Distinct roles of Smads and microRNAs in TGF-β signaling during kidney diseases

Hong Kong Journal of Nephrology (2013) 15, 14e21

Available online at www.sciencedirect.com

journal homepage: www.hkjn-online.com

REVIEW ARTICLE

Distinct roles of Smads and microRNAs in TGF-b signaling during kidney diseases Rong Li a,b,c, Hui Y. Lan a,d,e, Arthur C.K. Chung a,e,* a

Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong, China c Department of Nephrology, First People’s Hospital of Yunnan Province, Yunnan, China d Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, China e CUHK Shenzhen Research Institute, Shenzhen, China b

Available online 25 April 2013

KEYWORDS chronic kidney disease; microRNAs; Smads; transforming growth factor-b signaling

Summary Chronic kidney disease (CKD) is known to be the hallmark with fibrosis and inflammation that leads to end stage renal disease. Since the discovery over 2 decades ago of transforming growth factor (TGF)-b as a key mediator in CKD, studies of TGF-b signaling in the kidney have focused on fibrosis and inflammation. TGF-b exerts its cellular effect via Smad2 and Smad3 after binding to its receptors. Smad7, as an inhibitory Smad, provides a negative feedback loop to limit TGF-b action for maintaining homeostasis. Recently, the precise roles of individual Smads and receptors have been further characterized and the results reveal the complexity of TGF-b signaling during CKD. Although Smad3 plays a pathogenic role in CKD, Smad2 and Smad7 are protective. Furthermore, Smad4 enhances Smad3-mediated renal fibrosis as well as suppresses nuclear factorekB-driven renal inflammation in a Smad7-dependent manner. Emerging evidence demonstrates the ability of TGF-b/Smad3 signaling to regulate specific microRNAs, revealing that microRNAs are critical downstream effectors of TGF-b/Smad3 signaling in renal fibrosis and inflammation. Recent studies in animal models of kidney disease demonstrate the therapeutic potential of microRNA therapy, Smad3 inhibitor, and Smad7 agonist in CKD. Because the accumulation of extracellular matrix and infiltration of inflammatory cells, which are the major pathological consequences of CKD, are due to activation of TGF-b/Smad3 signaling pathways, targeting the downstream TGF-b/Smad3 signaling pathway by gene transfer of either Smad7 or Smad3-dependent microRNAs, and by applying Smad3 inhibitor and Smad7 agonist may offer a specific and effective therapeutic strategy for kidney disease. 纖維化及炎癥是慢性腎病(CKD)的主要病理變化，最終導致終末期腎病。自從在二十多年前TGF-b 被發現為CKD的關鍵中介物起，腎臟TGF-b訊息傳遞的相關研究便著眼於纖維化及炎癥方面。TGFb在與受體結合後，透過Smad2及Smad3以產生細胞效應。具抑制性的Smad7可透過負回饋機制，

* Corresponding author. Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, China. E-mail address: [email protected] (A.C.K. Chung). 1561-5413/$36 Copyright ª 2013, Hong Kong Society of Nephrology Ltd. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.hkjn.2013.03.003

TGF-b signaling during kidney diseases

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以限制TGF-b的作用，從而維持平衡狀態。近年的研究更確認了個別Smads及受體的具體角色，顯 示了CKD的TGF-b訊息傳遞複雜性。Smad3在CKD中具有致病角色，Smad2及Smad7則具保護性。再 者，Smad4可促進Smad3介導的腎纖維化，并通過Smad7依賴的方式抑制NF-kB介導的腎臟炎癥。 新證據更顯示，TGF-b/Smad3 訊息傳遞具調節特定microRNAs的能力，意味著microRNAs在腎纖維 化及炎癥的TGF-b/Smad3訊息傳遞中是重要的下游效應物。近年關於腎病的動物實驗更發現，microRNA療法、Smad3抑制劑、及Smad7作用劑對CKD具潛在療效。有鑑於CKD的主要病理變化，如 細胞外基質(ECM)的積聚及炎性細胞的浸潤，乃歸因於TGF-b/Smad3訊息傳遞途徑的活化，我們可 以透過Smad7-或Smad3-倚賴性microRNAs的基因轉移、及採用Smad3抑制劑或Smad7作用劑等，對 TGF-b/Smad3的下游訊息傳遞途徑作出干預，從而為腎病患者提供一個特定且有效的治療方案。

