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Crystallisation Kinetics and Aggregation in Bio-mineralisation: What bridges crystals in kidney stones?

PGR-P-754

Key facts

Type of research degree
PhD
Application deadline
Friday 17 April 2020
Project start date
Thursday 1 October 2020
Country eligibility
UK and EU
Funding
Competition funded
Source of funding
Research council
Supervisors
Dr Antonia Borissova Dimitrova
Schools
School of Chemical and Process Engineering, School of Chemistry
<h2 class="heading hide-accessible">Summary</h2>

The study of crystallisation kinetics and aggregation in kidney stones is a step to understand solidification in biological systems - a scientific challenge that can only be addressed by multi-disciplinary teams of scientists and clinicians. Surgical treatment of kidney stones does not stop their recurrence. Previous research1 proved that only microcrystals are produced which are normally discharged without causing pain. Kidney stones are a result of crystal aggregation the mechanism of which is still unknown. A hypothesis relates to the role of proteins in this process. Understanding their binding role is a major challenge. The aim is to study the bio-chemical composition and the structure of kidney stones to identify the &quot;glue&quot; for the crystals. The evolution of the complex chemical system of the nephron fluids, including ionic equilibria, protein interactions and phase transformations of the salts present has not been investigated as a holistic environment for stone formation and represents a significant challenge. The structural dynamics of the crystallising system at the levels of crystal nucleation, growth, aggregation and phase transformation is important to understand the mechanism of stone formation. The project will investigate the phenomena leading to stone formation by applying mathematical modelling and experimental methods. The project will be a collaboration within the Faculty of Engineering &amp; Physical Sciences (SCaPE and Chemistry) with input from the Urology Department of the Leeds Teaching Hospital. The approach has been tested in a summer placement project in 2019 and images revealing the complex kidney stone structure were obtained and analysed. The existence of an amorphous layer around crystals supported the hypothesis of a binder in the formation of the aggregates (Figure 1). The aim is to develop and test a methodology for predicting kidney stone formation. Through understanding of the &ldquo;glue&rdquo;, further work is expected to lead to the development of diagnostic tests and therapeutic intervention. The project will be interdisciplinary and be co-supervised by Dr Antonia Borissova (School of Chemical and Process Engineering) and Prof Bruce Turnbull (School of Chemistry). Urologists from the Leeds Teaching Hospital NHS Trust are willing to provide guidance. Candidates with degrees in Chemistry, Physics, Materials or Chemical Engineering will have the necessary background for this project. The experience gained will equip the candidate for a large variety of job opportunities. Industrial and academic employers value experience in interdisciplinary research as well as the experimental and computer modelling skills.

