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Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

Sik Lee, Sarah Huen, Hitoshi Nishio, Saori Nishio, Heung Kyu Lee, Bum-Soon Choi, Christiana Ruhrberg and Lloyd G. Cantley
JASN February 2011, 22 (2) 317-326; DOI: https://doi.org/10.1681/ASN.2009060615
Sik Lee
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Sarah Huen
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Hitoshi Nishio
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Saori Nishio
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Heung Kyu Lee
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Bum-Soon Choi
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Christiana Ruhrberg
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Lloyd G. Cantley
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    Figure 1.

    Macrophages switch phenotypes during kidney injury and repair. (A and B) Immunostaining for F4/80 (green) shows a progressive increase in macrophages surrounding the injured tubules from days 1 to 7. n = 4 to 8 mice/group, P < 0.001 for days 3 to 7 versus day 1. (C) Immunostaining for Cd11c and F4/80 shows that a small percentage of F4/80+ cells coexpress the dendritic cell marker Cd11c (arrows), whereas other F4/80+ cells do not express Cd11c (asterisks). (D) Quantification of F4/80+Cd11c+ cells in control mice and on day 7 after injury. **P < 0.01 versus day 0. (E and F) qPCR for iNos (E) and Arg-1 (F) from F4/80+ cells isolated from kidneys at indicated times after I/R. Values reported as ddCt relative to day 1. **P < 0.01, ***P < 0.001.

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    Figure 2.

    Macrophages induced in vitro and activated in vivo demonstrate similar expression patterns. (A) PCR of mRNA isolated from BMMs treated for 24 hours with either Ifnγ or IL-4. Ifnγ induces expression of the M1 marker iNos, whereas IL-4 induces expression of the M2 marker Arg-1. (B) RNA isolated from BMMs stimulated for 24 hours with either Ifnγ or IL-4 was used for real-time PCR of message levels for iNos, Arginase-1, or IL-12 relative to their expression in uninduced BMMs. A representative experiment is shown (from an n of 3). (C) F4/80+Cd11c− macrophages were isolated from kidneys at baseline (D0) and compared with F4/80+Cd11c−MR− macrophages 24 hours after I/R injury and F4/80+Cd11c−MR+ macrophages 7 days after I/R injury. A representative real-time PCR for iNos, Arg-1, and IL-12 relative to their expression in D0 cells is shown (from an n of 3).

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    Figure 3.

    Macrophages demonstrate plasticity in vitro and in vivo. (A) BMMs were stimulated with Ifnγ to induce iNos expression and labeled with the cytosolic tracker dye PKH26. Original magnification: ×1000. (B) PKH26 labeled M1 macrophages were injected intravenously at 3 days after I/R, followed on day 5 by immunofluorescence analysis of PKH26, iNos, and MR. Multiple PKH26+MR+ cells were detected in the injured kidneys on day 5 (arrowheads), whereas most PKH26+ cells had lost iNos expression (arrows). Original magnification: ×400. (C) Quantification of PKH26+iNos+ and PKH26+MR+ macrophages injected either on day 0 and analyzed on day 1 after I/R or injected on day 3 and analyzed on day 5 after I/R. n = 4 separate mice/group, ***P < 0.001 versus day 1. (D) BMM were stimulated for 48 hours with vehicle (BMM), Ifnγ (M1), IL-4 (M2), or vehicle followed by MPT coculture (BMM+MPT) or Ifnγ followed by MPT coculture (M1+MPT) and then immunostained for MR (green) or isotype control. Stimulation with IL-4 or coculture with MPT results in increased expression of MR (bottom three panels). Original magnification: ×400. (E) FACS analysis of MR expression by naive BMM versus IL-4–induced BMMs (M2), naive BMMs versus naive BMMs cocultured with MPT cells, and Ifnγ-activated BMMs (M1) versus M1 cocultured with MPT cells.

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    Figure 4.

