Systematic implantation of dedifferentiated fat cells ameliorated monoclonal antibody 1-22-3-induced glomerulonephritis by immunosuppression with increases in TNF-stimulated gene 6
© Maruyama et al.; licensee BioMed Central. 2015
Received: 18 August 2014
Accepted: 27 March 2015
Published: 16 April 2015
Implantation of mesenchymal stem cells (MSCs) has recently been reported to repair tissue injuries through anti-inflammatory and immunosuppressive effects. We established dedifferentiated fat (DFAT) cells that show identical characteristics to MSCs.
We examined the effects of 106 of DFAT cells infused through renal artery or tail vein on monoclonal antibody (mAb) 1-22-3-induced glomerulonephritis (as an immunological type of renal injury) and adriamycin-induced nephropathy (as a non-immunological type of renal injury) in rats. The mAb 1-22-3-injected rats were also implanted with 106 of DFAT cells transfected with TSG-6 siRNA through tail vein.
Although DFAT cells transfused into blood circulation through the tail vein were trapped mainly in lungs without reaching the kidneys, implantation of DFAT cells reduced proteinuria and improved glomerulosclerosis and interstitial fibrosis. Implantation of DFAT cells through the tail vein significantly decreased expression of kidney injury molecule-1, collagen IV and fibronectin mRNAs, whereas nephrin mRNA expression was increased. Implantation of DFAT cells did not improve adriamycin-induced nephropathy, but significantly decreased the glomerular influx of macrophages, common leukocytes and pan T cells. However, the glomerular influx of helper T cells, was increased. Implantation of DFAT cells decreased expression of interleukin (IL)-6 and IL-12β mRNAs and increased expression of TNF-stimulated gene (TSG)-6 mRNA in renal cortex from mAb 1-22-3-injected rats. The basal level of TSG-6 protein was significantly higher in DFAT cells than in fibroblasts. Expression of TSG-6 mRNA in MCs cocultured with DFAT cells was significantly higher than in mesangial cells or DFAT cells alone. Systematic implantation of DFAT cells with TSG-6 siRNA through tail vein did not improve proteinuria, renal dysfunction and renal degeneration in the mAb 1-22-3-injected rats.
Systematic implantation of DFAT cells effectively ameliorated mAb 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage infiltration and expression of IL-6, IL-10 and IL-12β, and increased production of serum and renal TSG-6 that improved the mAb 1-22-3-induced renal degeneration by the immunosuppressive effects of TSG-6. Thus DFAT cells will be suitable cell source for the treatment of immunological progressive renal diseases.
Despite the availability of long-term therapies, chronic renal failure caused by immunoglobulin A (IgA) nephropathy, diabetic nephropathy and glomerulosclerosis cannot be cured through current treatments. End-stage renal disease is an appropriate application for regenerative medicine. Regarding regenerative medicines for chronic renal failure, the implantation of cells, including stem cells and progenitor cells, has been experimentally applied in treatments for progressive renal diseases . To date, however, there have been no clinical trials of cell implantation for progressive renal diseases. This is because the complexity of the kidney structure prevents efficient regeneration in response to single-source cell implantation. As a source of cells for use in regenerative medicine, embryonic stem cells or inducible pluripotent stem cells possess a nearly unlimited capacity for self-renewal and have the potential to differentiate into virtually any cell type. Thus, mesenchymal stem cells (MSCs) have arisen to become a candidate cell source in regenerative medicine for kidney diseases.
Recent studies have shown that adipose tissue can provide an alternative source of MSCs . Adipose tissue contains nonadipocyte cells, known as the stromal-vascular fraction, which can be isolated by centrifugation of collagenase-digested adipose tissue, which is comprised of multipotent fibroblast-like cells, known as adipose-derived stromal cells (ASCs) .
We established an adipogenic progenitor cell line derived from mature adipocytes and named these cells as dedifferentiated fat (DFAT) cells . Clonally-expanded DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages, indicating that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application to cell-based therapies . In contrast to ASCs, which contain a variety of cell types, DFAT cells originate from a fraction of highly homogeneous mature adipocytes. This property of DFAT cells will likely lead to higher safety and efficacy for clinical cell therapies.
