Historical sequence of events

Distinct stratigraphic layering on the scale of 10’s to 100’s of nanometers (nano-layering) is revealed by auto-fluorescence (AF, emission of a specific fluorescence light without labels in response to a specific excitation wavelength)16,17 generated by changes in organic matter composition (Supplementary Fig. 1). We interpret the crystalline architecture of COD, COM and UA in kidney stones using the Law of Superposition (i.e., older layers at the bottom and younger layers at the top), proposed in 1667 by Nicholas Steno, a Danish physician and pioneering geobiologist2,19. Our observations are synthesized into a historical sequence of events (HSE, Fig. 1, a paragenetic sequence in geology)20.

Figure 1 Historical sequence of events (HSE) constructed from super-resolution auto-fluorescence (SRAF) images of the MP2 calcium oxalate (CaOx) kidney stone. (a) The HSE. (b) Sketch depicting individual HSE events. (c) Representative SRAF image composed of merged three pseudo-colored red, green and blue (RGB) channels. Brightness and contrast of the RGB channel intensities are adjusted to highlight the dark crystalline fabrics. Raw images with and without adjustments are presented in Supplementary Fig. 2. Full size image

The earliest stage of kidney stone growth begins with the precipitation of 5–250 μm-diameter perfectly geometrically formed (euhedral) COD and COM with internal concentric zonations consistent with free-floating growth in the renal calyx and/or pelvis (i.e., crystalline sediment or crystalluria)(COD FF and COM FF , HSE 1 in Figs. 1 and 2a)21,22. The outermost surfaces of larger COD FF have euhedral extensions reflecting crystal twinning21, including the adherence of smaller COD FF to other larger COD FF faces23 in the form of aggregates (HSE 2 in Figs. 1, 2a and Supplementary Fig. 2a). These outermost surfaces then dissolve (HSE 3 in Figs. 1 and 2a), cutting 10’s of microns down into internal concentric COD FF layers. Following dissolution, small <5–10 µm-diameter COM FF land on and encrust outer COD FF surfaces (Figs. 1c and 2a). The next generation of COM exhibits a dense nano-layered cortex (COM C ; HSE 4 in Figs. 1, 2a and Supplementary Fig. 2b)24,25 that encrusts both pristine and dissolved COD FF surfaces. In addition, some COM FF land on and are then encrusted by the same COM C (HSE 4 in Figs. 1 and 2a). COM C generally adopts the same crystallographic C-axis orientation as the COD FF and COM FF (Fig. 1c) they encrust (syntaxial overgrowths).

Figure 2 Evidence for in vivo dissolution and nano-layering from confocal auto-fluorescence (CAF) and SRAF imaging of the MP2 CaOx kidney stone. Specific areas of the MP2 stone from which these image enlargements are made are shown in Supplementary Fig. 1. (a) Tiled CAF image of merged pseudo-colored RGB channels with no image adjustments. (b,c) SRAF images of merged pseudo-colored RGB channels. The brightness and contrast of each image is individually adjusted to highlight the layered crystalline architecture (raw images with and without adjustments are presented in Supplementary Fig. 22). (d) SRAF image of COM C nano-layering from merged two-channel blue and red (pseudo-colored cyan) channels (Z-stack optical sections of all layers are presented in Supplementary Video 2). The SRAF green channel is identical to the red channel (pseudo-colored cyan) and therefore not included. (e) Individual ~140 nm-thick dark and light nano-layers (open and closed arrowheads) with enlargement (e). Radiating twinned crystals grow with their c-axis oriented perpendicular to each dark or light nano-layer (arrows). (f) Black and white circular polarization phase contrast (CPOLPC) image shows COM C dark organic matter-rich and light mineral-rich nano-layering with enlargement (g). The original color image of the same area is presented in Supplementary Fig. 18. Images (d,e) are displayed with best-fit intensity profiles. Images (f,g) are displayed with best-fit intensity profiles after a gamma correction of 0.4. Full size image

