TGF-␤ in Renal Injury and Disease

Erwin P. Böttinger, MD

Summary: Chronic progressive kidney diseases typically are characterized by loss of differ- entiated epithelial cells and activation of mesenchymal cell populations leading to renal fibrosis in response to a broad range of diverse renal injuries. Recent evidence has indicated that epithelial microinjury leads to unbalanced epithelial-mesenchymal communication to initiate the fibrotic response. Transforming growth factors ␤ constitute a large family of cytokines that control key cellular responses in development and tissue repair. Activation of autocrine and paracrine transforming growth factor-␤ signaling cascades in the context of epithelial microinjuries initiate a variety of cell type–dependent signaling and activity profiles, including epithelial apoptosis and epithelial-to-mesenchymal transition, that trigger fibrogenic foci and initiate progressive fibrogenesis in chronic renal injury. Semin Nephrol 27:309-320 © 2007 Elsevier Inc. All rights reserved. Keywords: Fibrosis, chronic kidney disease, cell biology, signal transduction, apoptosis

he transforming growth factor ␤ (TGF-␤) ple levels. TGF-␤ are synthesized as inactive family includes TGF-␤s, activins, and precursors and secreted as latent complexes, Tbone morphogenetic proteins (BMPs)/ requiring activation in a highly controlled man- growth and differentiation factors that are ner.7 structurally-related, secreted cytokines.1 More TGF-␤ initiate signaling across the plasma than 30 family members have been identified membrane into the cell by inducing hetero- (Fig. 1A). TGF-␤, the prototypical member of meric complexes of type I and type II receptors this family and the focus of this review, was with serine/threonine kinase activity (reviewed discovered more than 25 years ago as a factor in Derynck and Zhang,8 Shi and Massague,9 and produced by transformed cells that induced the ten Dijke and Hill10). The family of TGF-␤ re- anchorage-independent growth of normal rat ceptors includes 5 type II receptors and 7 type 2,3 kidney cells. Numerous studies have shown I receptors, also named activin receptor-like that TGF-␤ are pleiotropic molecules that regu- kinases (ALKs) (Fig. 1B). On ligand-induced late cell proliferation, differentiation, apoptosis, heteromeric complex formation, the constitu- migration, and adhesion of many different cell tively active type II receptor kinase phosphor- types.1,4,5 TGF-␤–family ligands exert pivotal ylates the type I receptor on specific serine and roles in embryogenesis, tissue repair, and in threonine residues in the juxtamembrane re- maintaining tissue homeostasis.6 In addition, disruption of TGF-␤ signaling has been linked to gion, leading to activation of type 1 receptor 11 various developmental disorders and numerous kinases (Fig. 2). ␤ human diseases including cancer, fibrosis, and The ligand-induced activation of TGF- –re- autoimmune diseases. Because of their extraor- ceptor complexes leads to the recruitment and ␤ dinary multifunctionality, activities of TGF-␤ activation of major TGF- signal transducers of family members are tightly controlled at multi- the Smad family where activated type I receptor kinases transiently interact with and phosphor- ylate receptor-regulated Smads (R-Smads) at Department of Medicine, Mount Sinai School of Medicine, New York, NY. Supported in part by National Institutes of Health grants RO1DK56077, their extreme C-terminal serine residues (re- RO1DK60043, P5ODK064236-01003, and UO1DK060995. viewed in Derynck and Zhang,8 Shi and Mas- Address reprint requests to Erwin Böttinger, Mount Sinai School of Medicine, 9 10 One Gustave L. Levy Pl, Box 1118, New York, NY 10029. E-mail: sague, and ten Dijke and Hill ). Smad2 and [email protected] Smad3 act downstream of the TGF-␤, activin, or 0270-9295/07/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2007.02.009 nodal type I receptors ALK4, ALK5, and ALK7,

Seminars in Nephrology, Vol 27, No 3, May 2007, pp 309-320 309 310 E.P. Böttinger

INHBA/EDF B ALK5/ TβR1 INHBB/ACTBB ALK4/ActR-IB INHBC/ACTBC ALK7 A INHBE/ACTBE ALK3/ BMPR1A type I receptors LFTB/BMP17 LFTA/BMP18 ALK6/ BMPR1B or ALKs EBAF ALK1 MIS/AMH ALK2 TGFB1 TGFB2 ActR-IIA TGFB3 ActR-IIB GDF8/myostatin type II receptors BMP11/GDF11 TβR-II BMP3B/GDF10 BMPR-II BMP3 MIS/AMHR-II GDF1 GDF3/Vgr2 BMP10 GDF2/BMP9 GDF5 C Smad1 GDF6 GDF7 Smad5 BMP2 BMP4 Smad8 R-Smad BMP5 BMP6/Vgr1 Smad2 BMP7/Op1 BMP8/Op2 Smad3 BMP8B Smad4 Co-Smad Nodal/BMP16 BMP15/GDF9B Smad6 GDF9 I-Smad PLAB/GDF15 INHA Smad7

Figure 1. (A) TGF-␤ family members. Phylogenetic analysis of the TGF-␤ family. ACT, activin; GDF, growth and differentiation factor; INH, inhibin; MIS/AMH, müllerian inhibiting substance/anti-müllerian hormone; OP, osteogenic protein. (B) TGF-␤ family type I and type II receptors. Phylogenetic analysis of mammalian type I and type II receptors. ActR, activin receptor; MIS/AMH, MIS/AMH receptor; BMPR, BMP receptor; T␤R, TGF-␤ receptor. (C) Smad family. Phylogenetic analysis of mammalian Smads shows 3 distinct subfamilies: R-Smads, receptor regulated Smads; Co- Smads, common-partner Smads; and I-Smads, inhibitory Smads. whereas Smad1, Smad5, and Smad8 act down- ways.13 In addition to the canonical Smad stream of BMP type I receptors ALK2, ALK3, pathways, TGF-␤ may signal through mitogen- and ALK6 (Fig. 1C). ALK1 is a type I receptor activated protein kinases, phosphoinositide for TGF-␤ that activates Smad1, Smad5, and 3-OH kinases, small guanosine triphosphatases, Smad8 in neurons and endothelial cells depen- and other mediators (reviewed in Derynk and dent on ALK5 kinase activity.12 Phosphorylated Zhang,8 and Bottinger and Bitzer14). However, R-Smads form heteromeric complexes with a the molecular mechanisms of Smad-indepen- common-partner Smad4 and translocate to the dent TGF-␤ signaling remain incompletely un- nucleus, where these heteromeric complexes derstood. can bind to DNA to control transcription of target genes through interaction with co-activa- ␤ tors and co-repressors (Fig. 2). Smad6 and TGF- IS SYNONYMOUS WITH Smad7 share a conserved Mad homology 2-, INFLAMMATION AND FIBROSIS dwarfin type (MH2) domain with R-Smads and A large body of evidence, accumulated during 2 Co-Smads and constitute the subgroup of inhib- decades of investigation, establishes TGF-␤ as a itory Smads. Inhibitory Smads interact with and major regulator of inflammation and fibrosis. recruit Smurf (Smad ubiquitination regulatory The fibrogenic activity of TGF-␤ originally was factor) ubiquitin ligases to ligand receptor com- identified in classic studies showing that TGF-␤ plexes, initiating Smurf-induced receptor degra- stimulated granulation tissue when injected dation via proteosomal and lysosomal path- subcutaneously into newborn mice and stimu- TGF-␤ in renal injury and disease 311

