Observation of the kidney stone surface

A kidney stone (Sample 1) is composed of several aggregates of typical COD crystals (Fig. 1a, Supplementary Fig. 1b, d) [29,30,31], and the COD crystals forming Sample 1 are found to have some transparency before the phase transition experiment (Fig. 1b). Scanning electron microscope (SEM) observation of the stone surface shows that the COD crystals are randomly aggregated regardless of their planes and orientations, and that the crystals vary in size (Fig. 1c, d). The surface of a few mm-sized COD crystals was covered with COD crystals of several tens µm in size (Supplementary Fig. 5a, b).

Fig. 1

Changes in the COD stone (Sample 1) surface during the experiment. (a) The stereoscopic image of the COD stone at the start of the experiment. The stone is formed via the aggregation of several COD crystal. (b) The enlarged image of the white dotted box area in (a). (c) The SEM image. (d)A model diagram of the white dotted box area in (c). (e) The stereoscopic image of the COD stone one week after the start of the experiment. Etch-pits formed due to dissolution of the COD crystal surface. (f) The enlarged image of the white dotted box area in (e). (g) The SEM image. (h) A model diagram of the white dotted box area in (g). (i) The stereoscopic image of the COD stone two weeks after the start of the experiment. The stone surface was covered with COM crystals. (j) The enlarged image of the white dotted box area in (i). (k) The SEM image. (l) A model diagram of the white dotted box area in (k)

When the COD stone was immersed in supersaturated calcium oxalate solution, their surfaces became whiter, and the roughness increased as time progressed (Fig. 1e, f). Detailed observation of the stone surface after one week of immersion by SEM revealed many etch pits on the COD crystal surfaces, which were caused by dissolution (Fig. 1g, Supplementary Fig. 5c, d). At the same time, multiple COM crystals of several µm in size nucleated and grew on the surface of the COD crystals with the b-axis facing outward (Fig. 1g, h). After immersion in the solution for another week, the entire stone surface was covered with fine COM crystals (Fig. 1i, j). The COM crystals were aligned and oriented on the surface (Fig. 1k, l, Supplementary Fig. 5e, f).

Changes inside the kidney stone

While the macrotextures appeared on the stone surface as described above, greater changes were observed in the inside of the stone. An example of a surgically removed stone (Sample 2) shown in Fig. 2a has COD crystal morphology, but most of the interior was filled with COM. Micro-CT images show that COM crystals with many voids exist inside of the COD crystals (Fig. 2b, c). Such stones have been seen frequently.

Fig. 2

Interior of a calcium oxalate stone removed from a patient (Sample 2) and changes in the COD stone (Sample 1) interior during in the experiment. (a) A stereoscopic image of a commonly reported COD stone (Sample 2). (b) The micro-CT image of the COD stone shows in Fig. 2(a) (Sample 2). (c)An image with the CT score originating from COD in blue and that originating from COM in orange. The micro-CT image of Sample 2 shows a difference in the density of components between the inside and the outside of the stone. The exterior stone is relatively low-density components (COD), and the interior is higher densities components (COM). (d) The micro-CT image of Sample 1 at the start of the experiment. (e) The micro-CT image of Sample 1 after one week. (f) The micro-CT image ofSample 1 after two weeks. (g) The mapping ofSample 1 at the start of the experiment. The COM region and COD region determined based on the CT score. (h) The mapping ofSample 1 after one week. (i) The mapping ofSample 1 after two weeks. The inside of the COD stone has developed voids and a mosaic-like structure, and the CT score indicates that the interior of the COD stone has changed to consist of mainly COM crystals

Another observed stone shown in Fig. 2d (Sample 1) also had COD crystal morphology, but originally composed mostly of COD with < 100 μm several COM domains in the inside of the COD single crystal. This crystal had almost no voids in the inside the crystal before the incubation experiment (Fig. 2d, g). It is common to find stones with COD in periphery region and COM in the center region [9, 21, 32,33,34]. By the incubation of this stone in the simulated urine solution for two weeks, periodic micro-CT images show the increase in the abundances and the sizes of the COM domains and the abundance of void areas in the inside of the COD. Finally, the incubated kidney stone (Sample 1) became closer to another kidney stone (Sample 2) with COD morphology filled with COM and voids (Fig. 2e, f, h, i).

