Comparative study of allogenic and xenogeneic mesenchymal stem cells on cisplatin-induced acute kidney injury in Sprague-Dawley rats

Abstract

Background: The paracrine and regenerative activities of mesenchymal stem cells (MSCs) may vary with different stem cell sources. The aim of the present study is to compare the effects of MSCs from different sources on acute kidney injury (AKI) induced by cisplatin and their influence on renal regeneration.

Methods: A single intraperitoneal injection of cisplatin (5 mg/kg) was used to induce AKI in 120 Sprague-Dawley rats. Rats were treated with either rat bone marrow stem cells (rBMSCs), human adipose tissue-derived stem cells (hADSCs), or human amniotic fluid-derived stem cells (hAFSCs). 5 × 10(6) MSCs of different sources were administered through rat tail vein in a single dose, 24 hours after cisplatin injection. Within each group, rats were sacrificed at the 4th, 7th, 11th, and 30th day after cisplatin injection. Serum creatinine, BUN, and renal tissue oxidative stress parameters were measured. Renal tissue was scored histopathologically for evidence of injury, regeneration, and chronicity. Immunohistochemistry was also done using Ki67 for renal proliferative activity evaluation.

Results: MSCs of the three sources were able to ameliorate cisplatin-induced renal function deterioration and tissue damage. The rat BMSCs-treated group had the lowest serum creatinine by day 30 (0.52 ± 0.06) compared to hADSCs and hAFSCs. All MSC-treated groups had nearly equal antioxidant activity as indicated by the decreased renal tissue malondialdehyde (MDA) and increased reduced glutathione (GSH) level and superoxide dismutase (SOD) activity at different time intervals. Additionally, all MSCs improved injury and regenerative scores. Rat BMSCs had the highest count and earliest proliferative activity in the renal cortex by day 7 as identified by Ki67; while, hAFSCs seem to have the greatest improvement in the regenerative and proliferative activities with a higher count of renal cortex Ki67-positive cells at day 11 and with the least necrotic lesions.

Conclusions: Rat BMSCs, hADSCs, and hAFSCs, in early single IV dose, had a renoprotective effect against cisplatin-induced AKI, and were able to reduce oxidative stress markers. Rat BMSCs had the earliest proliferative activity by day 7; however, hAFSCs seemed to have the greatest improvement in the regenerative activities. Human ADSCs were the least effective in the terms of proliferative and regenerative activities.

Keywords: Adipose-derived; Amniotic fluid-derived; Cisplatin nephrotoxicity; Mesenchymal stem cells.

Fig. 1

Appearance of different stem cells under inverse microscope. Under inverse microscopy, cultured rat bone marrow mesenchymal stem cells (rBMSCs, (a) human adipose tissue-derived mesenchymal stem cells (hADSCs, (b) and human amniotic fluid-derived mesenchymal stem cells (hAFSCs, (c) at passage 3 were morphologically defined by the fibroblast-like appearance (original magnification × 200)

Fig. 2

Analysis of hADSCs with flow cytometry. Flow cytometry analysis of hADSCs revealed that their expression of surface antigens such as CD29, CD90, CD105, and CD13 was positive; but, CD14 and CD34 was negative (passage 3)

Fig. 3

Analysis of hAFSCs with flow cytometry. Flow cytometry analysis of hAFSCs revealed that their expression of surface antigens such as CD105, CD90, CD13, Oct4, and CD29 was positive; but CD34 and CD14 was negative (passage 3)

Fig. 4

Analysis of rBMSCs with flow cytometry. Flow cytometry analysis of rat bone marrow mesenchymal stem cells revealed that their expression of surface antigens such as CD29, CD90, and CD44 was positive; but CD45 and CD34 was negative (passage 3)

Fig. 5

Photomicrographs of different stem cells’ colonies after 1 week of culture. Photomicrographs of rat bone marrow mesenchymal stem cells (rBMSCs, (a) human adipose tissue-derived mesenchymal stem cells (hADSCs, (b) and human amniotic fluid-derived mesenchymal stem cells (hAFSCs, (c) after 1 week of culture grew in colonies that contained heterogeneous small spindle-shaped fibroblastoid cells and more rounded cells (original magnification × 100)

Fig. 6

Photomicrographs showing osteogenic differentiation potential of different stem cells. Photomicrographs of rat bone marrow mesenchymal stem cells (rBMSCs, (a) human adipose tissue-derived mesenchymal stem cells (hADSCs, (b) and human amniotic fluid-derived mesenchymal stem cells (hAFSCs, (c) showing differentiation potential toward osteoblasts as indicated by the formation of calcium-rich hydroxyapatite detected with Alizarin Red and appearing as irregular red-orange (original magnification × 200)