Ligand

Extracellular

P SARA Intracellular P P R-Smad R-I R-II

I-Smad R-SmadP Co-Smad

R-SmadP Co-Smad

Nucleus P ad m ad -S m Gene R -S Co TF responses

Figure 2. The canonical TGF-␤/Smad signaling pathway. On ligand-binding, heteromeric complexes of type I and type II serine/threonine kinase receptors are stabilized. The constitutive type II receptor kinase phosphorylates the type I receptor on specific serine and threonine residues in the juxtamembrane region, enabling the type I receptor to transmit the signal inside the cell through phosphorylation of R-Smads at 2 extreme C-terminal serine residues. Access of R-Smads to the activated type I receptor is controlled through auxiliary proteins, including Smad anchor for receptor activation (SARA), and others. After phosphorylation, activated R-Smads form heteromeric complexes with Smad4. Smad complexes can bind to and regulate promoters of target genes in concert with numerous transcriptional regulators. lated collagen formation in fibroblasts.15 Other hypertension is induced by intrarenal activation seminal reports established that TGF-␤ regu- of the renin angiotensin system (RAS).26,27 lates immune function,16 chemotaxis, and in- A molecular link between intrarenal RAS and flammation,17 and induces its own synthesis.18 TGF-␤ was established in vitro and in vivo. For In experimental models of renal and pulmonary example, TGF-␤ is induced by angiotensin II fibrogenesis, inhibition of TGF-␤ with neutral- and is required for induction of hypertrophy in izing anti–TGF-␤ antisera ameliorates character- tubular epithelial cells and of matrix synthesis istic accumulation of extracellular matrix.19,20 in mesangial cells.28,29 TGF-␤1 also is up-regu- Together, these studies showed a remarkable lated in glomerular diseases30 and experimental ␤ multifunctionality of TGF- and established this and human diabetic nephropathy.31,32 In- growth factor as a putative key regulator in creased expression of TGF-␤ and TGF-␤ recep- 1,21,22 inflammation, tissue repair, and fibrosis. tors is a hallmark of virtually all human and experimental CKD (reviewed in Border and No- REGULATION OF TGF-␤ IN ble,33 and Bitzer et al34). Importantly, inhibitors KIDNEY INJURY AND DISEASE of the RAS reduce TGF-␤ expression in renal Since the 1980s, the so-called intraglomerular cells in vitro and in vivo,35 suggesting that re- hypertension model has been the classic para- duction of TGF-␤ isoform levels may underlie digm for the underlying cause of progression of the renoprotective effects of RAS blockade in chronic kidney disease (CKD).23-25 Glomerular diabetic and nondiabetic CKD.36 Independent 312 E.P. Böttinger of the RAS pathway, TGF-␤ also is induced di- that TGF-␤ and its signal transducers are central rectly by a broad range of products of metabo- mediators of the progression of CKD. lism, including glucose, advanced glycation products, free fatty acids, reactive oxygen spe- FIBROGENIC PROGRAMS cies, and others (reviewed in Ziyadeh37). In DOWNSTREAM OF TGF-␤ addition, mechanical stretch and shear stress TGF-␤ broadly controls messenger RNA levels increase TGF-␤ release and activation in renal and promoter activities of extracellular matrix and vascular cells.38-41 genes, including COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, COL6A3, COL7A1, and non- ␤ FUNCTIONAL ROLES FOR TGF- collagenous matrix genes such as fibronectin, IN KIDNEY INJURY AND DISEASE proteoglycans, and others (reviewed in Verrec- Numerous studies have shown that TGF-␤ inhi- chia and Mauviel,57 and Schnaper and Kopp58). bition attenuates functional and pathologic ab- TGF-␤ controls transcription of several extracel- normalities in experimental renal disease, sug- lular matrix (ECM) genes through Smad3-de- gesting a critical role for TGF-␤ in CKD and pendent mechanisms in which Smad3 interacts renal fibrosis (Table 1).19,42-48 Transgenic mice with activator protein 1 complexes or Sp1, re- overexpressing active TGF-␤1 develop progres- spectively, on CAGA motif Smad binding ele- sive glomerulosclerosis and tubulointerstitial fi- ments.59-62 Smad3 also is required for fibrogenic brosis, indicating that persistently increased activation of vascular smooth muscle cells by TGF-␤ activity is sufficient to induce progres- angiotensin II.63 In addition, microarray screens sive renal disease in mice.49-51 Interestingly, ap- in Smad2 and Smad3 knockout fibroblasts indi- optosis of glomerular epithelial cells (podo- cate a broad role for Smad3, but not Smad2, as cytes) is induced by TGF-␤1 and/or Smad7, and a critical mediator of fibrogenic signaling by precedes activation of mesangial cells and mes- TGF-␤ in mesenchymal cells.64 angial matrix deposition in TGF-␤1 transgenic Smad-independent pathways may have a role mice.52 Furthermore, knockout of the major in modulation of ECM gene expression signal transducer Smad3 in mice prevents renal by TGF-␤. For example, full activation of defects and fibrosis induced by experimental COL1A1 transcription is dependent on actin ureteral obstruction or diabetes mellitus.53,54 cytoskeleton-modulated signals and requires Transgenic overexpression of inhibitory Smad7 concomitant and interdependent activation of ameliorates fibrosis in ureteral obstruction and Smad3 and phosphoinositide 3-OH kinase/v-akt renal ablation models.55,56 These and numerous murine thymoma viral oncogene homolog 1 other studies consistently support the paradigm (AKT1) signaling, as well as p38 mitogen-acti-

