Inhibition of SIRT2 Alleviates Fibroblast Activation and Renal Tubulointerstitial Fibrosis via MDM2

Background/Aims: Renal tubular epithelial cells and fibroblasts are the main sources of myofibroblasts, and these cells produce the extracellular matrix during tubulointerstitial fibrosis (TIF). Histone deacetylases (HDAC) inhibitors exert an antifibrogenic effect in the skin, liver and lung. Sirtuin 2 (SIRT2), which is a class III HDAC, is an important member of NAD+-dependent protein deacetylases. The current study evaluated the role of SIRT2 in renal TIF. Methods: Immunohistochemical staining and Western blot were performed to evaluate SIRT2 expression in TIF patients and unilateral urethral obstruction (UUO) mice. Western blot was used to assess the protein levels of SIRT2, α-SMA, collagen III, fibronectin, and MDM2 in tubular epithelial cells and fibroblasts. The specific inhibitor AGK2 was used to inhibit SIRT2 activity, and targeted siRNA was used to suppress SIRT2 expression. Results: SIRT2 expression increased in the tubulointerstitium of TIF patients and UUO mice. SIRT2 inhibition ameliorated TIF in UUO mice. SIRT2 expression in tubular cells was unchanged after exposure to TGF-β1. The SIRT2-specifc inhibitor AGK2 did not attenuate TGF-β1-induced tubular epithelial-mesenchymal transition. However, SIRT2 was upregulated in fibroblasts, and fibroblasts were activated after TGF-β1 treatment. Genetic knockdown and chemical inhibition of SIRT2 attenuated TGF-β1-induced fibroblast activation. We also explored the downstream signaling of SIRT2 during fibroblast activation. Genetic knockdown and chemical inhibition of SIRT2 suppressed TGF-β1-induced increase in MDM2 expression, and inhibition of the MDM2-p53 interaction using Nutlin-3 did not suppress SIRT2 upregulation. Conclusion: Our results suggest that SIRT2 participates in the activation of fibroblasts and TIF, which is mediated via regulation of the MDM2 pathway, and the downregulation of SIRT2 may be a therapeutic strategy for renal fibrosis.

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Metformin attenuates renal tubulointerstitial fibrosis via upgrading autophagy in the early stage of diabetic nephropathy

Scientific Reports ◽

10.1038/s41598-021-95827-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Fengzhen Wang ◽

Haihan Sun ◽

Bangjie Zuo ◽

Kun Shi ◽

Xin Zhang ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Epithelial Cells ◽

Early Stage ◽

Tubulointerstitial Fibrosis ◽

Tubular Epithelial Cells ◽

Renal Tubular Epithelial Cells ◽

Sprague Dawley ◽

Mesenchymal Transition ◽

Renal Tubulointerstitial Fibrosis ◽

Renal Tubular

AbstractThis study aimed at comparing the effects of metformin on tubulointerstitial fibrosis (TIF) in different stages of diabetic nephropathy (DN) in vivo and evaluating the mechanism in high glucose (HG)-treated renal tubular epithelial cells (RTECs) in vitro. Sprague–Dawley (SD) rats were used to establish a model of DN, and the changes of biochemical indicators and body weight were measured. The degree of renal fibrosis was quantified using histological analysis, immunohistochemistry, and immunoblot. The underlying relationship between autophagy and DN, and the cellular regulatory mechanism of metformin on epithelial-to-mesenchymal transition (EMT) were investigated. Metformin markedly improved renal function and histological restoration of renal tissues, especially in the early stages of DN, with a significant increase in autophagy and a decrease in the expression of fibrotic biomarkers (fibronectin and collagen I) in renal tissue. Under hyperglycemic conditions, renal tubular epithelial cells inactivated p-AMPK and activated partial EMT. Metformin-induced AMPK significantly ameliorated renal autophagic function, inhibited the partial EMT of RTECs, and attenuated TIF, all of which effectively prevented or delayed the onset of DN. This evidence provides theoretical and experimental basis for the following research on the potential clinical application of metformin in the treatment of diabetic TIF.

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V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00278.2011 ◽

2012 ◽

Vol 302 (9) ◽

pp. F1121-F1132 ◽

Cited By ~ 15

Author(s):

Xueqin Cao ◽

Qiongqiong Yang ◽

Jing Qin ◽

Shili Zhao ◽

Xiaoyan Li ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cells ◽

Transforming Growth Factor ◽

Tubulointerstitial Fibrosis ◽

Dependent Manner ◽

Tubular Epithelial Cells ◽

Mesenchymal Transition ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular ◽

Tgf Β1

The ubiquitous vacuolar H+-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na+-independent pH recovery was significantly faster after an NH4Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.

