Research progress on the relationship between nanocrystals and calcium oxalate stones

Abstract

Urological stones are the common urological diseases in clinical practice. So far, the mechanisms of kidney stone formation still remain unclear. Among all of the mechanisms, the supersaturated crystallization theory focuses on the microcrystal size of stones. However, there are few researches on the formation mechanism of stones in nanoscale. This narrative review summarizes the current mainstream mechanisms of kidney stone formation. It also discusses the relationship between calcium oxalate (CaOx) nanocrystals and the formation of kidney stones from the perspective of the physicochemical properties of the nanocrystals. To further explore the role of CaOx nanocrystals in the formation of CaOx, this review lists some convenient and reliable methods for tracking nanocrystals. Currently, in addition to mainstream treatments like potassium sodium hydrogen citrate and thiazide diuretics, some researches indicate that polysaccharide drugs, selenium nanoparticles, and dietary can inhibit the formation of nanocrystals, ultimately preventing the formation of CaOx stones.

Introduction

Urological stones are the most common urological diseases in clinical practice, and China is one of the regions with a high incidence of urolithiasis in the world, with a prevalence rate of about 5%. The age of patients who have urolithiasis was between 32 and 45 years. In China, among patients with kidney stones, 88.9% have stones containing calcium oxalate, and 77.5% of these stones have a calcium oxalate content exceeding 50%. Although calcium oxalate stones are predominant in both male and female patients, the proportion of components in kidney stones varies significantly between genders. Specifically, the proportion of calcium oxalate in kidney stones is much higher in male patients than in female patients, while the proportions of infectious stones and cystine stones are considerably higher in female patients than in male patients. ¹ Therefore, the study of urinary stones has important clinical significance. This review summarizes several mainstream mechanisms of kidney stone formation and current research on tracking and inhibiting stones from a nanocrystal perspective, aiming to provide direction for future therapeutic strategies. This review is guided by the Scale for the Assessment of narrative review articles. ²

The mechanism of the formation of calcium oxalate stone

At present, the mechanism of the formation of CaOx stone is still unclear. The main mechanisms of stone formation are as follows (Figure 1).

Figure 1.

Mainstream mechanisms of kidney stone formation.

The supersaturated crystallization theory

Given that supersaturation acts as the chemical catalyst for urinary calculi creation, it's an essential prerequisite for the development of a crystal nucleus. Urine is a complex multi-ionic solution in which various ions interact to form diverse complexes. As a result, the supersaturation concentration of calcium oxalate in urine is not fixed or easily defined. A supersaturated solution that does not spontaneously precipitate solid phases is referred to as a metastable supersaturated solution. For a precisely saturated aqueous solution, the saturation level is 1, whereas the supersaturation level of urine can range from 2 to 8, depending on the stone-forming components. ³ This range is known as the “metastable zone.” Within the metastable zone, although the urine is supersaturated, new solid phases rarely form. Even if crystals appear in the urine of healthy individuals, they do not develop into stones. However, if the degree of urinary supersaturation exceeds the upper limit of the metastable zone, entering a state of hypersaturation, a large number of crystals will spontaneously form in the urine. This becomes a critical factor initiating stone formation. After the process of the crystal growth, aggregation and retention, the CaOx crystals eventually become CaOx stones. ⁴

The Randall's plaques theory

The Randall's plaques are a crystalline deposit that attaches to the renal papillary area and that is composed mainly of calcium phosphate. 80 years ago, Randall's plaques were originally discovered by Dr Alexander Randall when he biopsied the body's kidneys. At that time he proposed the hypothesis that Randall plaques (RPS) was the origin of idiopathic CaOx stones. Randall's plaques are calcium salt deposits that form in the interstitial tissue of the renal papilla. When renal tubular epithelial cells are damaged and expose the underlying Randall's plaque, calcium ions and oxalate in supersaturated urine can bind to the plaque, leading to crystal formation and the subsequent development of calcium oxalate stones. ⁵ Subsequently, the chemical leading to the creation of urinary calculi is supersaturation. Therefore, RPS is the key to the formation of CaOx stones. Clément used transmission electron microscope (TEM) and scanning transmission electron microscope to study the morphology and chemical composition of incipient RPS at the nanoscale and found that the incipient RPS consisted mainly of two types of nano-calcifications. ⁶ One type consists of nanoparticles of calcium phosphate and carbonate cores. The presence of calcium carbonate in the core of the large mineral particles (MPs) can serve as a natural catalyst for calcium phosphate accumulation, enhancing calcium levels. Another type is calcified organic vesicles. Membrane vesicles, made up of anionic-phospholipids and rich in proteins, have the ability to engage with calcium phosphate, triggering apatite precipitation in vitro. Consequently, MPs are frequently abundant on the inner side of kidney calcified vesicles. Additionally, the calcified vesicles grouped to create micro-calcifications through their with an organic fibrillary network.

