Nestin+ Cells Isolated From the Peritoneum Ameliorate Peritoneal Fibrosis Development

doi:10.21203/rs.3.rs-1398817/v1

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Nestin+ Cells Isolated From the Peritoneum Ameliorate Peritoneal Fibrosis Development

https://doi.org/10.21203/rs.3.rs-1398817/v1

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The intermediate filament protein Nestin, a specific marker for multifunctional, multilineage progenitor cells, is regulated during the progression of reparative and reactive fibrosis in the liver, heart and kidney. In this study, Nestin was found in both the mouse peritoneum and HPMCs derived from PD effluent (PDE) and HMrSV5. Moreover, these Nestin⁺PMCs showed extensive proliferation for more than 20 passages. The mice that received chronic peritoneal dialysis solution (PDS) infusions showed typical features of peritoneal fibrosis (PF), including substantially increased peritoneal thickness, excessive matrix deposition, increased peritoneal permeability, and increased expression levels of α-smooth muscle actin (α-SMA) and collagen I (Col I). Nestin⁺cells isolated from the peritoneum could significantly ameliorate these pathological changes. A parallel decrease in interleukin-33 (IL-33) and ST-2 accumulation in the peritoneum of Nestin⁺ cell-transplanted mice was observed. In addition, downregulation of IL-33 expression decreased

TGF-β1-induced accumulation of extracellular matrix (ECM) in the HPMCs. In conclusion, our findings discover a novel mechanism of Nestin⁺cells in PF, that is, Nestin⁺cells isolated from the peritoneum probably had a protective effect on high-glucose PDS-induced PF by suppressing IL-33/ST-2 signaling.

IL-33

Nestin

Peritoneal dialysis

Peritoneal fibrosis

Nestin is expressed in the peritoneum

IL-33 positively correlates with the extent of peritoneal fibrosis

Nestin⁺cells delay the process of peritoneal fibrosis probably by suppressing IL-33/ST-2 signaling

Peritoneal dialysis (PD) has been widely used as an effective treatment for end-stage renal disease (ESRD) in which the peritoneal mesothelium acts as a physical barrier with the capacity to trap bacteria and molecules [1, 2]. However, the prolonged exposure of peritoneal mesothelial cells (PMCs) in the peritoneal membrane to glucose-based PD solution (PDS) can cause PMC damage and loss, peritoneal fibrosis (PF), ultrafiltration failure and eventual discontinuation of PD [3–5]. Several therapeutic strategies for the treatment of PF, including the use of pharmacological strategies and the transplantation of mesenchymal stem cells to inhibit the mesothelial-to-mesenchymal transition (EMT), have shown promise [6–8]. However, these therapies cannot prevent PMC loss, which plays a critical role in causing PF [9]. Previous studies have suggested that PMCs showed strong labeling with antibodies against Nestin, which is a specific marker for multifunctional, multilineage progenitor cells, indicating that these cells represent a “young”, not entirely differentiated cell population [10]. The intermediate filament protein Nestin is widely used as a marker of neural stem cells [11]. More importantly, Nestin is also expressed in some adult stem/progenitor cell populations, indicating that Nestin might be a common marker of multipotent stem cells [12]. Under specific damage conditions, upregulation of Nestin expression was found in tubular cells, podocytes, interstitial cells and fibrotic rat hearts [13–15]. More importantly, Nestin⁺ cells isolated from the kidney showed the characteristics of mesenchymal stem cells (MSCs), which can repair acute kidney injury [16].

IL-33 is expressed in various tissues and cells in the human body, including endothelial cells, bronchial cells, and intestinal epithelial cells [17]. ST2 is the only known receptor for IL-33, recent reports have demonstrated that IL-33 promotes ST-2-dependent organ tissue fibrosis in several animal models, including lung [18], pancreas [19], and liver models [20].

However, the ability of Nestin⁺ cells isolated from the peritoneum to regulate PF has not been evaluated to our knowledge. In this study, cells isolated from the peritoneum of Nestin-GFP transgenic mice were transplanted into C57BL/6 wild-type mice, and the adeno-associated virus (AAV)-RNAi-IL-33 was used to infect to HPMCs. The effects and mechanism of Nestin⁺ cells were investigated in an established PF model.

