Ubiquitination and regulation of Smad7 in the TGF-β1/Smad signaling of aristolochic acid nephropathy

Abstract

Aristolochic acid I (AAI) affects TGF-β1/Smad signaling, which causes AA nephropathy (AAN), but the mechanisms are not fully understood. We aimed to clarify whether Arkadia and UCH37 participate in TGF-β1/Smad signaling via Smad7, and the regulatory mechanisms of Smad7. One side, mice and cultured mouse renal tubular epithelial cells (RTECs) were treated with various AAI doses and concentrations, respectively; on the other side, RTECs were transfected with small interfering RNA (siRNA) expression vectors against Arkadia and UCH37 and then treated with 10?µg/ml AAI. And then detect the mRNA and protein levels of Smad7, UCH37, Arkadia and any other relative factors by RT-PCR and Western blotting. In kidney tissues and RTECs, the mRNA and protein levels of Smad7 decreased with increasing AAI doses concentrations by real-time PCR and Western blotting, whereas those of Arkadia, UCH37, Smad2, Smad3 and TβRI increased. Cells transfected with the Arkadia siRNA expression vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3 and TβRI after AAI treatment, while those of CK18 and Smad7 increased compared with those of untransfected RTECs. Conversely, cells transfected with the UCH37 siRNA expression vector showed the opposite effect on analyzed signaling molecules after AAI treatment. Arkadia and UCH37 participate in TGF-β1/Smad signaling-mediated renal fibrosis, and Smad7 blocks TGF-β1 signaling by inhibiting Smad2/Smad3 phosphorylation and enhancing the degradation of TβRI.     

Aristolochic acid I-induced DNA damage and cell cycle arrest in renal tubular epithelial cells in vitro

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAI-treated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53-independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.     

Gremlin-mediated decrease in bone morphogenetic protein signaling promotes aristolochic acid-induced epithelial-to-mesenchymal transition (EMT) in HK-2 cells

Ingestion of aristolochic acid (AA) is associated with the development of aristolochic acid nephropathy (AAN), which is characterized by progressive tubulointerstitial fibrosis, chronic renal failure and urothelial cancer. Our previous study showed that bone morphogenetic protein-7 (BMP-7) could attenuate AA-induced epithelial-to-mesenchymal transition (EMT) in human proximal tubule epithelial cells (PTEC). However, how gremlin (a BMP-7 antagonist) antagonizes the BMP-7 action in PTEC remained unsolved. The aim of the current study was to investigate the role of gremlin in AA-induced EMT in PTEC (HK-2 cells). HK-2 cells were treated with AA (10 μmol/L) for periods up to 72 h. Cell viability was determined by tetrazolium dye (MTT) assay. Morphological changes were assessed by phase-contrast microscopy. Markers of EMT, including E-cadherin and α-smooth muscle actin (α-SMA) were detected by indirect immunofluorescence stains. The BMP-7 and gremlin mRNA and protein expression in HK-2 cells were analyzed by quantitative real-time PCR (real-time RT-PCR) and western blotting after exposure to AA. The level of phosphorylated Smad1/5/8, a marker of BMP-7 activity, was also determined by western blot analysis. Cells were transfected with gremlin siRNA to determine the effects of gremlin knockdown on markers of EMT following treatment with AA. Our results indicated that AA-induced EMT was associated with acquisition of fibroblast-like cell shape, loss of E-cadherin, and increases of alpha-SMA and collagen type I. Interestingly, exposure of HK-2 cells to 10 μmol/L AA increased the mRNA and protein expression of gremlin in HK-2 cells. This increase was in parallel with a decrease in BMP-7 expression and a down-regulation of phosphorylated Smad1/5/8 protein levels. Moreover, transfection with siRNA to gremlin was able to recover BMP-7 signaling activity, and attenuate EMT-associated phenotypic changes induced by AA. Together, these observations strongly suggest that gremlin plays a critical role in the modulation of reno-protective action of BMP, and that inhibition of gremlin will be a promising means of developmenting novel treatments for AAN.     

