Based on our findings, we have made the following series of suppositions and deductions.
The beginning of encrustation and CaOx stone
For CaOx stone, the traditional view is that metabolic stones are caused by metabolic disorders, caused by the supersaturation of mineral salts in urine that exceeds their solubility limits. Nevertheless, emerging evidence has suggested that metabolic stones may also be caused by infection28,29,30,31.
In fact, there is considerable evidence that bacteria influences the formation of stones23. The two most likely microbial active sites associated with stone formation are located in the gastrointestinal and urinary tracts. Since the urinary tract microbiome resides at or near the site of lithogenesis and will have a more direct impact on overall lithogenesis, it may influences lithogenesis more than the gut microbiome32,33.
Different from CaOx stone, the formation mechanism of infectious stone has been clarified and widely accepted. This process starts with bacteria. First, under the action of urease produced by some bacteria, urea in urine is decomposed into carbon dioxide and ammonia34. After ammonia is hydrolyzed to ammonium ion, it can synthesize ammonium hydroxide with water, which will significantly increase the urine pH level. When the urine pH reaches 7.2, ammonium ions can combine with phosphate and magnesium ions in urine to form MAP35. On the other hand, carbon dioxide is reduced to bicarbonate, which will combine with cations in urine to form CaAp under alkaline environment36. MAP and CaAp are constantly produced and mixed together to form infectious stone and be considered as infectious compositions37. Sometimes the amount of CaOx is more than that of MAP in infectious stone, and sometimes MAP may predominate29. In addition, in the case of infection and high pH, calcium hydrogen phosphate(CaHP) may also be generated and mixed with other compositions to form infectious stone38,39,40. Thus, CaHP is also associated with infection.
The encrustation of stent was a continuous process, and its main compositions changed continually in this process. We found that encrustations were mainly composed of infectious compositions at the beginning. Interestingly, the proportion of CaOx gradually increased with time, which was the main composition of the later encrustations41. Most notably, there was no single CaOx existed in the early stage. Furthermore, research reported that the proportion of infectious compositions in the inner layer of CaOx stone was higher than that in the outer layer42. Infectious compositions may be the first substances to appear whether in time or space during formation of encrustation and stone, that is to say, formation of CaOx stone began with infectious compositions.
Crystal nucleation for CaOx stone
Why isn't CaOx the initial composition of CaOx stone? We know that the formation process of urolithiasis requires three steps: crystal nucleation, crystal growth and crystal aggregation, and stone formation4,43. Nucleation is the initial step in crystallization in which a small number (dozens or hundreds) of molecules gather together to form crystal nucleus. It can be achieved by three ways including homogeneous nucleation, heterogeneous nucleation or epitaxy44,45. The prerequisite for nucleation is that some solute reaches supersaturation in urine. When the supersaturation reaches a certain degree, the same solute molecules spontaneously gather to form crystals, which is called homogeneous nucleation. Heterogeneous nucleation can be viewed as a catalyzed form of homogeneous nucleation in which the nuclei form on some foreign surface or particle, which can be epithelial cell fragments, various tubular or matrix. Moreover, if the nucleated crystal encounters another solute, and the lattice (the spatial structure after crystal formation) of this solute is similar to that of the nucleated crystal, this solute will grow on the formed crystal. This way can be thought of as a special form of heteronucleation and is known as epitaxy45.
Nucleation of CaOx occurs at a concentration of 0.58 mmol/L oxalate in solution46. However, the saturated concentration of oxalate in urine is 0.040-0.114 mmol/L (the saturated concentration will increase with the decrease of urine pH, 0.040mmol/L at pH6.5 and 0.114mmol/L at pH5.0)47, which means that single CaOx crystal requires 5-10 times of the supersaturated concentration of oxalate to nucleate. In fact, the oxalate concentration in urine is difficult to reach the level required for homogeneous nucleation, and such high oxalate concentration may exist in extremely serious acidosis that will endanger life48. Accordingly, homogeneous nucleation of CaOx in urinary tract is difficult to achieve, and heterogeneous nucleation may be the main way49.
