Articles in PresS. Am J Physiol Renal Physiol (January 6, 2016). doi:10.1152/ajprenal.00365.

2015

TGF- Signaling in the Kidney: Pro-fibrotic and Protective Effects

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Angara Sureshbabu1*, Saif A. Muhsin2*, and Mary E. Choi1,2#

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Joan and Sanford I. Weill Department of Medicine, Division of Nephrology & Hypertension,
Weill Cornell Medical College, and 2New York-Presbyterian Hospital-Weill Cornell Medical
Center, New York, NY 10065, USA

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Running Title: Pro-fibrotic and Protective Effects of TGF-

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*A. Sureshbabu and S.A. Muhsin contributed equally and are considered co-first authors

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Address correspondence to:

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Mary E. Choi, M.D.

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Joan and Sanford I. Weill Department of Medicine

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Division of Nephrology & Hypertension

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Weill Cornell Medical College

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525 East 68th Street, Box 3

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New York, NY 10065

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E-mail: mechoi@med.cornell.edu

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Copyright 2016 by the American Physiological Society.

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Abstract

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Transforming Growth Factor-beta (TGF-) is generally considered as a central mediator of

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fibrotic diseases. Indeed, much focus has been placed on inhibiting TGF- and its downstream

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targets as ideal therapeutic strategies. However, pharmacological blockade of TGF- has not yet

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translated into successful therapy for humans, which may be due to pleiotropic effects of TGF-

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signaling. Equally, TGF- signaling as a protective response in kidney injury has been relatively

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underexplored. Emerging body of evidence from experimental kidney disease models indicates

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multifunctionality of TGF- capable of inducing pro-fibrotic and protective effects. This review

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article discusses recent advances highlighting the diverse roles of TGF- in not only promoting

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renal fibrosis, but also protective responses of TGF- signaling. We review, in particular,

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growing evidence that supports protective effects of TGF- by mechanisms which include

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inhibiting inflammation and induction of autophagy. Additional detailed studies are required to

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fully understand the diverse mechanisms of TGF- actions in renal fibrosis and inflammation

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that will likely direct towards effective anti-fibrotic therapies.

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Keywords: TGF-, Smad, BMP, Kidney, Fibrosis, Autophagy, Apoptosis, Inflammation

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Introduction

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Transforming growth factor-beta (TGF-) is a multifunctional cytokine that is well

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recognized to regulate a broad spectrum of cellular processes such as growth, differentiation,

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apoptosis, wound repair, and the pathogenesis of fibrosis. It is a member of a superfamily of 38

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cytokines that include TGF-, bone morphogenetic proteins (BMP), growth differentiation

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factors, inhibins, and activins. TGF- superfamily members, in particular, TGF- and BMP have

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been implicated to play important and diverse roles in chronic kidney disease (CKD) (71, 100).

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The hallmark of progressive CKD is the excessive extracellular matrix (ECM) production

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leading to renal fibrosis and TGF- has been generally considered a potent pro-fibrotic mediator

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in this process (17, 48).

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Mammalian TGF- includes three major isoforms that are encoded by distinct genes

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(TGF-1, TGF-2, and TGF-3). The isoforms are synthesized as large precursor proteins (pro-

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TGF-) forming dimeric complexes in the endoplasmic reticulum, and are subsequently cleaved

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near the carboxy-terminus to yield mature 112 amino acid polypeptides, which share 60-80%

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conservation across the three TGF- isoforms (99). The mature TGF- dimer remains associated

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with cleaved latency peptide portion of the precursor as an inactive latent complex, and

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dissociation from the complex results in biologically active TGF- (76). Thus, newly synthesized

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TGF- bound to the latency associated peptide (LAP) forming a small latent complex (SLC) is

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biologically inactive and cannot bind to its receptors. Through the formation of disulfide bonds,

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this complex loosely binds to a latent TGF- binding protein (LTBP) to form a large latent

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complex (LLC). TGF- is then secreted in a latent state and is stored in the ECM (2, 76).

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Activation of TGF- involves release from the latent complex following exposure to a number of

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different factors including integrins, proteases, metalloproteinases, reactive oxygen species

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(ROS), plasmin, and acid, that allows binding to its cell surface receptors for initiation of TGF-

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signaling (25, 76, 81, 87).

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It is well established that activation of TGF- leads to pleiotropic cellular responses that

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are mediated by TGF- signaling via interactions with TGF- receptor type I (TRI) and TGF-

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receptor type II (TRII) and subsequent activation of intracellular Smad and non-Smad

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dependent signaling pathways (13, 16, 57). In this review, we describe emerging body of

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evidence from experimental kidney disease models demonstrating multifunctional nature of

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TGF- capable of inducing pro-fibrotic and protective effects. We provide an overview on recent

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advances highlighting the diverse roles of TGF- in not only promoting renal fibrosis but also

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protective responses, focusing on growing evidence for protective effects of TGF- by

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mechanisms which include inhibiting inflammation and induction of autophagy.

