Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.     

TGF-β-mediated upregulation of Sox9 in fibroblast promotes renal fibrosis

TGF-β signaling plays a principal role in renal fibrosis, but the precise mechanisms and the downstream factors are still largely unknown. Sox9 exhibits diverse roles in regulating the production of extracellular matrix proteins. Here we found that Sox9 was induced by TGF-β in the kidney fibroblast and acted as an important downstream mediator of TGF-β signaling in promoting renal fibrosis. TGF-β/Smad signaling mediated the upregulation of Sox9 in kidney fibroblast by binding to a conserved enhancer. In different mouse models of renal fibrosis, as well as in the kidney biopsy tissue from patients with renal fibrosis, Sox9 expression significantly increased. Immunostaining confirmed the upregulation of Sox9 in the kidney fibroblast during renal fibrosis. Delivery of Sox9 knockdown plasmid to the kidney by ultrasound microbubble–mediated gene transfer suppressed the unilateral ureteral obstruction (UUO) or folic acid-induced mouse renal fibrosis, whereas ectopic expression of Sox9 aggravated renal fibrosis. In addition, we identified Sox9 as a direct target of miR-30. Notably, miR-30 expression was significantly inhibited by TGF-β1 in the kidney fibroblast and the downregulation of miR-30 was observed in renal fibrosis. Mechanistically, inhibition of miR-30 independently strengthened the effect of TGF-β/Smad signaling on Sox9 upregulation. Adenovirus-mediated ectopic expression of miR-30 in kidney fibroblast greatly reduced UUO-induced renal fibrosis by targeting Sox9. These findings link Sox9 to intrinsic mechanisms of TGF-β signaling in renal fibrosis and may have therapeutic potential for tissue fibrosis.     

Loss of poly(ADP-ribose) polymerase 1 attenuates renal fibrosis and inflammation during unilateral ureteral obstruction

Poly(ADP-ribose) polymerase 1 (PARP1) contributes to necrotic cell death and inflammation in several disease models; however, the role of PARP1 in fibrogenesis remains to be defined. Here, we tested whether PARP1 was involved in the pathogenesis of renal fibrosis using the unilateral ureteral obstruction (UUO) mouse model. UUO was performed by ligation of the left ureter near the renal pelvis in Parp1-knockout (KO) and wild-type (WT) male mice. After 10 days of UUO, renal PARP1 expression and activation were strongly increased by 6- and 13-fold, respectively. Interstitial fibrosis induced by UUO was significantly attenuated in Parp1-KO kidneys compared with that in WT kidneys at 10 days, but not at 3 days, based on collagen deposition, α-smooth muscle actin (α-SMA), and fibronectin expression. Intriguingly, the UUO kidneys in Parp1-KO mice showed a dramatic decrease in infiltration of neutrophil and reduction in expression of proinflammatory proteins including intercellular adhesion molecule-1, tumor necrosis factor-α, inducible nitric oxide synthase, and toll-like receptor 4 as well as phosphorylation of nuclear factor-κB p65, but not transforming growth factor-β1 (TGF-β1) at both 3 and 10 days. Pharmacological inhibition of PARP1 in rat renal interstitial fibroblast (NRK-49F) cell line or genetic ablation in primary mouse embryonic fibroblast cells did not affect TGF-β1-induced de novo α-SMA expression. Parp1 deficiency significantly attenuated UUO-induced histological damage in the kidney tubular cells, but not apoptosis. These data suggest that PARP1 induces necrotic cell death and contributes to inflammatory signaling pathways that trigger fibrogenesis in obstructive nephropathy.     

Response gene to complement 32 interacts with Smad3 to promote epithelial-mesenchymal transition of human renal tubular cells

Previous studies demonstrate that response gene to complement 32 (RGC-32) mediates transforming growth factor-β(1)-induced epithelial-mesenchymal transition (EMT) of human renal proximal tubular cells. However, the mechanisms underlying RGC-32 function remain largely unknown. In the present study, we found that RGC-32 function in EMT is associated with Smad3. Coexpression of RGC-32 and Smad3, but not Smad2, induces a higher mesenchymal marker α-smooth muscle actin (α-SMA) protein expression as compared with RGC-32 or Smad3 alone, while knockdown of Smad3 using short hairpin interfering RNA blocks RGC-32-induced α-SMA expression. These data suggest that RGC-32 interacts with Smad3, but not Smad2, in the regulation of EMT. In addition to α-SMA, RGC-32 and Smad3 also synergistically activate the expression of extracellular matrix protein fibronectin and downregulate the epithelial marker E-cadherin. RGC-32 colocalizes with Smad3 in the nuclei of renal proximal tubular cells. Coimmunoprecipitation assays showed that Smad3, but not Smad2, physically interacts with RGC-32 in renal proximal tubular cells. Mechanistically, RGC-32 and Smad3 coordinate the induction of EMT by regulating the EMT regulators Slug and Snail. Taken together, our data demonstrate for the first time that RGC-32 interacts with Smad3 to mediate the EMT of human renal proximal tubular cells.     

