Year : 2006 | Volume
: 17 | Issue : 2 | Page : 137--148
Renal Fibrosis, Origin and Possible Interventions: A Time for Action
E Nigel Wardle
37 Princess Road, Camden, London NW1 8JS, United Kingdom
E Nigel Wardle
37 Princess Road, Camden, London NW1 8JS
|How to cite this article:|
Wardle E N. Renal Fibrosis, Origin and Possible Interventions: A Time for Action.Saudi J Kidney Dis Transpl 2006;17:137-148
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Wardle E N. Renal Fibrosis, Origin and Possible Interventions: A Time for Action. Saudi J Kidney Dis Transpl [serial online] 2006 [cited 2019 Dec 9 ];17:137-148
Available from: http://www.sjkdt.org/text.asp?2006/17/2/137/35781
Fibrosis in the kidneys arises when there has been damage to the glomeruli or the tubules, resulting in atrophy of normal epithelium and loss of surrounding heparan sulphate proteoglycans. Subsequently, fibroblasts form new extra cellular fibrous tissue expanding the extra cellular space. There are many causes of tubulointerstitial fibrosis (TIF), ranging from the effects of hypertension, glomerulonephritides and pyelonephritis, to conditions causing heavy proteinuria, and any process that incites glomeruli or proximal tubules to produce pro-inflammatory mediators as happens in acute or chronic allograft rejection. Following injury, both interstitial cells and proximal tubular epithelium are activated, and myofibroblasts (MFs) appear. MFs, defined by their expression of α - smooth muscle actin (SMA), generally correlate with the degree of renal impairment and histological damage  for they play a key role in development of glomerulosclerosis and interstitial fibrosis.  [Table 1] summarizes the processes that trigger fibrosis. One should be aware that therapeutic corticosteroids synergize with TGFE in causing tissue fibrosis.
The fibroblasts in murine kidneys stain with fibroblast specific protein FSP-1, as well as the mesenchymal cell markers, LI-SMA and Vim (for vimentin). In normal kidneys, there are Vim positive fibroblasts, as one might find in the dermis, but no myofibroblasts. The latter can be formed from mesangial cells  and probably from capillary endothelial cells  and certainly by the transformation of tubular epithelial cells. Thus, Abbate et al  showed that, when there is uptake of ultra filtered proteins by the proximal tubules of rats, LISMA positive cells and macrophages appeared in the renal interstitium prior to tubulointerstitial fibrosis.
There is good evidence that tubular epithelial cells trans-differentiate into MFs that express FSP-1, α-SMA and Vim. This process, called epithelial-mesenchymal transformation (EMT), occurs in four steps under the direction of the fibrosing cytokine, transforming growth factor beta (TGFβ).  Initially, there is loss of adhesion between epithelial cells, since TGFβ stops expression of E-cadherin molecules. Following this, the tubular cells undergo reorganization of their actin cytoskeleton, and they express αSMA. Subsequently, there is dissolution of the tubular basement membrane. Finally, the new MFs that have arisen from epithelial cells migrate and invade the renal interstitium. Clearly, MFs are morphologically intermediate between fibroblasts and smooth muscle cells. They have the ability to contract and to be motile. They form fibronectin and collagens to add to the extra cellular matrix. Their surface integrins could determine where and how they do all this. EMT is actually produced by TGFβ acting along with epidermal growth factor (EGF). 19 We also know that administration of PDGF-BB to rats induces a dose dependent proliferation of tubulointerstitial cells and there is then an associated expression of αSMA., IL-10 too will induce proximal tubule cell injury, α-SMA expression in fibroblasts and fibronectin production. It has always been suspected that IL-4 will produce fibrosis, and indeed in situations in which the Th2 cytokines IL4 and IL-13 are being produced, as in the respiratory tract and lungs, fibrosis will ensue. The local inflammatory cells may then be producing or responding to chemokines like CCL2 and CCL3.
Cell Signalling: TGFβ causing Fibrosis
When pharmacological mediators, growth factors or cytokines act on target cells, their responses like contraction, proliferation or growth are determined by intracellular signalling cascades that pass on information by calcium ion influx, coupled with activation of enzymes, that lead to activation of intranuclear transcription factors (TFs), so that genes are induced. A general scheme is shown in [Figure 1].
