A spatially anchored transcriptomic atlas of the human kidney papilla identifies significant immune injury in patients with stone disease.

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers.

Water Loading and Uromodulin Secretion in Healthy Individuals and Idiopathic Calcium Stone Formers.

BACKGROUND: Uromodulin is a protein made only by the kidney and released in urine, circulating in polymerizing and nonpolymerizing forms. This protein's multiple functions include inhibition of stone formation in the urine. The physiological determinants of uromodulin production are incompletely understood. METHODS: We investigated changes in uromodulin levels and key factors governing its production and release in urine and serum. We performed an experiment to determine whether water loading, a common intervention to prevent stone formation, will alter the rate of uromodulin production. During a 2-day period, 17 stone forming participants and 14 control participants were subjected to water loading (day 1) and normal fluid intake (day 2). Uromodulin levels were measured on timed hourly collections in urine and plasma during the period of the study. RESULTS: Water loading increased urinary uromodulin secretion (33±4 versus 10±4 µ g/min at baseline, P < 0.0001) in stone formers and control participants. Despite high urine volumes, most participants maintained relatively stable urinary uromodulin concentrations. Native Western blots for polymerizing and nonpolymerizing uromodulin suggest that polymerizing uromodulin was the predominant form at higher urinary flow volumes. Urine flow rates and sodium excretion were significant correlates of urinary uromodulin production. Water loading did not affect serum uromodulin levels, which were also not associated with urinary uromodulin. CONCLUSIONS: Water loading increases the secretion of polymerizing urinary uromodulin. This increased secretion reduces the variability of urinary uromodulin concentrations despite high urine volumes. Serum uromodulin levels were not affected by this treatment.

Integrated Cytometry With Machine Learning Applied to High-Content Imaging of Human Kidney Tissue for In Situ Cell Classification and Neighborhood Analysis.

The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.

Holmium Laser Enucleation of Prostate in Patients with Pre-Existing Localized Prostate Cancer, Dual Center Study.

Background: Holmium laser enucleation of the prostate (HoLEP) has been used as an effective minimally invasive technique for management of enlarged prostates. We aimed to report the role of HoLEP in prostate cancer (PCa) patients either on active surveillance with bothersome lower urinary tract symptoms (LUTS) or for prostate debulking before radiation therapy and the impact on PCa management plans. Methods: Prospectively maintained database in two institutions was reviewed for patients with localized PCa managed by HoLEP with at least a follow-up of 1 year. We assessed prostate-specific antigen (PSA) trends, effect on international prostate symptom score (IPSS) and further management of PCa. Results: Out of >2000 HoLEP patients, 117 patients with a median follow-up of 30 months were included. Mean (standard deviation) age was 72.3 (±8.3) years with median (interquartile range, IQR) IPPS of 22 (16-28) and median (IQR) PSA at 7.6 (5.3-14.9) ng/mL. Gleason grade group was 1, 2, 3, and 4 in 47 (73.2%), 32 (27.35%), 7 (5.9%), and 4 (3.4%) patients, respectively. Median (IQR) PSA has significantly dropped to 1.3 (0.6-3.1), 1.4 (0.75-2.9), and 1.7 (0.86-2.75) ng/mL at 6-week, 3-month, and 1-year follow-up, respectively (p < 0.001). IPSS scores post-HoLEP obviously improved with mean (IQR) IPSS of 10 (5-13), 7 (3-12), and 3 (2-5) at 6-week, 3-month, and 1-year, respectively (p < 0.001). Eighty-eight (72%) patients stayed on active surveillance, whereas 27 (23%) patients had radiotherapy ± androgen deprivation therapy for persistently high or relapsing PSA. Within 36 intermediate-risk patients, 15 (41.6%) and patients had radiotherapy, whereas 21 (58.3%) patients continued active surveillance. Conclusions: HoLEP is beneficial in debulking large prostate in PCa patients with bothersome LUTS on active surveillance or before radiotherapy. HoLEP reduces the contribution of large adenoma to PSA level, thus reflecting PSA level better and helping reduce overtreatment.