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.     

Reduced Klotho expression level in kidney aggravates renal interstitial fibrosis

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-β(1) (TGF-β(1)) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-β(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β(1) activity and is a cause of renal fibrosis. On the other hand, TGF-β(1) reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression.     

TGF-βII 型受体的RNA 干扰抑制肾纤维化

目的:研究针对TGF-βⅡ型受体的RNA干扰质粒对α-SMA表达的抑制作用,探讨RNA干扰TGF-βRII的方法对肾间质纤维化的抑制作用。方法:单侧结扎输尿管方法制备肾间质纤维化小鼠动物模型。分别经输尿管逆行注射a:RNA干扰质粒;b:错配对照质粒;c:空载体;d:生理盐水;e:正常对照。通过western-blot及免疫组织化学方法检测第3、5、10天后肾组织内TGF-βRⅡ、α-SMA,观察其对肾间质纤维化的抑制作用。结果:针对TGF-βRII的RNA干扰质粒,明显抑制肾组织内TGF-βRⅡ、α-SMA表达;几组对照未见相应作用。结论:针对TGF-βRⅡ的RNA干扰质粒,能够抑制肾间质纤维化的发生、发展,可能成为延缓肾功能减退的有效方法。     

The regulatory peptide apelin: a novel inhibitor of renal interstitial fibrosis

Epithelial–mesenchymal transition (EMT) of tubular epithelial cells is a key event in renal interstitial fibrosis and the progression of chronic kidney disease (CKD). Apelin is a regulatory peptide involved in the regulation of normal renal hemodynamics and tubular functions, but its role in renal fibrosis remains unknown. In this study, we examined the inhibitory effects of apelin on transforming growth factor-β1 (TGF-β1)-induced EMT in HK-2 cells, and evaluated its therapeutic efficacy in mice with complete unilateral ureteral obstruction (UUO). In vitro, apelin inhibited TGF-β1-mediated upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin. Increased levels of phosphorylated Smad-2/3 and decreased levels of Smad7 in TGF-β1-stimulated cells were reversed by apelin co-treatment. In the UUO model, administration of apelin significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin and laminin expression, and markedly suppressed expression of α-SMA, TGF-β1 and its type I receptor, as well as interstitial matrix components. Interestingly, in UUO mice, there was a reduction in the plasma level of apelin, which was compensated by upregulation of APJ expression in the injured kidney. Exogenous supplementation of apelin normalized the level of plasmatic apelin and renal APJ. In conclusion, our study provides the first evidence that apelin is able to ameliorate renal interstitial fibrosis by suppression of tubular EMT through a Smad-dependent mechanism. The apelinergic system itself may promote some compensatory response in the renal fibrotic process. These results suggest that apelin has potential renoprotective effects and may be an effective agent for retarding CKD progression.     

Sphingosine kinase 2 cooperating with Fyn promotes kidney fibroblast activation and fibrosis via STAT3 and AKT

Sphingosine kinases (Sphks) are the rate-limiting enzymes in the conversion of sphingosine to biologically active sphingosine-1-phosphate. The present study aimed to determine the role of Sphk2 and its downstream targets in renal fibroblast activation and interstitial fibrosis. In the kidney interstitium of patients with renal fibrosis, Sphk2^high-expressing cells (mainly interstitial fibroblasts) were significantly elevated and highly correlated with disease progression in patients. In a murine model of renal interstitial fibrosis, Sphk2 was upregulated in the kidney of wild-type mice in response to disease progression. Importantly, Sphk2-knockout (KO) mice exhibited significantly lower levels of extracellular matrix (ECM) production and a suppressed inflammatory response in the kidney tissues, compared to those in their wild-type counterparts, whereas the expression of TGF-β1 was unaffected. TGF-β1 effectively upregulated Sphk2 expression in the renal interstitial fibroblast line, NRK-49F, independent of canonical Smad signaling activation. Furthermore, siRNA-mediated Sphk2 knockdown or suppression of Sphk2 activity by ABC294640 exposure effectively attenuated AKT and STAT3 activation and ECM production, but had no effects on Smad2 and Smad3 activation. Sphk2 phosphorylated Fyn to activate downstream STAT3 and AKT, thereby promoting ECM synthesis. Therefore, our findings indicate that targeting Sphk2-Fyn-STAT3/AKT signaling pathway may be a novel therapeutic approach for renal fibrosis.     