Introduction Chronic kidney disease (CKD) is a condition characterized by a gradual loss of kidney function over time because of renal tissue injury and nephron loss. Eventually, CKD can lead to end-stage renal diseases. The patients may die due to secondary events, such as cardiovascular failure. The incidence of CKD is rising worldwide as the population pertension and diabetes increase in prevalence. Thus, CKD represents a significant human, clinical, and socioeconomic burden. Regardless of etiology, typical pathological phenotypes of CKD include fibrosis, infiltration of inflammatory cells, and nephron atrophy. Over 2 decades of studies have demonstrated a critical role for the cytokine transforming growth factor (TGF)-b as a key mediator of these pathological phenotypes. TGF-b contains three isoforms (TGF-b1, 2, and 3) that are pleiotropic cell signaling molecules involved in embryonic development and the regulation of tissue homeostasis.1e3 Among these isoforms, TGF-b1 is the central mediator in the fibrogenic process.1 TGF-b1 exerts its cellular effect via Smad2 and Smad3, after binding to TGF-b receptors II and I (TbRII and TbRI). Upon ligand binding, phosphorylated TbRII activates TbRI by phosphorylation. TbRI subsequently activates Smad2 and Smad3, which consequently interact with Smad4. Thus, an active Smad2/3/ 4 complex is formed and this translocates to the nucleus to regulate the target gene transcription.2 As an inhibitory Smad, Smad7 induces TbRI and Smads degradation via the ubiquitin proteasome degradation mechanisms4,5 to maintain homeostasis. By contrast, Smad7 is also capable of inducing IkBa to suppress nuclear factor (NF)ekB-driven inflammatory response (Fig. 1). In addition to TGF-b, advanced glycation end-products (AGEs), high glucose, angiotensin II (AngII) are also able to activate Smad signaling via mitogenactivated protein kinase (MAPK) to induce kidney injury. Recent studies demonstrate that microRNAs may be downstream mediators of TGF-b/Smad3 signaling during CKD. Thus, targeting TGF-b/Smad3 specific microRNAs could be an effective alternative to treat kidney disease. Studies of asiatic acid, a Smad7 agonist, and Smad3 inhibitor also reveal a possible therapeutic potential of these compounds.

Role of TGF-b signaling in kidney diseases Fibrosis TGF-b/Smad signaling Tubulointerstitial fibrosis and glomerulosclerosis are regarded as a central event in the progression of CKD