Removal of Small, Asymptomatic Kidney Stones and Incidence of Relapse.

BACKGROUND: The benefits of removing small (≤6 mm), asymptomatic kidney stones endoscopically is unknown. Current guidelines leave such decisions to the urologist and the patient. A prospective study involving older, nonendoscopic technology and some retrospective studies favor observation. However, published data indicate that about half of small renal stones left in place at the time that larger stones were removed caused other symptomatic events within 5 years after surgery. METHODS: We conducted a multicenter, randomized, controlled trial in which, during the endoscopic removal of ureteral or contralateral kidney stones, remaining small, asymptomatic stones were removed in 38 patients (treatment group) and were not removed in 35 patients (control group). The primary outcome was relapse as measured by future emergency department visits, surgeries, or growth of secondary stones. RESULTS: After a mean follow-up of 4.2 years, the treatment group had a longer time to relapse than the control group (P<0.001 by log-rank test). The restricted mean (±SE) time to relapse was 75% longer in the treatment group than in the control group (1631.6±72.8 days vs. 934.2±121.8 days). The risk of relapse was 82% lower in the treatment group than the control group (hazard ratio, 0.18; 95% confidence interval, 0.07 to 0.44), with 16% of patients in the treatment group having a relapse as compared with 63% of those in the control group. Treatment added a median of 25.6 minutes (interquartile range, 18.5 to 35.2) to the surgery time. Five patients in the treatment group and four in the control group had emergency department visits within 2 weeks after surgery. Eight patients in the treatment group and 10 in the control group reported passing kidney stones. CONCLUSIONS: The removal of small, asymptomatic kidney stones during surgery to remove ureteral or contralateral kidney stones resulted in a lower incidence of relapse than nonremoval and in a similar number of emergency department visits related to the surgery. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and the Veterans Affairs Puget Sound Health Care System; ClinicalTrials.gov number, NCT02210650.).

Functional and Morphological Changes Associated with Burst Wave Lithotripsy-Treated Pig Kidneys.

Purpose: Burst wave lithotripsy (BWL) is a new technique for comminution of urinary stones. This technology is noninvasive, has a low positive pressure magnitude, and is thought to produce minor amounts of renal injury. However, little is known about the functional changes related to BWL treatment. In this study, we sought to determine if clinical BWL exposure produces a functional or morphological change in the kidney. Materials and Methods: Twelve female pigs were prepared for renal clearance assessment and served as either sham time controls (6) or were treated with BWL (6). In the treated group, 1 kidney in each pig was exposed to 18,000 pulses at 10 pulses/s with 20 cycles/pulse. Pressure levels related to each pulse were 12 and -7 MPa. Inulin (glomerular filtration rate, GFR) and para-aminohippuric acid (effective renal plasma flow, eRPF) clearance was measured before and 1 hour after treatment. Lesion size analysis was performed to assess the volume of hemorrhagic tissue injury created by each treatment (% FRV). Results: No visible gross hematuria was observed in any of the collected urine samples of the treated kidneys. BWL exposure also did not lead to a change in GFR or eRPF after treatment, nor did it cause a measurable amount of hemorrhage in the tissue. Conclusion: Using the clinical treatment parameters employed in this study, BWL did not cause an acute change in renal function or a hemorrhagic lesion.

Aquablation therapy in large prostates (80-150 cc) for lower urinary tract symptoms due to benign prostatic hyperplasia: WATER II 3-year trial results.

