Understanding the pathogenesis of haemolytic uraemic syndrome: the role of the podocyte

Lead Research Organisation: University of Bristol
Department Name: Clinical Science at North Bristol

Abstract

Haemolytic uraemic syndrome (HUS) is the leading cause of acute kidney failure in children. 1-5%
of children with HUS die and some are left with chronic kidney damage. It is commonly caused by a
gastrointestinal infection (E.coli) that affects >1000 people/year in Britain. It produces toxins, which
in 10-15% of cases causes kidney and other organ damage. It is a public health concern. Currently,
we do not understand how HUS is caused and therefore management focuses on supportive care.
Recent work by our collaborators using a mouse model has provided a potential breakthrough in our
understanding of HUS.
This project will build on this and aims to investigate the kidney effects of HUS. It will study the
effects of the toxin on specific kidney cells. We will create a mouse model that closely resembles the
disease in humans. This part of the project is underway. We will also look at human kidney cells
using different techniques to see how the toxin affects them and, in particular, how the toxin affects
the interaction between these cells. Understanding how this disease affects the kidney will identify
potential ways of treating this condition and alleviate the suffering of affected children and their
families.

Technical Summary

Aims: To investigate the role of the podocyte in the renal pathogenesis of diarrhoea associated
haemolytic uraemic syndrome (D+HUS).
Hypothesis: The renal pathogenesis of D+HUS is caused by E.coli-0157 derived shigatoxin (stx)
binding to podocytes, via Gb3 receptors, and reducing VEGF-A secretion. This directly affects
glomerular endothelial cell function causing thrombotic microangiopathy (TMA).
Objectives/Design:
Both in vivo and in vitro techniques will be used. We will create a murine model of paediatric HUS.
There is no reliable HUS animal model due to species variations in Gb3 expression. Humans show
glomerular Gb3 expression while mice show tubular expression. To overcome this, a podocyte
specific inducible Gb3 expressing mouse will be created. Transgenic and wild type mice will be
injected with stx to induce HUS. Building on our preliminary in vitro work, we will investigate
changes to podocyte VEGF-A secretion in response to stx. We will investigate the effects of reduced
VEGF-A on glomerular endothelial cells (GEnC), in particular the glycocalyx and the effect this has
on complement factor H binding. We know this is affected in atypical (D-) HUS.
Methods:
1) In vivo: A construct, which has tetracycline tet-o-promoter linked to Gb3 synthase has been
developed to create an inducible podocyte-specific Gb3 expressing mouse. Cloned plasmids
will be injected into the single-cell embryo, transferred into pseudo-pregnant mice and
allowed to reach term. Breeding of tet-o-Gb3 mice with podocin-RtTA mice (already existing)
will create mice that, when given doxycycline, will express Gb3 on podocytes only. This
controls production of Gb3 synthase avoiding cellular toxicity. Thus, over-coming species
variation and creating a model of HUS. Transgenic and wild-type will have stx injected
inducing HUS. Mice will have daily biochemical tests. Kidneys will be examined histologically
(gross+EM). In situ hybridisation will assess Gb3 expression and VEGF-A production in HUS.
2) In vitro: Bristol is a leader in studying podocyte and GEn cell lines. Using this resource I will
investigate the effects of stx on these cells. Particularly looking for changes in podocyte
VEGF-A secretion that may change the endothelial glycocalyx or complement factor H
binding. This could link the pathogenesis of D+ and D- HUS. Improved disease understanding
may identify potential therapeutic targets to help treat HUS.
Opportunities: This project provides experience in in vivo mouse work and in vitro work on
immortalised cells lines. It will help explore an important area of paediatric nephrology whilst
providing a backdrop to learn transferable research skill

Publications

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Keir LS (2017) VEGF regulates local inhibitory complement proteins in the eye and kidney. in The Journal of clinical investigation

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Keir LS (2016) Complement and the kidney in the setting of Shiga-toxin hemolytic uremic syndrome, organ transplantation, and C3 glomerulonephritis. in Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis

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Keir LS (2015) Shiga toxin associated hemolytic uremic syndrome. in Hematology/oncology clinics of North America

 
Description British Association of Paediatric Nephrology travel award
Amount £500 (GBP)
Organisation British Association for Paediatric Nephrology (BAPN) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2012 
End 10/2012
 
