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• Research Paper Volume 14, Issue 8 pp 3633-3651

  Endothelin-1-mediated miR-let-7g-5p triggers interlukin-6 and TNF-α to cause myopathy and chronic adipose inflammation in elderly patients with diabetes mellitus

  Relevance score: 8.903031

  Chung-Huang Tsai, Pei-Ju Huang, IT Lee, Chien-Min Chen, Min Huan Wu

  Keywords: diabetes, sarcopenia, miRNA, endothelin-1 (ET-1), TNF-α, interleukin-6, hyperglycemia

  Published in Aging on April 25, 2022

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  Background: Diabetes and sarcopenia are verified as mutual relationships, which seriously affect the quality of life of the elderly. Endothelin-1 is well investigated, is elevated in patients with diabetes, and is related to muscle cellular senescence and fibrosis. However, the mechanism of ET-1 between diabetes and myopathy is still unclear. The aim of this study was to evaluate the prevalence of sarcopenia in the elderly with diabetes and to clarify its relationship with ET-1 molecular biological mechanism, progress as well as changes in muscle and fat.

  Methods: We recruited 157 type 2 diabetes patients over 55 years old and investigated the prevalence of sarcopenia in diabetes patients and examined the association of ET-1 alterations with HbA1c, creatinine, or AMS/ht2. Next, sought to determine how ET-1 regulates inflammation in muscle cells by western blot and qPCR assay. Using XF Seahorse Technology, we directly quantified mitochondrial bioenergetics in 3T3-L1 cells.

  Results: ET-1 was positively correlated with HbA1c, creatinine levels, and duration of disease, and negatively correlated with AMS/ht2. We found that ET-1 dose-dependently induces tumor necrosis factor-α (TNF-α) and interleukin (IL)-6β expression through the PI3K/AKT, and NF-κB signaling pathways in C2C12 cells. Also identified that TNF-α, IL-6β, and visfatin releases were found in co-cultured with conditioned medium of ET-1/C2C12 in 3T3-L1 cells. ET-1 also reduces the energy metabolism of fat and induces micro-environment inflammation which causes myopathy. ET-1 also suppresses miR-let-7g-5p expression in myocytes and adipocytes.

  Conclusion: We describe a new mechanism of ET-1 triggering chronic inflammation in patients with hyperglycemia.

• Priority Research Paper Volume 12, Issue 12 pp 11200-11223

  Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia

  Relevance score: 11.76063

  Elena Alcalde-Estévez, Ana Asenjo-Bueno, Patricia Sosa, Gemma Olmos, Patricia Plaza, María Ángeles Caballero-Mora, Diego Rodríguez-Puyol, María Piedad Ruíz-Torres, Susana López-Ongil

  Keywords: endothelin-1, fibrosis, senescence, aging, sarcopenia

  Published in Aging on June 22, 2020

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  Endothelial dysfunction, with increased endothelin-1 (ET-1) synthesis, and sarcopenia, characterized by the loss of muscular mass and strength, are two aging–related conditions. However, a relationship between them has not been already established. The aim of this study was to determine whether ET-1 induces senescence and fibrosis in cultured murine myoblasts, which could be involved in the development of sarcopenia related to aging. For this purpose, myoblasts were incubated with ET-1 to assess cellular senescence, analyzed by senescence associated β-galactosidase activity and p16 expression; and fibrosis, assessed by fibronectin expression. ET-1 induced myoblast senescence and fibrosis through ET_A receptor. The use of antioxidants and several antagonists revealed that ET-1 effect on senescence and fibrosis depended on ROS production and activation of PI₃K-AKT-GSK pathway. To stress the in vivo relevance of these results, circulating ET-1, muscular strength, muscular fibrosis and p16 expression were measured in male C57Bl6 mice from 5-18-24-months-old. Old mice shown high levels of ET-1 correlated with muscular fibrosis, muscular p16 expression and loss of muscle strength. In conclusion, ET-1 promotes fibrosis and senescence in cultured myoblasts, similar results were found in old mice, suggesting a potential role for ET-1 in the development of sarcopenia related to aging.

  

  Endothelin-1 induces senescence in mouse myoblasts (C₂C₁₂) through ET_A receptor. Cells were grown on coverslips (A, C) and incubated with 1 nM ET-1 at different times (A, B), or incubated with 10 μM Bosentan (Bos), 100 nM BQ-123 (BQ123) or 100 nM BQ-788 (BQ788) added 30 min before ET-1 (1 nM), and then incubated for 72h (C) or 48h (D). Then, senescence was tested measuring SA-ß-GAL activity (panel A, C) and protein content from p16 (panel B, D). Representative microphotographs are shown on the left with 40x magnification and the densitometric analysis is shown on the right panel A, C. Scale bar, 50 μm. A representative Western blot of p16 is shown next to the densitometric analysis on the panel B, D. Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells (C or time 0), and **p<0.05 vs ET alone.

