Compare the effects of intravenous and intraperitoneal mesenchymal stem cell transplantation on ultrafiltration failure in a rat model of chronic peritoneal dialysis

Abstract

Aim: The purpose of this study was to compare the possible healing effects of intraperitoneal (IP) and intravenous (IV) mesenchymal stem cell (MSC) transplantation on ultrafiltration failure (UFF) in a chronic rat model of peritoneal dialysis (PD). Methods: Rats were initially divided into two groups. The UFF-group received once-daily IP injections of 20 mL of 3.86% glucose PD solution for six weeks to stimulate the development of UFF, and a control group received no injections. The UFF group was sub-divided into four groups: an UFF-C group, a MSC-IP group, a MSC-IV group and a placebo (P) group. Peritoneal equilibration tests (PETs) and peritoneal biopsies were performed in the control and UFF-C groups. MSCs were administered by IP injection in the MSC-IP group and by IV injection in the MSC-IV group. The P group received IP injection of placebo. PETs and peritoneal biopsies were performed in the MSC-IP, MSC-IV and P groups at the three weeks after receiving MSCs or placebo. Results: When compared with the control group, ultrafiltration capacity significantly decreased, and the submesothelial thickness increased in the UFF-C and P group, but there were no differences between the control and MSC-IP and MSC-IV groups. The rate of glucose transport was high in the UFF-C and P group compared with the control group, and D/P_Cr rates in the UFF-C and P group were lower than in the control group. However, D/D0_glucose was higher and D/P_Cr was lower in the MSC-IP group than in the UFF-C and P groups, but D/D0_glucose rate of MSC-IV group similar to UFF-C and P groups and there was no difference between MSC-IV group and the other groups in terms of D/P_Cr rates. The MSC-IP, MSC-IV and P groups had significantly decreased tumor necrosis factor α concentrations compared with the UFF-C group. MSC-IP group had lower levels of TGF-β1 compared with the P group; MSC-IP group had also lower levels of interleukin-6 compared with UFF-C group. Conclusion: The UFF group had a high permeability UFF. These results showed that IV and IP MSC transplantation exerted positive effects on UFF in a chronic rat model of PD. However, healing effect of small solute transport in MSC-IP group was better than MSC-IV group. IP MSC transplantation may be more effective than IV MSC transplantation for the renewal of the peritoneum in chronic PD patients with UFF.

• Chronic peritoneal dialysis
• intraperitoneal MSC transplantation
• intravenous MSC transplantation
• ultrafiltration failure

Introduction

Peritoneal dialysis (PD) has been used as a beneficial treatment for end-stage kidney disease. However, peritoneal damage remains a serious complication of long-term PD that leads to marked functional and morphological alterations of the peritoneal membrane.1,2

Peritoneal fibrosis arises in response to a variety of injurious factors, including hyperosmolar and acidic bioincompatible dialysate components, uremic toxins, episodes of infectious peritonitis and chronic inflammation.3–5 These changes include loss of mesothelial cell mass, marked interstitial fibrosis and alterations in the structure and number of peritoneal blood vessels, and a progressive increase in the thickness of the submesothelial compact zone.6,7 These PD-induced changes can eventually result in a gradual increase in peritoneal small solute transport rates, resulting in ultrafiltration failure (UFF).1,2,8

The epithelial-to-mesenchymal transition as a possible mechanism has been identified.9,10 At the molecular level, there is increasing evidence that a number of cytokines [interleukin (IL)-6 and tumor necrosis factor α (TNF-α)], growth factors [TGF-β1 and vascular endothelial growth factor (VEGF)] and prostaglandins play key roles in regulating and sustaining these processes PD leading to failure of peritoneal membrane function.6 Studies to reduce peritoneal fibrosis have included the intraperitoneal (IP) or systemic application of some drugs.11–18 These therapies could be useful for treating peritoneal fibrosis, whereas clinically available treatment for peritoneal fibrosis in PD patients remains limited at present.19–22

Mesenchymal stem cells (MSCs) are a multipotent cell source that can be obtained from the bone marrow and many other tissues. They initiate the remodeling process by replacing damaged tissues and cells and appear to function through paracrine mechanisms.23,24 A few reports showed that IP MSCs transplantation could ameliorate functional and structural derangements of the peritoneal membrane by suppressing inflammation and TGF-β1 signaling in a paracrine manner.25,26

In this work, we compared the possible healing effects of IP and intravenous (IV) administration of MSCs on structural changes of peritoneal membrane and UFF in a rat model of chronic PD. To our knowledge, this study is the first to compare the effects of IP and IV MSC transplantation for the treatment of peritoneal UFF.

