Scanning Electron Microscopy and Energy Dispersive Spectroscopy Reveal Amorphous Apatitic Cores in Ductal plugs bearing Calcium Oxalate Stone Overgrowths

Scanning Electron Microscopy and Energy Dispersive Spectroscopy Reveal Amorphous Apatitic Cores in Ductal plugs bearing Calcium Oxalate Stone Overgrowths | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Scanning Electron Microscopy and Energy Dispersive Spectroscopy Reveal Amorphous Apatitic Cores in Ductal plugs bearing Calcium Oxalate Stone Overgrowths Victor Hugo Canela, Antonia Costa-Bauzá, Felix Grases, Sharon B. Bledsoe, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6596012/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Kidney stone prevalence is increasing, with high recurrence rates and associations with comorbidities such as metabolic syndrome and chronic kidney disease. The most common stones in nephrolithiasis are calcium oxalate (CaOx) stones, either monohydrate (COM), dihydrate (COD) or both, which can form as overgrowths on intratubular plugs of mineral that protrude from the terminal collecting ducts of the renal papilla. Although this is a recognized mode of stone formation, the underlying mechanisms remain poorly understood. In this study, we analyzed 11 ductal plug stones from four patients using stereoscopic microscopy, micro computed tomography (micro CT), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Detailed surface imaging revealed COM and COD overgrowths on apatite plugs, which often showed various crystallized phases and typically exceeded normal ductal luminal diameters (mean = 575 ± 440 µm). Sectioned plugs revealed amorphous nonconcentric apatitic cores and radially expanding mineral layers suggestive of mineral accretion. These findings support a model in which ductal plugs originate within the collecting duct lumen and grow asymmetrically beyond the duct wall, potentially contributing to epithelial injury and crystal retention. Further investigations using advanced imaging, molecular, and omics approaches are needed to elucidate the pathophysiology of plug formation and stone retention at the renal papilla. Biological sciences/Physiology/Kidney Biological sciences/Physiology/Kidney/Nephrons Health sciences/Urology/Urological manifestations Biological sciences/Physiology Health sciences/Nephrology Health sciences/Pathogenesis Health sciences/Urology Physical sciences/Chemistry Health sciences/Anatomy Health sciences/Anatomy/Kidney Health sciences/Anatomy/Urinary tract Stones Ductal plugs apatite nephron kidney disease inflammation calculi Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The incidence and prevalence of kidney stone disease have increased in recent decades (Scales CD, 2012; Thongprayoon C et al., 2020 ; Lang J, 2021). Calcium oxalate (CaOx) stones, either dihydrate (COD) or monohydrate (COM), are the most common types of kidney stones, responsible for significant morbidity worldwide (Worcester & Coe, 2010 ). Kidney stones tend to recur at high rates, and are also associated with various comorbidities, including chronic kidney disease, metabolic syndrome, hypertension, reduced bone mineral density and coronary artery disease (Vaughan LE, et al, 2018; Beara-Lasic L & Goldfarb DS, 2019). CaOx stones are retained and may grow at the renal papillary tip (Daudon M and Letavernier E., 2015; Khan SR et al. 2021 ). One widely accepted mechanism for CaOx stone retention in the renal calyx involves the papillary lesions known as Randall’s plaques. These plaques are subepithelial calcium phosphate (CaP) deposits in the interstitium of the papilla tip, initiating in the basement membranes of thin loops of Henle, collecting ducts or vasa recta (Evan AP 2003). In this mechanism of CaOx retention, it is hypothesized that the subepithelial layer becomes damaged or injured by unknown processes, allowing calyceal urine to interact with the plaque, facilitating the precipitation of CaOx crystals onto the CaP apatite surface. Another reported mechanism of retention involves CaOx stones forming as overgrowths on mineral plugs that emerge from the terminal collecting duct at the renal papilla (Bird VY & Khan SR 2017; Grases F & Söhnel O, 2017 ; Williams JC 2018; Williams JC 2024). The distal end of the plug extends into the calyx, where urine interacts with the apatite deposits, creating a nidus for early CaOx stone growth. Continuous exposure to calyceal urine allows for more CaOx crystal deposition and therefore progressive stone formation, which may eventually become symptomatic (Williams JC 2018). Endoscopic studies have described papillary plugging as yellow plaque deposits at the papillary tip (Coe F 2010; Borofsky MS, 2016; Williams JC 2018) (Williams JC 2024). These ductal plugs are composed of CaP apatite crystals within the lumens of inner medullary collecting ducts (IMCD) and ducts of Bellini (BD) (Evan AP 2007; Grases F et al 2016 ; Williams JC 2018). Patients with plugging often show dilation of the BD but with varying severity (Williams JC 2018). Several theories, including the fixed particle theory, have been proposed to explain stone retention, but the exact mechanisms pertaining to stone formation remain unclear (Finlayson B and Reid F 1978 ; Kok DJ & Khan S, 1994; Grases F et al 2016 ). The present study aims to characterize the morphology of CaOx stones grown on ductal plugs by leveraging an array of imaging techniques including stereoscopic microscopy, micro-computed tomography (micro CT), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). By applying these imaging and analytical techniques, we aim to describe the surface and internal microstructure of CaOx ductal plug stones. These techniques provide an avenue to identify key morphological patterns pertaining to their formation and growth. The findings highlighted here support previous findings and provide novel insights into the mechanisms of plug formation and the pathophysiology of kidney stone disease. Materials and Methods Ductal plug stones were collected from patients during surgery (percutaneous nephrolithotomy, stone removal by ureteroscopy or both; see Supplemental Table 1 ). A standard protocol for the study of kidney stone patients approved by the Indiana University (IU) Institutional Review Board (IRB) committee (IRB protocol #1010002261) was followed throughout the present study as previously described (Canela et al. 2021 ). The SEM and EDS analyses presented in this study were conducted according to the protocol established by the Laboratory of Renal Lithiasis Research and Biobank of Renal Calculi (BICUIB) of the University of the Balearic Islands. Renal papillary samples taken during surgery were fixed in 