Isolation of huMSCs
Human umbilical cords were collected from healthy infants and mothers at the University Hospital of the University of São Paulo, Brazil. Wharton’s jelly was surgically extracted less than 24 h after delivery. Small (1–5 mm) explants were plated in culture dishes and cultured with alpha-modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with sodium bicarbonate at pH 7.3 (Sigma-Aldrich), penicillin (300 U/ml; Thermo Fisher Scientific, Waltham, MA, USA), streptomycin (300 μg/ml; Thermo Fisher Scientific) and 20% foetal bovine serum (Sigma-Aldrich). Explants were incubated at 37 °C in 5% CO2, without changing the medium, for 10–15 days. When cells began to migrate from the explants, the medium was changed every 3–4 days; when they reached 80% confluence, the explants were removed. The cells were then treated with 0.25% trypsin–ethylenediaminetetraacetic acid (trypsin–EDTA; Thermo Fisher Scientific), to be seeded as first-passage (P1) cells. Animals were injected with cells from the third to the fifth passages (P3–P5 cells).
Isolation of aMSCs
To isolate aMSCs, we collected subcutaneous tissue from an adult patient undergoing elective lipoaspiration surgery. The tissue sample was washed with phosphate-buffered saline (PBS). After digestion with 0.1% collagenase type 1 for 60 min, cells were supplemented with 1% bovine serum albumin and 2 mM CaCl2. The stromal fraction was separated by centrifugation at 300 × g at room temperature. The aMSCs were cultured and expanded exactly as described for the huMSCs. Animals were injected with P3–P5 cells.
Cell immunophenotyping
Flow cytometry analysis was performed with allophycocyanin-conjugated, fluorescein isothiocyanate-conjugated, phycoerythrin-cyanine 7-conjugated or phycoerythrin-conjugated antibodies against CD45, CD34, human leukocyte antigen-D region, CD44, CD29, CD105, CD73 and CD90 (BD Biosciences Research, Franklin Lakes, NJ, USA), analysed in a FACSDiva flow cytometer with appropriate software (BD Biosciences Research).
Lineage differentiation of MSCs
Lineage differentiation was performed with P2 cells seeded in six-well plates at 1 × 105 cells per well. Adipogenesis, osteogenesis and chondrogenesis were tested over periods of 14, 21 and 14 days, respectively, with commercial differentiation kits (StemPro; Thermo Fisher Scientific). Differentiation media were changed every 3–4 days. Adipogenesis, osteogenesis and chondrogenesis were confirmed by staining with Oil Red O, Alizarin and Alcian blue, respectively.
Cell protein extraction
Total proteins were extracted from P7–P8 huMSCs and aMSCs. The culture medium was aspirated from the adherent cells, which were then washed twice in cold PBS. Radioimmunoprecipitation assay buffer (Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich) were added, after which the cells were scraped from the plates and sonicated. Samples were centrifuged at 4000 × g for 30 min at 4 °C, and supernatants containing total proteins were isolated. Total protein was quantified by bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA).
Rat model of renal IRI
Young (2–3 months old) male Wistar–Kyoto rats, weighing 200–300 g, were purchased from the University of São Paulo Biomedical Institute, and all protocols were in accordance with the University of São Paulo Guide for the Care and Use of Laboratory Animals.
Prior to IRI induction, rats were anesthetised with ketamine (70 mg/kg body weight (BW)) and xylazine (7 mg/kg BW). Both renal arteries were clamped for 45 min. At 6 h after reperfusion, some rats were injected intraperitoneally with 2 ml of saline diluted with 1 × 106 freshly recovered P3–P5 huMSCs (IRI + huMSC group) or 1 × 106 freshly recovered P3–P5 aMSCs (IRI + aMSC group), whereas other rats went untreated (IRI group). To establish the peak of the acute phase and the recovery of renal function, plasma urea levels were determined over a 7-day period (control group, n = 4; IRI group, n = 9; IRI + huMSC group, n = 5). On the basis of the data obtained, we chose to euthanise some animals on post-IRI day 2 (D2) to evaluate the peak, and some animals on D7 to evaluate the recovery. We also euthanised some animals on D49, in order to study the long-term effects on renal function (Fig. 1).