Table 1. Cell Type–Dependent Activities Induced by TGF-␤ In Vitro Cell Type Induced Response Epithelial cells Tubular epithelial cells Apoptosis, growth inhibition, EMT, hypertrophy Podocytes Apoptosis, growth inhibition, basement membrane turnover Endothelial cells Glomerular endothelial cells Apoptosis, growth inhibition, differentiation Mesenchymal cells Mesangial cells Myofibroblast activation, ECM turnover, apoptosis Fibroblasts Myofibroblast activation, ECM turnover, apoptosis Inflammatory cells Macrophages/monocytes Chemotaxis All cells Autoinduction TGF-␤ in renal injury and disease 313 vated protein kinase in mesangial cells.65-67 vascular cell compartments can initiate fibro- V-abl Abelson murine leukemia viral oncogene genic phenotypes characteristic of mesenchy- homolog (C-Abl) is required for TGF-␤–induced mal cells. In addition, the classic concept of ECM gene expression, myofibroblast transfor- fibrosis as the “dark side of tissue repair”33 nei- mation, and proliferation independently of any ther involves nor explains the apparent abnor- effect on Smad signaling in fibroblasts, and C- malities of epithelial and/or endothelial cells, Abl inhibition ameliorates pulmonary and renal such as atrophy and apoptosis of epithelial fibrosis in experimental models.68,69 Connec- and/or endothelial cells and loss of tubular ep- tive tissue growth factor, a secreted cysteine- ithelial and postglomerular vascular structures, rich domain protein, is induced by TGF-␤ in which are hallmarks of progressive CKD. mesenchymal cells, including hepatic stellate An extended hypothesis/concept for fibrosis cells (HSCs), mesangial cells, and fibroblasts, has been advanced recently in which epithelial and may be required for maximal matrix syn- or vascular microinjury initiates epithelial or thesis by TGF-␤.70-74 Although there is increas- vascular degeneration through epithelial or en- ing evidence for Smad-independent TGF-␤ sig- dothelial apoptosis (or possibly epithelial-to- naling mechanisms, Smad3 is considered a key mesenchymal transition [EMT]), which initiates signal transducer for TGF-␤–mediated ECM reg- myofibroblast-like cell phenotypes and fibro- ulation.75 genic foci with progressive ECM accumula- tion.14,80,81 Epithelial cell injury and apoptosis CROSS-TALK BETWEEN INFLAMMATORY are initial responses to various forms of renal PATHWAYS AND TGF-␤ SIGNALING injury52,82-85 (Fig. 3). TGF-␤ provides a plausible link between epithelial/endothelial injury and There is considerable evidence for cross-modu- mesenchymal cell activation. For example, the lation of TGF-␤/Smad signaling by several path- engulfment and phagocytosis of apoptotic bod- ways involved in regulating inflammatory and ies by myofibroblasts, mesangial cells, HSCs, fibrotic responses. For example, c-Jun N-termi- Kupffer cells, or macrophages directly stimu- nal kinase (JNK) mediates inhibition of Smad- lates increased synthesis and secretion of dependent activation of collagen genes by TGF-␤1 by the phagocytosing cells,86-89 suggest- proinflammatory cytokines such as tumor ing that cells that phagocytose apoptotic bodies necrosis factor ␣ in dermal fibroblasts.76 Other generate and secrete TGF-␤1(Fig. 3). TGF-␤ mechanisms of transmodulation of TGF-␤/ itself may act on epithelial cells to induce apo- Smad-dependent activation of ECM genes by proinflammatory cytokines tumor necrosis fac- ptosis in renal tubular epithelial cells and glo- tor ␣ or interferon ␥ involve nuclear factor ␬ B merular podocytes and thereby may promote or signal transducer and transactivator 1–medi- further epithelial injury, resulting in increased ated induction of inhibitory Smad7.77,78 In addi- phagocytic activity and alteration in epithelial- 84,90 tion, prostaglandin E2 inhibits TGF-␤1–induced mesenchymal cell communication. collagen synthesis, indicating extensive signal- Proof-of-concept studies show that preven- ing cross-talk in profibrotic signaling in HSCs.79 tion of epithelial apoptosis ameliorates tissue In summary, proinflammatory cytokine path- fibrosis in hepatic and pulmonary experimental ways negatively regulate ECM synthesis via mo- models and that engineered persistent epitheli- lecular cross-talk with TGF-␤/Smad signaling. al/parenchymal cell apoptosis is sufficient to induce progressive fibrotic responses. For ex- ample, prevention of TGF-␤–induced apoptosis ␤ EMERGING ROLE FOR TGF- of alveolar epithelial cells in mice deficient for IN EPITHELIAL APOPTOSIS AND the proapoptotic Bcl2 family protein Bid pro- INITIATION OF FIBROGENIC FOCI tects these mice against bleomycin-induced pul- Despite considerable progress in our under- monary apoptosis and fibrosis, but not inflam- standing of fibrogenic signaling mechanisms, it mation.91 In addition, pharmacologic or remains unclear how tissue injury that fre- transgenic inhibition of apoptosis is associated quently manifests itself initially in epithelial or with attenuation of experimental pulmonary fi- 314 E.P. Böttinger

