Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes

Bone marrow-derived mesenchymal stem cells (MSCs) have contributed to the improvement of diabetic nephropathy (DN); however, the actual mediator of this effect and its role has not been characterized thoroughly. We investigated the effects of MSC therapy on DN, focusing on the paracrine effect of renal trophic factors, including exosomes secreted by MSCs. MSCs and MSC-conditioned medium (MSC-CM) as renal trophic factors were administered in parallel to high-fat diet (HFD)-induced type 2 diabetic mice and streptozotocin (STZ)-induced insulin-deficient diabetic mice. Both therapies showed approximately equivalent curative effects, as each inhibited the exacerbation of albuminuria. They also suppressed the excessive infiltration of BMDCs into the kidney by regulating the expression of the adhesion molecule ICAM-1. Proinflammatory cytokine expression (e.g., TNF-α) and fibrosis in tubular interstitium were inhibited. TGF-β1 expression was down-regulated and tight junction protein expression (e.g., ZO-1) was maintained, which sequentially suppressed the epithelial-to-mesenchymal transition of tubular epithelial cells (TECs). Exosomes purified from MSC-CM exerted an anti-apoptotic effect and protected tight junction structure in TECs. The increase of glomerular mesangium substrate was inhibited in HFD-diabetic mice. MSC therapy is a promising tool to prevent DN via the paracrine effect of renal trophic factors including exosomes due to its multifactorial action.

harvested from femurs and tibias by flushing whole bone marrow with complete α-modified Eagle's medium (α-MEM; Invitrogen, Carlsbad, CA) containing 15% fetal bovine serum and 1% penicillin-streptomycin. Single cell suspensions were filtered through a 70-µm nylon filter (Becton Dickinson, Franklin Lakes, NJ) and plated in 75-cm 2 flasks. The cells were grown in complete α-MEM at 37°C and 5% CO 2 . After 72 h, the medium was replaced with fresh medium, and adherent cells grown to 80% confluency to obtain samples were defined as passage 0. Cells in passage 3 were used for experiments.

Detection of donor MSCs
HFD-and STZ-induced diabetic mice without bone marrow transplantation were administered MSCs isolated from SD-Tg (CAG-EGFP) (Sankyo Labo Service Corporation, Inc., Tokyo, Japan) rats or MSCs isolated from Lewis rats (Charles River Laboratories Japan, Inc., Yokohama, Japan) that were labeled with a PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St. Louis, MO). Mice administered MSCs derived from CAG-EGFP rats were sacrificed at 1, 2, or 4 weeks after MSC injection, and lung, liver, kidney, spleen, and bone were obtained. Each tissue was digested into single cells with collagenase (Sigma-Aldrich, St. Louis, MO) and the number of GFP-positive MSCs distributed in each organ was analyzed by flow cytometry. The organs obtained from mice, which were administered PKH26-labeled MSCs, were also immersed in 4% paraformaldehyde and bone was decalcified with 0.5 M EDTA (Wako, Osaka, Japan) for 2 days. Frozen sections of each organ were stained with DAPI (Dojindo Laboratories, Kumamoto, Japan) at 0.1 mg/mL. The distribution of MSCs expressing red fluorescence in each organ was observed by confocal laser scanning microscopy (LSM 510; Carl Zeiss, Oberkochen, Germany). The ratio of MSCs distributed in each organ was determined by counting PKH26-positive cells in 10 randomly selected visual fields at ×100 magnification per mouse (n = 3-5) and compensated for by the number of MSCs given to each mouse.

Transmission electron microscopic observation of exosomes
Exosome pellets isolated from MSC-CM were fixed for 24 h with 2.5% glutaraldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The samples were washed with PBS, fixed with 1% osmium tetroxide solution (TAAB Laboratories Equipment Ltd., Aldermaston, UK), and dehydrated with ethanol (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After soaking the samples in propylene oxide (KANTO KAGAKU, Tokyo, Japan), they were embedded with an epoxy resin (TAAB Laboratories Equipment Ltd.) and polymerized with heating. Ultrathin sections of the samples were prepared using an ultra-microtome (MT-X; RMC Boeckeler Instruments, Inc., Tucson, AZ). The sections were observed with a transmission electron microscope (H7650; Hitachi High-Technologies Corporation, Tokyo, Japan) after electron staining was performed.

Immunoblotting
The molecular content of kidney and exosome was analyzed by immunoblotting. Kidney tissues and exosome pellets isolated from MSC-CM were lysed in Radio Immuno Precipitation Assay buffer that included 20 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM EDTA (Sigma-Aldrich, St. Louis, MO), pH 8.0, 0.1% (w/v) sodium dodecyl sulfate, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 tablet of complete Mini™ (Roche Diagnostics, Mannheim, Germany) and Phos STOP (Roche Diagnostics). Five to 40 g of each lysate, as determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), were resolved on a 12% denaturing polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween 20, the membrane was incubated with primary antibodies (Supplemental Table 1). It was then incubated with horseradish peroxidase-conjugated secondary antibodies (Supplemental Table 2). Immuno-reactivity was developed using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).

SUPPLEMENTARY TABLE and FIGURE LEGENDS
Supplementary

Supplementary Table S2. Secondary antibodies
The secondary antibodies used for immunofluorescence are listed according to species, conjugate, and manufacturer. Abbreviations: Abs, antibodies; Gt, goat; Dnk, donkey.

Supplementary Figure S1. Immunophenotype of rat MSCs
Flow cytometry analysis of the expression of cell surface markers related to rat MSCs.
The green line shows the population stained with the target antibody. The purple line shows the population stained with the isotype control antibody.