Chronic hypoxia as a mechanism for progressive renal fibrosis

Drug Discovery Today: Disease Mechanisms

DRUG DISCOVERY

TODAY

Vol. 4, No. 1 2007

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA

DISEASE Renal diseases MECHANISMS

Chronic hypoxia as a mechanism for progressive renal fibrosis Jill T. Norman1,*, Leon G. Fine2 1

Centre for Nephrology, Department of Medicine, Department of Biochemistry and Molecular Biology, 2nd Floor, Royal Free and University College Medical School, Hampstead Campus, Rowland Hill Street, London NW3 2PF, UK 2 Cedars Sinai Department of Biomedical Sciences, Cedars Sinai Medical Center, 8700 Beverly Boulevard, Davis Building 5072, Los Angeles, CA 90048, USA

Renal fibrosis is the hallmark of chronic kidney disease (CKD) leading to organ failure and the need for renal replacement therapy. An increasing body of evidence

Section Editor: Michael S. Goligorsky – Renal Research Institute, New York Medical College, Valhalla, NY, USA

suggests that tissue hypoxia, which is sustained, among other things, by progressive microvascular loss, plays an important role in the pathogenesis of ongoing renal fibrosis via direct and indirect mechanisms. Therapeutic strategies that target chronic hypoxia may be of

high for affected individuals in terms of a reduced quality of life and shortened life expectancy. Current therapies are aimed at the control of known risk factors via a combination of lifestyle modification and pharmacologic intervention but there is a clear need for new approaches.

benefit in ameliorating this intractable disease. Chronic kidney disease and renal fibrosis Introduction Renal fibrosis is the hallmark of chronic kidney disease (CKD) of diverse aetiologies in which accumulation of extracellular matrix (ECM) disrupts normal tissue architecture leading to progressive renal dysfunction and end-stage renal disease (ESRD) necessitating renal replacement therapy (RRT), viz. dialysis or transplantation [1]. ESRD is increasing worldwide affecting millions of individuals. In developed countries the annual incidence of dialysis has doubled over the last decade and is highest in the United States (339 new patients per million of population [2]). A further doubling of the number of people receiving RRT is predicted over the next 10 years driven at least in part, by the aging population, the global epidemic of diabetes and the increasing incidence of hypertension and of obesity [1]. Treatment of ESRD imposes a substantial economic burden; in USA the annual expenditure is predicted to exceed $28 billion by 2010 [1]. Costs are also *Corresponding author: J.T. Norman ([email protected]) 1740-6765/$ ß 2007 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmec.2007.06.004

Chronic kidney disease presents a common pathology of glomerulosclerosis and tubulointerstitial fibrosis. Although CKD may initiate with glomerular injury, it is the degree of tubulointerstitial involvement that provides the best prognostic indicator of disease progression. Tubulointerstitial fibrosis displays several characteristic features [3]: (i) an inflammatory cell infiltrate, which results from both activation of resident inflammatory cells and recruitment of circulating inflammatory cells; (ii) an increase in interstitial cells due to increased proliferation and decreased apoptosis of resident interstitial cells as well as migration of cells into the tubulointerstitium; (iii) the appearance of myofibroblasts expressing the cytoskeletal protein a-smooth muscle actin (aSMA), which arise by differentiation of resident interstitial fibroblasts and infiltrating cells including bone marrow-derived progenitor cells and inflammatory cells, and via transdifferentiation of tubular epithelial cells [4,5]; 29

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(iv) tubular atrophy as a consequence of tubular cell apoptosis and epithelial-mesenchymal transdifferentiation (EMT); (v) obliteration of the peritubular capillaries; (vi) accumulation of ECM as the net result of both increased production and decreased turnover of ECM proteins. The accumulated matrix differs not only in amount and distribution but also in composition and in mechanical properties, for example, fibrillar collagens are more highly cross-linked. Matrix turnover is regulated primarily by the matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinase (TIMPs) and the plasmin-plasminogen activator-plasminogen activator inhibitor (PAI) cascade [3]. In vivo, fibrosis is characterised by increased levels of the inhibitors TIMP-1 and PAI-I [3]. Expression of a variety of pleiotropic cytokines, growth factors, vasoactive agents and their receptors is altered in the fibrotic kidney with an increase in proinflammatory, vasocontrictive and profibrotic factors and a decrease in antifibrotic factors such as hepatocyte growth factor (HGF) and bone morphogenetic protein-7 (BMP-7) [3]. Among the profibrotic factors, transforming growth factor-b1 (TGF-b) predominates, signalling via Smad 3 to induce a broad spectrum of fibrogenic effects in different renal cell types. Other factors include connective tissue growth factor (CTGF), plateletderived growth factor (PDGF), fibroblast growth factor (FGF)-2, endothelin (ET)-1 and angiotensin (Ang)-II.

