The pathology of fibrosis
There are many different causes of chronic kidney disease, diabetes, high blood pressure, toxins but what eventually leads to kidney failure is nearly always the same, fibrosis. Fibrosis is scarring but unlike other scars fibrosis doesn’t stop. It carries on in a progressive relentless manner replacing the functioning kidney with scar tissue. Our research seeks to understand why the scarring continues and hopefully to identify new drug targets to stop the process.
A number of our projects have identified a vicious cycle of fibrosis, continuously pushing the process on.
We have identified different points where we think it might be possible to break the vicious cycle, stopping the scarring and potentially allowing the kidney to recover.
Figure 1a Collagen deposition in Folic Acid Nephropathy
Figure 1b Collagen deposition in Folic Acid Nephropathy with N-Ras ASO treatment
Figure 2 BUN in FAN, negative control ASO in brown and N-Ras ASO in green
Inside the cell there are various signalling pathways regulating the cellular behaviour. The family of Ras proteins act as molecular switches within many of these pathways. Working with a team at King’s College London we have identified that N-Ras is a critical point in the vicious cycle.
Using an innovative technology, antisense oligonucleotide (ASO), we have demonstrated that if you reduce the amount of N-Ras protein in human proximal tubule epithelial cells it is possible to break the cycle.
We have gone on to demonstrate that the same approach can be used in a preclinical model of acute to chronic kidney disease, reducing the amount of scarring and stopping the progressive loss of kidney function
One of the key proteins in the scarring – fibrosis- in the kidney is fibronectin. We generally think of fibrosis as being made up of collagen fibres. However, in kidney fibrosis fibronectin is one of the early scar proteins to accumulate. It appears that fibronectin is required for collagen to form fibres in the kidney.
There is a single fibronectin gene but there are many fibronectin proteins. This is due to alternative splicing of fibronectin RNA. (The ability to make many proteins from a single gene is part of the explanation for humans having between 20,000 and 25,000 genes but 100,000 proteins.)
We recognised that in our models of fibrosis the type of fibronectin produced by human kidney cells was EDA fibronectin. EDA stands for Extra Domain A and is the result of the inclusion by alternative splicing of a single exon of 270 nucleotides.
This has also been observed in patients with glomerulosclerosis.
It transpires that EDA fibronectin is not some inert scar but a highly bioactive protein and we have identified that one its effects is to activate TGFβ1. TGFβ1 is powerful growth factor involved in cancer, inflammation and fibrosis. TGFβ1 is such a potent factor that it is highly regulated; it is secreted by cells and maintained in a latent form. EDA fibronectin can alter the latency complex and activate TGFβ1driving fibrosis including the production of more EDA fibronectin.
In collaboration with Ionis Pharmaceuticals in California we have invented a potential treatment for fibrosis that blocks the activation of latent TGFβ1. Using RNAse H independent ASO we can prevent the inclusion of EDA into fibronectin without reducing the total amount of fibronectin.
The CCN Proteins in Renal Interstitial Fibrosis
The CCN family of proteins are described as matricellular due to their ability to form direct communication lines between extracellular matrices (ECM) proteins and cells. In fact, they were among the first ECM proteins to be recognised as more than just structural support for cells. CTGF (CCN2) as first described in 1988. In 2006 SWTIRR were the first group to describe its expression in renal epithelial cells. Seen as a pro fibrotic factor downstream of TGF β1 CCN2/CTGF became a focus of fibrosis research. Research at SWTIRR identified a role for CCN2/CTGF as more of a modulator than a driver of tubulointerstitial fibrosis. The team at SWTIRR also proposed that the enzymatic cleavage of CCN proteins may be a key process in unmasking CCN protein function.
In physiology we often see a dynamic equilibrium, a balance between two opposing forces that can be adjusted to meet changing needs. If CCN2/CTGF acts to modulate towards a fibrotic end what counteracts this?
CCN3, formerly called Nov, is the 3rd member of the CCN family. It shares the 4 module structure with CCN2/CTGF, but reports from Bruce Riser's group and others suggested that CCN3 might be a natural antagonist to CCN2/CTGF.
Using an optimised human in vitro model we also observed anti-fibrotic actions of CCN3. Mirroring CCN2/CTGF, co-incubation with CCN3 reduces TGFβ induced fibronectin expression by proximal tubules epithelial cells.
The underlying mechanisms responsible for the counter regulatory nature of the CCN proteins is not understood but if the phenomenon is extended to other sites in the body such as the vasculature the Implications are substantial.
The actions of the CCN proteins and their contextual regulation by enzymatic cleavage remain an area of investigation at SWTIRR.
Glucose, the proximal tubule and diabetic nephropathy.
For over 20 years there have been two schools of thought regarding the roles of different areas of the kidney and the development of diabetic nephropathy. For much of that time the prevailing view considered diabetic nephropathy a glomerular disease. The minority view was that the proximal tubule played a central role. It is generally accepted that raised levels of the sugar d-glucose played an important role but the nature of that role remained unclear.
The development of a new class of drugs for the treatment of type 2 diabetes may help answer some of these questions. Under ‘normal’ conditions water and small molecules such as glucose pass through the glomerulus and are either excreted as urine or reabsorbed. Approximately 180 litres of water and 180 g of sugar pass through the glomerulus daily, most of which is reabsorbed by the proximal tubule. Approximately 90% of filtered glucose is reabsorbed by the sodium glucose transporter 2 (SGLT2). The gliflozin drugs reduce the activity of SGLT2. This results in reduced glucose reabsorption and more excreted in the urine. Gliflozins such as Canagliflozin, Dapagliflozin and Empagliflozin have proved extremely successful.
At SWTIR we developed a hypothesis that inhibition of SGLT2 could protect the proximal tubule cells and reduce the fibrotic signals produced by damaged proximal tubule epithelial cells (PTEC). With the help of Kidney Research UK we are investigating the role of extracellular glucose in PTEC expression of fibronectin and CCN2/CTGF. By determining the intracellular signalling cascades which mediate SGLT2 controlled fibrogenic processes we hope to be able to determine how the gliflozins work and whether a more efficient strategy could achieve the same ends.