regardless of etiology. These two pathological events lead to glomerular and tubular malfunction and degeneration as an excessive accumulation and deposition of extracellular matrix (ECM) components occurs within renal tubules and glomeruli. TGF-b is widely recognized as a strong inducer of fibrosis in renal structures during CKD because it can increase ECM production and alter the degradation of ECM components.3 Studies from both in human and experimental kidney diseases confirm that TGF-b1 is highly upregulated in the fibrotic kidney.6,7 The role of TGF-b1 in renal fibrosis has extensively reviewed6,8e10 and now we will focus on the recent findings of TGF-b signaling in fibrosis. Upon TGF-b binding, TbRII binds to TbRI to form a stable complex. TbRII then activates TbRI via phosphorylation. The activated TbRI receptor thus phosphorylates Smad2 and Smad3 at C-terminal serines. Receptor phosphorylation activates TGF-b-induced signaling and provides the basis for the roles of TbRII and TbRI kinases as signal transducers. TbRI has also been reported to participate in the pathogenesis of experimental intestinal fibrosis.11 During intestinal fibrosis, TbRI and intestinal wall collagen deposition are upregulated in experimental animal models but these effects are suppressed after treatment with a TbRI inhibitor, SD-208. Although TGF-b1 induces gene expression of TbRI and collagen 1 in isolated myofibroblasts, knockdown of TbRI expression by siRNA and treatment with SD-208 abolish these effects. Conditionally deleting TbRII in tubular epithelial cells of a mouse model of unilateral ureteral obstruction (UUO) inhibits severe tubulointerstitial fibrosis in the UUO kidneys.12 This inhibition is associated with the impairment of TGF-b/Smad3 signaling but not with the ERK/p38 MAP kinase pathway. Similarly, in vitro disruption of TbRII from kidney fibroblasts or tubular epithelial cells suppresses TGF-b1-induced Smad signaling and fibrosis. Thus, TbRII plays an important role in regulating renal fibrosis. Impairing TGF-b/Smad3, but not the noncanonical TGF-b signaling pathway, is the key mechanism by which disruption of TbRII protects against renal fibrosis. Smad2 and Smad3 are known to be two critical downstream mediators responsible for the biological effects of TGF-b1. Activation of Smad2 and Smad3 are also significantly increased in both experimental and human kidney diseases, such as obstructive kidney diseases, 13e17 remnant kidney disease,18,19 hypertensive nephropathy,20 diabetic nephropathy 21e25 and chronic aristolochic acid nephropathy.26 However, only Smad327,28 can directly bind to the specific DNA sequences, called Smad binding element; in the promoters of TGF-b target genes such as collagen

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Figure 1 Transforming growth factor (TGF)-b/Smads and its cross-talk pathways in renal fibrosis and inflammation. After binding with TGF-b1, TGF-b receptor II activates TGF-b receptor I kinase, which phosphorylates Smad2 and Smad3. Phosphorylated Smad 2 and Smad3 then form a complex with Smad4, which translocates into the nucleus to induce the target gene transcription, such as microRNAs and Smad7. As an inhibitory Smad, Smad7 inhibits TGF-b/Smad3-dependent fibrosis and suppresses nuclear factorekBdriven inflammatory response by upregulating nuclear factor of k light polypeptide gene enhancer in B cells inhibitor, alpha (IkBa). In addition, advanced glycation end-products and angiotensin II are able to directly activate Smad3 via the ERK/p38/mitogenactivated protein kinase (MAPK) cross-talk pathway.

(ColIa2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1)29 and Smad 7, suggesting a pathologic role for Smad3 in fibrosis. This critical role of TGF-b/Smad3 signaling during renal fibrosis is further confirmed by the findings that fibrosis is alleviated in employing Smad3 knockout (KO) in mouse models of diabetic nephropathy,21 obstructive nephropathy,11 and chronic aristolochic acid nephropathy.26 Active Smad 2 also forms a complex with Smad4 but its role in fibrosis has not been widely explored because deletion of Smad2 gene in mice causes embryonic lethality.30 However, a recent study shows that deletion of Smad2 gene conditionally from the kidney tubular epithelial cells significantly promotes fibrosis in response to UUO.31 Knockdown of Smad2 expression in tubular epithelial cells in vitro also induces expression of collagen I, collagen III, and TIMP-1 but reduces expression of the matrix-degrading enzyme MMP-2 after TGFb1 treatment. By contrast, overexpression of Smad2 inhibits TGF-b1-induced Smad3 phosphorylation and collagen I

matrix expression. Smad2 deletion enhances TGF-b/Smad3 signaling and fibrosis because deletion of Smad2 elevates Smad3 phosphorylation, nuclear translocation, promoter activity, binding of Smad3 to a collagen promoter (Col1a2), and autoinduction of TGF-b1.31 In conclusion, Smad3 signaling promotes renal fibrosis whereas Smad2 protects against TGF-b-mediated fibrosis by counteracting TGF-b/ Smad3 signaling. Smad4 is known as the common Smad in TGF-b/Smad signaling but its functional role in TGFeb1-driven renal fibrosis remains largely unclear. This may be largely associated with the lethality of Smad4 KO mice.32 A recent study shows that conditionally deletion of Smad4 inhibits renal fibrosis and TGFeb1-induced collagen I expression in fibroblasts.33 The mechanism of how deletion of Smad4 inhibits renal fibrosis is not attributed to the failure to activate Smad3 phosphorylation and nuclear translocation but to reduce the Smad3-mediated promoter activities and the binding of Smad3 to the Col1a2 promoter.33

