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NDT Advance Access originally published online on September 21, 2007
Nephrology Dialysis Transplantation 2007 22(12):3391-3407; doi:10.1093/ndt/gfm393
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


Treatment targets in renal fibrosis

Peter Boor1,2, Katarína Sebeková2, Tammo Ostendorf1 and Jürgen Floege1

1Division of Nephrology, RWTH University of Aachen, Aachen, Germany and 2Department of Clinical and Experimental Pharmacotherapy, Slovak Medical University, Bratislava, Slovakia

Correspondence and offprint requests to: Peter Boor, MD, Division of Nephrology, RWTH University of Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. Email: boor{at}email.cz

Keywords: animal models; kidney scarring; progressive renal disease; treatment options



   Introduction
Top
 Introduction
Conclusions
 Acknowledgements
 References
 
Renal fibrosis is the principal process underlying the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD). It is a relatively uniform response involving glomerulosclerosis, tubulointerstitial fibrosis and changes in renal vasculature (loss of glomerular and peritubular capillaries) (Figure 1). Of these, tubulointerstitial fibrosis has evolved as the most consistent predictor of an irreversible loss of renal function and progression to ESRD [1].


Figure 1
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  		Fig. 1. Severe tubulointerstitial fibrosis in an experimental rat model of progressive mesangioproliferative glomerulonephritis on day 100 after disease induction. The renal cortical section stained with periodic acid-Schiff (PAS) shows typical tubulointerstitial damage: accumulation of extracellular matrix (*), tubular atrophy (**) and inflammatory infiltrates (***). Glomerular damage is apparent as glomerulosclerosis (arrow) and proteinuria (double arrow). Magnification: 100x.

 
Mechanisms contributing to tubulointerstitial injury and tubular atrophy include glomerular proteinuria, chronic hypoxia, misdirected glomerular ultrafiltration, tubular protein leakage and direct toxic insults of e.g. drugs (reviewed in detail elsewhere [1–6]). Direct or indirect tubulointerstitial injury via oxidative stress and various effector molecules trigger cellular responses like (i) tubular epithelial cell (TEC) apoptosis, (ii) activation of fibroblasts and their phenotypic switch to myofibroblasts, (iii) influx and/or proliferation of lymphocytes/macrophages, fibrocytes (the circulating fibroblast precursors), fibroblasts as well as (iv) epithelial-to-mesenchymal transition (EMT) of TECs.

Renal fibrosis provides an excellent treatment target, since a large variety of pathophysiologically distinct diseases converge finally into this single process. However, we still do not have effective therapies, nor does such a therapy exist in most other types of organ fibrosis. Why? As part of the vital repair process, the regulation and redundancy in this system must be highly effective. The consequence is an amazingly complicated process that involves many cell types and mediators [1,2,4,5,7]. Not unexpectedly, mono-therapeutic approaches, or even a combination of therapies, fail to completely stop the progression of renal fibrosis [8–10]. In addition, not all combination therapies can be additive [11].

When identifying new targets or validating potential therapeutic options, we are confronted with several problems:

  • Rodent models often do not fully mimic the clinical situation. Apart from the obvious species differences, it is often difficult to distinguish whether the tested approach truly affected the phase of interstitial fibrosis or whether it ameliorated the underlying primary renal injury to an extent that halted the progression. This problem is particularly relevant to the present review.
  • Few approaches have been validated in multiple models.
  • In the experimental situation, treatment is rarely started at a time point of established fibrosis (Table 1).
  • Different parameters and techniques are used for the evaluation of fibrosis. Often histological changes are evaluated by semi-quantitative scoring rather than objective quantitative parameters. Standardized consensus approaches are urgently required.
  • Most studies compare intervention to no intervention rather than comparing different approaches amongst each other or in combination with one another.


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  		Table 1. Examples of experimental studies that showed effective antifibrotic treatment aproaches in established secondary renal fibrosis

 
In the following, we will first discuss antifibrotic approaches, that are clinically available or close to being so, and then discuss potential new targets in fibrosis therapy (Table 2).


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  		Table 2. Summary of reviewed targets and/or compounds for treatment of renal fibrotic disease

 
Renin–angiotensin–aldosterone (RAAS) and kallikrein–kinin system
Angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor type 1 blockers (ARB) are undisputedly the first line drugs in combating renal fibrosis. These drugs, however, are not able to halt the progression completely and in some conditions, like aristocholic acid-induced renal fibrosis in rats, they are not effective at all [12]. Combination therapy of ACEI and ARB [13] or high-dosage of ACEI [14] show potential in experimental studies to arrest or even regress renal fibrosis, at least in the early stages.