Rats were maintained on a 12-h/12-h light/dark cycle. On D1, D6 and D48, the rats were placed in metabolic cages and 24-h urine samples were collected. On D2, D7 and D49, animals were anaesthetised with ketamine (70 mg/kg BW) and xylazine (7 mg/kg BW), after which arterial blood was collected from the aorta. Animals were then euthanised with an overdose of anaesthesia. Kidneys were flushed with saline and cut into sections: half of the kidneys were fixed in methacarn for histological analysis; the remaining kidneys were stored at −80 °C for further studies.
Biochemical parameters
We used colourimetric methods to determine plasma creatinine by the Jaffé reaction (Labtest, Brazil) and plasma urea (Labtest, Brazil). To determine plasma sodium we used ion-selective electrodes (AVL 9140 Electrolyte Analyzer; Roche, Basel, Switzerland), and urinary sodium was determined using photometry (CELM, Brazil). Osmolality was measured with a wide-range osmometer (3 W2; Advanced Instruments, Norwood, MA, USA).
Kidney protein extraction
Kidney samples were homogenised in ice-cold HEPES–KOH buffer, pH 7.5, containing a protease inhibitor cocktail (Sigma-Aldrich) in a homogeniser (PT 10/35; Brinkmann Instruments, Westbury, NY, USA). Homogenates were centrifuged at 4000 × g for 30 min at 4 °C to remove nuclei and cell debris. Supernatants, containing total protein, were isolated. To study membrane proteins, the collecting duct water channel aquaporin 2 (AQP2) and Klotho, we performed a second centrifugation of supernatants, at 100,000 × g for 1 h at 4 °C, and the pellet was re-suspended in HEPES–KOH to obtain the membrane protein fraction. Total protein and membrane proteins were quantified by bicinchoninic acid protein assay kit (Pierce).
Electrophoresis and immunoblotting
Kidney and cell samples were run on polyacrylamide gels. Gels were run in duplicate and stained with Coomassie blue (0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid). Selected bands from those gels were scanned, and the density was determined. Gels not so stained were transferred by electroelution to nitrocellulose membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK), and blots were blocked with 5% non-fat dry milk in Tris-buffered saline. Blots were then incubated overnight with antibodies against β-galactosidase (β-gal) at 1:1000 (Sigma-Aldrich); against AQP2 at 1:10,000, Klotho at 1:500, p21Waf1/Cip1 (hereafter p21) at 1:500, p16INK4a (hereafter p16) at 1:1000, transforming growth factor beta 1 (TGF-β1) at 1:1000 and Actin at 1:2000 (all Santa Cruz Biotechnology, Dallas, TX, USA); against HO-1 at 1:1000 (Assay Designs, Ann Arbor, MI, USA); and against MnSOD at 1:4000 (Cayman Chemical, Ann Arbor, MI, USA). The labelling was visualised with horseradish peroxidase-conjugated IgG secondary antibody—anti-rabbit (1:2000), anti-mouse (1:2000) or anti-goat (1:10,000) (all Sigma)—and enhanced chemiluminescence detection (Amersham Biosciences; GE Healthcare). Densitometry was used to quantitatively analyse the antibodies, normalising the bands to Actin expression or to selected scanned bands from gels stained with Coomassie Blue (whichever we found to be most suitable for each membrane). Images were visualised with a transilluminator (Alliance 4.2; UVItec, Cambridge, UK) and analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Histopathology
Kidneys extracted from animals euthanised on D2 or D7 were processed in paraffin, cut into 4-μm sections and stained with periodic acid–Schiff for light microscopy. The proportional renal damage (tubular epithelial swelling, vacuolar degeneration, necrosis and desquamation) was graded, and a semi-quantitative score of tubular damage was determined as follows: 0, <10%; 1, 10–25%; 2, 26–50%; and 3, >50%.