<h2 class="heading hide-accessible">Full description</h2>

<p>Previous research<sup>1</sup> proved that only microcrystals are produced which are normally discharged without causing pain. Kidney stones are a result of crystal aggregation the mechanism of which is still unknown. A hypothesis relates to the role of proteins in this process. Understanding their binding role is a major challenge. The aim is to study the bio-chemical composition and the structure of kidney stones to identify the &quot;glue&quot; for the crystals.&nbsp;</p> <p>The evolution of the complex chemical system of the nephron fluids, including ionic equilibria, protein interactions and phase transformations of the salts present has not been investigated as a holistic environment for stone formation and represents a significant challenge. The structural dynamics of the crystallising system at the levels of crystal nucleation, growth, aggregation and phase transformation is important to understand the mechanism of stone formation. The project will investigate the phenomena leading to stone formation by applying mathematical modelling and experimental methods.&nbsp;</p> <p>The project will be a collaboration within the Faculty of Engineering &amp; Physical Sciences (SCaPE and Chemistry) with input from the Urology Department of the Leeds Teaching Hospital.&nbsp;</p> <p>The approach has been tested in a summer placement project in 2019 and images revealing the complex kidney stone structure were obtained and analysed. The existence of an amorphous layer around crystals supported the hypothesis of a binder in the formation of the aggregates.</p> <p>The aim is to develop and test a methodology for predicting kidney stone formation. Through understanding of the &ldquo;glue&rdquo;, further work is expected to lead to the development of diagnostic tests and therapeutic intervention.</p> <p>The project will be interdisciplinary and be co-supervised by Dr Antonia Borissova (School of Chemical and Process Engineering) and Prof Bruce Turnbull (School of Chemistry). Urologists from the Leeds Teaching Hospital NHS Trust are willing to provide guidance.<br /> Candidates with degrees in Chemistry, Physics, Materials or Chemical Engineering will have the necessary background for this project. The experience gained will equip the candidate for a large variety of job opportunities. Industrial and academic employers value experience in interdisciplinary research as well as the experimental and computer modelling skills.</p> <h5>Purpose of the Research</h5> <p>The purpose of the proposed investigation is to reveal the causes for the kidney stone formation applying a systems approach to the environment and the processes of stone genesis and development. Our aim is to reconstruct the liquid environment in the nephron during the stone formation (impossible to monitor in-vivo currently) analysing samples from recurrent kidney stone patients with a focus on the chemical and structural composition of the bridges between crystals. The analytical techniques applied to achieve this aim will be Scanning Electron Microscopy (SEM) with Energy-Dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), including synchrotron, X-ray Photoelectron Spectroscopy (XPS) and Fourier-Transform Infrared Spectroscopy (FTIR) methods. Oxalate, cystine and urate stones will be studied as some of the common re-current kidney stones.</p> <p>The compositional and structural changes in the nephron fluid preceding the formation of the kidney stones and thus the pre-cursors for kidney stones will be identified. It will be achieved through a literature study and theoretical work on the development of the biochemical equilibrium in the nephron leading to aggregation of micro-crystals to form stones. Analytical work to verify the hypotheses raised about possible &ldquo;binders&rdquo; will be done using kidney stones from patients. The research proposed will be the first attempt to reverse engineer the kidney stone applying the network and system approach to the mathematical models of the stone and the nephron environment.</p> <p>The confidence in the feasibility of this research originates also from the success of the reverse engineering the kidney work<span class="superscript_text">1</span> on calcium oxalate crystallisation in the nephron and the predictive capabilities of the model developed. An early task will be to modify the existing model to predict cystine and urate crystallisation.&nbsp; This will then be upgraded to include an aggregation kernel to simulate kidney stones.</p> <p>This 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 aim of the research is to establish the physico-chemical nature of the bridges in the crystal aggregates rather than creating an exhaustive list of protein binders. Understanding the nature of the bonds (bridges) will allow creating an in-silico molecular model of the core phenomena leading to kidney stone formation.</p> <p>Background</p> <p>Previous works, including one of the authors&#39; own, 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 mechanism of which is still unknown. The nephron fluid dynamics was represented as a crystalliser/separator series with changing volume to allow for water removal along the tubule. The model integrated crystallisation kinetics and crystal size distribution and allowed the prediction of the calcium oxalate concentration profile, nucleation and growth rates. The critical supersaturation ratio for the nucleation of calcium oxalate crystals has been estimated as 2 and the mean crystal size as 1 micron. The model allowed the exploration of the effect of varying the input calcium oxalate concentration and the rate of water extraction, simulating real-life stressors for stone formation such as dietary loading and dehydration. This model of crystallisation in kidneys needs further improvement to include an aggregation/ agglomeration kernel and increase the model predictive capacity. This will be achieved through the integration of the crystallisation and the biochemical equilibrium phenomena &ndash; a missing link in the understanding of the kidney stone disease. A chemical species distribution along each nephron segment will be produced and integrated with the crystallisation and agglomeration models to create an &ldquo;in-silico&rdquo; image of the nephron&rsquo;s operation. The proposed research will support achieving this aim.