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TGF- isoforms in the kidney

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Studies of human kidney specimens have confirmed that the three major isoforms TGF-

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1, TGF-2, and TGF-3 are expressed in the kidney (33). While functional redundancy between

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the TGF- isoforms has been long recognized, there is a growing body of evidence for the

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existence of non-redundant functions in inflammation and organ development (76). TGF-1, the

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major focus of this review, is the predominant and best-characterized isoform, while TGF-2 and

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TGF-3 are less known. Some clear differences among the isoforms have been noted. In the

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normal human adult kidney, glomerular expression of TGF-2 and TGF-3 is seen mainly in

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podocytes, whereas TGF-1 is primarily detected in the tubules but not in the glomeruli (33).

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Interestingly, glomerular expression of TGF-1, generally with TGF-2 and TGF-3, was

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detected in podocytes in kidney biopsy specimens from patients with proliferative

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glomerulonephritis and in mesangial cells in diabetic nephropathy and IgA nephropathy (33).

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Moreover, increased expression of TGF-1 was associated with development of severe

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glomerulonephritis and glomerulosclerosis (33).

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Biological actions of TGF- isoforms are mediated by ligand binding to its receptors for

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the initiation of signaling. Both TGF-1 and TGF-3 bind directly with TRII, whereas TGF-2

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requires the presence of a type III TGF- receptor (TRIII) for ligand binding to TRII (99).

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Given the differences in the expression patterns and the mechanism of ligand binding, together

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with apparent non-overlapping phenotypes of the three TGF- isoform knockout mice, it is not

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unreasonable that some cellular responses may differ among the TGF- isoforms.

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All three TGF- isoforms have been shown, in vitro, to induce ECM protein production

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in various renal cells, including glomerular mesangial cells, renal fibroblasts, and renal tubular

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epithelial cells (91, 99). While most studies have demonstrated similar pro-fibrotic effects of the

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TGF- isoforms, a number of recent studies have suggested that TGF-2 and TGF-3 can exert

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anti-fibrotic effects (72, 75, 96, 99). Studies in human podocytes demonstrated anti-fibrotic

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effects of TGF-2 through upregulation of sphingosine kinase-1 (SK-1) activity, thereby

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suppressing pro-fibrotic connective tissue growth factor (CTGF) expression (75). It is important

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to note that similar anti-fibrotic effects of TGF-2 were not observed in other kidney cell types,

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as all three isoforms equally induced upregulation of CTGF mRNA in cultured mesangial cells

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and glomerular visceral epithelial cells (33). Moreover, TGF-2 stimulated the expression of

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ECM proteins and induced EMT in tubular epithelial cells, whereas neutralizing antibody to

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TGF-2 or repression of TGF-2 expression inhibited renal fibrogenesis (91). Further

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investigations are warranted to clarify the seemingly opposite findings regarding the anti-fibrotic

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roles of TGF-2 and TGF-3, which carry important implications for therapeutic targeting

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strategy.

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Activation of TGF-1 Signaling

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TGF-1 signaling involves activation of complex intracellular networks to regulate

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pleiotropic biological functions. As depicted in Figure 1, the active form of TGF-1 transmits

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signals through type I and type II receptors, TRI and TRII. These cell surface receptors

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contain serine/threonine kinases that phosphorylate transcription factor proteins called Smads to

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initiate the canonical TGF- signaling pathway. TGF-1 ligand assembles a heteromeric

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complex through binding to TRII, which then phosphorylates the kinase domain of TRI, and

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leads to the downstream activation of the receptor-activated or regulatory Smads (R-Smads),

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namely Smad2 and Smad3. The activated R-Smads form an oligomeric complex with the

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common mediator Smad4 and undergo nuclear translocation to regulate transcription of target

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genes (57). On the other hand, inhibitory Smads (Smad6 and Smad7) bind to activated TRI and

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interfere with R-Smad activation. Smad7 has also been shown to negatively regulate TGF-

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signaling through recruitment of E3 ubiquitin ligases, Smurf1 and Smurf2, which target TGF-

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receptors and Smad7 to undergo degradation via proteasomal and lysosomal pathways (22, 36).

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Additionally, TGF-1 signaling is mediated via non-canonical, non-Smad pathways

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(Figure 1) including the mitogen-activated protein kinases (MAPKs), mediated by Ras-Raf

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activating MEK (also called MAPKK) and extracellular signal-regulated kinase (ERK), (16, 68,

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90) or TAK1 (TGF- activated kinase 1) pathway, the upstream MAPK kinase kinase (MKKK)

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activating MKK3-p38 and MKK4-JNK (c-Jun N-terminal kinase) signaling cascades (13, 37, 38,

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69). TGF-1 signaling also activates Rho family small GTPases, Rho, Rac, Cdc42 as well as

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Phosphatidylinositol 3 Kinase (PI3K)/AKT and Integrin Linked Kinase (ILK) (3, 23, 50, 66, 95,

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98).

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Although the precise mechanisms underlying progressive renal fibrosis are not

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completely understood, studies have shown that TGF-1, mediated via its canonical Smad

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signaling pathway, drives development of renal fibrosis (77). Recent investigations have also

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demonstrated that the non-canonical, non-Smad pathways can also mediate TGF-1 fibrogenic

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responses (13, 38). However, new findings show that blocking TGF-1 signaling in renal cells in

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vitro or in experimental animal models in vivo does not necessarily reduce renal fibrosis.