Description NIHR Clinical Lecturer
Amount £160,000 (GBP)
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 03/2013 
 
Description University of Bristol MRC Centenary award
Amount £50,000 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 07/2013 
End 10/2014
 
Description Walls Bursary
Amount £1,000 (GBP)
Organisation Renal Association 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 11/2012
 
Title Gb3 mouse podocyte expression 
Description A new conditionally immortalised mouse podocye cell line was created. This was characterised and then used for transfection with Gb3 synthase. The result was that the mouse podocytes express the cell receptor Gb3 which is not normally expressed by mouse podocytes. 
Type Of Material Cell line 
Provided To Others? No  
Impact This work is still in its infancy but by transfecting the mouse podocytes with Gb3 we hope to study them with our shiga toxin assay. This was a preliminary experiment to se if our Gb3 synthase construct would produce Gb3. We are now using this gene with the RtTa/tet-o system to create a podocyte specific Gb3 synthase expressing mouse. 
 
Title Immunofluorescent complement challenge assay 
Description A new assay was developed to functionally assess complement activation on cells in culture. Previously similar assays looked at cells in single cell suspension but glomerular endothelial cells do not normally exist in this way. Therefore the assay was adapted to allow cells to be studied whilst still in monolayer. IF was used to look for C3d and C4d deposits on the cell surface. Image J was then used to analyse and quantify the images obtained. Numerous control samples were also run alongside. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact This assay has allowed us to study glomerular endothelial cells in vitro and their response to complement. It has allowed us to study the effects of treatment on these cells and how they respond to complement. This work has been prepared for publication. 
 
Title Podocyte specific Gb3 synthase expressing mouse 
Description A new transgenic mouse line was created. A construct was made linking a tet-o promoter to the gb3 synthase gene. This was microinjected into the pronuclei of a mouse. The resultant offspring were screened for construct insertion. 5 founder lines were established and each are not being tested for expression of Gb3 synthase and its product Gb3. The tet-o Gb3 synthase mice have been bred with podocin Rtta mice to create a glomerular podocyte specific Gb3 synthase expression. It has confirmed podocyte Gb3 expression after 10 days doxycycline induction. It has been tested with Shiga toxin and preliminary results show that mutant mice do have a glomerular phenotype. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This model is still being testing. Preliminary results that these mice do develop a glomerular pheontype and so this could serve as a murine model for stx HUS, the most common cause of acute kidney failure in children. However, further investigation is required to work up its phenotype. Rescue experiments are also planned to test possible treatmnt options. 
 
Description CRISPR/Cas sytem to induce CFH mutations in RPE cells 
Organisation Sanford-Burnham Medical Research Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution I designed the primers to sequence the CFH gene and we worked together to design the CFH construct to be introduced into the new cell line.
Collaborator Contribution The stem cell core team carried out the cellular transfections of the CRISPR/Cas constructs and propagated the cells.
Impact Initial efforts to create new cell line with CRIPSR in RPE cells failed. A new approach was therefore sought. This work was superseded when a new source of iPS RPE was sourced from another collaborator.
Start Year 2014
 
Description Complement proteins expression by human podocytes 
Organisation University of Hamburg
Country Germany 
Sector Academic/University 
PI Contribution New research collaboration. Both groups were working separately to study the expression of complement proteins in human podocytes. This was noted during a poster presented at the International Paedaitric Nephrology Association in China. The decision was made to combine these studies for publication
Collaborator Contribution I carried out work on human podocytes to study the way they express protective complement regulators and have assessed this functionally. The collaborators have looked a the same cells but focused on their synthesis of activating complement proteins. The work is being combined for publication. I am writing the paper This work was delayed due to leave of absence of collaborator but has restarted as of 2019.
Impact Separate posters presented at ASN, IPNA meetings Working towards joint publcations.
Start Year 2013
 
Description Complement reagents 
Organisation University of Colorado
Department School of medicine; University of Colorado
Country United States 
Sector Academic/University 
PI Contribution My studies on complement protein expression in the kidney benefited from expert opinion of Dr Thurman and he provided reagents that helped us to extend our studies in different complement regulator expression.
Collaborator Contribution Dr Thurman provided complement reagents that helped further our complement regulator studies.
Impact Preliminary results presented at ASN 2014. Publication using experiment results is in preparation 2019.
Start Year 2013
 
