An inhibitor of Krüppel-like factor 5 suppresses peritoneal fibrosis in mice

Back ground: Krüppel-like transcription factor 5 (KLF5) is a transcription factor regulating cell proliferation, angiogenesis and differentiation. It has been recently reported that Am80, a synthetic retinoic acid receptor α-specific agonist, inhibits the expression of KLF5. In the present study, we have examined the expression of KLF5 in fibrotic peritoneum induced by chlorhexidine gluconate (CG) in mouse and evaluated that Am80, as an inhibitor of KLF5, can reduce peritoneal fibrosis. Methods: Peritoneal fibrosis was induced by intraperitoneal injection of CG into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. Control mice received only a vehicle (0.5% carboxymethylcellulose solution). After 3 weeks of treatment, peritoneal equilibration test (PET) was performed and peritoneal tissues were examined by immunohistochemistry. Results: The expression of KLF5 was less found in the peritoneal tissue of control mice, while KLF5 was expressed in the thickened submesothelial area of CG-injected mice receiving the vehicle. Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening, accompanied with the reduction of type III collagen expression. The numbers of transforming growth factor β-positive cells, α-smooth muscle actin-positive cells and infiltrating macrophages were significantly decreased in Am80-treated group. PET revealed the increased peritoneal permeability in CG mice, whereas Am80 administration significantly improved the peritoneal high permeability state. Conclusions: These results indicate the involvement of KLF5 in the progression of experimental peritoneal fibrosis and suggest that Am80 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of KLF5 expression.

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P1196AN INIHIBITOR OF KRUPPEL-LIKE FACTOR 5 SUPPRESSES PERITONEAL FIBROSIS IN MICE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1196 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Kumiko Muta ◽

Yuka Nakazawa ◽

Yoko Obata ◽

Hiro Inoue ◽

Kenta Torigoe ◽

...

Keyword(s):

Transforming Growth Factor ◽

Inflammatory Responses ◽

Retinoic Acid Receptor ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Treated Group ◽

Peritoneal Fibrosis ◽

Retinoic Acid Receptor Α ◽

Klf5 Expression

Abstract Background and Aims We presented previously that Am80, a synthetic retinoic acid receptor α specific agonist, inhibited the expression of Krüppel-like transcription factor 5 (KLF5) and reduced peritoneal fibrosis in mice. Now, we examined further detail about the mechanism to inhibit peritoneal fibrosis. Method Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. After 3 weeks of treatment, peritoneal tissues were examined using serial sections by immunohistochemistry to identify what kind of cells expressed KLF5. We also examined the effect of Am80 to inhibit peritoneal fibrosis in vitro. Results While KLF5 was expressed in the thickened submesothelial area of CG injected mice, Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening. The numbers of transforming growth factor β positive cells, α-smooth muscle actin (αSMA) or F4/80 positive cells were significantly decreased in Am80 treated group. KLF5 was expressed in αSMA, F4/80 or CD31 positive cells. Conclusion These results indicate the KLF5 might not only associate phenotypical differentiation from fibroblasts to myofibroblasts but also regulate inflammatory responses and angiogenesis in peritoneal fibrosis model. Am80 can suppress peritoneal fibrosis through inhibiting these mechanisms. In vitro experiments are ongoing.

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22-Oxacalcitriol Prevents Progression of Peritoneal Fibrosis in a Mouse Model

Peritoneal Dialysis International ◽

10.3747/pdi.2011.00234 ◽

2013 ◽

Vol 33 (2) ◽

pp. 132-142 ◽

Cited By ~ 17

Author(s):

Misaki Hirose ◽

Tomoya Nishino ◽

Yoko Obata ◽

Masayuki Nakazawa ◽

Yuka Nakazawa ◽

...

