Long Noncoding RNA Arid2-IR Is a Novel Therapeutic Target for Renal Inflammation

Increasing evidence shows that microRNAs play an important role in kidney disease. However, functions of long noncoding RNAs (lncRNAs) in kidney diseases remain undefined. We have previously shown that TGF-β1 plays a diverse role in renal inflammation and fibrosis and Smad3 is a key mediator in this process. In this study, we used RNA-sequencing to identify lncRNAs related to renal inflammation and fibrosis in obstructive nephropathy induced in Smad3 wild-type and knockout mice. We found that Arid2-IR was a Smad3-associated lncRNA as a Smad3 binding site was found in the promoter region of Arid2-IR and deletion of Smad3 abolished upregulation of Arid2-IR in the diseased kidney. In vitro knockdown of Arid2-IR from tubular epithelial cells produced no effect on TGF-β-induced Smad3 signaling and fibrosis but inhibited interleukin-1β-stimulated NF-κB-dependent inflammatory response. In contrast, overexpression of Arid2-IR promoted interleukin-1β-induced NF-κB signaling and inflammatory cytokine expression without alteration of TGF-β1-induced fibrotic response. Furthermore, treatment of obstructed kidney with Arid2-IR shRNA blunted NF-κB-driven renal inflammation without effect on TGF-β/Smad3-mediated renal fibrosis. Thus, Arid2-IR is a novel lncRNA that functions to promote NF-κB-dependent renal inflammation. Blockade of Arid2-IR may represent a novel and specific therapy for renal inflammatory disease.     

SKLB023 hinders renal interstitial fibrosis in obstructive nephropathy by interfering TGF-β1/Smad3 signaling

Renal fibrosis is the principal process underlying the progression of chronic kidney disease to end-stage renal disease. It is a relatively uniform response involving glomerulosclerosis, tubulointerstitial fibrosis and changes in renal vasculature. A considerable number of studies have confirmed that inducible nitric oxide synthase (iNOS) was highly expressed in renal interstitial fibrosis and the overexpression of iNOS played a negative role in kidney disease progression. In our previous study, SKLB023 as a novel small-molecule inhibitor of iNOS, blocked joint inflammation and cartilage destruction in arthritis. However, the pharmacological role and function of SKLB023 in renal fibrosis remained poorly understood. In the study, oral administration of SKLB023 (25 and 50 mg per kg per day) for 7 day exhibited potent anti-fibrotic effects against the model UUO using the pathological assessment of H & E and Masson''s trichrome staining. SKLB023 inhibited the expression of α-SMA, col I, col IV, fibronectin and further decreased iNOS expression as well as TGF-β1/Smad3 phosphorylation in the injured kidney tissues of UUO mice. Similarly, SKLB023 suppressed in vitro features of fibrosis in TGF-β1-induced NRK-49F by the inhibition of the corresponding fibrotic protein expression. These findings confirmed that SKLB023 hindered renal interstitial fibrosis by interfering with TGF-β1/Smad3 signaling, highlighting that SKLB023 has potential in therapeutic strategies.

The novel small-molecule inhibitor of iNOS (SKLB023) hindered renal interstitial fibrosis in vivo and in vitro by interfering with TGF-β1/Smad3 signaling, highlighting that SKLB023 has potential in the therapeutic strategy for renal fibrosis.     