Kallikrein cleaves the precursor kininogen to the active vasodilator kinin peptide, bradykinin, which via its B2 receptors mediates protective effects in hypertension, renal injury and fibrosis [15,16]. Gene delivery of kallikrein ameliorated renal scarring without affecting the blood pressure [17], and even reversed established kidney fibrosis [18]. ACE is the same enzyme as kininase II, which degrades active kinins. Thus, ACE inhibition leads to kinin accumulation, which may contribute to the beneficial effects of ACEI.

Aldosterone inhibition, e.g. with mineralocorticoid receptor blockers (spironolacton, eplerenone), also shows beneficial effects in experimental as well as in clinical studies (reviewed in [19,20]).

Renin inhibitors (aliskiren, enalkiren, zalkiren) are promising drugs for combating renal fibrosis as shown in severely hypertensive transgenic rats (dTGR) harbouring the human renin and angiotensinogen gene [21]. Clinical effects on renal fibrosis remain to be determined. The recently discovered receptor for renin and prorenin [(P)RR – (pro)renin receptor] was linked to glomerular fibrosis [22]. (P)RR blockers might be even more potent than renin inhibitors, since renin inhibitors do not block renin or prorenin binding to and activation of (P)RR [22].

Vasopeptidase inhibitors (e.g. AVE7688, omapatrilat) block both ACE and neutral endopeptidase, which results in a more pronounced blood pressure decrease, as compared with ACEI. AVE7688 potently retarded renal fibrosis in an animal model of Alport syndrome [23]. However, the higher incidence of angioedema with omapatrilat as compared with ACEI has limited clinical studies thus far.

Endothelin, sympathic nerve system
Endothelin, acting via its receptors ETA and ETB, is a potent vasoconstrictor and mediator of fibrotic response [2,24–26]. Although the dual inhibitor of ETA and ETB bosentan is in clinical use, its benefits vs side effects (mainly hepatotoxicity and fluid retention) in CKD patients remain largely to be evaluated.

Blockade of sympathic nerve activity with moxonidine reduced the progression of fibrosis in 5/6-nephrectomized rats without affecting blood pressure [27,28]. Selective {alpha} and β blockers in subantihypertensive dosages also ameliorated the development of renal fibrosis [29,30]. In cyclosporine-induced renal damage, however, kidney denervation had no effect on the development of fibrosis [31].

Environmental factors and metabolic syndrome
Smoking accelerates progression of kidney diseases, in part through blood pressure-mediated effects [32], but at least in experimental situations, also via a possible profibrotic effect [33,34]. Another independent factor that leads to progression of kidney disease is obesity [36]. Various pathways, including increased metabolic demands on the kidney, hyperinsulinaemia or leptin overproduction, were shown to induce or promote kidney scarring in obesity [37–40]. Diet-induced hypercholesterolaemia in rodents or pigs resulted in renal fibrosis, which was reversible by lipid-lowering dietary interventions [41,42]. Several experimental studies demonstrated the protective effect of statins on kidney scarring, which may relate to their lipid-lowering but also to their anti-inflammatory actions [43–45]. First clinical data confirmed the renoprotective effects of statins [46] and large trials in CKD patients are underway.

Immunosuppressive agents
Mycophenolate mofetil (MMF) ameliorated fibrosis in various experimental models [47–50]. These effects were comparable, but not additive, with those of enalapril or lisinopril [48,49]. Confirmatory data in transplanted patients has recently emerged [51]. Rapamycin attenuated renal fibrosis in patients with kidney transplantation [52] and in unilateral ureteral obstruction (UUO) in rats [53]. Dexamethasone reduced fibrosis in renin transgenic rats similiarly to MMF [55], but failed to reduce fibrosis in mercuric chloride-treated rats, whereas tacrolimus was effective in the latter [56].

Turnover and composition of the extracellular matrix
Matrix metalloproteinases (MMPs) were initially thought to be beneficial in renal fibrosis, given that they degrade extracellular matrix (ECM) proteins. Recent data suggest that their role in renal scarring may be much more complex [57] (Figure 2). In renal fibrosis, local delivery of MMP-1 reduced collagen content in streptozotocin-induced diabetic nephropathy in rats [58]. On the other hand, tubular MMP-2 overexpression in mice induced renal fibrosis [59] and selective pharmacological MMP-2 inhibition increased fibrosis in UUO [60].