Description Complement regulator study 
Organisation University of Edinburgh
Department Queen's Medical Research Institute Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Frequent meetings to share data and discuss and analyse results have help to shape the direction of the research. Together we are sharing different experiences and expertise to look at things from a new angle.
Collaborator Contribution Shared knowledge and advice on experimental design and data interpretation. The direction of the research has been guided through frequent meetings and discussion.
Impact Change in direction of the research at my host insitution. Poser presentation ASN, Philadelphia Nov 2011and at ASN San Diego Nov 2012 Oral Presentation at the Academic Paediatric Association of Great Britain and Ireland-Prize for best Scientific Presentation. Poster presentation at Gordons Research Conference, Tuscany, August 2012. Oral Presentation at European Society of Pediatric Nephrology (ESPN), September 2012-awarded BAPN travel award to attend. Work presented by oral talk to pediatric nephrology research meeting Boston Childrens Hospital BMJ opinion piece 2012. Gordons conference presentation 2014. ESPN presentation and best poster 2013. JCI paper 2017
Start Year 2009
 
Description Mouse model of glomerular disease-pod1 
Organisation Mount Sinai Hospital (Canada)
Department Samuel Lunenfeld Research Institute
Country Canada 
Sector Academic/University 
PI Contribution Working with the team has allowed me to share ideas and discuss projects. I have worked on a different project separate to my MRC funded project. This allowed me to gain hands on experience in a new field.
Collaborator Contribution Experience and knowledge on mouse models and techniques associated. Colony management, genotyping and a battery of techniques to study the phenotypes of the mice generated.
Impact New techniques have been developed by myself which I hope to take back to my host insitution. 3 poster presentations: Great Lakes conference, Ontario, April 2012, Gordons Research Conference, Italy August 2012, American Society of Nephrology meeting San Diego, November 2012 2013, ASN, Atlanta November. Poster presentation. Prepared for submission to Journal of the American Society of Nephrology (JASN): published 2014
Start Year 2011
 
Description NIH Intramural Programme: iPSC RPE 
Organisation National Eye Institute (NEI)
Country United States 
Sector Academic/University 
PI Contribution I established a collaboration with this group as we sought to use a human iPSC RPE cell line that carried the AMD associated genetic mutation to show human relevance of in vitro and in vivo murine observations related to complement protein production and function. Our efforts to make iPSC RPEs expressing this mutation using CRISPR were unsuccessful. Therefore this collaboration helped to secure an alternative source of cells and these were from phenotype patients which had an advantage.
Collaborator Contribution The collaborator sourced the patient samples, created the iPSC RPE cells and sent these to our lab for study.
Impact JCI publication 2017
Start Year 2013
 
Description Podocytes produce complement proteins 
Organisation University Medical Center Hamburg-Eppendorf
Country Germany 
Sector Hospitals 
PI Contribution Independently we made the observation that human podocyte synthesize and secrete complement proteins. I particularly focused on the protective proteins such as complement factor H and CD46. I developed an in vitro assay to study functional effects of these synthesized proteins. I also set up in situ hybirdation to show RNA expression of these proteins in tissue samples (human and mouse). This technique was shared and successfully transferred to my collaborator.
Collaborator Contribution Our German collaborators also found that human podocytes synthesize complement proteins but focused more activatory proteins such as C3. They set up and performed some murine studies looking at functional capacity of activator complement proteins.
Impact Poster presentation at ASN 2014. This work is also being prepared for publication. This work has been delayed due to leave of absence of collaborator but activities have restarted in 2019.
Start Year 2013
 
Description Shiga toxin Assay 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I visited the University of Warwick and carried out experiments on my cells with the help of the team. I initially contacted the Warwick team based on their extensive experience in this area.
Collaborator Contribution Provided advice and practicle help to carry out shiga toxin experiments on glomerular conditionaly immortalised cells. Protocols were shared. Also suplied advice on toxin purification and reagents to use.
Impact This collaboration has provided me with the chance to carry out techniques which are not available yet at my insitution. However, with the help and knowledge shared in this colaboration I am now in a position to set up techniques at my host insitution. Poster presentation Philadelphia American Society of Nephrology meeting Nov 2011 Oral presentation at the Academic Paediatric Association of Great Britain and Ireland-prize for the best scientific presentation awarded. Oral presentation at Royal College of Paediatric and child health meeting, Glasgow May 2012. Award prize for best oral presentation. Collaboration ended when Warwick toxin unit closed. However, their knowledge allowed me to set up toxin research capacity in Bristol (this included setting up cat three unit, police infection, necessary security and provisions for animal experiments including protocol for monitoring)
Start Year 2010
 