  
  
  

  

  Endothelin-1 increases FN expression in mouse myoblasts (C₂C₁₂) through ET_A receptor. Cells were incubated with 1 nM ET-1 at different times. Then, FN mRNA expression was assessed by RT-qPCR (panel A) and FN protein content by Western blot (panel B). To study the ET receptor implicated, cells were incubated with 100 nM BQ-123 (BQ123) or 100 nM BQ-788 (BQ788) added 30 min before ET-1 (1 nM), and then incubated for 24h. Then, FN protein content (panel C) as well as intracellular FN (in red) and extracellular FN (in green) expression (panel D) were studied by Western and immunofluorescence, respectively. In the experiments of the analysis of protein content, a representative Western blot is shown at the top with the densitometric analysis below (panel B,C). Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells (C), and **p<0.05 vs ET alone.

  
  
  

  

  Fibronectin induces senescence in mouse myoblasts (C₂C₁₂) through integrin/ILK activation. Cells were grown on coverslips (panels A, C) to test senescence measuring SA-ß-GAL activity and p16 protein content by Western blot (panels B, D). (A) Cells were incubated with 2.5 μg/ml FN at different times to assess SA-ß-GAL activity by confocal microscopy. (B) Cells were incubated at different doses of FN for 48h to analyze p16 protein content. (C) Cells were incubated with 2.5 μg/ml FN or 1 nM ET-1 in the presence or not of 50 μM Tirofiban (TF) for 48h to assess senescence by SA-ß-GAL activity (panel C) or by p16 protein content (panel D). Representative microphotographs are shown at the top with 40x magnification and the densitometric analysis is shown below. Scale bar, 50 μm. A representative Western blot of p16 is shown at the top and the densitometric analysis is shown below. In panels C and D closed bars represent data of FN treatment and stripped bars represent data of ET-1 treatment; lane 1: control cells; lane 2: FN or ET alone; lane 3: FN or ET plus TF; lane 4: TF alone. Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells (C or time 0), and **p<0.05 vs ET or FN alone. (E) Cells were transfected with siRNA against ILK or scrambled as siControl to assess senescence by p16 protein content upon 2.5 μg/mL FN treatment for 48h. A representative Western blot of ILK and p16 are shown on the left panel and the densitometric analysis is shown on the right. Values are the mean±SEM of 3 independent experiments, *p<0.05 vs. control cells (C from siControl).

  
  
  

  

  Role of ROS in endothelin-dependent fibrosis and cellular senescence. (A, B) Cells were incubated with 1 nM ET-1 at different times, some cells were incubated in the presence of 100 μM N-acetylcysteine (NAC) or 100 nM BQ123, and then 1 nM ET-1 was added and incubated for 16h (B). CellROX probe was added during the last 30 min of incubation. After being washed twice, in vivo cells were visualized by microscopy confocal to test ROS production in red. Representative microphotographs are shown at the top with 40x magnification, scale bar, 50 μm. The densitometric analyses are shown below. (C–E) Cells were incubated with 1 nM ET-1 in the presence of 100 μM NAC to assay FN protein expression by Western blot (C). Cellular senescence was assessed by measuring p16 protein content for 48h by Western blot (panel D) and SA-ß-GAL activity for 72h (panel E). A representative Western is shown on the top with the densitometric analysis below (panels C, D). Representative microphotographs are shown on the left panel E with 40x magnification and the densitometric analysis is shown on the right. Scale bar, 50 μm. Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells (C or time 0), and **p<0.05 vs ET alone.

