Superexpressão induzida de VEGF e HGF em células mesenquimais estromais derivadas líquido amniótico na inibição da fibrose intersticial após isquemia aguda em ratos

Abstract

Despite extensive research on an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Moreover, patients who survive AKI are at high risk for chronic progressive kidney disease. Mesenchymal stromal cells derived from human amniotic fluid (hAFSCs) are a new source of stem cells which express renal progenitor markers (CD24). The possibility of combining gene and cell therapy allows stem cells to be manipulated to overexpress vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), two of the most important growth factors for kidney regeneration. Therefore, the aim of this study was to evaluate whether hAFSCs overexpressing VEGF and HGF demonstrate a nephroprotective effect due to their mitogenic and anti-inflammatory effect, leading to a long term inhibition of fibrosis. In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples, characterized their immunophenotypic properties and differentiation capacity and selected a clonal lineage which expresses CD24 and CD117 markers. This lineage was subqequently transduced with lentiviral vectors (LV) encoding VEGF and HGF. In a second phase, renal ischemia and reperfusion (IR) injury was induced in a rat model by clamping the renal pedicle for 50 minutes in 50 male Wistar rats. Treatment groups (n = 10 per group) were assigned as follows: a control group treated with Chang Medium only; a group which received non-transduced AFSC (1x106 cells/animal); a group which received AFSC transduced with LV-VEGF (1x106 cells/animal); a group which received AFSC transduced with LV-HGF (1x106 cells/animal); and a group treated with AFSC transduced both with LV-VEGF (0,5x106 cells/animal) and AFSC transduced with LV-HGF (0,5x106 cells/animal). Serum creatinine was measured at 24 hours, 48 hours and 2 months after IR injury and histological analysis was performed to analyze following parameters: tubular necrosis and hyaline cast formation by PAS and H&E staining at 48 hours, and interstitial fibrosis by Masson s Trichrome and Picrosirius Red staining at 2 months. Additionally, the expression of KI-67, α-SMA and TGF-β1 was assessed by immunohistochemistry. The results showed a beneficial effect of AFSCs delivered to rats with IR injury, which was characterized by a faster improvement in renal function and a lower fibrotic index. However, administration of hAFSCs overexpressing VEGF and HGF resulted in an even better outcome compared to non-transduced AFSCs. As early as 24 hours after AFSC delivery, a nephroprotective effect was observed after both hAFSC and hAFSC VEGF + HGF treatment, which was characterized by significantly lower creatinine values compared to those of the control group. At 48 hours, all treatment groups still demonstrated a significant increase in creatinine values compared to sham animals, except in the hAFSC HGF + VEGF group. In hAFSC HGF + VEGF and hAFSC VEGF treatment groups, a significant increase in renal tubules proliferation was observed, measured by an increase in KI-67 expression, which is probably due to the effect of VEGF overexpression. Furthermore, we observed a decrease in α-SMA and TGF-β expression at 48 hours in the non-transduced hAFSC, hAFSC HGF and hAFSC VEGF + HGF groups. As TGF-β1 is involved in transdifferentiation of tubular epithelial cells to α-SMA-positive myofibroblasts which increases extracellular matrix deposition, the reduction in the expression of α-SMA and TGF-β indicates an inhibition of fibrosis. Although VEGF and HGF have both been described to have nephroprotective properties, we interestingly observed that the hAFSC expressing both VEGF and HGF resulted in more pronounced kidney damage compared to non-treated animals when treated with 1x106 cells/animal, which suggests that toxic side effects are possibly induced by high secretion levels of growth factors. In conclusion, cellular therapy using the combination of hAFSCs transduced with lentiviral vectors encoding VEGF and HGF, resulted in a stronger nephroprotective effect than non-transduced hAFSC delivered to rats with I/R injury, which was characterized by an increased mitosis index, an improved renal function and an inhibition of genes involved in fibrosis resulting in a lower fibrotic index at two months.