Methods

Animals

Fifty-one non uremic male Wistar albino rats weighing 250–350 g at 16 weeks of age were purchased from the Erciyes University Experimental and Clinical Research Center. The animals were also housed in the same center in a controlled environment at 22 ± 2 °C with a 12 hours light/dark cycle. Food and water were given ad libitum. They were allowed one week of acclimatization before the start of the experiments. The experimental protocol was approved by Animal Experiments Ethical Committee of Erciyes University.

PD rat model

Peritoneal UFF was induced by daily 20 mL 3.86% glucose PD solution (Dianeal 3.86%; Eczacibasi-Baxter Healthcare, Istanbul, Turkey), as previously described.25,27

Study design

Rats were randomly divided into two groups. The control group (C group, n = 10) did not receive any injection during the study and peritoneal UFF group (UFF group, n = 41) received once-daily IP injection of 20 mL 3.86% glucose PD solution. At the end of six weeks, two rats in the C group and seven rats in the UFF group were excluded from the study, and the UFF group was then divided into four groups; peritoneal UFF control group (UFF-C group, n = 8), intraperitoneal MSC group (MSC-IP, n = 9), intravenous MSC group (MSC-IV, n = 8) and placebo group (P group, n = 9).

A peritoneal equilibration test (PET) was applied to evaluate the peritoneal permeability function to the C and UFF-C groups at the end of six weeks. Then the rats were sacrificed, and tissue samples from abdominal peritoneum were taken to examine the histological changes of the peritoneal membrane, and samples of parietal peritoneum without muscle tissue were used for the measurement of inflammatory cytokines (VEGF, TNFα and TGF-β1) levels.

Peritoneal equilibrium test and peritoneal biopsy were also performed to the MSC-IP, MSC-IV and P groups at the third week after receiving MSC and placebo to evaluate healing effect of intraperitoneally and IV MSCs transplantation on UFF and histological changes of the peritoneal membrane. Study groups and study design shown in Figure 1.

Figure 1. Study groups and experimental design.

MSC transplantation

Green fluorescent protein (GFP)-labeled rat bone marrow MSCs were purchased from the Centre for Stem Cells and Gene Therapies Research and Practice, Kocaeli, Turkey. These stem cells were prepared as previously described.25 Third-passage cells were used for transplantation. The cells were administered by IP injections of 1.5 × 10⁶cells/kg rat to MSC-IP group and by IV injection to the tail vein of rats 1.5 × 10⁶cells/kg rat to MSC-IV group.

Peritoneal equilibration test

The ultrafiltration function of the peritoneal membrane was assessed by carrying out a 90-minute PET. A 90-minute PET was performed as previously described.25 After PET procedure, animals were sacrificed for pathological examination. Blood and dialysate fluid samples (2 mL) were collected for measurement of glucose at 0 and 90 min after initiation of PET. Serum and dialysate fluid levels of glucose, urea nitrogen, sodium, protein and creatinine were measured as previously described.25

Peritoneal solute transport analysis

Peritoneal membrane transport was quantified as the dialysate-to-plasma ratio (D/P) of urea nitrogen, creatinine, sodium and protein and D/D0glucose, where D is the glucose concentration in the dialysate after 90-min dwell, and D0 is the glucose concentration in the dialysis solution before instillation into the peritoneal cavity. The mass transfer of glucose out of the peritoneum was calculated using the formula: (initial dialysate glucose × initial infusion volume) − (final dialysate glucose × final drain volume).28 High D/P and low D/D0 glucose ratios indicate higher transport; low D/P and high D/D0 glucose indicate low transport.