  
  
  

  

  Endothelin-1 induces activation of PI₃K-AKT-GSK pathway in mouse myoblasts (C₂C₁₂) through ET_A receptor and ROS production. (A, B) Cells were incubated with 1 nM ET-1 at different times. Activation of PI₃K-AKT-GSK pathway was analyzed by Western blot measuring phosphorylation of AKT (P-AKT, panel A) and phosphorylation of GSK (P-GSK, panel B). (C, D) Cells were incubated in the presence of different antagonists to block PI₃K-AKT-GSK pathway (Wortmannin: 10 μM WTN; AKT inhibitor: 30 μM I-AKT), to block ET receptors (ET_A receptor antagonist: 100 nM BQ123; ET_B receptor antagonist: 100 nM BQ788) (C), and to block ROS production (antioxidant N-acetylcysteine: 100 μM NAC) (D). All of them were added at least 30 min before 1 nM ET-1 for 24h, to assay P-AKT (closed bars) or P-GSK (open bars). Representative Western blots are shown at the top of each panel. The densitometric analysis is shown below of each panel. Values are the mean±SEM of 5 independent experiments, *p<0.05 vs. control cells (C), and **p<0.05 vs ET alone.

  
  
  

  

  Role of PI₃K-AKT-GSK pathway in endothelin-dependent cellular fibrosis and cellular senescence. Cells were incubated in the presence of different antagonists to block PI₃K-AKT-GSK pathway (Wortmannin: 10 μM WTN; LY-294,002 hydrochloride: 50 μM LY; AKT inhibitor: 30 μM I-AKT). All of them were added at least 30 min before adding 1 nM ET-1 for 24h to assay FN expression by Western blot (panel A) or by Immunofluorescence (panel B), and to assay senescence by measuring p16 protein content for 48h by Western blot (panel C) and SA-ß-GAL activity for 72h (panel D). Representative Western blots are shown at the top of panels (A, C). Representative microphotographs are shown on the top of panel B, D with 40x magnification, scale bar, 50 μm. The densitometric analysis is shown below of each panel. Values are the mean±SEM of 6 independent experiments, *p<0.05 vs. control cells (C), and **p<0.05 vs ET alone.

  
  
  

  

  Aging mice present high circulating ET-1 levels, loss of muscle strength, fibrosis and senescence in gastrocnemius and tibialis anterior muscles. Animals were kept on a 12:12h light-dark cycle, at 24°C, and food and water were available ad libitum. Male C57Bl6 mice from 5, 18, and 24 month-old were used, 10 animals per group. (A) Serum ET-1 levels was measured by ELISA. (B) Muscle force was registered using the 4 limb grip test and data were corrected by body weight of each mouse. Values are the mean±SEM of 10 mice, *p<0.05 vs. 5-month-old; **p<0.05 vs 18 month-old. Mice of 5-month-old (Young-closed bars) and 24-month-old (Old-stripped bars) were used to measure Sirius red staining in sections of the gastrocnemius (GNM) and the tibialis anterior muscles to visualize fibrosis (C), to analyze FN protein expression by Western blot (D) and to assess senescence by measuring p16 protein content by Western blot (E) in those muscles of the same mice. (C) Sirius red staining (20x) is shown with the mean ± standard error below pictures. (D, E) A representative Western blot was shown above with the densitometric analysis below. Values are the mean±SEM of 20 mice, *p<0.05 vs. young mice. (F) Graphs of correlations based on data from young (5-month-old) and old (24-month-old) mice were shown: ET-1 levels and grip force (Spearman r= -0.5669, p= 0.0091), ET-1 levels and Sirius red in GNM (Spearman r= 0.7787, p= 0.0002), ET-1 levels and p16 expression in GNM (Spearman r= 0.6548, p= 0.0043), grip force and Sirius red in GNM (Spearman r= -0.7028, p= 0.0011) and grip force and p16 expression in GNM (Spearman r= -0.5789, p= 0.0118).

  
  
  

  

  Proposal mechanism of action of ET-1 on muscular fibrosis and senescence. Myoblast cells present both type of ET-1 receptors, ET_A and ET_B, which are inhibited by specific antagonists such as BQ123 and BQ788, respectively. The binding of ET-1 to ET_A receptor induces fibrosis and senescence through ROS production by activation of PI₃K-AKT-GSK pathway. The inflammation induced by ET-1 could be also implied. Several antagonists were used to inhibit both, ROS production with the antioxidant N-acetylcysteine (NAC), and the PI₃K-AKT-GSK pathway with AKT inhibitor, LY-294,002 and Wortmannin, to check the mechanism. Fibronectin could induce senescence through integrin receptor activation by joining to RGD sequence, and then trigger some downstream pathways through ILK activation. Tirofiban blocks the joining of FN to RGD sequence. Findings in aged mice are showed below which are similar to those found in myoblast cells induced by ET-1, suggesting that the appearance of fibrosis and senescence could be involved in the genesis of sarcopenia related to aging. Note: all antagonists or inhibitors are represented in an orange box and unexplored mechanisms with an arrow with dashed pink line.

