Crystallisation Kinetics and Phase Transformation in Biomineralisation (What bridges crystals in kidney stones?)

The mechanism of the kidney stone formation and the nephron system’s dynamics will be investigated using experimental and theoretical approaches. Surgical treatment of kidney stones does not stop their recurrence. The exact mechanism of their formation is still unknown. A hypothesis relates to the role of proteins in this process. Understanding their binding role is a major challenge that will be in the focus of this research. Previous works, including supervisor’s own, proved that the fluid residence time in a kidney's nephron is sufficient to produce microcrystals normally discharged without causing any pain. Kidney stones are a result of crystal aggregation. The evolution of the complex chemical system of the nephron fluid, including ionic equilibria, protein interactions and phase transformations of the salts present (phosphates, oxalates, urates etc.) has not been investigated as an environment for stone formation and represents a significant research challenge. At supersaturation conditions, a variety of salts form crystals that subsequently may aggregate and form kidney stones. The structural dynamics of the crystallising system at the levels of crystal nucleation, growth, aggregation and phase transformation is of utmost importance for the understanding of the mechanism of the stone formation. The project will investigate the phenomena leading to stone formation applying mathematical modelling and experimental methods. The aim will be to develop and test a methodology for predicting kidney stone formation and on that basis to suggest directions for design of new medicines to prevent it. Opportunities to apply the methods developed to studying other processes of biomineralisation e.g. tissue mineralisation both necessary (bones, teeth) and potentially deadly (arteries, brain, etc.), will be explored. The kidney's nephron will be reverse engineered to model the complex bio-chemical system with the simultaneously occurring processes of crystal nucleation and growth, aggregation and phase transformation in the nephron's fluid system. This integrated mathematical model will allow numerical simulation of the kidney stone formation. Scanning electron microscopy integrating Energy Dispersive X-ray spectroscopy, Raman spectroscopy and X-ray diffraction, including synchrotron will be applied to study the morphology, crystal structure and chemical composition of the kidney stones and laser diffraction - for the distribution of the crystals by size (volume) and to estimate the crystal size distribution in the aggregate. Evolutionary scanning of re-current patient samples will be used for model verification. The project will be run within the collaboration between the University of Leeds and the Urology Department in the Leeds Teaching Hospital.

<p><strong>Introduction</strong></p> <p>Urolithiasis/Nephrolithiasis remain a chronic disease and our fundamental interpretation of the pathogenesis of stones as well as their prevention and cure still remains rudimentary. The impact of preventing kidney stone formation on NHS provision and individuals is significant with the number of hospital episodes increasing by 70% over a 15-year period (Hospital Episode Statistics data). In Europe, up to 8% of the population have been affected, but this number is rising. It is estimated that at least 720,000 individuals currently have kidney stone problems in the UK. In US 10.6% of men and 7.1% of women are affected1. Surgical treatment of kidney stones does not stop their recurrence. There is still no recognised prevention for kidney stones and other illnesses caused by solidification. Stones represent the end-product of a long cascade of events. However, it is now apparent that once a calculus becomes dislodged from the papilla, it is too late. Increasing evidence suggests that nephrolithiasis is associated with systemic diseases like obesity, diabetes and cardiovascular disease. Nephrolithiasis places a significant burden on the health care system, which is likely to increase with time2.</p> <p>Previous works, including authors&rsquo; own3, proved that the fluid residence time in a nephron is sufficient to produce microcrystals normally discharged without causing any pain. Kidney stones are a result of crystal aggregation. The exact mechanism is still unknown. A hypothesis relates to the role of proteins in crystal&nbsp;aggregation and stone formation. Understanding their binding role is a major challenge. The evolution of the complex specie system of the nephron fluids, including ionic equilibrium and protein interactions, has not been investigated for stone formation and is a significant research challenge. An early detection and deactivation of the stone nuclei is a target for the treatment of urolithiasis and other solidification related diseases.