Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction

We investigated the role of Akt1, one of the three isoforms of Akt, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). We subjected wild type and Akt1−/− mice to UUO. The Akt1 gene was silenced in vitro using short hairpin RNA delivered via a lentiviral vector in human proximal tubular cells (HK2 cells) and kidney fibroblasts (NRK-49F cells). The obstructive kidneys of Akt1−/− mice showed more severe tubulointerstitial fibrosis than those of wild type mice. The expression of fibronectin and type I collagen was significantly increased in obstructed kidneys of Akt1−/− mice compared to those of wild type mice. The important finding was that the expression of transforming growth factor β1 (TGFβ1) was significantly increased in the Akt1−/− mice compared to the wild type mice. The knockdown of Akt1 enhanced the expression of TGFβ1 in HK2 cells. Interestingly, the upregulation of TGFβ1 due to genetic knockdown of Akt1 was associated with activation of signal transducer and activator of transcript 3 (STAT3) independently of the Smad pathway in NRK-49F and HK2 cells. Immunohistochemical staining also showed that expression of phosphorylated STAT3 was more increased in Akt1−/− mice than in wild type mice after UUO. Additionally, the deletion of Akt1 led to apoptosis of the renal tubular cells in both in vivo and in vitro studies. Conclusively, these results suggest that the deletion of Akt1 may contribute to renal fibrosis via induction of the TGFβ1/STAT3 pathway in a murine model of UUO.

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P0734THE DELETION OF AKT1 PROMOTES THE RENAL FIBROSIS IN THE MURINE MODEL OF UNILATERAL URETERAL OBSTRUCTION

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0734 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Il Young Kim ◽

Byung Min Ye ◽

Min Jeong Kim ◽

Dong Won Lee ◽

Soo Bong Lee ◽

...

Keyword(s):

Western Blot ◽

Murine Model ◽

Ureteral Obstruction ◽

Renal Fibrosis ◽

Caspase 3 ◽

Unilateral Ureteral Obstruction ◽

Wild Type ◽

Immunohistochemical Stain ◽

In The Wild ◽

Hk2 Cells

Abstract Background and Aims Previous studies have found the increased Akt activity in experimental renal fibrosis. We investigated the role of Akt1, one of the three Akt isoforms, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). Method In vivo, we subjected the wild type and Akt1−/− mice to UUO. In vitro, gene silencing of Akt1 was achieved using the short hairpin RNA delivered by the lentiviral vector in immortalized human proximal tubular cells (HK2 cells) and rat kidney fibroblasts (NRK-49F cells). Results In immunohistochemical stain, the expression of Akt1 was significantly higher in obstructed kidneys of wild type mice compared with control sham kidneys and increased gradually as UUO progressed. The fibronectin, type I collagen, and heat shock protein 47 (HSP47) were markedly more expressed in obstructed kidneys of Akt1−/− mice than in those of the wild type mice. Transforming growth factor β1 (TGFβ1) was highly induced within 1 day of UUO in obstructed kidneys of Akt1−/− mice and the expression of TGFβ1 was significantly higher in the Akt1−/− mice than in the wild type mice as UUO progressed. Western blot showed that silencing of Akt1 increased the expression of TGFβ1, which was enhanced by angiotensin II stimulation in HK2 cells, but not in NRK-49F cells. Immunohistochemical stain demonstrated that the expression of cleaved caspase-3 in renal tubules was significantly higher in the Akt1−/− mice than in the wild type mice. Western blot showed that silencing of Akt1 increased the expression of cleaved caspase-3 in HK2 cells, but not in NRK-49F cells. Conclusion Our findings suggest that the deletion of Akt1 might contribute to tubulointerstitial fibrosis and tubular apoptosis via TGFβ1 induction. We also showed that TGFβ1 upregulation by genetic deletion of Akt1 is associated with activation of STAT3 independently of the TGFβ1/Smad signaling pathway.