The COD FF , COM FF and COM C crystal complex then extensively and repeatedly dissolves (HSE 5 in Figs. 1, 2a), which is recorded by four types of fabrics that cross-cut the crystalline architecture (Supplementary Fig. 2a–d): (1) Micron-sized and larger crystals of COD FF partially dissolve from their center, creating irregular void spaces lined with remnants of original COD FF (white arrowheads in Figs. 1c, 2a, Supplementary Fig. 2c). (2) Bulk dissolution completely removes the original COM C -encrusted COD FF , leaving euhedral mold-shaped void spaces (moldic porosity, P in Figs. 1, 2 and Supplementary Fig. 2a–d). (3) Continued bulk dissolution cuts into COM C, creating canyon-like void spaces that cross-cut COM C nano-layering (Fig. 2b and Supplementary Fig. 2d. (4) Both irregular and euhedral moldic porosity within COD FF , COM FF , and COM C is then partially to completely filled with replacement COM (COM R ; HSE 6 in Figs. 1, 2a,b and Supplementary Fig. 2b,c,e–g). In addition, Ångstrom-scale dissolution and crystallization (mimetic replacement, COM M ) of COM C takes place, in which the original fine laminations of the cortex are completely to partially preserved (HSE 6 in Figs. 1, 2a and Supplementary Fig. 3).

The final stage of stone growth occurs when three separate stone fragments come into contact, locally dissolve at contact points and interlock to create a larger stone complex (Supplementary Fig. 1b,c and Video 1). At present, we cannot distinguish if the three stone fragments formed entirely by naturally occurring events within the patient’s kidney, or whether they broke apart as a result of previous medical intervention. The margins of each fragment are irregular, intertwined and exhibit large-scale 300–400 µm-scale truncation of COM C nano- layering (Supplementary Fig. 1b,d). These data indicate that dissolution continues as the margins of the stones come into contact. After the fragments merge into a stone complex, COM then grows on some outer stone surfaces as cements (COM CE ; lime green layers in Supplementary Fig. 1c, labeled COM CE in Fig. 2c and Supplementary Fig. 4a,b), which exhibit crystal-face specific differences in organic matter concentrations (sector zoning) (labeled SZ in Fig. 2c and Supplementary Fig. 4b)26. Uric acid cement crystals (UA CE ) then grow on outer surfaces of the stone complex (gray layers in Supplementary Fig. 1c, labeled UA CE in Fig. 2c and Supplementary Fig. 4a,b). Other outermost COM C stone surfaces continue to dissolve and are replaced by COD and COM (COD R and COM R , cyan in Supplementary Figs. 1c, 4c–f), which is consistent with previously observed “intimate COD and COM relationships6”.

COD FF aggregates, which reach 300 µm in diameter (dark blue S3 in Supplementary Fig. 1c), are consistent with previous observations of COD morphology within kidney stones and likely form under hypercalciuric conditions27. Their large size requires that crystals aggregate in urine collected in the renal calyx and pelvis as opposed to filtrate in the nephron collecting ducts, which only reach ~150 µm in diameter12,15. These large COD FF aggregates form the nucleus (nidus) that COM C encrusts (Fig. 1c). The symmetry of the COM C concentric layering indicates free-floating crystallization while completely bathed in urine. Growth while attached to tissue on one side would create discontinuous and asymmetric COM C layering around the COD FF nidus12,15. In addition, COM FF are observed to have landed on the growing surfaces of COM C . These crystals become entombed in, and encrusted by, the concentric COM C nano-layering (Figs. 1c and 2, Supplementary Fig. 4c,d) and are called protrusions28 into COM C .

The HSE (Fig. 1) of this representative stone has broad implications for understanding the growth history of other types of kidney stones, including those that originate in the renal papilla as interstitial deposits of calcium phosphate (apatite) called Randall’s plaques, or in the ducts of Bellini called Randall’s plugs29,30,31. Randall’s plaque commonly erupt into the renal calyx and pelvis, or are released through the nephron, and begin to accrete COD FF and eventually COM C (HSE 4) while bathed in urine12. Instead of the initial stages of apatite nidus growth32, CaOx kidney stones in the present study have a nidus composed of free-floating COD FF and COM FF (HSE 1–3, Fig. 1). The lack of apatite crystal dissolution fabrics within COD FF and COM C in the MP-series stone fragments implies that these CaOx stone fragments likely did not contain a precursor Randall’s plaque or plug nidus.

