Figure 1.
Culture, characterization and labeling of hAFSCs.
(A) Morphology of hAFSCs under high magnification. After 3 passages in culture, the cells were spindle-shaped with relatively large nuclei; conjugate nuclei were also present (bar, 100 µm). (B) Morphology of hAFSCs after 2 days of culture. The cells exhibited a needle-like shaped appearance and grew with no visible directionality (bar, 50 µm). (C) Morphology of hAFSCs after 10 days of culture. Cells grow as a vortex and a crater (bar, 50 µm). (D) The third passage hAFSCs stained with Oct-4. Cells stained positive for Oct-4, a stem cell surface marker (bar, 50 µm). (E) CM-DiI-labeled hAFSCs. (F) Double immunofluorescent staining of a kidney section taken from an animal injected with hAFSCs at day 14 post-injection. Positive HLA-class I staining confirms the presence of hAFSCs at day 14 post-transplantation in the frozen renal tissue sections (bar, 50 µm).
Figure 2.
Morphological analysis of renal histology.
HE and Masson's trichrome staining. (A, D) The normal control group at day 14 post-saline injection. No significant histological abnormalities were observed in the control group animals. (B, E) The UUO group at day 14 post-saline injection. Renal fibrosis was clearly visible in the UUO group animals at day 14. Increased numbers of collapsed vascellums and increased extracellular matrix production were observed. (C, F) The hAFSC group at day 14 post-hAFSCs injection. The number and the degree of destroyed tubules in the hAFSC group were less than the UUO group; further, renal fibrosis was reduced in the hAFSC group compared with the UUO group (bar, 50 µm). (J) Renal interstitium areas in each group. Fibrosis could be detected in the tubulointerstitium at day 7 and was more severe at day 14 in the UUO group. hAFSCs transplantation could significantly inhibit tubulointerstitial fibrosis (p<0.05). G-I: PAS staining. (G) The normal control group at day 14 post-saline injection. Normal morphology of a nu/nu mouse kidney was observed in the control group animals. The proximal and distal tubules as well as the glomeruli were intact. (H) The UUO group at day 14 post-saline injection. Marked disorganization of the structure of proximal and distal tubules with more cast formation and brush border disruption were observed. (I) The hAFSC group at day 14 post-hAFSCs injection. The tubular injury was observed, which showed less severe compared with the UUO group. (K) Results obtained with the tubular histology score. The loss of brush border, tubular dilation and apoptosis/necrosis of tubular cells were assessed. Values are presented as mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 3.
(A) The normal control group at day 14 post-saline injection. The PTCs of the control group were easily identified and were uniform in size and shape; also, they were regularly arranged throughout the majority of the interstitium. (B) The UUO group at day 14 post-saline injection. The number of PTCs was significantly reduced in the UUO group, especially in the areas around the severely atrophic tubules. (C) The hAFSC group at day 14 post-hAFSCs injection. The number of PTCs in the hAFSC group was also reduced compared with the control group; however, the number of PTCs in the hAFSC group was greater than the UUO group (p<0.05) (bar, 50 µm). (D) PTC density in each group. The PTC density was significantly decreased beginning at day 3 in the UUO group. However, PTC density in the hAFSC group at day 3 was significantly increased compared with the UUO group (p<0.05). Values are presented as the mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 4.
Double Immunostaining and Western Blot analysis of VEGF expression.
(A, J) The normal control group at day 14 post-saline injection. VEGF was most prominently expressed in glomerular podocytes and tubular epithelial cells in the control group. (B, K) The UUO group at day 14 post-saline injection. VEGF expression gradually decreased over time post-surgery, particularly in the areas of tubular injury. (C, L) The hAFSC group at day 14 post-hAFSCs injection. VEGF expression was markedly enhanced in the hAFSC group (bar, 50 µm). (N) The total IOD of VEGF in each group. The total IOD of each group reflected the protein level. VEGF expression was notably enhanced in the hAFSC group compared with the UUO group (p<0.05). (D, E, F) WT-1 positive staining showed the podocytes (red). (G, H, I) DAPI positive staining showed the nucleuses (blue). (M, O) Western blot analysis of VEGF protein expression. VEGF protein expression showed similar trends with the double immunostaining findings. hAFSCs transplantation increased VEGF expression associated with UUO. Con, the control group at day 14; U1, U3, U7 and U14, the UUO group at days 1, 3, 7 and 14, respectively; hA1, hA3, hA7 and hA14, the hAFSC group at days 1, 3, 7 and 14, respectively. Values are presented as the mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 5.
Immunohistochemical staining and Western blot analysis of HIF-1α expression.