Figure 3. Integrated model of TGF-␤ as a key signal linking epithelial microinjury and fibrogenesis. Epithelial microinjury may manifest as apoptosis or EMT, depending on the signaling context and cell state, and can be induced by a diverse spectrum of stimuli including angiotensin II, radiation, viral infection, stretch/pressure, TGF-␤, advanced glycation end products, high ambient glucose, reactive oxygen species, and others. Epithelial microinjury induces release and activation of TGF-␤ by apoptosing and neighboring epithelial cells. TGF-␤ also is released by cells recruited to phagocytose apoptotic epithelial cell bodies. Locally increased TGF-␤ itself can induce epithelial apoptosis and/or EMTs, depending on the signaling context. The TGF-␤–dependent epithelial microinjury promotes disintegration of differentiated/loss of epithelial cell layers, contributing to the epithelial degeneration characteristic of fibrotic condi- tions. In addition, locally increased TGF-␤ recruits inflammatory cells (macrophages/monocytes) through chemotaxis and stimulates proliferation and myofibroblast differentiation of local mesenchymal cells such as interstitial fibroblasts, hepatic stellate cells, or glomerular mesangial cells. External stimuli such as advanced glycation products, reactive oxygen species, glucose, stretch, or angiotensin II also can act directly on mesenchymal cells and myofibroblasts to release TGF-␤. Infiltrating inflammatory cells release TGF-␤ and additional cytokines, contributing further to increased local TGF-␤ levels and mesenchymal cell proliferation and myofibroblast differentiation. Increased local TGF-␤ activates fibrogenesis through synthesis of ECM and inhibits ECM degradation characteristic of myofibroblast-like mesenchymal cells. Mesenchymal cell expansion and fibrogenesis result in ECM accumulation and organization, leading to scar formation. TGF-␤ autoinduction contributes to persistently increased TGF-␤ activity, which promotes the progression of epithelial degeneration and interstitial cell expansion/scarring, leading to gradual organ destruction and loss of function characteristic of chronic fibrotic conditions. brosis.92-94 Mice with cre/lox-mediated hepato- gests that disturbance of the epithelial-mesen- cyte-specific deletion of the anti-apoptotic chymal cell balance causes progressive glomer- BcL-xL gene manifest spontaneous, prolonged ulosclerosis and/or interstitial fibrosis. For hepatocyte apoptosis associated with increased example, titratable ablation of podocytes in rat expression of TGF-␤1 in adjacent hepatocytes and mouse models is sufficient to initiate pro- and macrophages. In these animals, prolonged gressive focal segmental glomerulosclerosis and hepatocyte apoptosis leads to progressive he- renal failure.96,97 Type 1 diabetes, induced by patic fibrosis.95 streptozotocin in Renin2 transgenic rats, is as- Whether prevention of epithelial apoptosis sociated with increased TGF-␤1 expression and in experimental renal disease would ameliorate apoptosis in renal tubular epithelial cells depen- kidney injury and fibrosis similarly, however, dent on angiotensin II.98 Unilateral ureteral ob- remains to be established. Early evidence sug- struction is associated with increased tubular TGF-␤ in renal injury and disease 315 epithelial apoptosis in mice deficient for tion.101,102 Increased TGF-␤ expression is a decorin, a natural inhibitor of TGF-␤1, com- prominent feature in this model.105,106 Biopsy pared with control wild-type mice.99 In murine specimens of diseased human kidney show few models of progressive glomerulosclerosis, in- isolated epithelial cells in tubular structures cluding TGF-␤1 transgenic mice52 and CD2-as- which manifest molecular evidence for EMT by sociated protein knockout mice, a striking in- co-expression of epithelial and mesenchymal crease of apoptosis of podocytes is one of the markers.107-109 In addition, to date there is very first detectable cellular lesions.84 Interestingly, little evidence documenting EMT in experimen- TGF-␤1 expression is increased in podocytes at tal liver fibrosis and pulmonary fibrosis models the time of apoptosis.84 in vivo.110 Thus, the extent to which transition- These findings support a model to link initial ing epithelial cells contribute to tubular epithe- epithelial microinjury and apoptosis with local lial degeneration and/or initiation of mesenchy- generation of TGF-␤1 and activation of mesen- mal expansion by generating fibroblastic cells chymal cells as a trigger mechanism of fibrogen- that initiate fibrogenesis still remains unclear esis (Fig. 3). Fibrosis may occur at sites of (Fig. 3). sustained epithelial injury associated with apo- ptosis and TGF-␤ activation as a paracrine me- EMERGING ANTIFIBROTIC diator of mesenchymal cell activation and THERAPIES TARGET TGF-〉 SIGNALING phagocyte recruitment and/or gain of fibro- Large-molecule TGF-␤ antagonists and small- blasts.14,80,81 Thus, aberrant autocrine and para- molecule inhibitors of the TGF-␤–receptor type crine TGF-␤ signaling may have a central role in 1 kinase are under development for primary epithelial-fibroblast miscommunication, trigger- indications in fibrotic diseases, diabetic ne- ing fibrogenesis. phropathy, and possibly metastatic cancers (reviewed in Yingling et al111 and Laping112). ␤ TGF- AND EMT Intriguing recent reports have shown that long- EMT is an extreme manifestation of epithelial term inhibition of TGF-␤ in rodent models by plasticity in which polarized epithelial cells em- transgenic overexpression of soluble TGF-␤ re- bedded in organized cell layers convert into ceptor type 2 Fc chimera or the use of pan- motile fibroblastic cells (reviewed by Thi- neutralizing antibody 1D11 appear to be well ery100). In nonmalignant epithelial cells of renal, tolerated without evidence for increased carci- pulmonary, or hepatic origins, EMT manifesta- nogenesis or autoimmunity in these mod- tions may be triggered rather easily by diverse els.113,114 modes of stress and injury in vitro (reviewed in In addition, several antifibrotic compounds Kalluri and Neilson,101 Yang and Liu,102 and may exert their antifibrotic activities through Zavadil and Bottinger103). TGF-␤ is sufficient to down-modulation of TGF-␤ activity and signal- induce EMT in nonmalignant and malignant ep- ing. The low-molecular-weight plant alkaloid ithelial cells in vitro and is considered a major halofuginone inhibits extracellular matrix accu- factor promoting EMT in invasive and meta- mulation in several animal models of fibrotic static cancers in vivo (reviewed in Zavadil and disorders,115 associated with inhibition of Bottinger103). Smad2/Smad3 phosphorylation and up-regula- In contrast, proof of concept for EMT in tion of inhibitory Smad7.116 Pentoxifylline, a nonmalignant renal fibrosis, or hepatic and pul- nonselective phosphodiesterase inhibitor, at- monary fibrosis, has proven difficult. By using tenuates tubulointerstitial fibrosis by dual complex genetic engineering in mice, Iwano mechanisms, including inhibition of Smad3-/4- et al104 showed in vivo evidence for EMT in a activated transcription, and blockade of profi- model of tubulointerstitial fibrosis induced by brogenic effects of connective tissue growth unilateral ureteral obstruction. However, with factor.117 Pirfenidone is an antifibrotic agent few exceptions, in vivo evidence for EMT in that reduces ECM deposition in numerous fibro- fibrotic tissues is derived largely from the ex- sis models, possibly through reduction of perimental model of unilateral ureteral obstruc- TGF-␤1 synthesis.118 Finally, hepatocyte growth 316 E.P. Böttinger factor and BMP7 are thought to attenuate EMT ling the angiogenic switch: a balance between two and fibrosis in the unilateral ureteral obstruc- distinct TGF-b receptor signaling pathways. Trends tion renal injury model by antagonizing TGF-␤ Cardiovasc Med. 2003;13:301-7. 119-121 13. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu signaling. H, Thomsen GH, et al. Smad7 binds to Smurf2 to Chronic administration of antagonists of TGF-␤ form an E3 ubiquitin ligase that targets the TGF beta signaling in suitable patients with non–immune- receptor for degradation. Mol Cell. 2000;6:1365-75. mediated CKD will need to be guided by biomar- 14. Bottinger EP, Bitzer M. TGF-␤ signaling in renal dis- kers as surrogate readouts for efficacy.48,111 In ease. J Am Soc Nephrol. 2002;13:2600-10. addition to classic fibrosis markers, markers for 15. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, et al. Transforming growth factor epithelial and/or endothelial microinjury char- type beta: rapid induction of fibrosis and angiogen- acteristic of early and potentially reversible esis in vivo and stimulation of collagen formation in stages of CKD need to be developed. The con- vitro. Proc Natl Acad SciUSA.1986;83:4167-71. cept of using antagonists of TGF-␤ to prevent 16. Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, progression of CKD and stabilize life-sustaining Sporn MB, Fauci AS. Transforming growth factor functions of the kidney is promising if the ex- beta is an important immunomodulatory protein for human B lymphocytes. J Immunol. 1986;137: traordinary challenge of navigating their safe 3855-60. administration over long periods of time can be 17. Wahl SM, Hunt DA, Wakefield LM, McCartney-Fran- met. cis N, Wahl LM, Roberts AB, et al. Transforming growth factor type beta induces monocyte chemo- REFERENCES taxis and growth factor production. Proc Natl Acad 1. Roberts AB, Sporn MB. The transforming growth SciUSA.1987;84:5788-92. factors-␤. In: Sporn MB, Roberts AB, editors. Hand- 18. Van Obberghen-Schilling E, Roche NS, Flanders KC, book of experimental pharmacology. Peptide Sporn MB, Roberts AB. Transforming growth factor growth factors and their receptors. 95th ed. Berlin: beta 1 positively regulates its own expression in Springer-Verlag; 1990. p. 419-72. normal and transformed cells. J Biol Chem. 1988; 2. de Larco JE, Todaro GJ. Growth factors from murine 263:7741-6. sarcoma virus-transformed cells. Proc Natl Acad Sci 19. Border WA, Okuda S, Languino LR, Sporn MB, Ruo- U S A. 1978;75:4001-5. slahti E. Suppression of experimental glomerulone- 3. Roberts AB, Anzano MA, Lamb LC, Smith JM, Sporn phritis by antiserum against transforming growth MB. New class of transforming growth factors po- factor beta 1. Nature. 1990;346:371-4. tentiated by epidermal growth factor: isolation from 20. Giri SN, Hyde DM, Hollinger MA. Effect of antibody non-neoplastic tissues. Proc Natl Acad SciUSA. to transforming growth factor beta on bleomycin 1981;78:5339-43. induced accumulation of lung collagen in mice. Tho- 4. Moses HL, Serra R. Regulation of differentiation by rax. 1993;48:959-66. TGF-beta. Curr Opin Genet Dev. 1996;6:581-6. 21. Sporn MB, Roberts AB. Peptide growth factors and 5. Massague J. The transforming growth factor-beta inflammation, tissue repair, and cancer. J Clin Invest. family. Annu Rev Cell Biol. 1990;6:597-641. 1986;78:329-32. 6. Chang H, Brown CW, Matzuk MM. Genetic analysis 22. Moses HL, Yang EY, Pietenpol JA. TGF-beta stimula- of the mammalian transforming growth factor-beta tion and inhibition of cell proliferation: new mech- superfamily. Endocr Rev. 2002;23:787-823. anistic insights. Cell. 1990;63:245-7. 7. Rifkin DB. Latent transforming growth factor-beta 23. Hostetter TH, Olson JL, Rennke HG, Venkatachalam (TGF-beta) binding proteins: orchestrators of TGF- MA, Brenner BM. Hyperfiltration in remnant beta availability. J Biol Chem. 2005;280:7409-12. nephrons: a potentially adverse response to renal 8. Derynck R, Zhang YE. Smad-dependent and Smad- ablation. Am J Physiol. 1981;241:F85-93. independent pathways in TGF-beta family signalling. 24. Hostetter TH, Troy JL, Brenner BM. Glomerular he- Nature. 2003;425:577-84. modynamics in experimental diabetes mellitus. Kid- 9. Shi Y, Massague J. Mechanisms of TGF-beta signaling ney Int. 1981;19:410-5. from cell membrane to the nucleus. Cell. 2003;113: 25. Dunn BR, Anderson S, Brenner BM. The hemody- 685-700. namic basis of progressive renal disease. Semin 10. ten Dijke P, Hill CS. New insights into TGF-beta- Nephrol. 1986;6:122-38. Smad signalling. Trends Biochem Sci. 2004;29: 26. Anderson S, Rennke HG, Brenner BM. Therapeutic 265-73. advantage of converting enzyme inhibitors in arrest- 11. Wrana JL, Attisano L, Wieser R, Ventura F, Massague ing progressive renal disease associated with sys- J. Mechanism of activation of the TGF-beta receptor. temic hypertension in the rat. J Clin Invest. 1986; Nature. 1994;370:341-7. 77:1993-2000. 12. Goumans MJ, Lebrin F, Valdimarsdottir G. Control- 27. Anderson S, Brenner BM. Intraglomerular hyperten- TGF-␤ in renal injury and disease 317