2.1. Animal studies

The C57BL/6 mice were purchased from Wenzhou Medical University Laboratory Animal Center (Wenzhou, China) and all of the animal work had taken place there. Homozygous transgenic mice that expressed enhanced GFP under the control of a Nestin promoter (Nestin-GFP, on the C57BL/6 genetic background) were provided by Cyagen Biosciences. The mice had free access to a standard rodent diet and tap water and were anaesthetized with 40mg/kg pentobarbital sodium through an intraperitoneal injection before sacrifice. This study has been carried out in accordance with the World Medical Association Declaration of Helsinki, and that all subjects provided written informed consent.

We used male C57BL/6 mice at 8 weeks of age. C57BL/6 mice were divided into three groups: the control group, which received intraperitoneal administration of normal saline; the PD model mouse group without treatment, which received an intraperitoneal administration of PDS (4.25% Dianeal; Deerfield, IL, Baxter, USA) at 10 ml/100 g^− 1·d^− 1 for 4 weeks only; and the treatment group, PD model mice transplanted with Nestin⁺ cells.

A peritoneal equilibration test (PET) was applied to evaluate the peritoneal permeability function 28 days after modeling. The mice were instilled with PDS at 10 ml/100 g body weight before being killed. After 30 min, the peritoneal fluid was removed [8], and orbital sinus blood samples were collected. Peritoneal solute transport was calculated by D30/D0 and D/P_urea. D30 was defined as the glucose concentration in the dialysate sample at 30 min, and D0 was defined as the initial dialysate glucose concentration. D indicates the dialysate urea concentration, and P_urea indicates the plasma urea concentration. Then, the parietal peritoneum, omentum, and diaphragm were carefully dissected for use in western blots and immunofluorescence staining.

2.2. Isolation and Nestin-GFP⁺ cell transplantation

The peritoneum was dissected from 2-week-old Nestin-GFP mice and cut into small pieces. The PMCs were then dissociated from the visceral peritoneum by digestion with 0.25% pancreatin at 37°C for 15 min, and Eagle’s medium (DMEM)/F12 (Invitrogen) containing 10% fetal bovine serum (FBS, Gibco) was added to stop the pancreatin activity. The samples were centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was aspirated. The centrifugal sediment was resuspended and passed through a 70 µm strainer, which resulted in single cells. Flow cytometry was used to sort these PMCs expressing GFP from the suspension.

In the treatment group, after 1 week of intraperitoneal injection of PDS, Nestin-GFP⁺ PMCs (2×10⁶ cells) were suspended in 2.5 ml of 4.25% PDS and injected intraperitoneally. At 28 days of injection, the mice were sacrificed, and the peritoneal dialysis effluent(PDE), parietal peritoneum and omentum were collected.

2.3. Culture of PMCs from PDE and PMC lines

Human PMCs (HPMCs) harvested from PDE from patients with ESRD who underwent placement of PD catheters for less than 1 month were used for the culture. PDE was drained into a 50 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. Cell pellets were suspended in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco, NY, USA) with 10% fetal bovine serum (FBS, Gibco, NY, USA) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin, Gibco, NY, USA). Then, suspended cells were seeded into a 25-cm² culture flask (Corning, Armonk, NY, USA).

Several antibodies were used to examine every batch of initially isolated mesothelial cells to ensure that they were positive for the mesothelial markers cytokeratin 18 (CK 18, Abcam, Cambridge, MA, USA) and vimentin (Abcam, Cambridge, MA, USA). HPMCs were used at passages 3–5. The HPMC line (HMrSV5) was cultured in the same medium mentioned above.

The HPMCs were divided into four groups: a normal control group, with a normal glucose concentration (17.5 mM); a group treated with transforming growth factor β1 (TGF-β1, MCE, NJ, UAS) for varying durations (6, 12, 24 and 48 h) to identify the optimal time, after which the most effective concentrations were determined (1, 5, and 10 nM); a group with co-stimulation of TGF-β1 and AAV-RNAi-Vector infection and a group with co-stimulation of TGF-β1 and AAV-RNAi-IL-33. At the end of the treatments, the cells were collected and subjected to western blot analyses and immunofluorescence staining.