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Dapagliflozin Alleviates Renal Fibrosis by Inhibiting RIP1-RIP3-MLKL-Mediated Necroinflammation in Unilateral Ureteral Obstruction

Frontiers in Pharmacology ◽

10.3389/fphar.2021.798381 ◽

2022 ◽

Vol 12 ◽

Author(s):

Mei Ying Xuan ◽

Shang Guo Piao ◽

Jun Ding ◽

Qi Yan Nan ◽

Mei Hua Piao ◽

...

Keyword(s):

Cell Death ◽

Ureteral Obstruction ◽

Renal Fibrosis ◽

Apoptotic Cell ◽

Subsequent Development ◽

Unilateral Ureteral Obstruction ◽

Apoptotic Cell Death ◽

Inflammasome Activation ◽

Renal Tubular

Dapagliflozin, a sodium-glucose cotransporter-2 inhibitor, offers renoprotection in diabetes. However, potential for use in nondiabetic kidney disease remains unknown. Herein, we assessed whether dapagliflozin alleviates renal fibrosis by interfering with necroinflammation in a rat model of unilateral ureteral obstruction (UUO) and in vitro. After induction of UUO, rats were administered dapagliflozin daily for seven consecutive days. UUO induced significant renal tubular necrosis and overexpression of RIP1-RIP3-MLKL axis proteins; these coincided with NLRP3 inflammasome activation, and subsequent development of renal fibrosis. Oxidative stress caused by UUO is tightly associated with endoplasmic reticulum stress and mitochondrial dysfunction, leading to apoptotic cell death through Wnt3α/β-catenin/GSK-3β signaling; all of which were abolished by both dapagliflozin and specific RIP inhibitors (necrostatin-1 and GSK872). In H2O2-treated HK-2 cells, dapagliflozin and RIP inhibitors suppressed overexpression of RIP1-RIP3-MLKL proteins and pyroptosis-related cytokines, decreased intracellular reactive oxygen species production and apoptotic cell death, whereas cell viability was improved. Moreover, activated Wnt3α/β-catenin/GSK-3β signaling was inhibited by dapagliflozin and Wnt/β-catenin inhibitor ICG-001. Our findings suggest that dapagliflozin ameliorates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/β-catenin/GSK-3β signaling in UUO.

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P0518VNN1 MEDIATES RENAL MALADAPTIVE REPAIR AFTER AKI BY INDUCING PREMATURE SENESCENCE OF RENAL TUBUAR CELLS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0518 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Yani He ◽

Kehong Chen ◽

Jia Chen

Keyword(s):

Renal Fibrosis ◽

Sham Group ◽

Renal Tissue ◽

Premature Senescence ◽

Wild Type ◽

Injury Score ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Tubule Cells ◽

Renal Tubular

Abstract Background and Aims Renal maladaptive repair after acute kidney injury (AKI) can easily progress to chronic kidney disease. Sustained renal interstitial damage caused by accelerated senescence of renal tubular cells leads to renal fibrosis. Vanin-1 (VNN1) is an extracellular enzyme with panthenylmethylaminease activity that indirectly reduces the synthesis of glutathione, causing oxidative stress. The purpose of this study was to investigate the expression and of VNN1 in renal tissue and to study its role in senescence of renal tubular cells after ischemia reperfusion (I/R). Method hirty male wild BALB/c mice were randomly divided into control group, sham group and I/R group. In the I/R group, the bilateral renal pedicle was ligatured for 35 min followed by reperfusion. The expression of VNN1 and aging markers (P16, P21, SA-b-gal) were detected. Furthermore, wild type mice and VNN1 knockout mice were used to compare the degree of renal tissue and functional damage and the senescence of renal tubular cells after I/R injury. Results Compared with sham group, Scr, BUN and renal injury score increased significantly in I/R group at the early stage (3d) of renal injury. Renal fibrosis was observed in the later stage (28-42d). The expression of VNN1 in renal tubular cells of I/R group increased after I/R injury. VNN1 was coexpressed with P16, suggesting that VNN1 might be related to cellular stress senescence. The SCr, BUN of VNN1 KO mice was significantly lower than that of wild type mice at 7-28 d after renal reperfusion. The renal interstitial injury score of VNN1 KO mice was significantly lower than that of wild type mice, and the renal interstitial fibrosis level was significantly higher than that of wild type mice at 42d after reperfusion. The results suggest that VNN1 KO promotes renal repair of AKI. The ratio of P16 positive tubule cells in VNN1 KO mice was significantly higher than that in wild-type mice at 7d after renal reperfusion, suggesting that VNN1 could promote the senescence of renal tubule cells during AKI repair. Conclusion VNN1 mediates renal maladaptive repair after AKI by inducing premature senescence of renal tubular cells.