Histological assessment

After PETs, the rats were sacrificed and anterior parietal peritoneum was excised from the other sides of the injection points. Parietal peritoneum tissue was divided in two parts. One of peritoneal tissue was fixed with 10% formalin and embedded in paraffin. Tissue section (5 µm) were then stained with hematoxylin and eosin stain.

All sections were examined by light microscopy by the same pathologist who has not any information about groups. Inflammation, fibroblastic activity and neovascularization were evaluated semiquantitatively by counting inflammatory cells, fibroblasts and capillaries per high power field at ×400 magnification and classified normal or increased. Fibrosis also was scored as an evaluation of edema and collagen density. Mesothelial cells were counted as the mean of five different areas (cells/high power field at ×400 magnification) and classified as normal or decreased.

Submesothelial thickness was also measured from the inner surface of the muscle to the mesothelium. The thickness of submesothelial area was randomly measured at 10 points by two independent observers. Other peritoneal tissue taken from parietal peritoneum was used for immunofluorescence microscopy.

Immunofluorescence microscopy

Peritoneal tissue prepared for evaluate in immunofluorescence microscopy.25 To examine the survival of transplanted cells, GFP-positive cells were detected on serial sections using blue light in an immunofluorescence microscope. Preparations displaying green fluorescence along the mesothelial line were regarded as being positively marked, and images of the preparations were taken under the microscope.

Measurement of cytokines and growth factors in peritoneal tissues

Samples of parietal peritoneum without muscle tissue were used for the measurement of TNF-α, IL-6, VEGF and TGF-β1 levels. Cytokines levels were measured ELISA method, and total protein level was measured by Lowry’s method.29 Results were expressed as pg/mg protein wet weight of tissue for VEGF, TNF-α, IL-6 and TGF-β1.

Statistical analysis

Statistical analysis was performed using version 17.0 of SPSS for Windows (Chicago, IL). First, the distributions of all data were determined by using the Shapiro–Wilk test. The data with normal distribution were expressed as mean ± SD, and the data with non-normal distribution were expressed as median (min – max). The comparisons between the groups for the data with normal distribution were performed using ANOVA with the post hoc Tukey procedure and for the data with abnormal distribution were performed using the Kruskal–Wallis test. Then, when a statistical significance was found in Kruskal–Wallis test, differences between two groups were tested by the Mann–Whitney U-test. Comparison of the histopathological findings was performed by the chi-square test. All tests were considered as statistically significant if p values were <0.05.

Results

Histopathology of the peritoneum

The thickness of the peritoneum significantly increased with daily IP administration of PD fluid containing 3.86% glucose (p < 0.01; Table 1; Figure 2). Furthermore, the UFF-C group showed more inflammation and neovascularization in the peritoneal membrane than the C group (Table 1). The rats in the MSC-IP and MSC-IV groups had similar peritoneal membrane thicknesses to those in the C group. The peritoneal membrane thickness was also lower in the MSC-IP and MSC-IV groups than in the P group (p < 0.05; Table 1; Figure 2).

Figure 2. H&E staining of representative samples of peritoneal tissues. (A) In UFF-C group, there was thickening of the submesothelial area with inflammatory cell infiltration and neoangiogenesis. (B) Control rat peritoneum. (C) In the MSC-IP group, there was submesothelial thickness similar compared to C group. (D) In the MSC-IV group, morphological changes did not significantly differ in from C group. (E) In the P group, increased submesothelial thickness and peritoneal fibrosis with neovascularization was seen (×10).

Table 1. Evaluation of peritoneal membrane histopathology and permeability of all groups.

