P0972INHIBITION OF LYSINE63 UBIQUITINATION PREVENTS THE PROGRESSION OF RENAL FIBROSIS IN DIABETIC NEPHROPATHY IN VITRO AND IN VIVO

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Francesca Conserva ◽  
Paola Pontrelli ◽  
Rossella Menghini ◽  
Michele Rossini ◽  
Alessandra Stasi ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Drug Combination ◽  
Renal Fibrosis ◽  
Renal Damage ◽  
Mesenchymal Transition ◽  
Ubiquitinated Proteins ◽  
Collagen Iii ◽  
Sirius Red

Abstract Background and Aims Diabetic Nephropathy (DN) is the primary cause of end stage renal disease (ESRD). Our group demonstrated that in DN an accumulation of lysine63 (K63)-ubiquitinated proteins at tubular level is involved in the progression of renal damage, in particular renal fibrosis. Current treatments do not provide complete renoprotection and targeted therapies that prevent fibrosis or delay its progression are still lacking. Aim of the present study was to evaluate the renoprotective effect of specific drug and their combinations, including an inhibitor of K63 ubiquitination (K63Ub) and/or an anti-hypertensive agent, in vitro and in vivo in a murine model of DN. Method Renal Proximal Tubule Epithelial Cells (HK2) were pre-incubated with a specific inhibitor of K63Ub and/or with the ACE-inhibitor Ramipril. Accumulation of K63 ubiquitinated proteins along with α-sma expression, indicator of epithelial-to-mesenchymal transition (EMT), were analyzed through immunofluorescence and western blotting. The same drug combination was also tested in streptozotocin (STZ)-treated DBA/2J mice, a model of human DN. In mice, K63Ub was evaluated by IHC, while renal fibrosis was evaluated by Sirius red and Collagen III expression. Urinary albuminuria was measured by ELISA. Results We observed that the association of the specific K63Ub inhibitor with Ramipril was able to block hyperglycemia-induced EMT in HK2 cells by significantly reducing α-sma expression, when compared to single drugs alone (p<0.05).To demonstrate the efficacy of these drug combinations in reducing the progression of renal damage in DN we firstly confirmed the increased accumulation of K63 Ub proteins in DBA/2J STZ-treated mice (p=0.01). Interestingly, increased K63Ub in diabetic mice was also associated to increased tubular-interstitial fibrosis (p<0.05). Treatment of STZ-mice with the specific K63Ub inhibitor was able to reduce both K63Ub proteins accumulation and renal fibrosis, evaluated on kidney samples by IHC against Collagen III (p≤0.05) and by Sirius Red staining (p≤0.05) when compared to both untreated mice and mice treated with ramipril. Importantly, treatment with the K63Ub inhibitor alone did not reduce albuminuria (STZ-mice: 561.29±390.56; STZ+K63Ubinhibitor: 724.25±690.89; p=n.s.), while the drug combination including the specific K63Ub inhibitor and Ramipril, significantly reduced both K63Ub-related fibrosis and albuminuria (p=0.01), demonstrating an addictive and synergic effect of these molecules when used in combination. Conclusion Our data demonstrated and confirmed the importance of K63Ub in the progression of renal fibrosis in vitro and in vivo. We proposed and patented a novel combination of drugs that ameliorates both fibrosis and proteinuria in DN. Novel treatment regimens could represent an important goal for reducing the incidence of ESRD related to diabetes complication.

scholarly journals Inhibition of Lysine 63 Ubiquitination Prevents the Progression of Renal Fibrosis in Diabetic DBA/2J Mice

2021 ◽  
Vol 22 (10) ◽  
pp. 5194
Author(s):  
Paola Pontrelli ◽  
Francesca Conserva ◽  
Rossella Menghini ◽  
Michele Rossini ◽  
Alessandra Stasi ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Epithelial To Mesenchymal Transition ◽  
Selective Inhibition ◽  
Collagen I ◽  
Protein Accumulation ◽  
Mesenchymal Transition ◽  
Collagen Iii ◽  
Proximal Tubular Epithelial Cells

