Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation

Emily E Bowen*, Jennifer A Hurcombe, Fern Barrington, Lindsay S Keir, Louise K Farmer, Matthew D Wherlock, Carolina G Ortiz-Sandoval, Valentina Bruno, Arlette Bohorquez-Hernandez, Daniel Diatlov, Niyousha Rostam-Shirazi, Sara Wells, Michelle Stewart, Lydia Teboul, Abigail C Lay, Matthew J Butler, Robert J P Pope, Eva M S Larkai, B Paul Morgan, John MoppettSimon C Satchell, Gavin I Welsh, Patrick D Walker, Christoph Licht, Moin A Saleem, Richard J M Coward*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

Abstract

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear.

METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied.

FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype.

CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease.

FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

Original languageEnglish
Pages (from-to)761-777.e8
JournalMed (New York, N.Y.)
Volume4
Issue number11
Early online date16 Oct 2023
DOIs
Publication statusPublished - 10 Nov 2023

Bibliographical note

Funding Information:
We thank Professors Shamshad Cockcroft and Kevin Mills for their expert glycosphingolipid analysis advice; Mark Nicholas for instruction in use of the Sysmex XN-20 for blood sample analysis; Rosie Hillier at MRC Harwell for help with the breeding and genotyping of all mouse models; Dr. Rebecca Foster for discussions and interpretation of the data; Eva Coward, Alex Coward, and Joe Coward in constructing the context and significance statement; and staff in the Wolfson Bioimaging department at the University of Bristol for access to the Leica DM2000 microscope and Technai 12 transmission electron microscope. This work was supported by a Kidney Research UK Clinical Research PhD Fellowship Grant TF_007_20151127 (to E.E.B.), a UK Medical Research Council (MRC) Clinical Research Training Fellowship (G0901987) and MRC Centenary award (to L.S.K.), and an MRC Senior Research Fellowship Grant MR/K010492/1 (to R.J.M.C.). M.D.W. and L.K.F. were supported by Kidney Research Grants RP42/2012 and SP/FSGS1/2013, respectively. The Mary Lyon Center as part of the MRC Harwell Institute is funded by the MRC (A410). The study was conceived by R.J.M.C. and M.A.S. and developed by E.E.B. with expert input from G.I.W. S.C.S. and C.L. The in vitro and in vivo mouse experiments were performed by E.E.B. J.A.H. and F.B. A.C.L. and R.J.P.P. participated in C5 inhibitor mouse experiments. Original Tet-O-Gb3 constructs used to generate PodrtTA-Tet-O-Gb3 mice were made by M.D.W. and L.S.K. All mouse models were generated and genotyped by S.W. M.S. and L.T. at MRC Harwell. J.A.H. and E.M.S.L. optimized C3 and fibrinogen antibody conditions for ex vivo kidney immunofluorescence. L.S.K. optimized Gb3 detection techniques prior to this study. L.K.F. and E.E.B. performed electron microscopy experiments and analysis on mouse kidneys in both animal studies. M.J.B. provided expertise in cell culture, RT-PCR, and analysis of these experiments. Expert blood film analysis was provided by J.M. Complement expertise and antibodies against mouse C7 and C9 were provided by B.P.M. CFH binding assay studies were performed by E.E.B. C.G.O.-S. V.B. A.B.-H. and D.D. and permeability assays were performed by E.E.B. and N.R.-S. Histology expertise, light microscopy, and immunohistochemistry staining of human kidney biopsies were provided by P.D.W. E.E.B. and J.A.H. performed statistical analysis of the paper. E.E.B. J.A.H. and R.J.M.C. had unrestricted access to all of the data. The paper was initially written by E.E.B. and R.J.M.C. All authors read, commented on, and approved the final paper, taking full responsibility for its content. The authors declare no competing interests. We support inclusive, diverse, and equitable conduct of research.

Funding Information:
We thank Professors Shamshad Cockcroft and Kevin Mills for their expert glycosphingolipid analysis advice; Mark Nicholas for instruction in use of the Sysmex XN-20 for blood sample analysis; Rosie Hillier at MRC Harwell for help with the breeding and genotyping of all mouse models; Dr. Rebecca Foster for discussions and interpretation of the data; Eva Coward, Alex Coward, and Joe Coward in constructing the context and significance statement; and staff in the Wolfson Bioimaging department at the University of Bristol for access to the Leica DM2000 microscope and Technai 12 transmission electron microscope. This work was supported by a Kidney Research UK Clinical Research PhD Fellowship Grant TF_007_20151127 (to E.E.B.), a UK Medical Research Council (MRC) Clinical Research Training Fellowship ( G0901987 ) and MRC Centenary award (to L.S.K.), and an MRC Senior Research Fellowship Grant MR/K010492/1 (to R.J.M.C.). M.D.W. and L.K.F. were supported by Kidney Research Grants RP42/2012 and SP/FSGS1/2013 , respectively. The Mary Lyon Center as part of the MRC Harwell Institute is funded by the MRC (A410).

Publisher Copyright:
© 2023 The Author(s)

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