TGF-b signaling during kidney diseases Owing to the presence of a Smad binding element in the Smad7 promoter,34e36 TGF-b rapidly induces Smad7 expression37 as a safeguard mechanism to prevent prolonged activation of TGF-b signaling. In CKD, Smad7 mRNA expression is increased, and Smad7 protein is reduced despite the upregulation of renal TGF-b1 and high levels of activated Smad2/3.38 The reduction of Smad7 protein in diseased kidney is attributed to the activation of Smurfs and arkadia-dependent ubiquitineproteasome pathways which degrade Smad7 protein via a post-transcriptional modification mechanism.4,18,39,40 E3 ubiquitin ligases, termed Smurf1, Smurf2, and arkadia have been shown to physically interact with Smad7.40,41 After recruitment of the Smad7eE3 ubiquitin ligases complex to the activated TGF-b receptors, E3 ubiquitin ligases induce their degradation through the proteasomal-ubiquitin degradation pathways.4,5,39e41 Thus, aberrant expression of this intracellular regulator of TGF-b/Smad signaling, Smad7, results in promoting further activation of TGF-b/Smad signaling and progressive renal fibrosis as confirmed in various rodent models of kidney diseases.17,18,40,42,43 This is further evidenced in Smad7 KO mice in both UUO and diabetic models in which more severe renal fibrosis is developed.17,25 MAPK crosstalk Increasing evidence has shown that several factors interact with the TGFb/Smad signaling. Physical interactions of Smad3 allow signal crosstalk with other signaling pathways, such as the MAPK signaling pathway, which also plays a role in the pathophysiological processes of kidney diseases. In the context of renal fibrosis, Smad3, without TGF-b activation, can interact with MAPKs during AGE or AngII-mediated fibrotic response. In diabetic conditions, AGE is capable of activating Smad 2/3 both in vivo and in vitro via the ERK/p38 MAPK-dependent mechanism.21 Similarly, under hypertensive conditions, Smad3 signaling is activated by AngII via the AT1 receptormediated, ERK/p38 MAPK-Smad crosstalk pathway, in addition to the TGFeb-dependent mechanism.20,44 Furthermore, both AGEs and AngII are able to activate Smad2/3 to stimulate connective tissue growth factor (CTGF) expression in kidney cells lacking the TGF-b1 gene or TbRII, but not in those with blockade of MAPK signaling by specific inhibitors, or dominant negative adenovirus to ERK1/2 and p38.22,44 The identification of TGF-b-independent Smad pathways has advanced our understanding of complicated Smad signaling under disease conditions.

MicroRNAs Recent research into microRNAs has demonstrated that TGFb regulates several specific microRNAs (miRs) to control fibrogenesis in renal disease. TGF-b1 is able to upregulate miR-21, miR-192, miR-216a, and miR-377, but downregulate the miR-29 and miR-200 families (Table 1).45e47 These microRNAs play significant roles as downstream effectors in TGFeb-induced renal fibrosis. Both miR-192 and miR-21 play a pathological role in kidney injury and upregulation of their expression in disease condition promotes TGFeb-induced expression of fibrotic markers.48e51 In addition, miR-377 induces fibronectin in MCs,52 whereas miR-491-5p induces Par-3 degradation in TECs.53 MiR-382 participates in TGFeb1induced loss of E-cadherin in human renal epithelial cells.54