To evaluate the efficiency of cell therapy for progressive renal diseases, animal models of sustained renal failure are required. Proteinuria was maintained at a higher level and blood urea nitrogen (BUN) and serum creatinine levels were higher in rats with unilateral nephrectomy, after the administration of Thy-1.1 monoclonal antibody (mAb) 1-22-3. Morphologically, typical sclerotic changes were observed in the mAb 1-22-3 injected rats. These findings suggest that the renal lesions in the mAb 1-22-3 rats provide a suitable model for chronic progressive glomerulonephritis .
Implantation of MSCs has recently been reported to repair tissue injuries through their anti-inflammatory and immunosuppressive effects . Implantation of MSCs has been reported to suppress fibrosis of infarcted heart , bleomycin-induced lung fibrosis , liver fibrosis  and interstitial fibrosis of kidney . Moreover, MSCs have been established to have immunosuppressive effects. Systemic infusion of MSCs has been reported to suppress graft rejection in animal models, which has led to a number of clinical trials [12,13]. Implantation of MSCs has been developed in a clinical study in which MSCs were reported to effectively inhibit graft-versus-host disease in Phase II studies .
DFAT cells have the potential to differentiate into lineages of mesenchymal tissue and the cell surface antigen profile of DFAT cells has been shown to be very similar to that of bone marrow MSCs . We previously examined the effects of implantation of DFAT cells on habu snake venom-induced chronic renal dysfunction in mice and found improvement of glomerulosclerosis . Thus, DFAT cells may provide a source of cell therapy for severe progressive renal diseases. In the present study, to evaluate the ability of DFAT cells to serve as a cell source for progressive renal diseases, we examined the effects of implantation of DFAT cells in mAb 1-22-3-induced glomerulonephritis (as an immunological type of renal injury) and adriamycin-induced nephropathy (as a non-immunological type of renal injury) in a rat model.
Our investigation conformed to the Guide for the Care and Use of Laboratory Animals: Eighth Edition Washington, DC: The National Academies Press, 2011. The ethics committee of Nihon University School of Medicine approved all research protocols involving the use of living animals.
A hybridoma producing mouse anti-rat Thy 1.1 mAb 1-22-3 (IgG3) was prepared by immunization of BALB/c mice with collagenase-treated fresh rat glomeruli. Ascitic fluid containing mAb 1-22-3 was produced in BALB/c mice primed with 2,6,10,14-tetramethylpentadecane (Sigma Chemical, St. Louis, MO, USA) and injected intraperitoneally with the hybridoma. The obtained fluid was subjected to 50% ammonium sulfate precipitation and the obtained immunoglobulin-rich fraction was dialyzed against phosphate-buffered saline (PBS).
Preparation of the DFAT cells from adipose tissue
Approximately 1 g of subcutaneous adipose tissue from male Wistar rats was treated with collagenase and centrifuged. Adipocytes were isolated from the top layer. More than 99% of the isolated cells were mature lipid-filled adipocytes. The mature adipocytes floating on top of the culture medium attached to the upper surface of the culture flasks within a few days. Approximately 10% to 20% of the adherent cells flattened out by day 3 and changed to a spindle-shaped morphology by day 7. The cells subsequently entered a proliferative log-phase upon inversion of the flasks and changing of the media, and reached confluence by day 14. During this stage, the cells lose the lipid droplets completely and exhibit the fibroblast-like morphology of DFAT cells.
Distribution of DFAT cells
DFAT cells from Wistar rats were labeled with a Qtracker® Cell Labeling Kit (Molecular probes, Life Technology, Tokyo, Japan). A total 106 labeled DFAT cells was injected through the renal arteries or tail vein in Wistar rats. One week after the injection, kidney, aorta, liver and lungs were removed and fixed in 3% formalin in PBS and embedded in paraffin. Sections were observed under a fluorescence microscope. Images were obtained with a digital imaging system.