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Anti-TGF- therapy in Renal Fibrosis

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Based on significant pre-clinical evidence showing TGF-1 as a key mediator of fibrotic

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diseases, much effort had been placed on inhibiting TGF-1 as a therapeutic strategy. Several

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recent clinical trials using pharmacological blockade of TGF- in fibrotic kidney diseases are

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summarized in Table 2. Therapeutic approach using neutralizing anti-TGF- antibody has been

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explored in models of experimental diabetes induced by STZ and Leprdb/db diabetic mice,

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demonstrating significant reduction in TGF-1 levels and inhibition of glomerular mesangial

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matrix expansion with suppression of collagen and fibronectin expression (10, 80, 102).

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However, the recent multicenter phase II trial sponsored by Eli Lilly using LY2382770, a

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humanized neutralizing monoclonal antibody against TGF-1 for the treatment of diabetic

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nephropathy had to be prematurely terminated due to futility in efficacy (clinicaltrials.gov:

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NCT01113801). Fresolimumab (GC-1008, Genzyme) is a human monoclonal antibody that

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neutralizes all three isoforms (TGF-1, 2, and 3). A single-dose infusion was shown to be

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relatively safe and well tolerated in a phase I multicenter, open-label study conducted in patients

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with treatment-resistant primary FSGS. A subsequent phase II multicenter, double-blind,

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randomized study of fresolimumab in patients with steroid-resistant primary FSGS was recently

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completed but no study results have been reported to date (clinicaltrials.gov: NCT01665391).

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Pirfenidone is a small-molecular weight synthetic compound that has attracted much

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attention as an orally active anti-fibrotic agent recently approved by the U.S. Food and Drug

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Administration for the treatment of idiopathic pulmonary fibrosis (35). Pirfenidone effects are, in

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part, mediated via inhibition of TGF-1 synthesis (78). In the kidney, the effects of pirfenidone

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in suppressing renal fibrosis have been shown in several experimental models including diabetic

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nephropathy, UUO, and subtotal nephrectomy (9, 74, 82). An open-label, single-center pilot

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study demonstrated that pirfenidone significantly slowed renal function decline rate in patients

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with FSGS, with a median improvement of 25% in patients who have moderate to severe CKD

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and are already being treated with angiotensin antagonists (11). However, the phase II trial failed

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to show an improvement in proteinuria (clinicaltrials.gov: NCT00001959). Limitations of the

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trial included the small-scale, exploratory nature of the study and the lack of control group. A

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phase I/II randomized, placebo-controlled trial conducted in 77 patients with overt diabetic

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nephropathy who had albuminuria and reduced estimated glomerular filtration rate (eGFR) (20 to

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75 ml/min per 1.73 m2) demonstrated encouraging results of pirfenidone in improving kidney

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function, but without improvement in proteinuria (clinicaltrials.gov: NCT00063583). The mean

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eGFR increased in the pirfenidone (1200 mg/d) group (+3.3 8.5 ml/min per 1.73 m), whereas,

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the mean eGFR decreased in the placebo control group (-2.2 4.8 ml/min per 1.73 m) (79).

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The results from these clinical trials portend some promise of pirfenidone as an anti-

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fibrotic therapy to improve or slow the decline in kidney function associated with CKD. It should

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be noted, however, that in the above studies, the assessment of kidney function was by eGFR

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using the Modification of Diet in Renal Disease (MDRD) equation, and not by direct

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measurement of GFR. In addition, no serial kidney biopsies were performed to confirm

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histologic evidence of regression of renal fibrosis. Optimal dosage also may need to be further

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identified as well.

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Hence, anti-TGF- therapy has not yet translated into successful treatment in CKD

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patients, and unfortunately, many of the clinical trials that have been completed to date have

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been small and/or uncontrolled, and conclusive evidence regarding efficacy is limited and

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disappointing. Below, we describe studies that provide evidence for pro-fibrotic effects as well

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as evidence that supports protective effects of TGF-1.

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Pro-fibrotic Effects of TGF-1

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Renal fibrosis is characterized by excessive production and accumulation of ECM

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proteins in the kidney that leads to parenchymal scarring, renal dysfunction, and ultimately end-

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stage kidney disease. TGF-1 is the most extensively studied prototype member of the TGF-

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superfamily in the context of fibrosis. TGF-1 is well known as a central mediator of renal

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fibrosis and has long been considered to play potent roles in the progression of CKD (7, 17, 48).

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Increased expression of TGF-1 mRNA and protein is seen in patients with fibrotic kidney

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diseases, including IgA nephropathy, focal and segmental glomerulonephritis, lupus nephritis,

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and diabetic and human immunodeficiency virusassociated nephropathy (5). Moreover, urinary

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TGF-1 excretion is significantly elevated in patients with glomerular disease and heavy

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proteinuria (24).

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The overexpression of TGF-1 in transgenic mice with increased levels of circulating

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active TGF-1 induces progressive renal disease characterized by mesangial expansion,

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accumulation of ECM proteins, and development of glomerulosclerosis and tubulointerstitial

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fibrosis (44). The accumulation of ECM proteins is stimulated by TGF-1 acting as both an

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inducer of ECM synthesis and an inhibitor of ECM degradation by reduced synthesis of matrix

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metalloproteinases, and increased synthesis of proteinase inhibitors, such as plasminogen

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activator inhibitor-1 (PAI-1).

