understanding
the development of
renal failure
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In the body, hormones and hormone-like agents pass messages between cells and organs. These messages can either maintain health or, in disease states, aggravate the disease.

The hormones and hormone-like agents act on the outside of cells. The cells interpret the messages and react by producing complex networks of signal pathways inside the cell.

Because a single hormone or agent can induce a wide variety of outcomes, it is important that we identify the different signals inside the cell that regulate the different outcomes.

To this end, Principal Investigator Dr Mark Dockrell is supervising the work of Post Doctoral Research Fellow: Felicia Heidebrecht; Research Fellows: Dr Seema Jain and Dr Subash Somalanka; PhD students, Irbaz Badshah and Tarun Madne;and honorary research fellow Dr Rosa Montero into the causes of Renal Interstitial Fibrosis(scarring of the kidneys) and collaborating with the work of Dr Mysore Phanish.

We know that progressive scarring will eventually lead to chronic kidney disease.

Therefore, the research is aimed at finding out not only what causes the scarring but a way to safely stop, or considerably slow down, the formation of this damaging scar tissue.

For information on current projects, please click the links below:

The Expression and Regulation of Erk5 in Renal Cells

Podocyte Production, Splicing and Responses to Fibronectin

K Cadherin Expression in Interstitial Fibrosis and Tubular Atrophy in Renal Transplants: the Role of Calcineurin Inhibitors

The Role of N-Ras in a Folic Acid-Induced Model of Renal Fibrosis

Targeting RNA splicing in Renal Disease

The Role of Inflammatory cells in Diabetic Nephropathy

Investigating Biomarkers of Progressive Renal Disease and screening of Nephrotoxic Agents

Urine Retinol Binding Protein: Development of a new monoclonal antibody based assay to identify tubular proteinuria patients with kidney stones and diabetes

 

Dr Mysore Phanish performed his PhD studies at SWTIRR under the supervision of Dr Dockrell and Professor Bruce Hendry of King’s College London from 2002-2005. He gained his medical degree from Jawaharlal Nehru Medical College, Karnataka University, Dharwar, India. He was awarded gold medals for first rank to the University in his 2nd and final year MBBS aDr Mysore Phanishnd for obtaining first rank to the University in General Medicine. He was awarded the Pfizer Postgraduate Medical Award & Medal by Pfizer Pharmaceuticals Ltd for the year 1991 and the Dr R R Joshi Gold Medal for best outgoing medical student of Karnataka University, India, awarded by the Indian Medical Association for the year 1992.

After obtaining his medical degree (MBBS) in 1992, he obtained an MD degree in General Medicine from one of the premier medical institutions of India, the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh in 1996. He trained in Nephrology at the University Hospital of Wales, Cardiff, Bangor District General Hospital, St Helier Hospital and Guy’s Hospital, London. Dr Phanish is currently a consultant nephrologist at the St Helier Hospital, Epsom and St Helier University Hospitals NHS trust and Mayday University Hospital NHS Trust and Honorary Senior Research Fellow at SWTIRR.

Connective Tissue Growth Factor (CTGF CCN2) is a more recently identified protein that promotes scarring within the kidney. It is both a marker and mediator of kidney scarring. During his work at SWTIRR Dr Phanish was the first to identify CTGF production from human proximal tubule epithelial cells (PTECs). He later went on to dissect various cell signalling pathways that regulate the production of CTGF. He also studied cell signalling pathways regulating transition of tubule epithelial cells to scar producing myo-fibroblasts. The results from this work were published in peer-reviewed journals (Nephron-experimental nephrology and Biochemical Journal). He has also presented his work in several national and international renal meetings.

Dr. Phanish is a recognised expert in the CTGF field and his current work aims to investigate the effects of CTGF on human proximal tubule epithelial cells. He intends to investigate the mechanisms of pro-fibrotic effects of CTGF on human PTECs. Dr Phanish obtained a grant from Johnson and Johnson (as a co-applicant with Dr. Dockrell) to study urinary markers in diabetic kidney disease.
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The Expression and Regulation of Erk5 in Renal Cells

Cellular responses to external stimuli are mediated by a variety of intracellular signalling molecules including the all-important Mitogen Activated Protein Kinases (MAP kinases) which are activated by a diverse range of stimuli including, as the name suggests, growth factors and also stimuli such as stress; in turn they regulate a range of potential cellular responses from cell proliferation to apoptosis. Each MAP kinase is activated by dual tyrosine/threonine phosphorylation which is catalysed by its specific up-stream kinase. Erk5 (formerly Big MAP kinase1) is an atypical MAP kinase, which is distinctive both in its size and also the fact that it is activated by mitogens and by stress.

First discovered in the mid 1990s, Erk5 was initially described as being expressed primarily in muscle. Within a few years it was demonstrated to be expressed in endothelial cells where it could be activated by shear stress. In 2008 our group reported that Erk5 was expressed in human proximal tubule epithelial cells where it was activated by both EGF and the pro-fibrotic growth factor TGF-beta1.

