Research ArticleHEALTH AND MEDICINE

ROS-responsive chitosan-SS31 prodrug for AKI therapy via rapid distribution in the kidney and long-term retention in the renal tubule

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Science Advances  09 Oct 2020:
Vol. 6, no. 41, eabb7422
DOI: 10.1126/sciadv.abb7422
  • Fig. 1 Chemical structure of SC-TK-SS31 and the schematic illustration showing the relief of AKI by SC-TK-SS31.

    After intravenous administration, SC-TK-SS31 stays stably during delivery in blood circulation and accumulates in renal tubules of the kidney subject to AKI. Specifically expressed Kim-1 in injured TECs is associated with the internalization of SC-TK-SS31. Epithelial cell injury in AKI is characterized by increased formation of ROS. Mitochondria, as the major sites of ROS production, are first damaged and urgent for protection. ROS triggers the release of SS31 via cleavage of ROS-sensitive TK linker, therefore preventing the mitochondria damage and decreasing ROS production. The alleviation of oxidative stress further diminishes inflammation and apoptosis of TECs, thereby relieves AKI.

  • Fig. 2 Renal distribution and renal tubules accumulation of SC.

    (A) The chemical structures of SC and its analogies, C, PEG-C, and S-PEG-C. (B) Fluorescence images of the main organs (heart, lung, liver, spleen, and kidney) of mice at 4 or 12 hours after intravenous injection of Cy5-C, Cy5-PEG-C, Cy5-S-PEG-C, or Cy5-SC. One of three independent experiments is shown. Renal ischemia was induced by clamping of the bilateral renal pedicles for 30 min and then removed clamping to induce IR AKI. After the initiation of AKI, fluorescence-labeled SC and its analogies were administered intravenously. Four or 12 hours later, the main organs were harvested for fluorescence visualization. The healthy mice were treated with the same protocol as control. (C and D) Region of interest analysis of the kidney uptake at 4 and 12 hours after injection. The data are the means ± SD. n = 3 independent mice. (E) Representative confocal images of kidney sections after intravenous injection of Cy5-SC (red signal) for 4 hours. Blue indicates 4′,6-diamidino-2-phenylindole (DAPI) staining. White dashed circles denote glomeruli. Scale bars, 500 μm.

  • Fig. 3 Kim-1–associated endocytosis of SC in vitro and in vivo.

    (A) Flow cytometry assay of cellular uptake of Cy5-C and Cy5-SC in normal or H2O2-stimulated HK-2 cells for 2 or 6 hours. The data are the means ± SD. n = 3. (B) Western blotting analysis of Kim-1 expression using lysates from normal and H2O2-stimulated HK-2 cells. (C) Representative confocal images of HK-2 cells that were previously stimulated with H2O2 at a concentration of 500 μM for 4 hours and then treated with Cy5-C and Cy5-SC for another 4 hours (red signal). “+Blocking” indicates pretreatment with Kim-1 antibody at a concentration of 30 μg/ml for 0.5 hours before administration of Cy5-C or Cy5-SC. “−Blocking” indicates without treatment of Kim-1 antibody. Experiments were repeated three times. Nuclei, staining with DAPI, are shown in blue. Green indicates immunostaining for Kim-1. The bottom images correspond to magnified views of the boxed areas above. Scale bars, 20 μm. FITC, fluorescein isothiocyanate. (D) Representative confocal images of kidney sections from AKI-injured mice after intravenous injection of Cy5-SC (red signal) for 4 hours. Blue indicates DAPI staining. Immunostaining for Kim-1 is shown in green. Scale bar, 20 μm.

  • Fig. 4 H2O2-triggered cleavage of SC-TK-SS31 and ROS-sensitive drug release inside the H2O2-stimulated HK-2 cells.

    (A) Absorption peaks of SC-TK-SS31 in the presence of 10 mM H2O2 monitored by HPLC at a wavelength of 220 nm. SC-NO-SS31 (irresponsive prodrug) was treated as control. Peaks with an elution time of 4.1 min represent SS31. (B) The release profiles of SC-TK-SS31 and SC-NO-SS31 in the presence of 10 mM H2O2 monitored by HPLC. The data are the means ± SD. n = 3. (C) Schematic illustration of TK cleavage. (D) Representative confocal images of H2O2-stimulated HK-2 cells after incubated with SC-TK-SS19 for 12 hours. Blue indicates SS19, representing SS31. MitoTracker-stained mitochondria are shown in green. Scale bar, 40 μm. (E) Representative confocal images of colocalization of SS19 (the fluorescent analog of SS31, representing drug, blue signal) with Cy5-SC (representing carriers, red signal). Cy5-SC-NO-SS19 was treated as control. The white arrows denote the left side to right side of the intensity profiles in (F). Scale bar, 10 μm. (F) The level of overlay of SS19 and Cy5-labeled carriers.

