Diverse Role of TGF-β in Kidney Disease

1Increasing evidence shows that chronic kidney disease (CKD) is a global-burden-disease. The prevalence and incidence of CKD have risen by almost 90% over last 30 years. During the progression of CKD, renal function is impaired with a loss of nephrons and the development of renal fibrosis characterized by the excessive accumulation of extracellular matrix (ECM) components, reduction in glomerular filtration rate (GFR), and abnormal albuminuria. CKD eventually leads to the development of end-stage renal disease (ESRD). Fibrosis and inflammation are the two major features of CKD and prolonged renal inflammation promotes renal fibrosis as well. Physiologically, fibrosis is a repair and healing process in response to the initial renal insults. However, as the pathological condition prolongs, unresolved renal inflammation turns into a major driving force to promote renal scar formation via a progressive process of renal fibrosis.

2Transforming growth factor-β has been long considered as a master cytokine in the pathogenesis of renal inflammation and fibrosis. The TGF-β superfamily contains members of TGF-βs, activins, inhibins, growth and differentiation factors (GDFs), bone morphogenetic proteins (BMPs), and glial-derived neurotrophic factors (GDNFs). It is well established that there are three isoforms of TGF-β in mammals, the TGF-β1, 2 and 3. Of these, TGF-β1 has been considered as a profibrotic mediator in various kidney diseases. Newly synthesized TGF-β1 releases and binds to the latency-associated peptide (LAP) to form a latent complex which later binds to the TGF-β binding protein (LTBP) to form a larger complex. The latent complex is inactive and stored in the ECM until it is released by reactive oxygen species (ROS) and plasmin or acid. Once TGF-β1 is released from LAP and LTBP, it becomes active. Active TGF-β1 binds to Type II TGF-β receptor (TβRII), which recruits and activates Type I TGF-β receptor (TβRI) and downstream receptor-associated Smads (R-Smads), Smad2, and Smad3. The phosphorylated Smad2/3 then form an oligomeric complex with Smad4. Subsequently, the Smad2/3/4 complex translocate into the nucleus to regulate transcription of target genes, inducing α-smooth muscle actin (α-SMA), collagens, and inhibitory Smad7. Interestingly, Smad7 can antagonize TGF-β-mediated fibrosis, carcinogenesis and inflammation in various diseases. Smad7 negatively regulates TGF-β/Smad signaling by competing with the R-Smad binding to the TβRI (; Figure 1). Moreover, Smad7 also induces the IκBα, a NF-κB inhibitor, to suppress NF-kB-driven inflammatory response.

3In this review, the diverse roles of canonical TGF-β signaling, the distinct roles of downstream Smad proteins, and the potential therapeutic strategies for renal fibrosis and inflammation by targeting downstream TGF-β/Smad signaling are discussed.

4It is well accepted that TGF-β is a master regulator in renal inflammation and fibrosis. TGF-β exerts multifunctional effects on cell proliferation, apoptosis, migration, differentiation, and ECM production. TGF-β1 induces tubular and glomerular epithelial cell-to-mesenchymal transition (EMT) and excessive ECM production and deposition in glomeruli and tubulointerstitium. TGF-β1 is highly expressed in a wide range of kidney diseases associated with fibrosis. The functions of TGF-β1 on renal fibrosis and EMT were further confirmed by the findings that overexpression of active TGF-β1 in liver causes the development of severe renal fibrosis in mice. Whereas, anti-TGF-β treatments by using neutralizing antibodies, inhibitors against the TβRII, or antisense oligonucleotides to TGF-β1 halt the progression of renal fibrosis, suggesting a vital pathological role of TGF-β in CKD.

5Renal inflammation is driven by NF-κB-dependent mechanism. TGF-β is considered to be one of anti-inflammatory cytokines during the renal repair process in response to the injuries. A number of studies have reported that mice deficient TGF-β1 suffer from the lethal inflammation and the early death, suggesting a protective role for TGF-β in renal inflammation. Consistently, conditional deletion of TβRII from mice results in protection against TGF-β/Smad3-mediated renal fibrosis while enhancing NF-κB-driven renal inflammation. More importantly, TGF-β is also a master regulator of T cell immune responses in a variety of immune diseases, which makes TGF-β as a key regulator in renal inflammation.