Hypoxia and fibrosis The idea that hypoxia plays a role in renal fibrosis arose out of understanding of the mechanism(s) by which glomerular disease induces tubulointerstitial injury. The chronic hypoxia hypothesis [6,7] proposed that primary glomerular disease leads to downstream injury to the peritubular microvasculature. Compromise of the microvasculature creates a hypoxic environment that triggers a fibrotic response in tubulointerstitial cells (Fig. 1). This, in turn, impacts on adjacent capillaries and nephrons, setting up a self-perpetuating cycle of destruction, culminating in organ failure. In support of this hypothesis, a direct correlation between loss of the microvasculature and development of glomerular and tubulointerstitial scarring has been established [8]. The irreversible consequence of hypoxia is obliteration of the renal microvasculature. However, several other mechanisms may contribute to decreased tissue oxygenation; these include anaemia resulting in reduced oxygen delivery, increased vasoconstriction as result of overproduction of vasconstrictors such as Ang-II and ET-1 or decreased production of vasodilators such as nitric oxide, decreased capillary flow, increased metabolic demand as a result of hyperfiltration by uninjured nephrons and increased oxygen diffusion 30

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distances as ECM accumulates between blood vessels and adjacent cells [9,10] (Fig. 1). Several studies, using a variety of techniques to measure tissue oxygenation in animal models of CKD [reviewed in 11], have shown that hypoxia occurs early in progressive renal failure suggesting a causal relationship [9–11].

Oxygen sensing and hypoxic regulation of gene expression Cells have evolved complex systems to maintain oxygen homeostasis, and a number of different interrelated cellular oxygen-sensing signal cascades have been proposed based variously on an isoform of the neutrophil NADPH oxidase, components of the mitochondrial electron transport chain such as a specialised low pO2 affinity cytochrome c oxidase (aa3), haem-oxygenase-2, a subfamily of 2-oxoglutaratedependent dioxygenases known as the hypoxia-inducible factor (HIF) prolyl-hydroxylases (PHDs) and HIF asparaginyl hydroxylase called factor-inhibiting HIF (FIH-1) [12]. In response to low oxygen, cells regulate expression of a large number of genes, either directly by transcriptional activation or indirectly via secondary mediators, involved in cell survival and adaptation [13]. Transcriptional regulation is, in large part, mediated by the HIF family of transcription factors [13,14]. HIFs are heterodimeric transcription factors comprising a constitutively expressed b subunit and an oxygen-regulated a subunit. In normoxia, the a subunit is rapidly degraded; hydroxylation of proline residues (Pro402, Pro564) by the oxygen-dependent PHDs targets the protein for proteosomal degradation via binding to the Von Hippel Lindau (VHL) protein and ubiquitination. Transcriptional activity of HIF is further regulated by FIH-1; oxygen-dependent hydroxylation of asparagine blocks binding of HIF-a to the transcriptional coactivator CBP/p300, thereby inhibiting gene transcription. In hypoxia, HIF-a proteins are stabilised, dimerise with HIF-b and bind to hypoxia response elements (HREs) in target genes to activate transcription [13,14]. Three mammalian a subunits have been identified: HIF-1a, HIF-2a and HIF-3a/IPAS (inhibitory Per-Arnt-Sim protein). HIF-1a and HIF-2a appear to have both unique and overlapping functions [15,16]. Expression of these subunits is also selective with respect to cell type, tissue compartment and experimental conditions. In the kidneys of rats exposed to hypoxia, HIF-1a accumulates mainly in tubular cells whereas HIF-2a is induced in peritubular endothelial cells and fibroblasts [17]. The role of HIF-3a is less well characterised but it may act as an intrinsic repressor of the HIF system [18]. Although HIF-1 is considered a master regulator of the hypoxic response, several other factors have been implicated in the transcriptional response to hypoxia including Sp1, AP1, CREB, p53 and NFkB [14]. The mechanism(s) by which these transcription factors are regulated by hypoxia is unclear but might relate to changes in cellular redox potential and to

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Figure 1. Mechanisms of hypoxia-induced renal fibrosis. Primary glomerular disease leads to altered glomerular and peritubular haemodynamics inducing endothelial injury and microvascular insufficiency, creating a hypoxic tissue environment and triggering tubular injury, inflammation and fibrosis. This in turn affects adjacent capillaries and tubules. If the affected vessels or tubules are the outflow tracts of previously unaffected glomeruli, this will exacerbate glomerular injury setting up a progressive cycle of tissue damage, microvascular obliteration, fibrosis and, ultimately, organ failure. Additional mechanisms that could contribute to hypoxia include anaemia, increased oxygen diffusion distances due the accumulating ECM and an increased metabolic demand in tubular cells.

the diverse intracellular signalling pathways activated by hypoxia [14], many of which are also implicated in fibrosis. In addition to directly regulating gene expression, hypoxia can alter gene expression indirectly via secondary mediators. For example, hypoxia-induced growth factors, cytokines and their receptors can form autocrine or paracrine regulatory loops. Many of the growth factors induced by hypoxia have been implicated in fibrosis, in particular TGF-b, CTGF and PDGF [3]. Both direct and indirect regulation of gene expression by hypoxia are likely to play a role in renal fibrosis.

