Research ArticleKidney Disease

The protective role of macrophage migration inhibitory factor in acute kidney injury after cardiac surgery

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Science Translational Medicine  16 May 2018:
Vol. 10, Issue 441, eaan4886
DOI: 10.1126/scitranslmed.aan4886
  • Fig. 1 Elevated MIF is associated with reduced incidence of AKI and enhanced antioxidant capacity in cardiac surgery patients.

    (A) Percentage of patients with AKI within 72 hours after cardiac surgery, stratified by median serum concentration (n = 60). (B) Perioperative kinetics of serum MIF in patients without postoperative AKI compared to patients with AKI. (C) Incidence of AKI stratified by median urinary MIF concentration. (D) Perioperative kinetics of urinary MIF. (E) Inverse correlation between postoperative serum MIF and postoperative urinary NGAL 24 hours after cardiac surgery. (F and G) Antioxidant capacity of serum samples measured using the RedoxSYS Diagnostic System (Luoxis Diagnostics). Comparison of the antioxidant capacity in serum samples with high serum MIF (>median) with low serum MIF (≤median) at the corresponding time points. (H and I) Comparison of the antioxidant capacity in serum samples of patients with AKI with patients without AKI. NGAL served as a kidney injury marker. Data are means ± SEM. r, Pearson coefficient; R, goodness of fit. (A and C) P < 0.05 analyzed by Fisher’s exact test. (B and D) P < 0.05 versus other groups at the corresponding time point (difference between groups) analyzed by Mann-Whitney U test.

  • Fig. 2 Renal ischemia as a stimulus for MIF release into the bloodstream.

    (A) Kinetics of serum MIF in patients undergoing kidney tumor enucleation. Comparison of patients exposed to renal hypoxia (n = 18) by cross-clamping of the renal artery versus no hypoxia (n = 28). Blood samples were drawn 1 day before surgery (pre-OP), 5 min after tumor enucleation (intra-OP), in the recovery room shortly after the termination of surgery, and on POD1. (B) Association between the duration of renal ischemia and postoperative serum MIF on the first day after surgery. Data are means ± SEM; §§P < 0.01 versus other groups at the corresponding time point (difference between groups) and *P < 0.05 versus baseline analyzed by Mann-Whitney U test.

  • Fig. 3 Comparison of AKI and tubular injury in WT and Mif−/− mice in an experimental model of AKI induced by unilateral ischemia and 24 hours of reperfusion.

    (A) Schematic depicting the induction of acute kidney damage after 35 min of ischemia by cross-clamping of the renal artery and reperfusion for 24 hours in WT mice and Mif−/− mice (n = 5 both groups). (B) Serum creatinine [normalized to body weight (BW)] in WT and Mif−/− mice 24 hours after I/R injury. (C and D) Tubular necrosis was evaluated applying the tubular injury score. Immunohistochemical staining of apoptotic cells, cleaved caspase-3–positive cells, and necroptotic, pMLKL-positive tubules. +/+, WT mice; −/−, Mif−/− mice; hpf, high-power field. Scale bars, 100 μm. Data are means ± SD; *P < 0.05, **P < 0.01 analyzed by Student’s t test.

  • Fig. 4 Comparison of tubular injury in WT and Mif−/− mice in an experimental model of AKI induced by unilateral ischemia and 6 hours of reperfusion or by rhabdomyolysis.

    (A) Schematic depicting the induction of acute kidney damage after 35 min of ischemia by cross-clamping of the renal artery and reperfusion for 6 hours in WT mice and Mif−/− mice (n = 10 both groups). (B and C) Tubular necrosis was evaluated applying the tubular injury score. Immunohistochemical analysis of apoptotic cells, cleaved caspase-3–positive cells, and necroptotic, pMLKL-positive tubules. (D) Schematic depicting the induction of rhabdomyolysis by intramuscular (i.m.) glycerol injection in WT mice and Mif−/− mice (n = 10 both groups). (E and F) Histological analysis of tubular damage by tubular injury score and immunohistochemical staining of apoptotic cells, cleaved caspase-3–positive cells, and necroptotic, pMLKL-positive tubules in WT mice and Mif−/− mice. Scale bars, 100 μm. Data are means ± SD; *P < 0.05, **P < 0.01 analyzed by Student’s t test.