  
  
  

• Research Paper Volume 11, Issue 6 pp 1804-1820

  Endothelial cells secreted endothelin-1 augments diabetic nephropathy via inducing extracellular matrix accumulation of mesangial cells in ETBR^-/- mice

  Relevance score: 7.992157

  Hong-hong Zou, Li Wang, Xiao-xu Zheng, Gao-si Xu, Yunfeng Shen

  Keywords: endothelin B receptor-deficient mice, endothelin-1, diabetic nephropathy, mesangial cells, endothelial cells, NF-kapapB

  Published in Aging on March 29, 2019

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  Endothelin B receptor (ETBR) deficiency may contribute to the progression of diabetic nephropathy (DN) in a streptozotocin (STZ) model, but the underlying mechanism is not fully revealed. In this study, STZ-diabetic ETBR^-/- mice was characterized by increased serum creatinine and urinary albumin, enhanced glomerulosclerosis, and upregulated ET-1 expression compared with STZ-diabetic WT mice. In vitro, HG conditioned media (CM) of ETBR^-/- GENs promoted mesangial cell proliferation and upregulated ECM-related proteins, and ET-1 knockout in GENs or inhibition of ET-1/ETAR in mesangial cell suppressed mesangial cell proliferation and collagen IV formation. In addition, ET-1 was over-expressed in ETBR^-/- GENs and was regulated by NF-kapapB pathway. ET-1/ETBR suppressed NF-kappaB to modulate ET-1 in GENs. Furthermore, ET-1/ETAR promoted RhoA/ROCK pathway in mesangial cells, and accelerated mesangial cell proliferation and ECM accumulation. Finally, in vivo experiments proved inhibition of NF-kappaB pathway ameliorated DN in ETBR^-/- mice. These results suggest that in HG-exposed ETBR^-/- GENs, suppression of ET-1 binding to ETBR activated NF-kappaB pathway, thus to secrete large amount of ET-1. Due to the communication between GENs and mesangial cells in diabetes, ET-1 binding to ETAR in mesangial cell promoted RhoA/ROCK pathway, thus to accelerate mesangial cell proliferation and ECM accumulation.

  

  ETBR expression level was up-regulated in kidney tissue of DN. Ten weeks after the establishment of diabetic mice, the mice were sacrificed and kidneys were collected for further use. (A) mRNA levels of ETBR in kidney tissue from patients with DN (GEO database, GSE30528 and GSE111154) and healthy control. EDNRB, gene of ETBR. (B). HE staining of kidney tissue from STZ-diabetic mice and control mice was conducted to confirm the occurrence of diabetic nephropathy. (C). Protein level of ETBR in kidney tissue from STZ-diabetic mice at six and tenth weeks after the initial intraperitoneal injection of STZ. N=3.

  
  
  

  

  Severer diabetic nephropathy in ETBR^-/- mice. (A) Body weight and kidney weight were detected in control mice (WT), STZ-diabetic mice, ETBR^-/- mice and STZ-diabetic ETBR^-/- mice. **p<0.01, compared with control. N=5. (B) Serum glucose level, serum creatinine level and urinary albumin level were measure in control mice (WT), STZ-diabetic mice, ETBR^-/- mice and STZ-diabetic ETBR^-/- mice. **p<0.01, compared with control or STZ-diabetic WT mice. (C) Periodic acid-Schiff (PAS) staining of kidney tissues from control mice, STZ-diabetic WT mice, ETBR^-/- mice, and STZ-diabetic ETBR^-/- mice. **p<0.01, compared with control, STZ-diabetic WT, or ETBR^-/- mice. 1000×magnification. (D) MASSON staining of of kidney tissues from control mice, STZ-diabetic WT mice, ETBR^-/- mice, and STZ-diabetic ETBR^-/- mice. 1000×magnification. (E) Protein levels of ECM-related protein CTGF and p-p65 in control mice, STZ-diabetic WT mice, ETBR^-/- mice, and STZ-diabetic ETBR^-/- mice. *p<0.05, compared with WT, ETBR^-/-, or STZ mice. **p<0.01, compared with WT, ETBR^-/-, or STZ mice. N=3. (F-G) Serum ET-1/kidney ET-1 expressions and transcriptional level of EDN1 from kidney were detected in control mice, STZ-diabetic WT mice, ETBR^-/- mice, and STZ-diabetic ETBR^-/- mice. *p<0.05, **p<0.01, compared with WT, ETBR^-/-, or STZ mice. Bars depict the mean ± SD. N=5.