Effects of hypoxia on tubulointerstitial cells Although in vivo studies are suggestive, support for a causal role of hypoxia in fibrosis comes from in vitro studies of the

effect of hypoxia (1% O2) on tubulointerstitial cells, viz. microvascular endothelial cells, tubular epithelia and interstitial fibroblasts, which, as outlined above, undergo a series of well-defined changes in fibrosis. Although numerous studies have examined the response of a variety of endothelial cells to hypoxia, the hypoxic response in renal microvascular endothelial cells is largely unexplored. Loss of the cortical microvasculature in fibrotic kidneys [8] implies that the predominant response of renal endothelial cells to hypoxia might be cell death. Another intriguing possibility is that by analogy with tubular cells, endothelial cells possess the capacity to transdifferentiate to (myo)fibroblasts [19] and that hypoxia can drive this process leading to microvascular disruption, exacerbating tissue hypoxia and www.drugdiscoverytoday.com

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increasing the number of ECM-producing fibroblasts. Another hypoxia-sensitive component of the microvasculature, which can play an as yet unappreciated role in renal fibrosis, are the pericytes, contractile cells that surround the endothelial cells and stabilise the vessels [20]. These cells have the potential to contribute to pathological vasoconstriction and also to ECM accumulation because they possess the capacity to differentiate into synthetic (myo)fibroblast-like cells. The majority of in vitro studies of the effect of hypoxia on tubular epithelial cells have focussed on proximal tubular epithelial (PTE) cells. Hypoxia induces as complex transcriptional response in this cell type in vitro [21]. Overall the data suggest that hypoxia can induce changes consistent with a fibrogenic phenotype, promoting ECM accumulation with a switch to production of interstitial collagen type I and suppressing matrix turnover [22,23]. Hypoxia also downregulates secretion of the renoprotective, matricellular protein, osteopontin [24]. Hypoxia stimulates EMT of PTE to a myofibroblastic phenotype [25], a process increasingly implicated in fibrosis [3] and can induce PTE apoptosis [26] consistent with the loss of tubular cells in vivo. Exposure of PTE to hypoxia induces expression of fibrogenic factors including TGF-b [22] and ET-1 [27] as well as angiogenic factors such as, vascular endothelial cell growth factor (VEGF) [28] and angiopoeitin-4 [29] capable of acting as autocrine or paracrine mediators. Notably, although TGF-b is induced by hypoxia in PTE, the changes in ECM metabolism appear to occur via TGF-b1-independent mechanisms [22]. In addition to independent effects, there is evidence of synergism between hypoxia and other known fibrogenic stimuli [23,30]. Fibroblasts are the major ECM-producing cells in the tubulointerstitium [3–5]. In vitro hypoxia promotes a fibrogenic phenotype [31] with increased proliferation, enhanced myofibroblast differentiation and altered ECM metabolism, changes that are associated with sustained activation of MAPK signalling pathways [32]. Although not tested in renal fibroblasts, hypoxia can suppress apoptosis in other fibroblasts [33] consistent with in vivo studies suggesting that in CKD, interstitial fibroblast apoptosis is suppressed leading to accumulation of contractile myofibroblasts [3]. Hypoxia can also induce fibroblast production of proinflammatory factors although this might be context- and cell-type dependent [34,35]. Activation of the renin-angiotensin system (RAS) is central to the pathogenesis of renal fibrosis, and during fibrosis interstitial cells express components of the RAS [36]. In some (nonrenal) fibroblasts, hypoxia has been shown to upregulate both angiotensin-converting enzyme (ACE) and angiotensin-II receptor type 1 [37], suggesting that in the kidney, hypoxia might have similar effects. Exposure to low oxygen increases fibroblast ECM production, upregulating a variety of ECM proteins [31], many of which are increased in the fibrotic matrix in vivo [3]. In 32