sion: implications and drug treatment. Annu Rev 41. Sotoudeh M, Li YS, Yajima N, Chang CC, Tsou TC, Med. 1988;39:243-53. Wang Y, et al. Induction of apoptosis in vascular 28. Wolf G, Mueller E, Stahl RA, Ziyadeh FN. Angioten- smooth muscle cells by mechanical stretch. Am J sin II-induced hypertrophy of cultured murine prox- Physiol. 2002;282:H1709-16. imal tubular cells is mediated by endogenous trans- 42. Border WA, Noble NA, Yamamoto T, Harper JR, forming growth factor-beta. J Clin Invest. 1993;92: Yamaguchi Yu, Pierschbacher MD, et al. Natural 1366-72. inhibitor of transforming growth factor-beta pro- 29. Kagami S, Border WA, Miller DE, Noble NA. Angio- tects against scarring in experimental kidney dis- tensin II stimulates extracellular matrix protein syn- ease. Nature. 1992;360:361-4. thesis through induction of transforming growth 43. Isaka Y, Brees DK, Ikegaya K, Kaneda Y, Imai E, factor-beta expression in rat glomerular mesangial Noble NA, et al. Gene therapy by skeletal muscle cells. J Clin Invest. 1994;93:2431-7. expression of decorin prevents fibrotic disease in rat 30. Okuda S, Languino LR, Ruoslahti E, Border WA. kidney. Nat Med. 1996;2:418-23. Elevated expression of transforming growth factor- 44. Sharma K, Jin Y, Guo J, Ziyadeh FN. Neutralization beta and proteoglycan production in experimental of TGF-beta by anti-TGF-beta antibody attenuates glomerulonephritis. Possible role in expansion of kidney hypertrophy and the enhanced extracellular the mesangial extracellular matrix [published erra- matrix gene expression in STZ-induced diabetic tum appears in J Clin Invest 1990;86:2175]. J Clin mice. Diabetes. 1996;45:522-30. Invest. 1990;86:453-62. 45. Isaka Y, Akagi Y, Ando Y, Tsujie M, Sudo T, Ohno N, 31. Yamamoto T, Nakamura T, Noble NA, Ruoslahti E, et al. Gene therapy by transforming growth factor- Border WA. Expression of transforming growth fac- beta receptor-IgG Fc chimera suppressed extracellu- tor beta is elevated in human and experimental lar matrix accumulation in experimental glomerulo- diabetic nephropathy. Proc Natl Acad SciUSA. nephritis. Kidney Int. 1999;55:465-75. 1993;90:1814-8. 46. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-de la Cruz 32. Sharma K, Ziyadeh FN. Renal hypertrophy is associ- MC, Hong SW, Isono M, et al. Long-term prevention ated with upregulation of TGF-beta 1 gene expres- of renal insufficiency, excess matrix gene expres- sion in diabetic BB rat and NOD mouse. Am J sion, and glomerular mesangial matrix expansion by Physiol. 1994;267:F1094-101. treatment with monoclonal antitransforming growth 33. Border WA, Noble NA. Transforming growth factor factor-beta antibody in db/db diabetic mice. Proc beta in tissue fibrosis. N Engl J Med. 1994;331: Natl Acad SciUSA.2000;97:8015-20. 1286-92. 47. Rocco MV, Chen Y, Goldfarb S, Ziyadeh FN. Ele- 34. Bitzer M, Sterzel RB, Bottinger EP. Transforming vated glucose stimulates TGF-beta gene expression growth factor-beta in renal disease. Kidney Blood and bioactivity in proximal tubule. Kidney Int. 1992; Press Res. 1998;21:1-12. 41:107-14. 35. Ketteler M, Noble NA, Border WA. Transforming 48. Grygielko ET, Martin WM, Tweed C, Thornton P, growth factor-beta and angiotensin II: the missing Harling J, Brooks DP, et al. Inhibition of gene mark- link from glomerular hyperfiltration to glomerulo- ers of fibrosis with a novel inhibitor of transforming sclerosis? Annu Rev Physiol. 1995;57:279-95. growth factor-beta type I receptor kinase in puro- 36. Noble NA, Border WA. Angiotensin II in renal fibro- mycin-induced nephritis. J Pharmacol Exp Ther. sis: should TGF-beta rather than blood pressure be 2005;313:943-51. the therapeutic target? Sem Nephrol. 1997;17: 49. Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, 455-66. Imai E. Glomerulosclerosis induced by in vivo trans- 37. Ziyadeh FN. Mediators of diabetic renal disease: the fection of transforming growth factor-beta or plate- case for TGF-beta as the major mediator. J Am Soc let-derived growth factor gene into the rat kidney. Nephrol. 2004;15 Suppl 1:S55-7. J Clin Invest. 1993;92:2597-601. 38. Petermann AT, Hiromura K, Blonski M, Pippin J, 50. Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Monkawa T, Durvasula R, et al. Mechanical stress Wakefield L, et al. Hepatic expression of mature reduces podocyte proliferation in vitro. Kidney Int. transforming growth factor beta 1 in transgenic 2002;61:40-50. mice results in multiple tissue lesions. Proc Natl 39. Topper JN, Cai J, Qiu Y, Anderson KR, Xu YY, Deeds Acad SciUSA.1995;92:2572-6. JD, et al. Vascular MADs: two novel MAD-related 51. Bottinger EP, Kopp JB. Lessons from TGF-beta trans- genes selectively inducible by flow in human vascu- genic mice. Miner Electrolyte Metab. 1998;24: lar endothelium. Proc Natl Acad SciUSA.1997;94: 154-60. 9314-9. 52. Schiffer M, Bitzer M, Roberts IS, Kopp JB, ten Dijke 40. Cortes P, Riser B, Narins RG. Glomerular hyperten- P, Mundel P, et al. Apoptosis in podocytes induced sion and progressive renal disease: the interplay of by TGF-beta and Smad7. J Clin Invest. 2001;108: mesangial cell stretch, cytokine formation and extra- 807-16. cellular matrix synthesis. Contrib Nephrol. 1996; 53. Sato M, Muragaki Y, Saika S, Roberts AB, Ooshima A. 118:229-33. Targeted disruption of TGF-beta1/Smad3 signaling 318 E.P. Böttinger

