Therapeutic potential of stromal cells of non-renal or renal origin in experimental chronic kidney disease

Background Mesenchymal stromal cell (MSC)-based therapy is a promising strategy for preventing the progression of chronic kidney disease (CKD), with the potential to induce tissue regeneration. In search of the best cellular source we compared, in the rat model of adriamycin (ADR) nephropathy, the regenerative potential of human stromal cells of non-renal origin, such as bone marrow (bm) MSCs and umbilical cord (uc) MSCs, with that of newly discovered stromal cells of renal origin, the kidney perivascular cells (kPSCs) known to exhibit tissue-specific properties. Methods The therapeutic effect of repeated infusions of human bmMSCs, ucMSCs, kPSCs (1.5 × 106 cells/rats) or conditioned medium from ucMSCs was studied in athymic rats with ADR-induced nephropathy (7.9 mg/kg). The ability of the three stromal cell populations to engraft the damaged kidney was evaluated by detecting the presence of human nuclear antigenpos cells. Glomerular podocyte loss and endothelial damage, sclerotic lesions and inflammation were assessed at 14 and 28 days. In-vitro experiments with a transwell system were performed to investigate the effects of different stromal cell populations on parietal epithelial cells (PECs) activated or not with albumin or angiotensin II for 24 h. Results Infusions of non-renal and renal stromal cells resulted in a comparable engraftment into the kidney, in the peritubular areas and around the glomerular structures. All three cell populations limited podocyte loss and glomerular endothelial cell injury, and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis. Human ucMSCs had an anti-inflammatory effect superior to that of the other stromal cells, reducing macrophage infiltration and inducing polarisation towards the M2 macrophage phenotype. Conditioned medium from ucMSCs shared the same renoprotective effects of the cells. Consistent with in-vivo data, bmMSCs and kPSCs, but even more so ucMSCs, limited proliferation, migratory potential and extracellular matrix production of activated PECs, when cultured in a transwell system. Conclusions Our data indicate that either non-renal or renal stromal cells induce renal tissue repair, highlighting ucMSCs and their conditioned medium as the most reliable clinical therapeutic tool for CKD patients. Electronic supplementary material The online version of this article (10.1186/s13287-018-0960-8) contains supplementary material, which is available to authorized users.

Other organs as heart, liver and lung were also processed for quantification of non-renal or renal stromal cells positive for HNA (3 sections for each organ, n=3 animals).

Renal Morphology
Kidney samples were fixed in Duboscq-Brazil. Paraffin-embedded sections (3-µm) were stained with periodic acid-Schiff reagent. At least 15-20 glomeruli were examined for each rat, and the extent of lesions was expressed by giving a score from 0 to 4 related to the percentage of glomerular tuft occupied by lesions (0 = no lesions; 1 = lesions affecting ≤25% of the glomerulus; 2 = lesions affecting 26 to 50% of the glomerulus, 3 = lesions affecting 51 to 75% of the glomerulus, 4 = lesions affecting 76 to 100% of the glomerulus). The formation of synechiae was evaluated at 14 days after ADR injection and data were expressed as percentage of glomeruli with different degrees of lesions. The extent of glomerulosclerosis was evaluated at 28 days after ADR, and glomerular damage was expressed as the percentage of glomeruli affected by glomerulosclerosis (%GS).

Immunohistochemistry
For immunofluorescence experiments, sections (3-µm) from OCT or PLP-fixed kidney specimens were analysed as appropriate. After antigen unmasking and blocking of nonspecific sites, sections were incubated with the following primary antibodies: rabbit anti-Wilm´s tumour 1 (WT1, 2 µg/ml, Santa Cruz, CA, USA, https://www.scbt.com), goat anti-nephrin (0.2 µg/ml; Santa Cruz), Apoptosis was analyzed quantifying the glomerular cleaved caspase-3 expression, by using the analysis software ImageJ 1.40g. Digitised images were binarised using a threshold for areas of glomerular cleaved caspase-3 staining, and the values were expressed as percentage of area occupied by cleaved caspase-3 staining (n=3 sections for each animal).

TGF-β analysis
Serum TGF-b levels were measured using a commercial available rat ELISA kit (Abcam, Cambridge, U.K., http://www.abcam.com) employing internal controls, as proposed by the manufacturer and analyzed with an ELISA microplate reader at 450 nm. Serum samples were previously acidified to activate latent TGF-β to its immunoreactive form. The limit quantification was 8 pg/ml. The results were calculated in ng/ml by fitting to a standard curve, obtained using human recombinant TGF-β.

Morphometrical analysis
Glomerular podocytes were identified as cells positive for WT1. Estimation of glomerular volume (VG) was performed using a computer-based image analysis system (Mac OS09; Apple Computer) as previously described [4]. Mean value of VG and the estimation of the average number of podocytes per glomerulus were determined by the stereological method of particle density proposed by Weibel [5].
Glomerular capillary volume density (Vv) was quantified as RECA-1 positive vessels in 15-20 glomeruli per rats randomly acquired (n=3-7 rats for each group). Renal sections were digitalised using an inverted confocal laser microscopy (original magnification, x630; LSM 510 Meta; Carl Zeiss). By using the analysis software ImageJ 1.40g, digitised images were binarised using a threshold for areas of RECA-1 staining, and the values were expressed as percentage of area occupied by RECA-1 on total area of the acquired field.    Representative micrographs of renal sections from control and ADR rats receiving saline, bmMSCs, ucMSCs, kPSCs or CM-ucMSCs at 14 days showing cleaved-caspase 3 expression (red). Renal structures were stained with lectin (green) and nuclei with DAPI (blue). Scale bar 20 µm. ADR adriamycin, bmMSC bone marrow-derived mesenchymal stromal cell, CM-ucMSC conditioned medium obtained from umbilical cord-derived mesenchymal stromal cell, DAPI 4′,6diamidino-2-phenylindole, kPSC kidney perivascular stromal cell.