Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

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).

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.

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-4rα^tm1Sz 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.

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).