protects against renal tubulointerstitial fibrosis sion in response to transforming growth induced by unilateral ureteral obstruction. J Clin factor-beta1. J Biol Chem. 2004;279:2632-9. Invest. 2003;112:1486-94. 67. Wang L, Ma R, Flavell RA, Choi ME. Requirement of 54. Fujimoto M, Maezawa Y, Yokote K, Joh K, Koba- mitogen-activated protein kinase kinase 3 (MKK3) yashi K, Kawamura H, et al. Mice lacking Smad3 are for activation of p38alpha and p38delta MAPK iso- protected against streptozotocin-induced diabetic forms by TGF-beta 1 in murine mesangial cells. J Biol glomerulopathy. Biochem Biophys Res Commun. Chem. 2002;277:47257-62. 2003;305:1002-7. 68. Daniels CE, Wilkes MC, Edens M, Kottom TJ, Mur- 55. Terada Y, Hanada S, Nakao A, Kuwahara M, Sasaki S, phy SJ, Limper AH, et al. Imatinib mesylate inhibits Marumo F. Gene transfer of Smad7 using electropo- the profibrogenic activity of TGF-beta and prevents ration of adenovirus prevents renal fibrosis in post- bleomycin-mediated lung fibrosis. J Clin Invest. obstructed kidney. Kidney Int. 2002;61:94-8. 2004;114:1308-16. 56. Hou CC, Wang W, Huang XR, Fu P, Chen TH, 69. Wang S, Wilkes MC, Leof EB, Hirschberg R. Imatinib Sheikh-Hamad D, et al. Ultrasound-microbubble-me- mesylate blocks a non-Smad TGF-beta pathway and diated gene transfer of inducible Smad7 blocks trans- reduces renal fibrogenesis in vivo. FASEB J. 2005;19: forming growth factor-{beta} signaling and fibrosis in 1-11. rat remnant kidney. Am J Pathol. 2005;166:761-71. 70. Igarashi A, Okochi H, Bradham DM, Grotendorst GR. 57. Verrecchia F, Mauviel A. Control of connective tis- Regulation of connective tissue growth factor gene sue gene expression by TGF beta: role of Smad expression in human skin fibroblasts and during proteins in fibrosis. Curr Rheumatol Rep. 2002;4: wound repair. Mol Biol Cell. 1993;4:637-45. 143-9. 71. Lasky JA, Ortiz LA, Tonthat B, Hoyle GW, Corti M, 58. Schnaper HW, Kopp JB. Renal fibrosis. Front Biosci. Athas G, et al. Connective tissue growth factor 2003;8:e68-86. mRNA expression is upregulated in bleomycin-in- 59. Vindevoghel L, Lechleider RJ, Kon A, de Caestecker duced lung fibrosis. Am J Physiol. 1998;275:L365-71. MP, Uitto J, Roberts AB, et al. SMAD3/4-dependent 72. McCarron RM, Wang L, Racke MK, McFarlin DE, transcriptional activation of the human type VII col- Spatz M. Cytokine-regulated adhesion between en- lagen gene (COL7A1) promoter by transforming cephalitogenic T lymphocytes and cerebrovascular growth factor beta. Proc Natl Acad SciUSA.1998; endothelial cells. J Neuroimmunol. 1993;43:23-30. 95:14769-74. 73. Paradis V, Dargere D, Vidaud M, De Gouville AC, 60. Verrecchia F, Vindevoghel L, Lechleider RJ, Uitto J, Huet S, Martinez V, et al. Expression of connective Roberts AB, Mauviel A. Smad3/AP-1 interactions tissue growth factor in experimental rat and human control transcriptional responses to TGF-beta in a liver fibrosis. Hepatology. 1999;30:968-76. promoter-specific manner. Oncogene. 2001;20: 74. Mori T, Kawara S, Shinozaki M, Hayashi N, Kaki- 3332-40. numa T, Igarashi A, et al. Role and interaction of 61. Chung KY, Agarwal A, Uitto J, Mauviel A. An AP-1 connective tissue growth factor with transforming binding sequence is essential for regulation of the growth factor-beta in persistent fibrosis: a mouse human alpha2(I) collagen (COL1A2) promoter activ- fibrosis model. J Cell Physiol. 1999;181:153-9. ity by transforming growth factor-beta. J Biol Chem. 75. Roberts AB, Tian F, Byfield SD, Stuelten C, Ooshima 1996;271:3272-8. A, Saika S, et al. Smad3 is key to TGF-beta-mediated 62. Poncelet AC, de Caestecker MP, Schnaper HW. The epithelial-to-mesenchymal transition, fibrosis, tumor transforming growth factor-beta/SMAD signaling suppression and metastasis. Cytokine Growth Factor pathway is present and functional in human mesan- Rev. 2006;17:19-27. gial cells. Kidney Int. 1999;56:1354-65. 76. Verrecchia F, Tacheau C, Wagner EF, Mauviel A. A 63. Wang W, Huang XR, Canlas E, Oka K, Truong LD, central role for the JNK pathway in mediating the Deng C, et al. Essential role of Smad3 in angiotensin antagonistic activity of pro-inflammatory cytokines II-induced vascular fibrosis. Circ Res. 2006;98: against transforming growth factor-beta-driven 1032-9. SMAD3/4-specific gene expression. J Biol Chem. 64. Yang YC, Piek E, Zavadil J, Liang D, Xie D, Heyer J, 2003;278:1585-93. et al. Hierarchical model of gene regulation by trans- 77. Bitzer M, von Gersdorff G, Liang D, Dominguez- forming growth factor beta. Proc Natl Acad Sci Rosales A, Beg AA, Rojkind M, et al. A mechanism of U S A. 2003;100:10269-74. suppression of TGF-beta/SMAD signaling by NF-kap- 65. Hubchak SC, Runyan CE, Kreisberg JI, Schnaper paB/RelA. Genes Dev. 2000;14:187-97. HW. Cytoskeletal rearrangement and signal trans- 78. Ulloa L, Doody J, Massague J. Inhibition of trans- duction in TGF-beta1-stimulated mesangial cell col- forming growth factor-beta/SMAD signalling by lagen accumulation. J Am Soc Nephrol. 2003;14: the interferon-gamma/STAT pathway. Nature. 1999; 1969-80. 397:710-3. 66. Runyan CE, Schnaper HW, Poncelet AC. The phos- 79. Hui AY, Dannenberg AJ, Sung JJ, Subbaramaiah K, phatidylinositol 3-kinase/Akt pathway enhances Du B, Olinga P, et al. Prostaglandin E2 inhibits trans- Smad3-stimulated mesangial cell collagen I expres- forming growth factor beta 1-mediated induction of TGF-␤ in renal injury and disease 319