17 By contrast, members of the miR-29 and miR-200 families play a protective role in renal injury. During renal injury, downregulation of miR-29a and miR-29b promotes collagen expression55,56 and reduction of miR-200a expression in diseased condition enhances TGFeb-dependent epithelialto-mesenchymal transition by increasing expression of TGF-b2 and matrix proteins.57 These findings suggest that microRNAs play a vital role in TGF-b induced renal injury. How TGF-b signaling regulates microRNA expression is still underexplored. TGF-b signaling has been shown to promote the processing of primary transcripts of miR-21 into precursor miR-21 by the Drosha complex.58 Results from our laboratory demonstrate that the expression of miR-21, miR-192, and the miR-29 family during renal diseases are TGF-b1/Smad3 dependent.48,50,56 Smad3 mediates the expression of these microRNAs by physically interacting with Smad3-binding sites in their promoters.48,49,56 Binding of Smad3 on these sites in the promoters may either induce transcription and posttranscriptional processing of microRNAs (such as miR-21 and miR-192) or repress the expression (such as miR29b).48,49,56 In addition, regulating TGF-b/Smad3-mediated microRNAs via maintaining renal miR-29b but suppressing miR-192 and miR-21 is another mechanism of how Smad7 protects kidneys from fibrosis.46,49 More importantly, microRNAs can also affect the TGF-b/ Smad3 signaling. Overexpression of miR-200a reduces Smad3 activity and alleviates TGFeb1-driven fibrosis.57 In addition, both TGF-b1 and TGF-b2 reduce miR-200a expression in renal cells.57 This reduction is accompanied by an increase in TGF-b2 expression because the 30 UTR of TGF-b2 is found to be a target for miR-200a. These findings suggest a possible feedback between TGF-b2 and miR-200a. Furthermore, downregulating Smad7 is also shown to be one of mechanisms of how miR-21 induces fibrosis in kidney and lung.50,59 These findings indicate critical roles of microRNAs in TGFeb-induced renal fibrosis and the complexity between TGF-b/Smads and microRNAs under pathophysiological conditions.

Inflammation It is well known that TGF-b has both profibrotic and antiinflammatory properties. The profibrotic role of TGF-b/ Smad signaling in renal disease has been extensively studied but the role of TGF-b/Smad signaling in renal inflammation attracts little attention. One convincing piece of evidence for the involvement of TGF-b in inflammation is from the outcome of TGF-b1 KO mice, which suffer from lethal inflammation and die at 3 weeks after birth.60 Furthermore, conditional deletion of TGF-b1 or TbRII genes from T cells develops autoimmune diseases.61,62 However, overexpressing human latent TGF-b1 in mice dampens progressive renal inflammation and fibrosis in obstructive and immunologically-induced crescentic glomerulonephritis.13,63,64 These findings demonstrate the vital role of TGF-b in anti-inflammation. Recent studies show that deletion of TbRII in kidneys impairs the antiinflammatory properties of TGF-b1 as evidenced by promoting NF-kB signaling and proinflammatory cytokines.12 As mentioned above, TGF-b1 induces Smad7 expression to provide a negative feedback loop by causing the

18 Table 1

R. Li et al. Summary of the roles of various microRNAs during chronic kidney disease.

Treatment with TGF-b

MicroRNA

Cell type

Mouse model of kidney diseases

Experimental data

Refs

Upregulation

miR-21 miR-192

TECs MCs TECs MCs MCs MCs TECs TEC TECs MCs TECs

Enhances fibrosis and inflammation Increases collagen deposition, induces EMT Increases col1a2 expression Induces fibronectin expression Induces Par-3 degradation Suppresses E-cadherin Promotes collagen expression Enhances EMT

48,50

miR-216a miR-377 miR-491-5p miR-382 miR-29 miR-200

UUO, DN in db/db mice UUO, STZ induced DN, DN in db/db mice STZ induced DN, DN in db/db mice Spontaneous and STZ induced DN UUO (rat) e UUO STZ induced DN, adenine-induced

