Improved Protective Effect of Umbilical Cord Stem Cell Transplantation on Cisplatin-Induced Kidney Injury in Mice Pretreated with Antithymocyte Globulin

Abstract

Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone.

Figure 1

Shematic representation of the protocol and all control and experimental groups.

Figure 2

Representative images of kidney tissue in mice with cisplatin-induced AKI treated with DiI labeled MSCs (day 4). (a) DiI labeled MSCs (red fluorescence) in peritubular area. Original magnification: ×1000. (b) DiI labeled MSCs (red fluorescence) within proximal tubule epithelium. Original magnification: ×1000. Nuclei are stained with DAPI (blue fluorescence). Basal lamina of proximal tubule is marked with a gray line.

Figure 3

Schematic representation of survival curves in BALB/cOlaHsd cisplatin treated mice. The survival was estimated by Kaplan-Meier statistical analysis. Mice pretreated with ATG had an increased survival after MSCs treatment during their follow-up (log rank p = 0.012).

Figure 4

MSC treatment after ATG immunosuppression improves renal function and morphology as well as intestine morphology in BALB/cOlaHsd mice 4 days after cisplatin administration. (a) Representative micrographs of renal histology of healthy control mice and of mice treated with cisplatin and saline or MSCs and/or ATG (original magnification: ×200, periodic acid-Schiff (PAS) staining). (b) Serum concentration of blood urea nitrogen (BUN) and creatinine and caspase 3 score. Graphical results are expressed as mean ± SEM; ANOVA followed by Duncun multicomparison test: ^∗ p < 0.05 control versus all other groups. (c) Representative micrographs of intestine of healthy control mice and of mice treated with cisplatin and saline or MSCs and/or ATG (original magnification: ×200, Kreyberg-Jareg staining).

Figure 5

Kidney ultrastructure. (a) Ultrastructure of epithelial cell with large and rounded nucleus (N), numerous mitochondria (M), and pronounced microvilli (MV). (b) Necrotic epithelial cell (asterisk) with typical ultrastructural signs of necrosis such as electron pale and disintegrated cytoplasm, disrupted microvilli, and few mitochondria (M). L-lumen of proximal tubule. (c) Details of necrotic epithelial cell with strongly dilated perinuclear space (arrowhead), ruptured Golgi apparatus (asterisk), and highly demolished cytoplasmic integrity with hardly recognizable structures. (d) Epithelial cell with no brush border on apical surface, multilamellar bodies (arrowheads), and extremely large autophagic vacuoles (arrows) and fulfilling the entire cytoplasm. N-nucleus. (e) Apoptotic epithelial cell with condensed chromatin in nuclear fragments (N), lipid droplets (L), disappeared microvilli, and electron dense and condensed cytoplasm (asterisk). (f) Epithelial cells of proximal tubule with normal ultrastructure. Under basal lamina (arrow) of tubule, peritubular cell (asterisk) of connective tissue with typical signs of apoptosis such as cytoplasm condensation and chromatin fragmentation and condensation is present. Representative TEM images of kidney samples taken from control mice (a), mice receiving ATG and cisplatin (b–d), and mice receiving MSCs after ATG and cisplatin (e, f). Original magnifications: ×4400 (a), ×2600 (b), ×5800 (c), ×2600 (d), ×4400 (e), and ×2000 (f).

Figure 6

Gene expression of inflammatory and oxidative proteins in kidneys of 8–12-week-old male BALB/cOlaHsd mice after cisplatin-induced AKI treated with ATG and/or MSCs. Bars represent alterations in expression of cytokines, inflammatory, and oxidative proteins as measured by RT-PCR in kidney tissues of sacrificed mice. Results are expressed as mean ± SEM. ANOVA followed by Duncun multicomparison test: ^∗ p < 0.05; ^∗∗ p < 0.001; SAA3: serum amyloid A3, HO-1: heme oxygenase-1, GPx: glutathione peroxidase, CAT: catalase, SOD-1: superoxide dismutase-1, SOD-2: superoxide dismutase 2, IL-1β: interleukin 1β, TGF-β: transforming growth factor β, and ICAM-1: intercellular adhesion molecule 1.