Figure 2
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  		Fig. 2. Complexity of MMP driven proteolysis. Each of the 23 human and 24 murine MMPs can be regulated at different levels, ranging from RNA processing to activation of inactive MMPs (zymogens) and/or degradation of active MMPs by other proteases (e.g. other MMPs). The broad range of the MMPs targets resulting in a diversity of biological functions adds another level of complexity to this protease system. With respect to fibrosis, it seems obvious, that MMPs should not be viewed as merely ECM degrading ‘anti-fibrotic’ molecules. Adapted and modified from [285]. ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EMT, epithelial-to-mesenchymal transition; FGF, fibroblast growth factor; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein 1; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; TGF-β, transforming growth factor-β; TIMP, tissue inhibitor of MMPs; TNF-{alpha}, tumour necrosis factor-{alpha}; VEGF, vascular endothelial growth factor.

 
Tissue inhibitors of matrix metalloproteinases (TIMP-1 to -4) are natural MMP inhibitors. Genetic TIMP-1 deletion in mice had no influence on experimental kidney fibrosis [61,62], whereas overexpression of TIMP-1 increased age-related fibrosis [63].

Membrane-bound ‘a disintegrin and metalloprotease domain’ proteases (ADAMS) and secreted ‘ADAM with thrombospondin motifs’ proteases (ADAMTS) are two other important families of proteases (containing more than 50 members). ADAM-19 is abundant in fibrotic renal lesions in human biopsies [64]. ADAMTS-1-deficient mice exhibited kidney fibrosis in addition to renal malformations [65].

Some proteases of the fibrinolytic pathway, i.e. plasminogen activator inhibitor-1 (PAI-1) [66–69], tissue-type plasminogen activator (tPA) [70,71] and plasminogen [72,73] are important profibrotic factors. However, in anti-glomerular basement membrane (anti-GBM) nephritis, all three factors proved to be antifibrotic, which may relate to their involvement in the glomerular coagulation cascade, i.e. effects on the primary renal disease as opposed to direct effects on renal fibrosis [68,74,75]. Genetic urokinase-type plasminogen activator (uPA) deficiency had no effect on fibrosis in UUO or anti-GBM nephritis; deficiency of its receptor (uPAR) was antifibrotic in UUO but not in anti-GBM nephritis [66].

Tissue transglutaminase (Tg) stabilizes ECM by protein cross-linking. This leads, for example, to resistance of such modified collagens to proteolytic degradation by MMPs. Tissue Tg is upregulated and correlates closely with the severity of renal fibrosis in experimental models and in humans [76,77]. At least in vitro, inhibition of tissue TG reduced glucose-induced deposition of ECM proteins in renal proximal tubular epithelial cells [77].

Oral administration of a protease mixture (trypsin and bromelain with rutosid added as an antioxidant) reduced renal fibrosis in rats with 5/6-nephrectomy or Goldblatt hypertension independently of blood pressure [78,79]. Single proteases or their mixtures are used in human medicine.

Relaxin is a small peptide hormone with potent antifibrotic activity in the kidney [80–82]. Its benefit may derive in part from interference with transforming growth factor-β (TGF-β), a potent profibrotic cytokine [83]. Continuous subcutaneous infusion of relaxin showed promising results in a phase II clinical trial in patients with systemic sclerosis, but this effect was not confirmed in a phase III trial [84].

Pirfenidone is a potent inhibitor of ECM accumulation as shown in several experimental models of renal damage, e.g. mesangioproliferative GN, 5/6-nephrectomy or UUO [85,86], and in patients with idiopathic pulmonary fibrosis and advanced liver fibrosis. We lack clinical data in renal patients.

Integrins are heterodimeric cell receptors for the ECM. Integrin {alpha}1 chain-deficient mice developed more severe glomerulosclerosis in adriamycin-induced renal injury [87]. In contrast, genetic {alpha}1 chain-deficiency or antagonism of {alpha}1 chain reduced fibrosis in a mouse model of Alport syndrome [88] and in rats with crescentic GN [89] or mesangioproliferative GN [90]. Integrin {alpha}Vβ6 binds to and activates latent TGF-β. Mice deficient of the β6 integrin chain exhibited reduced fibrosis following UUO [91]. Deficiency of β6 or {alpha}Vβ6-blocking antibodies also reduced fibrosis in Alport mice [92]. In contrast, genetic deficiency of the {alpha}8-chain did not contribute to glomerulosclerosis or fibrosis [93]. Outside-in signalling of integrins involves, amongst other functions, activation of the integrin-linked kinase (ILK) pathway. ILK was suggested to participate in the development of renal fibrosis [94,95].

