J Korean Med Sci.  2017 Oct;32(10):1616-1625. 10.3346/jkms.2017.32.10.1616.

Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury

• Cho W
• Song JY
• Oh SW
• Kim MG
• Ko YS
• Lee HY
• Jo SK

• KMID: 2400433
• DOI: http://doi.org/10.3346/jkms.2017.32.10.1616

Abstract

Effective clearance of inflammatory cells is required for resolution of inflammation. Here, we show in vivo evidence that apoptosis and reverse transendothelial migration (rTEM) are important mechanisms in eliminating neutrophils and facilitating recovery following ischemia/reperfusion injury (IRI) of the kidney. The clearance of neutrophils was delayed in the Bax knockout (KO)BM → wild-type (WT) chimera in which bone marrow derived cells are partially resistant to apoptosis, compared to WTBM → WT mice. These mice also showed delayed functional, histological recovery, increased tissue cytokines, and accelerated fibrosis. The circulating intercellular adhesion molecule-1 (ICAM-1)+ Gr-1+ neutrophils displaying rTEM phenotype increased during the recovery phase and blockade of junctional adhesion molecule-C (JAM-C), a negative regulator of rTEM, resulted in an increase in circulating ICAM-1+ neutrophils, faster resolution of inflammation and recovery. The presence of Tamm-Horsfall protein (THP) in circulating ICAM-1+ neutrophils could suggest that they are derived from injured kidneys. In conclusion, we suggest that apoptosis and rTEM are critically involved in the clearance mechanisms of neutrophils during the recovery phase of IRI.

Keyword

Ischemia/Reperfusion; Neutrophil; Apoptosis; Reverse Transendothelial Migration; Recovery

MeSH Terms

Acute Kidney Injury*
Animals
Apoptosis
Bone Marrow
Chimera
Cytokines
Fibrosis
Inflammation
Intercellular Adhesion Molecule-1
Kidney
Mice
Neutrophils*
Phenotype
Transendothelial and Transepithelial Migration
Uromodulin
Cytokines
Intercellular Adhesion Molecule-1
Uromodulin

Figure

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  Fig. 1 Characteristics of Bax KO splenocytes. (A) Bax KO splenocytes are partially resistant to H2O2 induced apoptosis compared to WT splenocytes. (B) Cytokine production upon LPS stimulation was not different between Bax KO and WT splenocytes. (C) Migratory capacity of splenocytes toward MCP-1 was not different between Bax KO and WT. All experiments were conducted in triplicates. KO = knockout, WT = wild-type, LPS = lipopolysaccharide, MCP = monocyte chemotactic protein, TNF = tumor necrosis factor, IL = interleukin, PBS = phosphate-buffered saline, HPF = high-power field. *P < 0.05.

• 

  Fig. 2 Apoptotic neutrophils increased during the recovery phase of IRI. The number of Annexin V+ Gr-1+ apoptotic neutrophils out of 106 kidney cells was compared between days 1 and 2 of IRI (n = 4–5 per group). IRI = ischemia/reperfusion injury. *P < 0.05.

• 

  Fig. 3 Neutrophil infiltration persisted in Bax KOBM → WT chimera throughout the recovery phase of IRI and was associated with delayed functional, histological recovery. (A) Left: representative pictures of Gr-1 immunohistochemistry, × 100, Right: mean number of Gr-1+ neutrophils in kidneys on day 1, 3, 5 of IRI. (B) TNF-α, MCP-1, and IL-6 levels in the kidneys were persistently elevated on day 5 of IRI in Bax KOBM → WT chimera. (C) Serum creatinine. (D) Left: representative pictures of PAS stained kidney tissues, × 100, Right: semiquantitative scoring of tubular injury (n = 5–6 per group). KO = knockout, WT = wild-type, IRI = ischemia/reperfusion injury, TNF = tumor necrosis factor, MCP = monocyte chemotactic protein, IL = interleukin, WT = wild-type, PAS = periodic acid-Schiff. *P < 0.05.

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  Fig. 4 Persistent inflammation in Bax KOBM → WT chimera led to early fibrosis. (A) Upper: representative pictures of MT stained kidney tissues, × 40, lower: semiquantitative scoring of fibrosis. (B) Upper: representative pictures of F4/80 immunohistochemistry, × 100, lower: mean number of F4/80+ cells in kidneys on 14 days after IRI (n = 5–6 per group). KO = knockout, WT = wild-type, MT = Masson's trichrome, IRI = ischemia/reperfusion injury. *P < 0.05.

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  Fig. 5 Circulating ICAM-1+ neutrophils increased during the recovery phase of IRI. (A) Flow cytometry showed the increase of ICMA-1+ Gr-1+ reverse transmigrated neutrophils in peripheral blood on day 2 after IRI. (B) The number of circulating ICMA-1+ Gr-1+ neutrophils out of 106 kidney cells (n = 4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury. *P < 0.05 compared to sham. †P < 0.05 compared to I/R day 1.

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  Fig. 6 Substantial portion of circulating ICAM-1+ neutrophils during the recovery phase of IRI are positive for THP. (A) Flow cytometry show the increased percentage of THP+ ICAM-1+ neutrophils on day 2 after IRI. Neutrophils were gated first. (B) Percentage of THP+ ICAM-1+ neutrophils after IRI. (C) THP was increased in peripheral blood leukocytes during the recovery phase of IRI (n=4–5 per group). ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, THP = Tamm-Horsfall protein. *P < 0.05.

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  Fig. 7 Effect of JAM-C blocking antibody on the number of circulating ICAM-1+ neutrophils, kidney neutrophils, functional, and histological recovery following IRI. (A) Upper: Flow cytometry show that treatment with anti-mouse JAM-C blocking Ab resulted in increase of circulating ICAM-1+ neutrophils following IRI. Lower: percentage of circulating ICMA-1+ Gr-1+ neutrophils out of total neutrophils (n = 4–5 per group). (B) Treatment with anti-mouse JAM-C blocking Ab resulted in decreased number of infiltrating neutrophils on day 3 after IRI. Upper: representative pictures of Gr-1 immunohistochemistry, × 100, lower: mean number of Gr-1+ neutrophils in kidneys on day 3 of IRI (n = 4–5 per group). (C) Treatment with anti-mouse JAM-C blocking Ab resulted in faster functional recovery. (D) Treatment with anti-mouse JAM-C blocking Ab resulted in faster histological recovery. Upper: representative pictures of PAS stained kidney tissues, × 100, lower: semiquantitative scoring of tubular injury (n = 5–6 per group). JAM-C = junctional adhesion molecule-C, ICAM-1 = intercellular adhesion molecule-1, IRI = ischemia/reperfusion injury, Ab = antibody, PAS = periodic acid-Schiff. *P < 0.05 compared to day 1. † P < 0.05 compared to day 2. ‡ P < 0.05.

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