Modulation of Renal Parenchyma in Response to Allogeneic Adipose-Derived Mesenchymal Stem Cells Transplantation in Acute Kidney Injury

Abstract

Background and objectives: In regenerative medicine, mesenchymal stem cells derived from adipose tissues (Ad-MSCs) are a very attractive target to treat many diseases. In relation to nephrology, the aim of the current study is to investigate the effects of Ad-MSCs for the amelioration of acute kidney injury and to explore the mechanism of renal parenchymal changes in response to allogeneic transplantation of Ad-MSCs.

Methods and results: The nephrotoxicity was induced by cisplatin (CP) in balb/c mice according to RIFLE Class and AKIN Stage 3. PCR, qRT-PCR and fluorescent labeled cells infusion, histopathology, immunohistochemistry, functional analyses were used for genes and proteins expressions data acquisition respectively. We demonstrated that single intravenous infusion of 2.5×10⁷/kg mAd-MSCs in mice pre-injected with CP recruited to the kidney, restored the renal structure, and function, which resulted in progressive survival of mice. The renal tissue morphology was recovered in terms of diminished necrosis or epithelial cells damage, protein casts formation, infiltration of inflammatory cells, tubular dilatation, and restoration of brush border protein; Megalin and decreased Kim-1 expressions in mAd-MSCs transplanted mice. Significant reduction in serum creatinine with slashed urea and urinary protein levels were observed. Anti-BrdU staining displayed enhanced tubular cells proliferation. Predominantly, downgrade expressions of TNF-α and TGF-β1 were observed post seven days in mAd-MSCs transplanted mice.

Conclusions: Ad-MSCs exerts pro-proliferative, anti-inflammatory, and anti-fibrotic effects. Ad-MSCs transplantation without any chemical or genetic manipulation can provide the evidence of therapeutic strategy for the origin of regeneration and overall an improved survival of the system in functionally deprived failed kidneys.

Keywords: Acute kidney injury; Adipose-derived mesenchymal stem cells; Cisplatin; TGF-β1; TNF-α; Tubular injury.

Fig. 1

Morphometry and characterization of mAd-MSCs from Balb/c mice. (A) (a) mAD-MSCs with spindle-shaped morphology (b) The transcriptomic expressions of stem cells markers CD90 and CD44 on 2% agarose gel electrophoresis and (c) Semi-quantification of these markers relative to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) (d) Immunocytochemistry for positive expressions of stem cells markers CD90 and CD44 (raw1 & 2), and negative expressions of CD45 and CD34 (raw 3 & 4). All proteins are shown with 4′, 6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FIT-C) conjugated secondary antibody and merged images (20× Magnification). (B) (a~d) In vitro differentiation of mAd-MSCs into osteogenic lineage by the formation of calcium hydroxyapatite mineral positive cells, which were visualized by Alizarin Red S (orange-red) (b) and Von Kossa stain (brown to black pigments) (d) both are shown with respective controls (a & c). (f) Adipogenic lineage differentiation of mAd-MSCs for the formation of intracellular lipid vacuoles, which were visualized by Oil Red O stain and is shown with its control (e) (40× Magnification).

Fig. 2

mAd-MSCs localized to the kidney within 24~72 hours, which facilitate the regeneration of renal tubular cells by excessive proliferation rate. Fluorescence microscopic examinations of heart, lung, liver, and kidney are demonstrated for in vivo tracking or homing of mAd-MSCs within 24~72 hours in AKI mice and proliferation of injured kidney tissues post seven days of intravenous mAd-MSCs infusion in CP-treated mice. The viability and proliferation of cells were not affected by the dyes like CMFDA and Dil. In vivo transplanted mAd-MSCs were observed in kidneys of AKI produced by CP (18 mg/kg, s.c.). Localization of cells is shown by arrows. (A) CMFDA labeled mAd-MSCs in CP-induced AKI (B) Dil labeled mAd-MSCs in CP-induced AKI. (C) Merged images of Co-stained Dil labeled mAd-MSCs and fluorescein isothiocyanate (FIT-C) labeled anti-BrdU staining following injection of BrdU reagent to assess proliferation of cells in injured kidney slices. In each panel (a) Heart (b) Lung (c) Liver (d) Right Kidney (e) Left Kidney (20× Magnification). (D) Micrograph of the kidney slices of FIT-C labeled anti-BrdU staining. (a & b) CP-treated mice (96 hrs CP), (c & d) CP+mAd-MSCs transplanted kidney sections (20× Magnification).