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It has been shown that TGF-1 activation is required for its biological actions and

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important in the development of renal fibrosis. Active TGF-1 is released when the latent

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complex anchored to ECM is cleaved through proteolysis, following exposure to factors such as

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integrins (2). Integrin v6 is a heterodimeric matrix receptor expressed in renal epithelia which

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binds and activates latent TGF-1. Studies using an in vivo model of progressive renal fibrosis

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induced by unilateral ureteral obstruction (UUO) in 6 integrin-null mice have demonstrated that

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blockade of v6-mediated TGF-1 activation was associated with lower expression of type I

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and type III collagen, PAI-1 and collagen content, and protected against tubulointerstitial fibrosis

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(56). Thus, these studies provide evidence that local activation of TGF-1 is important in the

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development of renal fibrosis.

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A significant body of evidence exists in the literature that various approaches that disrupt

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TGF-1 signaling protect against renal fibrosis. Inhibition of TGF-1 by using the proteoglycan

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decorin which is a known inhibitor of TGF-1 or anti-TGF- antibodies abrogated development

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of renal fibrosis (32, 62). BMP-7 is a TGF- superfamily member that has been shown to play a

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protective role in renal fibrosis through anti-inflammatory, anti-oxidative, and anti-fibrotic

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effects by counteracting the effects of TGF-1 (100). BMP-7 signaling pathway and its role in

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fibrosis have been the subject of several recent reviews (6, 48, 71). In murine mesangial cells,

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BMP-7 decreases Smad3 accumulation and inhibits the transcriptional upregulation of Smad3

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targets such as plasminogen activator inhibitor-1 (PAI-1), thereby, suppresses the pro-fibrotic

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effects of TGF-1 (93). Administration of exogenous recombinant human BMP-7 reduced renal

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fibrosis in experimental models of renal injury in mice, including UUO and streptozotocin

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(STZ)-induced diabetic kidney disease (64, 84, 92). Thus, BMP-7 opposes TGF-1/Smad3-

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mediated renal fibrosis. Studies in Smad3-null mice revealed that targeted deletion of Smad3

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signaling resulted in reduced ECM protein accumulation and attenuation of tubulointerstitial

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fibrosis following UUO injury (31, 73, 77). They also showed that the numbers of

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myofibroblasts, macrophages, and CD4/CD8 T cells, and monocyte influx into the kidney after

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UUO were significantly decreased in the obstructed kidneys of Smad3-null mice (31, 77). These

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findings suggest that TGF-1/Smad3 signaling mediates development of renal fibrosis and

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inflammation.

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TGF-1 signaling via the non-Smad pathways also participates in mediating pro-fibrotic

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responses. Data from in vitro studies indicate that TGF-1-stimulated collagen expression in

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mesangial cells is mediated via TAK1/MKK3/p38 signaling cascade, and fibronectin expression

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in fibroblasts (13, 38, 69). Moreover, investigations using pharmacologic and genetic blockade in

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in vivo models of glomerular and tubulointerstitial injury in mice have shown that the

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TAK1/MKK3/p38 and JNK pathways promote renal fibrosis (13, 39). Both the p38 and JNK are

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downstream targets of TAK1 activation. Conditional Tak1 gene deletion in mice suppressed

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interstitial myofibroblast accumulation, collagen deposition, and expression of pro-fibrotic

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molecules following UUO injury (55). These studies strongly suggest that the non-Smad

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signaling pathway via TAK1 plays a critical role in ECM production and the pathogenesis of

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kidney fibrosis.

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Protective Effects of TGF-1

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Given the abundance of evidence from the preclinical studies, targeting TGF- signaling

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pathways seems logical in order to attenuate the development of renal fibrosis and progression of

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CKD. However, contrary to highly anticipated results, data from clinical trials to date have not

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been as strong as was hoped, and anti-TGF-1 treatment directed at pharmacological blockade of

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TGF-1 has not yet translated into successful therapy for humans. Recently emerging evidence

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suggests that it may be due, at least in part, to the fact that TGF-1 is capable of inducing not

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only pro-fibrotic effects, but also protective effects (Table 1).

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Numerous in vitro studies have shown that TGF-1 acts as a potent stimulator of ECM

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production, including the synthesis of type I collagen and fibronectin. However, a recent report

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by Neelisetty and colleagues demonstrated surprising findings that blocking TGF- signaling

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through deletion of TRII in matrix-producing renal interstitial cells failed to suppress renal

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fibrosis in mouse models kidney injury induced by UUO or aristolochic acid (67). The studies

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showed that TRII deleted renal interstitial cells had significantly reduced type I collagen

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production, but the overall renal fibrosis following kidney injury was not decreased in the

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conditional knockout mice (67). These findings indicate that inhibiting TGF- signaling in

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matrix-producing renal interstitial cells is not sufficient to protect against development of fibrosis

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after kidney injury.

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Interestingly, mice overexpressing latent (inactive) form of TGF-1 in keratinocytes

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demonstrated protection against renal fibrosis in experimental obstructive kidney disease and

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crescentic glomerulonephritis (28, 29). These mice had increased levels of latent, but not active,

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TGF-1 in plasma and kidney tissue, and upregulation of renal Smad7. The observed protective

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effects in the latent TGF-1 transgenic mice may be, at least in part, due to Smad7-mediated

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inhibition of nuclear factor-B (NF-B) as well as inhibiting TGF-1 activation, consequently

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protecting the kidney against inflammation and fibrosis.

