Inguinal adipose tissues of Balb/c mice were isolated in Hank’s Balanced Salt Solution (HBSS, cat.no.14025092, Life Technologies, USA) and washed twice with HBSS at 280 g to remove blood and liquid. Clear fat’s tissues were cut into 3 mm small fragments and incubated in the solution of HBSS contained 100 units/ml collagenase type I (cat.no.17100-017, Life Technologies, USA) along with 3 mM CaCl₂ for two hours at 37°C and 5% CO₂. After neutralization with fetal bovine serum (FBS, ref.no.16000-044, Gibco Life Technologies, USA), cells were centrifuged, the pellet was washed in HBSS, and filtered through 70 μm then 40 μm nylon mesh. mAd-MSCs were cultured in Minimum Essential Medium Eagle-Alpha Modification (α-MEM, cat.no.32561-037, Life Technologies, USA) containing 100 U/ml penicillin/streptomycin and 10% FBS in 25 cm² flasks. The flasks were incubated at 37°C in the humid atmosphere at 5% CO₂. Non-adherent cells were removed by medium replacement after every 2~3 days. Upon confluence at 70% density, cells were sub-cultured through 1X TrypLE select (cat.no.12563-011, Gibco Life Technologies, USA) in 1:3 ratio. Passage 2 mAd-MSCs were used for transplantation studies in AKI model (8).

mAd-MSCs were first analyzed by transcriptomic expressions of CD90 (Thy1) and CD44 (HCAM) (H-300) by RT-PCR. The probes are mentioned in Supplementary Table S1. Further, it was confirmed by positive expressions of these markers and negative expressions of CD45 (H-230) and CD34 (C-18), through immunocytochemistry (ICC). For ICC mAd-MSCs were cultured in 4 well chambered slides and fixed for 2 minutes in 4% paraformaldehyde, washed with Ca+, Mg+ PBS, and blocked with goat serum for an hour at room temperature. After being washed with PBS, the cells were incubated at 1:50 concentrations of rabbit polyclonal primary antibodies of CD90 (sc-9163), CD44 (sc-7946), CD45 (sc-25590), and CD34 (sc-7045) overnight at 4°C. Next day, after washing, goat anti-rabbit IgG-fluorescein isothiocyanate (FIT-C)-conjugated secondary antibody (sc-2012) was placed for an hour in dark at room temperature. 4′, 6-diamidino-2-phenylindole (DAPI; sc3598; 0.5 μg/ml) was used to stain nuclei. All the antibodies and DAPI were purchased from Santa Cruz Biotechnology. Inc., USA. The mAd-MSC phenotype and multipotency were further confirmed by the ability to differentiate into osteocytes and adipocytes. Osteogenesis (cat.no.SCR028, Australia) was carried out in inductive media containing 1 mM dexamethasone on vitronectin/collagen coated 24 well plates according to the manufacturer’s instructions. After 20 days, Alizarin Red S dye, and Von Kossa stains were used to observe calcified differentiated cells. For adipogenesis 100 nM dexamethasone was used in inductive media (cat.no.TM005, ABM, Canada) with 50 mg/ml indomethacin. After 21 days, differentiated cells were observed through Oil red O staining. Microscopic analyses were carried out at 20~40× magnifications.

The experimental animal procedures were approved by the ethical and the animal care and use committee of Sindh Institute of Urology and Transplantation (SIUT)-Karachi, according to international standards. 6~8 week old male Balb/c mice (25~30 g; n=70, 10/group) were maintained under pathogen-free conditions. They received standard food pellets and water ad libitum 12 hours light and 12 hours dark cycle, 22±2°C temperature, and 40~45% humidity. Mice were divided into seven groups according to the duration (days) post CP injection and harvesting of samples. The groups included were (i) Normal/Control; (ii) 24 hr; (iii) 48 hr; (iv) 72 hr; (v) 96 hr; (vi) 120 hr; (vii) CP+mAd-MSCs transplanted. AKI was induced by single subcutaneous infusion of 18 mg/kg Cisplatin (CP) (cat.no.3410101336-20504, Korea United Pharm. Inc., Korea) in physiological saline and control group received normal saline instead. Mice weighed daily and euthanized by anesthetic overdose at each time point of the experimental period. Blood serum and kidney tissues were collected between 24~120 hours of CP injection from CP groups, and CP+mAd-MSCs transplanted mice were euthanized after a week (total 12 days). Weight (gram) and survival of mice were observed.

