Evaluation of the effect of autologous mesenchymal stem cell injection in a large-animal model of bilateral kidney ischaemia reperfusion injury

Abstract

Objectives: Adult mesenchymal stem cells (MSC) have been proven to be of benefit to the kidney in different experimental models of renal injuries. All studies have been performed in valuable rodent models, but the relevance of these results to large mammals and ultimately, to humans remains unknown. Therefore, the aim of this study was to investigate the effect of MSC transplantation in an alternative ovine large-animal model of bilateral kidney ischaemia reperfusion injury.

Material and methods: Sheep were divided into three groups: one sham-operated group and two groups submitted to renal bilateral ischaemia for 60 min. Animals with ischaemia reperfusion injury were treated with injection of autologous MSCs or with vehicle medium.

Results: The model sheep presented with renal histological manefestations that closely resembled lesions seen in patients. Transplanted MSCs were found in glomeruli but not in tubules and did not express glomerular cell markers (podocin, von Willebrand factor), but functional evaluation showed no beneficial effect of MSC infusion. Morphological and molecular analyses corroborated the functional results. MSCs did not repair kidney parenchyma and failed to modulate cell death and proliferation or cytokine release (tumour necrosis factor-alpha, vascular endothelial growth factor alpha (VEGF-alpha), Bcl-2, caspase).

Conclusion: In this unique autologous large-animal model, MSCs did not exhibit reparative or paracrine protective properties.

Figure 1

Mesenchymal stem cells (MSC) in culture, in vitro differentiation and cytofluorometry analysis. MSCs were characterized by their property to adhere to cell culture plastic dish and exhibit spindle shape morphology in culture (a). They were further identified by their canonical ability to differentiate into osteocytes (b), chondrocytes (c) and adipocytes (d) in vitro. Cell surface markers of MSCs were studied using flow cytometry analysis (e). Sheep MSCs were positive for CD44 and CD58 whereas being negative for CD31 and CD45.Original magnification: ×200 in (a); ×100 in (b) and (d); ×400 in (c).

Figure 2

Mesenchymal stem cell (MSC) localization and counting in the kidney and phenotypical analysis. MSCs were tracked using cm‐DiI fluorescence (a). MSCs were present in glomeruli (white circle) whereas no MSCs could be found in tubules (a). MSC differentiation into glomerular cell type was investigated (b, c). Glomeruli were stained with an antibody against podocin (b) and von Willebrand factor (c) to assess podocyte or endothelial cell phenotype, respectively. No MSCs expressed either marker. MSCs did not differentiate into glomerular cells, and they remained located inside the capillary lumen. Counting MSCs showed that they were mainly present in the cortex localizing in glomeruli (d). Up to 85 ± 5.1% of the glomeruli presented MSCs. There was no significant difference when comparing outer (87 ± 9.2%) and inner cortex (81 ± 7.1%). There were approximately 4 MSCs per glomerulus section (e). No significant difference was observed in the number of MSCs per glomerulus in the outer or inner cortex. Original magnification: ×200 in (a), (b) and (c).

Figure 3

Blood urea nitrogen (BUN), plasma creatinine concentration and plasma renin activity (PRA). BUN and plasma creatinine values did not change in sham‐operated group. IRI medium‐treated group showed significant increase in BUN and plasma creatinine at days 1 and 2, with values returning to baseline by day 7. In IRI MSC‐treated group, values followed the same trend as in IRI medium‐treated. White bars = group A (sham‐operated group); grey bars = group B (IRI medium‐treated group); black bars = group C (IRI MSC‐treated group). §P < 0.05 vs. group A at corresponding time points. PRA values did not change in sham‐operated group. IRI medium‐treated group and IRI MSC‐treated group showed significant increase in PRA at 10 min and 30 min, with values returning to almost baseline at day 7. There was no significant difference observed after MSCs infusion in IRI sheep. White bars = group A (sham‐operated group); grey bars = group B (IRI medium‐treated group); black bars = group C (IRI MSC‐treated group). §P < 0.05 vs. group A at corresponding time points.

Figure 4

Urine Na/creatinine and Ca/creatinine ratios. Effect of mesenchymal stem cell (MSC) transplantation on urinary Na/creatinine (a) and Ca/creatinine (b) ratios. Ratios’ values did not change in the sham‐operated group. IRI medium‐treated group showed significant increase in either ratios at 10 min and 30 min, with values returning to almost baseline at day 7. In IRI MSC‐treated group values followed the same trend as in IRI medium‐treated group. White bars = group A (sham‐operated group); grey bars = group B (IRI medium‐treated group); black bars = group C (IRI MSC‐treated group). §P < 0.05 vs. group A at corresponding time points.

Figure 5

Weight of kidneys, renal injury scores and morphological analysis. In the sham‐operated group, no renal damage could be observed (a). In IRI medium‐treated group, typical ischaemia reperfusion‐induced damage was present with tubular dilatation (black arrows) and necrosis, epithelial cell shedding (white arrows), inflammatory cell infiltration (dashed black arrows), cast formation and cortical necrosis (b). In IRI MSC‐treated group, the type and extent of lesions were the same as in IRI medium‐treated group (c). Injury scores confirmed that there was no damage in sham‐operated group, and when comparing IRI medium‐treated group and IRI MSC‐treated group, no improvement of renal lesions could be observed after MSCs treatment (d). §P < 0.05 vs. group A. Original magnification: ×200 in (a), (b) and (c).

Figure 6

Renal cell apoptosis and proliferation. Renal cell apoptosis and proliferation was assed in all groups. Terminal deoxynucleotidyl transferase‐mediated digoxigenin‐deoxyuridine nick‐end labelling (TUNEL) staining was performed on kidneys at day 7 in sham‐operated group (a), medium‐treated IRI group (b) and MSC‐treated IRI group (c). Quantification of TUNEL‐positive cells/high‐power field was carried out (d). Proliferating cell nuclear antigen (PCNA) staining was performed on sections of kidneys at day 7 in sham‐operated group (e), medium‐treated IRI group (f) and MSC‐treated IRI group (g). Quantification of PCNA was performed (h). There was a significant increase in TUNEL and PCNA expression in IRI groups when compared to sham group, but no statistically significant difference was observed when MSCs were injected compared to medium treatment in IRI groups. §P < 0.05 vs. sham‐operated group. Original magnification: ×400 in (a–g).

Figure 7

Gene expression analysis. Comparative gene expression ratios in all groups. Four different regions of the kidney have been individually investigated: outer cortex, inner cortex, outer medulla and inner medulla. GAPDH was used as internal control. Black bars on all graphs illustrate gene expression ration of 1 (i.e. a value obtained when gene expression is identical in compared groups). Values lower or higher than 1 indicate gene expression is decreased or increased in one group when compared to the other. No significant difference in genes expression was observed after MSC treatment in IRI groups. *P < 0.05.