How does CaOx gather through heterogeneous nucleation? We found that no encrustations composed of single CaOx. Therefore, we consider that infectious compositions such as CaOx and CaHP always nucleates before CaOx due to its low required supersaturation, and becomes the basic core for CaOx to adhere and grow. CaOx always needs to be accompanied by these compositions and nucleates in the form of epitaxy. Infection, as the main source of infectious compositions, triggers this process. This is the reason why Randall plaque, the recognized precursor of CaOx stone, is mainly composed of CaAp, even calcium phosphate (CaP) or magnesium phosphate (MP)50. A research also confirmed this inference51, which revealed that Randall plaque can be found in the center of many CaOx stones, and further optical microscope scanning saw that CaOx crystals randomly inserted into the plaque composed of CaAp, and there was an obvious interface between them.
UA may also nucleate homogeneously and become the core for CaOx attachment, which may depend on its saturated concentration in urine is much higher than CaOx (up to 1.094mmol/L)47. This process has been confirmed in previous study52. Moreover, the bacteria that cause infection can act as the core to induce heterogeneous nucleation. Some studies have shown that there are nanobacteria(NB) in kidney stones, which can produce enough CaAp in the cell wall to cause pathological calcification and stone formation5. Damaged urothelium and cell residues also be used as the core of heterogeneous nucleation53.
Growth and aggregation of CaOx crystals
The volume of the initially formed CaOx crystal nucleus is very small, which is nanometer level50, and requires continuous solute deposition to grow up. However, this growth is too slow and the crystals are easy to be washed away by urine, so that crystals need to aggregate and adhere to the urinary tract in some ways44,45. Previous studies have shown that the damage of renal tubular epithelial cells provides a place for crystals to adhere. This damage is caused by inflammatory and immune factors, and may be related to the stimulation of CaOx crystals54,55,56,57. However, bacterial infection can also lead to inflammatory reaction and tissue damage. This may be another mechanism of renal tubular epithelial cell damage. Vitro experiments and proteomic analysis confirmed that Escherichia coli can promote the growth and aggregation of CaOx crystals58,59. Recent studies have confirmed that Escherichia coli enhances the adhesion of CaOx crystals to cells through flagellin mediated inflammation and oxidative damage to the kidneys, ultimately promoting the formation of CaOx stones60,61.
It is not clear how small crystals gather with each other to form larger crystals. However, we considered that since infectious compositions such as CaAp can become the core of CaOx attachment, it can be used as a bridge to connect CaOx various crystals as well. Therefore, there are different proportions of infectious compositions in various parts of CaOx stones42.
Crystals suspended in urine collide or disperse with each other as urine flows45, which is obviously not conducive to their aggregation. We found that purulent floccules can wrap crystals and form stone like masses, and their compositions basically consistent with that of the patient's primary stones, so we considered that these purulent floccules may be the material that fixes various CaOx crystals and accelerate their aggregation, which similar to glue. These purulent floccules can also covered the surface of the formed stone, forming a structure like a sugar coating. When enough crystals were absorbed and solidified by this coating, a new layer of CaOx stone was formed. This may be why the structure of some stones can be layered like concentric circles or annual rings. Some studies have shown that neutrophils tend to gather after infection, break and mix with fibrin to form a physical structure called neutrophil extracellular traps(NETs). This structure, like a spider web, is one of the main components of pus, which can stick and capture bacteria62,63,64. Similarly, this viscous structure may also be used to fix CaOx crystals, which needs further research to confirm.
Despite there are few studies focus on the composition of pus, the available evidence showed that some special components found in pus overlapped with the matrix components of CaOx stone, such as calgranulin, lactoferrin and osteopontin65,66,67,68. Calgranulin and osteopontin is believed to be related to the aggregation of CaOx crystals65,69,70,71.