The inhibiting and promoting factors

This mainly includes the disturbance of physical and chemical environment in vivo and the abnormality of macromolecular organic substrates. Both hypercalcemia and hyperoxaluria are common metabolic abnormalities in patients with CaOx stones. High concentrations of Ca²⁺ can form a state of calcium supersaturation in the urine, thereby directly elevating the likelihood of stone development. ⁷ In addition, elevated levels of both calcium and oxalate could trigger oxidative stress, potentially harming renal tubular epithelial cells and encouraging the accumulation of CaOx crystals. ⁸ Macromolecular organic substrates can inhibit or promote the formation of stones, mainly including macromolecular proteins and glycosaminoglycans (GAG). Within the realm of macromolecular organic substrates, nephrocalcin, osteopontin (OPN) and UPTF-1 are known to prevent the nucleation, growth and aggregation of CaOx crystals. ⁸ Tamm-Horsfall protein has a dual role, it not only inhibits the aggregation of crystals, but also promotes adhesion.^9,10 GAG plays a crucial role as a stone inhibitor in urine, being a polysaccharide composed of acid groups like sulfates and carboxylic groups. In the GAG, acid groups have the ability to bind with Ca²⁺, creating soluble complexes in urine and diminishing the excessive saturation of Ca²⁺ in a solution. Consequently, GAG might prevent the creation of CaOx crystals. Moreover, when GAG that contains a large number of anions absorbed on the surface of the crystals, it can make zeta potential become negative and increase the electrostatic repulsion between crystals. Through this mechanism, GAG can inhibit the aggregation and precipitation of crystals. Although GAGs can inhibit stone formation in urine, existing studies have shown that plant polysaccharides, despite their structural similarity to GAGs, can more effectively prevent cell-crystal interactions, thereby suppressing crystal formation. ¹¹ A detailed introduction to plant polysaccharides (such as tea polysaccharides (TPS), corn silk polysaccharides (CCSP), etc.) will be provided later in the text.

The pyroptosis of HK-2 cells

Pyroptosis is a new programmed cell death and a natural immune function of the body, which plays an important role in antagonizing infection and endogenous danger. ¹² In recent years, with the continuous deepening of research, many studies have revealed a strong correlation between CaOx stones and the pyroptosis of renal tubular epithelial (HK-2) cells. ¹⁰ It is mainly manifested in oxidative stress damage and increased crystal adhesion during the process of pyroptosis. Some studies have shown that in environments rich in oxalic acid, renal tubular epithelial cells may generate reactive oxygen species (ROS). At the same time, the inflammasome is activated and produces pro-inflammatory cytokines, and then it will initiate oxidative stress reactions leading to damage to renal tubular epithelial cells. Eventually, it could promote the formation of CaOx stones. ¹³

However, the supersaturation crystallization theory is limited to the level of microcrystalline. Current experiments have shown that the formation of CaOx stones is closely associated with nanocrystals in urine. Energy-dispersive spectrometer analysis revealed that CaOx, uric acid (UA) and calcium phosphide (CaP) are the primary constituents of urinary nanocrystals in individuals with CaOx stones. ¹⁴ Fast fourier transformation and selected area electron diffraction have shown that the central region of the urinary crystallites was mainly composed of CaP or UA and the peripheral area was calcium oxalate monohydrate (COM) or a CaP-COM mixture. The outcome substantiates the initiation of CaOx stone creation by UA or CaP crystals at the nanoscale level. ¹⁴ It has been reported that the extent of damage nanocrystals cause to renal tubular epithelial cells is more severe than that caused by microcrystals. ¹⁵ To date, there are few studies on the relationship between nanocrystals and the pathogenesis of kidney stones. This review focuses on this topic in the hope of providing direction for future research into the mechanisms of kidney stone disease from a nanoscopic perspective, as well as for the prevention and treatment of kidney stones.