Fig. 7

Photomicrographs showing adipogenic differentiation potential of different stem cells. Photomicrographs of rat bone marrow mesenchymal stem cells (rBMSCs, (a) human adipose tissue-derived mesenchymal stem cells (hADSCs, (b) and human amniotic fluid-derived mesenchymal stem cells (hAFSCs, (c) showing adipocyte differentiation potential visualized by highly refractive intracellular lipid vacuoles and droplets appear as cherry red spheres within the cells by Oil-red-O staining (original magnification × 100)

Fig. 8

Photomicrographs showing chondrogenic differentiation potential of different stem cells. Photomicrographs of rat bone marrow mesenchymal stem cells (rBMSCs, (a) human adipose tissue-derived mesenchymal stem cells (hADSCs, (b) and human amniotic fluid-derived mesenchymal stem cells (hAFSCs, (c) showing chondrogenic differentiation potential visualized by formation of glycosaminoglycans with a red color (original magnification × 100)

Fig. 9

Gel electrophoresis and RT-PCR gene expression of different stem cells. Gel electrophoresis for lipoprotein lipase showed that: lane M (DNA marker) made DNA ladder and lane 4d, 7d, 14d, and 21d showed bands present at suspected size and proved the formation of lipoprotein lipase (arrow) of rat bone marrow mesenchymal stem cells (BMSCs) (a), human adipose tissue-derived mesenchymal stem cells (hADSCs) (b), and human amniotic fluid-derived mesenchymal stem cells (hAFSCs) (c). RT- PCR gene expression showed about a tenfold increase in gene expression of PPAR-γ during adipocytic differentiation (d) and osteocalcin during osteocytic differentiation of MSCs (e) as indicated by the curves above the threshold green line

Fig. 10

Renal pathological changes of experimental groups at different time points. Pathological changes in cisplatin-injected rats sacrificed at day 4 (a) showed marked degenerative changes and atrophic tubules in OSOM (H&E, ×200). Pathological changes in cisplatin-injected and rat bone marrow stem cells (rBMSCs)-treated rats (b), human adipose tissue-derived stem cells (hADSCs)-treated rats (c), and human amniotic fluid-derived stem cells (hAFSCs)-treated rats (d) sacrificed at day 4 showed regenerative changes in the OSOM in the form of many regenerating tubules lined by large cells with large hyperchromatic nuclei with few solid sheets (H&E, B and D × 200, C × 40). Pathological changes in cisplatin-injected rats sacrificed at day 7 (e) showed combined degenerative and regenerative changes (H&E, ×200). Pathological changes in cisplatin-injected and rBMSCs-treated rats (f), hADSCs-treated rats (g), and hAFSCs-treated rats (h) sacrificed at day 7 showed less marked degenerative changes and many regenerative changes in the OSOM in the form of many interstitial solid sheets (H&E, F and H × 200, G × 100). Pathological changes in cisplatin-injected rats sacrificed at day 11 (i) showed more marked degenerative changes and mild interstitial round cell infiltrate (H&E, ×200). Pathological changes in cisplatin-injected and rBMSCs-treated rats (j), hADSCs-treated rats (k), and hAFSCs-treated rats (l) sacrificed at day 11 showed more regenerative changes, few necrotic tubules, and interstitial round cell infiltrates (H&E, J × 100, and K and L × 200). Pathological changes in cisplatin-injected rats sacrificed at day 30 (m) showed mild peritubular and perivascular fibrosis (H&E, ×100). Pathological changes in cisplatin-injected and rBMSCs-treated rats (n), hADSCs-treated rats (o), and hAFSCs-treated rats (p) sacrificed at day 30 showed less chronic changes as regard the tubular atrophy and the renal fibrosis (H&E, N and O × 100, P × 40)

Fig. 11

Proliferating cells/HPF identified by Ki67 immunohistochemistry in OSOM at day 11 of different experimental groups. Proliferating cells/HPF identified by Ki67 immunohistochemistry in OSOM at day 11 of cisplatin-injected rats (a), cisplatin-injected and rat bone marrow stem cells (rBMSCs)-treated rats (b), cisplatin-injected and human adipose-derived tissue stem (hADSCs)-treated rats (c), and cisplatin-injected and human amniotic fluid-derived stem cells (hAFSCs)-treated rats (d) as positive regenerating tubules and some scattered cells representing interstitial epithelial solid sheets (magnification × 200)