5% formaldehyde and dehydrated through graded ethanol concentrations and cleared in xylene as described in previous works (Evan AP, 2003; Evan AP 2014; Evan AP 2018). Tissues were embedded in paraffin, sections were cut at 4–6µm and stained with hematoxylin and eosin (H&E), periodic acid-Schiff hematoxylin (PASH), and picrosirius red (PSR). Mineral deposits were identified using the Yasue method stain. Briefly, the Yasue method uses 5% aqueous silver nitrate and rubeanic acid to produce a dark brown to black histochemical staining of calcium mineral deposits (Yasue, 1969 ; Evan, AP 2018; Canela, VH et al. 2020). Dry ductal plug stones were photographed and scanned using micro-computed tomographic (micro CT) imaging to verify and determine the presence of a calcium phosphate plug with an anchor of CaOx stone mineral. The stones were scanned using a Skyscan 1172 micro-CT system (Bruker-MicroCT, Kontich, Belgium) at 60kVp, 0.5-mm Al filter, for a final voxel size of 4–8 µm (Williams 2021). To estimate the cross-sectional diameter (width) of the ductal plug stones, ten measurements were taken across each stone using virtual slices from micro CT reconstructions. The measurements were obtained at various positions along the plug’s length to account for structural variation and were averaged to obtain a representative width per plug ( Supplemental table 2 ). Ductal plug stones were carefully sectioned, imaged and captured using stereoscopic microscopy and a scanning electron microscope (SEM; TM4000 Plus II, Hitachi, Tokyo, Japan) with microanalysis by X-ray dispersive energy (RX, Quantax 75 EDS microanalyzer, Bruker, Berlin, Germany) (Grases F and Costa-Bauzá, A 2021; Costa-Bauzá, A et al. 2023 ). Results Stones were analyzed by stereoscopic microscopy and micro CT to reveal the characteristic morphology of the ductal plug stones, as shown in Figs. 1 A-C, 2 D-E, 3 A and 4 A. Ductal plugs typically show a relatively thick width (575 ± 440 µm) and extended protrusions. The plugs were consistently composed of apatite, often capped at one end with a CaOx overgrowth composed of COM, COD or a combination of both (e.g., Fig. 1 D and Figs. 3 B-D). Mineral composition was confirmed by infrared spectroscopy (data not shown), micro CT, SEM and microanalysis by EDS ( Supplemental Figures S1 -S5 ). SEM analysis revealed distinct zones within the apatite plug, ranging from loosely packed, amorphous-like regions (Phase 1) to more densely compact structures (Phases 2 and 3). These findings support a model of CaP maturation radiating outward from a nonconcentric core. In sectioned ductal plugs, this internal nonconcentric region (e.g., Fig. 4 B), composed of loosely aggregated, amorphous CaP apatite, was consistently observed (Figs. 4 and 5 ). This region was surrounded by alternating layers of low and high compactness (Phases 1–3). The asymmetric morphology of the plug suggests a directional growth, potentially indicating that one side may have expanded more rapidly than the other (Fig. 4 C-D and Fig. 5 A- 5 B). Figure 2 A shows an endoscopic image from patient 3 ( Supplemental Table 1 ) with papillary tissue loss and visible mineral plugs (yellow arrowheads). Histological sections stained with PSR (Fig. 2 B), and Yasue stain (Fig. 2 C) demonstrate fibrosis, inflammation, and mineral retention consistent with ductal plugging. The PSR stained section (Fig. 2 B) highlights a mineral plug (black asterisk) in a longitudinal IMCD, and surrounding areas with intense red staining (yellow arrowheads), indicating increased collagen density likely associated with inflammation and fibrosis. In Fig. 2 C, Yasue stain of a consecutive section confirms the presence of mineral plug staining (yellow arrow) with dark brown and black staining in a collecting duct in longitudinal section. The Yasue-stained section also shows extensive tissue loss (acellular zones in the interstitium and tubules) consistent with previous reports of significant tissue injury in patients with plugged papillae (Evan, AP 2005; Williams JC et al. 2024). Discussion Our results demonstrate that stereoscopic microscopy, micro CT, SEM, and EDS together provide a robust method for analyses of ductal plug stones. Using this multimodal approach, we confirm that stones obstructing the papillary ducts are predominantly composed of CaP apatite. The distal end portion of the plug exposed to calyceal urine acts as a nidus for CaOx crystal overgrowth (Fig. 1 , Fig. 2 D- 2 E, Fig. 3 , Fig. 4 A). These findings support previous reports showing that CaOx stones, either COD or COM, can form as overgrowths on mineral plugs emerging from the terminal collecting ducts (Bird VY & Khan SR 2017; Grases F & Söhnel O, 2017 ; Williams JC 2018; Williams JC 2024). In sectioned plugs, we identified nonconcentric, loosely packed apatitic core structures. These structures suggest dynamic mineral accretion and may provide clues as to how CaP crystals expand outward, disrupt tubular epithelial integrity, obstruct the papillary ducts, and potentially damage surrounding nephrons structures during their development. Recent comparative studies of CaOx stone phenotypes show that ductal plugging is associated with more papillary tissue loss than papillae with extensive Randall’s plaque (Williams, JC. et al . 2022; Williams et al. 2024 ). Our results are consistent with these findings; radial plug expansion may drive tubular injury, epithelial detachment, and fibrosis, contributing to tubulointerstitial damage (Williams et al. 2024 ). Ductal plugs have been described during endoscopic stone removal procedures as yellowish plaque deposits obstructing dilated BD (Coe F 2010; Borofsky MS, 2016; Grases F et al 2016 ; Williams JC 2018; Williams JC 2024). We observed similar morphologies, with most plugs exceeding the expected diameter of terminal collecting ducts (575 ± 440 µm, Supplemental Table 2 ), possibly indicating chronic tubular obstruction and tubulointerstitial damage. The structural changes in the papilla are corroborated by histopathologic studies in stone formers with distal renal tubular acidosis (dRTA) and in patient with prior bariatric surgery. In these patients, CaP deposits in IMCD and BD are associated with epithelial injury and progressive tubular damage, cell loss and fibrosis (Evan, AP 2005; Evan, AP 2007; Coe, FL et al. 2010 ). Our case study (Patient 3) presents similar patterns with visible papillary tissue loss (Fig. 2 A), fibrosis and plug associated inflammation (Fig. 2 B- 2 C). Figure 2 shows a representative case: a 44-year-old female patient (Patient 3, Supplemental Table 1 ) without systemic disease who underwent percutaneous nephrolithotomy. Figure 2 A reveals classic