collagen alpha 1(I) in hepatic stellate cells. 94. Lee CG, Kang HR, Homer RJ, Chupp G, Elias JA. J Hepatol. 2004;41:251-8. Transgenic modeling of transforming growth factor- 80. Canbay A, Friedman S, Gores GJ. Apoptosis: the beta(1): role of apoptosis in fibrosis and alveolar nexus of liver injury and fibrosis. Hepatology. 2004; remodeling. Proc Am Thorac Soc. 2006;3:418-23. 39:273-8. 95. Takehara T, Tatsumi T, Suzuki T, Rucker EB III, 81. Selman M, Pardo A. The epithelial/fibroblastic path- Hennighausen L, Jinushi M, et al. Hepatocyte-spe- way in the pathogenesis of idiopathic pulmonary cific disruption of Bcl-xL leads to continuous hepa- fibrosis. Am J Respir Cell Mol Biol. 2003;29:S93-7. tocyte apoptosis and liver fibrotic responses. Gastro- 82. Savill J. Apoptosis and renal injury. Curr Opin Neph- enterology. 2004;127:1189-97. rol Hypertens. 1995;4:263-9. 96. Kim YH, Goyal M, Kurnit D, Wharram B, Wiggins J, 83. Kumar D, Robertson S, Burns KD. Evidence of apo- Holzman L, et al. Podocyte depletion and glomeru- ptosis in human diabetic kidney. Mol Cell Biochem. losclerosis have a direct relationship in the PAN- 2004;259:67-70. treated rat. Kidney Int. 2001;60:957-68. 84. Schiffer M, Mundel P, Shaw AS, Bottinger EP. A 97. Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hus- novel role for the adaptor molecule CD2-associated sain S, Filipiak WE, et al. Podocyte depletion causes protein in TGF-beta-induced apoptosis. J Biol Chem. glomerulosclerosis: diphtheria toxin-induced podo- 2004;279:37004-12. cyte depletion in rats expressing human diphtheria 85. Susztak K, Ciccone E, McCue P, Sharma K, Bottinger toxin receptor transgene. J Am Soc Nephrol. 2005. EP. Multiple metabolic hits converge on CD36 as 98. Kelly DJ, Cox AJ, Tolcos M, Cooper ME, Wilkinson- novel mediator of tubular epithelial apoptosis in Berka JL, Gilbert RE. Attenuation of tubular apopto- diabetic nephropathy. PLoS Med. 2005;2:e45. sis by blockade of the renin-angiotensin system in 86. Fadok VA, Bratton DL, Konowal A, Freed PW, West- diabetic Ren-2 rats. Kidney Int. 2002;61:31-9. cott JY, Henson PM. Macrophages that have in- 99. Schaefer L, Mihalik D, Babelova A, Krzyzankova M, gested apoptotic cells in vitro inhibit proinflamma- Grone HJ, Iozzo RV, et al. Regulation of fibrillin-1 by tory cytokine production through autocrine/ biglycan and decorin is important for tissue preser- paracrine mechanisms involving TGF-beta, PGE2, vation in the kidney during pressure-induced injury. and PAF. J Clin Invest. 1998;101:890-8. Am J Pathol. 2004;165:383-96. 87. Canbay A, Feldstein AE, Higuchi H, Werneburg N, 100. Thiery JP. Epithelial-mesenchymal transitions in de- Grambihler A, Bronk SF, et al. Kupffer cell engulf- velopment and pathologies. Curr Opin Cell Biol. ment of apoptotic bodies stimulates death ligand 2003;15:740-6. and cytokine expression. Hepatology. 2003;38: 101. Kalluri R, Neilson EG. Epithelial-mesenchymal tran- 1188-98. sition and its implications for fibrosis. J Clin Invest. 88. Canbay A, Taimr P, Torok N, Higuchi H, Friedman S, 2003;112:1776-84. Gores GJ. Apoptotic body engulfment by a human 102. Yang J, Liu Y. Dissection of key events in tubular stellate cell line is profibrogenic. Lab Invest. 2003; epithelial to myofibroblast transition and its implica- 83:655-63. tions in renal interstitial fibrosis. Am J Pathol. 2001; 89. Savill J, Smith J, Sarraf C, Ren Y, Abbott F, Rees A. 159:1465-75. Glomerular mesangial cells and inflammatory mac- 103. Zavadil J, Bottinger EP. TGF-beta and epithelial- rophages ingest neutrophils undergoing apoptosis. to-mesenchymal transitions. Oncogene. 2005;24: Kidney Int. 1992;42:924-36. 5764-74. 90. Arsalane K, Dubois CM, Muanza T, Begin R, Boud- 104. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, reau F, Asselin C, et al. Transforming growth factor- Neilson EG. Evidence that fibroblasts derive from beta1 is a potent inhibitor of glutathione synthesis in epithelium during tissue fibrosis. J Clin Invest. 2002; the lung epithelial cell line A549: transcriptional 110:341-50. effect on the GSH rate-limiting enzyme gamma-glu- 105. Kaneto H, Morrissey J, Klahr S. Increased expression tamylcysteine synthetase. Am J Respir Cell Mol Biol. of TGF-beta 1 mRNA in the obstructed kidney of rats 1997;17:599-607. with unilateral ureteral ligation. Kidney Int. 1993; 91. Budinger GR, Mutlu GM, Eisenbart J, Fuller AC, 44:313-21. Bellmeyer AA, Baker CM, et al. Proapoptotic Bid is 106. Morrissey J, Guo G, Moridaira K, Fitzgerald M, Mc- required for pulmonary fibrosis. Proc Natl Acad Sci Cracken R, Tolley T, et al. Transforming growth U S A. 2006;103:4604-9. factor-beta induces renal epithelial jagged-1 expres- 92. Kuwano K, Kunitake R, Maeyama T, Hagimoto N, sion in fibrotic disease. J Am Soc Nephrol. 2002;13: Kawasaki M, Matsuba T, et al. Attenuation of bleo- 1499-508. mycin-induced pneumopathy in mice by a caspase 107. Rastaldi MP, Ferrario F, Giardino L, Dell’Antonio G, inhibitor. Am J Physiol. 2001;280:L316-25. Grillo C, Grillo P, et al. Epithelial-mesenchymal tran- 93. Uhal BD, Rayford H, Zhuang J, Li X, Laukka J, sition of tubular epithelial cells in human renal bi- Soledad-Conrad V. Apoptosis-dependent acute lung opsies. Kidney Int. 2002;62:137-46. injury and repair after intratracheal instillation of 108. Vongwiwatana A, Tasanarong A, Rayner DC, Melk A, noradrenaline in rats. Exp Physiol. 2003;88:269-75. Halloran PF. Epithelial to mesenchymal transition 320 E.P. Böttinger