Downregulation

49,51

47 52 53 54 55,56 57

DN Z diabetic nephropathy; EMT Z epithelial-to-mesenchymal transition; MC Z mesangial cells; STZ Z streptozotocin; TEC Z tubular epithelial cells; TGF Z transforming growth factor; UUO Z unilateral ureteral obstruction.

degradation of TbRI and Smads. Under pathological conditions, reduced renal Smad7 protein not only enhances TGFb/Smad3-mediated renal fibrosis, but also induces renal inflammation by activating the NF-kB-dependent inflammatory response.25,63 These results were confirmed by Smad7 KO mice in the models of obstructive and diabetic nephropathy.22,25 The activation of NF-kB/p65 along with the levels of renal inflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor-a, monocyte chemotactic protein-1, intercellular adhesion molecule-1, and the macrophage infiltration in Smad7 KO mice with diabetic kidney disease or obstructive nephropathy are much higher than Smad7 wild type mice.22,25 By contrast, overexpression of Smad7 in T cells inhibits mouse crescentic glomerulonephritis.65 In addition, Smad7 is able to directly induce expression of IkBa, an inhibitor of NF-kB, suggesting that TGF-b may act by inducing Smad7 to stimulate IkBa expression for suppressing NF-kB activation.25,63 Smad4 also plays an important role in renal inflammation. Findings from our laboratory show that disruption of Smad4 gene in kidneys results in greater infiltration of CD45þ leukocyte and F4/80þ macrophage and elevation of IL-1b, tumor necrosis factor-a, monocyte chemotactic protein-1, and intercellular adhesion molecule-1 in the ligated kidney and in IL-1b-induced macrophages.33 This enhancement of inflammation may be due to the fact that loss of Smad4 impairs Smad7 expression because TGF-b1 upregulates Smad7 expression via direct binding of Smad3 and Smad4 to the Smad7 promoter.34,35 The reduction of Smad7 expression subsequently attenuates transcriptional regulation of Smad7 to induce IkBa for the suppression of NF-kB signaling.33

Therapeutic potential As discussed above, TGF-b/Smad signaling plays an important role in renal fibrosis and inflammation, therefore blocking TGF-b signaling or targeting the downstream of TGF-b/Smads should be effective therapeutic strategies to treat CKD. During the past decades, targeting TGF-b with neutralizing antibodies (such as antieTGF-b2 IgG4),66 antisense TGF-b oligodeoxynucleotides, or soluble human TGF-b type II receptor67 ameliorate the diabetic nephropathy by inhibiting renal fibrosis and reducing albuminuria.

Application of specific inhibitors of TGF-b receptor kinases, including GW788388 and IN-1130, is able to block TGF-b signaling and reduce renal fibrosis in rodent models.68,69 However, blockade of TGF-b signaling at the upstream levels may impair the potent anti-inflammation property of TGF-b and subsequently promote renal inflammation. Moreover, because of the existing intracellular Smad crosstalk pathways, targeting TGF-b and TGF-b receptor kinases may not be an optimal therapeutic approach. Because the central role of Smad3 in TGF-b-induced fibrosis and inflammation is widely recognized and the emerging role of microRNAs in renal fibrosis is being recognized, we thus proposed to specifically target the downstream of TGF-b/NF-kB pathways by exploring the therapeutic potential of Smad7 and TGF-b/Smad-dependent microRNAs (Fig. 2). In the past decade, our laboratory has developed an ultrasoundemicrobubble-mediated gene transfer system to target genes or microRNAs specifically to avoid the potential side-effects of virus-based gene therapy. As reported recently, ultrasound-mediated transfer of Smad7 overexpression plasmid is capable of suppressing renal fibrosis and inflammation in a number of rodent models including diabetes, obstructive nephropathy, remnant kidney, and autoimmune disease.25,26,38,46 Also, gene transfer of Smad7 is capable of protecting kidneys from fibrosis by regulating TGF-b/Smad3-mediated renal expression of miR-21, miR-192, and miR-29b.46 Most recently, by using same gene transfer technique, we demonstrate that overexpression of miR-29b or knockdown of miR-21 in a mouse model of obstructive and diabetic kidney diseases are capable of preventing or blocking renal fibrosis and inflammation.46,48,50,56 Recently, studies demonstrates that a specific inhibitor of Smad3 abrogates endothelialemesenchymal transition, reduces renal fibrosis, and retards progression of nephropathy, but does not attenuate albuminuria.70 By contrast, asiatic acid acts as an agonist of Smad7 to inhibit liver fibrosis by upregulating Smad7 expression.71 These novel agents, Smad3 inhibitor and asiatic acid, may offer an alternative treatment for renal fibrosis and inflammation. Thus, application of microRNAs, Smad7, Smad3 inhibitor, and Smad7 agonist in the treatment of kidney disease offers a novel, effective, and safe therapeutic approach to combat CKD.