  • Fig. 5 Renal inflammatory cell infiltration after I/R injury and rhabdomyolysis.

    Quantification of inflammatory cells in renal tissue after injury. The immune cell infiltration of granulocytes (A), monocytes and dendritic cells (B), and macrophages (C) in Mif−/− mice was analyzed after 35 min of ischemia and 6 hours of reperfusion (n = 10 both groups), 35 min of ischemia and 24 hours of reperfusion (n = 5 both groups), and 24 hours after the induction of rhabdomyolysis (n = 10 both groups). (D) Relative mRNA expression of Cxc1 in kidney tissue of Mif-deficient and WT mice after 6 hours of I/R injury. Cxcl1, chemokine (C-X-C motif) ligand 1; ErHr3, Er-Hematopoiesis related-3; F4/80, monocytes/macrophages marker; Ly6G, lymphocyte antigen 6 complex locus G6D. Scale bars, 100 μm. Data are means ± SD; *P < 0.05, **P < 0.01 analyzed by Student’s t test.

  • Fig. 6 Effects of rMIF on oxidative stress in vitro and oxidative stress in kidney tissue of WT and Mif−/− mice in vivo.

    (A and B) GSH in cell lysates of pmTECs after normoxia, H2O2, or hypoxia treatment (A) or in kidney tissue lysates (B) measured with the GSH/GSSG assay kit. (C and D) TBARS in cell lysates after hypoxia for 24 hours (C) or in kidney tissue lysates (D). TBARS was normalized to protein and to control [phosphate-buffered saline (PBS) or WT in normoxia conditions]. In vitro biological n = 3; 6 hours I/R n = 10; 24 hours I/R n = 5, rhabdomyolysis n = 10. Data are means ± SD; *P < 0.05, **P < 0.01 analyzed by Student’s t test.

  • Fig. 7 Effects of rMIF administration on cytotoxicity and oxidative stress in vitro and effects of rMIF treatment on AKI in vivo.

    (A to C) Morphology (A) and LDH release of WT (B) and Mif-deficient (C) pmTECs challenged with hypoxia (<1% O2) for >24 hours in full or starvation medium. Comparison of pretreatment with PBS and rMIF (100 ng/ml). LDH was normalized to 100% cell death/toxicity. (D and H) Schematic depicting the rMIF administration and acute kidney damage by cross-clamping of the renal artery for 35 min, contralateral nephrectomy, and reperfusion for 24 hours in WT mice and Mif−/− mice (n = 5 each group). Mice were injected with 16 μg of rMIF twice (30 min before cross-clamping and 6 hours after cross-clamping). (E and I) Serum creatinine levels (normalized to BW) 24 hours after I/R injury. (F, G, J, and K) Tubular necrosis was evaluated applying the tubular injury score. Immunohistochemical staining of apoptotic cells, cleaved caspase-3–positive cells, and pMLKL-positive tubules. i.p., intraperitoneally. Scale bars, 100 μm. Data are means ± SD; *P < 0.05, **P < 0.01 analyzed by Student’s t test.

  • Fig. 8 Effects of recombinant sCD74 on cytotoxicity and oxidative stress in vitro and on tubular cell injury in vivo.

    (A) LDH release of WT pmTECs challenged with hypoxia for >24 hours in starvation medium. Comparison of pretreatment with PBS, sCD74 alone (40 nM), or sCD74 (40 nM) plus rMIF (100 ng/ml). LDH was normalized to 100% cell death/toxicity. (B) GSH in cell lysates of pmTECs after normoxia, H2O2, or hypoxia treatment measured with the GSH/GSSG assay kit. (C) Schematic depicting recombinant sCD74 administration and acute kidney damage induced by cross-clamping of the renal artery for 35 min, contralateral nephretomy, and reperfusion for 24 hours in WT mice and Mif−/− mice (n = 5 each group). Mice were injected with 20 μg of sCD74 twice (30 min before cross-clamping and 6 hours after cross-clamping). (D) Serum creatinine normalized to BW 24 hours after I/R injury. (E and F) Tubular necrosis was evaluated applying the tubular injury score. Immunohistochemical staining of apoptotic cells, cleaved caspase-3–positive cells, and pMLKL-positive tubules. Data are means ± SD; *P < 0.05, **P < 0.01 analyzed by Student’s t test. ns, not significant.