Evaluation of thin section

In order to observe the internal structure in more detail, a thin section of Sample 1 was analyzed with a polarized microscope and Raman spectrometer (Fig. 3a, b, Supplementary Fig. 6). COM crystals formed by phase transition inside COD crystals. They present typical COM mosaic structures (Fig. 3c, d, e, f, g, h). The outline angle of the mosaic COM structures (orange dashed line in Supplementary Fig. 6b, e) ranged from 40 to 45°, which was similar to the face angle of COD crystals (Supplementary Fig. 1b, Supplementary Fig. 6b-f). This phenomenon is identical to “pseudocrystals” in natural minerals such as quartz [35]. Pseudocrystals occur when the original mineral gradually transforms into a different crystalline phase while maintaining its external shape.

Fig. 3

Analysis of a COD stone (Sample 1) section after the experiment. (a) Cross-Nicol image of a thin section of Sample 1. The degree of phase transition differs depending on regions. (b) Results of Raman spectroscopy. The crossed points in the cross-Nicol image were measured. (c, d) Enlarged cross-Nicol image of a thin section. The phase transition is less pronounced inside narrow grain boundaries or cracks. (e, f) Enlarged cross-Nicol image of a thin section. As the grain boundaries become wider, COD crystals nucleate within the grain boundary and the phase transition proceeds further inside. (g, h) Enlarged cross-Nicol image of a thin section. Phase transition is in full progress. (i) Cross-Nicol image of COD structure. (j) Mapping of OPN. (k) Mapping of RPTF-1. (l) Mapping of Cal A. (m) Cross-Nicol image of mosaic COM structure. (n) Mapping of OPN. (o) Mapping of RPTF-1. (p) Mapping of Cal A

Comparison of multi-IF staining fluorescence intensities of a thin section of Sample 1 shows that the calcium-binding proteins, osteopontin (OPN) and renal prothrombin fragment 1 (RPTF-1), are abundantly distributed in the COD crystals (Fig. 3i, j, k, m, Supplementary Fig. 7a, b, c, d) and the concentric COM structure (Supplementary Fig. 7e, f, g, h). Furthermore, apparent protein striations appeared in these structures. In contrast, in the mosaic COM structure (Fig. 3m), the relative protein distribution is low, and proteins distributed uniformly (Fig. 3n, o, p). Calgranulin A (Cal A) is faintly distributed in COD and COM structure (Fig. 3l, p). OPN and RPTF-1 proteins which can be incorporated into the crystals interior due to their strong affinity to the crystals [28]. OPN and RPTF-1 were partially exposed to the solution during the phase transition, and then reincorporated into the newly crystallized COM crystals. Note that no protein was added to the solution in this phase transition experiment. Since some of the proteins leaked into the solution during this process, the relative amount of protein inside the COM crystals decreased. The distribution of proteins in the stones after the phase transition experiment strongly supports that the mosaic COM structure did form in the experiment by the mechanism of solution-mediated phase transition.

Crystal defects affect phase transition

Upon close observation of a thin section of Sample 1, it becomes evident that the extent of phase transition within COD crystals is not uniform. The observed regions are characterized by a coexistence of areas exhibiting minimal phase transition (Fig. 3c, d) and areas where phase transition progressed remarkably (Fig. 3g, h). We posit that this variation in phase transition extent is intrinsically linked to the rate of phase transition.

When classified by phase transition rate, we found approximately three patterns. Firstly, in regions classified as slow phase transition rate, notably narrow grain boundaries or cracks are observable (Fig. 3c, d). Between these grain boundaries or cracks, COM crystals nucleate and impede solution penetration. Secondly, in regions classified as medium phase transition rate, wider grain boundaries are observed compared to those previously mentioned (Fig. 3e, f). Along these grain boundaries, the growth of COD crystals is apparent. The evidence of these grown crystals as COD is substantiated by their crystallographic facet orientations and the multi-IF staining outcomes. We reported the selective attachment of RPTF-1 to the face of COD crystals [28]. Between the grain boundaries in our results, the fluorescence intensity of RPTF-1 is notably heightened (Fig. 3o). As COD crystals grow to obstruct grain boundaries, phase transition to COM crystals progresses within the inner regions. Notably, this phase transition rate exceeds that observed within the narrower grain boundaries. Finally, in regions classified by the fastest phase transition rate, aggregations of numerous COD crystals were observed (Fig. 3g, h). There are a large number of grain boundaries in such the aggregations.

These observations substantiate the assertion that phase transition rate accelerates concomitantly with wider or a large number of grain boundaries. This thus implies that crystal defects, such as grain boundaries and cracks, significantly influence the phase transition phenomenon.

