Bi-functional KIT-PR1P peptides combine with VEGF to protect ischemic kidney in rats by targeting to Kim-1

Figures

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  Fig. 1. The bioactivity and targeting ability of KIT-PR1P. (A–D) The angiogenic ability of KIT-PR1P/VEGF and natural VEGF. N = 3. The Scale Bar = 200 μm ∗∗P < 0.01. (E) The targeting ability of KIT-PR1P/VEGF and PR1P/VEGF in hypoxic HK-2 cells was verified by immunofluorescence of the renal tubular injury marker Kim-1 and VEGF co-staining assay. VEGF (green), the renal tubular injury marker Kim-1 (red), DAPI (blue). Yellow staining indicates co-localization. The Scale Bar = 100 μm. (F) Fluorescence Integrated density of VEGF (35.34 ± 18.22, 17.48 ± 11.65, 4.89 ± 5.18) and Kim-1 (47.15 ± 23.47, 30.54 ± 15.99, 32.28 ± 17.85) in the KIT-PR1P/VEGF group, PR1P/VEGF group and PBS group. ∗∗∗∗P < 0.0001, ∗∗P < 0.01.All data are expressed as mean ± SD. (G) Fluorescence co-localisation analysis of Kim-1and VEGF in the KIT-PR1P/VEGF group. (H) Fluorescence co-localisation analysis of Kim-1 and VEGF in the PR1P/VEGF group. (I) Fluorescence co-localisation analysis of Kim-1 and VEGF in the PBS group.

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  Fig. 2. The targeting ability of KIT-PR1P/VEGF in vivo. (A–B) Immunofluorescence co-staining assay. for VEGF (green) and the renal tubular injury marker Kim-1 (red) at 6 h and 24 h post-administration. DAPI (blue). The Scale Bar = 50 μm. (C) Fluorescence Integrated density of VEGF (24.60 ± 12.33, 10.79 ± 7.95, 4.47 ± 1.91, 3.17 ± 3.54) and Kim-1 (18.63 ± 9.67, 13.61 ± 6.10,13.23 ± 6.75, 13.85 ± 6.62) in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001.All data are expressed as mean ± SD. (D) Fluorescence Integrated density of VEGF (42.82 ± 25.27, 7.13 ± 6.16, 8.94 ± 5.11, 3.61 ± 3.33) and Kim-1 (14.60 ± 7.62, 19.28 ± 10.51, 13.49 ± 6.20, 8.70 ± 6.06) in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 24 h. ∗∗∗∗P < 0.0001. All data are expressed as mean ± SD. (E) Fluorescence co-localisation analysis of Kim-1 and VEGF in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 6 h. (F) Fluorescence co-localisation analysis of Kim-1 and VEGF in the KIT-PR1P/VEGF group, PR1P/VEGF group, VEGF group and PBS group at I/R 24 h. (G)Quantitative ELISA assay for VEGF in kidney at 6 h and 24 h…

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  Fig. 3. The evaluation of renal function by serum Scr level after KIT-PR1P/VEGF intravenous injection. (A) Summary of Scr data for renal function after administration. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001. ∗∗P < 0.01. (B–D) Histogram of Scr assay for renal function after I/R injury and at 24 h, 72 h and 2w post-administration. All data are expressed as mean ± SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05. N = 6.

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  Fig. 4. Morphology evaluation of ischemic kidney after KIT-PR1P/VEGF intravenous injection. (A–C) Histopathological staining (H&E) for kidney pathological injury observation. Scale Bar = 20 μm. Red arrow indicated renal tubular injury; green asterisk indicated glomerulus. (D–F) Quantitative analysis of renal tubule injury based on H&E staining. (G–H) Masson staining of ischemic kidney after KIT-PR1P/VEGF intravenous injection. (I–K) Quantitative analysis of the area of renal interstitial fibrosis in obstructed kidneys. All data are expressed as mean ± SD. ∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01. N = 5.

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  Fig. 5. Immunofluorescence staining of CD31 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. Green fluorescence in this figure indicated areas stained positive for CD31, and blue fluorescence indicated DAPI. (A–C) Immunofluorescence staining for endothelial marker CD31 after I/R injury. Scale Bar = 20 μm. (D–F) Quantitative of Immunofluorescence staining for CD31. All data was expressed as mean ± SD.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5.

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  Fig. 6. TUNEL staining and Immunofluorescence staining of cleaved-Caspase3 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. (A–C) TUNEL staining for apoptosis cells assessment (green). Scale Bar = 100 μm. (D–F) Quantitative of kidney TUNEL-positive cells. All data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5. (H–J) Immunofluorescence staining of cleaved-Caspase3 in ischemic kidney after KIT-PR1P/VEGF intravenous injection. Red fluorescence indicated areas stained positive for cleaved-Caspase3, and blue fluorescence indicated DAPI. Immunofluorescence staining for cleaved-Caspase3 after I/R injury. Scale Bar = 50 μm. (K–M) Quantitative of Immunofluorescence staining for cleaved-Caspase3. All data was expressed as mean ± SD.∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5.

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  Fig. 7. Downstream pathway of KIT-PR1P/VEGF. (A) Immunofluorescence staining of p-VEGFR in ischemic kidney at 24 h intravenous injection of KIT-PR1P/VEGF after I/R injury and administration. Scale Bar = 20 μm. (B) Quantitative of IHF staining for p-VEGFR. All data are expressed as mean ± SD.∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. N = 5. (C) Western blotting assay of ischemia-reperfused kidneys for p-VEGFR, AKT, phospho-AKT, ERK, phospho-ERK at 24 h post administration. GAPDH was used as a control; (D) Statistical analysis of relative value for Western blot of p-VEGFR; (E) Statistical analysis of relative value for Western blot of AKT; (F) Statistical analysis of relative value for Western blot of p-AKT. (G) Statistical analysis of relative value for Western blot of ERK. (H) Statistical analysis of relative value for Western blot of p-ERK. N = 5. ∗∗∗∗p < 0 0.0001, ∗∗∗p < 0 0.001, ∗∗p < 0 0.01, ∗p < 0.05.

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