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addition, hypoxia upregulates enzymes involved in posttranslational modification of collagen [38,39], potentially producing a matrix with altered mechanical properties and more resistant to degradation. It seems that some aspects of the fibrogenic phenotype induced in renal fibroblasts by hypoxia may be due to the autocrine actions of hypoxiainduced growth factors or to synergistic interactions between hypoxia and such factors. However, in renal fibroblasts, transcriptional induction of interstitial collagen a1(I) (COL1A1) by hypoxia is independent of TGF-b [31]. Furthermore, this transcriptional activation is HIF-independent and regulated by the transcription factor, Sp1 [40]. In parallel with increased ECM production, hypoxia suppresses matrix degradation via decreased expression and activity of MMPs, in particular interstitial collagenase MMP-1, and increased expression of TIMP-1, this latter mediated by HIF-1regulated gene transcription [31]. The adamalysins (ADAMs) are another family of metalloproteases involved in processing of ECM proteins and shedding of cell surface molecules. The ADAMs have not been widely studied in renal pathophysiology; however, a recent study suggests that ADAM 17 is upregulated by Ang-II [41]. Members of ADAM family are regulated by hypoxia in renal fibroblasts (unpublished) and thus may have as yet unexplored roles in hypoxia-induced fibrosis. In parallel with changes in ECM metabolism, hypoxia alters regulatory cell–matrix interactions leading to increased adhesion, decreased migration and increased contraction via an increase in specialised adhesion complexes, focal adhesions, and sustained activation of the focal adhesion kinase and downstream signalling pathways including those mediated by the Ras GTPases and MAPK kinases [32], which have also been implicated in fibrosis [42,43]. Collectively, the in vitro data suggest that hypoxia can induce functional and phenotypic changes in renal epithelial cells and fibroblasts consistent with changes seen in these cells in fibrosis.

Hypoxia as an inflammatory stimulus In addition to direct fibrogenic effects on tubulointerstitial cells, hypoxia may be an important stimulus for the persistent inflammation which is suggested to be an intrinsic component of the fibrotic response [3] in that hypoxia provides a homing signal for inflammatory cells [44] which accumulate at sites of injury. In response to the low oxygen environment, these cells alter a wide array of genes involved in survival, tissue revascularisation and recruitment and activation of more inflammatory cells [44,45]. Further, oxygen consumption by macrophages themselves might exacerbate local hypoxia. Where tissue oxygenation is restored by neovascularisation, the inflammatory response is terminated, but in the absence of angiogenic repair, chronic hypoxia might potentiate an ongoing inflammatory response and stimulate fibrosis. In addition, it has been suggested that inflammatory

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cells have the potential to differentiate into fibroblasts and to contribute to the pathological ECM accumulation [46,47] although it remains to be tested whether hypoxia can drive this process.

Hypoxia as a stimulus for progenitor cell recruitment Several studies have demonstrated the incorporation of circulating cells into different tissue compartments of the kidney in response to injury that might contribute to fibrosis [48]. Although the mechanisms of cell recruitment and retention within the kidney have not been established, a role for hypoxia seems likely since progenitor cells preferentially home to ischaemic sites via interactions between the CXCR4 chemokine receptor and its ligand, stromal cell-derived factor-1 [49], both of which have been identified as HIF targets. It is interesting to speculate that once progenitor cells are recruited, the low oxygen environment may also affect cell differentiation potentially inducing a pathological phenotype [50]. Similarly, hypoxia might alter the differentiation of intrinsic progenitor stem cells, although such a population of pluripotent cells has yet to be identified in the adult kidney [51].

Hypoxia and the failure of the reparative angiogenesis in renal fibrosis One of the anomalies in considering hypoxia as a mechanism for renal fibrosis is the apparent failure of angiogenesis to repair/replace damaged vessels since hypoxia, classically, is a potent angiogenic stimulus via induction of a range of angiogenic factors. In models of progressive renal disease, although there is an early, transient increase in VEGF, levels of expression are reduced in advanced disease [8]. The mechanism for this reduction is unclear but it appears to correlate with inflammatory cell infiltration and might relate to tubular atrophy because PTEC are an important source of VEGF [28]. Similar to VEGF, levels of another angiogenic factor,

angiopoietin-1, are suppressed in the fibrotic kidney [52] suggesting an overall reduction in proangiogenic factors. In parallel, an increase in antiangiogenic factors can also contribute to failure of angiogenic repair. At least two matricellular proteins upregulated in fibrosis have antiangiogenic activity [53] and in some cell types, hypoxia increases expression of endostatin, an endogenous inhibitor of angiogenesis [54].

Chronic hypoxia as a therapeutic target in renal fibrosis Accumulating evidence supports an important role for hypoxia in renal fibrosis suggesting that therapeutic manipulation of the hypoxic response should be of benefit in ameliorating disease [9–11]. Therapeutic strategies can loosely be divided into those that ameliorate hypoxia and those that target its downstream effects, for example blockade of hypoxia-induced fibrogenic factors such as TGF-b [1] (Table 1), approaches include: (i) correction of anaemia by administration of erthyropoetic agents [55,56], such as erythropoietin (EPO), to improve oxygen delivery. These agents may have beneficial effects beyond their ability to stimulate erythrocyte production, for example EPO can stimulate VEGF and angiogenesis and mobilise endothelial progenitor cells; (ii) normalisation of vascular tone by alleviating vasoconstriction and/or enhancing vasodilation. Activation of the RAS has long been implicated in the pathogenesis of renal fibrosis, and the therapeutic efficacy of angiotensin-converting enzyme (ACE) inhibition and angiotensin receptor blockade (ARB) is well established. In the remnant kidney model, administration of an ARB restored cortical perfusion and reduced tissue hypoxia [9]. In addition, angiotensin blockade may have blood pressure-independent renoprotective effects [11];

Table 1. Targets and related therapiesa Target b

Anaemia

Vascular tone (i) Alleviate vasoconstriction

Strategic approach to target

Expected outcome of intervention at target

(i) Exogenous EPO or modified EPO. (ii) EPO mimetics (peptidic and nonpeptidic). (iii) HIF-a stabilization by: (a) PHD inhibitors; (b) blockade of FIH-1. (iv) Ex vivo gene therapy.