</p> <p>The work of Kim et.al.<span class="superscript_text">2-5</span> and other researchers<span class="superscript_text">6-8</span> is a cornerstone and will be used as the background for the proposed research. The &ldquo;detection of the organic matrix&rdquo;<span class="superscript_text">3</span> will use far better equipment than that available to Kim and collaborators. 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 agglomerate and the SEM analysis will allow us to monitor both phases: crystals as solids 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 both hypotheses will be considered. Based on the previous works <span class="superscript_text">2-3</span> 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 minimisation or destruction. The work of Finlayson, Khan and Hackett <span class="superscript_text">6-8</span> will be used in the design of the experiments.</p> <p>&nbsp;</p> <h5>The project plan includes the following major parts:</h5> <h6>Biochemical structural analysis</h6> <p>Literature review on the bio-chemical composition and the structure of kidney stones:</p> <p>The evolution of the complex species system of the nephron fluids, including ionic equilibrium and protein interactions, has not been investigated as an environment for stone formation and thus represents a significant research challenge. Biological fluids contain a complex mixture of salts and macromolecules. Polymers with anionic side chains such as poly (acrylic acid) have been shown to provide a simple means of mimicking certain characteristics of urinary proteins. These can directly affect the precipitation of the stone forming salts and their crystals (co-crystals). The number of kidney stone promoters in the group of proteins is constantly increasing.</p> <p>The aim of the research is to establish the physico-chemical nature of the bridges in the crystal aggregates rather than creating an exhaustive list of protein binders. Understanding the nature of the bonds (bridges) will allow the creation of an in-silico molecular model of the core phenomena leading to kidney stone formation. Numerous works studying particularly COM crystallisation, promoters and inhibitors of kidney stones with focus on the role of proteins as binders in crystal agglomeration have been published. The two main suggested mechanisms are free- and fixed particle mechanisms for kidney stone formation. SEM has been applied to study these phenomena. What makes the proposed research different from all previous ones is its fundamental character. The equation of the kidney stone formation i.e. Crystals + &ldquo;Glue&rdquo; = Kidney Stones is the overall one only. The individual steps of this complex &ldquo;reaction&rdquo;, their &ldquo;activation energies&rdquo; and process conditions are still unknown. Studying the physico-chemical nature of the bridges built in the process of kidney stone formation will allow their identification. A critical analysis of the published research on kidney stone formation will be conducted and a review article prepared for publication. Particular attention will be paid on the assumptions made, especially in the modelling, for example, neglecting the distribution of crystals by size in the modelling from Finlayson and Reid<span class="superscript_text">7</span> to Kok and Khan<span class="superscript_text">8</span>. The work of Robertson and Peacock<span class="superscript_text">9</span> models the crystal size distribution (CSD), but our aim is to make the additional step of relating crystal size with crystal aggregation and agglomeration through differentiating between the bonds created between crystals with different size. This would also allow identifying the part of the nephron where the free-particle mechanism of kidney stone formation starts. Another focus of the literature survey will be the phenomena related to the Randall&rsquo;s plaques and crystal-cell wall interactions, particularly in idiopathic calcium oxalate stone formers. The incidence of papillary plaques is significantly more common in patients with calcium oxalate (88%) and calcium phosphate stones (100%) than in patients without a history of the urinary stone disease (43%)<span class="superscript_text">10</span>. 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. In addition, SEM examination of the calcium oxalate stones developed on Randall&rsquo;s plaque revealed that plaque may also be made of tubules obstructed by calcium phosphate plugs. Hypercalciuria has been associated with Randall&rsquo;s plaque formation. However, several additional mechanisms may be involved resulting in increased tissue calcium phosphate saturation and the role of macromolecules in plaque formation. Nevertheless, calcium oxalate monohydrate stones developed on plaques are mainly related to low diuresis and an increased urine oxalate concentration, evidencing that the processes leading to plaque and stone formation, respectively, may differ<span class="superscript_text">11</span>.</p> <p>Kidney stone samples from patients with recurrent kidney stones (oxalate, cystine and uric acid) will be studied using a range of biochemical and structural techniques such as Scanning Electron Microscopy (SEM) and X-ray Diffraction methods (XRD). This work was carried out during a summer placement at the University of Leeds in 2019 and funded by Translate MedTech. Ethical approval was obtained for the analytical studies. A kidney stone was analysed using SEM/EDX and XRD techniques and it was confirmed that the stone was from a re-current patient with cystine stones. The work was presented at the Translate MedTech Conference in Leeds, 6th December 2019. To determine the elemental chemical composition of the samples SEM-EDX (Electron Dispersive Spectroscopy) methods will be used. The modern facilities in LEMAS will allow improving both the scope (range of bio-chemical compounds) and the depth (atomic and molecular level) of the results. The electron optics studies will face a series of challenges the greatest of which is the quantitative analyses of the spectra. Identifying which elements are present from an EDX spectrum is relatively routine. Improving the potential for quantitative analysis using EDX is possible by applying methods for correction of the X-ray intensities. One of the common corrections applied is for signal absorption. 