Hyaluronan (HA) assembly and its Role in Bone Morphogenic Protein-7 Driven Antagonism of Renal Fibrosis

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

Organ failure resulting from fibrosis is the predominant cause of chronic diseases in our population including kidney disease, liver disease, lung and heart disease. However no treatments exist that can halt or reverse fibrosis and prevent organ damage. This research aims to identify targets that can potentially alleviate or reverse fibrosis, with the aim of developing new therapies to prevent end-organndamage in at-risk patients.

Myofibroblast are the principle fibrosis-producing cells. They are produced by the circulating factor TGF-beta-1 through transformation of epithelial cells and fibroblasts. BMP7 is another circulating factor, which opposes TGF-beta-1-driven fibrosis. However, neither direct blocking of TGF-beta-1, nor treatment with BMP7 are viable therapies as they cause significant side effects. Thus we need to further study these two circulating factors to identify more targetted therapy with less side effects. Our department has previously shown that TGF-beta-1-driven formation of myofibroblasts is dependent on how cells make and assemble a third molecule, Hyaluronan (HA). We also know that BMP7 can assemble HA differently to TGF-beta-1. Our aim is to carry out detailed studies to determine the effects of BMP-7 on how fibroblasts/epithelial cells generate, assemble and handle HA; thus allowing us to differentiate between good and bad HA. This will allow us to prevent formation of myofibroblasts, thus enabling us to identify therapies that can prevent or reverse fibrosis in at risk patients.

Technical Summary

Hyaluronan (HA) is a matrix polysaccharide involved in regulation of cellular processes, and demonstrates increased expression in renal disease. TGF-beta-1 promotes epithelial mesenchymal transition and fibroblast to myofibroblast differentiation. These processes drive renal interstitial fibrosis, which predicts outcome in all renal diseases, and our previous studies have shown that both these processes are mediated by HA. BMP7 is a cytokine demonstrating reduced expression in renal disease. Evidence suggests it can both prevent and reverse TGF-beta-1-driven fibrosis. We have shown that BMP7 modifies cellular HA matrix and there is evidence that HA modulates BMP7 responses. The aim of this proposal is to investigate if the prevention and/or reversal of TGF-beta-1 responses by BMP7 are mediated by the effects of BMP7 on HA matrix.

The work will initially utilise fibroblast and renal epithelial cells models and encompass a variety of cell biology techniques to characterise BMP7-driven alterations in HA at a pericellular, cell-surface and cytoplasmic level and relate these changes to TGF-beta-1 driven regulation of cell phenotype. Furthermore, the in vivo applicability and relevance of our in vitro observations will then be investigated by identifying BMP7 driven alteration in HA and HA-related proteins in the kidney in an animal model of renal disease. This work will inform the next steps in terms of identifying suitable HA related targets for the development of knockout animal models, and aid in the development of new therapeutic strategies in progressive renal disease.

Planned Impact

Progressive fibrosis in tissues and organs is the pathological process that underlies a significant number of chronic diseases in our populations. Prevention and/or reversal of this process is a major goal of research in across a variety of research disciplines including research into cardiovascular, renal, pulmonary and liver disease. Furthermore, research in this area can also be applicable to other areas including wound healing research, immuno-regulatory and cancer research. The work proposed in this application will thus potentially benefit other academic and scientific institutions through direct collaboration, as well as through dissemination via peer reviewed publications and presentation at scientific meetings.

The work outlined is specifically designed to identify new pathways and processes that can be potentially targeted to prevent and/or reverse a pro-fibrotic myofibroblast phenotype. Furthermore, the proposed work will validate altered expression of these targets in an animal model of renal disease, and in prevention and/reversal of progressive fibrosis in this model. If successful this could lead to future interventional animal studies that could ultimately translate into the bedside thus promoting public health and well being, as well as improving costs for treatment of patients with chronic kidney disease and chronic disease in general. The award will provide me with dedicated research time, supervision and mentorship at two leading research institutions that will facilitate towards my career development as an independent researcher. It will also provide training and mentorship for a post-doctoral research assistant, who will obtain extensive experience and transferable skills in a range of scientific techniques and methodologies valued by biotechnology companies, thus providing a skilled worker into the workforce.

Through its dissemination and through public engagement we also hope to inspire a new generation of scientists and researchers in this field, and in the field of medical research in general. Our research will be disseminated through liaison with the Cardiff University Communications and International Relations Division. Furthermore, the Institute of Nephrology itself holds a variety of public engagement programs including open days attended by renal patients and their relatives and hosting visits for A-level and work-experience students. A number of these students are able to join our laboratory for a four-week summer project through the Nuffield Foundation Bursary Scheme and many have proceeded to study medicine or biological sciences at university.
 
