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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal
Open Access

Potent Therapy and Transcriptional Profile of Combined Erythropoietin-Derived Peptide Cyclic Helix B Surface Peptide and Caspase-3 siRNA against Kidney Ischemia/Reperfusion Injury in Mice

Yuanyuan Wu, Weiwei Chen, Yufang Zhang, Aifen Liu, Cheng Yang, Hui Wang, Tongyu Zhu, Yaping Fan and Bin Yang
Journal of Pharmacology and Experimental Therapeutics October 2020, 375 (1) 92-103; DOI: https://doi.org/10.1124/jpet.120.000092
Yuanyuan Wu
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Weiwei Chen
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Yufang Zhang
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Aifen Liu
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Cheng Yang
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Hui Wang
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Tongyu Zhu
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Yaping Fan
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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Bin Yang
Renal Group, Basic Medical Research Centre, Nantong University, Nantong, China (Y.W., Y.Z., A.L.); Leicester-Nantong Joint Institute of Kidney Science, Department of Nephrology, Affiliated Hospital of Nantong University, Nantong, China (W.C., H.W., Y.F., B.Y.); Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China (C.Y., T.Z.); Shanghai Key Laboratory of Organ Transplantation, Shanghai, China (C.Y., T.Z.); and Renal Group, Department of Cardiovascular Sciences, University of Leicester, University Hospitals of Leicester, Leicester, United Kingdom (Y.W., B.Y.)
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  • Fig. 1.
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    Fig. 1.

    CHBP and/or CASP3siRNA preserved renal function and structure in IR kidneys. (A) Schematic diagram of mouse renal IR models with the treatment of CHBP and/or CASP3siRNA. Bilateral kidney pedicles were occluded for 30 minutes, followed by 48-hour reperfusion. CASP3siRNA or NCsiRNA was injected via the tail vein at a dose of 0.03 mg/kg BW 2 hours before surgery. CHBP was given through intraperitoneal cavity at 24 nmol/kg BW 15 minutes postreperfusion. (B) The level of SCr was demonstrated for each group. There were six animals in each group, and the detection was repeated for three times independently. The experiment was performed three times independently. (C) Representative photomicrographs of H&E staining in renal cortex were shown for each group. Scale bar, 100 μm. (D) Semiquantitative analysis of TID score (n = 6). The sections were blindly scored by two researchers independently. Data were shown as means ± S.D. n = 6 animals per group; *P < 0.05; **P < 0.01. Statistical comparisons were calculated by ANOVA followed by post hoc LSD test.

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    Fig. 2.

    CHBP and/or CASP3siRNA ameliorated apoptosis in tubulointerstitial areas. (A) The method of ISEL fragmented DNAs was used to detect apoptotic cells in kidney tissues. AEC was used to develop color labeling. Representative photomicrographs of apoptotic cells (indicated by arrows) in cortical areas were shown in each group. Scale bar, 50 μm. (B) The average number of ISEL+ cells per field are demonstrated for each group (n = 6). The sections were blindly reviewed by two researchers independently. Data were shown as means ± S.D. n = 6 animals per group; *P < 0.05; **P < 0.01. Statistical comparisons were calculated by ANOVA followed by post hoc LSD test.

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    Fig. 3.

    CHBP and/or CASP3siRNA decreased the number of active 17 kDa caspase-3–positive cells. (A) Representative photomicrographs of cells positively stained with 17 kDa caspase-3+ were shown in the indicated groups. Detected by immunostaining, 17 kDa caspase-3 was labeled and revealed by AEC. Scale bar, 50 μm. (B) The average number of 17 kDa caspase-3+ cells per field are demonstrated in each group (n = 6). The sections were blindly reviewed by two researchers independently. Data were shown as means ± S.D. n = 6 animals per group; *P < 0.05; **P < 0.01. Statistical comparisons were calculated by ANOVA followed by post hoc LSD test.

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    Fig. 4.

    CHBP and/or CASP3siRNA decreased 17 kDa active caspase-3 and HMGB1 expression in IR kidneys. (A) The level of active caspase-3 was measured by Western blotting and typical bands were shown. (B) Semiquantitative analysis showed the expression of 17 kDa caspase-3 corrected by the endogenous control of β-actin in each group (n = 6). (C) Representative bands of HMGB1 were shown. (D) The level of HMGB1 protein corrected by β-actin was determined in each group (n = 6). The immunoblotting was performed three times independently. Data were shown as means ± S.D. n = 6 animals per group; *P < 0.05; **P < 0.01. Statistical comparisons were calculated by ANOVA followed by post hoc LSD test.

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    Fig. 5.

    Identified DEGs and qPCR validation. (A) Venn diagram illustrating the number of significant DEGs in the three comparisons. The sorting criteria was FC > 1.414 and P < 0.05. In four groups (n = 3), each kidney sample was analyzed separately. (B–E) The expression of SLC22A7, CFH, GREM1, and ANGPTL2 mRNA was detected by qPCR, which was done three times independently. n = 3 kidney samples from individual animals in each group; *P < 0.05. Statistical comparisons were calculated by two-tailed unpaired Student’s t test.

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    Fig. 6.

    GO analysis of DEGs in IR kidneys. The top 30 significantly enriched GO items of biologic processes were shown from three comparisons, modified by CHBP (A) and then further by CASP3siRNA (B) or NCsiRNA (C). The text on the left indicated the category of GO, and the bar chart indicated the enrich factor in each category. Underlined categories were particularly discussed in this study. n = 3 animals in each group.