  • Fig. 5 Relief of oxidative stress and apoptosis by SC-TK-SS31 in vitro.

    H2O2-stimulated HK-2 cells were treated with SS31, C-TK-SS31, SC-TK-SS31, or SC-NO-SS31 at an SS31 concentration of 20 μg/ml for 24 hours. (A) The experimental groups for evaluating therapeutic effect of SC-TK-SS31, pure SS31, and the analogies of SC-TK-SS31 including C-TK-SS31 and SC-NO-SS31. The chemical structures of SC-TK-SS31 and its analogies, C-TK-SS31 and SC-NO-SS31. (B) Fluorescence images of mitochondrial ROS analysis with MitoSOX in H2O2-stimulated cells after different treatments. Scale bar, 20 μm. (C) Fluorescence images of JC-1 assay to measure mitochondrial membrane depolarization in H2O2-stimulated cells after different treatments. Scale bar, 10 μm. (D) Western blotting analysis of HO-1 expression in H2O2-stimulated HK-2 cells after different treatments. (E) Flow cytometry–based apoptosis assay by the Annexin V–FITC Apoptosis Kit. PI, propidium iodide. (F) The MitoSOX mean fluorescence intensity of (B). (G) The mean ratio of fluorescence intensity of JC-1 aggregates and JC-1 monomers in (C). (H) Densitometric analysis of Western blotting of HO-1 expression in (D). (I) The mean percentage of apoptotic cells in (E). n = 3, *P < 0.05 and ***P < 0.001 as compared with H2O2 group. n = 3, ##P < 0.01 and ###P < 0.001 between groups as indicated.

  • Fig. 6 Amelioration of renal functions by SC-TK-SS31 in vivo.

    (A) After initiation of IR-induced AKI, different agents (SS31, C-TK-SS31, SC-NO-SS31, and SC-TK-SS31) were administered intravenously at an SS31 dosage of 2 mg/kg. Forty-eight hours later, blood and kidneys were collected for evaluation. (B and C) Serum analysis of creatinine and BUN in AKI mice after different treatments. (D) Cell necrosis score in outer stripe of the outer medulla (OSOM) of AKI mice after different treatments. (E) H&E staining of kidney tissues in the OSOM. Scale bar, 50 μm. Kidneys of sham-operated mice represented normal architecture with remarkable brush borders (shown as black arrowheads) in tubules. Serious necrosis was observed in kidneys of saline-treated mice. Red arrowheads donate hyaline casts and cell sloughing. Asterisks donate adhesion. Focal necrosis was indicated in SS31-, C-TK-SS31–, and SC-NO-SS31–treated groups. Cell sloughing and adhesion can be observed. Necrosis was rare in the SC-TK-SS31–treated mice with brush borders presented in tubules. The bottom images correspond to magnified views of the boxed areas above. Scale bar, 50 μm. All data are expressed as means ± SD. n = 5 and 6 in each group, *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with saline. n = 5 and 6 in each group, #P < 0.05, ##P < 0.01, and ###P < 0.001 between groups as indicated. n.s., no significant difference.

  • Fig. 7 SC-TK-SS31–protected mitochondria from damage and reduced the oxidative stress, inflammation, and apoptosis in AKI mice.

    (A) Bio-TEM image of mitochondria in outer medulla. Swollen mitochondria with disrupted cristae architecture are noted with arrowheads. Mitochondria with disruption of membranes and release of matrix materials into the cytosol are noted with arrowheads. N, nucleus. Scale bar, 1 μm. (B) Immunohistochemical staining (brown) with nitrotyrosine, a marker of peroxidation. Scale bar, 50 μm. (C) Macrophage immunostaining using anti-CD68 antibody. Scale bar, 50 μm. (D) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay for apoptosis cells staining (brown nuclei) in outer medulla in AKI mice with different treatments. Scale bar, 50 μm. (E and F) GSH and MDA level changes in AKI mice after different treatments. (G and H) Renal cytokine TNF-α and IL-6 alternation in AKI mice after different treatments. (I) The number of TUNEL+ cells per high-power field (hpf) in the sham or AKI group with different treatments. All data are expressed as means ± SD. n = 5 and 6 in each group, *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with saline. n = 5 and 6 in each group, #P < 0.05, ##P < 0.01, and ###P < 0.001 between groups as indicated.

Supplementary Materials

  • Supplementary Materials

    ROS-responsive chitosan-SS31 prodrug for AKI therapy via rapid distribution in the kidney and long-term retention in the renal tubule

    Di Liu, Gaofeng Shu, Feiyang Jin, Jing Qi, Xiaoling Xu, Yan Du, Hui Yu, Jun Wang, Mingchen Sun, Yuchan You, Minxia Zhu, Meixuan Chen, Luwen Zhu, Qiying Shen, Xiaoying Ying, Xuefang Lou, Saiping Jiang, Yongzhong Du

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