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Taken together, these studies suggest that TGF-1 is capable of exerting protective

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effects by opposing the pro-fibrotic pathway via negative feedback effects of Smad2 and Smad7.

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The maintenance of balance through this negative self-regulation of pro-fibrotic effects may be

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important for tissue homeostasis. Under pathological conditions, it is possible that the balance

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tips towards the pro-fibrotic pathway through dysregulation of the inhibitory mechanisms to

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cause renal fibrosis, rather than solely through excess TGF-1, and that this may perhaps account

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for the disappointing results of targeting TGF- in humans. Furthermore, there is emerging

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evidence for the protective effects of TGF- by mechanisms that include inhibiting inflammation

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and induction of autophagy, which will be discussed in the following sections.

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TGF- Signaling and Renal Inflammation

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Renal inflammation is implicated as a critical event in the initiation and progression of

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renal fibrosis in CKD. Inflammatory response to renal injury involves infiltrating immune cells

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as well as activated resident renal cells, which stimulate the production and release of pro-

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fibrotic cytokines and growth factors and drive the pro-fibrotic process. Interestingly, despite the

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established pro-inflammatory role, TGF-1 also possesses anti-inflammatory effects (49). Its

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anti-inflammatory properties are evidenced by findings that targeted deletion of TGF-1 gene

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results in profound multifocal inflammatory disease in mice (48, 83). Tgf-1-null mice develop a

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rapid wasting syndrome and early death by 3-4 weeks of age, and display excessive

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inflammatory responses with massive infiltration of lymphocytes and macrophages in many

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organs (45). These data indicate that TGF-1 is a potent anti-inflammatory molecule.

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It has been proposed that the reason why therapies directed against TGF-1 in general

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have yielded disappointing results is because blockade of TGF-1 signaling not only abrogates

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its pro-fibrotic effects, but also inhibits its anti-inflammatory effects, and thereby can promote

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renal fibrosis. Indeed, in vivo studies in mice have confirmed that conditional deletion of TRII

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from the kidney tubular epithelial cells enhanced NF-B signaling and renal inflammation, with

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upregulation of pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis

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factor- (TNF-) in the kidney following UUO injury (59). Similarly, disruption of TRII in

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cultured renal tubular epithelial cells and fibroblasts resulted in impairment of the anti-

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inflammatory effect of TGF-1 (59). Furthermore, conditional Smad4 knockout mice in which

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Smad4 was specifically deleted from the kidney tubular epithelial cells displayed significantly

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enhanced renal inflammation as evidenced by increased infiltration of CD45+ leukocytes and

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F4/80+ macrophages, and upregulation of pro-inflammatory cytokines IL-1, TNF-, monocyte

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chemoattractant protein-1 (MCP-1), as well as intercellular adhesion molecule-1 (ICAM-1) in

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the UUO kidney (60). Thus, inhibition of TGF- signaling through either disruption of TRII or

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Smad4 resulted in enhanced renal inflammation, suggesting that TGF- possesses anti-

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inflammatory effects in the kidney.

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Further evidence indicates that loss of Smad4 represses Smad7 transcription that leads to

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decreased IB (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor,

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alpha) expression but enhanced NF-B activation in the tubulointerstitium after UUO, thereby

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promoting renal inflammation (60). Moreover, Smad7 knockout mice display enhanced renal

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inflammation with increased NF-B/p65 phosphorylation and increased macrophage infiltration

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in models of UUO and STZ-induced diabetes (8, 14). In contrast, Smad7 gene transfer into the

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kidney of STZ-induced diabetic rats using an ultrasound microbubble-mediated technique

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significantly attenuated NF-kB/p65-driven renal inflammation and macrophage infiltration in

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diabetic rats (8). Latent TGF-1 overexpression in mice also protected against renal

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inflammation, via up-regulation of renal Smad7 and IB and suppression of NF-B activation

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in models of UUO and crescentic glomerulonephritis (29, 94). Thus, Smad7-mediated inhibition

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of NF-B activation via the induction of IB may be a central mechanism to abrogate renal

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inflammation (Figure 2).

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Recent investigations have also explored the role of TGF- signaling and macrophage

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differentiation in inflammation. Kidney injury stimulates the recruitment and induction of

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macrophages to undergo classical activation and differentiate into pro-inflammatory M1 and

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anti-inflammatory M2 macrophages (1, 47). More recently, M2 macrophages have been further

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subdivided into three subsets namely M2a, M2b and M2c macrophages. Regulatory M2c

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macrophages are induced by TGF- or IL-10 and recent evidence reported that this particular

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subset displayed protection against renal fibrosis (54, 65). While M2c phenotype is induced by

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TGF- and generally considered to be anti-inflammatory, M2c macrophages in turn produce

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TGF-, and it has been proposed that this might be a mechanism by which macrophages drive

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the development and progression of fibrosis (54). However, a recent study using conditional

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deletion of TGF-1 in macrophages suggest that macrophage-derived TGF- may not serve as a

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functionally important source of TGF- for renal fibrosis (30).