For single cell preparation, 90~95% viable mAd-MSCs were harvested using accutase (cat.no.A1110501, Fisher Scientific, Norway), washed thoroughly, and suspended in PBS in presence of antithrombin Eskinase (Streptokinase B.P, 1.5MIU, Pakistan). Cells were suspended in 700 μl 1X PBS contained 3 μl (0.0045MIU) Eskinase (~0.00130/200 μl PBS) and gently filtered through 70 μm then 40 μm nylon mesh. Prior to this, 1 ml syringes were triturated through heparin (HeptaRotex; 5,000 units/ml, Germany) and sodium bicarbonate (7.5%) (REXA-BiCARB, Pakistan). mAd-MSCs were filled in the syringe through 200 μl tips and kept at 37°C until transplantation within ten minutes. For in vivo tracking of cells within 24~72 hours fluorescence labeling dyes, 5-chloromethylfluorescein diacetate (Cell Tracker^®CMFDA, cat.no.C7025, Life Technologies, USA) (Ex.492/Em.517 nm) and Vybrant Dil (cat.no.V-22885 Molecular Probes, Inc., USA) (Ex.549/Em.565) (50 μg/ml) were standardized to mAd-MSCs before infusion. mAd-MSCs were administered to AKI mice after 72 hours of injury (group vii: CP+mAd-MSCs transplanted mice). In different experiments, 2.5×10⁷ cells/kg (0.5~1×10⁶/200 μl) body weight labeled and unlabeled cells were infused gently in the lateral tail vein of CP-treated Balb/c mice.

Harvested kidneys were rinsed in saline (0.9% sodium chloride) and perfused in 10% neutral buffered formalin. After appropriate dehydration, kidney slices were embedded in paraffin, deparaffinized with xylene, rehydrated, and sectioned at 4 μm thick slices using a microtome. It proceeded to stain via Hematoxylin and Eosin (H&E), Periodic Acid-Schiff’s staining (PAS), and Masson’s Trichrome staining (TRI). Corticomedullary fields were recorded from each kidney section by the light microscope (Nikon Eclipse Ti-S) equipped with a DS-L3 (DS-Fi2) camera. Tubular damage was identified by the loss of brush border, lumen dilatation or collapse, vacuolation, and desquamation of epithelial cells in renal tubules after CP treatment. Histopathology grading by light microscopy was considered for renal injury. The grades were given to tubular necrosis that loss of brush border or tubular epithelial cells, protein cast formation, tubular dilatation, and accumulation of inflammatory infiltrate in ten randomly chosen non-overlapping fields (40× Magnification) of kidney sections. The scores for grading were based on the percentage of the affected parameter, which is as follows: 0 (no change/normal architecture), 1 (minimal changes ≤ 15%), 2 (mild changes 15≤25%), 3 (moderate degeneration 25~50%), 4 (severe degeneration 50~75%), and 5 (extremely severe degeneration ≥75~100%) (9).