2.4. Immunofluorescence

Histology and immunofluorescence staining of 4-µm paraffin sections from the anterior or posterior peritoneal tissues were performed as described previously [21]. For identification of the HPMCs, the cells from the PDE were stained for CK 18 and vimentin. For confirmation of the expression of Nestin in the peritoneum, human peritoneum samples obtained from patients undergoing abdominal surgery were assessed by immunofluorescence staining. The following primary antibodies were used: rabbit monoclonal anti-vimentin antibody (Abcam, Cambridge, MA, USA), mouse monoclonal anti-CK 18 antibody (Abcam, Cambridge, MA, USA), rabbit polyclonal anti-collagen I (Col-I) antibody (Abcam, Cambridge, MA, USA), and anti-α-smooth muscle actin (α-SMA) antibody (Abcam, Cambridge, MA, USA).

2.5. Hematoxylin-eosin staining and Masson’s staining.

The parietal peritoneum specimens from the 3 groups of mice were soaked in paraformaldehyde solution and then prepared for hematoxylin-eosin and Masson’s staining as previously described [22].

2.6. Western blot analysis

Sample collection and immunoblotting were performed as previously described [21]. The primary antibodies used were rabbit polyclonal anti-Nestin antibody (Abcam, Cambridge, MA, USA), anti-α-SMA (Abcam, Cambridge, MA, USA), anti-Col I (ABclonal, Wuhan, China), anti-ST-2 (ABclonal, Wuhan, China), IL-33 (ABclonol, Wuhan, China) and mouse monoclonal anti-β-actin (Thermo Scientific, Waltham, USA). The secondary antibodies used in this study were horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Cell Signaling Technology, Danvers, MA, USA) antibody and goat anti-mouse immunoglobulin G (Cell Signaling Technology, Danvers, MA, USA).

2.7. RNA extraction and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR)

Total RNA from the peritoneum samples collected from different week-old Nestin-GFP mice was extracted using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Real-time PCR for Nestin and β-actin mRNA expression was performed using the FastStart Universal SYBR Green Master (ROX) kit (Roche, Basel, Switzerland) with an ABI Prism 7900HT Sequence Detection System (Life Technology, Carlsbad, CA, USA) as previously described [21].

The mRNA expression levels of Nestin were measured using β-actin as an internal reference gene. The primers included the following: mouse-Nestin forward 5′-TCG CCA GGG AGG AGG CCA TT-3′ and reverse 5′-CTC CCC AGC CCT CCC CAG AC-3′; mouse-β-actin forward 5′-AGA GGG AAA TCG TGC GTG AC-3′ and reverse 5′-CAA TAG TGA TGA CCT GGC CGT-3′. Relative expression was calculated using the 2^−ΔΔCT method.

2.8. Cytokine assays

IL-33 concentrations were determined in cell culture supernatants and PDS by ELISAs following the manufacturer’s instructions (Elabscience Biotechnology, Wuhan, China).

2.9. Statistical analysis

The results are presented as the mean ± SD for continuous variables. Comparisons were made using analysis of variance followed by Tukey’s post hoc test. SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. A p-value < 0.05 was considered statistically significant.

3.1. Identification of cells isolated from PDE as HPMCs

PDE was derived from 4 uremic patients who underwent peritoneal dialysis therapy in one month. Among these 4 patients, 1 had gout, 1 had chronic glomerulonephritis, and 2 showed hypertensive nephropathy. In PDE-derived cultures, human PMCs (HPMCs) detached from the PDE generally grew well in DMEM/F12 medium containing 10% FBS and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). Suspended cells adhered to the dish within 1 day of seeding. Active proliferation occurred if the cell number was sufficient for the neighboring adherent cells to be in contact with each other. A confluent monolayer was reached at approximately 1 week, comprising passage 1. Cells isolated from the PDE presented a stellate or fusiform appearance with good transparency prior to confluency (Fig. 1A) and developed a typical cobblestone appearance after 3 passages (Fig. 1B). The phenotype of the PMCs was confirmed by immunofluorescence staining. The immunofluorescence study revealed that a proportion of the cells derived from the PDE showed positive staining in their cytoplasm with both the anti-vimentin and anti-CK 18 antibodies (Fig. 1C). PDE-derived HPMCs could be passaged for more than 20 generations.