(A) The normal control group at day 14 post-saline injection. HIF-1α staining was faint in the cortical nuclei in the control group at day 14 post-saline injection; nuclear staining was not observed in the medulla. (B) The UUO group at day 14 post-saline injection. HIF-1α expression was most apparent in areas of interstitial fibrosis and tubular dilation in the UUO group at day 14. (C) The hAFSC group at day 14 post-hAFSCs injection. HIF-1α expression was suppressed in renal tissues after hAFSCs transplantation (bar, 50 µm). (D) The total IOD of HIF-1α in each group. The total IOD in each group reflected the levels of HIF-1α protein. HIF-1α expression was decreased in tubules after hAFSCs transplantation beginning at day 3 compared with the UUO group (p<0.05). (E, F) Western blot analysis of HIF-1α protein expression. HIF-1α protein expression was significantly reduced in the hAFSC group at day 3 post-surgery compared with the UUO group and remained low at days 7 and 14 post-surgery; these results are consistent with the immuno-histochemistry findings. hAFSCs transplantation alleviated HIF-1α expression associated with UUO. Con, the normal control group at day 14; U1, U3, U7 and U14, the UUO group at days 1, 3, 7 and 14, respectively; hA1, hA3, hA7 and hA14, the hAFSC group at days 1, 3, 7 and 14, respectively. Values are presented as the mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 6.
Immunohistochemical staining and Western blot analysis of TGF-β1 expression.
(A) The normal control group at day 14 post-saline injection. Faint TGF-β1 staining was observed in renal tissues from normal control group mice at day 14 post-saline injection. (B) The UUO group at day 14 post-saline injection. TGF-β1 was markedly upregulated in the interstitial fibrotic areas and tubular epithelial cells in the UUO group at day 14. (C) The hAFSC group at day 14 post-hAFSCs injection. TGF-β1 expression was suppressed following hAFSCs transplantation (bar, 50 µm). (D) The total IOD for TGF-β1 in each group. The total IOD in each group reflected the level of TGF-β1 protein expression. TGF-β1 protein levels in the UUO group began to increase at day 7 and remained elevated at day 14 compared with the hAFSC group (p<0.05). (E, F) Western blot analysis of TGF-β1 protein expression. Western blot analysis showed that TGF-β1 protein levels began to increase at day 7 and remained elevated at day 14; these results were consistent with the immunohistochemistry findings. hAFSCs transplantation reduced TGF-β1 expression associated with UUO. Con, the normal control group at day 14; U1, U3, U7 and U14, the UUO group at days 1, 3, 7 and 14, respectively; hA1, hA3, hA7 and hA14, the hAFSC group at days 1, 3, 7 and 14, respectively. Values are presented as the mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 7.
Immunohistochemical staining and Western blot analysis of E-cadherin expression.
(A) Normal control group at 14 days after saline solution injection: Positive staining of E-cadherin was seen in the tubuloepithelial cells. (B) UUO group at 14 days after saline solution injection: Rare expression of E-cadherin was seen at day 14 after surgery, and most renal tubules were destroyed and renal interstitial fibrosis was obvious. (C) hAFSC group at 14 days after hAFSCs injection: The expression of E-cadherin increased after hAFSCs transplantation. (bar, 50 µm). (D) The total IOD of E-cadherin in each group: The total IOD in each group reflected the level of the E-cadherin. There was no significant difference in the expression of E-cadherin among the UUO, hAFSC, and control groups 1 day after surgery. The expression of E-cadherin began to reduce from 3 days after surgery. Rare expression of E-cadherin was seen at 14 days after surgery in the UUO group. However, the expression of E-cadherin significantly increased in the hAFSC group compared to the UUO group(p<0.05). (E, F) Western blot analysis for E-cadherin protein expression. E-cadherin protein expression showed the similar trends as the immunohistochemistry findings. hAFSCs transplantation increased E-cadherin expression compared with UUO at day 3, day 7 and day 14. Con, the control group at day 14; U1, U3, U7 and U14, the UUO group at days 1, 3, 7 and 14, respectively; hA1, hA3, hA7 and hA14, the hAFSC group at days 1, 3, 7 and 14, respectively. Values are presented as mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 8.
Immunohistochemical staining and Western blot analysis of Collagen I expression.