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TGF- Signaling and Autophagy

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Recent evidence proposes a new mechanism by which TGF-1 can provide

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cytoprotective effects via induction of a highly conserved cellular process known as

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macroautophagy, hereafter referred to as autophagy. It has long been known that autophagy is an

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intracellular process that occurs in all eukaryotes and results in the degradation of sequestered

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cytoplasmic elements in lysosomes (12). The execution of autophagy begins with the

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establishment of protein complexes that function in the formation of the phagophore, also known

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as the isolation membrane. The next major step, elongation of the autophagosome includes two

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ubiquitin-like protein complexes, namely autophagy-related gene (ATG)5 ATG12 and

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microtubule-associated protein 1 light chain 3 (LC3) conjugation systems. These two major steps

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are sequentially followed by autophagosome-lysosomal fusion, lysosomal degradation and

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release of free amino acids and fatty acids (12, 85, 86).

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Accumulating data implicate critical roles of autophagy in health and kidney disease (17).

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TGF-1 stimulation induced the accumulation of autophagosomes and conversion of LC3 to the

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lipidated form, LC3-II and upregulated autophagy-related genes, ATG5, ATG7, LC3 and Beclin

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1 in renal tubular epithelial cells (19, 42, 97). We also recently reported that TGF-1 induced

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autophagy in glomerular mesangial cells (18, 40). Hence, these studies provide strong evidence

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that TGF-1 acts as an inducer of autophagy.

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Important advances have also been made in our understanding of the mechanisms of

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autophagy activation by TGF-1 signaling pathways (18, 40, 88). In primary mesangial cells, we

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further showed evidence that TGF-1 induces autophagy through the TAK1-MKK3-p38

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signaling pathway, and autophagy promotes intracellular degradation of collagen and aggregated,

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insoluble procollagen (40). Moreover, TGF-1 protects against serum deprivation-induced

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mesangial cell apoptosis through the induction of autophagy via TAK1 and AKT activation (18).

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The role of AKT-mTOR signaling pathway has also been demonstrated to mediate the activation

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of autophagy in vivo following UUO injury in rats (41).

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Autophagy is increasingly recognized as an important cellular defense against various

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stress stimuli, and dysregulated autophagy is implicated in diseases characterized by progressive

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kidney fibrosis (17). Upregulation of autophagy expression was noted in human kidney biopsies

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of patients with acquired proteinuric diseases such as focal segmental glomerulosclerosis (FSGS)

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(27). We and others have demonstrated induction of autophagy following UUO-induced kidney

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injury (19, 40, 41). Using GFP-LC3 transgenic mice, we confirmed that autophagy is induced in

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renal tubular epithelial cells of obstructed kidneys after UUO (19). Autophagy deficiency led to

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enhanced kidney fibrosis following UUO injury. For instance, mice deficient in autophagic

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protein Beclin 1 by heterozygous deletion of Beclin 1 displayed increased collagen deposition in

372

the kidney (40). Both Lc3b-null mice and Beclin 1-heterozygous mice displayed enhanced

373

collagen deposition in the obstructed kidneys after UUO (19). Treatment with autophagy

374

inhibitor 3-Methyladenine (3-MA) also enhanced renal tubular cell apoptosis and

375

tubulointerstitial fibrosis in the obstructed kidneys after UUO in rats (41).

376

Podocytes exhibit a high basal level of autophagy, important in cell survival, given that

377

they are terminally differentiated and have a very limited capacity for cell division and

378

replacement (27). Podocyte-specific deletion of the Atg5 gene resulted in increased susceptibility

379

to injury with more severe albuminuria, foot process effacement, loss of podocytes, and

380

glomerulosclerosis, following exposure to inducers of proteinuric glomerular injury, such as

381

puromycin aminonucleoside, adriamycin, or LPS (27). Induction of autophagy has also been

382

shown to be protective against cyclosporine-induced renal tubular cell death (70). Collectively,

383

these findings provide evidence that TGF-1-induced autophagy provides cytoprotective effects

384

that promote renal cell survival and negatively regulate and limit ECM accumulation in the

385

kidney, and thereby protect against kidney fibrosis (Figure 3).

17

386
387

Negative Feedback Regulation of TGF- Signaling

388

The diverse roles of TGF-1 necessitate that its signaling is tightly regulated at various

389

levels. TGF-1 induces Smad7, which is an inhibitory Smad that acts in a negative feedback loop

390

to negatively regulate TGF-1 (22). Overexpression of Smad7 has been shown to oppose TGF-

391

1-mediated renal fibrosis following UUO or STZ-induced diabetes, whereas deficiency of

392

Smad7 aggravated the severity of the renal fibrosis (8, 14, 46). Smad7-null mice display

393

enhanced TGF-/Smad3 signaling and worsened progressive renal fibrosis in a model of

394

angiotensin II-mediated hypertensive nephropathy (51), whereas treatment with Smad7

395

prevented angiotensin II-induced hypertensive nephropathy (52). Disruption of Smad7 also

396

exacerbated chronic aristolochic acid nephropathy (AAN) in mice, a progressive CKD related to

397

herb medicine (15). Furthermore, overexpression of Smad7 in the kidneys with established

398

chronic AAN attenuated progression of chronic AAN through inactivation of TGF-/Smad3

399

signaling (15).

