By homing to the kidney, activated macrophages potently exacerbate renal injury

Abstract

Macrophages are important mediators of injury in most types of human kidney diseases; however, the pathogenic importance of both macrophage number and activation status is unknown. To examine this question, severe-combined immunodeficient mice with adriamycin nephrosis, an experimental model of human focal segmental glomerulosclerosis, were treated intravenously with either resting (1 x 10(6) to 5 x 10(6)) or activated (1 x 10(3) to 1 x 10(6)) macrophages on day 6 postadriamycin administration, and the effects on kidney injury were examined. On day 28, renal injury was worse in the group that received activated macrophages at doses as low as 1 x 10(4) macrophages per mouse compared with control adriamycin nephrotic mice. However, treatment with resting macrophages at doses as high as 5 x 10(6) macrophages per mouse had no significant effect on either renal histology or function. The transferred activated macrophages homed to inflamed kidneys during the middle-to-late stages of the disease, but such homing was not observed for resting macrophages. This study of in vivo cell adoptive transfer supports the importance of macrophage activation status over macrophage number in causing renal injury. These data suggest that therapeutic strategies for treating progressive kidney diseases should target activated macrophages.

Figure 1

Cytokine expression of macrophages isolated from AN kidney at day 28 of mice transfused with resting (RM) or activated (AM) macrophages. The expression of CCL2, CCL3, iNOS, and TNF-α mRNA was assessed by real-time PCR. *P < 0.05 vs. non-asterisked groups.

Figure 2

A: In vivo tracking of transfused macrophages in AN kidney. Representative fluorescence photomicrographs showing transfused resting (RM) and activated (AM) macrophages in kidney at day 7, 14, and 28 after ADR, following macrophage transfusion at day 6. B: Quantitation of transfused resting and activated macrophages in spleen, liver, and kidney at day 7, 14, and 28 after ADR, following macrophage transfusion at day 6. There were no transfused AM seen in liver following transfusion at a dose of 10³ and 10⁴. Error bars are omitted for clarity (also see Table 3). Original magnification ×200.

Figure 3

A: Effect of macrophage transfusion on histological injury, macrophage accumulation, and fibrosis of AN. Representative Gomori trichrome, α-SMA, and F4/80 stained sections of renal cortices at day 28 (magnification ×200) from AN mice transfused with vehicle (a), 5 × 10⁶ resting macrophages (b), 10⁴ activated macrophages (c), 10³ activated macrophages (d) (n = 6 per group). The injury caused by 10⁵ and 10⁶ AM was no different to that caused by 10⁴AM. B: Semiquantitative assessment of kidney injury. Each evaluation represents the mean ± SEM. *P < 0.05 vs. other three groups (n = 6 per group).

Figure 4

CCL-2-mediated macrophage chemotaxis and expression of chemokine receptors. A: Macrophage chemotaxis toward CCL-2 was tested for 8 hours. 20,000 cells were used in each assay and fluorescence was determined as described in methods. (**P < 0.01 compared to RM group). B: mRNA expression of chemokine receptors in RM and AM was examined by microdissection and real-time PCR (*P < 0.01 and **P < 0.001 compared to RM group).

Figure 5

Expression of cytokines and chemokines in AN. A: CCL2 staining, left: normal kidney; right: AN kidney. B: mRNA expression of chemokines and cytokines in tubules of AN (*P < 0.001 compared with normal controls).

Figure 6

Serum creatinine and 16-hour urine protein at day 28. The values represent the mean ± S.E.M. of evaluations from each group (n = 6 per group). *P < 0.05 for activated macrophages at doses 10⁴, 10⁵, or 10⁶ versus other five groups.