Tables

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    TABLE 1

    The sequence of primers for qPCR

    Gene SymbolFull NamePrimers (5′-3′)
    SLC22A7Solute carrier family 22 member 7Forward: CTG​TCT​GCC​TGT​GTT​TAT​CC
    Reverse: CTT​CCC​CAA​ATG​CCA​CAG​CT
    CFHComplement factor HForward: ACT​TTC​TCA​GAT​TTT​CCT​GG
    Reverse: TGG​TTG​TTA​CAT​GCT​TTG​GG
    ANGPTL2Angiopoietin-like 2Forward: GGA​TGG​TTC​ACA​GAG​AGA​GTA​C
    Reverse: CTC​CTT​GGA​GTT​GAC​ACA​AAT​G
    GREM1Gremlin-1Forward: GCAAGTATCTGAAGVGAGATTG
    Reverse: CGT​CAT​GGT​GGT​GAA​CTT​CTT​G
    ACTBβ-actinForward: GAG​ACC​TTC​AAC​ACC​CCA​GC
    Reverse: ATG​TCA​CGC​ACG​ATT​TCC​C
    • View popup
    TABLE 2

    IR + CHBP vs. IR, top five upregulated and downregulated genes

    The gene in italic was discussed for its potential function in IR-induced AKI. P < 0.05.

    Accession NumberGene SymbolGene NameFold Change
    Upregulated
    AK0161054930553I04RIKRIKEN cDNA 4930553I04 gene8.559
    AK046501SACSSacsin5.261
    NM_013501CRYAACrystallin, alpha A4.357
    NM_011255RBP4Retinol binding protein 4, plasma4.213
    NM_009744BCL6B cell leukemia/lymphoma 63.622
    Downregulated
    NM_001167777ASXL3Additional sex combs like 3−24.005
    NM_029070CLDN26Claudin 26−5.985
    NM_001167746DNAH17Dynein, axonemal, heavy chain 17−5.922
    AK171340PITPNC1Phosphatidylinositol transfer protein, cytoplasmic 1−5.113
    NM_007501NEUROD4Neurogenic differentiation 4−5.094
    • View popup
    TABLE 3

    IR + CHBP + CASP3siRNA vs. IR + CHBP, top five upregulated and downregulated genes

    The genes in italic were discussed for their potential function in IR-induced AKI. P < 0.05.

    Accession NumberGene SymbolGene NameFold Change
    Upregulated
    NM_001001450SSXB2Synovial sarcoma, X member B, breakpoint 214.730
    NR_0281114930523C07RIKRIKEN cDNA 4930523C07 gene10.131
    NM_011414SLPISecretory leukocyte peptidase inhibitor8.478
    NM_009253SERPINA3MSerine (or cysteine) peptidase inhibitor, clade A, member 3M8.289
    NM_175309UPK3BUroplakin 3B5.483
    Downregulated
    AK0161054930553I04RIKRIKEN cDNA 4930553I04 gene−8.735
    NM_0297472410137M14RIKRIKEN cDNA 2410137M14 gene−5.134
    NM_147025OLFR380Olfactory receptor 380−4.354
    NM_134160MCOLN3Mucolipin 3−4.142
    NM_001163513DLG5Discs large MAGUK scaffold protein 5−3.700
    • View popup
    TABLE 4

    IR + CHBP + CASP3siRNA vs. IR + CHBP + NCsiRNA, top five upregulated and downregulated genes

    Genes in italic indicate analysis by qPCR and were discussed as biomarker candidates of IR-induced AKI. P < 0.05.

    Accession NumberGene SymbolGene NameFold Change
    Upregulated
    NM_011824GREM1Gremlin 115.987
    NM_001001450SSXB2Synovial sarcoma, X member B, breakpoint 214.817
    NM_026433TMEM100Transmembrane protein 1009.749
    NM_011825GREM2Gremlin 2 homolog, cysteine knot superfamily (Xenopus laevis)9.312
    NM_007729COL11A1Collagen, type XI, alpha 18.978
    Downregulated
    AK041888ANGPTL2Angiopoietin-like 2−24.390
    AF107847GNASGNAS (guanine nucleotide binding protein, alpha stimulating) complex locus−12.195
    AK081320ZFYVE9Zinc finger, FYVE domain containing 9−7.576
    NM_010258GATA6GATA binding protein 6−6.711
    NM_026648DNAAF1Dynein, axonemal assembly factor 1−6.579
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Journal of Pharmacology and Experimental Therapeutics: 375 (1)
Journal of Pharmacology and Experimental Therapeutics
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1 Oct 2020
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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

CHBP and Caspase-3 siRNA Protected IR Kidneys

Yuanyuan Wu, Weiwei Chen, Yufang Zhang, Aifen Liu, Cheng Yang, Hui Wang, Tongyu Zhu, Yaping Fan and Bin Yang
Journal of Pharmacology and Experimental Therapeutics October 1, 2020, 375 (1) 92-103; DOI: https://doi.org/10.1124/jpet.120.000092

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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

CHBP and Caspase-3 siRNA Protected IR Kidneys

Yuanyuan Wu, Weiwei Chen, Yufang Zhang, Aifen Liu, Cheng Yang, Hui Wang, Tongyu Zhu, Yaping Fan and Bin Yang
Journal of Pharmacology and Experimental Therapeutics October 1, 2020, 375 (1) 92-103; DOI: https://doi.org/10.1124/jpet.120.000092
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