6It should be pointed out that TGF-β signaling is not the sole pathway mediating the fibrotic process. Increasing evidence shows that TGF-β signaling can interact with other signaling pathways to mediate fibrosis. Among TGF-β signaling, both canonical and non-canonical TGF-β/Smad signaling pathways play a role in the renal fibrosis (Figure 2). Importantly, under disease conditions, Smad signaling can also be activated independently TGF-β1 by many stress molecules such as angiotensin II, and advanced glycation end products (AGE) via the ERK/p38/MAPK-Smad crosstalk pathway. TGF-β/Smad can also interact with other signaling pathways such as Wnt/β-catenin, Jagged1/Notch, and Hedgehog to regulate epithelial dedifferentiation, myofibroblast transformation and proliferation. In addition, TGF-β can induce renal fibrosis by transactivating epidermal growth factor receptor (EGFR) and p53 via proto-oncogene tyrosine-protein kinase Src (c-Src) and ROS-dependent mechanisms. TGF-β1 also induces phosphorylation and acetylation of p53 and promote formation of p53/Smad3 complexes during renal fibrosis. By contrast, BMP signaling via Smad1/5/8 complex is able to counter regulate TGF-β/Smad-mediated renal fibrosis. Thus, TGF-β may exert its diverse role in renal inflammation and fibrosis by interacting with many other signaling pathways and molecules.

7In canonical TGF-β signaling, Smad2, and Smad3 are two key downstream mediators that are highly activated in the fibrotic kidney. Although Smad2 and Smad3 bind together, their functional roles are distinct. In the context of fibrosis, Smad3 is pathogenic while Smad2 is protective. Smad3 can induce matrix deposition by directly binding to the promoter region of collagen-producing genes and tissue inhibitor of matrix metalloproteinases (TIMP) while reducing the activity of MMP-1 to inhibit ECM degradation. By contrast, role of Smad2 in fibrosis is not fully elucidated due to a lack of Smad2 knockout (KO) mice which is embryonic lethal. However, a recent finding that conditional deletion of Smad2 from TECs accelerates renal fibrosis reveals a protective role of Smad2 in renal fibrosis. In addition, FSP1-specific Smad2 knockout in renal tubular, endothelial, and interstitial cells is also reported to reduce renal fibrosis and epithelial-to-mesenchymal transition in murine streptozotocin (STZ)-induced diabetic nephropathy.

8Smad4 is a common Smad associated with nuclear translocation of Smad2/3 and Smad1/5/8 complexes in response to TGF-β and BMP signaling. Limited evidence has shown a direct role of Smad4 in renal fibrosis due to the lethality of Smad4 knockout mice. However, conditional deletion of Smad4 from TECs significantly reduces renal fibrosis in the obstructive kidney. Mechanistically, deletion of Smad4 inhibits renal fibrosis by suppressing Smad3 promoter activity and blocking the binding of Smad3 to the collagen promoter without affecting its phosphorylation and nuclear translocation. This finding is consistent with studies in Smad4 knockout mesangial cells and in the folic acid-induced rodent model. It is also reported that the formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. In contrast, conditional deletion of Smad4 promotes renal inflammation by impairing Smad7-mediated inhibition of NF-κB activation. Thus, Smad4 may play a diverse role in renal fibrosis and inflammation and may not be a specific therapeutic target for CKD.

9Smad7 is a vital negative regulator of both TGF-β/Smad and NF-κB signaling pathways. Indeed, although TGF-β1 induces Smad7 transcriptionally, Smad7 inhibits TGF-β signaling by directly binding to the TβRI and blocking the activation of R-Smads. Mechanistically, Smad7 interacts with E3 ubiquitin ligases, such as arkadia, Smurf1 or Smurf2 (Smad ubiquitination regulatory factors), and recruit them to the TRβI to cause its degradation, hence resulting in the inhibition of TGF-β/Smad signaling. Under fibrosis conditions, Smad7 is reduced while Smad3 is highly activated as seen in diabetic nephropathy, hypertensive nephropathy, and aristolochic acid-induced nephropathy. Thus, the imbalance between Smad3 and Smad7 signaling may be a key mechanism in fibrogenesis and rebalancing this pathway by overexpressing Smad7 and inactivating Smad3 may represent as a better therapeutic strategy for CKD.