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) 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 (again a recently installed capability available through the Royce Institute Leeds spoke). Recent attempts have been undertaken to analyse organic crystalline materials<span class="superscript_text">12</span>. We do not know how exactly the occluded liquid is present in the kidney stone but plan to create a map of the distribution of stone density and identify the different phases from it. The distribution of crystals by specie type, size and shape in the agglomerates 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>Patients with recurrent stones, particularly with oxalate, cystine and uric acid stones will form the basis of this project. Ethical approval will be sought to undertake data gathering on patient characteristics, clinical progression, laboratory biochemical analysis (such as urinary pH, metabolic screen and blood tests). The samples of kidney stones (fragments) will be collected from recurrent patients, either in the clinic or during stone removal intervention. All findings of the kidney stone formation and its mechanisms will be systematised and a database with the relevant structure populated. The database work, including its structure and the software used, will start at a later stage of the project when sufficient information will have been accumulated.</p> <h6>Mathematical modelling</h6> <p>The mathematical modelling approach will be multi-scale: molecular, nephron and kidney stone levels. The aim of the molecular modelling is to study the solid-liquid interactions in the nephron based on calcium oxalate monohydrate (COM) as a model system. Quantum Modelling (QM) and Density Functional Theory (DFT) will be applied. The meso-level modelling will include modelling of nucleation, crystal growth and aggregation in the nephron.</p> <p>The work will be based on the mathematical model developed applying chemical engineering approach in representing the nephron as a plug flow reactor and a series of crystallisers and separators to simulate the water removal through the walls of the nephron for calcium oxalate<span class="superscript_text">1</span>. This software is available and will be modified for cystine and uric acid on the basis of the new findings from the experimental work. The update will include a higher precision in the kinetic parameters determination e.g. constants of crystal nucleation and growth, mass transfer through the membrane wall as well as developing of the aggregation kernel in the model.</p> <p>Using the results from the analytical studies, the macro-modelling will represent the evolving kidney stone as a network of crystals and bridges between them. The kidney stone bridges will be modelled for the first time. The model will simulate the nephron functioning thus predicting parameters impossible to monitor e.g. concentration profile along the nephron, prediction of the on-set of crystallisation, distribution of crystals and aggregates by size etc. The models developed will be applied to simulate the oxalate, cystine and uric acid crystallisation in the kidney&#39;s nephron and predict a mean crystal size.</p> <p>The models will be verified using both own data and data from literature.</p> <p>A microfluidic rig developed in previous student project at SCAPE will be used verify the crystallisation kinetics. The rig has only been used for crystallisation experiments (COM crystallisation from aqueous solution), but will be tested for cystine and uric acid crystallisation too.</p> <p>The task to reconstruct the liquid environment from its solid product (outcome) i.e. the kidney stone and understand the possible causes of the stone formation is a challenging one that will require further research, particularly in the application of the project outcome to developing new drugs and treatments.</p> <h5>References</h5> <p><span class="superscript_text">1</span> Borissova A, Goltz G E, Kavanagh J. Wilkins T A, Reverse Engineering the Kidney - Modelling Calcium Oxalate Monohydrate Crystallization in the Nephron. Medical &amp; Biological Engineering &amp; Computing, 2010, Vol 48, 35-43</p> <p><span class="superscript_text">2</span> Kim KM, Johnson FB. Calcium oxalate crystal growth in human urinary stones, Scan Electron Microsc., 1981, (Pt 3), 147-154</p> <p><span class="superscript_text">3</span> Kim KM. The Stones, Scan Electron Microsc., 1982, (Pt 4), 1635-1660</p> <p><span class="superscript_text">4</span> Kim KM, Resau J, Chung J. Scanning electron microscopy of urinary stones as a diagnostic tool, Scan Electron Microsc., 1984, (Pt 4), 1819-1831</p> <p><span class="superscript_text">5</span> Kim KM, Alpaugh HB, Johnson FB. X-ray microanalysis of urinary stones, a comparison with other</p> <p>methods, Scan Electron Microsc., 1985, (Pt 3), 1239-1246</p> <p><span class="superscript_text">6</span> Finlayson B, Khan SR, Hackett RL. Mechanisms of stone formation &ndash; An overview, Scan Electron</p> <p>Microsc. 1984;(Pt 3):1419-25. Review.</p> <p><span class="superscript_text">7</span> Finlayson B, Reid F. Expectation of free and fixed particles in urinary stone disease, Investig Urol, 1978, 15, 442-448</p> <p><span class="superscript_text">8</span> Kok DJ, Khan, SR. Calcium oxalate nephrolithiasis, a free or fixed particle disease, Kidney International, 46, 1994, 847-854</p> <p><span class="superscript_text">9</span> Robertson WG, Peacock M. Calcium Oxalate Crystalluria and Inhibitors of Crystallization in Recurrent Renal Stone-Formers, 1972, Clinical science 43(4):499-506</p> <p><span class="superscript_text">10</span> Low RK, Stoller ML. J Urol. 1997 Dec; 158(6):2062-4.</p> <p><span class="superscript_text">11 </span>Daudon M, Bazin D, Letavernier E. Urolithiasis 2015, 43 (Suppl. 1), 5&ndash;11.</p> <p><span class="superscript_text">12</span> Brydson R, Eddlestone ME, Jones W, Seabourne CR, and Hondow NJ. Phys. Conf. Proc., 2014, 522, 012060.</p> <p>&nbsp;</p>