Increase levels of EPO, stimulate erthyropoiesis, prevent/reduce anaemia.

(i) RAS: inhibit ACE or block angiotensin receptors.c

Decrease Ang-II and Ang-II-mediated signaling, inhibit vasoconstriction, restore capillary perfusion and suppress direct fibrogenic effects of Ang-II. Decrease in ET-mediated vasoconstriction; block direct fibrogenic effects of ET. Increase vasodilation.

(ii) ET-1: antagonize ET-1 or its receptors.d (ii) Enhance vasodilation

Enhance NO signaling.

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Table 1 (Continued ) Target

Strategic approach to target

Expected outcome of intervention at target

HIF

Stabilisation of HIF-a by: (i) PHD inhibitors.e (ii) Inhibition of FIH-1. (iii) Chemical hypoxia mimetics, e.g. CoCl2.f (iv) Gene transfer.

Activation of HIF, ameliorate fibrosis via multiple cell-type-dependent mechanisms including promoting angiogenesis and increasing erythropoiesis.

Microvascular repair/angiogenesis

(i) Administration of angiogenic factors, e.g. VEGF, angiopoeitin.g

Preserve microvasculature, repair and stabilize damaged vessels and suppress endothelial apoptosis.

(ii) Inhibition of antiangiogenic factors. Circulating progenitor cells

Inflammation i

EMT of tubular cells

(i) Prevent homing, retention and/or differentiation of fibrogenic precursor cells. (ii) Hypoxic targeting of engineered progenitor cells expressing antifibrotic genes. (iii) Targeting of endothelial precursor cells to hypoxic regions.

Decrease fibrogenic cells.

Anti-inflammatory agents.h

Reduce inflammation, ameliorate fibrosis.

(i) Block TGF-b expression, activation and signaling.

Decrease numbers of fibroblasts; maintain tubular integrity and function.

(ii) Increase HGF expression and signaling. (iii) Increase BMP-7 expression and signaling. Apoptosisj

(i) Block apoptosis of tubular epithelial cells.

Matrix remodelling (i) Matrix turnover

(ii) Matrix production

Intracellular signaling pathways

Increase vascular repair, angiogenesis.

(iii) Enhance apoptosis of myofibroblasts.

Reduce tubular cell death; maintain tubular integrity and function. Reduce endothelial cell death and loss of peritubular capillaries. Increase clearance of ECM-producing cells.

Block induction, activation and signaling of TGF-b, CTGF, ET-1, PDGF.k

Reduce inflammation, decrease number of myofibroblasts and ECM accumulation.

(i) Inhibit TIMP-1 expression and activity. (ii) Upregulate expression and activity of MMPs (MMP-1).l (iii) Inhibit PAI expression and activity.m (i) Inhibit collagen transcription and post-translational modification, e.g. inhibit cross-linking by tissue transglutaminase. (ii) Inhibit production of other ECM components.

Increase matrix turnover. Increase matrix turnover.

(ii) Block apoptosis of endothelial cells.

Fibrogenic cytokines

Decrease fibrosis.

Inhibit signaling cascades.n

Increase activity of PA; increase matrix turnover. Decrease collagen production; increase susceptibility to degradation; normalize cell–matrix interactions. Decrease ECM production/accumulation. Inhibit expression of proinflammatory and profibrotic factors.