  • Table 1 Association of serum MIF and incidence of AKI after cardiac surgery.

    AKIN, Acute Kidney Injury Network. P values were calculated using the Fisher’s exact test.

    Serum MIFRelative risk reduction
    (95% confidence interval)
    P value
    ≤ Median> Median
    0 hours post-OP≤16.9 ng/ml>16.9 ng/ml
      AKI within 72 hours, n (%)8 (14)6 (10)0.250 (−0.900–0.704)0.552
      AKIN stage
      022 (37)24 (40)
      12 (3)6 (10)
      2 5 (8)0 (0)
      31 (2)0 (0)
      0/124 (40)30 (50)0.024
      2/36 (10)0 (0)
    6 hours post-OP≤19.1 ng/ml>19.1 ng/ml
      AKI within 72 hours, n (%)10 (17)4 (7)0.600 (−0.135–0.8591)0.1253
      AKIN stage
      020 (33)26 (43)
      15 (8)3 (5)
      24 (7)1 (2)
      3 1 (2)0 (0)
      0/125 (42)29 (48)0.800 (−0.611–0.975)0.195
      2/35 (8)1 (2)
    12 hours post-OP≤15.5 ng/ml>15.5 ng/ml
      AKI within 72 hours, n (%)11 (18)3 (5)0.727 (0.120–0.915)0.03
      AKIN stage
      019 (32)27 (43)
      15 (8)3 (5)
      25 (8)0 (0)
      31 (2)0 (0)
      0/124 (40)30 (50)0.024
      2/36 (10)0 (0)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/441/eaan4886/DC1

    Materials and Methods

    Fig. S1. Flowchart.

    Fig. S2. MIF and NGAL concentrations in cardiac surgery patients.

    Fig. S3. Protective effects of MIF in an experimental model of AKI induced by bilateral ischemia and 24 hours of reperfusion.

    Fig. S4. Up-regulated MLKL expression in Mif−/− mice.

    Fig. S5. Cytotoxicity of 18 hours of hypoxia followed by 6 hours of normoxia in pmTECs.

    Fig. S6. MIF receptors and phosphorylation of MLKL in tubular epithelial cells.

    Fig. S7. Scheme of the proposed action of MIF in the setting of cardiac surgery–related AKI.

    Table S1. Baseline characteristics of patients undergoing cardiac surgery.

    Table S2. List of primary and secondary antibodies for Western blot, immunohistochemistry, and immunofluorescence staining.

    References (5357)

  • Supplementary Material for:

    The protective role of macrophage migration inhibitory factor in acute kidney injury after cardiac surgery

    Christian Stoppe,* Luisa Averdunk, Andreas Goetzenich, Josefin Soppert, Arnaud Marlier, Sandra Kraemer, Jil Vieten, Mark Coburn, Ana Kowark, Bong-Song Kim, Gernot Marx, Steffen Rex, Akinobu Ochi, Lin Leng, Gilbert Moeckel, Andreas Linkermann, Omar El Bounkari, Alexander Zarbock, Jürgen Bernhagen,* Sonja Djudjaj, Richard Bucala, Peter Boor*

    *Corresponding author. Email: christian.stoppe{at}gmail.com (C.S.); juergen.bernhagen{at}med.uni-muenchen.de (J.B.); pboor{at}ukaachen.de (P.B.)

    Published 16 May 2018, Sci. Transl. Med. 10, eaan4886 (2018)
    DOI: 10.1126/scitranslmed.aan4886

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Flowchart.
    • Fig. S2. MIF and NGAL concentrations in cardiac surgery patients.
    • Fig. S3. Protective effects of MIF in an experimental model of AKI induced by bilateral ischemia and 24 hours of reperfusion.
    • Fig. S4. Up-regulated MLKL expression in Mif−/− mice.
    • Fig. S5. Cytotoxicity of 18 hours of hypoxia followed by 6 hours of normoxia in pmTECs.
    • Fig. S6. MIF receptors and phosphorylation of MLKL in tubular epithelial cells.
    • Fig. S7. Scheme of the proposed action of MIF in the setting of cardiac surgery–related AKI.
    • Table S1. Baseline characteristics of patients undergoing cardiac surgery.
    • Table S2. List of primary and secondary antibodies for Western blot, immunohistochemistry, and immunofluorescence staining.
    • References (5357)

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