(A) Normal control group at 14 days after saline solution injection: Light staining of Collagen I was seen in renal interstitium. (B) UUO group at 14 days after saline solution injection: The expression of Collagen I significant increased at day 14 after surgery in the UUO group, and the renal interstitial fibrosis were more severe. (C) hAFSC group at 14 days after hAFSCs injection: The expression of Collagen I reduced after hAFSC transplantation. (bar, 50 µm). (D) The total IOD of Collagen I in each group: The total IOD in each group reflected the level of the Collagen I. There was no significant difference in the expression of Collagen I among the UUO, hAFSC, and control groups at 1 day and 3 days after surgery. The expression of Collagen I significantly increased at 7 and 14 days after surgery in the UUO group and hAFSC group compared to the control group, respectively. However, hAFSC transplantation significantly resulted in a reduction of the expression of Collagen I compared to the UUO group. (E, F) Western blot analysis of Collagen I protein expression. Western blot analysis showed that Collagen I protein levels began to increase at day 7, and remained elevated at day 14; these results were consistent with the immunohistochemistry findings. hAFSCs transplantation reduced Collagen I expression associated with UUO. Con, the normal control group at day 14; U1, U3, U7 and U14, the UUO group at days 1, 3, 7 and 14, respectively; hA1, hA3, hA7 and hA14, the hAFSC group at days 1, 3, 7 and 14, respectively. Values are presented as the mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group. Values are presented as the mean±SEM. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 9.
Immunohistochemical staining and Western blot analysis of MCP-1 expression.
(A) Normal control group at 14 days after saline solution injection: Light staining of MCP-1 was seen in the renal tissue and a few inflammatory cells and fibrosis were seen in tubulointerstitial. (B) UUO group at 14 days after saline solution injection: The expression of MCP-1 significant increased in the UUO group at 14 days after surgery. (C) hAFSC group at 14 days after hAFSCs injection: The expression of MCP-1 reduced after hAFSCs transplantation. (bar, 50 µm). (D) The total IOD of MCP-1 in each group: The total IOD in each group reflected the level of the MCP-1 protein. The expression of MCP-1 in the UUO group and hAFSC group at day 1 after surgery was not obvious. There was no significant difference in the expression of MCP-1 among the UUO, hAFSC, and control groups at 1 day after surgery. However, the expression of MCP-1 significantly increased in the UUO group and hAFSC group at 3, 7 and 14 days after surgery compared to the control group, respectively. hAFSCs transplantation significantly reduced the tubuloepithelial cell expression of MCP-1 compared to the UUO group. (E, F) Western blot analysis of MCP-1 protein expression. Western blot analysis showed that MCP-1 protein levels began to increase at day 3, and remained elevated at day 14; These results were consistent with the immunohistochemistry findings. hAFSCs transplantation reduced MCP-1 expression associated with UUO(P<0.05). Con, the normal control group at day 14; U1, U3, U7 and U14, the UUO group at days 1, 3, 7 and 14, respectively; hA1, hA3, hA7 and hA14, the hAFSC group at days 1, 3, 7 and 14, respectively. Values are presented as the mean±SD. *p<0.05 vs. control group; #p<0.05 vs. UUO group. Values are presented as the mean±SEM. *p<0.05 vs. control group; #p<0.05 vs. UUO group. Values are presented as the mean±SEM. *p<0.05 vs. control group; #p<0.05 vs. UUO group.
Figure 10.
Proliferation and apoptosis evaluation.
(A, E) Normal control group at 14 days after saline solution injection: The proliferation activity in tubular cells showed no obvious increase in the control group. (B, F) UUO group at 14 days after saline solution injection: The proliferation activity in tubular cells showed significant increased in the UUO group at 14 days after surgery. (C, G) hAFSC group at 14 days after hAFSCs injection: Significantly more proliferation in tubular cells could be seen in the hAFSC group. (bar, 50 µm). (D, H) PCNA-positive cells and Ki67-positive cells in renal tubules in each group. The proliferation activity among the three experimental groups showed no significant difference at day 1 post-surgery. Animals that were injected with a saline solution and animals that received hAFSCs after UUO demonstrated a significant increase in cell proliferation beginning at day 3 post-surgery. The hAFSC group had significantly more proliferation than the UUO group. Although the proliferation activity of the hAFSC group at day 14 post-surgery showed an obvious decrease than that at day 7 post-surgery, it still showed a significant increase than the UUO group at day 14. (I) Normal control group at 14 days after saline solution injection: The apoptosis activity in tubular cells showed no obvious increase in the control group. (J) UUO group at 14 days after saline solution injection: The apoptotic cells showed significant increased in UUO group at 14 days after surgery. (K) hAFSC group at 14 days after hAFSCs injection: The apoptotic cells were significantly decreased in animals in the hAFSC group (bar, 50 µm). (L) TUNEL-positive cells in renal tubules in each group. The apoptosis activity among the three experimental groups showed no significant difference at day 1 post-surgery. Animals that were injected with a saline solution and animals that received hAFSCs after UUO demonstrated a significant increase in cell apoptosis beginning at day 3 post-surgery. Although the apoptosis activity of the hAFSC group at day 14 post-surgery showed an obvious increase than that at day 7 post-surgery, it still showed a significant decrease than the UUO group at day 14. Values are presented as the mean±SEM. *p<0.05 vs. control group; #p<0.05 vs. UUO group.