a Abbreviations: ACE-I: angiotensin-converting enzyme inhibitor; Ang-II: angiotensin-II; BMP-7: bone morphogenetic protein-7; CTGF: connective tissue growth factor; EMT: epithelialmesenchymal transdifferentiation; EPO: erythropoietin; ET-1: endothelin-1; FIH-1: ‘factor-inhibiting HIF-1’, HIF-a-specific asparaginyl hydroxylase; HGF: hepatocyte growth factor; MAPK: mitogen-activated protein kinases; MMPs: matrix metalloproteinases; PA: plasminogen activators; PAI-1: plasminogen activator inhibitor-1; PDGF: platelet-derived growth factor; PHDs: prolyl-hydroxylases; RAS: renin-angiotensin system; TIMP-1: tissue inhibitor of metalloproteinase-1; TGF-b: transforming growth factor-b; VEGF: vascular endothelial cell growth factor. b The therapeutic efficacy of EPO and its derivatives and various strategies for increasing erythropoesis to correct anaemia have recently been reviewed [56]. c ACE inhibitors and ARBs are widely used in the management of CKD. d Selective and nonselective ET receptor antagonists have been developed and produce promising results in animal models. It seems that dual antagonism of ET-A and ET-B receptors may have maximal benefit. e Orally active PHD inhibitors have been developed and show promise in treating fibrotic disease [56]. f Although CoCl2 has been used to demonstrate the benefit of activation of the hypoxic response in ameliorating disease, toxicity issues limit its therapeutic potential. g Administration of exogenous angiogenic factors VEGF and stabilised angiopoietin have been shown to be beneficial in models of progressive renal fibrosis [8,52]. h Anti-inflammatory agents include steroids, retinoids and some cytokines. i The mechanism by which hypoxia induces EMT of tubular epithelial cells remains to be clarified, but one of the major drivers of EMT is TGF-b which is antagonized by HGF and by BMP-7 thus inhibiting TGF-b and/or promoting expression of antagonists might be effective in blocking transdifferentiation. j Because different cell populations show opposite apoptotic responses in fibrosis it will be important to develop strategies to target specific cell types within the kidney to inhibit or promote cell death. k There are numerous reagents at various stages of development targeting growth factors including agents that inhibit synthesis and activation, inhibit intracellular signaling and enhance inhibitory pathways, which can be relevant in blocking the actions of hypoxia-induced fibrogenic cytokines. l There are several agents which upregulate MMPs including the small molecule halofunginone but given the complexity of the role of MMPs in fibrosis [3], selective activators of particular enzymes are likely to be crucial to the success of this approach. m Selective anti-PAI-1 reagents are being developed but the ideal antifibrotic remains to be discovered. n Many profibrotic stimuli, including hypoxia, activate common intracellular signaling pathways; in animal models inhibition of signaling cascades has been shown to be effective in reducing fibrosis [1].

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(iii) preservation of the tubulointerstitial microvasculature by administration of exogenous angiogenic factors such as VEGF [8] and angiopoeitin-1 [52]. Although this approach has proved effective in experimental models, it remains to be established whether this will translate to human therapy; (iv) activation of HIF in the tubulointerstitium. Although hypoxia induces fibrogenic changes in renal cell in vitro at least some of which are mediated by HIF, stabilisation of HIF-a chemically (e.g. by the hypoxia mimetic, cobalt), using PHD inhibitors or by gene transfer [9,10] appears to ameliorate disease in experimental models and PHD inhibitors show promise in treating human disease [56]; (v) manipulation of hypoxia-induced cell homing. An intriguing possibility with therapeutic potential is that hypoxia-mediated recruitment and differentiation mechanisms can be used to selectively direct progenitor cells and genetically engineered precursors to the kidney and to sites of injury to either inhibit fibrosis or promote regression. Conversely, understanding the homing of injurious cell types might allow this process to be blocked.

Summary and conclusions In CKD progressive renal impairment correlates with tubulointerstitial fibrosis characterised by inflammation, interstitial expansion with accumulation of ECM, tubular atrophy and vascular obliteration. Loss of the microvasculature implies a hypoxic milieu and data in animal models of progressive renal disease show that hypoxia precedes ECM accumulation suggesting an important role for hypoxia in inducing and promoting fibrosis. Perhaps surprisingly, given the link between hypoxia, VEGF and angiogenesis in tumour growth and expansion, in renal fibrosis chronic hypoxia fails to induce a sustained angiogenic response; rather, there is a progressive rarefaction of the peritubular capillaries. In addition to overt microvascular obliteration, a variety of complementary mechanisms might contribute to hypoxia including anaemia, decreased capillary flow, increased vasoconstriction, increased metabolic demand and increased O2 diffusion distances owing to ECM accumulation. In vitro studies suggest that hypoxia can induce profibrotic changes in tubular epithelial cells and interstitial fibroblasts consistent with changes observed in the fibrotic kidney in vivo. A wide variety of genes, including many fibrogenic factors, are regulated by hypoxia acting primarily through the HIF family of transcription factors although these are not the sole transcriptional regulators of the response. Additional roles for hypoxia in fibrosis are postulated in the sustained inflammatory response characteristic of progressive disease, in the recruitment, retention and differentiation of circulating progenitor cells that contribute to the fibrogenic population and in altering the function of intrinsic stem cell populations.

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The identification of hypoxia as an important component of the fibrotic response and elucidation of the mechanisms underlying the hypoxic response provide several potential therapeutic targets (Table 1). Strategies include inter alia, correction of anaemia, normalisation of microvascular tone, administration of exogenous proangiogenic factors to restore microvasculature integrity, activation of HIF and hypoxiamediated targeting and mobilisation of intrinsic and engineered progenitor cells.