Collectively these data suggest that some systems, such as MMPs, the fibrinolytic pathway and integrins, may be difficult to interfere with clinically, given their complex nature, their redundancy and their sometimes opposing biological effects. However, some attractive therapeutic options such as Tg- antagonists are starting to emerge.

Complement system
The terminal complex of complement, C5b-9 (or membrane attack complex—MAC) is formed at sites of tubulointerstitial injury [1], its depletion in experimental nephropathy reduces proteinuria [96] and inhibition of MAC formation in proteinuric animal models ameliorated tubulointerstitial injury [1,97–99]. However, genetic C6 deficiency, which precludes MAC formation, did not affect the severity of non-proteinuric models of tubulointerstitial injury [100], suggesting that MAC mediates tubulointerstitial damage in proteinuric renal diseases only [100].

Besides C5b-9, complement C5 and in particular C5a may also become a target in renal fibrosis: in experimental murine immune-complex GN, genetic deficiency of the C5a receptor (C5aR) led to reduced interstitial cell infiltration and tubulointerstitial damage without affecting the glomerular injury. This pointed to a role of the anaphylatoxin C5a, a small peptide released from C5, in tubulointerstitial injury [105]. In the mouse UUO model, genetic C5 deficiency or pharmacological inhibition of C5aR potently reduced tubulointerstitial fibrosis [106]. C5aR deficiency or antagonism also attenuated the course of lupus nephritis in mice [107,108]. C5aR antagonists and anti-C5 antibodies (eculizumab) were/are being evaluated in clinical trials.

Complement regulatory proteins such as Crry, which inhibits C3 activation, or CD59, which inhibits C5b-9 formation, may also be of interest: Local deficiency of Crry aggravated tubular damage and fibrosis in puromycin-induced nephropathy (PAN) and in models of renal transplantation [101,102]. Crry and CD59 were equally therapeutic in PAN [103]. Treatment with Crry also decreased renal ECM accumulation in murine lupus nephritis [104], but here, effects on the immune system vs direct, antifibrotic effects are difficult to distinguish.

Cytokines
Few data are available on the role of interleukins (IL) in renal fibrosis. One exception is IL-1β, whose profibrotic properties in experimental renal disease have been established [109–113]. Although an IL-1 receptor antagonist (anakinra) is already in clinical use, it has not been tested so far in patients with renal fibrosis. In mice lacking the IL-8 receptor, which mediates transepithelial migration, trapped neutrophils lead to tissue destruction and renal fibrosis after experimental pyelonephritis [114]. An antifibrotic action of IL-10 was described in 5/6-nephrectomized rats [115]. IL-10 is also anti-inflammatory and anti-atherogenic and systemic treatment was well tolerated in psoriasis patients [116]. However, systemic treatment with IL-10 was ineffective in patients with rheumatoid arthritis or Crohn's disease [116], unless locally delivered in the gut of Crohn's patients. Thus, local delivery in renal patients could become a limiting factor. It also should be stressed that in systemic lupus erythematosus IL-10 seems to be a harmful factor [116]. Early IL-4 administration in rats with crescentic GN diminished tubulointerstitial fibrosis in the later course of the disease [117]. Although IL-5, -6 and -13 are involved in the pathogenesis of some renal diseases, no data currently exist on their role in renal scarring.

Interferon-{gamma} (IFN-{gamma}) has antifibrotic properties in vitro [118] and administration to 5/6-nephrectomized rats decreased kidney fibrosis [119]. However, in rats with mesangiopoliferative GN, IFN-{gamma} treatment decreased mesangial cell proliferation but not glomerular ECM accumulation [120]. IFN-{alpha} reduced interstitial fibrosis in carbon tetrachloride-induced nephrotoxicity [121]. Although IFN treatment long-term clinical therapy is feasible, the case for such treatment in renal fibrosis patients is not particularly strong.

Tumour necrosis factor-{alpha} (TNF-{alpha}) is a well-recognized promoter of fibrosis in the kidney [122–124]. Although anti-TNF-{alpha} antibodies are highly effective in patients with rheumatoid arthritis, side effects such as serious infections and malignancies [125], or increased mortality in patients with chronic heart failure [126], may limit clinical trials in renal fibrosis.