</p> <p><strong>Background of the research</strong></p> <p>There are two main suggested mechanisms for kidney stone formation i.e. free- and fixed-particle mechanisms. Randall&rsquo;s Plaques and crystal-cell wall interactions play an important role in the stone formation, particularly in idiopathic calcium oxalate stone formers. Recent studies based upon kidney biopsies demonstrated that apatite deposits on the plaque originate from the basement membranes of thin loops of Henle and then spread in the surrounding interstitium. Different mechanisms resulting in increased tissue saturation may apply, but the understanding of the mechanism of crystal&nbsp;aggregation requires considering the role of the macromolecules present in the formation of the condensed phase.</p> <p>Crystallisation, particularly of calcium oxalate monohydrate (COM) has been extensively studied, but many simplifications, particularly related to the distribution of crystals by size and shape have been made e.g. the work of Finlayson and Reid4, Kok and Khan5, Robertson and Peacock6. Our aim is to make the additional step of relating crystal size with crystal&nbsp;aggregation through differentiating between the bonds created between crystals of different size. This would also allow identifying the part of the nephron where the free-particle mechanism of kidney stone formation starts.</p> <p>Recent attempts have been undertaken to analyse organic crystalline materials7. The &ldquo;detection of the organic matrix&rdquo;8 will be done using far better equipment than previously. This will require extensive electron microscopy work as well as sophisticated in-silico modelling using thermodynamic, kinetic and fluid flow relationships between the parameters of the nephron system. There is always liquid occluded between the individual crystals in a crystal&nbsp;aggregate and the SEM analysis will allow us to monitor both phases: crystals and the occluded liquid as a new phase. We do not yet know whether the liquid forms chemical or physical bridges between the crystals, but will consider both hypotheses.</p> <p>Based on the previous works5,9 the role of a variety of possible binders in the stone formation will be studied and their identification in the samples will be a target. The expectations are that the list of known binders will be enriched with ones that were impossible to monitor before. The novelty will be in revealing the type of bridges the binders create. This would allow identifying the way for their effective destruction and prevention.</p> <p>The aim of the research is establishing the physico-chemical nature of the bridges in the crystal aggregates to allow creating of an in-silico molecular model of the phenomena leading to the kidney stone formation. Studying the chemical structure and composition of the bridges between crystals in a kidney stone would permit reconstructing of the liquid environment in the nephron during the period of the stone formation (impossible to monitor in-vivo currently) using SEM analyses of kidney stones.</p> <p><strong>Research Methodology</strong></p> <p>The facilities of the Leeds Centre for Microscopy and Spectroscopy (LEMAS) based in the School of Chemical and Process Engineering will be used for studying the structure and the chemical composition of kidney stone specimens in order to reveal the role of the complex organic-inorganic, molecular-ionic chemical system in the kidney&rsquo;s nephron on the stone formation. The analysis of the bio-chemical equilibrium in the samples will allow an insight into the mechanism of kidney stone formations and the role of the proteins as binders in the process of aggregation.</p> <p>The project plan includes the recapitulation of a large number of SEM studies. The samples (from recurrent stone formers) will be submitted to the necessary sample preparation to extract the information needed at different scales. Quantitative EDX analysis will be applied. With the use of modern windowless Silicon Drift Detectors, EDX in the SEM can now give useful information on light element composition particularly when accurate absorption corrections are included in the quantitative ZAF corrections software routines available in the Oxford Instruments Aztec software package. Both these facilities are available in LEMAS11. Identifying which elements are present from an EDX spectrum is relatively routine. Improving the potential for quantitative analysis using EDX is possible applying methods for correction of the X-ray intensities. One of the common corrections applied is for signal absorption. A recent EDX quantitative method called the z (zeta)-factor approach has been developed which incorporates both absorption correction and also the fluorescence correction to determine compositions10. Sample holders constructed of graphite or beryllium reduces absorption too.