Acknowledgements We would like to acknowledge the many investigators in the field whose work, owing to space constraints, we were unable to cite. Work from our laboratory was supported by the British Heart Foundation (PG/96045), National Institutes of Health USA (D98-001), Royal College of Surgeons London and the Medical Research Council (G78/773).

References 1 El Nahas, A.M. and Bello, A.K. (2005) Chronic kidney disease: the global challenge. Lancet 365, 331–340 2 US Renal Data System USRDS Annual Data Report: Atlas of End-Stage Renal Disease in the United States, National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, Maryland (2006) http://www.usrds.org/2006 3 Eddy, A.A. (2005) Progression in chronic kidney disease. Adv. Chronic Kidney Dis. 12, 353–365 4 Iwano, M. et al. (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J. Clin. Invest. 110, 341–350 5 Liu, Y. (2004) Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J. Am. Soc. Nephrol. 15, 1–12 6 Fine, L. et al. (1998) Progressive renal disease: the chronic hypoxia hypothesis. Kidney Int. 53 (Suppl. 65), S74–S78 7 Fine, L. et al. (2000) Is there a common mechanism for the progression of different types of renal disease other than proteinuria? Towards the unifying theme of chronic hypoxia Kidney Int. 57 (Suppl. 75), S22–S26 8 Kang, D.H. et al. (2002) Role of microvascular endothelium in progressive renal disease. J. Am. Soc. Nephrol. 13, 806–816 9 Nangaku, M. (2005) Chronic hypoxia and tubulointerstitial injury: a final common pathway to end-stage renal failure. J. Am. Soc. Nephrol. 17, 17–25 10 Eckardt, K-U. et al. (2005) Role of hypoxia in the pathogenesis of renal disease. Kidney Int. 68 (Suppl. 99), S46–S51 11 Norman, J.T. and Fine, L.G. (2006) Intrarenal oxygenation in chronic renal failure. Clin. Exp. Pharmacol. Physiol. 33, 989–996 12 Acker, T. et al. (2006) The good the bad and the ugly in oxygen sensing: ROS, cytochromes and prolyl hydroxylases. Cardiovasc. Res. 71, 195–207 13 Semenza, G.L. (2003) Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 3, 721–732 14 Cummins, E.P. and Taylor, C.T. (2005) Hypoxia-responsive transcription factors. Pflugers. Arch. 450, 363–371 15 Warnecke, C. et al. (2004) Differentiating the functional role of hypoxiainducible factor (HIF)-1a and HIF-2a (EPAS1) by the use of RNA interference: erythropoeitin is a HIF-2a target gene in Hep3B and Kelly cells. FASEB J. 18, 1462–1464 16 Raval, R.R. et al. (2005) Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol. Cell. Biol. 25, 5675–5686 17 Rosenberger, C. et al. (2002) Expression of hypoxia-inducible factor1alpha and factor-2alpha in hypoxic and ischemic rats. J. Am. Soc. Nephrol. 13, 1721–1732 18 Makino, Y. et al. (2001) Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414, 550–554 www.drugdiscoverytoday.com

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O’Riordan, E. et al. (2007) Chronic NOS inhibition actuates endothelialmesenchymal trasndifferentiation. Am. J. Physiol. Heart Circ. Physiol. 292, H285–H294 Armulik, A. et al. (2005) Endothelial/pericyte interactions. Circ. Res. 97, 512–523 Leonard, M.O. et al. (2003) The role of HIF-1a in transcriptional regulation of the proximal tubular epithelial cell response to hypoxia. J. Biol. Chem. 278, 40296–40304 Orphanides, C. et al. (1997) Hypoxia stimulates proximal tubular cell matrix production via a TGF-beta1-independent mechanism. Kidney Int. 52, 637–647 Li, X. et al. (2005) Synergistic effect of hypoxia and TNF-alpha on production of PAI-1 in human proximal renal tubular cells. Kidney Int. 68, 569–583 Hampel, D.J. et al. (2003) Osteopontin traffic in hypoxic renal epithelial cells. Nephron Exp. Nephrol. 94, 66–76 Manotham, K. et al. (2004) Transdifferentiation of cultured tubular cells induced by hypoxia. Kidney Int. 65, 871–880 Tanaka, T. et al. (2003) Hypoxia induces apoptosis in SV40-immortalised rat proximal tubular cells through the mitochondrial pathways, devoid of HIF-1 mediated up-regulation of Bax. Biochem. Biophys. Res. Commun. 309, 222–231 Ong, A.C. et al. (1995) An endothelin-1 mediated autocrine growth loop involved in human tubular regeneration. Kidney Int. 48, 390–401 Kim, B.S. et al. (2002) VEGF expression in hypoxia and hyperglycemia: reciprocal effect of branching angiogenesis in epithelial-endothelial cocultures. J. Am. Soc. Nephrol. 13, 2027–2036 Yamakawa, M. et al. (2004) Expression of angiopoietins in renal epithelial cell and clear cell carcinoma cells: regulation by hypoxia and participation in angiogenesis. Am. J. Physiol. Renal Physiol. 287, F649–F657 Nakagawa, T. et al. (2004) Differential regulation of VEGF by TGF-beta and hypoxia in rat proximal cells. Am. J. Physiol. Renal Physiol. 287, F658–F664 Norman, J.T. et al. (2000) Hypoxia promotes fibrogenesis in human renal fibroblasts. Kidney Int. 58, 2351–2366 Thayalan, M. and Norman, J. (2005) Hypoxia activates integrin signalling: a role in fibrosis? J. Am. Soc. Nephrol. 16, 66 Eul, B. et al. (2006) Impact of HIF-1 alpha and HIF-2 alpha on proliferation and migration of human pulmonary artery fibroblasts in hypoxia. FASEB J. 20, 163–165 Galindo, M. et al. (2001) Hypoxia induces expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and IL-8 in human dermal fibroblasts. Clin. Exp. Immunol. 123, 36–41 Safronova, O. et al. (2003) Effect of hypoxia on monocyte chemotactic protein-1 (MCP-1) gene expression induced by Interleukin-1b in human synovial fibroblasts. Inflamm. Res. 52, 480–486 Okada, H. et al. (2002) Interstitial fibroblast-like cells express reninangiotensin system component in fibrosing murine kidney. Am. J. Pathol. 160, 765–772