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Intravoxel incoherent imaging of renal fibrosis induced in a murine model of unilateral ureteral obstruction

Magnetic Resonance Imaging ◽

10.1016/j.mri.2015.07.012 ◽

2015 ◽

Vol 33 (10) ◽

pp. 1324-1328 ◽

Cited By ~ 20

Author(s):

Tiffany Hennedige ◽

Tong San Koh ◽

Septian Hartono ◽

Yet Yen Yan ◽

In Chin Song ◽

...

Keyword(s):

Murine Model ◽

Ureteral Obstruction ◽

Renal Fibrosis ◽

Unilateral Ureteral Obstruction ◽

Incoherent Imaging

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Proximal tubule PPARα attenuates renal fibrosis and inflammation caused by unilateral ureteral obstruction

AJP Renal Physiology ◽

10.1152/ajprenal.00309.2013 ◽

2013 ◽

Vol 305 (5) ◽

pp. F618-F627 ◽

Cited By ~ 42

Author(s):

Shenyang Li ◽

Nithya Mariappan ◽

Judit Megyesi ◽

Brian Shank ◽

Krishnaswamy Kannan ◽

...

Keyword(s):

Proximal Tubule ◽

Ureteral Obstruction ◽

Renal Fibrosis ◽

Transforming Growth Factor ◽

Obstructive Uropathy ◽

Kidney Tissue ◽

Unilateral Ureteral Obstruction ◽

Tubulointerstitial Fibrosis ◽

Wild Type ◽

Arginase 1

We examined the effects of increased expression of proximal tubule peroxisome proliferator-activated receptor (PPAR)α in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO), PPARα expression was significantly reduced in kidney tissue of wild-type mice but this downregulation was attenuated in proximal tubules of PPARα transgenic (Tg) mice. When compared with wild-type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule transforming growth factor (TGF)-β1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-smooth muscle actin, and reduced tubulointerstitial fibrosis. UUO-mediated increased expression of microRNA 21 in kidney tissue was also reduced in PPARα Tg mice. Overexpression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid-mediated increased production of TGF-β, demonstrating PPARα signaling reduces epithelial TGF-β production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of proinflammatory cytokines including IL-1β, IL-6, and TNF-α in wild-type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild-type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulointerstitial fibrosis and inflammation associated with obstructive uropathy.

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HuangQi Decoction Ameliorates Renal Fibrosis via TGF-β/Smad Signaling Pathway In Vivo and In Vitro

Cellular Physiology and Biochemistry ◽

10.1159/000443115 ◽

2016 ◽

Vol 38 (5) ◽

pp. 1761-1774 ◽

Cited By ~ 16

Author(s):

Jie Zhao ◽

Li Wang ◽

Ai-li Cao ◽

Ming-Qian Jiang ◽

Xia Chen ◽

...

Keyword(s):