To assess morphology, kidney specimens were snap-frozen at −20°C in optimum cutting temperature (O.C.T.) solution. For detection of proliferating cells in kidney sections, 5-Bromo-2′-Deoxyuridine+5-Fluoro-2′ Deoxyuridine (BrdU; 00-0103) was intraperitoneally injected (100 mg/kg) in mice for an hour before being sacrificed. AlexaFluor^®BrdU-Mouse Monoclonal (Clone MoBU-1; Invitrogen) antibody (Ex.495/Em.519 nm) was used in 1:500 dilution in the sections, which were already transplanted with Vybrant Dil (Ex.549/Em.565 nm) labeled mAd-MSCs. The kidney sections were adsorbed on polylysine-coated glass slides, air-dried, de-paraffinized, and then placed in a water bath. The sections were treated with heating for antigen retrieval. For peroxidase-linked immunostaining, endogenous peroxidase was removed by 0.05 M hydrogen peroxide (H₂O₂) for five minutes. Non-specificities were blocked by 5% bovine serum albumin. The kidney specimens were incubated with rabbit polyclonal Megalin (sc25470, Santa Cruz Biotechnology. Inc., USA) and Kidney Injury Molecule 1 (Kim-1) (ab47635, abcam, UK) against mouse at 1:1000 and 1:1500 dilutions respectively. Biotinylated horse (DSP-999) and Biotinylated goat (DGP-999) (Spring Bioscience Corp., USA) anti-polyvalent horseradish peroxidase (HRP) secondaries were used against Megalin and Kim-1 primary antibodies respectively. 3, 3′-diaminobenzidine (DAB) was used as a chromogen. The sections were counterstained with hematoxylin and mounted with DPX (cat.no.4458, SIGMA). Fluorescence microscope (Nikon Eclipse Ti-S) was used to acquire images at 20~40× magnifications.

Serum creatinine (sCr) (cat.no.CR510), blood urea (cat.no.UR107), and total urinary protein (cat.no.UP 1570) were measured in aforementioned groups. These kits were purchased from Randox Laboratories Limited, UK. Change in biochemical parameters was considered for renal functions’s assessment and the survival of mice.

Renal tissues were homogenized, sonicated, and total RNA was isolated using Ambion kit (cat.no.12183018A, PureLink TM RNA Mini Kit, Invitrogen, Life Technologies, USA) for the biological and technical triplicates and 0.5 μg RNA was used to synthesized cDNA according to the manufacturer’s instructions (part no.4368813, High Capacity cDNA Revert Transcriptase Kit, Applied Biosystems Inc., USA). It was amplified under the following PCR conditions: 94°C for five minutes, 30 cycles of PCR (94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec) 72°C for seven minutes and then at 4°C using Master Mix (cat.no.10572-063, PCR Supermix, Invitrogen, USA). Samples were electrophoresed at 2% agarose gel. For a standard reaction in real-time PCR, cDNA in 1:10 dilution was used as a PCR template. cDNA was mixed together with 2.5 nM of each primer in a final volume of 25 μl. Real-time PCR was performed using LightCycler TaqMan Master Mix (Roche, Germany) through manufacturer’s guidelines. An endogenous ‘housekeeping’ gene, 18S rRNA was quantified and used to normalize the results. All the genes were compared with control (normal adult mice) and relative genes expressions were quantified through 2^−ΔΔCt method. The probe sequences are given in Supplemental Table 1.

Quantitative data were expressed as means±s.e.m., or means±s.d. ANOVA was performed, followed to Post Hoc Tucky’s correction (SPSS 20.0 for Windows, SPSS Inc., USA; or MedCalc version 17.9.5) to compare multiple groups. Kaplan-Meier test was performed for survival function analysis. 95% confidence interval (*p≤0.05) was considered statistically significant.

mAd-MSCs were shown to be adherent to the plastic surface, spindle-shaped, and fibroblast appearance with colony forming properties, which were observed through light microscopy. mAd-MSCs rarely expressed CD45 and CD34 expressions, which are specific surface markers for hematopoietic cells while CD90 and CD44 are positive markers of stem cells, and were also positively detected by RT-PCR and ICC. Osteogenic and adipogenic differentiation assays confirmed the multipotential capacity of isolated mAd-MSCs by the formation of hydroxyapatite minerals and fatty depots respectively (Fig. 1).