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

scholarly journals miR-98-5p Alleviated Epithelial-to-Mesenchymal Transition and Renal Fibrosis via Targeting Hmga2 in Diabetic Nephropathy

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Yingchun Zhu ◽  
Jiang Xu ◽  
Wenxing Liang ◽  
Ji Li ◽  
Linhong Feng ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Renal Fibrosis ◽  
Epithelial To Mesenchymal Transition ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
High Dose ◽  
Mrna Levels ◽  
Mesenchymal Transition

Recently, microRNAs have been recognized as crucial regulators of diabetic nephropathy (DN) development. Epithelial-to-mesenchymal transition (EMT) can play a significant role in tubulointerstitial fibrosis, and it is a hallmark of diabetic nephropathy progression. Nevertheless, the function of miR-98-5p in the modulation of EMT and renal fibrosis during DN remains barely investigated. Hence, identifying the mechanisms of miR-98-5p in regulating EMT and fibrosis is of huge significance. In our present research, decreased miR-98-5p was demonstrated in db/db mice and mice mesangial cells treated with the high dose of glucose. Meanwhile, activated EMT and increased fibrosis was accompanied with the decrease of miR-98-5p in vitro and in vivo. Additionally, to further find out the roles of miR-98-5p in DN development, overexpression of miR-98-5p was applied. Firstly, in vivo investigation exhibited that elevation of miR-98-5p restrained proteinuria, serum creatinine, BUN, the EMT process, and fibrosis. Furthermore, high glucose was able to promote mice mesangial cell proliferation, EMT process, and induced renal fibrosis, which could be prevented by overexpression of miR-98-5p. Moreover, high mobility group A (HMGA2) can exhibit an important role in diverse biological processes. Here, HMGA2 was investigated as a target of miR-98-5p currently. Luciferase reporter assay was conducted and the correlation of miR-98-5p and HMGA2 was validated. Moreover, it was displayed that HMGA2 was remarkably elevated in db/db mice and mice mesangial cells. Furthermore, miR-98-5p strongly depressed HMGA2 protein and mRNA levels in mice mesangial cells. Overall, these revealed miR-98-5p could suppress the EMT process and renal fibrosis through targeting HMGA2 in DN.

scholarly journals MiR-27b-3p inhibits the progression of renal fibrosis via suppressing STAT1

Human Cell ◽  
2021 ◽  
Vol 34 (2) ◽  
pp. 383-393
Author(s):  
Lin Bai ◽  
Yongtao Lin ◽  
Juan Xie ◽  
Yiyuan Zhang ◽  
Hongwu Wang ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Transforming Growth Factor ◽  
Luciferase Assay ◽  
Mesenchymal Transition ◽  
Collagen Iii ◽  
Tgf Β1 ◽  
Active Caspase

AbstractRenal fibrosis is a pathologic change in chronic kidney disease (CKD). MicroRNAs (miRNAs) have been shown to play an important role in the development of renal fibrosis. However, the biological role of miR-27b-3p in renal fibrosis remains unclear. Thus, this study aimed to investigate the role of miR-27b-3p in the progression of renal fibrosis. In this study, HK-2 cells were stimulated with transforming growth factor (TGF)-β1 for mimicking fibrosis progression in vitro. The unilateral ureteric obstruction (UUO)-induced mice renal fibrosis in vivo was established as well. The results indicated that the overexpression of miR-27b-3p significantly inhibited epithelial-to-mesenchymal transition (EMT) in TGF-β1-stimulated HK-2 cells, as shown by the decreased expressions of α-SMA, collagen III, Fibronectin and Vimentin. In addition, overexpression of miR-27b-3p markedly decreased TGF-β1-induced apoptosis in HK-2 cells, as evidenced by the decreased levels of Fas, active caspase 8 and active caspase 3. Meanwhile, dual-luciferase assay showed that miR-27b-3p downregulated signal transducers and activators of transcription 1 (STAT1) expression through direct binding with the 3′-UTR of STAT1. Furthermore, overexpression of miR-27b-3p attenuated UUO-induced renal fibrosis via downregulation of STAT1, α-SMA and collagen III. In conclusion, miR-27b-3p overexpression could alleviate renal fibrosis via suppressing STAT1 in vivo and in vitro. Therefore, miR-27b-3p might be a promising therapeutic target for the treatment of renal fibrosis.