Fig. 3

mAd-MSCs transplantation restores the kidney architecture. (A) Histopathological improvement in AKI model of balb/c mice in response to cellular therapy by mAd-MSCs. The model was developed by CP nephrotoxicity (18 mg/kg b.w. s.c.). Time course representative kidney sections were stained with Hematoxylin & Eosin (H&E) (raw1), Periodic Acid Schiff’s (PAS) (raw2), and Masson’s trichrome staining (TRI) (raw3), and examined by light microscope. Control (saline) showed the normal architecture of tubules and glomeruli. All changes are shown by arrows. Thick arrows show tubular protein casts formation; thin arrows show degeneration of tubules while double arrows show dilatation of tubules. Congestion (erythrocyte trapping) was displayed in the H&E stain of 24~48 hrs. Some rectangular features of tubules were also observed after 72~120 hrs of CP infusion. Shrinkage of glomeruli was observed in 96 hrs of CP infusion. Both are shown in H&E and PAS stains respectively. Accumulation of nuclei is shown in H&E and PAS stains of 72 hr. PAS stains displayed some sign of fibrosis post 72~120 hrs. All stains exhibited structural improvement post mAd-MSCs transplantation, as well as some clear areas (PAS), were also observed. a=control (saline); b=24 hr; c=48 hr; d=72 hr; e=96 hr; f=120 hr; g=CP+mAd-MSCs. CP: cisplatin, G: Glomerulus, PT: Proximal tubules, DT: Distal tubules (40× Magnification). (B) Representatives graphs of histopathological scoring through H&E and PAS stains for the presence of (a) Tubular epithelial cells’s necrosis or degeneration, (b) Tubular protein casts formation, (c) Infiltration of inflammatory cells, and (d) Renal tubular dilatation. The graphical results are presented as mean±s.d. ANOVA followed by Tucky-Kramer test. *p<0.05, **p<0.01 and ***p<0.001 were the levels of significance for all groups. [1=24 hr; 2=48 hr; 3=72 hr; 4=96 hr; 5=120 hr; 6=CP+mAd-MSCs transplanted]. (C) Immuno changes in Megalin (raw1) and Kim-1 (raw2) expressions, which were enhanced and reduced respectively in mAd-MSCs transplanted mice. These changes exposed the recovery of the renal tubular brush border and reduced necrosis in the renal tissues post cellular therapy. Protein expressions in these sections are represented by arrows. (a) Control (saline); (b) 72 hr; (c) 96 hr; (d) CP+mAd-MSCs transplant (40× Magnification).

Fig. 4

mAd-MSCs ameliorate kidney failure by restoring its functions, which improved the survival of mice. Functional improvement of kidneys with the survival of balb/c mice was observed in AKI model by CP (18 mg/kg, s.c.) in post transplanted mAd-MSCs. (A) Serum Creatinine, (B) Serum Urea, (C) Total Urinary Protein, (D) Weight dynamics, (E) Survival function for a cumulative survival rate of balb/c mice in all groups was recorded from day 0~12. Data is presented as mean±s.e.m. ANOVA followed by Post-Hoc Tucky’s test for comparison was used. *p<0.5, **p<0.1, and ***p<0.001 were the levels of significance for all groups.

Fig. 5

Transcriptomic features demonstrate the anti-fibrotic and anti-inflammatory effects of mAd-MSCs. Quantitative transcriptomic expressions profiling of the genes in renal tissues of sacrificed balb/c mice by TaqMan qRT-PCR was evaluated in pre (24-120 hours) and post-transplantation of mAd-MSCs. The data was evaluated by the 2^−ΔΔCt method for fold change in genes expressions, which were used for multiple comparisons in ANOVA followed by Tucky’s test. The levels of significance *p<0.05, **p<0.01, and ***p<0.001 were used [log10 scale for Pax2, TGFβ-1, and Ksp]. (A) Tumor necrosis factor alpha (TNF-α). (B) Paired box gene 2 (Pax2). (C) Transforming growth factor beta 1 (TGFβ1). (D) Mitogen-activated protein kinase 1 (MAPK1). (E) Jagged1. (F) Solute carrier family 4 member 2 (SLC4a2). (G) Kidney-specific protein (Ksp-cadherin). [1=24 hr; 2=48 hr; 3=72 hr; 4=96 hr; 5=120 hr; 6=CP+mAd-MSCs transplanted].