Experiment 2: Effects of implantation of DFAT cells on adriamycin-induced nephropathy. In all male Wistar rats weighing 250 g, the right kidney was nephrectomized. Control rats were observed without any further injections. Other rats were injected with 1.0 mL saline containing 4 mg/kg body weight adriamycin (Wako Junyaku Inc. Tokyo, Japan) through the tail vein at seven days after nephrectomy without DFAT cells (Saline) or with 106 DFAT cells in 1.0 mL saline. Rats were injected through the renal artery (DFAT ia) or through the tail vein (DFAT iv). Thirty-five days after the nephrectomy, all rats were killed and the left kidney was removed (Figure 1).
Experiment 3: DFAT cells (2 x 105 cells) from Wistar rats were transfected with 20 nM rat TNF-stimulated gene (TSG)-6-small interfering (si) RNA or 20 nM control siRNA purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA in siRNA Transfection Medium (Santa Cruz). To confirm sufficient inhibition of expression of TSG-6 protein, we performed ELISA analysis for siRNA transfected DFAT cells 24 hours after transfection. In male Wistar rats weighing 250 g, the right kidney was nephrectomized. Rats were injected with 0.5 mg mAb 1-22-3 and 106 DFAT cells transfected with TSG-6 siRNA or control siRNA through the tail vein. Fourteen days after the injections, all rats were killed and the left kidney was removed (Figure 1). Urinary protein excretion was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Serum BUN and creatinine were measured by SRL Inc. (Wako, Saitama, Japan).
Morphological and immunohistological analysis
The 3-mm paraffin sections of removed renal cortex were stained with hematoxylin and eosin (H & E). Renal cortical thickness was measured under high magnification (×400). The glomerular injury score (GIS) was obtained using the following formula: ((0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4))/50. To semi-quantify the tubulointerstitial area, 20 areas of renal cortex were randomly selected. The percentage of each area that showed sclerofibrotic change was estimated and assigned a score of 0, normal; 1, involvement of <10% of the area; 2, involvement of 10% to 30% of the area; 3, involvement of 30% to 50% of the area; or 4, involvement of 50% of the area. The tubulointerstitial injury score (TIS) was calculated as ((0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4))/20.
Deparaffinized 5-μm sections were briefly incubated with 3% H2O2 and then with primary antibody for 60 minutes, rinsed with Tris-buffered saline containing 0.1% Tween 20, and incubated with a secondary antibody for 30 minutes. The primary antibodies used for immunohistochemical analysis were as follows: ED1 (IgG1) recognizing pan monocytes/macrophages was purchased from Chemicon International Inc. (Temecula, CA, USA); OX1 recognizing CD45+ common leukocytes was purchased from Santa Cruz Biotechnology (Dallas, TX, USA); Mouse anti-rat monoclonal antibody OX19 (IgG1) recognizing rat CD5 antigen was used to detect and deplete pan T cells ; OX38 (IgG2a) recognizing CD4+ helper T cells was used to detect and deplete CD4+ T cells . OX-19 and OX-38 were precipitated from ascites using the corresponding hybridoma (European Collection of Animal Cells, Porton Down, Salisbury, UK). Counterstaining was then performed before examination under a light microscope.
Determination of mRNA expression
Total RNA was extracted from renal cortices and cultured cells with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed into cDNA with random 9-mers with a Takara RNA PCR Kit (AMV) Ver. 3.0 (Takara Bio, Ohtsu, Japan). Real-time quantitative PCR was performed with diluted cDNA using a FastStart TaqMan Probe Master (Roche Applied Science, Basel, Switzerland) and an SYBR Select Master Mix (Life Technologies) in an ABI 7500 sequence detector (Life Technologies) according to the manufacturer’s instructions. All assay-on-demand primers and probes (Kim-1, collagen IV, fibronectin, nephrin, TSG-6, TNF-α, IL-6, IL-10, IL-12β and TGF-β1) were purchased from Life Technologies. Real-time PCR data were analyzed with standard curves and normalized to 18S ribosomal RNA with its specific primer sets (5’ and 3’ primers: 5’-CGGCTACCACATCCAAGGAA-3’ and 5’-GCTGGAATTACCGCGGCT-3’) as described previously . Correlation coefficients for the standard curves were all >0.90.