Signaling Pathway ◽

Ureteral Obstruction ◽

Interstitial Fibrosis ◽

Unilateral Ureteral Obstruction ◽

Type I ◽

Renal Interstitial Fibrosis ◽

Smad Signaling ◽

Smad Signaling Pathway ◽

Tgf Β1

Objective: Traditional Chinese Medicine compound HuangQi decoction is widely used in clinical treatment of chronic kidney disease, but its role on renal interstitial fibrosis and the underlying mechanism remains unclear. The aim of this study is to investigate the effect of HuangQi decoction on renal interstitial fibrosis and its association with the TGF-β/Smad signaling pathway Methods: A total of 120 C57/BL mice were randomly divided into six groups: sham group, sham plus high-dose HuangQi decoction (1.08g/kg) group, unilateral ureteral obstruction (UUO) model group, and UUO model plus low to high doses of HuangQi decoction (0.12g/kg, 0.36g/kg and 1.08g/kg respectively) groups. Animals were sacrificed 14 days after the administration and ipsilateral kidney tissue was sampled for pathologic examinations. Immunohistochemistry, PCR and western blot were used to detect the expressions of related molecules in the TGF-β/Smad signaling pathway. TGF-β1 was used in in vitro experiments to induce human kidney proximal tubule epithelial cells (HK2). Results: HuangQi decoction improved ipsilateral kidney fibrosis in UUO mice and downregulated the expressions of TGF-β1, TβRI, TβRII, Smad4, Smad2/3, P-Smad2/3, α-SMA, collagen type I, III and IV in a dose-dependent manner while upregulated the expression of Smad7 in the same fashion. Similar results were found in in vitro studies. Conclusion: The protective effect of HuangQi decoction for unilateral ureteral obstruction kidney damage in mice was mediated by downregulating the TGF-β/Smad signaling pathway.

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Increasing cGMP-dependent protein kinase activity attenuates unilateral ureteral obstruction-induced renal fibrosis

AJP Renal Physiology ◽

10.1152/ajprenal.00657.2013 ◽

2014 ◽

Vol 306 (9) ◽

pp. F996-F1007 ◽

Cited By ~ 17

Author(s):

Wenpeng Cui ◽

Hasiyeti Maimaitiyiming ◽

Xinyu Qi ◽

Heather Norman ◽

Qi Zhou ◽

...

Keyword(s):

Transgenic Mice ◽

Renal Fibrosis ◽

Cell Function ◽

Wild Type ◽

Dependent Protein Kinase ◽

Tubular Cells ◽

Cgmp Dependent Protein Kinase ◽

Proximal Tubular ◽

Dependent Protein

Our previous studies support the protective effect of cGMP and cGMP-dependent protein kinase I (PKG-I) pathway on the development of renal fibrosis. Therefore, in the present studies, we determined whether pharmacologically or genetically increased PKG activity attenuates renal fibrosis in a unilateral ureteral obstruction (UUO) model and also examined the mechanisms involved. To increase PKG activity, we used the phosphodiesterase 5 inhibitor sildenafil and PKG transgenic mice. UUO model was induced in wild-type or PKG-I transgenic mice by ligating the left lateral ureteral and the renal fibrosis was observed after 14 days of ligation. Sildenafil was administered into wild-type UUO mice for 14 days. In vitro, macrophage and proximal tubular cell function was also analyzed. We found that sildenafil treatment or PKG transgenic mice had significantly reduced UUO-induced renal fibrosis, which was associated with reduced TGF-β signaling and reduced macrophage infiltration into kidney interstitial. In vitro data further demonstrated that both macrophages and proximal tubular cells were important sources of UUO-induced renal TGF-β levels. The interaction between macrophages and tubular cells contributes to TGF-β-induced renal fibrosis. Taken together, these data suggest that increasing PKG activity ameliorates renal fibrosis in part through regulation of macrophage and tubular cell function, leading to reduced TGF-β-induced fibrosis.

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Liproxstatin-1 attenuates unilateral ureteral obstruction-induced renal fibrosis by inhibiting renal tubular epithelial cells ferroptosis

Cell Death and Disease ◽

10.1038/s41419-021-04137-1 ◽

2021 ◽

Vol 12 (9) ◽

Author(s):

Bo Zhang ◽

Xiang Chen ◽

Feng Ru ◽

Yu Gan ◽

Bingsheng Li ◽

...