Objective: The objective of this study is to determine if Aquablation therapy can maintain its effectiveness in treating men with lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) with large-volume (80-150 cc) prostates at 3 years. Subjects and Methods: One hundred one men with moderate-to-severe BPH symptoms and prostate volumes between 80 and 150 cc were enrolled in a prospective, nonrandomized, multicenter, international clinical trial in late 2017. Baseline, procedural, and follow-up parameters were recorded at baseline and scheduled postoperative visits. IPSS, Qmax, and treatment failure are reported at 3 years. Results: The mean prostate volume was 107 cc (range 80-150). Mean IPSS improved from 23.2 at baseline to 6.5 at 3 years (16.3-point improvement, p < 0.0001). Mean IPSS quality of life improved from 4.6 at baseline to 1.1 at 3 years (improvement of 3.4 points, p < 0.0001). Maximum urinary flow increased from 8.7 to 18.5 cc/s. At 3 year follow-up, 6% of treated patients needed BPH medication and an additional 3% required surgical retreatment for LUTS. Conclusions: Three-year follow-up demonstrates a sustained symptom reduction response along with low irreversible complications to Aquablation in men with LUTS due to BPH and prostates of 80-150 cc. Current treatment options available for men with prostates of this size have similar efficacy outcomes but are burdened with high rates of irreversible complications. There are now numerous clinical studies with Aquablation used in various prostates sizes, and it should be offered as an option to men with LUTS due to BPH.

Fragmentation of Stones by Burst Wave Lithotripsy in the First 19 Humans.

PURPOSE: We report stone comminution in the first 19 human subjects by burst wave lithotripsy (BWL), which is the transcutaneous application of focused, cyclic ultrasound pulses. MATERIALS AND METHODS: This was a prospective multi-institutional feasibility study recruiting subjects undergoing clinical ureteroscopy (URS) for at least 1 stone ≤12 mm as measured on computerized tomography. During the planned URS, either before or after ureteroscope insertion, BWL was administered with a handheld transducer, and any stone fragmentation and tissue injury were observed. Up to 3 stones per subject were targeted, each for a maximum of 10 minutes. The primary effectiveness outcome was the volume percent comminution of the stone into fragments ≤2 mm. The primary safety outcome was the independent, blinded visual scoring of tissue injury from the URS video. RESULTS: Overall, median stone comminution was 90% (IQR 20, 100) of stone volume with 21 of 23 (91%) stones fragmented. Complete fragmentation (all fragments ≤2 mm) within 10 minutes of BWL occurred in 9 of 23 stones (39%). Of the 6 least comminuted stones, likely causative factors for decreased effectiveness included stones that were larger than the BWL beamwidth, smaller than the BWL wavelength or the introduction of air bubbles from the ureteroscope. Mild reddening of the papilla and hematuria emanating from the papilla were observed ureteroscopically. CONCLUSIONS: The first study of BWL in human subjects resulted in a median of 90% comminution of the total stone volume into fragments ≤2 mm within 10 minutes of BWL exposure with only mild tissue injury.

A Randomized Trial Evaluating the Use of a Smart Water Bottle to Increase Fluid Intake in Stone Formers.

OBJECTIVE: The aim of this study is to evaluate if the use of a smart water bottle improves urine volume in stone forming patients. METHODS: Adults with nephrolithiasis and low urine volume (<1.5 L) documented on a 24-hour urinalysis (24 hr U) were randomized to receive either standard dietary recommendations to increase fluid intake (DR arm), or DR and a smart water bottle (HidrateSpark®; Hydrate Inc., Minneapolis, MN) that recorded fluid intake, synced to the user's smartphone, and provided reminders to drink (SB arm). Participants completed baseline surveys assessing barriers to hydration. They then repeated a 24 hr U and survey at 6 and 12 weeks, respectively. RESULTS: Eighty-five subjects (44 DR, 41 SB) were enrolled. The main baseline factor limiting fluid intake was not remembering to drink (60%). Follow-up 24 hr Us were available for 51 patients. The mean increase in volume was greater in the SB arm (1.37 L, 95% confidence interval -0.51 to 3.25) than the DR arm (0.79 L, 95% confidence interval -1.15 to 2.73) (P = .04). A smaller percentage of subjects in the SB arm reported not remembering to drink as the main factor limiting fluid intake in the follow-up questionnaire compared to baseline (45.4% vs. 68.4%, P < .05). This was not true for the DR arm (40.0% vs. 51.2%, P = .13). CONCLUSIONS: Difficulty remembering to drink is a barrier to achieving sufficient fluid intake in stone formers. The use of a smart bottle was associated with greater increases in 24 hr U volumes and less difficulty remembering to drink.

Improving Burst Wave Lithotripsy Effectiveness for Small Stones and Fragments by Increasing Frequency: Theoretical Modeling and Ex Vivo Study.