<h2 class="heading">How to apply</h2>

<p>Formal applications for research degree study should be made online through the&nbsp;<a href="https://eps.leeds.ac.uk/chemical-engineering-research-degrees/doc/apply">University&#39;s website</a>. Please state clearly in the research information section&nbsp;that the research degree you wish to be considered for is &ldquo;Crystallisation Kinetics and Aggregation in Bio-mineralisation: What bridges crystals in kidney stones?&rdquo; as well as&nbsp;<a href="https://eps.leeds.ac.uk/chemical-engineering/staff/234/dr-antonia-borissova">Dr Antonia Borissova Dimitrova</a>&nbsp;as your proposed supervisor.</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><em>We welcome applications from all suitably-qualified candidates, but UK black and minority ethnic (BME) researchers are currently under-represented in our Postgraduate Research community, and we would therefore particularly encourage applications from UK BME candidates. All scholarships will be awarded on the basis of merit.</em></p>

<h2 class="heading heading--sm">Entry requirements</h2>

Applicants to research degree programmes should normally have at least a first class or an upper second class British Bachelors Honours degree (or equivalent) in an appropriate discipline.

<h2 class="heading heading--sm">English language requirements</h2>

The minimum English language entry requirement for research postgraduate research study is an IELTS of 6.0 overall with at least 5.5 in each component (reading, writing, listening and speaking) or equivalent. The test must be dated within two years of the start date of the course in order to be valid.

<h2 class="heading">Funding on offer</h2>

<p><strong>UK/EU</strong>&nbsp;&ndash;&nbsp;Engineering &amp; Physical Sciences Research Council Studentship&nbsp;for 3.5 years. A full standard studentship consists of academic fees (&pound;4,600 in Session 2020/21), together with a maintenance grant&nbsp;paid at standard Research Council rates (&pound;15,285&nbsp;in Session 2020/21). UK applicants will be eligible for a full award paying tuition fees and maintenance. European Union applicants will be eligible for an award paying tuition fees only, except in exceptional circumstances, or where residency has been established for more than 3 years prior to the start of the course.&nbsp;Funding is awarded on a competitive basis.</p>

<h2 class="heading">Contact details</h2>

<p>For further information please contact the Graduate School Office<br /> e:&nbsp;<a href="mailto:phd@engineering.leeds.ac.uk">phd@engineering.leeds.ac.uk</a>, t: +44 (0)113 343 5057.</p>


<h3 class="heading heading--sm">Linked funding opportunities</h3>
<h3 class="heading heading--sm">Linked research areas</h3>