One will wish to understand how the fibrosing cytokine TGFβ exerts its actions. Only by understanding the individual steps in signalling will appropriate blocking agents be devised. TGFβ is held dormant in tissues attached to latent TGFβ binding protein or Latency Associated Protein (LAP).  This strategy is important because TGFβis (a) an immunosuppressive agent, which can cause apoptosis of cells and, (b) it is the fibrosing cytokine. Glomerular cells produce a large size latent TGFβ1, whilst tubular cells produce a small latent TGFβ1. The integrin αVβ6 binds to, and activates latent TGFβ1; thrombospondin is a major activator  and so are the enzymes furin  or plasmin. Remember that angiotensin II in chronic renal failure stimulates extra cellular matrix protein synthesis through the induction of TGFβ1 expression in rat mesangial cells. Lysophosphatidic acid (LPA), from platelets often provokes TGFβ formation. [Table 2] lists various agents that stimulate TGFβ release.
With regard to the formation of extra cellular matrix by TGFβ, one discerns that (i) TGFβ inhibits enzymes of matrix degradation, for example by upregulating, plasminogen activator inhibitor (PAI-1), (ii) it promotes cell surface integrins pertinent to matrix organization, and it promotes synthesis of cell surface proteoglycans like heparan sulfates, and, (iii) as the principal fibrosing cytokine, it promotes the synthesis of collagens and fibronectin.
TGFβ binds firstly to its type II receptors, and then type I receptors are recruited, so that the TGFβ1/II-I complexes are able to initiate signals  The standard TGFβ signal pathway involves activation of SMAD protein TFs, which home into nuclei onto SBE, Smad Binding Elements, on the DNA. Oxidative stress aids the response.
The type I TGFβ receptors phosphorylate receptor Smads 1,2,3,5,8. As they translocate to cell nuclei, R-Smads form homo-oligomers and hetero-oligomers with each other, as well as hetero oligomers with facilitatory Smad 4. On the other hand, there are Inhibitor ISmads , which confer a degree of feedback control over the intensity and duration of TGFβ signalling. So, there are three groups of Smads: (i) There are receptor Smads, (ii) there is facilitatory Smad4, and (iii) there are I-Smads 6 and 7. Significantly, in scleroderma a decrease of I-Smad 7 means that there is enhanced TGFβ signalling and so marked fibrosis.  Normally, Smad 7 (I) will inhibit the fibrotic action of TGFβ on renal tubular epithelial cells by blocking the activation of R-Smad 2.  The anti-fibrotic agent decorin causes the phosphorylation of Smad 2, and so sequestration in the nuclei.  The antifibrotic agent halofuginone works by inhibition of Smads 2 and 3.  In some way, hyaluronan attenuates TGFβ1-mediated signalling in proximal tubular cells,  and the proteoglycan syndecan 2 also regulates the TER receptors. 
Although the scheme in [Figure 2] represents basic understanding of how fibrosis occurs, it does not represent the whole picture. Smads alone do not activate transcription, but they help in the recruitment of other TFs to gene promoters. Furthermore, TGFβ can activate other signalling pathways like the MAP (ERK) kinases.  One Smad-independent pathway of TGFβ signalling is through the TF c-jun/ c-fos that is called AP-1. By this means, there is formation of fibronectin and of PAI-1.  Also, by using its specific inhibitor, it has been shown how signalling through p38 MAPK is implicated in fibrosis.  Sp1 TF mediates stimulation of TGFβ on type IV collagen. TGFβ first stimulates the enzyme protein kinase CK2.  Then, one has to consider how the Rho GTPases will affect the αSMA of myofibroblasts and their motility.
As mentioned, interleukin 4 can up regulate type I collagen expression,  and interleukin 13 would be expected to behave likewise. Studies are now being reported on the excretion of TGFD in urine. Its excretion is elevated in lupus nephritis  and in diabetic nephropathy,  and in IgA nephropathy and other GNs. 
Connective Tissue Growth Factor (CTGF), is implicated along with TGFβ, and often independently of TGFβ in the formation of fibrous tissue.  CTGF up regulates extra cellular matrix proteins. In kidney cells, it is induced by TGFβ, by mechanical strain, by high glucose or by the AGEs that accumulate in diabetes. We now understand the modular structure of CTGF.  IGFBP/von WF/TSP1/C-terminal module, and how these units influence signalling. The insulin-like growth factor binding domain stimulates IGF-1. The von Willebrand factor-like type C domain stimulates TGFβ but inhibits BMP. The thrombospondin TSP-1 module inhibits VEGF. The C-terminal module interacts with integrins or with heparin sulphate proteoglycans on cell membranes.
