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

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.

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