Introduction and Objective: In clinical trial NCT03873259, a 2.6-mm lower pole stone was treated transcutaneously and ex vivo with 390-kHz burst wave lithotripsy (BWL) for 40 minutes and failed to break. The stone was subsequently fragmented with 650-kHz BWL after a 4-minute exposure. This study investigated how to fragment small stones and why varying the BWL frequency may more effectively fragment stones to dust. Methods: A linear elastic theoretical model was used to calculate the stress created inside stones from shock wave lithotripsy (SWL) and different BWL frequencies mimicking the stone's size, shape, lamellar structure, and composition. To test model predictions about the impact of BWL frequency, matched pairs of stones (1-5 mm) were treated at (1) 390 kHz, (2) 830 kHz, and (3) 390 kHz followed by 830 kHz. The mass of fragments >1 and 2 mm was measured over 10 minutes of exposure. Results: The linear elastic model predicts that the maximum principal stress inside a stone increases to more than 5.5 times the pressure applied by the ultrasound wave as frequency is increased, regardless of the composition tested. The threshold frequency for stress amplification is proportionate to the wave speed divided by the stone diameter. Thus, smaller stones may be likely to fragment at a higher frequency, but not at a lower frequency below a limit. Unlike with SWL, this amplification in BWL occurs consistently with spherical and irregularly shaped stones. In water tank experiments, stones smaller than the threshold size broke fastest at high frequency (p = 0.0003), whereas larger stones broke equally well to submillimeter dust at high, low, or mixed frequencies. Conclusions: For small stones and fragments, increasing frequency of BWL may produce amplified stress in the stone causing the stone to break. Using the strategies outlined here, stones of all sizes may be turned to dust efficiently with BWL.

Label-free imaging of non-deparaffinized sections of the human kidney to determine tissue quality and signatures of disease.

Label-free fluorescence imaging of kidney sections can provide important morphological information, but its utility has not been tested in a histology processing workflow. We tested the feasibility of label-free imaging of paraffin-embedded sections without deparaffinization and its potential usefulness in generating actionable data. Kidney tissue specimens were obtained during percutaneous nephrolithotomy or via diagnostic needle biopsy. Unstained non-deparaffinized sections were imaged using widefield fluorescence microscopy to capture endogenous fluorescence. Some samples were also imaged with confocal microscopy and multiphoton excitation to collect second harmonic generation (SHG) signal to obtain high-quality autofluorescence images with optical sectioning. To adjudicate the label-free signal, the samples or corresponding contiguous sections were subsequently deparaffinized and stained with Lillie's allochrome. Label-free imaging allowed the recognition of various kidney structures and enabled morphological qualification for adequacy. SHG and confocal imaging yielded quantifiable high-quality images for tissue collagens and revealed specific patterns in glomeruli and various tubules. Disease specimens from patients with diabetic kidney disease and focal segmental glomerulosclerosis showed distinctive signatures compared to specimens from healthy controls with normal kidney function. Quantitative cytometry could also be performed when DAPI is added in situ before imaging. These results show that label-free imaging of non-deparaffinized sections provides useful information about tissue quality that could be beneficial to nephropathologists by maximizing the use of scarce kidney tissue. This approach also provides quantifiable features that could inform on the biology of health and disease.

Human jackstone arms show a protein-rich, X-ray lucent core, suggesting that proteins drive their rapid and linear growth.