Administered cells were detected by fluorescence microscopy in kidney sections. mAd-MSCs were identified by CMFDA (green) and Dil (red) labeled cells in the renal parenchyma within 24~72 hours following systemic administration of 0.5~1×10⁶ mAd-MSCs. It demonstrates localization and engraftment potential of mAd-MSCs under injury cues. Most cells were localized in the cortex and outer medulla in the region of proximal tubular cells; the most prominent region of CP-induced renal injury. mAd-MSCs were not observed in the heart, some observed in the liver, and many cells were observed in the lungs (Fig. 2A~C). Nuclear stains via anti-BrdU displayed proliferating tubular epithelial cells in the cortical region as compared to control and other body organs of the same mouse. BrdU is incorporated into newly synthesized DNA during the S phase of the cell cycle, which demonstrates enhanced multiplication or proliferation of cells in renal tubules. Most glomeruli contained a few BrdU-retained cells (Fig. 2C, D).

H&E, PAS staining, and histopathological grading displayed significant (***p≤0.001) severe tubular cell necrosis, brush border cells loss, and inflammatory infiltrates with luminal casts formation in animals following exposure to CP (18 mg/kg, s.c.) from 24~120 hours as compared to the normal architecture of renal cells in control mice. After 24 hours, the main features were vacuolization in tubules, sloughing of cells, and removal of nuclei from tubules while other organs, heart, lung, and liver remain in normal architecture (data not shown). Most of the renal parenchyma was indistinguishable. Atrophied cells and the injuries were found to be more severe after 72~96 hours of CP, and mortality was high between 96~120 hours. As a result of these changes, segmental hyalinization was observed in the cortical region. Glomeruli were also affected; atrophied, segmental shrinkage in Bowmen’s capsules were observed in 96 hours of CP-treated sections. H&E staining showed that congestion was started from 24 hours of CP infusion, and low congestion was found in the group that received mAd-MSCs. Prominent casts were observed after 48 hours of CP infusion while hexagonal or rectangular shape tubular dilatations were observed after 96 hours of CP infusion. Histopathology also revealed the accumulation of nuclei after patchy or diffused denudation and cellular blabbing of renal epithelial cells post 96 hours of CP injection in Balb/c mice. The renal sections of 24~48 hours of CP exposure executed no fibrosis and did not grade as it was not significant (≤15%) and found in a patch in some mouse in the corticomedullary regions post 72~120 hours. Glomerulosclerosis was not observed in any of the sections in all groups after CP exposure. The autopsy of dead mice after 120 hours of CP infusion evidenced complete grade 5 (≥75~100% fibrosis) (image not shown) collagen deposition. Hence, mild acute tubular necrosis (ATN) was observed after 24 hours, and severe degenerations were observed after 72 hours of CP infusion. Nearly Grade 4 resulted in extreme damage, and it covered 50% of the damaged area of the corticomedullary region. These parenchymal changes were not observed in mAd-MSCs transplanted mice. mAd-MSCs infusion demonstrated the clear, intact structure of tubular epithelial cells. The damage was attenuated, which was observed with scarce protein casts formed and low inflammatory infiltrates post seven days (***p≤0.001) as compared to controls. Although after mAd-MSCs transplantation, histopathology did not show complete regeneration of all renal tubular cells, and renal tubular dilatation was not significantly diminished, but with a lesser extent of damage survival of mice was increased as compared to CP-treated mice. Some whitish fatty or unfilled empty areas were observed around some tubules, which gave a smooth appearance, and it looked like something had been cleared off in mAd-MSCs treated mice (Fig. 3A, B). The brush border protein loss in AKI was condensed as it was observed with the sub-normal re-expression of Megalin post mAd-MSCs transplantation. Megalin is a specific protein in the brush border of proximal tubular cells. Kim-1 protein expression was reduced after cellular therapy, which is known to express in development and an injury in the kidney. Both expressions were tested by histopathologists in a blind study. It demonstrated that mAd-MSCs infusion protected the kidney to an extent from further severe AKI damage, and these cells can protect from the complete failure of the organ (Fig. 3C).