Smad3-mediated myocardin silencing: a novel mechanism governing the initiation of smooth muscle differentiation

Both TGF-β and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-β-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-β did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-β treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-β-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-β stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-β to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-β induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.     

糖尿病大鼠血糖控制前后肾小管上皮细胞VEGF、TGF-β1及Smad蛋白表达的动态研究

目的动态观察链脲佐菌素（STZ）诱导的糖尿病大鼠血糖控制前后肾小管上皮细胞（TEC）中血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、Smad2/3、Smad4的表达情况,探讨四者在糖尿病大鼠TEC表型转变和肾间质纤维化中可能发挥的作用及相互关系。方法实验动物随机分为5组,依病程长短分为①A组（2周组）,②B组（4周组）,③C组（8周组）,④D组（16周组）,⑤E组（24周组）,每组分别设有正常对照组（N组）和糖尿病组（a组）;另外,16周、24周两组加设胰岛素治疗组（b组）。采用尾静脉注射STZ法复制糖尿病大鼠模型;免疫组织化学方法检测肾小管VEGF、TGF-β1、Smad2/3、Smad4及α-平滑肌肌动蛋白（-αSMA）和纤连蛋白（FN）的表达;Western blot检测肾皮质VEGF和TGF-β1蛋白;PAS染色光镜观察肾小管基底膜变化及细胞外基质沉积情况等形态学改变;生化方法测定血糖、血肌酐及24小时尿蛋白量。结果正常对照组VEGF、TGF-β1及Smad2/3、Smad4在肾小管均有少量表达,-αSMA在肾小管无表达;糖尿病组肾小管前述四者的表达均显著高于正常对照组,且从16周开始肾小管上皮细胞可见α-SMA蛋白阳性表达;糖尿病16周时肾小管VEGF、TGF-β1、Smad2/3、Smad4两两之间呈正相关;随糖尿病进展,α-SMA及FN在肾小管表达增多,24h尿蛋白增多,肾脏肥大指数增大,而VEGF、TGF-β1二者都分别和-αSMA、FN、24h尿蛋白及肾脏肥大指数呈正相关性;胰岛素治疗后,VEGF、TGF-β1、Smad2/3、Smad4及FN的表达都比糖尿病组明显下降,且各指标之间的正相关性依然存在,-αSMA蛋白则呈阴性表达。结论糖尿病肾病大鼠肾小管上皮细胞表达的VEGF、TGF-β1及Smad2/3、Smad4参与了TEC表型转变和肾间质纤维化的发生,并且VEGF和TGF-β1相互作用,共同促进了肾脏损害。胰岛素对DN大鼠TEMT和肾间质纤维化的影响可能部分是通过间接阻断VEGF、TGF-β1和Smad2/3、Smad4在TEC中的合成来实现的。     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

Is cytochrome P450 3A4 regulated by menstrual cycle hormones in control endometrium and endometriosis?

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.     

The Notch γ-secretase inhibitor ameliorates kidney fibrosis via inhibition of TGF-β/Smad2/3 signaling pathway activation

Kidney fibrosis is a common feature of chronic kidney disease (CKD). A recent study suggests that abnormal Notch signaling activation contributes to the development of renal fibrosis. However, the molecular mechanism that regulates this process remains unexplored. Unilateral ureteral obstruction (UUO) or sham-operated C57BL6 mice (aged 10 weeks) were randomly assigned to receive dibenzazepine (DBZ, 250 μg/100 g/d) or vehicle for 7 days. Histologic examinations were performed on the kidneys using Masson's trichrome staining and immunohistochemistry. Real-time PCR and western blot analysis were used for detection of mRNA expression and protein phosphorylation. The expression of Notch 1, 3, and 4, Notch intracellular domain (NICD), and its target genes Hes1 and HeyL were upregulated in UUO mice, while the increase in NICD protein was significantly attenuated by DBZ. After 7 days, the severity of renal fibrosis and expression of fibrotic markers, including collagen 1α1/3α1, fibronectin, and α-smooth muscle actin, were markedly increased in UUO compared with sham mice. In contrast, administration of DBZ markedly attenuated these effects. Furthermore, DBZ significantly inhibited UUO-induced expression of transforming growth factor (TGF)-β, phosphorylated Smad 2, and Smad 3. Mechanistically, Notch signaling activation in tubular epithelial cells enhanced fibroblast proliferation and activation in a coculture experiment. Our study provides evidence that Notch signaling is implicated in renal fibrogenesis. The Notch inhibitor DBZ can ameliorate this process via inhibition of the TGF-β/Smad2/3 signaling pathway, and might be a novel drug for preventing chronic kidney disease.     

Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.