  
  
  

  

  HG conditioned media (CM) of ETBR^-/- GENs promoted mesangial cell proliferation and ECM formation. (A) After 24 h of HG (25mM) treatment, ET-1 level in primary GENs of ETBR^-/- mice and WT mice was detected by ELISA. CM was collected for the culture of SV40 MES13 cells. **p<0.01 compared with control or HG WT. N=3. (B-C) WT or ETBR^-/- CM GEN was used to cultivate SV40 MES13 cells for 24 h. SV40 MES13 cells in control group was cultured in HG serum-free medium. The proliferation of SV40 MES13 cells was detected in control, WT normal CM, WT HG CM, ETBR^-/- normal CM and ETBR^-/- HG CM groups by MTT assay. RhoA level on SV40 MES13 cells membrane and Collagen IV secretionwere detected in control, WT normal CM, WT HG CM, ETBR^-/- normal CM and ETBR^-/- HG CM groups by western blot. GTP-RhoA level (the activity of Rho) was detected using Rhotekin RBD-agrose by Rho-pull down assay. **p<0.01 compared with control or normal WT CM or HG WT CM or normal ETBR^-/- CM. N=3. (D-E) GENs were transfected with 50 nM si-EDN1 for 18 h, and HG medium was used to culture GENs for 24 h, then the CM was collected for the culture of mesangial cells. 25 μM ABT-627 (blocking agent of ET-1/ETAR pathway) or 25μM A192621 (blocking agent of ET-1/ETBR pathway) was added to the medium for the culture of mesangial cells. The proliferation of mesangial cells was detected by MTT assay, and mem RhoA, cyt RhoA, collagen IV protein levels were detected by western blot. **p<0.01 compared with si-ET-1 or si-ET-1+ABT-627 or si-ET-1+A192621. Bars depict the mean ± SD. N=3.

  
  
  

  

  ET-1 modulated mesangial cell proliferation and ECM through RhoA/ROCK pathway. (A) Under HG serum-free condition, mesangial cell proliferation was detected after the treatment of ET-1 (0.5 nM, 1 nM, 2 nM) for 24 h. **p<0.01, compared with 0nM. (B) Under HG serum-free condition, ECM-related proteins (Collagen IV, Fibronectin and CTGF) and RhoA on mesangial cell membrane were detected after the treatment of ET-1 (0.5 nM, 1 nM, 2 nM) for 24 h. ET-1 (1nM, 2500pg/ml) was used for the following experiments. **p<0.01, compared with 0nM. (C-F) Under HG serum-free condition, mesangial cells were treated with ET-1 (1 nM), or ET-1 (1 nM)+Y-27632 (30 μM, RhoA/ROCK inhibitor) for 24 h. The proliferation, cell apoptosis, cell cycle and ECM-related proteins were detected in ET-1 and ET-1+Y-27632 groups using MTT, flow cytometry and western blot assay. **p<0.01, compared with ET-1. Bars depict the mean ± SD. N=3.

  
  
  

  

  ET-1 promoted RhoA/ROCK pathway in mesangial cells through ETAR. Mesangial cells were treated with 1 nM ET-1, ABT-627(25μM, inhibitor of ETAR pathway) or A192621 (25μM, inhibitor of ETBR pathway) for 24 h. (A-C) Mesangial cell proliferation, RhoA/ROCK and ECM-related proteins, and cell apoptosis were detected in control, ABT-627, A192621, ET-1, ET-1+ ABT-627, ET-1+A192621 groups. **p<0.01 compared with ET-1 group. (D) Expression quantity from gene transcription level of EDNRA and EDNRB in mouse mesangial cells SV40 MSE 13 and mouse primary mesangial cells. **p<0.01 compared with ETAR. (E) ETAR and ETBR expressions on mesangial cell membrane were measured in ET-1 treated SV40 MSE 13 cells and primary mesangial cells. Bars depict the mean ± SD. N=3.