MicroRNA-29b Inhibits Diabetic Nephropathy in db/db Mice

Inflammation and its consequent fibrosis are two main features of diabetic nephropathy (DN), but target therapy on these processes for DN remains yet ineffective. We report here that miR-29b is a novel therapeutic agent capable of inhibiting progressive renal inflammation and fibrosis in type 2 diabetes in db/db mice. Under diabetic conditions, miR-29b was largely downregulated in response to advanced glycation end (AGE) product, which was associated with upregulation of collagen matrix in mesangial cells via the transforming growth factor-β (TGF-β)/Smad3-dependent mechanism. These pathological changes were reversed by overexpressing miR-29b, but enhanced by knocking-down miR-29b. Similarly, loss of renal miR-29b was associated with progressive diabetic kidney injury, including microalbuminuria, renal fibrosis, and inflammation. Restored renal miR-29b by the ultrasound-based gene therapy was capable of attenuating diabetic kidney disease. Further studies revealed that inhibition of Sp1 expression, TGF-β/Smad3-dependent renal fibrosis, NF-κB–driven renal inflammation, and T-bet/Th1-mediated immune response may be mechanisms associated with miR-29b treatment in db/db mice. In conclusion, miR-29b may play a protective role in diabetic kidney disease and may have therapeutic potential for diabetic kidney complication.     

Smad7 suppresses renal fibrosis via altering expression of TGF-β/Smad3-regulated microRNAs

Blockade of transforming growth factor-β (TGF-β) signaling by Smad7 gene therapy is known to prevent experimental renal fibrosis. This study investigated whether Smad7 suppresses renal fibrosis via altering the renal expression of fibrosis-related microRNAs. Application of gene therapy into diseased kidneys of obstructive nephropathy and kidney cells by overexpressing Smad7 restored miR-29b but inhibited the expression of miR-192 and miR-21, resulting in blockade of renal fibrosis. Furthermore, Smad7 overexpression also suppressed advanced glycated end products- and angiotensin II-regulated expression of these microRNAs. In contrast, disruption of Smad7 gene in mice demonstrated opposite results by enhancing the loss of miR-29b and upregulation of miR-192 and miR-21, resulting in promotion of renal fibrosis in ligated kidneys of a model of obstructive nephropathy. More importantly, treatment with anti-miR-29b, miR-21 and miR-192 mimics in Smad7 overexpressing tubular epithelial cells abrogated the suppressive function of Smad7 on renal fibrosis, suggesting that these microRNAs act downstream of Smad7 to override the Smad7 function. In conclusion, Smad7 protects kidneys from fibrosis by regulating TGF-β/Smad3-mediated renal expression of miR-21, miR-192, and miR-29b. Restored renal miR-29b but suppressed miR-192 and miR-21 may be a mechanism by which gene therapy with Smad7 inhibits renal fibrosis.     

miR-29b as a Therapeutic Agent for Angiotensin II-induced Cardiac Fibrosis by Targeting TGF-β/Smad3 signaling

Loss of miR-29 is associated with cardiac fibrosis. This study examined the role and therapeutic potential of miR-29 in mouse model of hypertension induced by angiotensin II (AngII). By using microRNA microarray, in situ hybridization, and real-time polymerase chain reaction, we found that AngII-induced cardiac fibrosis in the hypertensive heart and in cultured cardiac fibroblasts were associated with downregulation of miR-29a-c via a Smad3-dependent mechanism. In vitro knockdown of miR-29b enhanced but overexpression of miR-29b inhibited AngII-induced fibrosis, revealing a protective role of miR-29b in cardiac fibrosis in response to AngII. This was further demonstrated in vivo by the ability of overexpressing miR-29b in the mouse heart to prevent AngII-mediated cardiac fibrosis and cardiac dysfunction. Importantly, we also found that restored miR-29b in the established hypertensive heart was capable of blocking progressive cardiac fibrosis and improving cardiac dysfunction, demonstrating a therapeutic potential of miR-29b for chronic heart disease. Further studies revealed that targeting the transforming growth factor (TGF)-β1 coding sequence region, thereby inhibiting TGF-β/Smad3 signaling, could be a new mechanism by which miR-29b inhibited AngII-induced cardiac fibrosis. In conclusion, miR-29b plays a protective role in AngII-mediated cardiac remodeling and may be a therapeutic agent for cardiac fibrosis by targeting the TGF-β/Smad3 pathway.     