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Krick, S. et al. (2005) Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross talk between HIF-1 alpha and an autocrine angiotensin system. FASEB J. 19, 857–859 Hofbauer, K.H. et al. (2003) Oxygen tension regulates the expression of a group of pro-collagen hydroxylases. Eur. J. Biochem. 270, 4515–4522 Brinckmann, J. et al. (2005) Interleukin 4 and prolonged hypoxia induce a higher gene expression of lysyl hydroxylase 2 and an altered cross-link pattern: important pathogenetic steps in early and late stage of systemic scleroderma. Matrix Biol. 24, 459–468 Shakib, K. et al. (2001) Sp1 is an important mediator of hypoxia-induced collagen a1(I) gene transcription. J. Am. Soc. Nephrol. 12, 716 Lautrette, A. et al. (2005) Angiotensin II and EGF receptor cross-talk in chronic kidney diseases: A new therapeutic approach. Nat. Med. 11, 867–874 Hendry, B.M. and Sharpe, C.C. (2003) Targeting Ras genes in kidney disease. Nephron Exp. Nephrol. 93, 129–133 Nagayota, K. et al. (2002) Y27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int. 61, 1684– 1695 Kong, T. et al. (2004) Leucocyte adhesion during hypoxia is mediated by HIF-1-dependent induction of b2 integrin gene expression. Proc. Nat. Acad. Sci. U S A 101, 10440–10445 Murdoch, C. et al. (2005) Hypoxia regulated macrophage functions in inflammation. J. Immunol. 175, 6257–6263 Jabs, A. et al. (2005) Peripheral blood mononuclear cells acquire myofibroblastic characteristics in granulation tissue. J. Vasc. Res. 42, 174–180 Postlethwaite, A.E. et al. (2004) Cellular origins of fibroblasts: Possible implications for organ fibrosis in systemic sclerosis. Curr. Opin. Rheumatol. 16, 733–738 Ricardo, S.D. and Deane, J.A. (2005) Adult stem cells in renal injury and repair. Nephrol. 10, 276–282 Ceradini, D.J. and Gurtner, G.C. (2005) Homing to hypoxia: HIF-1 as a mediator of progenitor cell recruitment to injured tissue. Trends Cardiovasc. Med. 15, 57–63 Gustaffson, M.V. et al. (2005) Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev. Cell. 9, 617–628 Al-Awqati, Q. and Oliver, J.A. (2002) Stem cells in the kidney. Kidney Int. 61, 387–395 Kim, W. et al. (2006) COMP-Angiopoietin-1 ameliorates renal fibrosis in a unilateral ureteral obstruction model. J. Am. Soc. Nephrol. 17, 2474–2483 Bornstein, P. and Sage, E.H. (2002) Matricellular proteins: extracellular modulators of cell function. Curr. Opin. Cell Biol. 14, 608–616 Paddenberg, R. et al. (2006) Hypoxia-induced increase of endostatin in murine aorta and lung. Histochem. Cell. Biol. 125, 1–12 Rossert, J. et al. (2005) Anemia management and chronic renal failure progression. Kidney Int. 68 (Suppl. 99), S76–S81 (Pflugers Arch. Eur. J. Physiol. 450, 363–371) Jelkmann, W. (2007) Erythropoeitin after a century of research: younger than ever. Eur. J. Haematol. 78, 183–205