	GROUPS
	Control n = 8	UFF-C n = 8	MSC-IP n = 9	MSC-IV n = 8	Placebo n = 9
Submesothelial thickness (µm)	66.3 ± 24.8	132.9 ± 40.1^a	99.0 ± 57.1^c	108.3 ± 44^c	208.2 ± 115^a,b,d,e
Inflammation
Increased	0	4^a	1	3	3^a
Normal	8	4	8	5	6
Neovascularization
Increased	1	6^a	2	3	6^a,d
Normal	7	2	7	5	3
Fibrosis
Increased	1	3	2	2	6^a,d,e
Normal	7	5	7	6	3
Mesothelial cells count
Decreased	1	6^a	3	4	7^a
Normal	7	2^a	6	4	2
UF (mL) median (min – max)	4 (−2 – 10)	−3 (−24 – 6)^a	7.8 (5 – 10)^b,c	3.3 (−16 – 14)^b	1 (−17 – 12)
D/Purea (mean ± SD)	0.40 ± 0.05	0.59 ± 0.14^a	0.57 ± 0.11^a	0.49 ± 0.04	0.58 ± 0.09^a
D/PCr (mean ± SD)	0.65 ± 0.07	0.88 ± 0.15^a	0.73 ± 0.15^b,c	0.77 ± 0.16	0.85 ± 0.12^a
D/Pprotein (mean ± SD)	114.8 ± 6.9	125.38 ± 37.5^a	119.4 ± 8.4	118.2 ± 15	129.9 ± 10.6^a
D/PNa (mean ± SD)	0.78 ± 0.02	0.84 ± 0.03^a	0.82 ± 0.03^a	0.77 ± 0.2	0.81 ± 0.03
D/D0glucose (mean ± SD)	1.45 ± 0.22	0.65 ± 0.35^a	1.31 ± 0.41^b,c	0.92 ± 0.04^a	0.90 ± 0.12^a
Glucose mass transfer (mean ± SE)	−9649 ± 7985.6	12005 ± 5911^a	−5170 ± 2759^b,c	5500 ± 1684^a,d	1752 ± 4006

^ap < 0.05, versus control group.

^bp < 0.05, versus UFF-C group.

^cp < 0.05, versus placebo group.

^dp < 0.05, versus MSC-IP group.

Neovascularization, fibrosis and submesothelial thickness were significantly increased in P group compared to C group (p < 0.03 and p < 0.0001, respectively). Remarkable increase of submesothelial thickness was observed in the P group compared with that in the MSC-IP and MSC-IV groups (p < 0.0001). In the MSC-IP and MSC-IV groups, submesothelial thickness tended to increase compared with the C group but not significant (p = 0.07).

Compared with the UFF-C group, rats in the MSC-IP group had fewer inflammatory cells in the peritoneal membrane (p < 0.05), but MSC-IV and P groups had similar inflammatory cells number to UFF-C group. Following IP and IV injection of GFP-labeled MSCs, peritoneum sections taken from the MSC-IP and MSC-IV groups were examined directly via immunofluorescent microscopy. Fluorescence flashing was observed along the mesothelium line in the MSC-IP and MSC-IV groups but was not observed in the placebo group (Figure 3).

Figure 3. The immunofluorescence microscope images show the peritoneum slides, MSC-IP group (A) MSC-IV group and placebo group (C). GFP labeled-MSCs were observed along the mesothelium line in the MSC-IV and IP group (A) and (B) but were absent in the placebo group (B).

Peritoneal function

UF capacity

UF capacity was significantly higher in the MSC-IP group than in the UFF-C and placebo groups (p < 0.05). UF capacity of the MSC-IV group was also significantly higher than the UFF-C (p < 0.05), but there was no significant difference compared with the placebo group.

Glucose transport

The D/D0 glucose rate was significantly higher, and the glucose mass transfer level was significantly lower in the MSC-IP group than the placebo and UFF-C groups (p < 0.05), D/D0 glucose rates and glucose mass transfer levels of MSC-IV group were similar to the UFF-C and placebo groups and were different from the C group.

Solute permeability

Peritoneal permeabilities to sodium, urea and protein in the MSC-IP group were similar to those of the UFF-C and P groups. The D/P_Cr level in the MSC-IP group was lower than in the UFF-C and P groups (p < 0.05). However, peritoneal permeabilities to sodium, urea, creatinine and protein in the MSC-IV group were similar to those of the UFF-C and P groups. These results indicated that the MSC-IV, UFF-C and P groups had high peritoneal membrane permeability.