普罗布考对甲基胍诱导纤维化相关因子在HK-2细胞表达的影响

目的研究降脂药普罗布考对小分子尿毒素甲基胍诱导纤维化相关因子在人肾小管上皮细胞表达的影响。方法 HK-2细胞分3组培养,A组（正常对照,不加干预因素）;B组培养基中加入0.5mmol.L-1甲基胍;C组培养基中加入0.5mmol.L-1甲基胍和20μmol.L-1普罗布考。各组细胞培养48h后用免疫细胞化学、RT-PCR检测TGF-β1、CTGF和FN,并用流式细胞仪对以上因子作定量检测,以观察其在HK-2细胞中的表达。结果在甲基胍刺激后,细胞免疫化学染色,HK-2细胞质中着色,显示TGF-β1、CTGF和FN均有表达。正常对照组HK-2细胞质中无着色。测定结果显示甲基胍组TGF-β1、CTGF和FN显著高于正常对照组（P〈0.01）,加入普罗布考后,表达显著下降（P〈0.01）。结论甲基胍能促进纤维化相关因子在肾小管上皮细胞中表达;普罗布考能抑制甲基胍诱导纤维化相关因子在肾小管上皮细胞的表达。     

Ganoderic acid hinders renal fibrosis via suppressing TGF-β/Smad and MAPK signaling pathways

OBJECTIVE To investigatethe anti-renal fibrotic effect of Ganoderic acid(GA) and the related mechanisms in vivo and in vitro. METHODS We investigated whether GA could hinder renal fibrosis and the related mechanisms in a unilateral ureteral obstruction(UUO) mouse model and TGF-β1-stimulated HK-2 cells model. The C57 mice were randomly devided into sham group, sham with GA administration group, UUO group and UUO with GA treatment group. After UUO surgery,the mice were treated with GA(3.125, 12.5 and 50 mg·kg~(-1) per day, ip) for 14 d. Then the mice were sacrificed for collecting blood and kidneys. The kidney functions were revealed by blood urea nitrogen and serum creatinine.The fibrosis and tubules injury levels were detected by Masson staining and HE staining. The epithelial-mesenchymal transition(EMT) level was detected by Western blotting and immunohistochemical staining. The changes of related signaling pathways and underlying mechanisms were demonstrated by Western blotting. The cell model was adopted to identify the potent inhibitor of renal fibrosis. RESULTS After 14 d treatment, GA dosedependently attenuated UUO-induced tubular injury and renal fibrosis; GA(50 mg·kg~(-1)) significantly ameliorated renal disfunction during fibrosis progression. We further revealed that GA treatment significantly decreased the expression of mesenchymal cell markers alpha-smooth muscle actin(α-SMA) and vimentin, and up-regulated E-cadherin expression in the kidney, suggesting the suppression of tubular EMT. Further study showed that GA downregulated both TGF-β/Smad and MAPK(ERK,JNK, P38) signaling pathways both in vivo and in vitro.GA-A, a GA monomer, was identified as a potent inhibitor on renal fibrosis in TGF-β1-stimulated HK-2 cell model.CONCLUSION GA inhibits renal interstitial fibrosis induced by UUO, mostly by suppressing EMT progression and inhibiting TGF-β/Smad and MAPK signaling pathways. In conclusion, our results suggest that GA and its purified monomer GA-A might be developed as a potential therapeutic agent in the treatment of renal fibrosis.     