3.2. Nestin is expressed in the PMCs and the peritoneum

As a representative of Nestin expression, GFP fluorescence was observed in the peritoneum of Nestin-GFP transgenic mice via microscopic examination (Fig. 1D). Nestin was expressed on the HPMCs derived from the PDE (Fig. 1E) and HMrSV5, as shown by immunofluorescence (Fig. 1F). The Nestin mRNA levels were also observed using RT-PCR analyses of the peritoneum of 1-, 2-, 4-, and 12-week-old mice (Fig. 1G). Thus, Nestin is continuously present in neonates and adults in the peritoneum.

3.3. Treatment with Nestin⁺ cells isolated from the peritoneum attenuates peritoneal fibrosis in mice

Hematoxylin-eosin staining (Fig. 2A) was used to evaluate changes in cell density, and Masson’s staining (Fig. 2B) was used to assess peritoneal thickening. We found that an intraperitoneal administration of 4.25% PDS at 10 ml/100 g^− 1·d^− 1 for 4 weeks induced an increase in the cellularity of the submesothelial compact zone (84.67 ± 6.51 vs. 23.33 ± 6.11 cells×100/mm², P < 0.001) and thickness of this zone on day 28 (127.07 ± 20.94 vs. 42.93 ± 9.19 µm, P < 0.001). Both the cell density of the collagenous compact zone (52.00 ± 6.08 vs. 84.67 ± 6.51 cells×100/mm², P < 0.001) and the thickness of this zone (88.97 ± 7.52 vs. 127.07 ± 20.94 µm, P = 0.015) were significantly decreased in mice treated with Nestin⁺ cells (2x10⁶ cells) injected intraperitoneally 1 week after 4.25% PDS stimulation compared with mice given the PDS only(Fig. 2C, D).

3.4. Nestin⁺ cells isolated from the peritoneum suppress the expression of Col I and α-SMA in mice

Peritoneal fibrosis is typically characterized by increased expression levels of α-SMA and Col I.After intraperitoneal administration of 4.25% PDS, both α-SMA and Col-I were increased in the submesothelial compact zone on day 28 (Fig. 3). However, treatment with Nestin⁺ cells isolated from the peritoneum significantly reduced the area in which α-SMA and Col-I accumulated on day 28 compared with treatment with PDS only, as shown by western blots.

3.5. Nestin⁺ cells isolated from the peritoneum reduce the functional impairments of the peritoneal membrane in mice with PF

A PET was performed on day 28 to assess the functional alteration of the peritoneal membrane. The absorption rate of glucose from the dialysate (0.20 ± 0.04 vs. 0.46 ± 0.05, P < 0.001) (Fig. 4A)and the transport rate of blood urea from the plasma (1.12 ± 0.15 vs. 0.62 ± 0.06, P = 0.001) (Fig. 4B) were significantly higher in mice given the PDS only than in control mice, whereas these parameters were significantly improved in PDS-injected mice treated with Nestin⁺ cells isolated from the peritoneum (Fig. 4A, B) (0.42 ± 0.04 vs. 0.20 ± 0.04, P = 0.001; 0.75 ± 0.07 vs. 1.12 ± 0.15, P = 0.004).

3.6. Nestin+ cells isolated from the peritoneum suppress the expression of IL-33 and ST-2 in mice with PF

The fibrosis-related IL-33 was significantly increased in the PDE of mice with PDS-induced PF by day 28 compared with that of the control mice (116.50 ± 16.97 vs. 11.83 ± 3.25 pg/ml, p < 0.001) (Fig. 4C). Meanwhile, the expression of ST-2 was increased obviously in the mice with PDS only (Fig. 3). Treatment with Nestin⁺ cells isolated from the peritoneum significantly suppressed the secretion of IL-33 (49.33 ± 9.98 vs. 116.50 ± 16.97 pg/ml, p = 0.001) and expression of ST-2 compared with treatment with PDS only, it indicates Nestin + cells probably can suppress IL-33/ST-2 signaling in mice with PF.