signs of ductal plugging and papillary tissue loss (Fig. 2 A). Histological sections confirmed interstitial fibrosis and localized inflammation by PSR (Fig. 2 B) and confirmed tubular obstruction in IMCD by Yasue stain (Fig. 2 C ) . SEM, micro CT, and EDS analyses of the extracted ductal plug stone (Fig. 2 D) confirmed CaP composition with COM overgrowth studded with traces of COD crystals (Fig. 2 E). These structural and mineral features were consistent across our specimens (Fig. 1 B, Fig. 3 A, Fig. 4 A and Supplemental Figures S1 -S5 ). A key question in ductal plug formation is how crystals are retained within the tubular lumen. Finlayson and Reid first proposed the fixed particle theory, which postulates that mineral retention in a tubule occurs at an attachment site on the luminal surface of the epithelium. They contrasted this with a ‘free particle’ concept, in which crystals or crystal aggregates could grow rapidly enough within the renal tubule that the resulting mass would become lodged in the tubule, even obstructing flow. Finlayson and Reid (and later, Grases and Söhnel ) concluded that flow was too fast for mineral to grow to a size that could obstruct the lumen (Grases F & Söhel O, 2017). Kok and Khan concluded otherwise, calculating that mineral agglomerations could grow fast enough to become lodged in place within a terminal collecting duct. The ductal plugs we studied might have initiated by such rapid growth within the tubular fluid, but we note that the calculations of Kok and Khan are based on calcium oxalate crystal growth, while the plugs we have seen are all composed of apatite (Kok DJ & Khan S, 1994). Retention of apatite crystals within a tubule may be facilitated by underlying tubular and interstitial pathology, such as tubulointerstitial nephritis (TIN) (Joyce E, et al. 2016). TIN includes a group of inflammatory diseases, often triggered by drugs, metabolic disorders or immune mediated injury. In our case study (Patient 3), histology showed epithelial and basement membrane loss in affected ducts, along with surrounding fibrosis and inflammation (Fig. 2 B and Fig. 2 C). The features observed in our case study resemble a TIN-like inflammatory state or injury. We propose that tubular damage, along with an elevated urinary pH and cellular debris, may provide an environment promoting cast and apatite formation, resulting in ductal obstruction (Costa-Bauzá et al. 2023 ). Inflammatory pathways, triggered by necrotic epithelial cells and hypoxia-induced damage, may further enhance tubulointerstitial injury, fibrosis, and progressive loss of nephron function (Degterev A, 2005; Akcay, A. et al. 2009 ; Bonventre JV and Yang L., 2011; Sarhan M et al. 2018 ; Eddy, AA 2019). Advanced multiplexed imaging of a CaOx stone former with ductal plugs has revealed epithelial disruption and remarkable inflammatory infiltration. Williams et al. reported loss of aquaporin-2 and Na⁺/K⁺-ATPase expression in plug-affected ducts and identified inflammatory cells expressing MPO, CD68, CD201 and CD3, suggesting neutrophil, macrophage, and T cell involvement. Intraepithelial CD3⁺ T cells in neighboring collecting ducts, suggest tubulitis (an immune mediated injury) and an inflammatory process reminiscent of TIN injury at the papilla (Evan AP, et al. 2005 ; Williams, JC et al. 2024; Williams, JC and El-Achkar, TM, 2025). Given these findings, the authors of the study raised the possibility that ductal plugging may contribute to accelerated decline in renal function (Williams, JC et al. 2024). Our results support this hypothesis. If plugs grow large enough to obstruct only a small number of papillary ducts, and expand radially to cause tubulointerstitial injury, then significant nephron damage may ensue. Each terminal collecting drains an estimated 2750 nephrons, and obstruction of just four ducts could impact over 10,000 nephrons, potentially triggering localized tubulointerstitial stress, inflammation and injury (Kriz W and Kaissling B, 2008 ; Charlton, JR 2021; Williams, JC et al. 2024). The cumulative effect of this kind of obstruction over time could be clinically significant, considering that some idiopathic CaOx stone formers present with multiple ductal plugs in every papilla (Williams, JC et al. 2024). Conclusions Our results suggest that ductal plugs composed of CaP apatite may begin as loosely packed deposits within the IMCD or BD but progressively expand by breaching the tubular epithelium and extending asymmetrically into the surrounding interstitial space. The presence of a nonconcentric, loosely packed apatitic core in sectioned plugs supports a model of asymmetric growth and may indicate significant tubular disruption and interstitial injury. These findings hint at the potential for ductal plugs to inflict localized nephron damage and contribute to papillary pathology. Further studies validating our results and incorporating high resolution imaging, molecular profiling and spatial omics approaches are necessary to uncover the cellular and molecular mechanisms driving plug formation and growth in the renal papilla. Declarations Conflicts of interest: None Ethics approval statement: All patient specimens were obtained via Internal Review Board approved patient consent (Indiana University IRB protocol #1010002261). Author Contribution V.H.C. wrote the main manuscript text and prepared all figures. V.H.C, A.C.B., F.G. and J.C.W. conceptualized, visualized, designed and conducted formal analysis of the data. S.B. B. assisted in methodology and processing of tissues, patient data and project administration. J.E.L. provided patient data, tissues and samples for analysis. All authors reviewed and edited the manuscript. Acknowledgement The authors thank Drs. Marcos Oliveira and Robert Soltis from the College of Pharmacy and Health Sciences at Butler University for encouragement and support in the development of this work. We express our special gratitude to the Renal Lithiasis and Pathological Calcification Group at the University of Balearic Islands for their support during the origins and development of this work. Data Availability The authors confirm that the summary data supporting the findings of this study are available within the article and its supplementary materials. All underlying data will be made available to anyone upon request. References Scales, C. D. Jr, Smith, A. C., Hanley, J. M. & Saigal, C. S. Urologic Diseases in America Project. Prevalence of kidney stones in the United States. Eur. Urol. 62 (1), 160–165. 10.1016/j.eururo.2012.03.052 (2012). Thongprayoon, C., Krambeck, A. E. & Rule, A. D. Determining the true burden of kidney stone disease. Nat. Rev. Nephrol. 16 (12), 736–746. 