</p> <p>For more accurate analyses including extraction of light element chemical bonding information, thin site-specific cross-sections (under cryo-conditions using a newly installed dual beam FIB/SEM)) could be taken for TEM analyses and electron energy loss spectroscopy (EELS) in the TEM used to interrogate the biochemical glue. Further surface specific analysis could be carried out using near ambient pressure X-ray photoelectron spectroscopy.</p> <p>We do not know how exactly the occluded liquid is present in the kidney stone but plan to create a distribution map of stone density and identify the different phases from it. The distribution of crystals by specie type, size and shape in the&nbsp;aggregates will also be investigated in relation to the determination of the kidney stone pre-cursors. The SEM/TEM facilities available will permit qualitative (chemical compounds) and quantitative analyses of the bridging (size of crystals and their distribution, surface and structural parameters, chemical composition). The state-of-art equipment and required expertise available in LEMAS is a major strength of the project.</p> <p>The PhD student will be based in LEMAS, but meetings and laboratory studies will be also conducted in St James&rsquo;s Hospital in Leeds. There will be fortnightly progress meetings led by the urologists. Topical seminars i.e. List of potential &ldquo;binders&rdquo;, Image analyses, Stone variability etc. will be organised in St. James&rsquo;s Hospital and LEMAS with the participation of clinicians and scientists from both institutions. To maximise cross-fertilisation, the urologists will participate in some of the experimental work to help identify areas of interest.&nbsp;</p> <p>The work is the first step towards the fundamental understanding of the kidney stone using it as a fingerprint of the liquid environment from which it originated. The confidence in achieving the aim is based on the expertise involved and the sophisticated state-of-art equipment allowing monitoring the structure of the stone at an atomic/ molecular level. The project is a unique integration of medical, biological and engineering approaches.</p> <p><strong>References:</strong></p> <ol> <li>Patient Information. What I tell my patients about kidney stones? BJRM, 2013; Vol 18 No 1, pp.15-18</li> <li>Ziemba J.B and Matlaga B.R., Epidemiology and economics of nephrolithiasis, Investig Clin Urol. 2017 Sep; 58(5): 299&ndash;306</li> <li>Borissova, A., Goltz, G.E., Kavanagh, J., Wilkins, T. Reverse Engineering the Kidney - Modelling Calcium Oxalate Monohydrate Crystallization in the Nephron, Medical &amp; Biological Engineering &amp; Computing, 2010, 48, 649-659</li> <li>Finlayson B, Khan SR, Hackett RL. Mechanisms of stone formation &ndash; An overview, Scan Electron Microsc. 1984;(Pt 3):1419-25. Review</li> <li>Kok DJ, Khan, SR. Calcium oxalate nephrolithiasis, a free or fixed particle disease, Kidney International, 46, 1994, 847-854</li> <li>Robertson WG, Peacock M. Calcium Oxalate Crystalluria and Inhibitors of Crystallization in Recurrent Renal Stone-Formers, 1972, Clinical science 43(4):499-506</li> <li>Ward MB, Hondow N, Brown AP, Brydson R. Electron Energy-loss Spectroscopy and Energy-dispersive X-ray Analysis, Nanocharacterisation: Edition 2, Editors: Angus I Kirkland, Sarah J Haigh, Ref: http://dx.doi.org/10.1039/9781782621867-00108</li> <li>Kim KM, Johnson FB. Calcium oxalate crystal growth in human urinary stones, Scan Electron Microsc., 1981, (Pt 3), 147-154</li> <li>Viswanathan P, Rimmer JD, Kolbach AM, Ward MD, Kleinman JG, Wesson JA. Calcium oxalate monohydrate agglomeration induced by agglomeration of desialylated Tamm-Horsfall protein, Urol Res, 2011, 39, 269-282</li> <li>Ryall RL, Chauvet M, Grover PK. Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals, 2005, BJU International, 654-663</li> <li>Brydson R, Eddlestone ME, Jones W, Seabourne CR, and Hondow NJ. Phys. Conf. Proc., 2014, 522, 012060.</li> </ol> <p>&nbsp;</p>