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Inhibition of ROCK2 alleviates renal fibrosis and the metabolic disorders in the proximal tubular epithelial cells

Clinical Science ◽

10.1042/cs20200030 ◽

2020 ◽

Vol 134 (12) ◽

pp. 1357-1376 ◽

Cited By ~ 1

Author(s):

Ran You ◽

Wei Zhou ◽

Yanwei Li ◽

Yue Zhang ◽

Songming Huang ◽

...

Keyword(s):

Epithelial Cells ◽

Renal Fibrosis ◽

Tubulointerstitial Fibrosis ◽

Related Factor ◽

Tubular Epithelial Cells ◽

Renal Tubulointerstitial Fibrosis ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular ◽

Tgf Β1

Abstract Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson’s trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-β1 (TGF-β1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-β1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.

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Formin mDia1 contributes to migration and epithelial‐mesenchymal transition of tubular epithelial cells exposed to TGF‐β1

Journal of Cellular Biochemistry ◽

10.1002/jcb.29508 ◽

2019 ◽

Vol 121 (8-9) ◽

pp. 3861-3870 ◽

Cited By ~ 1

Author(s):

Yu‐Ying Li ◽

Guo‐Tao Jiang ◽

Li‐Jie Chen ◽

Yan‐Hong Jiang ◽

Jun‐Dong Jiao

Keyword(s):

Epithelial Cells ◽

Epithelial Mesenchymal Transition ◽

Tubular Epithelial Cells ◽

Mesenchymal Transition ◽

Tgf Β1

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Human Cytomegalovirus Induces TGF-β1 Activation in Renal Tubular Epithelial Cells after Epithelial-to-Mesenchymal Transition

PLoS Pathogens ◽

10.1371/journal.ppat.1001170 ◽

2010 ◽

Vol 6 (11) ◽

pp. e1001170 ◽

Cited By ~ 26

Author(s):

Masako Shimamura ◽

Joanne E. Murphy-Ullrich ◽

William J. Britt

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Epithelial To Mesenchymal Transition ◽

Tubular Epithelial Cells ◽

Renal Tubular Epithelial Cells ◽

Mesenchymal Transition ◽

Renal Tubular ◽

Tgf Β1

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Interaction between V-ATPase B2 and (Pro) renin Receptors in Promoting the progression of Renal Tubulointerstitial Fibrosis

Scientific Reports ◽

10.1038/srep25035 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 2

Author(s):

Yun Liu ◽

Sujun Zuo ◽

Xiaoyan Li ◽

Jinjin Fan ◽

Xueqin Cao ◽

...

Keyword(s):

Epithelial Cells ◽

Smooth Muscle Actin ◽

Tubulointerstitial Fibrosis ◽

Dependent Manner ◽

Tubular Epithelial Cells ◽

Atpase Inhibitor ◽

Renal Tubulointerstitial Fibrosis ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular ◽

Atpase Subunits

Abstract To investigate the levels of (Pro) renin receptor [(P) RR], α-smooth muscle actin (α-SMA), fibronectin (FN), and vacuolar H+-ATPase (V-ATPase) subunits (B2, E, and c) in rat unilateral ureteral obstruction (UUO) models and rat proximal tubular epithelial cells (NRK-52E) treated with prorenin to elucidate the role of V-ATPase in these processes by activating the (P) RR. UUO significantly upregulated (P) RR, V-ATPase subunits, α-SMA and FN expression in tubulointerstitium or tubular epithelial cells. A marked colocalization of (P) RR and the B2 subunit was also observed. Prorenin treatment upregulated α-SMA, FN, (P) RR, and V-ATPase subunits and activity in NRK52E cell in a dose- and time-dependent manner. The V-ATPase inhibitor bafilomycin A1 partially blocked prorenin-induced (P) RR, FN, and α-SMA expression. Co-immunoprecipitate and immunofluorescence results demonstrated that the V-ATPase B2 subunit bound to the (P) RR, which was upregulated after prorenin stimulation. Either siRNA-mediated (P) RR or B2 subunit knockdown partially reduced V-ATPase activity and attenuated prorenin-induced FN and α-SMA expression. From the data we can assume that activation of (P) RR and V-ATPase may play an important role in tubulointerstitial fibrosis with possible involvement of interaction of V-ATPase B2 subunit and (P)RR.

