GAM, glyoxylic acid monohydrate.

The porous Se@SiO₂ NPs reduced kidney injury, CaOx crystal deposition, and osteoblastic-associated proteins in the hyperoxaluria mouse model.

The H&E staining showed significant kidney histological injuries at the junction of the medullary and cortex in the GAM group, but these injuries were significantly ameliorated in the GAM + Se@SiO₂ group (Figure 3A, left). Von Kossa staining revealed that the CaOx crystal deposition mass was higher in the GAM group than in the control group. Following the porous Se@SiO₂ treatment, the CaOx crystal deposition mass was significantly lower in the GAM + Se@SiO₂ group than in the GAM group (Figure 3A, right). The levels of Scr and blood urea nitrogen (BUN) were lower in the GAM + Se@SiO₂ group than in the GAM group (Figure 3B). We further detected the expression of the osteogenic proteins OPN and bone morphogenetic protein 2 (BMP2), which are closely related to the formation of CaOx nephrolithiasis in the mice kidney tissues (19). The qRT-PCR assays showed that the level of OPN and BMP2 in the mice kidney tissues was much lower in the GAM + Se@SiO₂ group than in the GAM group (Figure 3C).

Figure 3 Porous Se@SiO₂ nanocomposites inhibit hyperoxaluria-induced CaOx crystal deposition and function impairment on renal. (A) H&E staining (left, magnification ×0.5 for full view, magnification ×200 for local zoom) and Von Kossa staining (right, magnification ×0.5 for full view, magnification ×200 for local zoom) of the mice kidney tissues. The arrows point to CaOx crystal deposition mass. Scale bar: 50 µm. (B) The levels of Scr and BUN in the serum of the mice. (C) The RNA expression level of OPN and BMP2 in the kidney tissues of the mice. *, P<0.05; **, P<0.01. H&E, Hematoxylin and eosin; GAM, glyoxylic acid monohydrate; BUN, blood urea nitrogen; OPN, osteopontin; BMP2, bone morphogenetic protein 2.

Porous Se@SiO₂ NPs reduced oxidative stress and apoptosis both in vivo and in vitro

We detected the ROS levels in the mice kidney tissues after the GAM treatment. Compared to the control group, GAM significantly increased the ROS levels. However, ROS production was significantly decreased in the GAM + Se@SiO₂ group (Figure 4A). We then measured the levels of MDA and SOD, which are biomarkers of oxidative stress, in the kidney tissues of the mouse model using the MDA and SOD assay kits. The results showed that GAM significantly increased the level of MDA and decreased the activity of SOD. However, the porous Se@SiO₂ NPs significantly decreased the level of MDA and increased the activity of SOD in the GAM + Se@SiO₂ group (Figure 4B). Further, the TUNEL assays showed that the positive rate of apoptosis was markedly increased in the GAM group, while the apoptotic level of the kidney tissue was significantly decreased in the GAM + Se@SiO₂ group (Figure 4C). We also conducted fluorescent dichlorofluorescein (DCF) assays and flow cytometry in the TCMK-1 cells after the oxalate treatment. The in vitro results were consistent with the in vivo results (Figure 4D,4E).

Figure 4 Porous Se@SiO₂ nanocomposites reduced oxidative stress and apoptosis. (A,C) Representative images of ROS and TUNEL assays in mice kidney tissues observed under a fluorescence microscope. Scale bar: 250 µm in A, 500 µm in C. (B) The levels of MDA and SOD in the mice kidney tissues. (D) Representative images of ROS assays in the TCMK-1 cells observed under a fluorescence microscope. (E) Apoptosis was detected by an Annexin V-FITC + PI kit and the cell apoptosis rate (%). *, P<0.05; **, P<0.01. ROS, reactive oxygen species; GAM, glyoxylic acid monohydrate; MDA, maleic dialdehyde; SOD, superoxide dismutase; FITC, fluorescein isothiocyanate; PI, propidium iodide; DCF, dichlorofluorescein.