The formation of kidney stones induced by nanocrystals

Physical and chemical properties of nanocrystals

Shape of nanocrystals

Both individuals in good health and those suffering from kidney stones exhibit nanocrystals in their urine; however, the shapes of these nanocrystals differ in their urine. Through the use of SEM and TEM, Huang found that most of the nanocrystals in the urine of patients with stones have sharp edges and corners. ¹⁶ The presence of inhibitors in the urine of healthy people helps preserve the equilibrium of Ca²⁺ dissociation on the surface of CaOx crystals, and the outgoing process of precipitation and dissolution leads to the edges and corners of the crystals to become dull. As the concentration of inhibitor in the urine becomes less, the dynamic equilibrium of precipitation-dissolution weakens, so the crystals still maintain their angular shape, so the crystals remain sharp.

The shape of external particles plays a crucial role in determining their biological safety and practical uses. Variations in how toxic particles of diverse forms can be affected by elements like the strength of their adhesion to cells, the rate of internalization and the kinetics of vascular margins. In urine of stone patients, the toxicity of different shapes of nanocrystals to proximal tubule epithelial cells is also different. A study comparing the cytotoxicity of nano-calcium oxalate dihydrate (nano-COD) crystals of different shapes on HK-2 cells showed that nano-COD with elongated bipyramidal shape (COD-EBD) had the largest active surface. ¹⁷ Such a configuration enlarges the interaction zone between the crystals and the surface of the cell membrane, thereby exacerbating the damage to the cell membrane. Therefore, it has the most serious damage to HK-2 cells, leading to a large amount of lactate dehydrogenase (LDH) release and ROS production and inducing apoptotic cell death. From a hydrodynamic point of view, the flow of urine through the renal tubules is laminar, with a small flow velocity close to the epithelium, thus increasing the contact time between the crystals and the epithelial cells. Due to the presence of Stokes resistance, COD-EBD has the largest specific surface area, the greatest resistance and the slowest flow rate. Therefore, the nanocrystals can be attached to the surface of epithelial cells in large numbers. And the nanocrystals with the sharp edges can scratch the epithelial cells, causing the cells to have an inflammatory response. ¹⁷

Besides, Dewansh found that nanocrystals can grow into micro-crystallites and that UA particles with sharp edges and higher torsion densities grow faster than relatively smooth particles, suggesting that the growth rate is dependent on the morphology of the particles. ¹⁸

Therefore, if the formation of calcium oxalate nanocrystals can be reduced, the formation of CaOx stones can be inhibited.

Size and distribution of nanocrystals

Kidney stones may develop due to crystals forming and remaining in the renal tubules. Crystal adhesion to renal tubular epithelial cells is responsible for its retention. And, crystal adhesion is also induced when the renal tubular epithelial cells are damaged. Sun found that when hydroxyapatite (HAP) crystals interact with HK-2 cells, cell survival and LDH secretion are influenced by both dosage and size. ¹⁵ With the rise in crystal concentration, there was a corresponding decline in cell survival. In different sizes of HAP crystals, the intracellular ROS level increased to 41.1% in the HAP-40 nm treated group, while it only increased to 10.2% in the HAP-2um group. High levels of ROS cause mitochondrial damage, ultimately leading to cell death or apoptosis. Among all of the HAP crystals, the smallest HAP crystal (40 nm) was the most toxic. This is mainly related to cytotoxicity caused by oxidative stress. ¹⁵ DNA damage from oxidative stress results in a halt of the cell cycle, thereby hastening the process of cell death. Crystals of nano-HAP are capable of being directly absorbed into the cell's core. The process of endocytosis results in the rearrangement of actin, affecting intracellular skeletal tissues, and ultimately large amounts of ROS are released, causing mitochondrial damage and apoptosis. ¹⁹