Mechanism of phase transition

The urinary environment is always highly supersaturated for COM and COD crystals [36, 37]. If supersaturation is maintained, it should be difficult for COD crystals to undergo a solution-mediated phase transition to COM crystals because they do not dissolve. Still, the results of this study showed a phase transition from inside of the COD stones to COM crystals. Therefore, it is necessary to consider where COD crystals can dissolve within the body. Generally, the center area of a stone is isolated from urine. When grain boundaries or cracks connect the inside of the stones to the urinary environment, urine can enter the stones (Fig. 4a). The supersaturated solution that penetrating these grain boundaries promotes the nucleation of crystals (either COD or COM) between these boundaries (Fig. 4b). Subsequently, the nucleated crystals grow in the penetrated solution. Over time, these growing crystals enclose grain boundaries, creating a barrier that isolates the inside of the stone from the urinary (Fig. 4c). Thus, the inside of the stone assumes an almost enclosed state, in other words a semi-closed system. Although solution exist in the semi-closed system, the solutes (comprising calcium ions and oxalate ions) are consumed by the growth of crystals between grain boundaries, resulting in a lower solute concentration. Consequently, the solution inside the stone becomes undersaturated for COD crystals. Only COM crystals can nucleate or grow within the semi-closed system (Fig. 4d). As COM crystals grow, a localized undersaturated environment for COD crystals is formed, which, as a result, dissolves the interior COD crystals (Fig. 4e). This dissolution of COD crystals induces the growth of COM crystals (Fig. 4f). Furthermore, the water released during the phase transition (from COD to COM crystals) decreases the calcium oxalate concentration inside of the stone, accelerating this process. Note that such solution-mediated phase transition proceeds gradually through very minute amounts of the solution phase. If stones have more grain boundaries, such semi-closed systems effectively form, and the phase transition progresses preferentially. Sivaguru et al. reported that during the phase transition from COD to COM crystals, voids, which are crystal defects, are formed inside the stone due to the loss of water and decrease in volume [19]. The formation of voids can be a new path connecting the inside of the stone to the urine, an element of further continuous progression of the phase transition. As phase transition advances, mosaic COM structure form.

Fig. 4

The mechanism of phase transition inside COD stones. (a) Grain boundaries composed of COD crystals. (b) Nucleation of crystals between the grain boundary. (c) Growth of crystals between the grain boundary. (d) The growth of crystals blocks the grain boundary, creating a semi-closed system inside the stone. In this semi-closed system, the supersaturation of calcium oxalate is lower compared to the external urine environment, rendering the COD crystals undersaturated and allowing for the nucleation of COM crystals. (e) The presence of more thermodynamically stable COM crystals inside the stone causes dissolution of the internal COD crystals. (f) Continuous nucleation of COM crystals, thereby progressing the phase transition

Solution-mediated phase transition stops in an environment where the COD crystals are constantly supersaturated. The boundary condition maintains a balance between solute consumption and supply near the interface where the COD crystals are in contact with urine. Therefore, COD crystals remain in the periphery area of the stone, surrounding the COM crystals.

In addition to grain boundaries, we believe that inclusion is also related to the progression of the phase transition. An inclusion is solution and/or crystals that are trapped inside a mother crystal during its formation, and it is thermodynamically unstable; thus, such defects will be good starting points for phase transition [38, 39]. Once COM crystals, the stable phase, nucleate from the solution entrapped within inclusions, as previously described, the progression of phase transition proceeds rapidly.

It is hypothesized that stone formation rates increase when the total amount of urine in a patient’s body is small, such as in summer or at night. The faster the crystal grows, the more inclusions develop in the crystal [38]. Therefore, the quality of COD crystals is highly dependent on the patient’s internal environment (season, time of day, etc.), creating regions of high and low density of inclusions [9]. The inclusion distribution probably caused a heterogeneous phase transition in the kidney stone.

Variations in crystal size

Here, we will mention why the crystals that makeup kidney stones vary so widely in size. The euhedral CODs that initially composed the kidney stone nucleated and grew in the renal pelvis. Because they are constantly exposed to high supersaturated conditions of urine and aggregate after growing in a place with no spatial constraints, there is a wide variation in crystal size, ranging from a few micrometers to millimeter-order, as seen in this study.