Other proteins of the TGFβ superfamily are relevant to formation of extra cellular matrix, especially as this is one of their functions in embryonic development. Activin A shares features with TGFβ. In particular, it has been shown to be a pro-fibrotic cytokine in rat anti-Thy1 glomerulonephritis. Both IL-1 and bFGF promote activin A production in mesangial cells. Conversely, there are the BMPs, bone morphogenetic proteins, which like TGFβ signal through types I/II serinethreonine kinase receptors and downstream by means of Smads 1,5,8. Some BMPs antagonize fibrosis. Indeed BMP7 prevents epithelial to mesenchymal cell transition as a prelude to renal fibrosis. It does this by stimulating formation of E-cadherins, and hence epithelial cell differentiation. BMP7 can partially offset hypertrophy of the renal tubular cells in diabetes (an effect attributable to TGFβ), and it reduces glomerulosclerosis,  since it opposes signalling by TGFβ in mesangial cells. BMP represses basal and TNFα stimulated expression of pro-inflammatory cytokines IL-6 and IL-1β and release of chemokines IL-8 and MCP-1 in the proximal tubules.
Approaches to the Prevention of Fibrosis
I wish that I could present herewith a list of effective anti-fibrotic drugs, but unfortunately that stage has not yet been reached. Looking at the mode of action of TGFβ in [Figure 2], one will realise that Smad 3 is an important mediator of the fibrotic reaction. Indeed Smad 3 deficiency in Smad 3 (-/-) null mice attenuates renal fibrosis after unilateral ureteric obstruction.  Smad 3 is essential for TGFO1 induced EMT and for the autoinduction of TGFL1. The small MW alkaloid halofuginone is a Smad 3 inhibitor.  In cultures of PTECs, proximal tubule epithelial cells, over-expression of the I-Smad 7 blocks the actions of TGFβ.
What about natural inhibitors of fibrosis? Nitric oxide (NO), which in theory can be boosted by oral L-arginine or its infusion, is anti-fibrotic  as demonstrable in antiThy.1 glomerulonephritis. In fact, NO down regulates CTGF.  A low NO is evident in many cases of CRF with hypertension and in IgA nephropathy. However, in cases of immune mediated renal injury like lupus nephritis, iNOS may be boosted and be a cause of tissue damage.  In trials with patients, L-arginine supplementation (15 Gms/day) has not conferred benefit.
Salt sensitive hypertension is accompanied by oxidative stress, in large part due to enhanced activity of vascular NADH/NADPH oxidase, exactly as found with angiotensin II infusion. As was indicated in relation to [Figure 2], ROS induced in cultured mesangial cells enhance the synthesis of TGFβ and promote deposition of extra cellular matrix proteins., By kallikrein gene transfer, enhancement of low NO and reduction of tissue oxidative stress, and glomerular and tissue fibrosis has been demonstrated. Tissue kallikrein reduces NADPH oxidase levels in vessels. The antifibrotic agent pirfenidone, that is being used for pulmonary fibroses or ureteric obstruction, probably works by reducing tissue ROS.A beneficial effect has been demonstrated in anti-GBM nephritis in rats,  but this drug can cause side-effects in man.
Whilst on the topic of oxidative stress that arises from the action of activated NAD (P) H oxidases, 12- or 15-lipoxygenases and xanthine oxidase which all generate superoxide or by the uncoupling of NO synthase,, we should consider that 5-lipoxygenases promote fibrosis, as is evident from the inhibitory effect of montelukast. Lipoxygenase inhibitors can be used to treat mesothelioma. This means that they could reduce peritoneal fibrosis too. They could be useful in diabetic nephropathy (to follow).
What the experimentalists envisage will in due course produce clinical rewards. Clearly antisense molecules can be used to thwart the action of TGFβ and that of CTGF. Alternatively, in experiments, neutralising anti-TGFβ antibody (αT) could be used to offset progressive (anti-thymocyte serum) nephritis. Also, ECM accumulation in anti-Thy.1 GN has been ameliorated by use of an antibody to the TGFβ type II receptor. The tyrosine kinase inhibitor imatinib mesylate is now being used for therapy of chronic myeloid leukaemia. Actually, it also stops the nonreceptor tyrosine kinase c-Abl contributing to lung or renal  fibrosis in animals by a Smad independent means. Undoubtedly it will be tried in man. Similarly, a PDGF receptor kinase blocker attenuates experimental interstitial fibrosis produced by ureteric obstruction.  When it becomes available, there is one antibody that should be a great success, and that is antibody to macrophage migration inhibitory factor (MIF). K N Lai and colleagues in Hong Kong  showed that in experimental IgA nephropathy, anti-MIF results in decreased expression of TGFβ! More results are awaited with interest.