Jackstone calculi, having arms that extend out from the body of the stone, were first described over a century ago, but this morphology of stones has been little studied. We examined 98 jackstones from 50 different patient specimens using micro-computed tomography (micro CT) and infrared (IR) spectroscopy. Micro CT showed that jackstone arms consisted of an X-ray lucent core within each arm. This X-ray lucent core frequently showed sporadic, thin layers of apatite arranged transversely to the axis of the arm. The shells of the jackstones were always composed of calcium oxalate (CaOx), and with the monohydrate form the majority or sole mineral. Study of layering in the shell regions by micro CT showed that growth lines extended from the body of the stone out onto jack arms and that the thickness of the shell covering of jack arms often thinned with distance from the stone body, suggesting that the arms grew at a faster radial rate than did the stone body. Histological cross-sections of decalcified jackstone arms showed the core to be more highly autofluorescent than was the CaOx shell, and immunohistochemistry showed the core to be enriched in Tamm-Horsfall protein. We hypothesize that the protein-rich core of a jack arm might preferentially bind more protein from the urine and resist deposition of CaOx, such that the arm grows in a linear manner and at a faster rate than the bulk of the stone. This hypothesis thus predicts an enrichment of certain urine proteins in the core of the jack arm, a theory that is testable by appropriate analysis.

Stone Morphology Distinguishes Two Pathways of Idiopathic Calcium Oxalate Stone Pathogenesis.

Introduction: About 1 in 11 Americans will experience a kidney stone, but underlying causes remain obscure. The objective of the present study was to separate idiopathic calcium oxalate stone formers by whether or not they showed positive evidence of forming a stone on Randall's plaque (RP). Materials and Methods: In patients undergoing either percutaneous or ureteroscopic procedures for kidney stone removal, all stone material was extracted and analyzed using micro-CT imaging to identify those attached to RP. Twenty-four-hour urine samples were collected weeks after the stone removal procedure and patients were off of medications that would affect urine composition. The endoscopic video was analyzed for papillary pathology (RP, pitting, plugging, dilated ducts, and loss of papillary shape) by an observer blinded to the data on stone type. The percent papillary area occupied by RP and ductal plugging was quantified using image analysis software. Results: Patients having even one stone on RP (N = 36) did not differ from non-RP patients (N = 37) in age, sex, BMI, or other clinical characteristics. Compared with the non-RP group, RP stone formers had more numerous, but smaller, stones, more abundant papillary RP formation, and fewer ductal plugs, both by quantitative measurement of surface area (on average, three times more plaque area, but only 41% as much plug area as in non-RP patients) and by semiquantitative visual grading. Serum and blood values did not differ between RP and non-RP stone formers by any measure. Conclusions: Growth of many small stones on plaque seems the pathogenetic scheme for the RP stone-forming phenotype, whereas the non-RP phenotype stone pathogenesis pathway is less obvious. Higher papillary plugging in non-RP patients suggests that plugs play a role in stone formation and that these patients have a greater degree of papillary damage. Underlying mechanisms that create these distinctive phenotypes are presently unknown.

Collagen fibrils and cell nuclei are entrapped within Randall's plaques but not in CaOx matrix overgrowth: A microscopic inquiry into Randall's plaque stone pathogenesis.

Calcium oxalate (CaOx) stones can grow attached to the renal papillary calcification known as Randall's plaque. Although stone growth on Randall's plaque is a common phenomenon, this mechanism of stone formation is still poorly understood. The objective of this study was to investigate the microenvironment of mature Randall's plaque, explore its molecular composition and differentiate plaque from CaOx overgrowth using multimodal imaging on demineralized stone sections. Fluorescence imaging showed consistent differences in autofluorescence patterns between Randall's plaque and calcium oxalate overgrowth regions. Second harmonic generation imaging established the presence of collagen only in regions of decalcified Randall's plaque but not in regions of CaOx overgrowth matrix. Surprisingly, in these stone sections we observed cell nuclei with preserved morphology within regions of mature Randall's plaque. These conserved cells had variable expression of vimentin and CD45. The presence of nuclei in mature plaque indicates that mineralization is not necessarily associated with cell death. The markers identified suggest that some of the entrapped cells may be undergoing dedifferentiation or could emanate from a mesenchymal or immune origin. We propose that entrapped cells may play an important role in the growth and maintenance of Randall's plaque. Further characterization of these cells and thorough analyses of the mineralized stone forming renal papilla will be fundamental in understanding the pathogenesis of Randall's plaque and CaOx stone formation.

Randomized Placebo-Controlled Trial of Reloxaliase in Enteric Hyperoxaluria.