Critical role of organic anion transporters 1 and 3 in kidney accumulation and toxicity of aristolochic acid I

Ingestion of aristolochic acid (AA), especially its major constituent aristolochic acid I (AAI), results in severe kidney injury known as aristolochic acid nephropathy (AAN). Although hepatic cytochrome P450s metabolize AAI to reduce its kidney toxicity in mice, the mechanism by which AAI is uptaken by renal cells to induce renal toxicity is largely unknown. In this study, we found that organic anion transporters (OATs) 1 and 3, proteins known to transport drugs from the blood into the tubular epithelium, are responsible for the transportation of AAI into renal tubular cells and the subsequent nephrotoxicity. AAI uptake in HEK 293 cells stably transfected with human OAT1 or OAT3 was greatly increased compared to that in the control cells, and this uptake was dependent on the AAI concentration. Administration of probenecid, a well-known OAT inhibitor, to the mice reduced AAI renal accumulation and its urinary excretion and protected mice from AAI-induced acute tubular necrosis. Further, AAI renal accumulation and severe kidney lesions induced by AAl in Oat1 and Oat3 gene knockout mice all were markedly suppressed compared to those in the wild-type mice. Together, our results suggest that OAT1 and OAT3 have a critical role in AAl renal accumulation and toxicity. These transporters may serve as a potential therapeutic target against AAN.     

辛伐他汀通过抑制Smad7表达的肾保护作用机制探讨

目的研究阿霉素肾病大鼠肾小管Smad7表达改变与细胞凋亡的关系,并探讨辛伐他汀通过影响Smad7表达和细胞凋亡的肾保护作用机制。方法建立大鼠阿霉素肾病模型,RT-PCR半定量分析肾皮质中Smad7mRNA的表达水平,免疫组化分析Smad7蛋白的定位和表达,用TUNEL法分析细胞凋亡。结果肾病组肾小管中Smad7mRNA、蛋白的表达均上调,与肾小管上皮细胞凋亡呈正相关。辛伐他汀可抑制Smad7表达上调、减少肾小管上皮细胞凋亡。结论阿霉素肾病大鼠肾小管Smad7表达增加与肾小管上皮细胞凋亡相关。辛伐他汀可以通过抑制Smad7表达而降低肾小管上皮细胞凋亡而实现肾保护作用。     

Tubastatin A suppresses renal fibrosis via regulation of epigenetic histone modification and Smad3-dependent fibrotic genes

Inflammation and fibrosis are implicated in the pathogenesis of hypertensive kidney damage. We previously demonstrated that a nonspecific histone deacetylase (HDAC) inhibitor attenuates cardiac fibrosis in deoxycorticosterone acetate-salt hypertensive rats, which induces HDAC6 protein and enzymatic activity. However, the HDAC inhibitor's effect and mechanism have not yet been demonstrated. We sought to determine whether an HDAC6-selective inhibitor could treat hypertension and kidney damage in angiotensin II-infused mice. Hypertension was induced by infusion of ANG in mice. Tubastatin A, an HDAC6 selective inhibitor, did not regulate blood pressure. Hypertensive stimuli enhanced the expression of HDAC6 in vivo and in vitro. We showed that the inhibition of HDAC6 prevents fibrosis and inflammation as determined by quantitative real-time PCR, western blot, and immunohistochemistry. Small interfering RNA (siRNA) against HDAC6 or Smad3 attenuated hypertensive stimuli-induced fibrosis and inflammation, whereas Smad2 siRNA failed to inhibit fibrosis. Interestingly, the combination of the HDAC6 inhibitor and Smad3 knockdown synergistically blocked transforming growth factor β (TGF-β) or ANG-induced fibrosis. We also demonstrated for the first time, to our knowledge, that acetylation of collagen type I can be regulated by HDAC6/p300 acetyltransferase. The chromatin immunoprecipitation assay revealed that the HDAC6 inhibitor suppressed TGF-β-induced acetylated histone H4 or phospho-Smad2/3 to Smad3 binding elements in the fibrosis-associated gene promoters including collagen type I. These results suggest that HDAC6 may be a valuable therapeutic target for the treatment of hypertension-induced kidney fibrosis and inflammation.     