Currently, Irbaz Badshah is continuing to investigate the expression and activation of Erk5 in renal tubule epithelial cells and podocytes. He has identified the expression of 3 of the splice variants and is investigating their activation by a range of stimuli involved in renal disease, expanding on the TGF-beta work and including other stimuli of diabetic nephropathy. With the help of colleagues from Boehringer Ingelheim he has been able to selectively inhibit Erk5 activation and examine its role in cell survival and phenotype maintenance. Further collaborations with Bristol University will hopefully identify whether Erk5 also has a role in podocyte motility.

Irbaz is registered for a PhD at St George's University of London and is supervised by Dr Mark Dockrell and Dr Debbie Baines.

If you'd like to know more about this project you can email either irbaz.badshah@esth.nhs.uk or mark.dockrell@swtirr.org.uk
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Podocyte Production, Splicing and Responses to Fibronectin

The glomerular basement membrane (GBM) is a specialised layer of extracellular matrix lying between the endothelial cells of capillaries and the podocytes. The GBM can be seen as a fusion of the basement membranes of the endothelial cells and the podocytes with both cell types contributing to the continued maintenance of its composition and integrity. In turn, receptors on the cells, such as integrins, respond to the proteins of the basement membrane. The GBM in known to primarily contain laminin, type IV collagen, nidogens, and heparan sulfate proteoglycan (HSPG); however recent work from our collaborator Rachel Lennon in Manchester has identified a far greater number of proteins including fibronectin. Although there remains some controversy over the source of GBM fibronectin there is increasing evidence for podocyte-derived fibronectin.

The mRNA transcript from the fibronectin gene is highly susceptible to alternative splicing. Three regions of variable splicing occur along the length of the fibronectin promoter. There are two regions susceptible to exon skipping, the EDA and EDB on exons 33 and 25 respectively and a third variable region V. One or both of the extra type III modules (EDA and EDB) may be present in cellular fibronectin, but they are never present in plasma fibronectin. The presence of EDA endows fibronectin with a number of distinct functions such as adhesion to different integrins and receptors as well as increased VEGF production.

This project is lead by Tarunkumar Madne, a PhD student funded by the Maharashtra State government and supervised by Drs Mark Dockrell, Mysore Phanish and Iain MacPhee seeks to investigate alternative splicing of fibronectin in human podocytes and the autocrine effect of EDA fibronectin on podocyte morphology and biological responses.

If you would like further information on this project please contact tarun.madne@esth.nhs.uk or mark.dockrell@swtirr.org.uk

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K Cadherin Expression in Interstitial Fibrosis and Tubular Atrophy in Renal Transplant: the Role of Calcineurin Inhibitors

Earlier research from SWTIRR confirmed that the widely studied junctional protein E-Cadherin is not actually expressed in the proximal tubule of the human kidney; it is in the rat and the mouse but not in man. In man we have K-and N-Cadherin. This is important because the proximal tubules are targets for a lot of kidney toxins and damage as well as a primary source for renal cell carcinoma and a central area associated with fibrogenesis. So, if we want to study the damage of the human proximal tubule and how this might relate to fibrosis we have to know more about K-Cadherin.

Previous work by Dr Nilesh Shah demonstrated how the loss of K-Cadherin can be a marker of diabetic nephropathy, now Dr Seema Jain is studying the loss of K-Cadherin in renal transplant patients and how its loss may be mediate by the immunosuppressant drugs given to many transplant recipients, calcineurin inhibitors.

Preliminary work looked at biopsies from patients with, what was formerly called, Chronic Allograft Nephropathy and compared them to healthy controls. The patients' biopsies showed a marked loss of K-Cadherin. Seema's work takes two parallel approaches. The first is a clinical study recruiting transplant patients and assessing whether changes in their renal function correlates with urinary K-Cadherin. The second is an in vitro study investigating whether Cyclosporine (Ciclosporin) and Tacrolimus (FK-506) cause loss of K-Cadherin in primary cultures of human proximal tubule epithelial cells and investigating the mechanism.

Dr Seema Jain is the South West Thames Renal Unit Research Fellow.
If you would like further information on this project please contact Seema.Jain@esth.nhs.uk or mark.dockrell@swtirr.org.uk

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The Role of N-Ras in a Folic Acid-Induced Model of Renal Fibrosis

Renal fibrosis is the consequence of a dysregulated progressive accumulation of extracellular matrix protein which, untreated, will lead to loss of renal function. The progressive loss of renal function and the consequent development of established renal failure (ERF) correlate strongly with the degree of tubulointerstitial fibrosis (TIF). It is widely accepted that one of the key driving factors in TIF is Transforming Growth Factor beta1 (TGF-beta1); however a systemic reduction in TGF-beta1 is likely to have deleterious side effects. Consequently, selective local targeting of TGF-beta1 and its subsequent effectors is likely to be a more successful strategy in reducing renal fibrosis.