  
  
  

  

  ET-1 was overexpressed in ETBR knockout GENs and was regulated by NF-kapapB pathway. GENs was cultured in HG medium, and the supernatant and GENs were collected at 6 h, 12 h, 16 h, 20 h, 24 h after cultivation. (A) Under the HG condition, ET-1 expression (in the supernatant)in WT and ETBR knockout GENs groups was detected at 6 h, 12 h, 16 h, 20 h, 24 hGENGEN . **p<0.01, compared with WT. There was significant difference in ET-1 production rate between ETBR knockout and WT GENs since 16 h, and the difference increased with time. ET-1 production rate (n)= ET-1(n)/ET-1(n-4). N represented the time point of sample collection. ET-1 represented the production of ET-1 in mesangial cells. *p<0.05 compared with WT. **p<0.01, compared with WT. (B) mRNA expressions of EDN1 in WT GENs and ETBR^-/- GENs groups were detected at 6 h, 12 h, 16 h, 20 h, 24 h. **p<0.01, compared with 6h. mRNA expression of ET-1 was increased in ETBR^-/- GENs within 24 h. **p<0.01, compared with 6h. (C) Protein levels of p-p65 in WT GENs and ETBR^-/- GENs groups were measured at 6 h, 12 h, 16 h, 20 h, 24 h. Bars depict the mean ± SD. N=3.

  
  
  

  

  p65 promoted the transcription of EDN1, and ET-1/ETBR modulated ET-1 through NF-kappaB. (A) WT GENs or ETBR^-/- GENs were treated with HG or 10 μM Bay or HG+10 μM Bay for 24 h. mRNA expressions of EDN1 in WT or ETBR^-/- GENs were detected in control, Bay, HG, HG+Bay groups. *p<0.05, compared with control, Bay, or HG group. **p<0.01, compared with control, Bay, or HG group. (B) Extracellular secretion of ET-1 was detected in HG, HG+Bay and HG+si-p65 groups. **p<0.01, compared with HG or HG+Bay group. (C) CHO cells were co-transfected with p65-expressing vector and pGL3 vector carrying different promoters of EDN1, and empty pGL3 basic was used as control. After 48 h of transfection, EDN1 promoter activity was detected by dual-Luciferase Reporter Assay System. (D) WT or ETBR^-/- GENs were treated with HG or HG+10 μM Bay for 24 h. ChIP assay showed p65 could bind with the segments at this region in HG-treated GENs. Under HG condition, inhibition of NF-kappaB significantly decreased the binding efficiency of this region. **p<0.01, compared with HG group. (E) After GENs treated with HG for 6 h, 1 nM ET-1 was added into GENs and cultured for 18 h. So, GENs was treated with HG for 24 h in total. ET-1 secretion in GENs was detected in control and si-EDN1 group. p-p65 expression was detected in HG group, HG+ET-1 group, HG+si-EDN1 group, and HG+ET-1+si-EDN1 group. **p<0.01, compared with control group. (F) After WT GENs treated with HG for 24 h, mRNA level of ETAR and ETBR were detected in HG treated WT GENs. ETAR and ETBR protein levels were detected in the membrane of WT GENs. **p<0.01, compared with ETBR. (G-I) WT GENs were treated with HG, ABT-627 (25 μM), or A192621 (25 μM) for 24 h. Or WT GENs were transfected with 100 nM si-ETBR, then treated with HG for 24 h. p-p65 and p65 protein levels, ET-1 secretion level and EDN1 mRNA expression were detected in control, ABT-627, A192621 and si-ETBR groups.**p<0.01, compared with control or A192621 group. Bars depict the mean ± SD. N=3.

  
  
  

  

  Inhibition of NF-kappaB pathway ameliorated DN in ETBR-/- mice in vivo. (A-C) C57BL/6 mice, ETBR^-/- mice were intraperitoneally injected with 50 mg/kg STZ every day for five days to establish STZ-diabetic mice model. Bay (1 mg/kg, Bay 11-7082) was dissolved in normal saline, and injected intraperitoneally twice a week between seventh and tenth weeks after STZ treatment. Y27632 (5 mg/kg, Rhoa/Rock inhibitor) was injected intraperitoneally twice a week between seventh and tenth weeks after STZ treatment. Serum creatinine, urinary albumin, serum ET-1 and kidney ET-1 were detected in WT, WT+Bay, WT+Y27632, ETBR^-/-, ETBR^-/-+Bay and ETBR^-/-+Y27632 mice groups. **p<0.01 compared with WT or ETBR^-/-. Bars depict the mean ± SD. N=6. (D) PAS staining showed that enlargement of glomeruli was observed in STZ-diabetic mice, and glomerulosclerosis was relieved in Bay and Y27632 treated WT or ETBR^-/- mice. **p<0.01 compared with STZ WT or STZ ETBR^-/-. 1000×magnification. (E) MASSON staining showed that collagen was produced in glomeruli in STZ-diabetic mice, and the formation of collagen was relieved in Bay and Y27632 treated WT or ETBR^-/- mice. 1000×magnification. (F) Cascade diagram of signaling pathways.