Particle collisions are the basis for crystal aggregation. The driving force for collision in a quiescent solution is the Brownian motion and settling mechanism of particles, which then combine together through van der Waals forces, binding with viscous proteins, or forming solid calcium bridges. The size of the crystal is an important factor in promoting aggregation. Different-sized nanocrystals also have different rates of aggregation. Shapur found that smaller crystals formed aggregates more readily compared to larger ones, and those with smaller starting sizes tended to be larger than those with larger initial sizes. ²⁰ Lou showed that nanocrystals in the urine of stone patients were significantly larger in size compared to healthy controls, and that they were heterogeneous in size and had a wide distribution. This indicates increased crystal collisions within the urinary system of stone patients, suggesting an unstable environment more prone to stone formation. As the time of placement increases, the degree of nanocrystal aggregation in the urine of patients becomes more apparent, forming larger volumes that are more conducive to the formation of urinary stones. ²¹

Zeta potential of nanocrystals

The zeta potential of urinary crystals correlates with the concentration of anion inhibitors in urine and the ability of urinary crystals to adsorb anions. The higher the negative zeta potential of the surface of the urinary nanocrystals, the more energy is required for them to approach each other. ²² Therefore, the nanocrystals are less likely to collide and adhere to each other, and the risk of stone formation is reduced. Small size nanocrystals have many lattice defects, so they are less able to adsorb anions, resulting in a smaller absolute value of its zeta potential. Small size nanocrystals are more likely to form kidney stones than microcrystals. ²³

Time dependence of nanocrystals

It has been shown that the cytotoxicity of nanocrystals on HK-2 cells is also time-dependent, with nanocrystals damaging organelles more rapidly than micrometric crystals. Sun found that when the treatment time for Vero cells was increased from 1 h to 12 h, the cell viability in the COM-50 nm treated group decreased to 50.74%, which was an increase of 39.45% over COM-10 um. ²⁴ This is due to the fact that nanocrystals possess a smaller size than microcrystals, and in addition to disrupting cell membranes, increasing membrane permeability, and facilitating LDH release, they can also be internalized into the cell, attacking organelles directly, and even interacting directly with the nucleus to cause DNA damage. In addition, internalized nanocrystals can induce ROS production by mitochondria, which in turn causes DNA damage and ultimately apoptosis. ¹⁹ Apoptosis is accompanied by phosphatidylserine (PS) ectopic and these act as attachment sites for microcrystals, thereby promoting adhesion and aggregation of urinary microcrystals. ²⁵

Given the current limited research on the correlation between kidney stones and nanocrystals, this article, based on the physicochemical properties of nanocrystals, summarizes the mechanisms by which nanocrystals may promote kidney stone formation, aiming to provide new perspectives for future studies on nanocrystals in renal calculi.

Detection and tracking of nanocrystals

Recent studies have shown that nanocrystals cause more severe damage to renal tubular epithelial cells compared to microcrystals, making early detection of nanocrystals particularly important. ¹⁵ Traditional techniques for identifying and analyzing crystals expelled in urine encompass polarized light microscopy, scanning electron microscopy, urine filtration, evaporation, and centrifugation. However, these methods are not effective for measuring the size and number of nanocrystals in urine. There are several methods available regarding the detection of nanocrystals in urine.

Nanoparticle tracking analysis

Nanoparticle tracking analysis (NTA) employs laser technology to gauge nanoparticles in a solution, utilizing Brownian motion principles. Numerous studies have employed NTA to measure the size of particles and count the number of nanoparticles like exosomes, liposomes, microspheres, and vesicles found in a very variety of biological specimens, such as blood, breast milk, and urine. Fluo-4, known for its intense fluorescence when attached to calcium as a calcium-binding probe, is commonly employed to identify free calcium in biological entities. Kumar found that the ability and sensitivity of NTA to detect calcium-containing nanocrystals could be enhanced by using the fluorescent dye Fluo-4. ²⁶ NTA can effectively detect nanocrystalline urine as well as the size of the nanocrystals, suggesting that NTA may be an early detection method for patients with kidney stones. ²⁷ Using Fluo-4 for NTA of kidney stones offers high sensitivity but suffers from low specificity. Although this method enables early detection of kidney stones—facilitating early prevention and improved prognosis—it cannot distinguish between crystal types or identify the location of crystal formation within the urinary tract. As a result, additional examinations are required when selecting appropriate drug therapies or surgical interventions. ²⁶