Previous work from our group and others, particularly the King's College London Renal Research Group, has identified a significant role for Ras GTPases in this process. The Ras family of proteins operate as intracellular master switches regulating an array of both upstream and downstream signalling cascades to modify cellular function. Recent work using cell culture models of renal fibrosis published by us has identified Neuronal Ras (N-Ras) in the control of two critical pro-fibrotic events downstream of TGF-beta; autoinduction and the production of Connective Tissue Growth Factor (CTGF).

Through a collaborative project with King's College & ISIS Pharmaceuticals we have obtained highly specific antisense oligonucleotides (ASO) designed to target N-Ras mRNA and prevent expression of the protein. In this project we aim to investigate the role of N-Ras in folic acid-induced renal fibrosis in parallel with cell culture studies. To this aim we will:

1) Determine N-Ras expression in the kidney and other key organs.

2) Characterise the effects of infusion of N-Ras ASO on renal and extra-renal tissues in the absence of disease, including markers of inflammation, cellular proliferation and apoptosis.

3) Investigate changes of the progression of fibrosis in the presence of N-Ras ASO, looking at expression TGF-beta, CTGF, collagen/fibronectin deposition, markers of inflammation, tubule cell death & proliferation and renal function..

Cell Biology studies will focus on identifying down-stream signalling cascaded that are regulated by N-Ras in the presence and absence of TGF-beta and activation of gene transcription using PCR arrays designed to monitor markers and mediators of fibrosis

Dr Subash Somalanka is the South West Thames Kidney Fund Clinical Research Fellow and he is driving this collaboration through his work at KCL and here at SWTIRR

If you would like further information on this project please contact Subash.Somalanka@esth.nhs.uk or mark.dockrell@swtirr.org.uk

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Targeting RNA splicing in Renal Disease

The constitutive splicing of pre-mRNA to remove transcripts of introns is one of the normal steps of the production of protein from a gene. Alternative splicing is the highly regulated process that enables the potential production of multiple proteins from a single gene by selecting the sequence that will form the mRNA and subsequently be translated to protein.

It is estimated that the pre-mRNA products of more than 75% of human genes can be alternatively spliced giving rise to multiple protein products and this is believed to be one of the key mechanisms for the production of ~100 000 proteins from ~ 25 000 genes. Many of these alternative products can have opposing roles.

The human extracellular matrix protein Fibronectin (FN) can exist in multiple forms with differing functions due to 3 distinct alternative splicing sites. The form of FN containing the Extra Domain A from exon 32, EDA+ FN, is extensively expressed at sites of renal fibrosis with little or none expressed in the healthy kidney. Evidence indicates that EDA+ FN expression occurs early in fibrogenesis and accelerates the process.

In this project we have set out to establish whether RNAse H-independent Antisense Oligonucleotides can be used to direct alternative splicing of FN pre-mRNA so as to alter the balance away from the expression of the EDA+ isoform in collaboration with ISIS Pharmaceuticals. From this we are investigating whether reduced EDA+ expression results in a reduction in fibrogenesis in our in vitro model using primary human cells.

Dr Felicia Heidebrecht is currently coordinating this project at SWTIRR and working closely with colleagues at ISIS Pharmaceuticals to develop this work.

If you would like further information on this project please contact Felicia.Heidebrecht@esth.nhs.uk, or mark.dockrell@swtirr.org.uk

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The Role of Inflammatory cells in Diabetic Nephropathy

Diabetic Nephropathy (DN) is the leading cause of kidney disease in the Western World. Traditionally DN has been seen as vascular disease with high circulating sugar levels causing progressive damage to the microvascular beds in the kidney in a similar way to that seen in the eye during diabetic retinopathy. Subsequently it was observed that the persistent and intermittent elevated sugar levels directly affected resident cells in the kidney such as the tubule epithelial cells and the podocytes. However, even this improved perception of the disease fails to explain all of its features.

More recently an hypothesis about the role of inflammation in the progression of DN has emerged. This theory has arisen due to the presence of macrophages in the kidneys of patients suffering from DN. Macrophages are important immune cells involved in the regulation of inflammation. Evidence has now established a correlation between the presence of these cells in the kidney and the decline in renal function. However, it is not clear how macrophages communicate and interact with resident kidney cells and how this is affected by the diabetic milieu – such as high sugar levels.

In order to investigate this further and identify whether these cells might represent a new target in the fight against DN we are undertaking a pilot study in human primary proximal tubule cells with human macrophages. By isolating monocytes, the parent cell of macrophages, from the blood of our cohort of healthy volunteers and carefully culturing these cells so that they develop into macrophages we are then able to incubate them with human kidney cells. From this we will be able to measure changes in cell phenotype, cytokine, pro-fibrotic factors and extracellular matrix proteins.

If you would like further information on this project please contact mark.dockrell@swtirr.org.uk
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