Mosaic COM crystals formed in phase transitions were observed with grain sizes ranging from a few micrometers to tens of micrometers, which is large for COM crystals. In a semi-closed system, COM nucleation proceeds slowly in an environment close to equilibrium conditions. Because they coexist and grow together, the number of crystals becomes small, and each develops relatively large. The semi-closed system makes COM crystals snarled because of the spatial constraints. In contrast, the COM crystals that make up concentric COMs, observed in detail in papers [40] and [41], are aggregates of nanometer to semi-micrometer order crystals. Concentric COM is a primary structure that nucleates and grows directly without undergoing a phase transition [9]. We have also recently shown that where calcium phosphate (CaP) crystals are present, COM crystals preferentially nucleate rather than COD crystals, and grow directly on CaP [42]. As many COMs nucleate and grow on the CaP surface, the size of each crystal becomes small due to the competition of solute. On the other hand, since they elongate and grow toward a wide space to form spherulite, faceted shapes are observed in the crystals’ elongation direction (c-axis direction). Thus, the process by which the microstructures comprising the stone are formed significantly affects the size and shape of the crystals.

Phase transition hardens kidney stones

To investigate whether these phase-transformed stones would be a problem in the medical field, we evaluated on the hardness of kidney stones and conducted stone-crushing experiments using a medical extracorporeal shock wave lithotripter (ESWL) (Fig. 5a, Supplementary Fig. 4a-c). When the stones were repeatedly subjected to shock waves, the brittle parts of the stones were quickly crushed into smaller pieces, but the hard parts remained as relatively large fragments (Fig. 5b, c, d). When we examined the phase and microstructure of these fragments, we found that most of them consisted of concentric COM structures or densely agglomerated mosaic COM structures (Fig. 5e-k). It means that the mosaic COM structure, composed after the phase transition, is rigid and difficult to crush, just like the concentric COM structure [43]. As the phase transition progresses, the stones gradually become harder and more difficult to crush in stone treatment.

Fig. 5

Investigation of the stone (Sample 2) hardness by a medical extracorporeal shock wave lithotripter. (a) Experimental system for crushing stone. A water tank was placed above the device, and stones were placed in a cage attached to the top of the tank and irradiated with shock waves. (b) Stone crushing process. The 50th shock wave irradiation. (c) The 100th shock wave irradiation. (d)The 200th shock wave irradiation. (e) Cross-Nicol image of the largest volumetric fragment of the crushed fragments. (f) An enlarged cross-Nicol image of Concentric COM region. (g) Raman spectra obtained in concentric COM region. (h) An enlarged cross-Nicol image of COD region. (i) Raman spectra obtained in COD region. (j) An enlarged cross-Nicol image of mosaic COM region. (k) Raman spectra obtained in mosaic COM region. Only a few COD areas were observed. Most of the crushed fragments consisted of concentric or mosaic COM

For example, crustaceans demonstrate a remarkable ability to rapidly construct robust hard tissues by effectively controlling the assembly of amorphous or metastable phases [1,2,3]. This controlled phase transition serves as an intelligent survival strategy for these organisms. In ironic contrast, the process by which the organism endeavors to build harder structures exacerbates the formation of kidney stones, leading to detrimental consequences.

What controls the phase transition rate

We observed the formation of thin, shell-like COM crystals on the surface of the stone. Such structures are not commonly observed in stones that form in the human body. The reason why the structure was created in this experiment can be attributed to the experimental conditions.

At the start of the experiment, the solution was supersaturated for COD phase. However, as phase transition inside the stone progressed, the overall concentration of the solution decreased (Supplementary Fig. 3). It is reasonable to consider that this temporary decrease in solution concentration caused the phase transition at the outermost surface. If the solution had been maintained at high levels of supersaturation, as in real urine, phase transition would have predominantly proceeded in the center regions of the stone. The observed phase transition rate inside the stone averaged several tens of micrometers per day, between the start and two weeks after the experiment. In a patient’s body, the phase transition rate of COD stones is not precisely known but COD crystal components in kidney stones often persist for several months to years. Comparing the fact and our experimental results, the phase transition rate (several tens of micrometers per day) is notably fast. The implication drawn from these findings is that a reduction in the calcium oxalate supersaturation in urine results in a significantly accelerated phase transition. When evidence of stone dissolution within the body (specifically, COD crystals) was reported, it led to affirmative discussions regarding the potential for in vivo stone dissolution therapy [19]. However, in reality, in an environment where stones dissolve, denser, harder, and more troublesome COM stones are newly formed, exacerbating the condition. Therefore, while lowering urine supersaturation reduces nucleation and growth of new crystals, it may cause stone disease more refractory by expediting phase transition. This supports the necessity of early intervention in stone treatment.