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Identification of Tisp40 as an Essential Regulator of Renal Tubulointerstitial Fibrosis via TGF-β/Smads Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000477887 ◽

2017 ◽

Vol 42 (2) ◽

pp. 697-712 ◽

Cited By ~ 5

Author(s):

Cheng-cheng Xiao ◽

Jie Zhang ◽

Peng-cheng Luo ◽

Cong Qin ◽

Yang Du ◽

...

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Renal Fibrosis ◽

Molecular Mechanisms ◽

Ischemia Reperfusion ◽

Critical Role ◽

Tubulointerstitial Fibrosis ◽

Mesenchymal Transition ◽

Renal Tubulointerstitial Fibrosis

Background: Tisp40, a transcription factor of the CREB/CREM family, is involved in cell proliferation, differentiation and other biological functions, but its role in renal tubulointerstitial fibrosis is unknown. Methods: In our study, we investigated the effects of Tisp40 on extracellular matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT) and the underlying molecular mechanisms in transforming growth factor-β (TGF-β)-stimulated TCMK-1 cells by quantitative real-time polymerase chain reaction (qPCR), Western blot analysis and immunofluorescence in vitro, and further explored the role of Tisp40 on renal fibrosis induced by ischemia-reperfusion (I/R) by qPCR, Western blot analysis, hydroxyproline analysis, Masson trichrome staining and immunohistochemistry staining in vivo. Results: The data showed that Tisp40 was upregulated in a model of renal fibrosis induced by I/R injury (IRI). Upon IRI, Tisp40-deficient mice showed attenuated renal fibrosis compared with wild-type mice. Furthermore, the expression of α-smooth muscle actin, E-cadherin, fibronectin, and collagen I was suppressed. Tisp40 overexpression aggravated ECM accumulation and EMT in the TGF-β-stimulated TCMK-1 cell line, whereas the opposite occurred in cells treated with small interfering RNA (siRNA) targeting Tisp40. Importantly, it is changes in the Smad pathway that attenuate renal fibrosis. Conclusion: These findings suggest that Tisp40 plays a critical role in the TGF-β/ Smads pathway involved in this process. Hence, Tisp40 could be a useful therapeutic target in the fight against renal tubulointerstitial fibrosis.

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MDM2 mediates fibroblast activation and renal tubulointerstitial fibrosis via a p53-independent pathway

AJP Renal Physiology ◽

10.1152/ajprenal.00528.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. F760-F768 ◽

Cited By ~ 8

Author(s):

Chen Ye ◽

Hui Tang ◽

Zheng Zhao ◽

Chun-Tao Lei ◽

Chao-Qun You ◽

...

Keyword(s):

Critical Role ◽

Tubulointerstitial Fibrosis ◽

Podocyte Injury ◽

Fibroblast Activation ◽

Downstream Signaling ◽

Renal Tubulointerstitial Fibrosis ◽

Inflammatory Processes ◽

Tgf Β1

It is well recognized that murine double minute gene 2 (MDM2) plays a critical role in cell proliferation and inflammatory processes during tumorigenesis. It is also reported that MDM2 is expressed in glomeruli and involved in podocyte injury. However, whether MDM2 is implicated in renal fibrosis remains unclear. Here we investigated the role of MDM2 in tubulointerstitial fibrosis (TIF). By immunohistochemical staining and Western blotting we confirmed that MDM2 is upregulated in the tubulointerstitial compartment in patients with TIF and unilateral urethral obstruction (UUO) mice, which mainly originates from myofibroblasts. Consistently, in vitro MDM2 is increased in TGF-β1-treated fibroblasts, one of the major sources of collagen-producing myofibroblasts during TIF, along with fibroblast activation. Importantly, genetic deletion of MDM2 significantly attenuates fibroblast activation. We then analyzed the possible downstream signaling of MDM2 during fibroblast activation. p53-dependent pathway is the classic downstream signaling of MDM2, and Nutlin-3 is a small molecular inhibitor of MDM2-p53 interaction. To our surprise, Nutlin-3 could not ameliorate fibroblast activation in vitro and TIF in UUO mice. However, we found that Notch1 signaling is attenuated during fibroblast activation, which could be markedly rescued by MDM2 knockdown. Overexpression of intracellular domain of Notch1 (NICD) by plasmid could obviously minimize fibroblast activation induced by TGF-β1. In addition, the degradation of NICD is strikingly suppressed by PYR-41, an inhibitor of ubiquitin-activating enzyme E1, and proteasome inhibitor MG132. Taken together, our findings provide the first evidence that MDM2 is involved in fibroblast activation and TIF, which associates with Notch1 ubiquitination and proteasome degradation.

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