during late deterioration of human kidney 115. Pines M, Nagler A. Halofuginone: a novel antifibrotic transplants: the role of tubular cells in fibrogenesis. therapy. Gen Pharmacol. 1998;30:445-50. Am J Transplant. 2005;5:1367-74. 116. Xavier S, Piek E, Fujii M, Javelaud D, Mauviel A, 109. Oldfield MD, Bach LA, Forbes JM, Nikolic-Paterson Flanders KC, et al. Amelioration of radiation-induced D, McRobert A, Thallas V, et al. Advanced glycation fibrosis: inhibition of transforming growth factor- end products cause epithelial-myofibroblast transdif- beta signaling by halofuginone. J Biol Chem. 2004; ferentiation via the receptor for advanced glycation 279:15167-76. end products (RAGE). J Clin Invest. 2001;108: 117. Lin SL, Chen RH, Chen YM, Chiang WC, Lai CF, Wu 1853-63. KD, et al. Pentoxifylline attenuates tubulointerstitial 110. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, fibrosis by blocking Smad3/4-activated transcription Crandall ED, du Bois RM, et al. Induction of epithe- and profibrogenic effects of connective tissue lial-mesenchymal transition in alveolar epithelial growth factor. J Am Soc Nephrol. 2005;16:2702-13. cells by transforming growth factor-beta1: potential 118. Shihab FS, Bennett WM, Yi H, Andoh TF. Pirfeni- done treatment decreases transforming growth fac- role in idiopathic pulmonary fibrosis. Am J Pathol. tor-beta1 and matrix proteins and ameliorates fibro- 2005;166:1321-32. sis in chronic cyclosporine nephrotoxicity. Am J 111. Yingling JM, Blanchard KL, Sawyer JS. Development Transplant. 2002;2:111-9. of TGF-beta signalling inhibitors for cancer therapy. 119. Mizuno S, Kurosawa T, Matsumoto K, Mizuno- Nat Rev Drug Discov. 2004;3:1011-22. Horikawa Y, Okamoto M, Nakamura T. Hepatocyte 112. Laping NJ. ALK5 inhibition in renal disease. Curr growth factor prevents renal fibrosis and dysfunc- Opin Pharmacol. 2003;3:204-8. tion in a mouse model of chronic renal disease. 113. Yang YA, Dukhanina O, Tang B, Mamura M, Letterio J Clin Invest. 1998;101:1827-34. JJ, MacGregor J, et al. Lifetime exposure to a soluble 120. Zeisberg M, Bottiglio C, Kumar N, Maeshima Y, TGF-beta antagonist protects mice against metastasis Strutz F, Muller GA, et al. Bone morphogenic pro- without adverse side effects. J Clin Invest. 2002;109: tein-7 inhibits progression of chronic renal fibrosis 1607-15. associated with two genetic mouse models. Am J 114. Ruzek MC, Hawes M, Pratt B, McPherson J, Led- Physiol. 2003;285:F1060-7. better S, Richards SM, et al. Minimal effects on 121. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Ch- immune parameters following chronic anti-TGF- arytan D, Strutz F, et al. BMP-7 counteracts TGF- beta monoclonal antibody administration to nor- beta1-induced epithelial-to-mesenchymal transition mal mice. Immunopharmacol Immunotoxicol. and reverses chronic renal injury. Nat Med. 2003;9: 2003;25:235-57. 964-8.