10.1038/s41581-020-0320-7 (2020). Lang, J., Narendrula, A., El-Zawahry, A., Sindhwani, P. & Ekwenna, O. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6596012","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":470755382,"identity":"a2bc0443-2fd0-4fe9-852f-babbbbdacdff","order_by":0,"name":"Victor Hugo Canela","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYNCCAgY5AwYGxgNgzgGitBgwGBswMDOQpiVxA9FaDI4ffvbgg4Fd+naJ/AMHPu5hkOO7kUBAy5k0c8MZBsm5O2ckMxyc8YzBWJKQFskZDGbSPAbMuRtuJDMc5jkAdCFhLezfpP8Y1KcbgLT8OcBQT1ALvwSPmTSDweEEsBag14EMQlp4csokewyOG+7seWxwsOeAhOHMMw/wa2FjP75N4kdFtbw5e+LDBz8O2MjzHSdgCzqQIE35KBgFo2AUjALsAADEbEZa1hsvGQAAAABJRU5ErkJggg==","orcid":"","institution":"Butler University","correspondingAuthor":true,"prefix":"","firstName":"Victor","middleName":"Hugo","lastName":"Canela","suffix":""},{"id":470755383,"identity":"9f037281-dcb7-4298-84ef-a53b002780e5","order_by":1,"name":"Antonia Costa-Bauzá","email":"","orcid":"","institution":"University of the Balearic Islands","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"","lastName":"Costa-Bauzá","suffix":""},{"id":470755384,"identity":"d21fc2b9-e340-4841-ba73-005d4f99625a","order_by":2,"name":"Felix Grases","email":"","orcid":"","institution":"University of the Balearic Islands","correspondingAuthor":false,"prefix":"","firstName":"Felix","middleName":"","lastName":"Grases","suffix":""},{"id":470755385,"identity":"8b82b2fa-c99c-4d5b-9b1d-340aa087cb1f","order_by":3,"name":"Sharon B. Bledsoe","email":"","orcid":"","institution":"Indiana University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sharon","middleName":"B.","lastName":"Bledsoe","suffix":""},{"id":470755386,"identity":"81dc85ec-a71d-4af6-9e2f-666715402913","order_by":4,"name":"James E. Lingeman","email":"","orcid":"","institution":"Indiana University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"E.","lastName":"Lingeman","suffix":""},{"id":470755387,"identity":"b0d960b8-e61e-4dd3-a0b6-ba23c648e5ec","order_by":5,"name":"James C. Williams","email":"","orcid":"","institution":"Indiana University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"C.","lastName":"Williams","suffix":""}],"badges":[],"createdAt":"2025-05-05 16:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6596012/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6596012/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84810242,"identity":"255082ba-c9d6-4be4-8ee1-166d3b319fd6","added_by":"auto","created_at":"2025-06-17 14:47:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2072986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative ductal plug stone with an overgrowth containing calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD); Patient 4, stone #4. (A) \u003c/strong\u003eStereoscopic microscope image of a representative ductal plug stone, showing an apatite plug appearing white or ivory in color. The brown region corresponds to the COM overgrowth, with a small visible area of COD crystals. (\u003cstrong\u003eB) \u003c/strong\u003eMicro CT reslice of the same stone from (A), revealing the ductal plug as calcium phosphate (CaP) apatite and the overgrowth mostly composed of COM. (\u003cstrong\u003eC) \u003c/strong\u003eScanning electron microscopy (SEM) image showing the detailed morphology of apatite, COM, and COD crystals. (\u003cstrong\u003eD) \u003c/strong\u003eMagnified view of (\u003cstrong\u003eC) \u003c/strong\u003econfirming the presence of these crystals. The white asterisks (*) indicate COD crystals, while the yellow arrowhead marks an interface region where COM crystal plates anchor to the apatite plug. Additional confirmation of crystal composition was obtained via energy-dispersive X-ray spectroscopy (EDS), as shown in the Supplementary Data.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/a410763ff5b7b6fb162d0eac.png"},{"id":84810246,"identity":"7e40d398-75a7-45a6-92c3-29e5284ba987","added_by":"auto","created_at":"2025-06-17 14:47:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":648448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative imaging of a plugged renal papilla from patient 3, shown by endoscopy, histopathology, and a calcium oxalate (CaOx) ductal stone (stone #1 from patient 3). (A) \u003c/strong\u003eEndoscopic view captured during surgery, showing a representative renal papilla from patient 3. The papilla shows significant tissue loss and multiple mineral plugs (yellow arrowheads) with a yellowish appearance. A papilla-anchored CaOx stone (ductal) is indicated by the yellow arrow. \u003cstrong\u003e(B and C) \u003c/strong\u003eHistological sections of a renal papilla biopsy from patient 3, stained with picrosirius red (PSR)(B) and Yasue stain (C). (\u003cstrong\u003eB\u003c/strong\u003e) The PSR stained section highlights a mineral plug (black asterisk) and surrounding areas with intense red staining (yellow arrowheads), indicating increased collagen density associated with inflammation and fibrosis. \u003cstrong\u003e(C) \u003c/strong\u003eYasue\u003cstrong\u003e \u003c/strong\u003estain of a consecutive section confirms the presence of mineral plug staining (yellow arrow) with dark brown and black staining in a collecting duct in longitudinal section. The section also shows extensive tissue loss. \u003cstrong\u003e(D) \u003c/strong\u003eStereoscopic microscope image of the ductal plug stone from (A), showing an apatite plug with a brown overgrowth region composed of COM studded with traces of COD crystals.\u003cstrong\u003e (E) \u003c/strong\u003eMicro CT reslice of the same stone from (D), revealing the ductal plug as calcium phosphate (CaP) apatite with an overgrowth predominantly composed of COM.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/48eb45fb0409bbb1e193c5f3.jpeg"},{"id":84810245,"identity":"08ebb4da-81ef-4657-85d9-ab0bef5489ab","added_by":"auto","created_at":"2025-06-17 14:47:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":855116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM) analysis of ducal stone #1 from patient 3 (corresponding to Figure 2D).\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e SEM image of stone 1 from patient 3, revealing detail of its overall structure. \u003cstrong\u003e(B)\u003c/strong\u003e Magnification of region b from (A) reveals COM crystal plates (white arrowheads) composing the stone. White arrows indicate areas where COD crystals are undergoing dissolution, losing water as they transition to COM. A central region of this inset contains an area of calcium phosphate crystals, suggesting an elevated urinary pH. \u003cstrong\u003e(C and D)\u003c/strong\u003e Higher magnification images highlighting the interface between calcium apatite and COM (white arrowheads) from (A). These panels show COM crystals anchoring onto the crystals of apatite, presumably marking the initial site of COM stone growth.