10Smad7 can also induce expression of IκBα, an inhibitor of NF-κB, to negatively regulate NF-κB-driven renal inflammation. Furthermore, Smad7 can interact with NF-κB directly as Smad7 promoter contains a putative NF-κB regulatory site. Under CKD conditions, loss of renal Smad7 is associated with activation of NF-κB signaling and severe renal inflammation as reported in hypertensive nephropathy and aristolochic acid-induced nephropathy. In contrast, overexpression of Smad7 suppresses both renal fibrosis and inflammatory in these disease models, making Smad7 as an promising therapeutic strategy for CKD.

11MicroRNAs (miRNAs) are small (approximately 20–22 nucleotides in length) non-coding single stranded RNAs. More than 200 miRNAs have been identified in renal cells and tissues so far. These miRNAs regulate a wide range of biological processes, including fibrosis and inflammation. Increasing evidence has demonstrated that TGF-β1/Smad3 signaling regulates various miRNAs during the renal pathological processes. As a transcriptional factor, Smad3 can bind and upregulate or downregulate miRNAs to promote renal inflammation and fibrosis. It is now clear that Smad3, but not Smad2, regulates these miRNAs by physically interacting with Smad binding site (SBE) located in their promoters to either increase (such as miR-21 and miR-192) or inhibit their transcription (such as miR-29 and miR-200 families). In addition, Smad7 may inactivate Smad3 to protect kidneys from fibrosis by upregulating renal miR-29b but suppressing miR-192 and miR-21. Among these miRNAs, miR-21 is well characterized as a profibrotic miRNA. miR-21 is upregulated in renal fibrosis in the patients with CKD as well as AKI. Mice deficient miR-21 or administration of anti-miR-21 oligonucleotides are able to protect against renal fibrosis. Expression of miR-21 is positively regulated by Smad3 but negatively by Smad7. Overexpression of miRNA-21 promotes renal fibrosis by targeting PTEN and Smad7. Thus, knockdown of miR-21 restores renal Smad7 levels and blocks both TGF-β/Smad3 and NF-κB signaling, thereby inhibiting progressive renal fibrosis and inflammation in mouse models of obstructive and diabetic nephropathy. However, miR-21 may be also protective in kidney disease as miR-21-deficient TGF-β(1)-transgenic mice show increased proteinuria and glomerular injury in streptozotocin-induced diabetic mice, suggesting a diverse role of miR-21 as a feedback inhibitor of TGF-β/Smad3 signaling.

12MiR-29 family is another well-documented miRNA in fibrotic diseases. The miR-29 family consists of miR-29a, b, c. All family members are encoded by two distinct genomic loci in both human and rodent genomes. As all members have the same seed binding sequence, they all bind to the same set of target genes. Renal miR-29b is decreased in association with activation of TGF-β/Smad3 signaling and progressive renal fibrosis in kidney diseases. miR-29b is negatively regulated by Smad3, but not Smad2, in response to TGF-β1, AGE, and angiotensin II. Overexpression of miR-29 inhibits renal fibrosis and inflammation by targeting TGF-β and Sp1/NF-κB signaling. Interestingly, miR-29b can also target T-bet, a master transcriptional factor for Th-1 T cell immune response. Therefore, overexpression of miR-29b is also capable of inhibiting T cell-mediated type-2 diabetic nephropathy in db/db mice. Notably, miR-29 also acts as a urinary exosome biomarker of renal fibrosis. Intramuscular injection of exosome-encapsulated miR-29 has been shown to inhibit renal fibrosis and muscle atrophy.

13Moreover, miR-93, miR-216a, miR-217, miR-377, miR-382, miR-491-5p, miR-433 and miR-17-5p are also demonstrated to be TGF-β1/Smad3-regulated profibrotic miRNAs, whereas miR-let-7, miR-15b, miR-101, and miR-130b exert their antifibrotic effects by inhibiting the expression and activity of TβRI, thus limiting transduction of downstream TGF-β-mediated signals. Other miRNAs such as miR-19b, miR-26a, miR-29, and miR-30 inhibit the TGF-β1/Smad signaling by targeting Smads or fibrotic transcriptional factors. All these findings imply that TGF-β may regulate miRNAs to exert its diverse roles in renal inflammation and fibrosis as shown in Table 1 and Figure 3.