<p>Formal applications for research degree study should be made online through the&nbsp;<a href="https://www.leeds.ac.uk/research-applying/doc/applying-research-degrees">University&#39;s website</a>. Please state clearly in the Planned Course of Study section that you are applying for <em><strong>PHD Chemical &amp; Process Engineering FT,&nbsp;</strong></em>in the research information section&nbsp;that the research degree you wish to be considered for is <strong><em>Crystallisation Kinetics and Phase Transformation in Biomineralisation (What bridges crystals in kidney stones?)</em></strong>&nbsp;as well as&nbsp;<a href="https://eps.leeds.ac.uk/chemical-engineering/staff/234/dr-antonia-borissova">Dr Antonia Borissova</a>&nbsp;as your proposed supervisor&nbsp;and in the finance section, please state clearly&nbsp;<em><strong>the funding that you are applying for, if you are self-funding or externally sponsored</strong></em>.</p> <p>If English is not your first language, you must provide evidence that you meet the University&#39;s minimum English language requirements (below).</p> <p style="margin-bottom:11px"><em>As an international research-intensive university, we welcome students from all walks of life and from across the world. We foster an inclusive environment where all can flourish and prosper, and we are proud of our strong commitment to student education. Across all Faculties we are dedicated to diversifying our community and we welcome the unique contributions that individuals can bring, and particularly encourage applications from, but not limited to Black, Asian, people who belong to a minority ethnic community, people who identify as LGBT+ and people with disabilities. Applicants will always be selected based on merit and ability.</em></p> <p class="MsoNoSpacing">Applications will be considered on an ongoing basis. &nbsp;Potential applicants are strongly encouraged to contact the supervisors for an informal discussion before making a formal application. &nbsp;We also advise that you apply at the earliest opportunity as the application and selection process may close early, should we receive a sufficient number of applications or that a suitable candidate is appointed.</p> <p>Please note that you must provide the following documents in support of your application by the closing date of 3 April 2024 for Leeds Opportunity Research Scholarship or 8 April 2024 for Leeds Doctoral Scholarship:</p> <ul> <li>Full Transcripts of all degree study or if in final year of study, full transcripts to date</li> <li>Personal Statement outlining your interest in the project</li> <li>CV</li> </ul> <p>&nbsp;</p>

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<p><strong>Self-Funded or externally sponsored students are welcome to apply.</strong></p> <p><strong>UK&nbsp;</strong>&ndash;&nbsp;The&nbsp;<a href="https://phd.leeds.ac.uk/funding/209-leeds-doctoral-scholarships-2022">Leeds Doctoral Scholarships</a> and <a href="https://phd.leeds.ac.uk/funding/234-leeds-opportunity-research-scholarship-2022">Leeds Opportunity Research Scholarship</a> are available to UK applicants (open from October 2023). <a href="https://phd.leeds.ac.uk/funding/60-alumni-bursary">Alumni Bursary</a> is available to graduates of the University of Leeds.</p> <p><strong>Non-UK</strong> &ndash;The&nbsp;<a href="https://phd.leeds.ac.uk/funding/48-china-scholarship-council-university-of-leeds-scholarships-2021">China Scholarship Council - University of Leeds Scholarship</a>&nbsp;is available to nationals of China (now closed for 2024/25 entry). The&nbsp;<a href="https://phd.leeds.ac.uk/funding/73-leeds-marshall-scholarship">Leeds Marshall Scholarship</a>&nbsp;is available to support US citizens. <a href="https://phd.leeds.ac.uk/funding/60-alumni-bursary">Alumni Bursary</a> is available to graduates of the University of Leeds.</p> <p><strong>Important:</strong>&nbsp; Any costs associated with your arrival at the University of Leeds to start your PhD including flights, immigration health surcharge/medical insurance and Visa costs are <strong>not</strong> covered under these studentships.</p> <p>Please refer to the <a href="https://www.ukcisa.org.uk/">UKCISA</a> website for information regarding Fee Status for Non-UK Nationals.</p>

<p>For further information about your application, please contact Doctoral College Admissions by email to&nbsp;<a href="mailto:phd@engineering.leeds.ac.uk">phd@engineering.leeds.ac.uk</a></p> <p>For further information about this project, please contact Dr Antonia Borissova by email to&nbsp;<a href="mailto:A.Borissova@leeds.ac.uk">A.Borissova@leeds.ac.uk</a></p>