Keyword(s):

Vitamin D ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Treated Group ◽

The Body ◽

Type Iii Collagen ◽

Peritoneal Fibrosis ◽

Type Iii ◽

Peritoneal Tissue ◽

Vitamin D Levels

Objective Vitamin D plays an important role in calcium homeostasis and is used to treat secondary hyperparathyroidism among dialysis patients. The biologic activity of vitamin D and its analogs is mediated by vitamin D receptor (VDR), which is distributed widely throughout the body. Recent papers have revealed that low vitamin D levels are correlated with severe fibrosis in chronic diseases, including cystic fibrosis and hepatitis. The aim of the present study was to evaluate the protective effects of vitamin D against the progression of peritoneal fibrosis. Methods Peritoneal fibrosis was induced by injection of chlorhexidine gluconate (CG) into the peritoneal cavity of mice every other day for 3 weeks. An analog of vitamin D, 22-oxacalcitriol (OCT), was administered subcutaneously daily from initiation of the CG injections. The peritoneal tissue was excised at 3 weeks. Changes in morphology were assessed by hematoxylin and eosin staining. Expression of VDR, alpha smooth muscle actin (as a marker of myofibroblasts), type III collagen, transforming growth factor β (TGF-β), phosphorylated Smad2/3, F4/80 (as a marker of macrophages), and monocyte chemoattractant protein-1 (MCP-1) was examined by immunohistochemistry. Southwestern histochemistry was used to detect activated nuclear factor κB (NF-κB). Results In the CG-injected mice, immunohistochemical analysis revealed expression of VDR in mesothelial cells, myofibroblasts, and macrophages in the thickened submesothelial zone. Treatment with OCT significantly prevented peritoneal fibrosis and reduced the accumulation of type III collagen in CG-treated mice. Among the markers of fibrosis, the numbers of myofibroblasts, cells positive for TGF-β, and cells positive for phosphorylated Smad2/3 were significantly decreased in the OCT-treated group compared with the vehicle-treated group. Furthermore, OCT suppressed inflammatory mediators of fibrosis, as shown by the reduced numbers of activated NF-κB cells, macrophages, and MCP-1-expressing cells. Conclusions Our results indicate that OCT attenuates peritoneal fibrosis, an effect accompanied by reduced numbers of myofibroblasts, infiltrating macrophages, and TGF-β–positive cells, suggesting that vitamin D has potential as a novel therapeutic agent for preventing peritoneal sclerosis.

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Nanotechnology and adeno-associated virus-based decorin gene therapy ameliorates peritoneal fibrosis

AJP Renal Physiology ◽

10.1152/ajprenal.00653.2013 ◽

2014 ◽

Vol 307 (7) ◽

pp. F777-F782 ◽

Cited By ~ 16

Author(s):

Kunal Chaudhary ◽

Harold Moore ◽

Ashish Tandon ◽

Suneel Gupta ◽

Ramesh Khanna ◽

...

Keyword(s):

Gene Therapy ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Mesothelial Cell ◽

Peritoneal Fibrosis ◽

Tissue Samples ◽

Adeno Associated Virus ◽

Peritoneal Tissue ◽

Stage Renal Disease

Peritoneal dialysis (PD) is a life-sustaining therapy for end-stage renal disease (ESRD), used by 10–15% of the dialysis population worldwide. Peritoneal fibrosis (PF) is a known complication of long-term PD and frequently follows episodes of peritonitis, rendering the peritoneal membrane inadequate for dialysis. Transforming growth factor (TGF)-β is an inducer of fibrosis in several tissues and organs, and its overexpression has been correlated with PF. Animal models of peritonitis have shown an increase in expression of TGF-β in the peritoneal tissue. Decorin, a proteoglycan and component of the extracellular matrix, inactivates TGF-β, consequently reducing fibrosis in many tissues. Recently, gold nanoparticles (GNP) have been used for drug delivery in a variety of settings. In the present study, we tested the possibility that GNP-delivered decorin gene therapy ameliorates zymosan-mediated PF. We created a PF model using zymosan-induced peritonitis. Rats were treated with no decorin, GNP-decorin, or adeno-associated virus-decorin (AAV-decorin) and compared with controls. Tissue samples were then stained for Masson's trichrome, enface silver, and hematoxylin and eosin, and immunohistochemistry was carried out with antibodies to TGF-β1, α-smooth muscle actin (α-SMA), and VEGF. Animals which were treated with GNP-decorin and AAV-decorin gene therapy had significant reductions in PF compared with untreated animals. Compared with untreated animals, the treated animals had better preserved peritoneal mesothelial cell size, a significant decrease in peritoneal thickness, and decreased α-SMA. Quantitative PCR measurements showed a significant decrease in the peritoneal tissue levels of α-SMA, TGF-β, and VEGF in treated vs. untreated animals. This study shows that both GNP-delivered and AAV-mediated decorin gene therapies significantly decrease PF in vivo in a rodent model. This approach has important clinical translational potential in providing a therapeutic strategy to prevent PF in PD patients.