Hirudin improves renal interstitial fibrosis by reducing renal tubule injury and inflammation in unilateral ureteral obstruction (UUO) mice

Renal interstitial fibrosis (RIF) often occurs in many chronic kidney diseases (CKD). Hirudin now is applied to treat fibrosis in some organs. In this study, we verified the treatment effects of hirudin on RIF in vivo and in vitro with the underlying mechanism. The RIF in vivo was the unilateral ureteral obstruction (UUO) model and RIF in vitro was the renal tubular epithelial cells induced by TGF-β. The renal pathological changes and renal fibrosis were observed by hematoxylin and eosin (H&E) staining and Masson staining. The α-SMA in renal tissues was detected by immunohistochemistry. The inflammatory factors were analyzed by the ELISA assay. The cell apoptosis was observed by TUNEL assay. The related proteins of fibrosis, epithelial-mesenchymal transition (EMT) and apoptosis were assessed by western blot analysis. The experimental data demonstrated that hirudin decreased fibrosis, EMT, inflammation and cell apoptosis in renal tissues of UUO rats and TGF-β-induced renal tubular epithelial cells. Furthermore, hirudin also reduced the expression of collgen-I, FN, α-SMA, N-cad, slug, E-cad, IL-1β, IL-6 and TNF-α in mice serum and TGF-β-induced renal tubular epithelial cells. The apoptosis related proteins (pro-caspase3, pro-caspase9, bcl2 and bax) expression was also down-regulated in renal tissues of UUO rats. In conclusion, hirudin depressed the fibrosis in renal tissues and renal tubular epithelial cells by inhibiting the inflammation, regulating the related proteins of fibrosis and ETM and decreasing the apoptosis of renal tubular epithelial cells. These findings may offer an effective treatment method for RIF.     

熊果酸调控TGF-β1/Smads信号通路对肾纤维化的干预作用

目的 观察熊果酸对单侧输尿管梗阻（unilateral ureteral obstruction,UUO）小鼠肾纤维化和肾小管上皮-间质转分化（epithelial-mesenchymal transdifferentiation,EMT）的影响,探索TGF-β1/Smads信号通路在其中的作用机制。方法 将50只ICR♂小鼠随机分为5组：假手术组,模型组,熊果酸低、中、高剂量组,每组10只。通过单侧输尿管结扎手术造小鼠肾纤维化模型,术后连续灌胃不同剂量的熊果酸7 d。给药结束后留取小鼠血清,测定小鼠血清生化指标（肌酐和尿素氮）;处死小鼠留取左侧梗阻肾组织标本,病理切片染色（HE和Masson染色）观察肾组织纤维化程度,采用免疫组化染色和Western blotting测定各组小鼠肾组织中EMT标志蛋白（E-cadherin和α-SMA）、TGF-β1、p-Smad2/3的表达。结果 与模型组相比,不同剂量熊果酸组小鼠血清肌酐及尿素氮水平呈现显著降低趋势;胶原沉积程度显著减轻,炎性细胞浸润程度显著改善;E-cadherin表达水平显著升高、α-SMA表达水平显著下降、TGF-β1和p-Smad2/3表达水平显著降低。结论 熊果酸减轻肾脏纤维化和EMT程度,其保护作用机制可能与熊果酸抑制TGF-β1/Smads信号通路有关。     

Hederagenin inhibits high glucose-induced fibrosis in human renal cells by suppression of NLRP3 inflammasome activation through reducing cathepsin B expression