Description The relationship between VEGF-A and complement regulator expression in the eye 
Organisation Scripps Research Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution Visit to the lab in California in 2012. Immunofluorescent staining carried out for complement regulators in mice and human tissue sections and cells. This pilot data was used to apply for a one year University of Bristol MRC Centenary award. This application was successful and I have spent one year at Scripps further exploring the role of complement regulators in the normal eye and in the disease Age related macular degeneration. This has results in presentation at ARVO and the Gordons Research conference in endothelial cell phenotypes in health and disease.
Collaborator Contribution Collaborators provided human, mouse and rat tissue and cells for this project. I am contributing to the project with complement knowledge, assays and reagents. We are working together to create a new iPS RPE cell line from patients with factor H polymorphisms
Impact Walls Bursary award from Renal Association-personal award University of Bristol MRC Centenary award-personal award with consumables. Lowy Medial Research Institute grant NIH grant also supported this work-PI was collaborator. Collaboration resulted in: Poster presentation at ARVO 2013, oral presentation at ARVO 2014, Oral presentation at Gorgons conference 2014. JCI paper 2017.
Start Year 2012
 
Description Clinican information (HUS) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Health professionals
Results and Impact Clinician information on how to diagnose, manage and treat Shiga toxin HUS. It is hope that this will standardize care in this patient group.

Positive feedback from colleagues.
Year(s) Of Engagement Activity 2013
URL http://rarerenal.org/clinician-information/haemolytic-uraemic-syndrome-clinician-information/
 
Description Kids Kidney Research UK 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact My supervisor Professor Saleem had received a start up grant from Kids Kidney UK which led to generation of preliminary data which helped to fund my project. As such they wanted to publise our work. Prof Saleem was asked to write a short script detailsing our project which could be understood by teenagers and children. I wrote this script. The charity now plans to record the script and put it on the internet to help tell children and thier families about the disease and the research we are carrying out.

Our project will be publised to children and their families who have an interest in kidney diseases.
Year(s) Of Engagement Activity 2011
 
Description MRC Centenary pop up science festival M Shed (University of Bristol) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Local people were invited to attend the M shed museum for a pop up science festival. MRC funded reseachers attended and presented their work to the public to illustrate their research. Small group sessions could be selected by the public to hear about a specific research area. I ran one of these and explained how eating undercooked meat can lead to Shiga toxin E.coli infections which can cause haemolytic uraemic syndrome and kidney injury.

Positive feedback obtained. Stronger relationship with the public engagement office. Will participate in such events in the future.
Year(s) Of Engagement Activity 2013
URL http://www.bristol.ac.uk/news/2013/9471.html
 
Description Parent/Patient information (infoKid HUS) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This online resource is designed to disseminate information about Shiga toxin HUS to parents and patients and other members of the public.

Good feedback was received from the parent/patient group HUSH.
Year(s) Of Engagement Activity 2014
URL http://www.infokid.org.uk/STEC-HUS
 
Description School visit (Mangotsfield) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact 5 groups of 20 childrens aged 11-15yrs attended session about The kidney. Three practical activities were devised to teach them about the normal role of the kidney in humans and what happens in childhood diseases. They all had to write down questions during the session and some were chosen to ask these questions at the end.

Positive feedback from the children and science teachers. Plans to repeat the experience in future years.
Year(s) Of Engagement Activity 2013
URL http://www.bristol.ac.uk/news/2013/9471.html
 
Description Stx HUS working group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact HUSH patient group were approached to join in the Stx HUS working group and in turn we were asked to help update their patient/parent advice leaflets.

Provide closer links with health care professionals and patients and parents. Ensure that accurate up to date information is available to patients and parents.
Year(s) Of Engagement Activity 2012