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/05801367384b5daf110f230b.jpeg"},{"id":84810250,"identity":"52f4389c-7e04-4876-a11f-bca510c936e2","added_by":"auto","created_at":"2025-06-17 14:47:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2386120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM analysis of a sectioned ductal plug stone from patient 1, stone #4 (A) \u003c/strong\u003eA ductal stone with an overgrowth portion of calcium oxalate monohydrate (COM), sectioned at the apatite-rich ductal plug region. \u003cstrong\u003e(B)\u003c/strong\u003e Top view of the sectioned portion of the ductal plug. The dagger (†) marks the corresponding portion of the plug from panel (A) as a fiduciary marker. Note the presence of a nonconcentric white region within the plug. \u003cstrong\u003e(C)\u003c/strong\u003e SEM image showing the internal region of the sectioned ductal plug, highlighting the amorphic nonconcentric white region within the plug. \u003cstrong\u003e(D) \u003c/strong\u003eHigher magnification of the plug from (C) reveals a portion adjacent to the nonconcentric region, where apatite crystals exhibit loosely packed, amorphous-like regions (Phase 1) to more densely compact structures (Phases 2-3). This morphology suggests outward plug growth relative to the amorphic nonconcentric region.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/01064f5e05d46fa834dadbf5.png"},{"id":84811596,"identity":"85998c74-5d43-4ada-87fb-f020b7fac97b","added_by":"auto","created_at":"2025-06-17 14:55:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2133076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM analysis of a sectioned ductal plug stone from patient 4, stone #2. (A)\u003c/strong\u003e SEM image showing the internal region of a sectioned ductal plug, including a nonconcentric region with loosely packed amorphic apatite crystals (E). \u003cstrong\u003e(B)\u003c/strong\u003e Higher magnification of region b from panel (A), showing the outward-growing patterns of the apatite crystals observed in Figure 4, panel D. \u003cstrong\u003e(C and D)\u003c/strong\u003e Higher magnification of region c from panel (A), showing convergent crystal growth (D, black arrows) in an area of apatite agglomeration within the nonconcentric region of the plug. \u003cstrong\u003e(E) \u003c/strong\u003eHigher magnification of a region of the nonconcentric area, showing loosely packed amorphic apatite structures.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/88eb0e5af18e1cc6f3ecec4b.png"},{"id":88948237,"identity":"7f35477e-cd67-4075-9a44-1ef50bcf423f","added_by":"auto","created_at":"2025-08-13 05:33:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9937782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/cf8df373-a407-4240-9ec2-df063a6a8581.pdf"},{"id":84810266,"identity":"8afa290c-fde0-4602-b04b-ae7a01577df2","added_by":"auto","created_at":"2025-06-17 14:47:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":11005862,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialSR.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6596012/v1/fd687afbfe0dc4f896b85913.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Scanning Electron Microscopy and Energy Dispersive Spectroscopy Reveal Amorphous Apatitic Cores in Ductal plugs bearing Calcium Oxalate Stone Overgrowths ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe incidence and prevalence of kidney stone disease have increased in recent decades (Scales CD, 2012; Thongprayoon C et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lang J, 2021). Calcium oxalate (CaOx) stones, either dihydrate (COD) or monohydrate (COM), are the most common types of kidney stones, responsible for significant morbidity worldwide (Worcester \u0026amp; Coe, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Kidney stones tend to recur at high rates, and are also associated with various comorbidities, including chronic kidney disease, metabolic syndrome, hypertension, reduced bone mineral density and coronary artery disease (Vaughan LE, et al, 2018; Beara-Lasic L \u0026amp; Goldfarb DS, 2019).\u003c/p\u003e \u003cp\u003eCaOx stones are retained and may grow at the renal papillary tip (Daudon M and Letavernier E., 2015; Khan SR et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One widely accepted mechanism for CaOx stone retention in the renal calyx involves the papillary lesions known as Randall\u0026rsquo;s plaques. These plaques are subepithelial calcium phosphate (CaP) deposits in the interstitium of the papilla tip, initiating in the basement membranes of thin loops of Henle, collecting ducts or vasa recta (Evan AP 2003). In this mechanism of CaOx retention, it is hypothesized that the subepithelial layer becomes damaged or injured by unknown processes, allowing calyceal urine to interact with the plaque, facilitating the precipitation of CaOx crystals onto the CaP apatite surface.\u003c/p\u003e \u003cp\u003eAnother reported mechanism of retention involves CaOx stones forming as overgrowths on mineral plugs that emerge from the terminal collecting duct at the renal papilla (Bird VY \u0026amp; Khan SR 2017; Grases F \u0026amp; S\u0026ouml;hnel O, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Williams JC 2018; Williams JC 2024). The distal end of the plug extends into the calyx, where urine interacts with the apatite deposits, creating a nidus for early CaOx stone growth. Continuous exposure to calyceal urine allows for more CaOx crystal deposition and therefore progressive stone formation, which may eventually become symptomatic (Williams JC 2018).