Therapeutic Effect of Three-Dimensional Cultured Adipose-Derived Stem Cell-Conditioned Medium in Renal Ischemia-Reperfusion Injury

Abstract

Background and objectives: We evaluated the effect of adipose-derived stem cell-derived conditioned medium (ADSC-CM) on the renal function of rats with renal ischemia-reperfusion injury (IRI)-induced acute kidney injury.

Methods and results: Forty male Sprague-Dawley rats were randomly divided into four groups: sham, nephrectomy control, IRI control, ADSC-CM. The ADSC-CM was prepared using the three-dimensional spheroid culture system and injected into renal parenchyme. The renal function of the rats was evaluated 28 days before and 1, 2, 3, 4, 7, and 14 days after surgical procedures. The rats were sacrificed 14 days after surgical procedures, and kidney tissues were collected for histological examination. The renal parenchymal injection of ADSC-CM significantly reduced the serum blood urea nitrogen and creatinine levels compared with the IRI control group on days 1, 2, 3, and 4 after IRI. The renal parenchymal injection of ADSC-CM significantly increased the level of creatinine clearance compared with the IRI control group 1 day after IRI. Collagen content was significantly lower in the ADSC-CM group than in the IRI control group in the cortex and medulla. Apoptosis was significantly decreased, and proliferation was significantly increased in the ADSC-CM group compared to the IRI control group in the cortex and medulla. The expressions of anti-oxidative makers were higher in the ADSC-CM group than in the IRI control group in the cortex and medulla.

Conclusions: The renal function was effectively rescued through the renal parenchymal injection of ADSC-CM prepared using a three-dimensional spheroid culture system.

Keywords: Acute kidney injury; Conditioned medium; Ischemia-reperfusion injury; Renal function; Three-dimensional cell culture.

Fig. 1

Characterization of ADSC. (A) Flow cytometry of ADSC from rats in the ADSC-CM group. Results are expressed as mean±standard error (n=9). (B) Appearance of ADSC from rats in the ADSC-CM group after three weeks of induction of adipogenic, osteogenic, or chondrogenic differentiation. Magnification, ×100 in adipogenesis and osteogenesis and ×400 in chondrogenesis (n=3). ADSC: adipose-derived stem cell, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 2

Effects of ADSC-CM on renal function. (A) Changes in serum BUN, (B) serum creatinine, and (C) CrCl of each group during the experiment. Blue-colored stars indicate statistically significant differences in the ADSC-CM group compared with the IRI control group; **p<0.01; ***p<0.001. Three times per rat, ten rats per group, but nine rats in the ADSC-CM group. BUN: blood urea nitrogen, CrCl: creatinine clearance, IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 3

Hematoxylin and eosin staining of kidney tissue. (A) Histopathological score in random non-overlapping fields. (B) Representative photomicrographs of cortex and medulla. Magnification ×40. Scale bar 50 μm. NS, p>0.05; ***p<0.001 compared with the IRI control group. Ten fields per rat, ten rats per group, but nine rats in the ADSC-CM group. NS: not significant, IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 4

Sirius red staining of kidney tissue. (A) The ratio of collagen-positive stained areas to random cortex and medulla areas. (B) Representative photomicrographs of cortex and medulla. Magnification ×40. Scale bar 50 μm. ***p<0.001 compared with the IRI control group. Four fields per rat, ten rats per group, but nine rats in the ADSC-CM group. IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 5

TUNEL assay of kidney tissue. (A) The ratio of TUNEL-positive stained areas to random cortex and medulla areas. (B) Representative photomicrographs of cortex and medulla. Magnification ×40. Scale bar 50 μm. ***p<0.001 compared with the IRI control group. Four fields per rat, ten rats per group, but nine rats in the ADSC-CM group. TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling, IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 6

Ki67 immunohistochemistry of kidney tissue. (A) The ratio of Ki67-positive stained areas to random cortex and medulla areas. (B) Representative photomicrographs of cortex and medulla. Magnification ×40. Scale bar 50 μm. ***p<0.001 compared with the IRI control group. Four fields per rat, ten rats per group, but nine rats in the ADSC-CM group. IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 7

GR immunohistochemistry of kidney tissue. (A) The ratio of GR-positive stained areas to the random cortex and medulla areas. (B) Representative photomicrographs of cortex and medulla. Magnification ×40. Scale bar 50 μm. ***p<0.001 compared with the IRI control group. Four fields per rat, ten rats per group, but nine rats in the ADSC-CM group. GR: glutathione reductase, IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.

Fig. 8

GPx immunohistochemistry of kidney tissues. (A) The ratio of GPx-positive stained areas to random cortex and medulla areas. (B) Representative photomicrographs of cortex and medulla. Magnification ×40. Scale bar 50 μm. ***p<0.001 compared with the IRI control group. Four fields per rat, ten rats per group, but nine rats in the ADSC-CM group. GPx: glutathione peroxidase, IRI: ischemia-reperfusion injury, ADSC-CM: adipose-derived stem cell-conditioned medium.