Chemokines
Several chemokines and their receptors were shown to act in a profibrotic manner, mainly via recruitment of inflammatory cells into the tubulointerstitium [6]. These profibrotic molecules include monocyte chemoatractant protein-1 (MCP-1/CCL2) and its receptor CCR2 [127–133], RANTES (CCL5) and its receptor CCR1 (but not CCR5) [134–137], macrophage-colony stimulating factor (M-CSF) [138], osteopontin [139–141] and fractalkine receptor 1 (CX3CR1) [142]. The secondary lymphoid tissue chemokine (SLC/CCL21) and its receptor CCR7 have been implicated in the regulation of fibrocyte infiltration of the renal interstitium and promotion of fibrosis [143]. Several problems render a translation of these results into clinical trials difficult [6]: (i) There are considerable species differences in the expression of chemokines and their receptors. (ii) Although profibrotic in most disease models, chemokines might be also beneficial; e.g. lack of CCR1 or CCR2 was antifibrotic in UUO but aggravated renal injury in crescentic GN [144,145]. (iii) Chemokine receptor antagonists developed for clinical use, e.g. antagonists of CCR2 and CCR5, cross-react with other closely related G-protein-coupled receptors, resulting in insufficient specificity in humans. An alternative approach could be the inhibition of chemokine signalling via phosphatidylinositol-3-kinase-{gamma} (PI3K{gamma}). Indeed, a PI3K{gamma} inhibitor ameliorated the severity of lupus nephritis in mice [146].

TGF-β, TGF-β signalling molecules, p38 MAPK, bone morphogenic protein-7 (BMP-7)
TGF-β, a central mediator of fibrosis, exerts its biological, in particular immunological, functions via complex signalling pathways [147]. In view of its potent action as an endogenous immunosuppressant, complete blockade of TGF-β signalling could have serious adverse consequences. Thus, TGF-β-deficient mice die of a multifocal inflammatory syndrome [148]. However, the complexity of TGF-β regulation and its downstream pathways provide possibilities for more specific treatment targets (e.g. BMP-7, HGF, CTGF, Smads) (Figure 3). It should be mentioned that while most of the research focuses on the TGF-β1 isoform, the other isoforms, β2 and β3, also have profibrotic effects on kidney cells [149].


Figure 3
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  		Fig. 3. TGF-β signalling and it modulation. The simplified scheme shows Smad signalling of TGF-β and BMP-7 and mainly encompasses molecules discussed in the text. Different molecules modify extracellularly the activity of TGF-β or BMP-7 (note: CTGF effects on BMP-4 have been shown, but its effects on BMP-7 signalling are less clear). TGF-β or BMP-7 binding to their receptors induces heteromeric receptor complexes with kinase activation (ALK5 and ALK3, respectively) that leads to recruitment and phosphorylation of Smads (Smad2/3 and Smad1/5, respectively). These phospho-Smads (pSmad) form heteromers with Smad4 and are transported to the nucleus where they regulate gene expression. Antagonistic effects of BMP-7 on TGF-β signalling are depicted in the example of collagen type I expression and on nuclear shuttling and phosphorylation of Smad3. Non-Smad TGF-β signalling, shown in the right side of the picture, can involve activation of p38 MAPK, JNK and Rho. The modulation of TGF-β-induced gene transcription by Smad co-repressors Ski and SnoN in nucleus is also shown. Arrows indicate stimulatory effects and blunted lines inhibitory effects. ‘P’ stands for phosphorylation. ALK3, BMPR-IA kinase; ALK5, TβRI kinase; BMP-7, bone morphogenic protein-7; BMPR-IA, BMP receptor IA; BMP-RII, BMP receptor II; JNK, c-Jun amino-terminal kinase; KCP, kielin/chordin-like protein; p38 MAPK, p38 mitogen-activated protein kinase; TβR, TGF-β1 receptor; TGF-β, transforming growth factor-β; USAG-1, uterine sensitization-associated gene 1.

