Bone marrow‑derived mesenchymal stem cell‑conditioned medium attenuates tubulointerstitial fibrosis by inhibiting monocyte mobilization in an irreversible model of unilateral ureteral obstruction

Abstract

Mesenchymal stem cell‑conditioned medium (MSC‑CM) contains various cytokines (osteopontin and macrophage colony stimulating factor 1) secreted by MSCs and may modulate the immune response in tubulointerstitial fibrosis. The aim of the present study was to investigate whether MSC‑CM treatment may affect B cell‑dependent immune responses, which have previously been reported to facilitate the renal fibrotic processes following unilateral ureteral obstruction (UUO). In the present study, histological analysis, flow cytometry, western blotting and reverse transcription‑quantitative polymerase chain reaction were performed. MSC‑CM treatment was observed to impede renal infiltration of B lymphocytes and the expression of CC chemokine ligand‑2. Additionally, UUO suppressed the subsequent recruitment of monocytes/macrophages to the kidney, limited local inflammation and attenuated renal fibrosis. The findings of the present study identified a potential mechanism of MSC‑CM in ameliorating the UUO‑kidney.

Figure 1.

MSC-CM treatment attenuates UUO-induced renal fibrosis. (A) Representative images of renal Masson's trichrome-stained sections in Sham + PBS, Sham + MSC-CM, UUO + PBS, and UUO + MSC-CM mice 2 weeks following surgery. Collagen is visible as the blue color. Scale bar=50 µm. (B) Representative images of Sham + PBS, Sham + MSC-CM, UUO + PBS and UUO + MSC-CM mice 2 weeks following surgery. Collagen is visible as the red stain. Scale bar=50 µm. (C) Quantitative analysis of renal Masson's trichrome-stained sections. (D) Quantitative analysis of Sirius red-stained renal sections. *P<0.05 vs. Sham + PBS group; ^#P<0.05 vs. Sham + MSC-CM group; ^&P<0.05 vs. UUO + PBS group. Data are presented as the mean ± standard deviation (n=6–8). MSC-CM, mesenchymal stem cell-conditioned media; Sham + PBS, sham-operated and PBS treated; Sham + MSC-CM, sham-operated and MSC-CM treated; UUO, unilateral ureteral obstruction; UUO + PBS, UUO model and PBS treated; UUO + MSC-CM, UUO model and MSC-CM treated.

Figure 2.

MSC-CM treatment ameliorates UUO-induced renal inflammation. (A) Representative western blotting images and (B) quantitative analysis of the expression levels of key proinflammatory factors in Sham + PBS, Sham + MSC-CM, UUO + PBS, and UUO + MSC-CM mice 3 days following surgery. *P<0.05 vs. Sham + PBS group; ^#P<0.05 vs. UUO + PBS group. ICAM-1, intercellular adhesion molecule 1; IL, interleukin; TNF-α, tumor necrosis factor-α; MSC-CM, mesenchymal stem cell-conditioned media; Sham + PBS, sham-operated and PBS treated; Sham + MSC-CM, sham-operated and MSC-CM treated; UUO, unilateral ureteral obstruction; UUO + PBS, UUO model and PBS treated; UUO + MSC-CM, UUO model and MSC-CM treated.

Figure 3.

MSC-CM treatment alters chemotactic responses following UUO. (A) Representative images and (B) quantitative analysis of F4/80 staining in Sham + PBS, Sham + MSC-CM, UUO + PBS and UUO + MSC-CM mice 3 days following surgery. Scale bar=50 µm. Data are presented as the mean ± standard deviation (n=7–8). (C) Representative western blotting images and (D) quantitative analysis for CCL-2, 3 days following surgery. Data are presented as the means ± standard deviation (n=6). (E) Reverse transcription-quantitative polymerase chain reaction analysis of CCL-2 mRNA in kidneys from Sham + PBS, Sham + MSC-CM, UUO + PBS and UUO + MSC-CM mice 3 days following surgery. Data are presented as the means ± standard deviation (n=6). *P<0.05 vs. Sham + PBS group; ^$P<0.05 vs. UUO + PBS group. CCL-2, CC chemokine ligand-2; MSC-CM, mesenchymal stem cell-conditioned media; Sham + PBS, sham-operated and PBS treated; Sham + MSC-CM, sham-operated and MSC-CM treated; UUO, unilateral ureteral obstruction; UUO + PBS, UUO model and PBS treated; UUO + MSC-CM, UUO model and MSC-CM treated.

Figure 4.

MSC-CM treatment alleviates mature B lymphocyte infiltration into the kidneys following UUO. (A) Representative images of Sham + PBS, Sham + MSC-CM, UUO + PBS and UUO + MSC-CM mice 3 days following surgery. Scale bar=50 µm. Data are presented as the mean ± standard deviation (n=6–7). (B) Representative flow cytometry images of renal B220⁺CD19⁺ B lymphocyte cytometry of Sham + PBS, Sham + MSC-CM, UUO + PBS and UUO + MSC-CM mice 3 days following surgery. (C) Quantitative analysis of B220 staining and (D) quantitative analysis of renal B220⁺CD19⁺ B lymphocyte cytometry. Data are presented as the means ± standard deviation (n=6). *P<0.05 vs. Sham + PBS group; ^$P<0.05 vs. UUO + PBS group. MSC-CM, mesenchymal stem cell-conditioned media; Sham + PBS, sham-operated and PBS treated; Sham + MSC-CM, sham-operated and MSC-CM treated; UUO, unilateral ureteral obstruction; UUO + PBS, UUO model and PBS treated; UUO + MSC-CM, UUO model and MSC-CM treated.