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P1226MITOCHONIC ACID 5 (MA-5) AMELIORATES CG-INDUCED PERITONEAL FIBROSIS IN MICE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1226 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Hiro Inoue ◽

Yoko Obata ◽

Takehiro Suzuki ◽

Miki Torigoe ◽

Kenta Torigoe ◽

...

Keyword(s):

Kidney Disease ◽

Mitochondrial Dysfunction ◽

Transforming Growth Factor ◽

Therapeutic Potential ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Parietal Peritoneum ◽

Disease Models ◽

Peritoneal Fibrosis ◽

Chemotactic Protein

Abstract Background and Aims Peritoneal fibrosis is one of important complications induced by long-term peritoneal dialysis. Mitochondrial dysfunction causes an increase of oxidative stress and depletion of ATP. Thus, it may be associated with a variety of disease including fibrosis in several organs. Recently, mitochonic acid 5 (MA-5) was synthesized and its therapeutic potential for mitochondrial dysfunction in kidney disease models has been reported. In this study, we investigated the effect of MA-5 for peritoneal fibrosis model in mice. Method Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3 weeks in C57/BL6 mice. MA-5 was administered at 2 mg/kg by gavage every day from the initiation of CG injection. Control mice received only a vehicle (distilled water). After 3 weeks of treatment, the animals were sacrificed and the peritoneal tissues were collected. The peritoneal sections were stained with Masson’s trichrome for light microscopic examination and the fibrotic thickening of parietal peritoneum was measured on the randomly selected different regions on each section. The expressions of F4/80, which is a marker of macrophages, monocyte chemotactic protein 1 (MCP1), α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) were evaluated by immunohistochemistry. Results The fibrotic thickening of parietal peritoneum was significantly attenuated in MA-5 treated mice compared with control mice (the thickness of submesothelial area: 100.24 +/- 13.67 vs 54.78 +/- 7.43 μm (p<0.05)). The numbers of TGF-β positive cells, α-SMA positive cells, MCP1 positive cells and infiltrating macrophages were significantly decreased in MA-5 treated mice than those in control mice. Conclusion These results suggest that MA-5 may have a therapeutic potential in the progression of peritoneal fibrosis as well as kidney disease models.

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Mineralocorticoid receptor blockade ameliorates peritoneal fibrosis in new rat peritonitis model

AJP Renal Physiology ◽

10.1152/ajprenal.00565.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. F1084-F1093 ◽

Cited By ~ 34

Author(s):

Hayato Nishimura ◽

Yasuhiko Ito ◽

Masashi Mizuno ◽

Akio Tanaka ◽

Yoshiki Morita ◽

...

Keyword(s):