Diabetic nephropathy is a major complication of diabetes mellitus and is related to dysfunction of renal cells. Hederagenin is a triterpenoid saponin from some Chinese herbs with anti-inflammatory and anti-diabetic activities. However, its role in diabetic nephropathy progression is still obscure. This study aimed to explore the effects of hederagenin on renal cell dysfunction in vitro. Human renal mesangial cells (HRMCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) were cultured under high glucose (HG) conditions to mimic diabetic nephropathy-like injury. Cell proliferation was evaluated by CCK-8. mRNA and protein levels were determined by qRT-PCR and western blotting, respectively. The secretion levels of fibrosis-related biomarkers were analyzed by ELISA. Results showed that hederagenin reduced HG-induced proliferation increase in HRMCs and HRPTEpiCs. Hederagenin attenuated HG-induced increase in mRNA and protein expression of NLRP3, ASC, and IL-1β. Hederagenin also suppressed HG-induced increase in mRNA and secretion levels of FN, Col. IV, PAI-1, and TGF-β1. NLRP3 inhibitor MCC950 attenuated HG-induced fibrosis of renal cells, and its activator nigericin reversed the suppressive effect of hederagenin on HG-induced fibrosis. Bioinformatics analysis predicted cathepsin B (CTSB) as a target of hederagenin to modulate NOD-like receptor (NLR) pathway. Hederagenin decreased CTSB level, and CTSB overexpression reversed the suppressive effect of hederagenin on HG-induced NLRP3 inflammasome activation and fibrosis in HRMCs and HRPTEpiCs. In conclusion, hederagenin attenuates HG-induced fibrosis of renal cells by inhibiting NLRP3 inflammasome activation via reducing CTSB expression, indicating a therapeutic potential of hederagenin in diabetic nephropathy.     

非布司他对高糖诱导的人肾小管上皮细胞增殖、凋亡及氧化应激的影响

目的 探讨不同浓度非布司他对高糖诱导的人肾小管上皮细胞(HK-2)增殖、凋亡及胞内信号通路的影响.方法 通过CCK8法和流式细胞术观察不同浓度非布司他对高糖诱导的HK-2增殖与凋亡的作用.采用蛋白免疫印迹法检测p22、p47磷酸化蛋白表达水平.结果 100 μmol/L非布司他具有最佳的逆转高糖抑制HK-2细胞增殖的作用,可改善高糖诱导的HK-2细胞凋亡,降低高糖介导的HK-2的p22、p47磷酸化蛋白表达水平(P＜0.01).结论 非布司他可通过抑制p22、p47磷酸化蛋白表达,改善高糖诱导的HK-2凋亡,以100 μmol/L浓度效果最优.     

Therapeutic activity of the histone deacetylase inhibitor SB939 on renal fibrosis

Fibrosis is the final pathological outcome of many chronic kidney diseases and is quite common. Thus, development of effective anti-fibrotic agents is urgently needed. Although histone deacetylases (HDACs) have been reported to be involved in renal fibrosis, current HDAC inhibitors are unsatisfactory anti-fibrosis drugs. Therefore, more potentially relevant anti-renal fibrosis HDAC inhibitors are needed. We initially found that non-cytotoxic concentrations of SB939 (pracinostat) had strong anti-fibrotic activity, drastically decreasing TGF-β1-induced alpha smooth muscle actin (α-SMA) expression in the NRK renal fibroblast cell line. Similar anti-fibrotic activity of SB939 on epithelial-to-mesenchymal transition (EMT) was confirmed using the HK-2 human renal proximal tubular epithelial cell line. SB939 inhibited Smad-independent TGF-β signaling involving the MAPK and PI3K/AKT pathways. To evaluate in vivo anti-fibrotic activity, we administered SB939 in a unilateral ureteric obstruction (UUO) model. SB939 treatment markedly inhibited the accumulation of α-SMA and tissue injury. Inflammatory and pro-fibrotic cytokines in the obstructed kidney were also significantly decreased by SB939 treatment. Our results suggest that SB939 might be a promising therapeutic drug for preventing renal fibrosis.     

Protective effect of BMP-7 against aristolochic acid-induced renal tubular epithelial cell injury