3.7. Effects of different TGF-β1 concentrations and durations on IL-33 secretion in HPMCs

HPMCs were stimulated by different concentrations of TGF-β1 (0, 1, 5, and 10 nM) for 24 h. The levels of IL-33 increased significantly when HPMCs (both from PDE and HMrSV5) were stimulated with 5 nM and 10 nM (Fig. 5A,) compared with those of the control group. Then, HPMCs were stimulated with TGF-β1 for different durations (6, 12, 24 and 48 h) at a concentration of 5 nM. The levels of IL-33 showed the strongest changes in HPMCs stimulated with TGF-β1 at 5 nM for 48 h compared with those of the control group (Fig. 5B). Thus, a concentration of 5 nM and a duration of 48 h were selected for further use.

3.8. Downregulation of IL-33 expression decreases TGF-β1-induced EMT of HPMCs

After HPMCs were stimulated with 5 nM/ml TGF-β1 for 48 h, western blot (Fig. 6) and immunofluorescence analyses (Fig. 7A, B and C) showed that the levels of α-SMA and Col-I were significantly increased compared with those of the control group. Furthermore, in the AAV-RNAi-IL-33 group, α-SMA and Col-I were decreased significantly compared with those of the AAV-RNAi-vector group. There were no significant changes in the levels of α-SMA and Col-I between the HPMCs incubated with TGF-β1 only and cells incubated with the AAV-RNAi-vector.

3.9. Downregulation of IL-33 expression may downregulates TGF-β1 induced IL-33/ST2 signaling in HPMCs

HPMCs from PDE were infected with AAV-RNAi-vector and AAV-RNAi-IL-33 and then stimulated with TGF-β1 at 5 ng/ml for 48 h. Compared to the control group, the levels of IL-33 (Fig. 6, 7B) and ST-2 (Fig. 6) were significantly increased following TGF-β1 stimulation. There were no significant changes in the levels of IL-33 and ST-2 between the HPMCs incubated with TGF-β1 only and the cells incubated with the AAV-RNAi-vector. AAV-RNAi-IL-33 resulted in a stronger decrease in these levels than AAV-RNAi-vector (Fig. 6).

This study provided the first evidence that Nestin⁺ cells isolated from the peritoneum have a clear protective effect on high-glucose PDS-induced PF through suppression of IL-33 secretion. Intraperitoneal administration of Nestin⁺ cells isolated from the peritoneum of Nestin-GFP mice through flow cytometric sorting ameliorated PDS-induced PF in mice, as evidenced by a significant reduction in peritoneal thickening and collagen deposition. In addition, Nestin⁺ cells significantly decreased IL-33 secretion in mice with PF, indicating that PF was reduced through suppression of IL-33. Moreover, transplanted Nestin⁺ cells obviously reduced the functional impairments of the peritoneal membrane in mice with PF. Furthermore, downregulation of IL-33 expression could decrease the expression of ST-2, α-SMA and Col-I in HPMCs, indicating that Nestin delays the course of EMT in HPMCs induced by TGF-β1. These results probably indicate that the delivered Nestin⁺ cells isolated from the peritoneum could protect mice from PF impairment and against peritoneal function damage after PDS injury by suppressing IL-33/ST-2 signaling.

Nowadays, the culture method of primary HPMCs is mainly isolated from peritoneal tissue of clinical patients; however, it is difficult to obtain the surgical materials and it involves problems of medical ethics[30, 31]. The other method to obtain HPMCs is from the dialysate of peritoneal dialysis patients[31, 32], which is simple and easy to popularize and apply. In this study, we collected peritoneal exudates from uremia patients within 1 month after peritoneal dialysis catheterization and cultured in vitro, and established a method for extracting HPMCs from peritoneal dialysis solution. The HPMCs extracted in our study came from the patients who underwent peritoneal dialysis catheterization recently, so their HPMCs can maintain good functional status, which is assured more conducive to its pathophysiological study. Previous studies have found that these HPMCs from peritoneal effusion adhering to the wall were more slowly than the primary cells from the omentum, and the cells will lose their vitality and cannot fuse after four passages[30]. In this study, we found that HPMCs derived from peritoneal effusion could be passaged for more than 20 generations, which may be related to the fact that the primary HPMCs in this study came from peritoneal exudates of patients with peritoneal dialysis catheterization recently and the cells in this period have better functional status. Thus, peritoneal dialysis effluent can represent a good quality and reliable source for the culture of HPMCs.