\u003c/p\u003e \u003cp\u003eEndoscopic studies have described papillary plugging as yellow plaque deposits at the papillary tip (Coe F 2010; Borofsky MS, 2016; Williams JC 2018) (Williams JC 2024). These ductal plugs are composed of CaP apatite crystals within the lumens of inner medullary collecting ducts (IMCD) and ducts of Bellini (BD) (Evan AP 2007; Grases F et al \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Williams JC 2018). Patients with plugging often show dilation of the BD but with varying severity (Williams JC 2018). Several theories, including the fixed particle theory, have been proposed to explain stone retention, but the exact mechanisms pertaining to stone formation remain unclear (Finlayson B and Reid F \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Kok DJ \u0026amp; Khan S, 1994; Grases F et al \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study aims to characterize the morphology of CaOx stones grown on ductal plugs by leveraging an array of imaging techniques including stereoscopic microscopy, micro-computed tomography (micro CT), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). By applying these imaging and analytical techniques, we aim to describe the surface and internal microstructure of CaOx ductal plug stones. These techniques provide an avenue to identify key morphological patterns pertaining to their formation and growth. The findings highlighted here support previous findings and provide novel insights into the mechanisms of plug formation and the pathophysiology of kidney stone disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eDuctal plug stones were collected from patients during surgery (percutaneous nephrolithotomy, stone removal by ureteroscopy or both; see \u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e). A standard protocol for the study of kidney stone patients approved by the Indiana University (IU) Institutional Review Board (IRB) committee (IRB protocol #1010002261) was followed throughout the present study as previously described (Canela et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The SEM and EDS analyses presented in this study were conducted according to the protocol established by the Laboratory of Renal Lithiasis Research and Biobank of Renal Calculi (BICUIB) of the University of the Balearic Islands.\u003c/p\u003e \u003cp\u003eRenal papillary samples taken during surgery were fixed in 5% formaldehyde and dehydrated through graded ethanol concentrations and cleared in xylene as described in previous works (Evan AP, 2003; Evan AP 2014; Evan AP 2018). Tissues were embedded in paraffin, sections were cut at 4\u0026ndash;6\u0026micro;m and stained with hematoxylin and eosin (H\u0026amp;E), periodic acid-Schiff hematoxylin (PASH), and picrosirius red (PSR). Mineral deposits were identified using the Yasue method stain. Briefly, the Yasue method uses 5% aqueous silver nitrate and rubeanic acid to produce a dark brown to black histochemical staining of calcium mineral deposits (Yasue, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Evan, AP 2018; Canela, VH \u003cem\u003eet al.\u003c/em\u003e 2020).\u003c/p\u003e \u003cp\u003eDry ductal plug stones were photographed and scanned using micro-computed tomographic (micro CT) imaging to verify and determine the presence of a calcium phosphate plug with an anchor of CaOx stone mineral. The stones were scanned using a Skyscan 1172 micro-CT system (Bruker-MicroCT, Kontich, Belgium) at 60kVp, 0.5-mm Al filter, for a final voxel size of 4\u0026ndash;8 \u0026micro;m (Williams 2021).\u003c/p\u003e \u003cp\u003eTo estimate the cross-sectional diameter (width) of the ductal plug stones, ten measurements were taken across each stone using virtual slices from micro CT reconstructions. The measurements were obtained at various positions along the plug\u0026rsquo;s length to account for structural variation and were averaged to obtain a representative width per plug (\u003cb\u003eSupplemental table 2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eDuctal plug stones were carefully sectioned, imaged and captured using stereoscopic microscopy and a scanning electron microscope (SEM; TM4000 Plus II, Hitachi, Tokyo, Japan) with microanalysis by X-ray dispersive energy (RX, Quantax 75 EDS microanalyzer, Bruker, Berlin, Germany) (Grases F and Costa-Bauz\u0026aacute;, A 2021; Costa-Bauz\u0026aacute;, A et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eStones were analyzed by stereoscopic microscopy and micro CT to reveal the characteristic morphology of the ductal plug stones, as shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Ductal plugs typically show a relatively thick width (575\u0026thinsp;\u0026plusmn;\u0026thinsp;440 \u0026micro;m) and extended protrusions. The plugs were consistently composed of apatite, often capped at one end with a CaOx overgrowth composed of COM, COD or a combination of both (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D). Mineral composition was confirmed by infrared spectroscopy (data not shown), micro CT, SEM and microanalysis by EDS (\u003cb\u003eSupplemental Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSEM analysis revealed distinct zones within the apatite plug, ranging from loosely packed, amorphous-like regions (Phase 1) to more densely compact structures (Phases 2 and 3). These findings support a model of CaP maturation radiating outward from a nonconcentric core. In sectioned ductal plugs, this internal nonconcentric region (e.g., Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), composed of loosely aggregated, amorphous CaP apatite, was consistently observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This region was surrounded by alternating layers of low and high compactness (Phases 1\u0026ndash;3). The asymmetric morphology of the plug suggests a directional growth, potentially indicating that one side may have expanded more rapidly than the other (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows an endoscopic image from patient 3 (\u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e) with papillary tissue loss and visible mineral plugs (yellow arrowheads). Histological sections stained with PSR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and Yasue stain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) demonstrate fibrosis, inflammation, and mineral retention consistent with ductal plugging. The PSR stained section (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) highlights a mineral plug (black asterisk) in a longitudinal IMCD, and surrounding areas with intense red staining (yellow arrowheads), indicating increased collagen density likely associated with inflammation and fibrosis. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Yasue stain of a consecutive section confirms the presence of mineral plug staining (yellow arrow) with dark brown and black staining in a collecting duct in longitudinal section. The Yasue-stained section also shows extensive tissue loss (acellular zones in the interstitium and tubules) consistent with previous reports of significant tissue injury in patients with plugged papillae (Evan, AP 2005; Williams JC et al. 2024).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results demonstrate that stereoscopic microscopy, micro CT, SEM, and EDS together provide a robust method for analyses of ductal plug stones. Using this multimodal approach, we confirm that stones obstructing the papillary ducts are predominantly composed of CaP apatite. The distal end portion of the plug exposed to calyceal urine acts as a nidus for CaOx crystal overgrowth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings support previous reports showing that CaOx stones, either COD or COM, can form as overgrowths on mineral plugs emerging from the terminal collecting ducts (Bird VY \u0026amp; Khan SR 2017; Grases F \u0026amp; S\u0026ouml;hnel O, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Williams JC 2018; Williams JC 2024).