    MPT coculture induces a hybrid activation state in macrophages that is IL-4 independent. BMMs were stimulated with Ifnγ for 48 hours (M1), followed by culture for 48 hours in the presence of Ifnγ, vehicle, IL-4, or MPT coculture, followed by RNA harvest and quantitative RT-PCR, normalized to Hprt expression. (A) Expression of iNos, Arg1, and MR after coculture of M1-induced cells with MPT was not statistically different from that seen after stimulation with IL-4. (B) Ym1 and Igf1 expression are significantly lower in M1+MPT compared with M1+IL-4, whereas IL-1β and Msr1 mRNA expression in M1+MPT are significantly upregulated. Expression is presented relative to M1-activated BMMs treated with vehicle. n = 4, *P < 0.05, **P < 0.01, and ***P < 0.001 for M1+IL-4 versus M1+MPT. (C) Quantitative RT-PCR analysis of Arg1 and MR expression in IL-4rαtm1Sz BMMsw (IL-4R KO) induced with IL-4 for 24 hours relative to that in IL-4–stimulated strain-matched Balb/c wild-type BMMs. (D–G) IL-4R KO BMMs or WT BMMs stimulated with Ifnγ for 24 hours (M1) were cocultured with (D and E) MPT cells (M1+MPT) or (F and G) primary tubular epithelial cells isolated from wild-type Balb/c kidneys (M1+PTEC) for 48 hours. ddCt expression shown relative to M1-induced KO and WT BMMs, respectively. A representative experiment is shown for each condition (from an n of 3). (H and I) Wild-type (Balb/c) or IL-4Rα–null mice were subjected to unilateral ischemia/reperfusion and contralateral nephrectomy. Sections from the ischemically injured kidney were stained for MR on day 7 after injury (H), and samples were obtained for BUN on the indicated days (I). The infiltration of MR-positive cells and the course of BUN after I/R were indistinguishable in the two groups of mice. n = 11 mice/group.

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    Figure 5.

    Proinflammatory macrophages promote tubule injury. Mice treated for 2 days with liposomal vehicle (LV) or liposomal clodronate (LC) were subjected to unilateral I/R and contralateral nephrectomy, followed by infusion of 1 × 106 M1 or M2 macrophages immediately after I/R. (A) BUN values are shown 24 hours after I/R ± macrophage infusion. n = 4 to 8 animals/group, **P < 0.01 versus I/R+LC alone, P = not significant versus I/R+LV alone. *P < 0.05 versus I/R+LC+M1, P = not significant versus I/R+LC alone. (B) Histology of mice treated as in A shows increased tubular injury in the LV control and LC+M1 group compared with LC or LC+M2. Original magnification: ×400. (C) Blinded scoring of tubular injury shows increased injury in the mice receiving M1 macrophages (n = 4 mice/group, ***P < 0.001 LC versus LV, P < 0.001 LC+M1 versus LC, P < 0.001 LC+M2 versus LC+M1, P = not significant versus LC+M2 versus LC).

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    Figure 6.

    Alternatively activated macrophages promote tubule proliferation and kidney repair. (A) Transwell coculture of ATP-depleted MPT cells with M2 macrophages results in increased cell numbers compared with coculture with M1 macrophages or MPT cells alone (n = 3, P < 0.01 for M2 versus M1 and M2 versus control; P = not significant for M1 versus control). (B) Mice were subjected to unilateral I/R and contralateral nephrectomy followed 48 and 72 hours later by injection of LV or LC. Quantitation of F4/80 and/or MR expressing cells/400× field in the outer medulla on day 5 after injury. n = 3, **P < 0.01 versus LV. (C and D) Proliferating epithelial cells on day 5 after injury were detected by Ki-67 immunostaining (C, 1000×) and quantified/400× field in the outer medulla (D, n = 3, ***P < 0.001). (E) BUN determination shows less improvement in GFR at 5 and 7 days after injury in the LC-treated group (***P < 0.001 versus LV at days 5 and 7). (F) Histology on day 5 after I/R of kidneys from mice treated on days 2 and 3 with LC or LV shows that the majority of injured tubules in LV-treated animals are undergoing repair with increasing numbers of cells/tubule (yellow arrows). In contrast, many tubules from LC-treated mice continue to have small numbers of highly simplified cells lining the basement membrane (green arrows). Original magnification: ×400.

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Journal of the American Society of Nephrology: 22 (2)
Journal of the American Society of Nephrology
Vol. 22, Issue 2
1 Feb 2011
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Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair
Sik Lee, Sarah Huen, Hitoshi Nishio, Saori Nishio, Heung Kyu Lee, Bum-Soon Choi, Christiana Ruhrberg, Lloyd G. Cantley
JASN Feb 2011, 22 (2) 317-326; DOI: 10.1681/ASN.2009060615

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Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair
Sik Lee, Sarah Huen, Hitoshi Nishio, Saori Nishio, Heung Kyu Lee, Bum-Soon Choi, Christiana Ruhrberg, Lloyd G. Cantley
JASN Feb 2011, 22 (2) 317-326; DOI: 10.1681/ASN.2009060615
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