Nestin is widely known as a marker of neural stem cells [12]. More importantly, this protein labels adult proliferating and stem/progenitor cell populations, indicating that Nestin is a specific marker of multifunctional progenitor cells [12]. Rats PMCs showed strong labeling with this antibody [10]. Similarly, in the present study, both HPMCs and mouse peritoneum strongly expressed Nestin, indicating that these cells represent a ‘young’, not entirely differentiated cell population. We also confirmed that the cultured PMCs from the PDE grew well even after 20 passages, which implied that these cells possessed a strong proliferative ability. Nestin expression was upregulated in cardiac, [15] renal [23] and lung ([24]) fibrosis. However, few studies have investigated the effects of Nestin⁺ PMCs on PF. Furthermore, we prospectively isolated Nestin⁺ cells from the peritoneum based on the GFP fluorescence intensity of Nestin-GFP mice. Excitingly, Nestin⁺ cells could suppress the expression of Col I and α-SMA and reduce the functional impairments of the peritoneal membrane in mice with PF, eventually leading to delayed PF progression.Therefore, Nestin⁺ cells isolated from the peritoneum can exert a protective effect on high-glucose PDS-induced PF.

IL-33 is a major cytokine in the modulation of fibrosis [25]. Furthermore, recent reports have confirmed that IL-33 promotes ST-2-dependent organ tissue fibrosis, such as lung [26], liver [27], and kidney fibrosis [28]. Conversely, IL-33 prevented global myocardial periarteriolar fibrosis in pressure-overloaded left ventricles [29]. However, whether and how this cytokine can modulate PF remains unclear. To address the possible mechanism of PF induced by PDS, we detected the levels of IL-33 in different groups and found that secretion of IL-33 was obviously increased in mice with PF, indicating that IL-33 positively correlates with the extent of PF. Furthermore, Nestin⁺ cells could reduce the secretion of IL-33, eventually leading to the delayed progression of PF. In addition, downregulation of Nestin expression decreased TGF-β1-induced EMT in HPMCs. Therefore, Nestin⁺ PMCs could protect against high-glucose PDS-induced PF by depressing IL-33 secretion. Moreover, a parallel decrease in IL-33 and ST-2 accumulation in the peritoneum of Nestin + cell-transplanted mice was observed. Therefore, the possible mechanism underlying the protective effects of Nestin + cells on peritoneal dialysis solution-induced PF is suppressing IL-33/ST-2 signaling,but the evidence is not sufficient and needs to be further studied.

In this study, we have applied Nestin + cells isolated from the peritoneum with the ability to suppress IL-33. The application of Nestin + cells isolated from the peritoneum effectively delayed the progression of high-glucose PDS-induced PF demonstrated by attenuating peritoneal fibrosis and the reduced expression of fibrotic markers. Nestin + Cells isolated from the peritoneum rise as a promising platform to enhance stem cell-based therapeutic strategy, which might be an effective approach for PF therapy.

PDS: peritoneal dialysis solution; PD: peritoneal dialysis; PF: peritoneal fibrosis; HPMCs: human peritoneal mesothelial cells; ECM: extracellular matrix; PDE: peritoneal dialysis effluent; α-SMA: α-smooth muscle actin; Col I: collagen I; IL-33:interleukin-33; ESRD: end-stage renal disease; PMCs: peritoneal mesothelial cells; PDS: peritoneal dialysis solution; EMT: mesothelial-to-mesenchymal transition; MSCs: multipotent stem cells; PET: peritoneal equilibration test; DMEM: Dulbecco's modified Eagle medium; FBS: fetal bovine serum; CK 18:cytokeratin 18; PCR: polymerase chain reaction; FBS: fetal bovine serum; TGF-β:transforming growth factor-β.

Acknowledgements

We thank the First Affiliated Hospital of Wenzhou Medical University for providing the PDE. The authors are also grateful to this article’s reviewers and editor.

Author’s contributions

Performed the experiments and analyzed the data: SMY, NJ and SQM. Participated in manuscript preparation: SMY, NJ and SMY. Detection of immunofluorescence, western blot and real-time PCR: YZ, YPST, NJ and ZYZ. Detection of HE staining and Masson’s staining: XYL, HC, DCY and HCJ. Analyzed and interpreted the data: CSC, CFZ and YHB. Participated in the experimental design and coordination: CSC and CFZ. Conceived the project, supervised the experiments, and analyzed data: CSC and CFZ. Wrote the manuscript: SMY, NJ and SQM.