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Figure 2 Schematic diagram of current therapeutic strategies for combating chronic kidney disease by suppressing transforming growth factor (TGF)-b-induced renal fibrosis and inflammation. These strategies include three different levels: Firstly, the suppression of the upstream TGF-b signaling by employing inhibitors for TGF-b receptor kinases (including GW788388 and IN-1130), antisense TGF-b oligodeoxynucleotides, neutralizing antibodies (antieTGF-b2 IgG4), and soluble human TbRII (sTbRII.Fc). Secondly, the blockade of Smad signaling by using Smad3 inhibitor, Smad7 agonist, and overexpression of Smad7 plasmids. Lastly the inhibition of downstream effectors of TGF-b signaling by anti-192, antiemicroR-21, and by restoring microR-29b and microR-200.

Summary and perspectives In general, Smad3 as a key mediator of TGF-b/Smad signaling plays a pathogenic role in both renal inflammation and fibrosis. Smad3 may also regulate its downstream specific microRNAs, such as miR-21, miR-192, and miR-29, leading to involvement in renal fibrosis and inflammation by directly interacting with Smad-binding sites in their promoters. At the same time, microRNAs may regulate Smad3 activity by targeting Smad7 and TGF-b. By contrast, Smad2 is renoprotective and suppresses renal fibrosis by competitively inhibiting Smad3 phosphorylation and nuclear translocation. Smad4 plays a diverse role in promoting Smad3-mediated renal fibrosis but suppressing NF-kBdriven renal inflammation. Smad7 is a negative regulator of TGF-b/Smad signaling in renal inflammation and fibrosis. However, reduction of Smad7 protein in the diseased kidney results in an imbalance within and between the TGF-b/ Smad and NF-kB signaling pathways to induce renal fibrosis

and inflammation. Taken together, the current understanding of the specific role of individual Smads, TGFeb/ Smad-regulated microRNAs and the molecular mechanisms of TGF-b/Smad-driven renal fibrosis and inflammation should provide us valuable opportunities to search for the better therapeutic strategy to fight kidney diseases.

Acknowledgments This work was supported by grants from the Research Grant Council of Hong Kong (RGC GRF 468711, 469110, 768409, CUHK5/CRF/09, and CUHK3/CRF/12R to H.Y.L.; GRF 463612, 464010, 763908, and 764109 to A.C.C.), the Focused Investment Scheme A, the Focused Investment Scheme B (1902061), and by direct grants from the Chinese University of Hong Kong (2010.2.025, 2011.1.076, 2012.1.021 to A.C.C.), 2011 Research Grant from Hong Kong Society of Nephrology (6903213 to A.C.C.), National Natural Science Foundation of

20 China (General Program, 81170681 to A.C.C.), Municipal Science and Technology Research and Development funding of basic research, Shenzhen (General Program C.02.12.00501 to A.C.C.), and Major State Basic Research Development Program of China (973 program, No.2012CB517700 to H.Y.L).

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