Nanoscale flow cytometry urinalysis

Compared to traditional ways of detecting crystals, such as electron microscopy and polarized light microscopy, nanoscale flow cytometry urinalysis has the advantages of high sensitivity, high throughput, and ease of use. Carson coupled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) for nanoscale flow cytometry to enumerate nanocrystals and compared the results with petrographic thin sectioning of calculi, finding a high degree of agreement between them. ²⁸ This result indicates that nanoscale flow cytometry urinalysis can improve the accuracy of stone subtype diagnosis and determine the stone composition and load more precisely. However, Alendronate-fluorescein and Alendronate-Cy5 are not yet mass-produced. Moreover, this technique also fails to determine the site of stone formation within the urinary tract, limiting its utility in surgical decision-making, and it does not offer prognostic information.

Carbon dots

Carbon dots (CDs) represent a novel category of carbon nanomaterials, generally under 10 nm in dimension. CDs, in contrast to conventional fluorescent substances, boast benefits such as affordability in production, ease of preparation, eco-friendliness, adjustable fluorescence, substantial quantum efficiency, and high compatibility with biological systems. ²⁶ Moreover, it has been shown that the use of CDs as fluorescent probes can help to increase the sensitivity of the assay and enable the dual imaging channel to minimize background interference. ²⁹ Wang stabilized the solid-state green fluorescence of CDs based on the precipitation-dissolution mechanism and the theory of non-homogeneous nucleation of crystals to enable CDs to enter into single particles of the nano-HAP lattice in the form of heterogeneous nuclei. ³⁰ Utilizing laser scanning copolymer microscopy allows for a distinct observation of the positioning and spread of CDs-HAP within cells, enabling the tracking of nano-HAP's metabolic routes in these cells. When renal tubular epithelial cells are damaged and expose Randall's plaque, calcium salts can deposit on these sites and initiate stone formation. The main component of Randall's plaque is HAP. ⁶ HAP has the ability to infiltrate cells through endocytosis and gather in lysosomes, resulting in an increase in intracellular ROS levels and the compromise of lysosomal integrity. ³¹ Eventually, this leads to cell necrotic death and exacerbate the adhesion and aggregation of CaOx crystals. ³¹ CDs-HAP can integrate into HAP structures and, by leveraging the fluorescent properties of CDs, enable in vivo tracking of HAP metabolism and formation pathways. This approach further facilitates the study of the formation mechanisms of Randall's plaque. Therefore, tracking CDs-HAP is beneficial in exploring the mechanism of HAP in the pathogenesis of kidney stones.

Manganese dioxide (MnO₂) nanoparticles possess a high extinction coefficient and abundant redox properties, making them an excellent sensing platform. ³² Furthermore, studies have shown that MnO₂ can quench the fluorescence of CDs. ³³ Based on the characteristic of oxalate reducing MnO₂ to MnO²⁺, the fluorescence of CDs can be restored. Therefore, the nanocomposite of CDs and MnO₂ can be used for the determination of oxalate content in urine. Fluorescence data indicates that the nanocomposite of CDs and MnO₂ can successfully measure oxalate content in the range of 1 to 50 μM, with a detection limit as low as 690 nM, and the results align well with clinical CT images. This method can accurately measure oxalate content in real clinical urine samples, with the advantages of low cost, high sensitivity, and good selectivity. However, its application is limited to specific stone types. Since kidney stones can also consist of UA, cystine, and other components, CDs-MnO₂ has inherent limitations in comprehensively classifying stone varieties.

Prevention of nanocrystal formation

Drug prevention

Currently, the main drugs used in the clinic for the treatment or prevention of kidney stones are thiazide diuretics, citrate supplements, and cysteine-coupled drugs. Nonetheless, the outgoing high rate and reemergence of stones could be attributed to the ineffectiveness of current medications. ³⁴ Several drugs are known to inhibit nanocrystals and thereby reduce the possibility of kidney stone formation.