Description Kidney Research UK Project Grant
Amount £196,662 (GBP)
Funding ID RP_047_20170303 
Organisation Kidney Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2018 
End 04/2021
 
Title CD44 alternative splicing bichromatic minigene reporter 
Description We have developed a fluorescent CD44v7/8 minigene reporter. This minigene contains a bichromatic reporter wherein a frame-shift caused by CD44v7/8 exon inclusion results in the generation of a STOP codon that leads to the translation of a protein with a red fluorescent tag (dsRED). Alternatively if exons for CD44v7/8 are spliced out, the frame-shift does not occur leading to green fluorescence-tagged (EGFP) protein. Hence red fluorescence highlights factors that favour induction of CD44 alternative splicing, whereas green fluorescence indicates factors that favour CD44s expression, and exon exclusion. Using this reporter we are able to investigate factors that regulate CD44 alternative splicing events. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Useful technique to study CD44 alternative splicing, and can be modulated to investigate pre-mRNA splicing of other genes of interest. 
 
Description Collaboration with Lerner Institute Cleveland Clinic Foundation 
Organisation Cleveland Clinic
Department Lerner Research Institute
Country United States 
Sector Academic/University 
PI Contribution Collaboration set up to investigate the relevance of Hyaluronan and associated matrix proteins in in vivo models of kidney disease. Successful visit to the Lerner Research Institute in November 2015 with firm ideas fixed for collaborative work. Will move to Lerner Research Institute in Cleveland for one year to carry out this work from November 2016 until November 2017.
Collaborator Contribution They will provide some funds towards the ongoing work and have also provided intellectual ideas to further the joint research.
Impact The partnership has led to 3 clear projects which will be undertaken on sabbatical to the Cleveland Lerner Institute later this year.
Start Year 2016
 
Description HA dependent regulation of CD44 alternative splicing events 
Organisation University of Exeter
Department Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration to develop the dichromatic CD44 mini gene reporter to be used in kidney proximal tubule epithelial cells. Collaboration to investigate the role of Hyal2 in alternative splicing and in the regulation of long noncoding RNA. All the experiments undertaken and ideas for the work came from my group in Cardiff.
Collaborator Contribution The technical and methodological knowhow was provided by Dr Oltean's group. Dr Oltean also reviewed the manuscript and grant applications jointly submitted.
Impact 1 publication in Science Signaling. 1 grant awarded from Kidney Research UK as a consequence of this work.
Start Year 2016
 
Description The role of CD44 variants and different HAS isoforms on prevention and/or promotion of kidney fibrosis 
Organisation Rush University
Country United States 
Sector Academic/University 
PI Contribution The direction of research, experiments and analysis of results are conducted by my group in Cardiff.
Collaborator Contribution The collaborators were involved in research idea generation. They also provided the in vivo tissue, which was shipped to Cardiff for analysis.
Impact A manuscript is currently under preparation for this work. Further joint grant funding applications are being/have been submitted.
Start Year 2016
 
Description The role of HA in regulation of Vascular Smooth Muscle Cell phenotype and vascular calcification in CKD patients 
Organisation Philadelphia University
PI Contribution We were approached by this team from USA to see if a protein of interest that they had been investigating was relevant to our studies in the prevention and promotion of fibrosis. They sent us cells, antibodies and primers to investigate this protein. Important research findings were identified as a result of experiments that were undertaken in Cardiff.
Collaborator Contribution The USA group from Philadelphia approached us with the ideas to begin with. They sent the cells and samples for us to investigate.
Impact A manuscript is under preparation and a grant application has been submitted (but unsuccessful). We are currently in process of generating further preliminary data prior to resubmission of the grant.
Start Year 2016
 
Description Media Release related to published article 
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 Publication related to work funded by the MRC Fellowship was released as a media release on the Cardiff University website.
Year(s) Of Engagement Activity 2018
URL https://www.cardiff.ac.uk/news/view/1091780-preventing-fibrosis
 
Description National Science week engagement for primary schools 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Engagement with primary school children on National Science Week
Year(s) Of Engagement Activity 2018
 
Description Wales Kidney Research Unit Open Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact Wales Kidney Research Unit open day. The findings and outcomes of the MRC funded Fellowship was communicated to interested patient groups and their thoughts and questions addressed.
Year(s) Of Engagement Activity 2018
URL http://kidneyresearchunit.wales/en/news-and-events.htm?newsid=65