Kidney stones dissolve In vivo

Results from the current study modify the long-held working assumption that COM is strongly insoluble in vivo3,12,15, except perhaps within some individual cell organelles (phagolysosomes)33. In addition, although the entombment of organic matter has been previously documented12,15, the clinical importance of these biomass-rich nano-layers remains undetermined28. Our results indicate that kidney stones repeatedly dissolve in vivo (Figs. 1 and 2, Supplementary Figs. 1–4; summarized as dissolution of COD FF outer surfaces [HSE 3], dissolution of COD FF , COM FF and COM C via four types of cross-cutting crystal fabrics [HSE 5], and mimetic replacement of COM C by COM M [HSE 6]) and highlight the intrinsic relationship between organic-rich and mineral-rich nano-layering.

The repeated dissolution, crystallization and resultant remodeling of crystalline architecture that takes place during CaOx stone growth is analogous to the commonly observed post-depositional physical, chemical and biological alteration observed in natural mineral deposits (diagenesis)24. The sector zoning in COM CE indicates that these crystals may be more soluble and thus more susceptible to diagenesis than expected with respect to calculated urine supersaturation states25, as controlled by the differential entrapment of organic matter on specific COM crystal faces34,35. The biomolecules present within the human kidney may also play a major role in driving the multiple events of dissolution as recorded in the HSE (Fig. 1). These are normal constituents of urine chemical composition36, and could plausibly include biomolecules derived from a resident microbial community (microbiome)13. However, the composition and potential effects of these biomolecules are currently unknown37.

COM C nano-layers

We use fast Fourier transform (FFT) frequency analyses to compare the nano-layering within individual COM C , COD FF and COM R (Supplementary Fig. 5). Both COM C and COD FF exhibit highest frequencies and thinnest nano-layers (Supplementary Fig. 5a–d,g–j). The crystalline architecture of COM C , COD FF and COM R exhibit different patterns in three distinct optical modalities applied (POLPC, CPOL, SRAF, Supplementary Fig. 11) indicating that multi-modal approaches are required to investigate kidney stone crystalline fabrics (Supplementary Fig. 5). Nano-layering is the most volumetrically dominant component of COM C (Figs. 1 and 2) and occurs in well-defined organic matter- and mineral-rich couplets12,15,38. However, a comprehensive understanding of the mechanisms controlling the abrupt switch between deposition of each organic matter-rich and mineral-rich nano-layer is unknown9,28,39,40. Under SRAF imaging, bright AF indicates organic matter-rich layers, while dim AF represents the adjacent mineral-rich couplet layers (Fig. 2d,e and Supplementary Figs. 5 and 12). In contrast, when observed under transmitted-light polarization and phase contrast (CPOLPC), the brighter layers are crystal-rich and the dimmer layers are organic matter-rich (Fig. 2f,g). While SRAF imaging reveals sharp well-defined COM C layer couplets at a spatial resolution as fine as ~140 nm (Fig. 2e), previous transmission electron microscopy imaging of other kidney stones has detected even finer layering at ~50 nm in thickness41. If these ~50 nm-thick layers are present in the six Mayo Clinic patient stones, they would not have been optically resolved with SRAF in the present study, but instead, would optically average into ~140 nm-thick layers within each couplet. As a result, the actual number of COM C layer couplets and their frequencies in the present study could be a 2–3-fold underestimate. Given these detection limits, the observed COM C is composed of ~140–250 nm couplets based on the optical resolution of our microscope system.

An initial interpretation of these COM C couplets (Fig. 2d–g) is that the organic matter-rich nano-layers are films of biomolecules (peptides, proteins, etc.) entombed between mineral-rich nano-layers28. Potential reasons for these rapid nano-layer shifts might include frequent changes in human host and kidney physiology, urine biochemistry and perhaps even microbiome ecology and activity. Alternatively, these layers are also comparable to the oscillatory zoning found in many minerals, where it is believed that a kinetic feedback mechanism results in periodic oscillations of crystal growth and impurity occlusion independent of biological activity42. Further analysis of the composition and concentration of organic matter entrapped in each cortex nano-layer will be required to establish a mechanistic hypothesis for their deposition. Since the exact amount of time required to form any given kidney stone is difficult to constrain, it is uncertain precisely how long it takes to deposit each nano-layer couplet. However, previously published observational data regarding how long it takes CaOx stones to grow43,44 implies that several thousands of nano-layers could possibly form within weeks or months. Given these rough estimates, each nano-layer may have formed on a sub-daily basis of hours or in some cases even minutes. If correct, kidney stones could be “read” in the future under clinical conditions as an unprecedented ultrahigh-sensitivity record of in vivo human renal function and dynamic biogeochemical reactions.