400

Similarly, studies utilizing other approaches to inhibit TGF- signaling, downstream of

401

the cell-surface expressed TGF- receptors, have also shown failure to protect against kidney

402

fibrosis. Findings in mice with targeted conditional deletion of Smad2 in the kidney show that

403

blockade of Smad2 signaling pathway not only failed to protect against the development of

404

fibrosis after UUO injury but actually enhanced renal fibrosis (58). Moreover, knockdown of

405

Smad2 in cultured renal tubular epithelial cells and fibroblasts resulted in enhanced ECM

406

production in response to TGF-1 stimulation, with increased expression of type I and type III

407

collagen, and TIMP-1, and decreased expression of MMP-2, a major matrix-degrading enzyme

408

(58). Smad2 deletion was associated with increases in Smad3 phosphorylation, nuclear

18

409

translocation, and Smad3 binding to collagen (COL1A2) promoter, and autoinduction of TGF-1

410

(58). These studies indicate that inhibition of Smad2 promotes renal fibrosis via enhanced Smad3

411

signaling. On the other hand, overexpression of Smad2 resulted in attenuation of TGF-1-

412

induced Smad3 phosphorylation and type I collagen production in renal tubular epithelial cells

413

(58). Taken together, these findings suggest that Smad2 functions to protect against development

414

of renal fibrosis through countering the pro-fibrotic function of Smad3 signaling.

415

Our recent investigations have uncovered a novel mechanism that autophagy regulates

416

TGF-1 expression and suppresses kidney fibrosis induced by UUO. Increased levels of mature

417

TGF-1 were seen in the obstructed kidneys of LC3 deficient mice upon UUO injury (19).

418

Similarly, mature TGF-1 levels were increased in LC3 deficient renal tubular epithelial cells,

419

and in cells treated with autophagy inhibitor bafilomycin A1, without alterations in TGF-1

420

mRNA (19). These findings suggest a novel feedback mechanism to limit TGF- signaling

421

through autophagic degradation and suppress kidney fibrosis.

422

A number of endogenous modulators of TGF- signaling has been described, such as

423

secreted Klotho, which may serve as a promising therapeutic target against renal fibrosis. Klotho

424

is a transmembrane protein expressed in renal tubular epithelial cells, and its extracellular

425

domain is secreted by ectodomain shedding (4). Secreted Klotho inhibited TGF-1-induced EMT

426

in cultured cells, and administration of secreted Klotho protein to mice suppressed renal fibrosis

427

induced by UUO (20). Interestingly, it has been suggested that the Klotho-mediated

428

renoprotective effects may be, at least in part, due to upregulated autophagy flux (4). Secreted

429

Klotho protein directly binds to TRII, thereby inhibiting TGF-1 binding to cell surface

430

receptors, and inhibit TGF-1 signaling (20). In contrast, high plasma levels of Lipoprotein(a)

431

[Lp(a)], a low-density lipoprotein (LDL)-like particle, can inhibit the activation of latent TGF-1

19

432

by competing with the binding of plasminogen to cell or matrix surfaces (26, 43). The

433

proteoglycan decorin is another natural antagonist of TGF-1 that binds active TGF-1 and can

434

neutralize the biological effects of TGF-1 (32). Retinoic acid, particularly all-trans retinoic acid

435

(ATRA), has also been shown to suppress renal expression of TGF-1 and TRII, leading to

436

decreased ECM accumulation and inhibit progression of renal fibrosis (53, 63, 101). Other

437

studies have indicated that ATRA exacerbates kidney injury with increased glomerular ECM,

438

mesangial cell activation, glomerular macrophage influx, and immune complex deposition in a

439

model of membranoproliferative glomerulonephritis in mice (34, 101). Thus, the therapeutic

440

effects of retinoic acid in fibrotic kidney disease remain somewhat controversial, suggesting that

441

caution is needed in applying retinoid therapy to human disease. No published clinical studies

442

using soluble Kotho protein or ATRA administration in patients with CKD have been reported to

443

date, despite the preclinical data supporting their therapeutic potential. Recently, a phase I/II trial

444

has been initiated to evaluate the safety and efficacy of isotretinoin (13-cis retinoic acid)

445

treatment in patients with biopsy-proven FSGS, minimal change disease, or collapsing

446

glomerulopathy (clinicaltrials.gov: NCT00098020). The results of this clinical study will be of

447

great interest.

448
449

Concluding Remarks

450

Renal fibrosis represents the common pathway in kidney injury of most progressive CKD

451

leading to renal failure and end-stage kidney disease. Unfortunately, there are no effective

452

therapies that prevent progressive renal fibrosis. Current treatment for patients with CKD

453

primarily relies on inhibition of the renin-angiotensin-aldosterone system (RAAS) by

454

angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs)

20

455

(61). However, the magnitude of the renoprotective effects of RAAS blockade is estimated to be

456

only about a 20% risk reduction of progression (21).

457

TGF-1 remains an attractive target for treating fibrotic kidney diseases, to counter its

458

potent pro-fibrotic effects. However, as this review highlights its opposing protective effects,

459

indiscriminate complete blockade of TGF- functions is not sufficient to reduce fibrosis, and

460

may actually aggravate the disease in some pathological settings. Perhaps, there are also

461

interspecies differences suggesting that results derived from preclinical studies in mice and in

462

vitro may not necessarily translate to humans. TGF-1 actions depend on multiple factors

463

including the involved cell type and context, the dose, isoform, and the distinct Smad and non-

464

Smad signaling pathways. Therefore, therapeutic targeting of TGF-1 requires more optimal

465

strategies such as those selectively directed at specific signaling pathway(s), and future

466

investigations to better understand the precise molecular mechanisms of TGF-1 signaling are

467

warranted.

