Angiopoietin 1 influences ischemic reperfusion renal injury via modulating endothelium survival and regeneration

Fig. 1. a Design of inducible whole body Angpt1 knockout mice. Angpt1 exon 3 floxed Angpt1flox/flox mice were mated with UBC/CreErt2(Tg) mice to generate the UBC-CreErt2(Tg)/Angpt1flox/flox mice. After the induction with tamoxifen, exon 3 of Angpt1 was excised. The truncated Angpt1 mRNA was expressed and degraded or abnormal Angpt1 protein was translated. b Design of renal tubule specific inducible Angpt1 over-expression mice. Pax8-rtTA(Tg) mice were mated with pTre-hAngpt1(Tg) mice to generate the Pax8-rtTA(Tg)/pTre-hAngpt1(Tg) mice. After the induction with doxycycline, the renal tubule began to generate human Angpt1 mRNA

Fig. 2. a Expression of Angpt1 mRNA 1, 3, 7, and 14 days after IRI in wild mice. The expression of Angpt1 increased 7 days and 14 days after IRI. (N = 6 per sham and IRI groups at each time point) b The expression of Angpt1 in kidney was significantly suppressed in the Angpt1 knockout mice on the day of IRI and 1, 3, or 7 days after IRI compared to that in the control mice (N = 6, 6, 12, and 17 in knockout group on day 0, 1, 3, and 7 days respectively, N = 6, 6, 12, and 15 in control group on day 0, 1, 3, and 7 days respectively, P < 0.05 or P < 0.01) c & d Western blotting of mouse Angpt1 3 days after IRI. The Angpt1 protein level was lower in the knockout than control mice. (N = 5 for each group, P < 0.05) e Serum creatinine level was higher in the Angpt1 knockout mice 3 and 7 days after IRI. (N = 17 in knockout group, N = 15 in control group, N = 5 in the sham group, *P < 0.05, **P < 0.01) f Serum BUN level was higher in Angpt1 knockout mice 3 days after IRI. (N = 17 in knockout group, N = 15 in control group, N = 5 in the sham group, *P < 0.05) g PAS staining of renal tissue in control, knockout and sham mice. The remaining tubular necrosis was more severe and the renal tubular…

Fig. 3. a & b CD31 endothelial staining and quantitative analysis of CD31 positive area 3 days after IRI in the control and knockout mice. The CD31 staining was less pronounced in the Angpt1 knockout mice. (N = 9 in the control group, N = 12 in the knockout group) c & d VE-cadherin protein expression 3 days after IRI in the control and knockout mice. The protein expression of VE-cadherin was lower in the Angpt1 knockout mice (N = 4 per group)

Fig. 4. a M1 inflammatory genes expression 3 days after IRI in the whole kidney. There was no difference of TNF-α, IL-1β, MCP-1, IL-6, CCR2 and CCL3 genes expression between the Angpt1 knockout and control mice. (N = 9 per group) b M2 inflammatory genes expression 3 days after IRI in the whole kidney. There was no difference of CX3CR1, CCL17 and Arginase 1 genes expression between the Angpt1 knockout and control mice. (N = 6 per group) c & d CD68 positive cells infiltration 3 days after IRI in the kidney. There was no difference of the amount of CD68 positive cells between the Angpt1 knockout and control mice. Arrow indicates CD68 positive cells (N = 6 per group)

Fig. 5. a & b Ki67 staining and semi-quantitative analysis of Ki67 positive tubular cells 3 days after IRI in the control and knockout mice. Fewer Ki67 positive cells were observed in the knockout mice. Arrow indicates Ki67 positive cells (N = 8 per group, P = 0.002) c Growth factor genes expression in the whole kidney. No difference of the gene expression of HGF, EGF, IGF-1, PDGF-A and PDGF-B was observed between the Angpt1 knockout and control mice (N = 6 per group)

Fig. 6. a X-gal staining in the Angpt1 over-expression and control mice. Positive staining was observed in the tubular cells 7 days after the initiation of supplement of doxycycline in the Angpt1 over-expression mice. The presence of positive staining indicates that the teteracycline responsive element promoter activity of Angpt1. The β-galactosidase activity in the over expression mice was activated. b ∆Ct (difference in the threshold cycle between Angpt1 and GAPDH) of human Angpt1 mRNA by quantitative RT-PCR analysis of control and human Angpt1 over expression mice 1 day after IRI. The ∆Ct in the control mice was much higher than that in the over expression mice indicating significant over-expression of human Angpt1 mRNA was observed in the over-expression mice (N = 20 per group) c Renal tissue human Angpt1 concentration in over-expression and control mice. The over-expression mice had higher renal human Angpt1 concentration than control mice (N = 8 for each group)

Fig. 7. a PAS staining of renal tissue 1 day after IRI in control and over expression mice. The tubular necrosis was less prominent in the over expression mice 1 days after IRI. Arrow indicates necrotic tubule. b Serum creatinine level in the control and over expression mice 1, 3 and 7 days after IRI. The serum creatinine level was lower in the over expression mice 1 and 3 days after IRI. (N = 19 in the control group, N = 23 in the over-expression group, N = 5 in the sham mice, *P = 0.004, **P = 0.015) c Serum BUN level in the control and over mice expression 1, 3 and 7 days after IRI. The serum BUN level was lower in the over expression mice 1 and 3 days after IRI. (N = 19 in the control group, N = 23 in the over-expression group, N = 5 in the sham mice, *P < 0.001, **P = 0.016) d Semi-quantitative analysis of tubular necrosis 1 day after IRI in the control and over expression mice. The tubular necrosis score was less in the over expression mice. (N = 19 in the control group, N = 23 in the over-expression group, N = 5 in the sham group)

Fig. 8. a & b CD31 endothelial staining and semi-quantitative analysis of the CD31 positive area 1 days after IRI in the control and over expression mice. The CD31 staining was more prominent in the over expression mice. (N = 15 per group, P = 0.033) c & d VE-cadherin protein expression 1 days after IRI in the control and over expression mice. The protein expression of VE-cadherin did not differ between the control and over expression mice (N = 4 per group)

Fig. 9. a & b Inflammatory genes expression 1 day after IRI in the whole kidney. The mRNA expression of TNF-α, CCR2 and CX3CR1 was higher in the over-expression than control mice. The gene expression of MCP-1, IL-6, IL-1β, CXCL2, CCL17 and arginase 1 did not differ between the over-expression and control mice. (N = 8 per group) c & d CD68 positive cells infiltration 1 day after IRI in the kidney. The infiltration of CD68 positive cells was not different between the over-expression and control mice. Arrow indicates CD68 positive cells (N = 6 per group)

Fig. 10. a Growth factor genes expression in the whole kidney. The mRNA expression of EGF, HGF and IGF-1 was higher in the over-expression mice. There was no difference of PDGF-A and PDGF-B genes expression between the over-expression and control mice. (N = 8 per group) b Apoptotic tubular cells revealed by TUNNEL staining in the kidneys. There were more apoptotic tubular cells in the control than the over-expression mice. Arrow indicates apoptotic cell. c Semi-quantitative analysis of the apoptotic cells (N = 6 per group)