 
Overexpression of Smad7, an inhibitory factor in the TGF-β signalling pathway, reduced renal fibrosis in 5/6-nephrectomy and UUO [150–152]. Alternatively, genetic deletion of the agonistic signalling molecule, Smad3, protected kidneys from interstitial fibrosis [153,154]. The plant alkaloid halofuginone is an inhibitor of collagen I synthesis, recently shown to also inhibit Smad3 [155]. Halofuginone was successfully used for topical treatment of fibrosis in patients with scleroderma, had good tolerability after oral administration and is currently being tested in cancer patients. Snail is a transcription factor involved in EMT and is strongly activated by TGF-β. Activation of Snail in adult transgenic mice induced renal fibrosis and Snail was found to be overexpressed in human fibrotic kidney [156]. Other targets in renal scarring might be the antifibrotic Smad co-repressors, Ski and SnoN [157,158]. Inhibition of the TGF-β1 receptor kinase (ALK5) alone [159,160] or in combination with inhibition of p38 mitogen-activated protein kinase (MAPK) [161] ameliorated renal fibrosis. Inhibition of p38 MAPK also reduced fibrosis in UUO and unilateral kidney ischaemia–reperfusion [162,163], and p38 MAPK inhibitors are currently tested in clinical trials in patients with inflammatory diseases, e.g. asthma. Lack of decorin, a proteoglycan involved in ECM assembly and a TGF-β antagonist, elevated interstitial fibrosis in UUO [164] and delivery of decorin by gene therapy prevented fibrosis in glomerulonephritic rats [165].

BMP-7 was shown to reduce (more potently than enalapril) or even reverse renal interstitial fibrosis in various experimental models [166–170]. Furthermore, treatment with BMP-7 had beneficial effects on renal osteodystrophy and reduced vascular calcification in uraemia [171,172]. However, in the protein overload model in rats, treatment with BMP-7 protein showed no significant effects on renal fibrosis [173]. BMP-7 therapy in patients has not yet been reported but the results of ongoing studies are eagerly awaited. Kielin/chordin-like protein (KCP) and uterine sensitization-associated gene-1 (USAG-1) are an endogenous BMP-7 agonist and antagonist, respectively. Mice lacking KCP were more susceptible to development of renal fibrosis [174], whereas mice lacking USAG-1 were protected [175].

Hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF; CCN2)
HGF was shown to have antifibrotic properties in different animal models via inhibitory effects on Smad2/3 and activation of SnoN, which binds and inactivates Smad2 [176–179]. However, HGF overexpression or its glomerular ultrafiltration in diabetic nephropathy rats with proteinuria have also been implicated in the development of renal fibrosis [180,181]. The pro-carcinogenic HGF effects [182] raise safety concerns for clinical trials.

CTGF, a potent profibrotic molecule, is at least in part a direct downstream mediator of TGF-β. It decreases vascular endothelial growth factor (VEGF) signalling, enhances signalling of several growth factors [e.g. TGF-β itself, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF)] and directly regulates cell functions (adhesion, migration, proliferation) via binding to integrins and ECM [183,184]. CTGF is a direct mediator of profibrotic effects of AGEs, is up-regulated in humans with renal fibrosis and its inhibition resulted in a potent reduction of fibrosis in experimental diabetic nephropathy, UUO and 5/6-nephrectomy [183–188]. Anti-CTGF therapy is now in clinical trials in diabetic nephropathy.

Vascular endothelial growth factor (VEGF)
VEGF is a potent angiogenic factor, which is also involved in fibrosis [189]. In rats with 5/6-nephrectomy, cyclosporine nephropathy and thrombotic microangiopathy, administration of VEGF121 reduced fibrosis and renal damage [189–191]. Consistent with this, VEGF antagonism accelerated renal damage in mice with progressive crescentic GN [192] and in rats with mesangioproliferative GN [193]. However, the action of VEGF may depend on the biological context, since, in contrast to the above data, a VEGF neutralizing antibody ameliorated early renal injury, in particular glomerular hypertrophy, in 5/6-nephrectomized rats, in mice fed with a high-protein diet and in rats with diabetic nephropathy [194–197]. Various anti-VEGF aproaches are approved for use in patients, and renal side effects include proteinuria and hypertension [198]. Given the above data, the complexity of the VEGF system (five family members with alternative splicing, four different receptors) and potential adverse pro-angiogenic effects of VEGF-therapy, it appears uncertain whether VEGF administration or blockade will become a useful approach in patients with renal fibrosis.