scholarly journals Effect of Huai Qi Huang on Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells through miR-200a

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jinyun Pu ◽  
Yu Zhang ◽  
Jianhua Zhou
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanism ◽  
Renal Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Mesenchymal Transition ◽  
Renal Tubular

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is a vital mechanism of renal fibrosis. Mounting evidence suggests that miR-200a expression decreases in tubular epithelial cells in unilateral ureteral obstruction (UUO) rats. Moreover, it has been demonstrated that Huai Qi Huang (HQH) can ameliorate tubulointerstitial damage in adriamycin nephrosis and delay kidney dysfunction in primary glomerular disease. However, the effect of HQH on EMT of tubular epithelial cells in UUO rats and its molecular mechanism is unclear. In order to explore the effect of HQH on EMT and its molecular mechanism in renal fibrosis,in vitroandin vivoexperiments were performed in our study. Our results showed that HQH increased miR-200a expression in UUO rats and in TGF-β1 stimulated NRK-52E cells. Meanwhile, HQH decreased ZEB1 and ZEB2 (the transcriptional repressors of E-cadherin),α-SMA expression in renal tubular epithelial cellsin vitroandin vivo. Furthermore, we found that HQH protected kidney from fibrosis in UUO rats. The results demonstrated that HQH regulated miR-200a/ZEBs pathway and inhibited EMT process, which may be a mechanism of protecting effect on tubular cells in renal fibrosis.

scholarly journals MicroRNA-27a targets Sfrp1 to induce renal fibrosis in diabetic nephropathy by activating Wnt/β-Catenin signalling

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
MingJun Shi ◽  
PingPing Tian ◽  
ZhongQiang Liu ◽  
Fan Zhang ◽  
YingYing Zhang ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Renal Fibrosis ◽  
Negative Control ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Expression Levels ◽  
Stage Renal Disease

Abstract Diabetic nephropathy (DN) commonly causes end-stage renal disease (ESRD). Increasing evidence indicates that abnormal miRNA expression is tightly associated with chronic kidney disease (CKD). This work aimed to investigate whether miR-27a can promote the occurrence of renal fibrosis in DN by suppressing the expression of secreted frizzled-related protein 1 (Sfrp1) to activate Wnt/β-catenin signalling. Therefore, we assessed the expression levels of miR-27a, Sfrp1, Wnt signalling components, and extracellular matrix (ECM)-related molecules in vitro and in vivo. Sfrp1 was significantly down-regulated in a high-glucose environment, while miR-27a levels were markedly increased. A luciferase reporter assay confirmed that miR-27a down-regulated Sfrp1 by binding to the 3′ untranslated region directly. Further, NRK-52E cells under high-glucose conditions underwent transfection with miR-27a mimic or the corresponding negative control, miR-27a inhibitor or the corresponding negative control, si-Sfrp1, or combined miR-27a inhibitor and si-Sfrp1. Immunoblotting and immunofluorescence were performed to assess the relative expression levels of Wnt/β-catenin signalling and ECM components. The mRNA levels of Sfrp1, miR-27a, and ECM-related molecules were also detected by quantitative real-time PCR (qPCR). We found that miR-27a inhibitor inactivated Wnt/β-catenin signalling and reduced ECM deposition. Conversely, Wnt/β-catenin signalling was activated, while ECM deposition was increased after transfection with si-Sfrp1. Interestingly, miR-27a inhibitor attenuated the effects of si-Sfrp1. We concluded that miR-27a down-regulated Sfrp1 and activated Wnt/β-catenin signalling to promote renal fibrosis.

scholarly journals circRNA_010383 acts as a sponge for miR-135a and its downregulated expression contributes to renal fibrosis in diabetic nephropathy

2020 ◽  
Author(s):  
Ada Admin ◽  
Fenfen Peng ◽  
Wangqiu Gong ◽  
Shuting Li ◽  
Bohui Yin ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Renal Fibrosis ◽  
Transient Receptor Potential ◽  
Mesangial Cells ◽  
Receptor Potential ◽  
Vascular Complication ◽  
Diabetic Patients