Peritoneal Dialysis ◽

Mineralocorticoid Receptor ◽

Transforming Growth Factor ◽

Enzyme Inhibitor ◽

Smooth Muscle Actin ◽

Late Phase ◽

Type Iii Collagen ◽

Receptor Blockade ◽

Peritoneal Fibrosis ◽

Pai 1

Peritoneal fibrosis (PF) is an important complication of long-term peritoneal dialysis. Although mineralocorticoid and mineralocorticoid receptor (MR) have attracted increasing attention in the field of vascular injury, including the heart, kidney, and vessels, little is known about the role of mineralocorticoid in PF. This work was designed to explore the effects of MR blockade on PF. We developed a new model of PF in rats based on mechanical scraping of the peritoneum. This model is characterized by acute-phase inflammation (neutrophil and macrophage infiltration on days 0–3) and late-phase PF (α-smooth muscle actin-positive fibroblast infiltration, type III collagen accumulation, and neoangiogenesis on days 7–14). Peritoneal thickening peaked on day 14. MR was expressed in rat peritoneum and a rat fibroblast cell line. Expression of its effector kinase [serum- and glucocorticoid-induced kinase-1 (Sgk1)], transforming growth factor-β (TGF-β), plasminogen activator inhibitor-1 (PAI-1), and CD31-positive vessels increased during the course of PF. Rats were treated with spironolactone, angiotensin receptor blockade (ARB), or angiotensin-converting enzyme inhibitor (ACEI)-ARB-spironolactone starting at 6 h after peritoneal scraping. All parameters, including peritoneal thickening, number of macrophages and CD31-positive vessels, and expression of monocyte chemoattractant protein-1, TGF-β, PAI-1, and Sgk1, were significantly suppressed by spironolactone (10 mg·kg−1·day−1). The effects of spironolactone (10 and 20 mg·kg−1·day−1) were very similar to those of triple blockade. ARB, but not ACEI, significantly reduced peritoneal thickening. Furthermore, peritoneal function assessed by peritoneal equilibration test was significantly improved by spironolactone. Our results suggest that MR is a potential target to prevent inflammation-induced PF in patients on peritoneal dialysis.

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Nitro-oleic acid inhibits the high glucose-induced epithelial-mesenchymal transition in peritoneal mesothelial cells and attenuates peritoneal fibrosis

AJP Renal Physiology ◽

10.1152/ajprenal.00425.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. F457-F467

Author(s):

Wenyan Su ◽

Haiping Wang ◽

ZiYan Feng ◽

Jing Sun

Keyword(s):

Peritoneal Dialysis ◽

Oleic Acid ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Muscle Actin ◽

Peritoneal Fibrosis ◽

Mesenchymal Transition ◽

Peritoneal Mesothelial Cells

As an electrophilic nitroalkene fatty acid, nitro-oleic acid (OA-NO2) exerts multiple biological effects that contribute to anti-inflammation, anti-oxidative stress, and antiapoptosis. However, little is known about the role of OA-NO2 in peritoneal fibrosis. Thus, in the present study, we examined the effects of OA-NO2 on the high glucose (HG)-induced epithelial-mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs) and evaluated the morphological and immunohistochemical changes in a rat model of peritoneal dialysis-related peritoneal fibrosis. In in vitro experiments, we found that HG reduced the expression level of E-cadherin and increased Snail, N-cadherin, and α-smooth muscle actin expression levels in HPMCs. The above-mentioned changes were attenuated by pretreatment with OA-NO2. Additionally, OA-NO2 also inhibited HG-induced activation of the transforming growth factor-β1/Smad signaling pathway and NF-κB signaling pathway. Meanwhile, OA-NO2 inhibited HG-induced phosphorylation of Erk and JNK. The results from the in vivo experiments showed that OA-NO2 notably relieved peritoneal fibrosis by decreasing the thickness of the peritoneum; it also inhibited expression of transforming growth factor-β1, α-smooth muscle actin, N-cadherin, and vimentin and enhanced expression of E-cadherin in the peritoneum. Collectively, these results suggest that OA-NO2 inhibits the HG-induced epithelial-mesenchymal transition in HPMCs and attenuates peritoneal dialysis-related peritoneal fibrosis.