Enzyme-linked immunosorbent assay
Levels of TNF-stimulated gene-6 (TSG-6) in culture medium were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA). Conditioned medium for 106 DFAT cells or 106 fibroblasts was collected for 24 hours incubation. The supernatants of all specimens were detected by a multidetection microplate reader using a double-antibody sandwich ELISA kit according to the manufacturer’s protocols. The concentrations of TSG-6 were normalized to the total protein content.
Coculture of DFAT and mesangial cells
Glomeruli were isolated from kidneys of four-week-old male stroke prone spontaneous hypertensive (SHR-SP) rats. Glomeruli were isolated with a graded-sieve technique as described previously . Mesangial cells (MCs) from SHR-SP were plated at 4,000 cells per cm2 in the bottom well of a six-well transwell coculture system (Corning Incorporated, Corning, NY, USA). DFAT cells from Wistar rats were plated separately at 1,000 cells per cm2 on the upper inserts of six-well transwell coculture system and cultured in RPMI + 20% fetal bovine serum (FBS) for seven days. Levels of TSG-6 in culture medium were determined using ELISA. RNA was extracted from MCs from SHR-SP or DFAT cells (RNeasy Mini Kit; Qiagen, Valencia, CA, USA) and assayed for TSG-6 by real-time quantitative PCR.
The values are reported as the mean ± SE. Student’s t-test was used for unpaired data. Two-way analysis of variance (ANOVA) with the Bonferroni/Dunn procedure as post-test was also used. P <0.05 was considered to be statistically significant.
Distribution of implanted DFAT cells
Effects of implantation of DFAT cells on mAb 1-22-3-induced glomerulonephritis
Effects of implantation of DFAT cells on adriamycin-induced nephropathy
Effects of implantation of DFAT cells on leukocyte accumulation in glomeruli in mAb 1-22-3-injected rats
Effects of implantation of DFAT cells on expression of cytokines in renal cortex in mAb 1-22-3-injected rats
Effects of implantation of DFAT cells on generation of TNF-stimulated gene 6 in DFAT cells in mAb 1-22-3-injected rats
Production of TNF-stimulated gene 6 from mesangial cells by transwell coculture of DFAT cells
Effects of implantation of DFAT cells with TSG-6 siRNA on mAb 1-22-3-induced glomerulonephritis
As an animal model for renal diseases, anti Thy-1.1 antibody induces transient glomerulonephritis with spontaneous improvement , whereas mAb 1-22-3 induces glomerulonephritis with significant proteinuria and progressive mesangial injury. In this study, one injection of mAb 1-22-3 induced mesangiolysis and glomerular inflammatory cell infiltration in unilaterally nephrectomized rats. In addition, expression of Kim-1, collagen IV and fibronectin mRNAs was increased, and expression of nephrin mRNA was decreased in renal cortex from rats injected with mAb 1-22-3, indicating that mAb 1-22-3 induced acute mesangial injury as well as podocyte injury. These results suggest that mAb 1-22-3-induced glomerulonephritis is an ideal animal model for progressive immune-mediated nephropathy.
We examined the effects of implantation of DFAT cells on mAb 1-22-3-induced glomerulonephritis in rats. DFAT cells can be obtained from small amounts of adipose tissue and express HLA-A, −B, and -C, but not HLA-DR. Thus, DFAT cells may be an attractive cell source for allogenic implantation. In response to specific culture conditions, DFAT cells can differentiate into adipocytes, osteoblasts, chondrocytes, myofibroblasts, skeletal myocytes, and cardiomyocytes [21-23]. Thus, DFAT cells are considered to be a suitable cell source for regenerative medicine for the mesenchymal organs. In the present experiments, implantation of DFAT cells decreased proteinuria and improved glomerular injury as well as interstitial degeneration of the kidney in mAb 1-22-3-injected rats. Notably, the systematic implantation of DFAT cells through the tail vein was more effective in reducing proteinuria and renal degeneration than direct implantation. Labeled DFAT cells implanted through the tail vein were trapped mainly in the lung and not delivered to the kidney. These findings indicate the possibility that DFAT cells trapped in the lung release some anti-inflammatory and/or immunosuppressive substances to improve renal injury in mAb 1-22-3 injected rats.