\u003c/p\u003e \u003cp\u003eIn sectioned plugs, we identified nonconcentric, loosely packed apatitic core structures. These structures suggest dynamic mineral accretion and may provide clues as to how CaP crystals expand outward, disrupt tubular epithelial integrity, obstruct the papillary ducts, and potentially damage surrounding nephrons structures during their development.\u003c/p\u003e \u003cp\u003eRecent comparative studies of CaOx stone phenotypes show that ductal plugging is associated with more papillary tissue loss than papillae with extensive Randall\u0026rsquo;s plaque (Williams, JC. \u003cem\u003eet al\u003c/em\u003e. 2022; Williams et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our results are consistent with these findings; radial plug expansion may drive tubular injury, epithelial detachment, and fibrosis, contributing to tubulointerstitial damage (Williams et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuctal plugs have been described during endoscopic stone removal procedures as yellowish plaque deposits obstructing dilated BD (Coe F 2010; Borofsky MS, 2016; Grases F et al \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Williams JC 2018; Williams JC 2024). We observed similar morphologies, with most plugs exceeding the expected diameter of terminal collecting ducts (575\u0026thinsp;\u0026plusmn;\u0026thinsp;440 \u0026micro;m, \u003cb\u003eSupplemental Table\u0026nbsp;2\u003c/b\u003e), possibly indicating chronic tubular obstruction and tubulointerstitial damage.\u003c/p\u003e \u003cp\u003eThe structural changes in the papilla are corroborated by histopathologic studies in stone formers with distal renal tubular acidosis (dRTA) and in patient with prior bariatric surgery. In these patients, CaP deposits in IMCD and BD are associated with epithelial injury and progressive tubular damage, cell loss and fibrosis (Evan, AP 2005; Evan, AP 2007; Coe, FL et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our case study (Patient 3) presents similar patterns with visible papillary tissue loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), fibrosis and plug associated inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows a representative case: a 44-year-old female patient (Patient 3, \u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e) without systemic disease who underwent percutaneous nephrolithotomy. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA reveals classic signs of ductal plugging and papillary tissue loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Histological sections confirmed interstitial fibrosis and localized inflammation by PSR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and confirmed tubular obstruction in IMCD by Yasue stain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. SEM, micro CT, and EDS analyses of the extracted ductal plug stone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) confirmed CaP composition with COM overgrowth studded with traces of COD crystals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These structural and mineral features were consistent across our specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cb\u003eSupplemental Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eA key question in ductal plug formation is how crystals are retained within the tubular lumen. Finlayson and Reid first proposed the fixed particle theory, which postulates that mineral retention in a tubule occurs at an attachment site on the luminal surface of the epithelium. They contrasted this with a \u0026lsquo;free particle\u0026rsquo; concept, in which crystals or crystal aggregates could grow rapidly enough within the renal tubule that the resulting mass would become lodged in the tubule, even obstructing flow. Finlayson and Reid (and later, Grases and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eS\u0026ouml;hnel\u003c/span\u003e) concluded that flow was too fast for mineral to grow to a size that could obstruct the lumen (Grases F \u0026amp; S\u0026ouml;hel O, 2017). Kok and Khan concluded otherwise, calculating that mineral agglomerations could grow fast enough to become lodged in place within a terminal collecting duct. The ductal plugs we studied might have initiated by such rapid growth within the tubular fluid, but we note that the calculations of Kok and Khan are based on calcium oxalate crystal growth, while the plugs we have seen are all composed of apatite (Kok DJ \u0026amp; Khan S, 1994).\u003c/p\u003e \u003cp\u003eRetention of apatite crystals within a tubule may be facilitated by underlying tubular and interstitial pathology, such as tubulointerstitial nephritis (TIN) (Joyce E, et al. 2016). TIN includes a group of inflammatory diseases, often triggered by drugs, metabolic disorders or immune mediated injury. In our case study (Patient 3), histology showed epithelial and basement membrane loss in affected ducts, along with surrounding fibrosis and inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The features observed in our case study resemble a TIN-like inflammatory state or injury.\u003c/p\u003e \u003cp\u003eWe propose that tubular damage, along with an elevated urinary pH and cellular debris, may provide an environment promoting cast and apatite formation, resulting in ductal obstruction (Costa-Bauz\u0026aacute; et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Inflammatory pathways, triggered by necrotic epithelial cells and hypoxia-induced damage, may further enhance tubulointerstitial injury, fibrosis, and progressive loss of nephron function (Degterev A, 2005; Akcay, A. et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bonventre JV and Yang L., 2011; Sarhan M et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Eddy, AA 2019).\u003c/p\u003e \u003cp\u003eAdvanced multiplexed imaging of a CaOx stone former with ductal plugs has revealed epithelial disruption and remarkable inflammatory infiltration. Williams et al. reported loss of aquaporin-2 and Na⁺/K⁺-ATPase expression in plug-affected ducts and identified inflammatory cells expressing MPO, CD68, CD201 and CD3, suggesting neutrophil, macrophage, and T cell involvement. Intraepithelial CD3⁺ T cells in neighboring collecting ducts, suggest tubulitis (an immune mediated injury) and an inflammatory process reminiscent of TIN injury at the papilla (Evan AP, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Williams, JC et al. 2024; Williams, JC and El-Achkar, TM, 2025).