Effects of MSC treatment on cytokines and growth factors

The all groups had similar peritoneal tissue VEGF levels. Although VEGF concentrations in the MSC-IP group was lower than UFF-C group, there was no statistically significant difference (p = 0.06). The MSC-IP, MSC-IV and P groups had significantly decreased TNF-α concentrations compared with the UFF-C group (p < 0.05; Table 2). MSC-IP group had lower levels of TGF-β1 compared with the P group, and TGF-β1 levels of P group were significantly higher than C and UFF-C groups. MSC-IP group had also lower levels of IL-6 compared with UFF-C group.

Table 2. Cytokines levels in the parietal peritoneum.

	GROUPS
	Control n = 8	UFF-C n = 8	MSC-IP n = 9	MSC-IV n = 8	Placebo n = 9
IL-6 (pg/mg protein) Mean ± SD	1080 ± 231	1473 ± 325	726 ± 274^a	1092 ± 376	1195 ± 285
TGF-β (pg/mg protein) Median (min – max)	882.7 (207–2126)	1811.1 (533–4107)	788 (68–1996)^b	1415 (487–3206)	4568 (341–7006)^c,d
VEGF (pg/mg protein) Mean ± SD	42 ± 20	39.8 ± 27.3	18.6 ± 7.3	22.5 ± 21	20.8 ± 15.6
TNF-α (pg/mg protein) Median (min – max)	69.4 (24–123)	105.9 (79.7–223.9)	40.4 (26.8–61.7)^a	52.08 (37.9–9.4)^a	63.4 (35.3–98.5)^a

^ap < 0.05 versus UFF-C group.

^bp < 0.05 versus placebo group.

^cp < 0.05 versus control group.

^dp < 0.05 versus MSC-IP group.

Discussion

This study has provided the evidence that PD solution induced peritoneal damage and UFF in rats, as evidenced by a significant decrease of peritoneal thickening, loss of peritoneal mesothelial cells, increased number of inflammatory cells, neovascularization and fibrosis, which was ameliorated by IP and IV injections of bone marrow-derived MSCs.

In addition, intraperitoneally, MSCs reduced the functional impairments of the peritoneal membrane, as described by decreased UF capacity and impaired small solute transport. Although, IV MSCs transplantation diminished peritoneal thickness and improved UF, it did not improve small solute transport in rats with peritoneal injury. Furthermore, IP MSC transplantation normalized IL-6 and TGF-β1 level in damaged peritoneum, but IV MSC transplantation did not improve these inflammation markers. It was expected the decreased inflammation in placebo group due to peritoneal rest. Whereas, in our study as interesting, levels of TNF-α in placebo group were significant higher than UFF-C group, and TGF β1 levels in P group were higher than UFF-C group, although there were not statistically significant between two groups. Perhaps, this condition can be explained as inflammation may be continued even if peritoneal rest.

These results indicate that the useful effects of IP MSCs transplantation on damaged peritoneum include reductions in local pathology, as well as improvements in peritoneal function, which was the better than IV injections of MSCs.

During the long-term PD therapy, some structural, morphological and functional changes occurred due to multiple factors, especially exposure to non-biocompatible dialysis solutions, or PD catheter.30,31 The most important factor of the PD solutions that are responsible for peritoneal deterioration seems to be glucose degradation products, which stimulate the production of extracellular matrix components as well as the synthesis of cytokines and growth factors, including IL-6, TNF-α, TGF-β1 and VEGF.2,32 The results of many studies and also our study results clearly show that PD treatment, by itself, strongly impacts peritoneal injury.2,32–34

Protection of the peritoneal membrane or healing of a damaged membrane is of crucial importance for patients with end-stage renal disease having no alternatives for renal replacement therapies, except PD. In many studies, drugs, peptides and gene therapy targeting fibrosis and/or angiogenesis could be useful for treating peritoneal fibrosis,19–22 whereas clinically available treatment for peritoneal fibrosis in PD patients remains limited at present. Contrary to expectations, some of these studies reported poor outcomes.7,11–16,35–37