Keyword(s):

Lipid Peroxidation ◽

Cell Death ◽

Epithelial Cells ◽

Ureteral Obstruction ◽

Renal Fibrosis ◽

Unilateral Ureteral Obstruction ◽

Tubular Epithelial Cells ◽

Renal Tubular Epithelial Cells ◽

Renal Tubular ◽

Hk2 Cells

AbstractRenal fibrosis is a common pathological process that occurs with diverse etiologies in chronic kidney disease. However, its regulatory mechanisms have not yet been fully elucidated. Ferroptosis is a form of non-apoptotic regulated cell death driven by iron-dependent lipid peroxidation. It is currently unknown whether ferroptosis is initiated during unilateral ureteral obstruction (UUO)-induced renal fibrosis and its role has not been determined. In this study, we demonstrated that ureteral obstruction induced ferroptosis in renal tubular epithelial cells (TECs) in vivo. The ferroptosis inhibitor liproxstatin-1 (Lip-1) reduced iron deposition, cell death, lipid peroxidation, and inhibited the downregulation of GPX4 expression induced by UUO, ultimately inhibiting ferroptosis in TECs. We found that Lip-1 significantly attenuated UUO-induced morphological and pathological changes and collagen deposition of renal fibrosis in mice. In addition, Lip-1 attenuated the expression of profibrotic factors in the UUO model. In vitro, we used RSL3 treatment and knocked down of GPX4 level by RNAi in HK2 cells to induce ferroptosis. Our results indicated HK2 cells secreted various profibrotic factors during ferroptosis. Lip-1 was able to inhibit ferroptosis and thereby inhibit the secretion of the profibrotic factors during the process. Incubation of kidney fibroblasts with culture medium from RSL3-induced HK2 cells promoted fibroblast proliferation and activation, whereas Lip-1 impeded the profibrotic effects. Our study found that Lip-1 may relieve renal fibrosis by inhibiting ferroptosis in TECs. Mechanistically, Lip-1 could reduce the activation of surrounding fibroblasts by inhibiting the paracrine of profibrotic factors in HK2 cells. Lip-1 may potentially be used as a therapeutic approach for the treatment of UUO-induced renal fibrosis.

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Protein arginine methyltransferase 1 mediates renal fibroblast activation and fibrogenesis through activation of Smad3 signaling

AJP Renal Physiology ◽

10.1152/ajprenal.00487.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. F375-F387 ◽

Cited By ~ 2

Author(s):

Yu Zhu ◽

Chao Yu ◽

Shougang Zhuang

Keyword(s):

Ureteral Obstruction ◽

Renal Fibrosis ◽

Collagen Type ◽

Collagen Type I ◽

Unilateral Ureteral Obstruction ◽

Type I ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Fibroblast Activation ◽

Protein Arginine Methyltransferase 1

Protein arginine methyltransferase 1 (PRMT1), which primarily causes asymmetric arginine methylation of histone and nonhistone proteins, has been found to activate gene expression and mediate multiple pathological processes. Its role in renal fibrosis, however, remains unclear. In the present study, we observed that PRMT1 and its specific epigenetic marker, asymmetric di-methylated histone 4 arginine 3 (H4R3Me2a), were highly expressed in cultured renal interstitial fibroblasts. Treatment of PRMT1 with AMI-1, a selective inhibitor of PRMT1, or silencing PRMT1 with siRNA inhibited serum-induced and transforming growth factor (TGF)-β1-induced expression of α-smooth muscle actin (α-SMA) and collagen type I, two hallmarks of renal fibroblast activation, in a dose-dependent and time-dependent manner. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, PRMT1 expression and H4R3Me2a were also upregulated, which was coincident with increased expression of α-SMA, collagen type I, and fibronectin. Administration of AMI-1 reduced PRMT1 and H4R3Me2a expression, attenuated extracellular matrix protein deposition, and inhibited renal fibroblast activation and proliferation. Moreover, AMI-1 treatment inhibited Smad3 phosphorylation and TGF-β receptor I expression but prevented Smad7 downregulation both in the kidney after unilateral ureteral obstruction injury and in cultured renal interstitial fibroblasts exposed to TGF-β1. Collectively, these results demonstrate that PRMT1 may mediate renal fibroblast activation and renal fibrosis development through activation of the TGF-β/Smad3 signaling pathway. They also suggest that PRMT1 inhibition may be a potential therapeutic approach for the treatment of fibrotic kidney disease.

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