Improved survival of AKI balb/c mice attributed to the recovery of renal functions. CP infusion caused a marked increase in serum creatinine, urea, and total urinary protein concomitant to the aforementioned histopathological events. These parameters confirmed the successful development of severely injured renal parenchymal cells in balb/c mice. Significant attenuated (**p≤0.001) mean levels of serum creatinine from <3 fold to~1.5 fold were observed in CP-induced AKI and after mAd-MSCs infusion at day 12 respectively (Fig. 4A). Blood urea and total urinary protein, although did not reach to the significance levels, but they achieved 50% reduction in these levels in post mAd-MSCs transplanted balb/c mice in contrast to CP treatment groups at 96 and 120 hours (Fig. 4B, C). CP toxicity results in 4~8 gram reduction in body weight of Balb/c mice. Most mice died between 96~120 hours, and the percent survival was decreased 80~90% after 96 hours in 18 mg/kg CP-treated mice. Mortality was significantly driven down in mice received mAd-MSCs. Survival of balb/c mice increased, which was observed untill day12 after transplantation of mAd-MSCs and it exposed the efficiency of cell therapy, which is associated with recovery of renal structure and its function (Fig. 4D, E).

Because MSCs are known to secrete growth factors and have immunomodulatory properties. We screened kidneys by TaqMan real-time quantitative RT-PCR. The changes in the expressions of inflammatory mediator, Tumor necrosis factor alpha (TNF-α); developmental protein, Paired box 2 (Pax2); pro-fibrotic cytokine, Transforming growth factor beta 1 (TGF-β1); survival protein, Mitogen-activated protein kinase 1 (MAPK1); Notch signaling ligand, Jagged1 (Jag1); epithelial markers, Solute carrier family 4 member 2 (SLC4a2); and Kidney specific protein, Ksp-cadherin (Ksp) were evaluated pre and post mAd-MSCs infusion. Inflammatory kidneys of CP-treated animals post 48 hours showed ~7 fold induction of the proinflammatory cytokine; TNF-α. Post mAd-MSCs transplantation demonstrated a marked reduction in TNF-α as compared to pretreated CP infusion. Many studies presented that fibrogenesis was shrunk by decreasing TGF-β 1. TGF-β1 promotes negative regulation in the expression of Pax2 protein through the post-transcriptional mechanism in which it abolished the stability of Pax2 mRNA and consequently, its protein. During embryogenesis, Pax2 is involved in mesenchymal to epithelial transition (MET), i.e. is tubular epithelial cells differentiation, but only confined to collecting ducts in healthy adult kidneys. Pax2 is regulated by TGF-β1. It plays role in tubular cells regeneration, proliferation, tubulogenesis, and apoptosis. Pax2 is re-expressed in proximal tubular cells after injury. In our study peak levels of Pax2 (1×10⁸ fold) significantly up-regulated (***p≤0.001) in 96 hours and when the cells were injected its levels were reduced significantly (***p≤0.001). During CP injury TGF-β1 level was also optimal in 96 hours and exhibited competitive expression vs Pax2. mAd-MSCs presence down-regulated TGF-β1 as well as Pax2 levels (10). Overall MAPK1 advanced to near 40 fold as compared to control, and it attained a normal level post mAd-MSCs infusion. Jagged1 displayed 8 fold changes with respect to control mice. However, there is a very low fold change in Jagged1 of the renal tissues after mAd-MSCs transplantation. Grossly some fold change differences were executed in the expressions levels of SLC4a2 and Ksp. It might be due to the other epithelial cells, which were not affected by CP infusion and were greater than the number of cells in proximal tubules. Hence, these results revealed potential mechanisms whereby administered mAd-MSC exerted their protective effects, which led to significant and prompt renoprotection as a first step (Fig. 5).