Platelet-derived growth factor (PDGF)
PDGF is a key mediator of fibrosis in different organs, including the kidney (Table 3 and Figure 4). The PDGF–B and –D isoforms mediate glomerular ECM accumulation in GN [199]. Their main receptor, i.e. PDGFR-β (Figure 4), is upregulated in the fibrotic tubulointerstitium [199]. PDGF-B also exerts its profibrotic effects in the tubulointerstitial compartment [200,201]. PDGF-D expression increases in obstructive uropathy in both humans and mice [202]. In rats with progressive mesangioproliferative GN, treatment with PDGF-D-neutralizing antibody ameliorated the early glomerular damage and the subsequent tubulointerstitial fibrosis [203]. Furthermore, this treatment prevented progression and fibrosis, even if initiated at the stage of established tubulointerstitial damage [204]. In a phase I clinical trial, a PDGF-D-neutralizing antibody had promising safety/tolerability profile, pharmacodynamic properties and long half-life (Hahne et al., J Am Soc Nephrol 2005; 16: Abstract SA-PO1017).


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  		Table 3. Experimental in vivo modulation of PDGF signalling in renal fibrosis

 

Figure 4
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  		Fig. 4. Simplified scheme of PDGF signalling and its regulation. Only the main molecules relevant for this review are listed. PDGF -AA, -AB and -BB are secreted in an active form whereas PDGF -DD and -CC are activated extracellularly via proteolytic cleavage of CUB-domain by urokinase plasminogen activator (tPA) or tissue plasminogen activator (uPA), respectively. Binding of PDGF isoforms results in autophosphorylation of the appropriate PDGF receptor. This subsequently leads to recruitment and activation of downstream signalling pathways such as Jak/STAT, PI3K, PLC-{gamma} or MAPK, which via regulation of gene expression mediate the biological functions of PDGF isoforms, e.g. proliferation, chemotaxis, migration or ECM production. The signalling pathways depicted are common to all three PDGF receptors and the signal transduction results in overlapping yet distinct biological effects depending on the receptor and cell type (e.g. via different binding affinities of the signalling molecules to phosphorylated receptors). An example of a relevant profibrotic cross-talk is the cooperation of PDGF and integrin signalling. Binding of ECM components (e.g. fibronectin, laminin) to integrins activates outside-in signalling via the focal adhesion kinase (FAK) pathway that enhances PDGF induced MAPK signalling. This results in enhanced cell proliferation and migration. Integrins also cooperate with other receptors for growth factors and directly activate signalling pathways such as PI3K, JNK and ILK. Various other regulatory steps might be involved in PDGF-signalling in fibrosis, e.g. regulation of PDGF and PDGFR expression, PDGF degradation or binding to non-signalling molecules (e.g. SPARC or heparin-sulphate proteoglycans). ECM, extracellular matrix; FAK, focal adhesion kinase; ILK, integrin linked kinase; JAK, Janus kinases; JNK, c-Jun amino-terminal kinase; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphatidyl-inositol-3-kinase; PLC-{gamma}, phospholipase C-{gamma}; STAT, Signal transducers and activators of transcription; tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator.

 
The PDGFR-{alpha} ligand PDGF-C (Figure 4), also seems to be an important mediator of fibrogenic stimuli. PDGF-C is expressed de novo in the fibrotic interstitium of rat and human kidneys [205,206] and its specific antagonism in the mouse UUO reduced fibrotic changes (Eitner et al., submitted for publication).

Imatinib (STI-571) is widely used in cancer therapy as a multi-kinase blocker of the c-abl, c-kit and PDGFR tyrosine kinases. Imatinib ameliorated renal fibrosis in UUO [207] and retarded the development of experimental diabetic [208] and chronic allograft nephropathy [209]. It remains uncertain whether this beneficial effect of imatinib was indeed mediated via reduction of PDGF signalling or whether it related to effects on c-abl kinase [207]. In clinical studies, side effects of imatinib include effects on haematopoesis, heart [210] and bone [211].

Other growth factors
The role of basic fibroblast growth factor-2 (FGF-2) in renal fibrosis is well established, at least in vitro [212–214]. FGF-1 and its receptor were also identified in human interstitial fibrotic lesions [215]. Specific interventions have not been performed in either case in renal fibrosis.

Inhibition of epidermal growth factor receptor (EGFR) is protective against the development of fibrosis in models of hypertensive renal damage and renal mass reduction [216,217]. Expression of a dominant negative EGFR prevented renal lesions induced by chronic Ang II infusion [218].

Similarly protective in the above model were the genetical deletion of TGF-{alpha} or pharmacological inhibition of its activating sheddase TACE (ADAM17) [218]. Anti-EGFR treatment is approved for treatment of some cancers.