Polysaccharide

The formation of CaOx stones is intimately linked to the excessive saturation of Ca²⁺, nucleation, the growth of crystals, and their clustering. Urine inhibitors are crucial in the development of stones. In contrast to those suffering from urological stones, individuals in good health exhibit a broader range of urinary inhibitors, characterized by increase levels and more potent activity. The list of these inhibitors encompasses minor inorganic salts like citrate and pyrophosphate, along with major urine macromolecules including GAGs, nephrocalcin, Tamm-Horsfall protein, and UPTF-1. Below, I will introduce several polysaccharide substances that can inhibit the formation of calcium oxalate nanocrystals.

It has been shown that polysaccharides extracted from TPS have the ability to repair damaged renal epithelial cells. ¹¹ Cellular repair is a passive treatment that repairs damaged kidney epithelial cells to prevent the formation of kidney stones. In contrast, for undamaged cells, early in vivo protection against oxidative damage from uric crystals or oxalic acid is an active and effective method of preventing renal stone formation, with a greater clinical value than passive repair. Zhao found that TPS successfully mitigated the damage of COM to renal proximal tubular epithelial cells in the human kidney by addressing the adhesion variances between calcium oxalate nanoparticles and HK-2 cells, each with varying molecular weights of TPS. ³⁵ Of the various molecular weights of TPS, the one weighing 4.82 kDa demonstrated superior cytoprotective properties and the most potent inhibitory impact on crystal adhesion.

Typically, porphyra yezoensis polysaccharides (PYPs) are acidic mucopolysaccharides, rich in sulfonic acid groups, and bear a significant structural resemblance to urethane GAGs. ³⁶ This substance is capable of the surface of crystals, potentially hindering their adhesion and enhancing cellular endocytosis. Within 30 min, CaOx crystals sticking to the cell surface were capable of being internalized into cells by microvilli, thus diminishing their toxic effects on renal tubular cells. Endocytosis, a process reliant on energy, involves vesicles formed via the plasma membrane absorbing substances from the surrounding environment. Currently, the function of cells absorbing external crystals during the formation of stones remains ambiguous. PS contains oxalate, a dicarboxylate with both positive (amino) and negative (carboxyl) groups, capable of interacting with PS's amino group or its carboxyl group, especially when suitable ‘bridging’ cations like calcium are available.^25,37 Exposure to oxalate alone might lead to a rearrangement of PS within the plasma membrane, facilitating the attachment of CaOx crystals to the exterier of renal epithelial cells. OPN frequently appears as an adhesion molecule on the surface of cells. Zhang found that PYP can reduce the adhesion of nanocrystals to cells by reducing the diffusion of PS on the cell surface and inhibiting the expression of OPN protein. ³⁸ In addition, PYP can also repair the morphology of cells damaged by CaOx and enhance their ability to endocylate nanocrystals to inhibit the adhesion of nanocrystals. Also, among all of the PYPS, PYP4 (4.02 kDa), which had the lowest molecular weight, exhibited the best biological activity.

Nonetheless, there is a positive correlation between the cytotoxic effects of CaOx and the quantity of endocytosed crystals at elevated concentrations. Crystals absorbed by endocytosis might engage directly with the lysosome in the cell, leading to its rupture, the release of substantial quantities of Ca²⁺ and Ox²⁻, and subsequent cellular harm. Research has shown that corn silk has a beneficial effect in treating urological stones. It has diuretic activity, increases the pH value of urine, and enhances the citrate content in the urine. ³⁹ Zhang found that CCSP attenuated the damage of nanocrystal to HK-2 cells by inhibiting cellular endocytosis of nanocrystals and reducing oxidative stress. ⁴⁰ When CCSPs attach to the surface of nano-COM, it amplifies the repelling force between the crystal and the negatively charged cell membrane, hinders the crystal's attachment to the cell, and consequently prevents the cell from endocytizing the crystal. As the content of −COOH in CCSPs increased, the negative charge of the COM crystal increased. Thus, among all experimental groups, CCSP3 with the highest hydroxyl content (containing 16.38%) had the strongest inhibitory ability. What's more, CCSPs have the potential to diminish damage from lipid peroxidation and enhance cellular resistance to free radicals. An increase in the −COOH levels in CCSPs enhances the impact of antioxidant abilities.

In addition, sulfated laminaria polysaccharide (SLP), an anionic polysaccharide abundant in sulfonic acid groups, has the ability to adhere to crystal surfaces, potentially hindering crystal adhesion and endocytosis. ⁴¹ Yu found that SLPs has a similar mechanism of action to PYPs. ⁴² Also, the ability of SLPs to shield cells from harm and prevent the endocytosis of crystals in cells is amplified when there is a rise in the −OSO3− levels in SLPs.