Discussion

The incidence of urolithiasis depends on several factors like geography, ethnicity, diet or genetics. The prevalence of urinary stone development varies from 4% to 20% (20). Several countries like the United States of America showed an increased prevalence of kidney stones, which is likely due to unhealthy lifestyle habits, such as high-protein/high-sugar/high-salt diets, smoking, drinking, and less physical activity/obesity (21-23). However, while these unfavorable factors might elevate the concentrations of oxalate, calcium, and uric acid in the urine, kidney stone formation is not a simple physicochemical disorder.

The majority of research suggests that stone formation is a form of pathological biomineralization or ectopic calcification, involving epithelial to mesenchymal transition, epithelial to osteoblast transformation, and the remodeling of the extracellular matrix in nephrons, especially in TECs (24-26). Notably, ROS-associated oxidative stress-induced TEC injury and inflammation have been considered the initial triggers of kidney stone formation (27,28). It is also well established that oxidative stress-induced injury is involved in the pathogenesis of a variety of chronic diseases, such as cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and diabetes (27,29-31). Thus, these diseases are risk factors for nephrolithiasis (32,33). Further, numerous clinical and experimental studies have confirmed that antioxidant treatments are effective in the therapy of these diseases, including nephrolithiasis (34-37).

Selenium is a recognized health-benefiting element that could play an important role in protecting cells against oxidative stress injury (14). Our previous studies showed that our Se@SiO₂ NPs protect cells from chemotherapy-induced oxidative stress injury by attenuating doxorubicin-induced cardiotoxicity and attenuating cisplatin-induced acute kidney injury (38,39). Due to the controllable release of Se, our porous Se@SiO₂ NPs have good biosafety in vivo. In this study, the CCK-8 assays first showed that the Se@SiO₂ NPs had almost no cytotoxicity on the mouse kidney TECs in vitro. In the hyperoxaluria mouse model, our results showed that there were no significant differences in the body weight of the mice in the control, GAM, and GAM + Se@SiO₂ groups. The levels of mice kidney Se concentration in the Se@SiO₂ and GAM + Se@SiO₂ groups were significantly higher than those of the control and GAM groups. Further, the H&E and Von Kossa staining showed that the Se@SiO₂ NPs significantly reduced renal injuries and CaOx crystal deposition in the hyperoxaluria mouse model.

Previous studies have indicated that upregulated expressions of BMP2 and OPN are involved in kidney stone formation during excess ROS-induced kidney injury and inflammation (40,41). In this study, the qRT-PCR assays showed that the levels of OPN and BMP2 in the mice kidney tissues were much lower in the GAM + Se@SiO₂ group than in the GAM group. A study has introduced that high oxalate and CaOx crystals induced the TECs to generate excess ROS, which severely damages cell structure and function through oxidation reactions with lipids, carbohydrates, proteins, and nucleic acids (27). It is well-known that the cells are equipped with mechanisms to decrease ROS availability, such as SOD. In this study, the DCF assay results showed that hyperoxaluria increased the ROS level dramatically, while the Se@SiO₂ NPs significantly inhibited the production of ROS induced by hyperoxaluria in the kidney tissue of the mouse model. The enzyme-linked immunoassays showed that the Se@SiO₂ NPs significantly reversed the increased level of MDA and the decreased activity of SOD induced by hyperoxaluria. Further, the TUNEL assays showed that the apoptotic level of the kidney tissue was significantly more decreased in the GAM + Se@SiO₂ group than in the GAM group. In vitro, the DCF assays and flow cytometry assays also showed that the Se@SiO₂ NPs alleviated the ROS level and cell apoptosis induced by oxalate.

Conclusions

In summary, the Se@SiO₂ NPs may decrease hyperoxaluria-induced kidney damage and kidney stone formation via their antioxidant benefits. Thus, Se@SiO₂ NPs might be a potential drug for the treatment of nephrolithiasis. Further studies are warranted to validate its applicability in humans.

Acknowledgments

Funding: The study was supported by Fund of Shanghai Hongkou District Health Committee (hong wei 2022-29).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-23-511/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-511/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-23-511/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-23-511/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The study was approved by the Animal Care and Use Committee of Shanghai Changhai Hospital (hospital project license, No. 2020QH08). All the animals were kept under specific pathogen-free conditions, in compliance with Guide for the Care and Use of Laboratory Animals (Eighth Edition). A protocol was prepared before the study without registration.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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