Aristolochic acid nephropathy (AAN) is regarded as a kind of rapidly progressive renal fibrosis caused by the ingestion of herbal remedies containing aristolochic acid (AA). Recent studies showed that bone morphogenetic protein-7 (BMP-7) exerts beneficial effects on acute and chronic kidney injuries induced by different pathological conditions. We examined whether BMP-7 protects human renal tubular epithelial cells (HK-2) against AA-induced injury in vitro. HK-2 cells were cultured with different concentrations of AA and BMP-7 for 48 h. Cell viability was determined by Cell Counting Kit-8 assay and lactate dehydrogenase (LDH) release. The apoptosis rate and the activity of caspase 3 protease were also examined. Epithelial-to-mesenchymal transition (EMT) was determined by cell morphology, E-cadherin and α-smooth muscle actin (α-SMA) protein expression, and TGF-β₁ and collagen III secretion. Additionally, the effect of anti-TGF-β1 antibody on AA-induced EMT was assessed. Our results indicated that BMP-7 significantly increased cell proliferation, decreased apoptosis rate and attenuated activation of caspase-3, resulting in the protection of HK-2 cells from AA-induced cytotoxicity. In addition, studies on EMT revealed that BMP-7 could inhibit AA-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner. It was partially through reducing the activation of a myofibroblast phenotype and production TGF-β1. Treatment with neutralizing anti-TGF-β1 antibody also blocked AA-induced EMT and collagen III secretion. Together, these observations strongly suggest that BMP-7 is a potent inhibitor of AA-induced renal tubular epithelial cell injury and might be a promising agent for aristolochic acid-induced kidney damage.     

阿米卡星对肾小管上皮细胞的损伤及热休克蛋白70表达的影响

目的:观察阿米卡星对肾小管上皮细胞的损伤及热休克蛋白70表达的影响。方法:常规培养人肾小管上皮细胞系HK-2细胞,给予阿米卡星(100μg·mL-1)损伤或MnCl2(2μg·mL-1)保护,于24,48,72,96 h时分光光度法测定细胞增殖,乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)、丙二醛(MDA)、N-乙酰-β氨基葡萄糖苷酶(NAG)含量,逆转录多聚酶链式反应(RT-PCR)法检测HSP70 mRNA表达,Western-blot检测HSP70蛋白表达。结果:阿米卡星损伤HK-2细胞后,细胞增殖显著减少,LDH释放量、NAG含量、MDA含量增加显著,SOD活力明显下降,HSP70 mRNA及其蛋白的表达显著增加(与对照组比,P<0.01);给予MnCl2干预后,HK-2细胞的细胞增殖明显有所恢复,LDH释放量、NAG含量、MDA含量有所下降,SOD活力有所增加,HSP70 mRNA及其蛋白的表达有所下降(与损伤组比,P<0.01)。结论:阿米卡星的肾脏毒性与HSP70的表达存在密切的联系。     

马兜铃酸体内毒性的产生及防治研究进展

马兜铃酸广泛存在于马兜铃科马兜铃属中草药中,具有。肾毒性和致癌致突变性。其在体内经一系列硝基还原酶代谢活化后与DNA共价结合形成加合物而诱发癌变,并通过诱导肾小管上皮细胞凋亡及间质纤维化而导致马兜铃酸肾病。综述马兜铃酸体内毒性产生的分子机制和防治研究进展,探讨相关药物的研发,为马兜铃酸肾病的治疗提供新思路。     

醛固酮对人近端肾小管上皮细胞结缔组织生长因子mRNA表达的影响

目的探讨醛固酮(ALD)在体外是否能够促进人近端肾小管上皮细胞株(HK2)结缔组织生长因子(CTGF)的表达.方法采用体外培养的HK2细胞,分别观察不同浓度(10-11、10-10、10-9、10-8、10-7 mol/L)ALD处理48 h,或用10-9 mol/L ALD处理不同时间(24 h、48 h、72 h)对HK2细胞CTGF表达的影响,应用RT-PCR法检测CTGFmRNA水平的表达.结果HK2细胞培养48 h后,空白对照组即有CTGF的基础表达,ALD不同浓度组CTGF mRNA的表达均有增加,与空白对照组相比较差异有统计学意义(P＜0.05或P＜0.01);以10-9 mol/L浓度的ALD作用于HK2细胞,随着作用时间的延长,CTGF mRNA的表达量明显增加,与对照组相比较差异有统计学意义(P＜0.05或P＜0.01).结论ALD能够促进HK2细胞CTGF mRNA水平的表达,并具有剂量依赖性和时间依赖性,ALD可能通过促进CTGF的分泌,发挥其促进肾间质纤维化的作用.