Selenium nanoparticles

Selenium (Se) is considered to be one of the essential trace minerals that are vital for maintaining a healthy and well-maintained body. ⁴³ Selenium binds to proteins to form essential antioxidant enzymes-selenoproteins (SPs). Using scanning electron microscopy, Liang that selenium nanoparticles (SeNPs) inhibit the formation and aggregation of calcium oxalate nanocrystal. ⁴⁴ When the concentration of SeNPs increases, the nanocrystals are more dispersed and SeNPs promote the transformation of COM to spherical COD, which reduces the likelihood of kidney stone development. Therefore, SeNPs could be used as a potential inhibitor of urinary tract stones.

Alvares first discovered that Haloferax alexandrinus GUSF-1 (KF796625) is capable of converting selenite into pentagonal SeNPs in vitro. ⁴⁵ They also found that SeNPs can regulate the shape of COM in vitro. As the concentration of SeNPs increased from 50/100 ug ml-1 to 100 ug ml-1, the average surface area of CaOx crystals decreased and their morphology changed from irregular rectangles to irregular spheres. These findings further support the significant role of SeNPs in the drug research of urinary system stones. Using Haloferax alexandrinus GUSF-1 to cultivate SeNPs not only ensures a sterile environment, but also greatly reduces the cost of cultivation. From the perspective of SeNPs, it can provide an economically feasible and environmentally friendly synthesis pathway for studying drug related to urological stones.

Heparin

Heparin (HP) represents a form of linear GAGs. The ability of GAGs to hinder the nucleation, growth, and aggregation of CaOx crystals is intimately linked to their molecular configuration, particularly the anionic segments of GAGs molecules. Among the eight components of GAGs, HP has the highest proportion of anionic groups such as -OSO3− and -COO−. In HP molecules, the presence of sulfate and urate groups with negative charges can attach to Ca2+, leading to a decrease in the supersaturation of CaOx solution and hindering the nucleation, expansion, and clustering of CaOx crystals. Ou found that HP rich in -OH groups can adsorb on the surfaces of nanocrystals COM or COD, increasing the surface negative charge, causing a decrease in zeta potential, and enhancing the dispersion of nanocrystals COM and COD in the suspension, thereby facilitating the inhibition of CaOx stone formation. ⁴⁶

Dietary prevention

Urine contains minerals such as calcium, oxalates, and phosphates from dietary sources or endogenous metabolism. Oxalate crystals form in the renal tubules of patients with hyperoxaluria. The build-up of oxalate may lead to blockages in the urinary tract. This element plays a crucial role in the formation of renal crystal. The accumulation of oxalate within the excretory system may result in alteration in kidney cell activity and the development of kidney stones. ⁴⁷ An excessive oxalate diet can promote the formation of stones. In the human body, the GOX1 gene is responsible for the oxalate metabolism. Studies have shown that primary hyperoxaluria caused by GOX1 variations is significantly associated with kidney stones. These related genes include GG(rs6086287), TT(rs2235250), GG(rs2255183), and CC(rs2294303). ⁴⁸ By comparing nanocrystals in urine before and after subjects undergoing an oxalate diet, Kumar found that increased oxalate dietary loads resulted in an increased number of nanocrystals and increased aggregation in the urine. ⁴⁹ Therefore, reducing the intake of foods high in oxalate and normalizing dietary calcium intake is a practical way to reduce the production of nanocrystals in the urine and thus reduce the formation of urinary stone.

Conclusion

In this paper, the relationship between nanocrystals and the formation and development of kidney stones is reviewed through their physicochemical properties as well as their cytotoxicity. Meanwhile, this paper summarizes the mechanisms by which nanocrystals promote the formation of kidney stone, methods for effective tracking and detection of nanocrystals, and potential urinary stone inhibiting drugs that inhibit nanocrystals. There are very few studies exploring the mechanisms of kidney stone formation from a nanocrystal perspective. It is hoped that this review will help elucidate the relationship between nanocrystals and kidney stone formation in order to develop new therapeutic and preventive methods.