Nitric oxide, NF-{kappa}B and Rho/Rho kinase
Enhanced production of nitric oxide (NO) following L-arginine administration prevented progression of renal fibrosis in several models [219–221] but accelerated it in mice with lupus nephritis [222] and adriamycin nephropathy [223]. In two different models of non-immune progressive kidney damage, pharmacological inhibitors of inducible NO synthase (NOS) [223] or endothelial NOS [224] reduced renal fibrosis. Studies in the UUO model using iNOS-deficient mice or using liposome-mediated iNOS gene transfer confirmed the protective role of iNOS [225,226], and genetic eNOS deficiency in mice led to progressive focal kidney scarring [227]. A second messenger of NO, cGMP, is synthesized by soluble guanylate cyclase (sGC) and degraded by phosphodiesterases (PDE). Stimulation of sGC slowed fibrosis development in progressive mesangioproliferative GN [228]. The PDE-5 inhibitor, sildenafil, effectively prevented the developmet of fibrosis, but it was ineffective in reversing established lesions [229]. Another PDE inhibitor, pentoxifiline, was antifibrotic in UUO and, combined with an ACEI, after 5/6 nephrectomy [230,231], but was less effective in a head-to-head comparison with a compound stimulating sGC [231]. In UUO, however, neither PDE-4 inhibition nor an A2A adenosine receptor agonist were able to reduce kidney damage [232].

Nuclear factor-{kappa}B (NF-{kappa}B) activation is a central convergence point of many proinflammatory pathways and a downstream effector of profibrotic molecules such as AGEs, the RAAS system or Smad7 [233]. Reduced renal fibrosis was noted following inhibition of NF-{kappa}B, e.g. with curcumin (diferuloylmethane) [233,234]. First clinical trials with curcumin in patients with malignancies showed no dose-dependent toxicity and phase II trials are underway.

Signalling via the G protein Rho and its associated Rho-kinase (ROCK) has been linked with renal fibrosis using two Rho-kinase inhibitors, Y-27632 and fasudil [235–238]. Both were already successfully used in clinical trials in patients with cardiovascular diseases [239]. Nevertheless, genetic deletion of ROCK1 had no effect on renal fibrosis in UUO [240]. The involvement of Rho/ROCK in human renal disease is largely unkown.

Stem cells
Stem cells hold great promise in acute renal failure and possibly even in chronic, fibrosing renal failure [241]. Indeed, three recent studies showed a beneficial effect of bone marrow-derived cells in a mice model of Alport syndrome [242–244]. However, very few studies have so far addressed the latter situation, and the challenge is to prevent stem cells receiving profibrotic signals from their environment and/or to prevent their maldifferentiation. Thus, it has recently been shown that bone marrow-derived myofibroblasts will contribute to renal fibrosis following ischaemia reperfusion injury [245]; and we observed that administration of mesenchymal stem cells to rats with progressive mesangioproliferative GN initially improved acute renal failure, but in the long-term, these cells adopted an adipocyte-like phenotype in glomeruli and induced an intense fibrotic response [246].

Other treatment targets and approaches
There is a number of targets that might be potentially effective in treatment of renal fibrosis (Table 2). Some of them were already tested in several experimental and/or small clinical studies, e.g. tranilast, 1,25 dihydroxyvitamin D, erythropoietin or glitazones, whereas others are single-study reports or descriptive studies without functional links to renal fibrosis (Table 2).



   Conclusions
Top
 Introduction
 Conclusions
Acknowledgements
 References
 
In the past years, many promising targets for the treatment of renal fibrosis have been validated in various animal models, and even more new targets have been identified. For several of the targets reviewed, substances/drugs have already been developed, are being tested, or are already being therapeutically employed in patients with non-renal indications. Renal fibrosis, in contrast, remains a largely uncharted territory in clinical trials. The reasons for this are certainly multifactorial and may include long study durations if hard endpoints, i.e. loss of GFR, are to be aimed for and, in particular, the lack of non-invasive markers or diagnostic tools to assess kidney scarring, and thus, monitor therapy. However, the industry has noted the enormous potential market, given the possibility of developing antifibrotic therapy that might be of benefit in many different types of organ fibrosis. Furthermore, there is hope that with a large consortia search for biomarkers and advancing ultrasound, or through MR-based or molecular-imaging techniques, even monitoring of the disease process may become feasible in the near future.



   Acknowledgements
Top
 Introduction
 Conclusions
 Acknowledgements
References
 
We apologize to all authors whose important work we could not cite due to space limitations. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) OS 196/1-1 (T.O.) and SFB 542, project C7 (T.O., J.F.).

Conflict of interest statement. None declared.



   References
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 Conclusions
 Acknowledgements
 References
 

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