Effect of adipose-derived mesenchymal stem cell transplantation on vascular calcification in rats with adenine-induced kidney disease

Abstract

Previous studies have investigated the use of mesenchymal stem cells (MSCs) to treat damaged kidneys. However, the effect of adipose-derived MSCs (ASCs) on vascular calcification in chronic kidney disease (CKD) is still poorly understood. In the present study, we explored the potential of ASCs for the treatment of CKD and vascular calcification. CKD was induced in male Sprague-Dawley rats by feeding them a diet containing 0.75% adenine for 4 weeks. ASCs transplantation significantly reduced serum inorganic phosphorus (Pi) as compared to that in the control. The histopathology of the kidneys showed a greater dilation of tubular lumens and interstitial fibrosis in the control group. Calcium and Pi contents of the aorta in the ASCs transplantation group were lower than those in the control group. Von Kossa staining of the thoracic aorta media revealed that ASCs transplantation suppressed vascular calcification. Thus, this study revealed that autogenic ASCs transplantation inhibits kidney damage and suppresses the progression of vascular calcification in the CKD rat model, suggesting that autogenic ASCs transplantation is a novel approach for preventing the progression of CKD and vascular calcification.

Figure 1

Histopathology of the kidneys in adenine-fed rats. (A) The average kidney weight in the control group was higher than that in the MSC group (P < 0.005). (B–E) Histopathological analysis of kidneys by haematoxylin–eosin staining and Masson-trichrome staining. Distention of tubular lumens (P < 0.01), interstitial fibrosis (P < 0.005), and reduction in glomerular numbers (P < 0.005) were observed in the control group as compared to those parameters in the MSC group. Data are expressed as the mean ± SEM. Black scale bars: 500 μm, white scale bars: 100 μm.

Figure 2

Immunohistopathology of kidneys from adenine-fed rats. (A) Immunohistopathological expression of AQP1 and ED1. (B) Mesenchymal stem cells (MSCs) transplantation significantly increased the AQP1-positive area (P < 0.0001) and (C) reduced the ED1-positive area (P < 0.0005) as compared to the those in control group. Data are expressed as the mean ± SEM. Black scale bars: 500 μm, white scale bars: 100 μm.

Figure 3

Serum parameters in adenine-fed rats. (A–G) Serum levels of blood urea nitrogen (BUN), creatinine (Cre), inorganic phosphorus (Pi), and calcium (Ca) in adenine-fed and control rats. (A,B) Mesenchymal stem cells (MSCs) transplantation significantly reduced serum BUN (P < 0.05) and Cr (P < 0.05) levels on day 42. (C,D,G) MSCs significantly reduced serum Pi (P < 0.05), FGF23 (P < 0.05), and intact parathyroid hormone (PTH) (P < 0.05) on day 56 as compared to those in the control group. (D,F). Serum levels of Ca and 1–25(OH)₂D₃ on day 56 did not significantly differ between the MSC and control groups. Data are expressed as the mean ± SEM.

Figure 4

Urinary parameters in adenine-fed rats. (A–C) Mesenchymal stem cells (MSCs) transplantation significantly elevated 24-h creatinine clearance (24-h CCr) (P < 0.05), urine creatinine (Cr) (P < 0.005), and the tubular reabsorption of phosphate (%TRP) (P < 0.005) on day 56 as compared to those in the control group. (D–F) Urinary levels of blood urea nitrogen (BUN), calcium (Ca), and inorganic phosphorus (Pi) on day 56 did not significantly differ between the MSC and control groups. Data are expressed as the mean ± SEM.

Figure 5

Effect of mesenchymal stem cells (MSCs) transplantation on calcification of the aortic media. (A) Von Kossa staining of the thoracic aorta media revealed that MSCs transplantation suppressed vascular calcification as compared to that in the control group. (B,C) MSCs transplantation reduced calcium (P < 0.0005) and phosphorus (P < 0.01) contents in the thoracic aorta as compared to those in the control group. (D) Calcification scores in the MSC group were significantly lower than those in the control group (P < 0.05). Data are expressed as the mean ± SEM. Black scale bars: 100 μm.

Figure 6

Expression of osteopontin (OP), Runx2, and fibronectin in the thoracic aorta. (A) Expression of OP, Runx2, and fibronectin in the thoracic aorta was detected by immunohistochemistry. (B–D) OP (P < 0.05), Runx2 (P < 0.05), and fibronectin (P < 0.05) mRNA expression were significantly suppressed in the MSC group as compared to the control group. Data are expressed as the mean ± SEM. Black scale bars: 100 μm.

Figure 7

In vivo imaging of luciferase positive (Luc+) mesenchymal stem cells. (A) Luminescence was observed bilaterally in the lungs, but not in the kidneys. The signal disappeared on the third day. (B) Expression of luciferase was not detected by immunohistochemistry.

Figure 8

Correlation between the inorganic phosphorous (Pi) or calcium (Ca) content of the thoracic aorta and serum Pi or the Ca × Pi product at time of sacrifice. (A,B) Linear correlation between the Pi or Ca content of the thoracic aorta and the Ca × Pi product at the time of sacrifice (P < 0.005, P < 0.001, respectively). (C,D) Linear correlation between the Pi or Ca content of the thoracic aorta and serum Pi (P < 0.0005, P < 0.001, respectively).