Kidneys extracted from animals euthanised on D49 were processed in paraffin, cut into 4-μm sections and stained with Masson’s trichrome. A semi-quantitative renal damage score was calculated based on the proportion of the kidney occupied by fibrosis, oedema or inflammatory cells, as follows: 0, none; 1, 1–5%; 2, 6–10%; 3, 10–25%; 4, 26–50%; 5, >50%.
To minimise bias in the morphometric analysis, the observer was blinded to the treatment groups and all microscopic fields were analysed. The mean scores were calculated by rat and by group.
Immunohistochemistry
We performed immunohistochemical reactions in 4-μm kidney tissue sections from D2 and D7, using antibodies against proliferating cell nuclear antigen (PCNA) and CD3 (1:200 and 1:50, respectively; Dako, Glostrup, Denmark), as well as against CD68 (1:100; Serotec, Hercules, CA, USA). Reaction products were detected by the avidin–biotin–peroxidase complex method (Vector Laboratories, Burlingame, CA, USA), and the colour reaction was developed in 3,3-diaminobenzidine (Sigma-Aldrich) and hydrogen peroxide. Counterstaining was with Harris’ haematoxylin. We analysed 30 juxtamedullary fields. The results of the immunoreactions were quantified by counting the number of positive cells per 0.087-mm2 field and averaging the number of cells per field in each section.
miR analysis
Experiments were performed on kidneys harvested on D2. We extracted miRs using an isolation kit (mirVana; Thermo Fisher Scientific), and we used total RNA enriched with miRs. For reverse-transcriptase reaction, 5 ng of RNA was used, employing the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). The miRs studied were miR-29a, miR-29b, miR-335 and miR-34a (Applied Biosystems; Thermo Fisher Scientific). RNU48, RNU44, U47 and U6 (Applied Biosystems; Thermo Fisher Scientific) were tested as possible housekeeping genes; we found U6 to be the best suited and used it in our analysis.
To perform a quantitative polymerase chain reaction (qPCR), we used TaqMan Universal PCR Master Mix II (Thermo Fisher Scientific). The data were analysed with DataAssist software (Applied Biosystems; Thermo Fisher Scientific) and relative gene expression calculated as 2–ΔΔCt, where Ct is the threshold cycle.
DNA extraction
Frozen whole kidney tissue digested in proteinase K was used in order to extract genomic DNA with a commercial kit (QIAamp™ DNA mini kit; Qiagen, Venlo, the Netherlands) and the DNA was then quantified using a NanoDrop microvolume spectrophotometer (Thermo Fisher Scientific). The absence of degradation was identified by electrophoresis on an agarose gel containing ethidium bromide.
Southern blotting to assess telomere length
A 2-mg aliquot of DNA was digested with Fast Digest HinfI (4 μl/2 mg of DNA; Thermo Fisher Scientific) at 37 °C for 2 h. Digested DNA samples were loaded onto 0.8% agarose gels and were run at 80 V for 4 h. To assess telomere length, Southern blotting was performed with a commercial kit (TeloTAGGG Telomere Length Assay; Roche), in which a 5′-TTAGGG-3′ digoxigenin-labelled telomere probe is used and visualised by linking to a chemiluminescent substrate. Blots were imaged in an image analyser (ImageQuant 350; GE Healthcare). We compared two different methods of analysing telomere shortening: the more established method, based on the average telomere lengths (i.e. the mean number of terminal restriction fragments (TRFs)); and a newer method, in which telomeres are stratified by length and the proportions of short (<8.6 kb), medium (8.6–21.2 kb) and long (>21.2 kb) telomeres are determined.
Telomerase activity
Total kidney protein was used to assess telomerase activity with a commercial kit (TeloTAGGG telomerase PCR ELISA; Roche), which combines PCR and enzyme-linked immunosorbent assay techniques (Additional file 1: Figure S1). To determine the intensity of telomerase activity, we assessed luminescence in a microplate reader (Thermo Fisher Scientific).
Statistical analysis
Results are expressed as mean ± standard error of the mean. Differences among groups were analysed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA), using analysis of variance followed by Tukey’s post test. When comparisons between two groups were made, unpaired t tests were used. When categorical variables were considered, histomorphometry data were also analysed with chi-square tests. Values of p ≤ 0.05 were considered significant.