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Type of MRI contrast, tissue gadolinium, and fibrosis

AJP Renal Physiology ◽

10.1152/ajprenal.00379.2014 ◽

2014 ◽

Vol 307 (7) ◽

pp. F844-F855 ◽

Cited By ~ 24

Author(s):

Catherine Do ◽

Jeffrey L. Barnes ◽

Chunyan Tan ◽

Brent Wagner

Keyword(s):

Contrast Agents ◽

Nephrogenic Systemic Fibrosis ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Treated Group ◽

Mri Contrast ◽

Cultured Fibroblasts ◽

Low K

It has been presupposed that the thermodynamic stability constant ( Ktherm) of gadolinium-based MRI chelates relate to the risk of precipitating nephrogenic systemic fibrosis. The present study compared low- Ktherm gadodiamide with high- Ktherm gadoteridol in cultured fibroblasts and rats with uninephrectomies. Gadolinium content was assessed using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy in paraffin-embedded tissues. In vitro, fibroblasts demonstrated dose-dependent fibronectin generation, transforming growth factor-β production, and expression of activated myofibroblast stress fiber protein α-smooth muscle actin. There were negligible differences with respect to toxicity or proliferation between the two contrast agents. In the rodent model, gadodiamide treatment led to greater skin fibrosis and dermal cellularity than gadoteridol. In the kidney, both contrast agents led to proximal tubule vacuolization and increased fibronectin accumulation. Despite large detectable gadolinium signals in the spleen, skin, muscle, and liver from the gadodiamide-treated group, contrast-induced fibrosis appeared to be limited to the skin and kidney. These findings support the hypothesis that low- Ktherm chelates have a greater propensity to elicit nephrogenic systemic fibrosis and demonstrate that certain tissues are resistant to these effects.

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Increased expression of epimorphin in a peritoneal fibrosis mouse model

Peritoneal Dialysis International ◽

10.1177/08968608211051572 ◽

2021 ◽

pp. 089686082110515

Author(s):

Muneharu Yamada ◽

Yohei Hirai ◽

Dan Inoue ◽

Shuhei Komatsu ◽

Takahiro Uchida ◽

...

Keyword(s):

Growth Factor ◽

Real Time ◽

Transforming Growth Factor ◽

Cultured Cells ◽

Smooth Muscle Actin ◽

Growth Factor Receptor ◽

Transforming Growth Factor Β ◽

Rt Pcr ◽

Peritoneal Fibrosis ◽

Compact Zone

Background: Long-term peritoneal dialysis results in functional and histopathological alterations of the peritoneal membrane, leading to peritoneal fibrosis (PF). The mechanism of PF has not been fully elucidated, and at present there is no effective therapy for PF. Epimorphin is a mesenchymal protein that not only regulates morphogenesis in organ development but is implicated in tissue repair. However, the role of epimorphin in PF has not yet been clarified. Methods: PF was induced in C57/Bl6 mice by intraperitoneal injection of chlorhexidine gluconate (CG-injected mice) three times a week for 3 weeks. The parietal peritoneum was subsequently dissected and assessed by Masson’s trichrome staining, and epimorphin expression was analysed by immunohistochemistry and real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, epimorphin-positive regions were analysed by multiple immunofluorescence staining using fibrosis-associated markers. In addition, normal rat fibroblast cells (NRK-49F) were treated with transforming growth factor-β (TGF-β) in the presence or absence of epimorphin. The expression of fibrosis-associated markers was assessed by real-time RT-PCR. Results: In CG-injected mice, Masson’s trichrome staining showed marked thickening of the submesothelial compact zone. Weak epimorphin expression was observed in the narrow submesothelial compact zone beneath the mesothelial cells in control mice; however, epimorphin expression was stronger in the submesothelial compact zone in CG-injected mice. Epimorphin expression was observed mainly in α-smooth muscle actin (α-SMA)-positive myofibroblasts. Epimorphin suppressed the TGF-β-induced upregulation of α-SMA and platelet-derived growth factor receptor-β in cultured cells. Conclusions: Our results suggest that epimorphin may be a therapeutic target for fibrotic diseases of the peritoneum.

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The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22041861 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1861

Author(s):

Jemima Seidenberg ◽

Mara Stellato ◽

Amela Hukara ◽

Burkhard Ludewig ◽

Karin Klingel ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Beclin 1 ◽

Type I ◽

Alpha Smooth Muscle Actin

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. Methods and Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

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