14However, the off-target effects, non-specificity, and toxicity of miRNAs are unavoidable. Thus, research into long non-coding RNAs (lncRNAs) is more promising for a better understanding of the pathogenic mechanisms of kidney diseases. Compared to miRNAs, lncRNAs are transcripts with lengths exceeding 200 nucleotides without protein-coding functions and are highly tissue-and-cell-type-specific. lncRNA regulates both target DNAs/RNAs and proteins transcriptionally or post-transcriptionally. By using the high-throughput RNA sequencing, 21 TGF-β/Smad3-dependent lncRNAs have been identified in an immunologically induced anti-glomerular basement membranous glomerulonephritis (anti-GBM GN) and obstructive nephropathy. Of these, the Arid-IR is a novel and Smad3-related lncRNA as a Smad3 binding site is found in its promoter region. It has been proven that knockdown of Arid2-IR in TECs improves renal inflammation in vivo and in vitro by inhibiting NF-κB-dependent inflammatory transduction without affecting Smad3-mediated fibrosis. In contrast, Erbb4-IR is another novel Smad3-dependent lncRNA capable of inhibiting renal fibrosis by targeting miR-29b and Smad7 in both obstructive nephropathy and type II diabetic nephropathy, respectively. A recent study also reveals the pathogenic role and mechanism of LRNA9884 in type II diabetic nephropathy. LRNA9884 is tightly regulated by Smad3 in response to TGF-β and AGEs and functions to trigger MCP-1 production by directly binding to the MCP-1 promoter, thereby promoting inflammation-driven type II diabetic nephropathy. In addition, several TGF-β/Smad3-associated lncRNAs are found to be associated with renal fibrosis. TCONS_00088786 and TCONS_01496394 are TGF-β/Smad3-associated lncRNAs as they contain potential binding sites for Smad3 and silencing TCONS_00088786 inhibits renal interstitial fibrosis by targeting miR-132. lncRNA-ATB is highly upregulated in patients with acute renal allograft rejection and renal carcinoma and is able to promote EMT. lncRNA uc.412 is able to induce mesangial cell proliferation in vitro although the underlying mechanisms are unclear. Lnc RNA-H19 is associated with TGF-β2-induced fibrosis in vivo and in vitro. lncRNA ENST00000453774.1 (LncRNA 74.1) is significantly down-regulated in TGF-β-treated TECs and in fibrotic kidney. Interestingly, a recent study also revealed that decreased human lnc-TSI (TGF-β/Smad3-interacting long non-coding RNA) correlates with the degree of renal fibrosis in patients with IgA nephropathy and treatment with lnc-TSI inhibits renal fibrosis by blocking its binding to the MH2 domain of Smad3.

15Taken together, TGF-β may diversely regulate renal fibrosis and inflammation via Smad3-dependent miRNAs/lncRNAs as shown in Table 2 and Figure 3.

16Theoretically, TGF-β is a key mediator for renal fibrosis and thus targeting TGF-β signaling could be a good therapeutic strategy for CKD. There are many approaches to develop anti-TGF-β treatment for CKD clinically (Table 3). It has been shown that treatment with Pirfenidone, a non-specific antifibrotic effect of TGF-β, can improve eGFR in the trials of DN and focal segmental glomerulosclerosis (FSGS). Disappointingly, a recent clinical trial study using a humanized monoclonal neutralizing antibody against TGF-β1 (LY2382770) for treatment of patients with diabetic nephropathy has been proven no efficacy on the improvements of serum creatinine, estimated GFR (eGFR), and proteinuria. In addition, the use of another humanized monoclonal antibody, Fresolimumab that inhibits all three isoforms of TGF-β, also fails to achieve the endpoints of proteinuria reduction in patients with FSGS, demonstrating targeting on the upstream of TGF-β signaling may not be a good therapeutic strategy for CKD. It is possible that blockade of the general effect of TGF-β1, including latent form of TGF-β1, may attribute to the failure of these clinical trials. Our previous studies in latent TGF-β transgenic mice explain this notion since mice overexpressing latent TGF-β1 are protected against renal inflammatory and fibrosis in unilateral ureteral obstructive (UUO) nephropathy and anti-GBM glomerulonephritis model. Thus, the latent form of TGF-β1 is renal protective while its active form is pathogenic. As most circulating TGF-β1 is latent form, thus, the use of anti-TGF-β1 antibodies may largely block the protective effect of latent TGF-β1, resulting in progressive renal injury as seen in these clinical trials. Results from these studies also suggest that treatment against renal fibrosis in patients with CKD should specifically target the downstream TGF-β signaling molecules, rather than to block the general effect of TGF-β1.