We examined the effects of implantation of DFAT cells on adriamycin-induced glomerulonephropathy. Adriamycin induces focal segmental glomerulosclerosis with severe proteinuria with interstitial degeneration of the kidney . Adriamycin induces injury by direct toxic damage to the glomerulus with subsequent tubulointerstitial injury with damages of the glomerular filtration barrier including glomerular endothelial cells, glomerular basement membrane and podocytes . Subsequently, adriamycin-induced nephropathy shows severe tubulointerstitial inflammation with marked infiltration by T and B lymphocytes and macrophages . However, it has been reported that adriamycin can induce structural and functional injury in immunodeficient mice without infiltration by T and B lymphocytes. These reports suggest adriamycin directly induces degeneration of glomerulus with tubulointerstitial injury without immunological mechanisms, which accompanies subsequent immunological injury of kidney with infiltration of lymphocytes. The implantation of DFAT cells did not improve the adriamycin-induced glomerular degeneration and rather increased the proteinura. It is, therefore, considered that the systematic implantation of DFAT cells mainly improved the renal degeneration of mAb 1-22-3-induced glomerulonephritis through immunosuppressive mechanisms.
It has recently been established that MSCs exert immunosuppressive effects through the inhibition of activated T cells and natural killer cells. MSCs can inhibit immune responses in non-human leukocytes in an antigen-restricted manner [27,28]. Implantation of MSCs has been reported to suppress systematic lupus erythematosus  and inflammatory bowel diseases . The complex mechanisms underlying the immunosuppressive effects of MSCs are reported to be the increases in regulatory T cells through the induction of prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase , and the release of several cytokines  and hepatocyte growth factor (HGF) . HGF has been reported to have immunosuppressive effects on reactions caused by transplantation and autoimmune diseases . In the present experiments, the immunostaining of HGF was not different in renal cortex from control rats, mAb 1-22-3-injected rats injected with saline or DFAT cell-implanted rats, indicating that HGF was not involved in the improvement of renal degeneration in mAb 1-22-3 injected rats as a result of DFAT cell implantation (Additional file 1: Figure S1).
We examined the effects of DFAT cell implantation on leukocyte influx into the kidney in mAb 1-22-3-injected rats. Infiltration of ED1+ pan monocytes/macrophages, OX1+ common leukocytes and OX19+ pan T cells was increased in the glomeruli in kidney from mAb 1-22-3-injected rats. DFAT cell implantation significantly decreased ED1+, OX1+ and OX19+ cells, whereas OX38+ helper T cells were significantly increased. These findings indicate the suppression of macrophage infiltration into the kidney and the increase in helper T cells in mAb 1-22-3-induced glomerulonephritis might be mechanisms underlying the systematic implantation-induced immunosuppression. Furthermore, expressions of IL-6, IL-10 and IL-12β mRNAs, which were suppressed by the implantation of DFAT cells, were increased in renal cortex from mAb-1-22-3-injected rats. IL-6, IL-10 and IL-12β have been reported to be mainly produced from macrophages , indicating that the implantation of DFAT cells ameliorated the immunological glomerulonephritis by suppression of macrophage infiltration into the kidney with inhibition of IL-6, IL-10 and IL-12β. However, mechanisms underlying the improvement of mAb 1-22-3-induced glomerulonephritis, when DFAT cells were trapped in the lung, remain to be elucidated.