\u003c/p\u003e \u003cp\u003eGiven these findings, the authors of the study raised the possibility that ductal plugging may contribute to accelerated decline in renal function (Williams, JC et al. 2024). Our results support this hypothesis. If plugs grow large enough to obstruct only a small number of papillary ducts, and expand radially to cause tubulointerstitial injury, then significant nephron damage may ensue. Each terminal collecting drains an estimated 2750 nephrons, and obstruction of just four ducts could impact over 10,000 nephrons, potentially triggering localized tubulointerstitial stress, inflammation and injury (Kriz W and Kaissling B, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Charlton, JR 2021; Williams, JC et al. 2024). The cumulative effect of this kind of obstruction over time could be clinically significant, considering that some idiopathic CaOx stone formers present with multiple ductal plugs in every papilla (Williams, JC et al. 2024).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results suggest that ductal plugs composed of CaP apatite may begin as loosely packed deposits within the IMCD or BD but progressively expand by breaching the tubular epithelium and extending asymmetrically into the surrounding interstitial space. The presence of a nonconcentric, loosely packed apatitic core in sectioned plugs supports a model of asymmetric growth and may indicate significant tubular disruption and interstitial injury. These findings hint at the potential for ductal plugs to inflict localized nephron damage and contribute to papillary pathology. Further studies validating our results and incorporating high resolution imaging, molecular profiling and spatial omics approaches are necessary to uncover the cellular and molecular mechanisms driving plug formation and growth in the renal papilla.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest:\u003c/h2\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003estatement: All patient specimens were obtained via Internal Review Board approved patient consent (Indiana University IRB protocol #1010002261).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eV.H.C. wrote the main manuscript text and prepared all figures. V.H.C, A.C.B., F.G. and J.C.W. conceptualized, visualized, designed and conducted formal analysis of the data. S.B. B. assisted in methodology and processing of tissues, patient data and project administration. J.E.L. provided patient data, tissues and samples for analysis. All authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors thank Drs. Marcos Oliveira and Robert Soltis from the College of Pharmacy and Health Sciences at Butler University for encouragement and support in the development of this work. We express our special gratitude to the Renal Lithiasis and Pathological Calcification Group at the University of Balearic Islands for their support during the origins and development of this work.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe authors confirm that the summary data supporting the findings of this study are available within the article and its supplementary materials. All underlying data will be made available to anyone upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eScales, C. D. Jr, Smith, A. C., Hanley, J. 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Elsevier, New York, 479\u0026ndash;563 (2008).\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Stones, Ductal plugs, apatite, nephron, kidney disease, inflammation, calculi","lastPublishedDoi":"10.21203/rs.3.rs-6596012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6596012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKidney stone prevalence is increasing, with high recurrence rates and associations with comorbidities such as metabolic syndrome and chronic kidney disease. The most common stones in nephrolithiasis are calcium oxalate (CaOx) stones, either monohydrate (COM), dihydrate (COD) or both, which can form as overgrowths on intratubular plugs of mineral that protrude from the terminal collecting ducts of the renal papilla. Although this is a recognized mode of stone formation, the underlying mechanisms remain poorly understood. In this study, we analyzed 11 ductal plug stones from four patients using stereoscopic microscopy, micro computed tomography (micro CT), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). Detailed surface imaging revealed COM and COD overgrowths on apatite plugs, which often showed various crystallized phases and typically exceeded normal ductal luminal diameters (mean\u0026thinsp;=\u0026thinsp;575\u0026thinsp;\u0026plusmn;\u0026thinsp;440 \u0026micro;m). Sectioned plugs revealed amorphous nonconcentric apatitic cores and radially expanding mineral layers suggestive of mineral accretion. These findings support a model in which ductal plugs originate within the collecting duct lumen and grow asymmetrically beyond the duct wall, potentially contributing to epithelial injury and crystal retention. Further investigations using advanced imaging, molecular, and omics approaches are needed to elucidate the pathophysiology of plug formation and stone retention at the renal papilla.\u003c/p\u003e","manuscriptTitle":"Scanning Electron Microscopy and Energy Dispersive Spectroscopy Reveal Amorphous Apatitic Cores in Ductal plugs bearing Calcium Oxalate Stone Overgrowths","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 14:47:22","doi":"10.21203/rs.3.rs-6596012/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c071b886-5ff7-4036-a296-54625e80cd1a","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49993038,"name":"Biological sciences/Physiology/Kidney"},{"id":49993039,"name":"Biological sciences/Physiology/Kidney/Nephrons"},{"id":49993040,"name":"Health sciences/Urology/Urological manifestations"},{"id":49993041,"name":"Biological sciences/Physiology"},{"id":49993042,"name":"Health sciences/Nephrology"},{"id":49993043,"name":"Health sciences/Pathogenesis"},{"id":49993044,"name":"Health sciences/Urology"},{"id":49993045,"name":"Physical sciences/Chemistry"},{"id":49993046,"name":"Health sciences/Anatomy"},{"id":49993047,"name":"Health sciences/Anatomy/Kidney"},{"id":49993048,"name":"Health sciences/Anatomy/Urinary tract"}],"tags":[],"updatedAt":"2025-08-13T05:08:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 14:47:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6596012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6596012","identity":"rs-6596012","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}