Mechanism and process of CaOx stone formation
Based on the previous evidence and inference, we have deduced the whole formation process of CaOx stone(Fig.8): In the first stage, urease producing bacteria enter the urinary tract and cause infection, As a consequence, the urine pH rises, CaAp, CaHP and MAP begin to form. If the infection is not controlled, these substances will be generated rapidly and infectious stones will be formed in a very short time36,38. In the second stage, if the infection is controlled by the intervention of the immune and inflammatory reaction of the body. Then the urine pH gradually decreased and infectious stone will no longer form and begin to dissolve at pH<6.5 7. Since CaOx aggregation requires an acidic environment (evidence shows that pH<6.5 is a necessary condition for the formation of CaOx stone)47,72, the epitaxy will be occurred if there is supersaturated CaOx in urine. On the other hand, patients with UA supersaturation can form UA cores by homogeneous or heterogeneous nucleation, and CaOx can also attached to them by epitaxy to form crystals, as another way of CaOx crystal formation.
In the third stage, the injury of urethral epithelium and renal tubular epithelium caused by immunity and inflammation provides the base of crystal adhesion and prevent them from being washed away by urine. Then purulent floccules formed by inflammation absorb and wrap CaOx crystals, combine with some organic debris (including cell residues, bacteria, mucus proteins) to form sediment, and become stones due to the loss of water53.
The nascent stone can also become a site for bacteria and purulent floccules to adhere and grow, making the stone further enlarged. Each stage exists and alternates in different parts of urinary tract, and eventually forms CaOx stone that we can see.
In addition, the nucleation and growth of CaOx crystals will be prevented by some substances, such as citric acid, which is widely considered as an inhibitor of CaOx stone formation and used to prevent the recurrence of CaOx stone73,74. Many bacteria can metabolize citrate in urine, thereby reducing the inhibitory activity of urine on CaOx and CaP, including some urease producing bacteria, such as proteus mirabilis and klebsiella pneumoniae75,76. This also promotes the formation of CaOx stone.
Infection and inflammation are crucial in the formation of CaOx stone. stones are always exists in the place where connecting the human body with the outside world including the urinary tract, digestive tract and oral cavity. For this reason, it is difficult to produce dense and solid encrustations in vitro77. In addition, some special cavities may also have stones if lead to the outside, such as renal caliceal diverticulum. One of the key points in distinguishing renal calyceal diverticulum from renal cyst is that there may be stones in the renal calyceal diverticulum, while the renal cyst does not78.
Catalysis of DJ stent in encrustations formation
Our analysis result showed that infection stone and urease-producing bacteria before surgery were the risk factors for early encrustations, which indicated that the catalytic effect of DJ stent on the formation of stone may be related to bacteria and infection. Research have shown that bacteria can form biofilm on the surface of DJ stents, which in turn become a place for bacteria to hide and breed79,80. The DJ stent can also be used as a material for purulent floccules or crystal adhesion, and urine is drained through the stent, which reducing the probability of crystals being washed away. As a result, the formation of stones are accelerated and appear in the form of encrustation.
CaOx stones in other parts of the body
Interestingly, the crystals we collected from the broken toe joint of a gout patient were composed of sodium urate monohydrate, CaOx dihydrate, and MAP hexahydrate. Staphylococcus aureus, a urease producing bacterium, was found in pus on the surface of the wound. At the same time, we have collected samples of dental calculus form this patient for compositional analysis, and found that it also contains CaOx. It means that stones containing CaOx may be generated in any place that meets the conditions for CaOx stone formation.
Summary and reflection
The formation of stone is a continuous process, which has different manifestations in different periods. Infectious stone was seen in the acute and early stages, while CaOx stone were seen in the chronic and late stages. Therefore, urolithiasisis not just a stone, it should be defined as a disease caused by metabolic disorder and infection.
Historically, most staghorn stones are composed of infectious compositions. However, recent studies have shown that metabolic stones, such as CaOx stones, account for an increasing proportion of staghorn stones. There are no clear explanations for the shift in staghorn stone composition from infection to metabolic stones, and it has been hypothesized to be due to dietary and lifestyle changes accompanying improved living standard81,82. Now, based on the evidence of this study, we speculate that this may be due to the extensive use of antibiotics in recent years. After the infection is controlled, the disease course enters the later stage, and CaOx stones increase.
In general, human beings are fighting with nature all the time, and the urinary tract is one of the front lines of the campaign. CaOx stone, like a mess on the battlefield after the war, is the product of this battle.
The formation of stone is a huge and complex process, although the detailed mechanism remains to be explored, our hypothesis described the whole process of CaOx stone formation and provides direction for further research.