468
469
470
471

Acknowledgements

472

This work was supported in part by the National Institutes of Health grants R01 DK57661 and

473

R01 HL079904 to M.E.C.

474

21

475

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767
768

28

769

FIGURE LEGENDS

770

Figure 1. Smad and non-Smad TGF-1 signaling pathways. (A) TGF-1 activation occurs

771

with the release from latent TGF- binding protein (LTBP) complex by proteases. TGF-1

772

signaling is initiated upon binding of active TGF-1 with TRII and forming TRI-TRII

773

heteromeric complex, leading to phosphorylation of Smad2/3, oligomerization with Smad4, and

774

subsequent nuclear translocation to regulate the transcription of ECM genes. Smad7 serves as a

775

negative regulator of TGF-1 signaling. TGF-1 also activates Smad-independent signaling such

776

as the MAPK, mediated by Ras-Raf-MEK-ERK, pathway, and TGF--activated kinase 1

777

(TAK1) mediated by TAK1- TAK1-binding protein 1 (TAB1) pathway. TGF-1-induced TAK1

778

activation leads to signaling via MKK4-JNK and MKK3-p38 pathways and activation of

779

transcription factors activator protein-1 (AP-1) and activating transcription factor 2 (ATF-2),

780

respectively, and activation of NF-B to mediate pro-fibrotic responses.

781
782

Figure 2. An overview of Smad signaling and inflammatory pathways of TGF-1 signaling.

783

Binding of Smad4 to phosphorylated Smad2/3 leads to the nuclear translocation of Smad

784

complex to regulate gene transcription including Smad7. Smad7 induces IB expression which

785

inhibits phosphorylation of p65/p50 proteins of NFB signaling complex to prevent NFB-

786

driven renal inflammation.

787
788

Figure 3. An overview of pro-fibrotic and protective pathways of TGF-1 signaling.

789

Activation of TGF-1 in response to kidney injury can signal both pro-fibrotic and protective

790

effects via Smad and non-Smad signaling pathways. Overexpression of Smad2 attenuates TGF-

29

791

1-induced Smad3 phosphorylation and type I collagen (Col-11) expression, whereas inhibition

792

of Smad2 promotes renal fibrosis via enhanced TGF-1/Smad3 signaling. TGF-1-induced

793

autophagy negatively regulates TGF-1-stimulated collagen accumulation in the kidney by

794

promoting collagen degradation. Autophagy also negatively regulates mature TGF-1

795

expression, thereby protects against kidney fibrosis.

30

Table 1. Protective roles of TGF- signaling in chronic kidney diseases.

Strategy and Results
Conditional deletion of TRII in renal interstitial cells
significantly reduces collagen production, but does not
ameliorate overall renal fibrosis

Disease Model
UUO,
Aristolochic acidinduced
nephropathy
UUO

(58)

Overexpression of latent TGF-1 in keratinocytes reduces renal

fibrosis

UUO

(28)

Overexpression of latent TGF-1 in keratinocytes protects

against glomerular crescentic formation and severe
tubulointerstitial damage
Overexpression of Smad7 suppresses renal fibrosis, whereas
deficiency of Smad7 aggravates the severity of renal fibrosis

Crescentic
glomerulonephritis

(29)

Diabetic
nephropathy

(8)

Smad7 overexpression in kidneys suppresses renal fibrosis and

disruption of Smad7 enhances renal fibrosis

UUO

(14, 46)

Smad 7 deficiency aggravates angiotensin II-mediated renal

fibrosis, whereas Smad7 treatment prevents progressive renal
injury

Hypertensive
nephropathy

(51, 52)

Smad7 disruption exacerbates nephropathy, whereas Smad7

overexpression in kidneys attenuates nephropathy

Aristolochic acidinduced
nephropathy

(15)

Conditional deletion of TRII in renal tubular epithelial cells

and renal fibroblasts enhances NF-B signaling and renal
inflammation

UUO

(59)

Conditional deletion of Smad4 in renal tubular epithelial cells

enhances renal inflammation

UUO

(60)

Treatment with autophagy inhibitor 3-Methyladenine enhances

renal tubular cell apoptosis and tubulointerstitial fibrosis

UUO

(41)

Heterozygous deletion of Beclin 1 and LC3b-null mice

enhances collagen deposition

UUO

(19, 40)

Conditional deletion of Atg5 in podocytes increases

susceptibility to renal injury

Puromycin,
Adriamycin,
Lipopolysaccharide

(27)

Conditional deletion of Smad2 in renal tubular epithelial cells

enhances renal fibrosis

References
(67)

Table 2. Recent clinical trials using anti-TGF- therapy in fibrotic kidney diseases.

Compound
LY2382770

Target
TGF-1

Company
Eli Lilly

Fresolimumab TGF-1,2,3 Genzyme

(GC1008)

Pirfenidone

TGF-1,2,3 InterMune

Phase

Disease

Phase II

Diabetic
nephropathy

Phase I

Focal Segmental
Glomerulosclerosis

References or
NCT
Identifier
NCT01113801
(89)

Phase II

NCT01665391

Phase II

Focal Segmental
NCT00001959
Glomerulosclerosis, (11)

Phase I/II

Diabetic
nephropathy

NCT00063583
(79)

Figure 2

Figure 3