Surely, there are simpler approaches that any clinician might try. A group in Taiwan showed in 2001 that dipyridamole inhibits TGFD activation of the ERK pathway. Dipyridamole has often been used as adjunctive therapy for GNs, but an anti-fibrotic action was never anticipated. On reflection, any agent that inhibits platelets will reduce the release of PDGF.  Clopidogrel was shown to limit glomerular matrix expansion in anti-Thy.1 GN in rats,  but an ACE inhibitor was more effective. Heparins have an antiplatelet action. Heparin infusion can maintain the GFR when there is rapid deterioration of renal function due to glomerulonephritis or malignant hypertension. It was always thought that an anti-complement action was relevant. We now appreciate that heparin can reduce the expression of TGFβ.  Considering [Figure 3] again, there is an inhibitor of Rho-ROCK signalling in MFs that could turn out to be useful , Under its influence there is reduced CISMA expression and a reduction of infiltration by macrophages. Bosentan is an antagonist of endothelin-1  that can also reduce fibrosis. Indeed, it has been used for digital ulcers in patients with scleroderma. Alternatively, statin therapy can offset Et-1. Statins can reduce fibrosis in particular circumstances , but you will know that they are principally anti-inflammatory agents, and there can be side-effects.
We need to look to old friends. Angiotensin II receptor blockade reduces the effects of TGFβ.  Certainly, a combination of anti-TGFβ with enalapril reduced matrix deposition by 80% in anti-Thy.1 GN in rats. 82 Blockade of TGFβ signalling early in the course of anti-GBM nephritis in rats by means of soluble TGFβ receptor fused to IgG1 (called Tβ-ExR) ameliorated histological progression 
We do not have anti-TGFβ for use in humans: it would have side-effects like liability to autoimmune disease. However, losartan can prevent severe renal fibrogenesis caused by UUO. Furthermore, in patients with IgA nephropathy, losartan not only reduces proteinuria but also the urinary excretion of TGFβ.  In a mouse model of renal fibrosis, both an ACE inhibitor (ramipril) and an AT1 antagonist (candesartan) conferred substantial protection. 
Many of you will have been using thiazolidinediones in diabetic practice. The TZDs have metabolic and immune effects.  Troglitazone will suppress the formation of collagen by mesangial cells.  The uptake of albumin by renal tubular cells results in NF-kappa B activation and a proinflammatory  and fibrotic response. The latter is suppressed by pioglitazone.  Hence, therapy with TZDs can be expected to offset diabetic glomerulosclerosis.
Sometimes, an imaginative leap brings reward. The hormone relaxin is produced in pregnancy and it weakens the ligaments of the symphysis pubis, and it remodels the female genital tract. Relaxin increases synthesis of collagenase and it restructures the lattices of type I collagen. It down regulates activation of fibroblasts, as shown by a decrease of ESMA.  since it negates the effects of TGFβ. Accordingly, relaxin decreases renal interstitial fibrosis. The commercial production of this agent could have an impact.
Hepatocyte Growth Factor (HGF) was known originally as "scatter factor" because by acting on their Met Receptors, it can transform epithelial cells. This sounds a note of warning. HGF is detected in many cancers. It is a mitogen for endothelial and epithelial cells. Interestingly, on the edges of gastric ulcers, cytoprotective prostaglandins PGE1/2 provoke release of HGF from local fibroblasts and the ulcers will heal. Generally, HGF reduces fibrosis, since HGF inhibits ILK [Figure 2].  HGF is deficient in idiopathic pulmonary fibrosis. Cyclic AMP elevating agents like PGE2 turn on an anti-fibrotic phenotype in pulmonary fibroblasts.  We shall return to this theme. Meantime, note that in animals, shortterm administration of HGF ameliorates the progression of renal interstitial fibrosis , HGF enhances the formation of the anti-fibrotic mediator called decorin  Moreover, HGF antagonizes the action of TGFβ1 in mesangial cells by stabilizing a Smad corepressor called TGF Interacting Factor (TGIF). 
Is it possible that judicious use of PGE2 would release a controlled amount of HGF that would modulate fibroblasts? Certainly a while ago, PGE1 or PGE2 were infused for their anti-inflammatory/immunosuppressive potential in conditions like rapidly advancing lupus nephritis,  anti-GBM nephritis or vasculitides.  This is expensive therapy for selected patients. Yet we know now that PGE2 stops formation of collagen within the liver, and we know that PGE2 generally inhibits formation of myofibroblasts.  Do we have a simpler agent that raises intracellular cAMP and mimics the E-prostaglandins? Yes, there is pentoxifylline that we use normally to suppress formation of TNFa. It is known to reduce proteinuria in several situations. It has been noted by experimentalists to inhibit fibroblast proliferation.
We shall only find out how effective such agents might be, if clinical groups around the world will do investigative studies on their patients. Measurements of urinary TGFβ will aid the monitoring. Already it is clear that in diabetic nephropathy 12/15-lipoxygenases are promoting the release of TGFβ.
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