The mesothelium is an important component of the peritoneal membrane and is responsible for regulating inflammatory responses as well as microcirculation and fibrin homeostasis.37–39 Few authors reported that transplanted mesothelial cells tend to collect in denudated areas of the peritoneum surface. Most of these studies did not evaluate peritoneal permeability.35,40

MSCs are known to migrate preferentially to the sites of inflammation following tissue injury when infused into animals.41 Although the underlying molecular mechanism of homing and engraftment by these cells is not yet fully understood, expression of specific growth factors, chemokines and extracellular matrix receptors on the surfaces of MSCs may facilitate their trafficking, adhesion and infiltration into sites of injury.42,43 MSCs are presently being studied for prophylaxis and therapy for a variety of diseases.44–49

More recently published few reports and our previously study25,26,50 showed that following MSCs injection into the damaged peritoneum, the increased submesothelial thickness, inflammation, TGF-β1 level and impaired parameters of peritoneal function clearly improved compared with the rats of peritoneal fibrosis. These studies suggest that MSCs may have reduced submesothelial edema and inflammation and the injected MSCs were detected on the peritoneal surface in rats with peritoneal fibrosis but were not seen in rats without peritoneal fibrosis. These findings suggest that intraperitoneally injected MSCs migrated to the sites of peritoneal injury and displayed both immunomodulatory and antifibrotic effects of the administered cells.

IP application of MSC therapy in clinical practice may not be possible, especially in young children and infants, that PD is not able to take a break due to hemodialysis cannot be done cause of unavailability of the vasculature and technical difficulties.

In this study, we evaluated whether systemic IV MSC treatment has demonstrated healing effect, as IP injection of MSCs for structural and functional defects in the peritoneal membrane caused by PD fluid, and compared with the results of IP MSC and IV MSCs. To our knowledge, this is the first study to examine this issue.

Findings of our study indicate that although IP MSCs transplantation may have decreased glucose transfer while increasing UF capacity and creatinine clearance, but had no effect on urea, protein or Na permeability because of probably there was not enough time for improving of these parameters. In summary, we believe that peritoneum permeability in the MSC-IP group was similar to the control group, and high permeability was continued in MSC-IV and placebo groups, which together indicated that IP MSCs exerted healing effects on peritoneum permeability but not IV MSCs. In addition, there are significant difference between the MSC-IP and placebo groups in terms of fibrosis and neovascularization.

In this study, retention of IP and IV administered GFP labeled MSCs into the peritoneal tissues were demonstrated in MSC-IP and IV group, whereas MSCs were not detected in placebo group. These findings suggest that the MSCs migrated to the sites of PD-induced peritoneal damage.

Recently, Sekiguchi et al.50 reported that bone marrow-derived cells accumulated and were maintained in the submesothelium in a mouse model of CG-induced peritoneal fibrosis. Their results suggest that bone marrow-derived cells have an important role in the process of peritoneal repair and mesothelial remodeling.

Ueno et al.26 administered IP GFP-labeled MSCs to rat with CG-induced peritoneal fibrosis and showed that retention of bone marrow-derived MSCs expanded ex vivo was only observed until day 3. However, the anti-inflammatory and antifibrotic effects in the peritoneum were prolonged until day 14. They suggest that such effects may be independent of transdifferentiation of MSCs into functional peritoneal mesothelial cells and are more likely to be due to paracrine activities of MSCs.

Previous studies have suggested that paracrine mechanisms involving the production of growth factors and anti-inflammatory cytokines by MSCs are associated with these properties.51,52

To identify an anti-inflammatory factor, we performed enzyme-linked immunosorbent assay to detect anti-inflammatory interleukin-6 and TNF-α in peritoneal tissue. The mechanism of the anti-inflammatory effect of MSCs in our study was probably the anti-inflammatory paracrine action may affect host macrophages and peritoneal mesothelial cells.

In conclusion, our results suggest that IP MSCs transplantation ameliorate PD fluid-induced peritoneal damage include reductions in local pathology, as well as improvements in peritoneal function, which was the better than IV injections of bone marrow-derived MSCs by probably anti-inflammatory paracrine action of IP administration of MSCs.

Declaration of interest

All the authors have declared no competing interest.