It has been demonstrated that intravenously infused MSCs were mostly trapped as emboli in the lung and that they secrete an anti-inflammatory protein, TSG-6, which decreases inflammatory responses and improves injured organs . TSG-6, a 30 kDa protein, is induced by a number of signaling molecules, principally TNF-α and IL-1 and may be induced by mechanical stimuli in mesenchymal cells. It is found to be correlated with proteoglycan synthesis and aggregation . Wang et al.  recently demonstrated that MSCs trapped in the lung produce TSG-6 after intravenous injection, which induced anti-inflammatory and immunosuppressive effects. Interestingly, they also demonstrated that intravenous injection of conditioned medium of cultured MSCs increased serum levels of TSG-6 as well as tissue levels of TSG-6 in injured peritoneum . In the present experiments, the systematic implantation of DFAT cells increased serum levels of TSG-6 as well as the expression of TSG-6 in the kidney of mAb 1-22-3-injected rats. In addition, the systematic implantation increased expression of TNF-α mRNA in renal cortex from mAb 1-22-3-injected rats, which may stimulate the expression of TSG-6 in renal cortex. Moreover, we found that production of TSG-6 protein was higher in DFAT cells than in fibroblasts and that the implantation of DFAT cells increased serum levels of TSG-6 protein in rats. These findings suggest that the implanted DFAT cells abundantly produced TSG-6, which ameliorated the mAb 1-22-3-induced glomerulonephritis.
To investigate whether separately placed DFAT cells can increase TSG-6 in MCs, we employed a transwell coculture system. Production of TSG-6 in conditioned medium from MCs with DFAT cells was higher than MCs or DFAT cells alone. Expression of TSG-6 mRNA in MCs cocultured with DFAT cells was higher than in MCs or DFAT cells alone, indicating that the separately placed DFAT cells increased TSG-6 in MCs. Recently, Roddy et al.  demonstrated that MSCs are activated by proinflammatory signals to introduce two phases: 1) the activated MSCs secrete PGE2, which drives resident macrophages of type 1 proinflammatory phenotype toward type 2 anti-inflammatory phenotype macrophages; and 2) the activated MSCs secrete TSG-6, which interacts with CD44 on resident macrophages to decrease toll-like receptor 2. TSG-6 from MSCs inhibits the initial activation of resident macrophages by modulating toll-like receptor 2/CD44/NF-kB signaling and thereby decreases the phase II inflammatory response. These findings are identical to our own in that the systematic implantation of DFAT cells reduced macrophage infiltration into the kidney with decreases the expression of IL-6, IL-10 and IL-12β mRNAs in mAb 1-22-3-injected rats.
In order to explore the contribution of TSG-6 in the amelioration of the mAb 1-22-3-induced glomerulonephritis with implantation of DFAT cells, we examined effects of implantation of DFAT cells transfected with TSG-6 siRNA. Implantation of DFAT cells with control siRNA through the tail vein reduced the increases in proteinuria. Systematic implantation of DFAT cells with TSG-6 siRNA through the tail vein did not reduce proteinuria, increases in serum BUN and creatinine and renal degeneration in the mAb 1-22-3-injected rats. These results suggest that the DFAT cells trapped in the lung produced TSG-6 in serum as well as in the kidney, which improved the renal degeneration and the renal dysfunction in rats with the mAb 1-22-3.
Systematic implantation of DFAT cells effectively ameliorated mAb 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage infiltration and expression of IL-6, IL-10 and IL-12β, and increased production of serum and renal TSG-6 that improved the mAb 1-22-3-induced renal degeneration by the immunosuppressive effects of TSG-6. Thus DFAT cells will be a suitable cell source for the treatment of immunological progressive renal diseases.
adipose-derived stromal cells
blood urea nitrogen
enzyme-linked immunosorbent assay
glomerular injury score
hepatocyte growth factor
- H & E:
hematoxylin and eosin
kidney injury molecule
mesenchymal stem cells
stroke prone spontaneous hypertensive
small interfering RNA
transforming growth factor
tubulointerstitial injury score
tumor necrosis factor
We acknowledge the support of this study by financial grants from the ‘Strategic Research Base Development’ Program for Private Universities subsidized by MEXT (S0801033), and by a financial grant from the Japan Science and Technology Agency (08030216). The authors thank Megumi Watanabe for her technical support of our experiments.
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