Metabolic changes induced by TGF-β1 via reduced expression of phosphatidylserine decarboxylase during myofibroblast transition

Metabolic alteration is increasingly recognized as an important pathogenic process that underlies fibrosis across many organ types, and metabolically targeted therapies could become important strategies for reducing fibrosis. In present study, target enzymes that are involved in changes in phospholipid metabolism during fibroblast-to-myofibroblast transition induced by transforming growth factor beta 1 (TGF-β1) were examined. Different amounts of phospholipids were found in the 2 groups. In response to TGF-β1 stimulation, 17 lipids decreased and 17 increased. The latter included the phospholipids phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Furthermore, among the rate-limiting enzymes that regulate these phospholipids, phosphatidylserine decarboxylase (PISD), which controls conversion of PS to PE and is localized in mitochondria, decreased in response to TGF-β1. Knockdown of PISD alone without TGF-β1 stimulation increased expression of α-smooth muscle actin mRNA and production of total collagen. Taken together, these results indicate that PISD is involved in the mechanism of fibrogenesis by regulating phospholipid metabolism.     

Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis

Idiopathic pulmonary fibrosis is a progressive and fatal fibrotic disease of the lungs with unclear etiology. Prior efforts to treat idiopathic pulmonary fibrosis that focused on anti-inflammatory therapy have not proven to be effective. Recent insight suggests that the pathogenesis is mediated through foci of dysregulated fibroblasts driven by profibrotic cytokine signaling. TGF-β and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-β–induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-β by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-β–induced ECM gene expression, morphologic transformation, and cell proliferation independently of any effect on Smad signaling. Further, using a mouse model of bleomycin-induced pulmonary fibrosis, we found a significant inhibition of lung fibrosis by imatinib. Thus, Abl family members represent common targets for the modulation of profibrotic cytokine signaling.     

Polydatin ameliorates pulmonary fibrosis by suppressing inflammation and the epithelial mesenchymal transition via inhibiting the TGF-β/Smad signaling pathway

Pulmonary fibrosis is a chronic and progressive lung disease which results in a loss of pulmonary function and eventually respiratory failure. Inflammation and epithelial mesenchymal transition (EMT) play important roles in the pathogenesis of pulmonary fibrosis. This study aimed to investigate the therapeutic effect of polydatin (PD) in bleomycin-induced pulmonary fibrosis. A bleomycin-induced pulmonary fibrosis animal model used SD rats. Morphological changes were analyzed by hematoxylin-eosin staining. RT-qPCR and western blot were used for the detection of the expression of TGF-β1, collagen I, collagen III, E-cadherin, fibronectin and the ratios of p-Smad2/Smad2, p-Smad3/Smad3. The concentrations of PICP, PIIINP, TNF-α, IL-1β, IL-6 and IL-17 were measured by enzyme linked immunosorbent assay (Elisa) assay. Results showed that PD attenuated bleomycin-induced pulmonary fibrosis. The beneficial effect of PD was possibly related to the inhibition of inflammation and EMT through suppressing the TGF-β/Smad signaling pathway. Our findings suggested that PD might be a potential therapeutic candidate in the treatment of pulmonary fibrosis.

Pulmonary fibrosis is a chronic and progressive lung disease which results in a loss of pulmonary function and eventually respiratory failure.     

The inhibitory effect of heat treatment against epithelial-mesenchymal transition (EMT) in human pancreatic adenocarcinoma cell lines

Epithelial-mesenchymal transition (EMT) plays a crucial role in cancer metastasis. In this study, we evaluated the effect of heat treatment on tumor growth factor-β1 (TGF-β1)-induced EMT in pancreatic cancer cells and tried to ascertain the mechanism related to any observed effects. Human pancreatic cancer cell lines (BxPC-3, PANC-1 and MIAPaCa-2) were stimulated by TGF-β1, and evaluated for morphological changes using immunofluorescence and EMT-related factors (i.e., E-cadherin, Vimentin, Snail or ZEB-1) using RT-PCR. To examine the effect of heat on EMT, the cancer cells were heat-treated at 43°C for 1 h then stimulated with TGF-β1. We then evaluated whether or not heat treatment changed the expression of EMT-related factors and cell migration and also whether Smad activation was inhibited in TGF-β signaling. After being treated with TGF-β1, pancreatic cancer cells resulted in EMT and cell migration was enhanced. Heat treatment inhibited TGF-β1-induced changes in morphology, inhibited the expression of EMT-related factors, and attenuated TGF-β1-induced migration in pancreatic cancer cells. Additionally, we observed that heat treatment blocked TGF-β1-induced phosphorylation of Smad2 in PANC-1 cells. Our results suggest that heat treatment can suppress TGF-β1-induced EMT and opens the possibility of a new therapeutic use of hyperthermia as a potential treatment for cancer metastasis.     