Funding

This work was supported by grants from the Wenzhou Municipal Science and Technology Bureau (Y20210162), the Natural Science Foundation of Zhejiang Province (LQ19H050002), and the National Natural Science Foundation of China (31900685 and 81772264).

Conflict of interests

None.

Ethics approval and consent to participate

The use of laboratory animals and human PD effluent (PDE) in this study was approved by the Ethical Committee of Wenzhou Medical University and Laboratory Animal Center, Wenzhou Medicine University, according to the licenses for use of experimental animals issued by the Zhejiang Ministry of Justice.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated for this study are available on request from the corresponding author.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Nestin+ Cells Isolated From the Peritoneum Ameliorate Peritoneal Fibrosis Development

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials And Methods

2.1. Animal studies

2.2. Isolation and Nestin-GFP⁺ cell transplantation

2.3. Culture of PMCs from PDE and PMC lines

2.4. Immunofluorescence

2.5. Hematoxylin-eosin staining and Masson’s staining.

2.6. Western blot analysis

2.7. RNA extraction and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR)

2.8. Cytokine assays

2.9. Statistical analysis

3. Results

3.1. Identification of cells isolated from PDE as HPMCs

3.2. Nestin is expressed in the PMCs and the peritoneum

3.3. Treatment with Nestin⁺ cells isolated from the peritoneum attenuates peritoneal fibrosis in mice

3.4. Nestin⁺ cells isolated from the peritoneum suppress the expression of Col I and α-SMA in mice

3.5. Nestin⁺ cells isolated from the peritoneum reduce the functional impairments of the peritoneal membrane in mice with PF

3.6. Nestin+ cells isolated from the peritoneum suppress the expression of IL-33 and ST-2 in mice with PF

3.7. Effects of different TGF-β1 concentrations and durations on IL-33 secretion in HPMCs

3.8. Downregulation of IL-33 expression decreases TGF-β1-induced EMT of HPMCs

3.9. Downregulation of IL-33 expression may downregulates TGF-β1 induced IL-33/ST2 signaling in HPMCs

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Status:

Version 1

Nestin+ Cells Isolated From the Peritoneum Ameliorate Peritoneal Fibrosis Development

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials And Methods

2.1. Animal studies

2.2. Isolation and Nestin-GFP+ cell transplantation

2.3. Culture of PMCs from PDE and PMC lines

2.4. Immunofluorescence

2.5. Hematoxylin-eosin staining and Masson’s staining.

2.6. Western blot analysis

2.7. RNA extraction and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR)

2.8. Cytokine assays

2.9. Statistical analysis

3. Results

3.1. Identification of cells isolated from PDE as HPMCs

3.2. Nestin is expressed in the PMCs and the peritoneum

3.3. Treatment with Nestin+ cells isolated from the peritoneum attenuates peritoneal fibrosis in mice

3.4. Nestin+ cells isolated from the peritoneum suppress the expression of Col I and α-SMA in mice

3.5. Nestin+ cells isolated from the peritoneum reduce the functional impairments of the peritoneal membrane in mice with PF

3.6. Nestin+ cells isolated from the peritoneum suppress the expression of IL-33 and ST-2 in mice with PF

3.7. Effects of different TGF-β1 concentrations and durations on IL-33 secretion in HPMCs

3.8. Downregulation of IL-33 expression decreases TGF-β1-induced EMT of HPMCs

3.9. Downregulation of IL-33 expression may downregulates TGF-β1 induced IL-33/ST2 signaling in HPMCs

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Status:

Version 1

2.2. Isolation and Nestin-GFP⁺ cell transplantation

3.3. Treatment with Nestin⁺ cells isolated from the peritoneum attenuates peritoneal fibrosis in mice

3.4. Nestin⁺ cells isolated from the peritoneum suppress the expression of Col I and α-SMA in mice

3.5. Nestin⁺ cells isolated from the peritoneum reduce the functional impairments of the peritoneal membrane in mice with PF