Biomineralization in natural and engineered environments

The alternating organic matter- and mineral-rich nano-layers comprising COM C are strikingly similar to those seen in other modern and ancient sedimentary deposits. These include marine stromatolites, ooids and oyster shells and pearls, as well as terrestrial hot-spring travertines and cave speleothems, among many other deposits45,46,47. Previous geobiology studies of these natural deposits have only partially deciphered the relative influence of the physical, chemical and biological factors that are active at the time of layered deposition. COM C nano-layer couplets represent a previously unknown template for understanding the mechanisms that fundamentally control shifts between biotic and abiotic processes during biomineralization. These mechanisms are directly applicable to understanding biomineralized deposits common to other natural and engineered environments in fields that range from environmental sustainability and energy production, to medical discovery and space exploration.

BF microscopy with a theoretical optical resolution of approximately 1-μm was used to compare the nano-layer couplets in kidney stones (Supplementary Fig. 6a–c,j) with travertine formed within ancient Roman aqueducts45,47 (Supplementary Fig. 6d–f,k) and cave limestone deposits (speleothems, Supplementary Fig. 6g–i,l)45,46. Out of necessity, these analyses are completed at a micrometer-scale resolution instead of the nanometer-scale since the cave and aqueduct systems have orders-of-magnitude higher crystal growth rates than those in the kidney (Supplementary Fig. 13). These higher rates of crystal growth dramatically increase the thickness of each layer, which makes only one or two layers fill an entire frame at super-resolution, making them incompatible for frequency analysis (Supplementary Fig. 13a–d). Requirement of lower magnification imaging indicates that CaOx kidney stone layers are 10-times higher frequency (~1.6 µm/layer) than those in the cave deposits (~16 µm/layer), and 3-times higher frequency than those in the aqueduct travertine (~5 µm/layer; Supplementary Fig. 6m). Although not yet proven, the significantly thinner and higher frequency nano-layers in kidney stones (Supplementary Fig. 6) may be the result of the short-time scales over which human renal function and biochemistry can change (i.e., seconds to hours). This may also reflect the abundant and diverse sources of inhibitors in the renal environment compared to those in other geological and engineered settings. In addition, the ~140 nm-scale and finer nano-layering in CaOx kidney stones is significantly smaller than the µm-size of whole microbial cells and their associated microbial mats that directly influence layering in other geological deposits such as stromatolites45,48. As a result of these size constraints, kidney stone biomineralization must be controlled by some combination of human host and/or kidney microbiome derived biomolecules rather than whole cells.

CaOx kidney stones occur throughout the animal kingdom49. While euhedral CaOx crystals similar to COD FF and COM FF are also common in plants, no COM C has yet been reported50. This lack of COM C in plants likely reflects the absence of the type of high flow-through hydrodynamic environment present in the kidney, which is required to consistently deliver dissolved ions to the site of COM C crystallization. CaOx crystallization in terrestrial plants serves to store carbon and H 2 O for later use in times of reduced carbon availability and drought50,51. By analogy, it is also possible that COD FF , COM FF and even COM C crystallization in animals may serve to store water for later use when the kidney ecosystem is stressed. In vitro batch reactor experiments52, as well as our own microfluidics experimentation (Supplementary Figs. 7 and 17) to test CaOx growth dynamics, have successfully grown free-floating polymorphic COD FF , COM FF and their aggregates (equivalent to HSE1, Supplementary Fig. 8). While COD-to-COM and apatite-to-COM conversion has been demonstrated convincingly, no previous experimentation has grown COM C with nano-layer couplets3,8,53,54.

Clinical and future implications