Epithelial cell α3β1 integrin links β-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis

Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell–specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654–β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654–β-catenin/pSmad2 complexes and showed accumulation of pY654–β-catenin in myofibroblasts. These findings demonstrate epithelial integrin–dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.     

Anti–microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways

MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti–miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti–miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-β–induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.     

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-β–like signaling pathway

The four-and-a-half LIM (FHL) proteins belong to a family of LIM-only proteins that regulate cell proliferation, differentiation, and apoptosis. The exact functions of each FHL protein in cancer development and progression remain unknown. Here we report that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, in a TGF-β–independent manner. Casein kinase 1δ, but not the TGF-β receptor, was required for the FHL-mediated TGF-β–like responses, including increased phosphorylation of Smad2/3, interaction of Smad2/3 and Smad4, nuclear accumulation of Smad proteins, activation of the tumor suppressor gene p21, and repression of the oncogene c-myc. FHL1–3 inhibited anchorage-dependent and -independent growth of a human hepatoma cell line in vitro and tumor formation in nude mice. Further analysis of clinical samples revealed that FHL proteins are often downregulated in hepatocellular carcinomas and that this correlates with decreased TGF-β–like responses. By establishing a link between FHL proteins and Smad proteins, this study identifies what we believe to be a novel TGF-β–like signaling pathway and indicates that FHL proteins may be useful molecular targets for cancer therapy.     

The polysaccharides from Grifola frondosa attenuate CCl4-induced hepatic fibrosis in rats via the TGF-β/Smad signaling pathway

The TGF-β1/Smad signaling pathway has been linked to hepatic fibrosis. Previous studies have shown that yellow polysaccharide can prevent the development of hepatic fibrosis. However, it is unclear whether the polysaccharide affects the TGF-β1/Smad signaling pathway. In this experiment, 50 experimental rats were randomly divided into a normal control group, model group, low GFP dose group (50 mg kg⁻¹), medium GFP dose group (100 mg kg⁻¹), and high GFP dose group (200 mg kg⁻¹). A cirrhotic portal hypertension rat model was established by a CCl₄ compound method. After 12 weeks of intragastric administration, the liver index of the medium dose and high dose group was significantly lower than that of the model group. The hepatic fibrosis lesions of rats in each dose group were improved to different extents, and the effect was most significant in the high dose group. The contents of ALT, AST, TBIL and CIV, PCIII, LN and HA in serum were significantly decreased. The activity of SOD and GSH-Px in the liver tissue of GFP medium and high dose groups was significantly increased and the content of MDA was significantly decreased. The contents of TNF-α, IL-1β and IL-6 were significantly decreased. The western blot results showed that the expressions of p-Smad 2/3, Smad4, PAI-1, Imp7 and Imp8 in medium dose and high dose groups were significantly lower than those in the model group, while the expression of Smad7 was significantly higher than that of the model group. The GFP-treated group was able to reduce the expression level of mi R-154 in liver tissue and increase the expression level of miR-146a. GFP has a significant intervention effect on rat hepatic fibrosis, and its mechanism may inhibit the progression of hepatic fibrosis by inhibiting oxidative stress and inflammatory response and regulating TGF-β1/Smad signaling pathway and mi RNA expression.

The TGF-β1/Smad signaling pathway has been linked to hepatic fibrosis.