Reloxaliase in Enteric HyperoxaluriaPatients with enteric hyperoxaluria received reloxaliase or placebo with food for 4 weeks. Urinary oxalate excretion decreased from 83.2 to 67.4 mg/24 hr during weeks 1 to 4 with reloxaliase compared with 84.2 to 78.1 mg/24 hr with placebo (P=0.004). Adverse events occurred for 69% of reloxaliase-treated patients versus 53% of those receiving placebo.

Using micro computed tomographic imaging for analyzing kidney stones.

Stone analysis is a critical part of the clinical characterization of urolithiasis. This article reviews the strengths and limitations of micro CT in the analysis of stones. Using micro CT alone in a series of 757 stone specimens, micro CT identified the 458 majority calcium oxalate specimens with a sensitivity of 99.6% and specificity of 95.3%. Micro CT alone was also successful in identifying majority apatite, brushite, uric acid, and struvite stones. For some minor minerals-such as apatite in calcium oxalate or calcium salts in uric acid stones-micro CT enables the detection of minute quantities well below 1%. The addition of a standard for calibrating X-ray attenuation values improves the ability of micro CT to identify common stone minerals. The three-dimensional nature of micro CT also allows for the visualization of surface features in stones, which is valuable for the study of stone formation.

Renal Protection Phenomenon Observed in a Porcine Model After Electromagnetic Lithotripsy Using a Treatment Pause.

Purpose: Pretreating the kidney with 100 low-energy shock waves (SWs) with a time pause before delivering a clinical dose of SWs (Dornier HM-3, 2000 SWs, 24 kV, and 120 SWs/min) has been shown to significantly reduce the size of the hemorrhagic lesion produced in that treated kidney, compared to a protocol without pretreatment. It has been assumed that a similar reduction in injury will occur with lithotripters other than the HM-3, but experiments to confirm this assumption are lacking. In this study, we sought to verify that the lesion protection phenomenon also occurs in a lithotripter using an electromagnetic shock source and dry-head coupling. Materials and Methods: Eleven female pigs were placed in a Dornier Compact S lithotripter where the left kidney of each animal was targeted for lithotripsy treatment. Some kidneys received 2500 SWs at power level (PL) = 5 (120 SWs/min) while some kidneys were pretreated with 100 SWs at PL = 1, with a 3-minute time pause, followed immediately by 2500 SWs at PL = 5 (120 SWs/min). Lesion size analysis was performed to assess the volume of hemorrhagic tissue injury created by each treatment regimen (% functional renal volume). Results: Lesion size fell by 85% (p = 0.01) in the 100 SW pretreatment group compared to the injury produced by a regimen without pretreatment. Conclusions: The results suggest that the treatment pause protection phenomenon occurs with a Dornier Compact S, a machine distinctly different from the Dornier HM-3. This result also suggests that this phenomenon may be observed generally in SW lithotripters.

Demineralization and sectioning of human kidney stones: A molecular investigation revealing the spatial heterogeneity of the stone matrix.

The molecular mechanisms by which kidney stones grow are largely unknown. Organic molecules from the urine combine with mineral crystals to form stones, but analysis of the stone matrix has revealed over a thousand different proteins, with no clues as to which are important for stone growth. Molecules that are present in every layer of a stone would be candidates for having an essential function, and thus the analysis of the stone matrix at a microscopic level is necessary. For this purpose, kidney stones were demineralized, sectioned, stained, and imaged by microscopy, using micro CT for precise orientation. Histological staining demonstrated heterogeneity in the density of adjacent layers within stones. Additional results also showed brilliant and unique autofluorescence patterns in decalcified nephroliths, indicating heterogeneous organic composition in adjacent layers. Regions of calcium oxalate (CaOx) stones were dissected using laser microdissection (LMD) for protein analysis. LMD of broad regions of demineralized CaOx stone sections yielded the same proteins as those found in different specimens of pulverized CaOx stones. These innovative methodologies will allow spatial mapping of protein composition within the heterogeneous stone matrix. Proteins that consistently coincide spatially with mineral deposition would be candidates for molecules essential for stone growth. This kind of analysis will be required to assess which of the thousand proteins in the stone matrix may be fundamental for stone growth.