Diabetic nephropathy (DN), a vascular complication of diabetes mellitus, is the leading cause of death in diabetic patients. The contribution of aberrantly expressed circRNAs to diabetic nephropathy <i>in vivo</i> is poorly understood. Integrated comparative circRNA microarray profiling was used to examine the expression of circRNAs in diabetic kidney of db/db mice. We found that circRNA_010383 expression was markedly downregulated in diabetic kidneys, mesangial cells and tubular epithelial cells cultured in high-glucose conditions. circRNA_010383 colocalized with microRNA-135a (miR-135a) and inhibited miR-135a function by directly binding to miR-135a. <i>In vitro,</i> the knockdown of circRNA_010383 promoted the accumulation of extracellular matrix (ECM) proteins <a></a><a>and </a>downregulated the expression of transient receptor potential cation channel, subfamily C, member (TRPC1), which is a target protein of miR-135a. Furthermore, <a></a><a>circRNA_010383 overexpression</a> effectively inhibited the high-glucose-induced accumulation of ECM and increased TRPC1 levels <i>in vitro</i>. More importantly, the kidney-target of circRNA_010383 overexpression inhibited <a></a><a>proteinuria</a> and renal fibrosis in db/db mice. Mechanistically, we identified that a loss of circRNA_010383 promoted proteinuria and renal fibrosis in DN by acting as a sponge for miRNA-135a. This study reveals that circRNA_010383 may be a novel therapeutic target for DN in the future.

Lysine-specific demethylase 1 induced epithelial–mesenchymal transition and promoted renal fibrosis through Jagged-1/Notch signaling pathway

2021 ◽  
pp. 096032712110387
Author(s):  
Huali Zhang ◽  
Jiaming Xing ◽  
Lingwei Zhao
Keyword(s):  
Cell Invasion ◽  
Renal Fibrosis ◽  
Epithelial Mesenchymal Transition ◽  
Notch 1 ◽  
Mesenchymal Transition ◽  
Lysine Specific Demethylase ◽  
Tgf Β1 ◽  
E Cadherin

Objective TGF-β1-induced excessive deposition of extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) process of tubular epithelial cells play critical roles in the progression of renal fibrosis. We are aimed to explore the effects of lysine-specific demethylase 1 (LSD1) in TGF-β1-treated HK-2 cells and in rats with unilateral ureteral obstruction (UUO), and to investigate the underlying molecular mechanism. Methods TGF-β1-treated HK-2 cells and UUO-treated rats were used to establish the model of renal fibrosis in vitro and in vivo, respectively. Protein expression of LSD1, E-cadherin, a-smooth muscle actin (a-SMA), Vimentin, Jagged-1, Notch-1 and Notch-2 were detected by Western blot. The concentrations of type I collagen (Col-I) and Fibronectin (FN) were measured by ELISA. Transwell assay were used to assess cell invasion. Results LSD1 was dramatically increased in TGF-β1-stimulated HK-2 cells. Knockdown of LSD1 decreased the TGF-β1-induced secretion of Col-I and FN, and suppressed TGF-β1-induced expression of E-cadherin,α-SMA and Vimentin, while suppressed cell invasion. Consistent with the in vitro data, the severe histopathological damage, collagen deposition and reduced E-cadherin, increased α-SMA induced by UUO was abated by the knockdown of LSD1 in vivo. Moreover, knockdown of LSD1 suppressed TGF-β1-induced expression of Jagged-1, Notch-1 and Notch-2. Furthermore, we found that inhibition of Notch signaling by a γ-secretase inhibitor RO4929097 almost recapitulated the effects of LSD1 knockdown in TGF-β1-induced HK-2 cells, and at least in part reversed the effects of LSD1 overexpression on EMT and ECM deposition in HK-2 cells. Conclusions Taken together, LSD1 significantly impact on the progression of TGF-β1-mediated EMT and ECM deposition in HK-2 cells, and it may represent novel target for the prevention strategies of renal fibrosis.

Sign in / Sign up

Export Citation Format

Share Document