17Given the diversity and the complexity of TGF-β in renal fibrosis and inflammation, direct targeting TGF-β or receptors may not be an ideal tactic due to its involvement in various vital biological processes. Although general blockade of the upstream TGF-β signaling may reduce fibrosis, it can also promote renal inflammation and cause unexpected renal injuries (Figure 4a). Because the imbalance of TGF-β/Smad3 signaling with overreactive Smad3 and reduced Smad7 is a key mechanism leading to renal fibrosis and inflammation, rebalancing Smad3/Smad7 signaling may serve as effective strategies to treat renal fibrosis and inflammation (Figure 4b). SIS3, a specific Smad3-inhibitor, has been shown to inhibit renal fibrosis in STZ-induced diabetic nephropathy and in obstructive nephropathy. Overexpression of renal Smad7 is also capable of inhibiting Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in various kidney diseases, including diabetic and hypertensive nephropathy, obstructive nephropathy, remnant kidney disease, crescentic glomerulonephritis, and chronic aristolochic acid nephropathy. Interestingly, treatment of CKD with two Traditional Chinese Medicine compounds, Naringenin from fruits as a Smad3 inhibitor and Asiatic acid derived from Centella asiatica as a Smad7 agonist, is capable of restoring the balance of Smad3/Smad7 signaling and thus additively inhibits renal fibrosis in rodent obstructive nephropathy. Similarly, the combination of Ginsenoside Rg1 from Panax ginseng C. A. Mey and Astragaloside IV from Radix astragali have also improved fibrosis and inflammation in STZ-induced diabetic nephropathy by inhibiting TGF-β/Smad2/3 while enhancing Smad7 signaling. Asperulosidic acid, a bioactive iridoid glycoside, can also exert renal protective effects by inactivating both TGF-β/Smad and NF-κB signaling pathways. Similar therapeutic effects are also found in other studies with herbal medicines.

18Targeting Smad3-dependent non-coding RNAs could be another therapeutic approach to treat renal fibrosis and inflammation (Figure 4c). Of Smad3-dependent miRNAs (Figure 3), inhibition of miR-21, miR-192, miR-433, and overexpression of miR-29 and miR-200 have been shown to have therapeutic effects on obstructive nephropathy and diabetic nephropathy. However, the off-target effect of anti-miRNA therapies raises concern and new therapeutic approach by targeting Smad3-dependent lncRNAs is sought. Targeting Arid2-IR and LRNA9884 can specifically inhibit renal inflammation while targeting Erbb4-IR can specifically inhibit renal fibrosis in obstructive and diabetic nephropathy. Furthermore, delivery of a human lncRNA lnc-TSI into the UUO kidney also inhibits Smad3-mediated renal fibrosis. All these findings highlight the therapeutic potentials by targeting downstream TGF-β signaling molecules including Smad3, Smad7, and non-coding RNAs in renal fibrosis and inflammation.

19Transforming growth factor-β plays diverse roles in renal fibrosis and inflammation. Blockade of upstream TGF-β signaling may not be a good therapeutic strategy, which has been proved by unsatisfied clinical trials. TGF-β may specifically regulate renal fibrosis and inflammation via downstream Smad-dependent mechanisms involving Smad3, Smad4, Smad7, and particularly Smad3-dependent non-coding RNAs. Targeting downstream TGF-β/Smad signaling by rebalancing Smad3/Smad7 or by specifically inhibiting or overexpressing Smad3-dependent non-coding RNAs related to fibrosis or inflammation may be a better therapeutic approach. Further studies to understand the diverse role of TGF-β signaling in kidney diseases may promote the translation from bench into clinical settings.

20Y-YG, X-SL, and X-RH wrote and revised the manuscript. X-QY and H-YL revised and edited the manuscript. All authors contributed to the manuscript conception development, data collection and analysis, and discussion on the manuscript writing and revising.

21The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.