Type 1 angiotensin receptors on macrophages ameliorate IL-1 receptor-mediated kidney fibrosis

Abstract

In a wide array of kidney diseases, type 1 angiotensin (AT1) receptors are present on the immune cells that infiltrate the renal interstitium. Here, we examined the actions of AT1 receptors on macrophages in progressive renal fibrosis and found that macrophage-specific AT1 receptor deficiency exacerbates kidney fibrosis induced by unilateral ureteral obstruction (UUO). Macrophages isolated from obstructed kidneys of mice lacking AT1 receptors solely on macrophages had heightened expression of proinflammatory M1 cytokines, including IL-1. Evaluation of isolated AT1 receptor-deficient macrophages confirmed the propensity of these cells to produce exaggerated levels of M1 cytokines, which led to more severe renal epithelial cell damage via IL-1 receptor activation in coculture compared with WT macrophages. A murine kidney crosstransplantation concomitant with UUO model revealed that augmentation of renal fibrosis instigated by AT1 receptor-deficient macrophages is mediated by IL-1 receptor stimulation in the kidney. This study indicates that a key role of AT1 receptors on macrophages is to protect the kidney from fibrosis by limiting activation of IL-1 receptors in the kidney.

Figure 1. AT1A receptor deficiency on macrophages exacerbates kidney fibrosis induced by UUO.

(A) Representative kidney sections from LysM-Cre⁺ mT/mG mice 7 days after UUO with contralateral unobstructed kidney on left and obstructed kidney on right. (B) Polarized images of representative sections from obstructed WT and Macro KO kidneys stained with picrosirius red for collagen fibrils at day 7 after UUO. Original magnification, ×20. (C) Western blot for Col I in whole kidney at day 7 after UUO. (D) mRNA expression of ColI, Pai1, and Tgfb1 in obstructed kidney at day 7 after UUO. (E) Gene expression of M1 markers Il1b, Il1r1, Tnfa, Il12b, and Ccl2 and M2 markers Arg1, Fizz1, Ym1, and Il1r2 in obstructed kidneys at 3 and 7 days after UUO. At day 3, *P < 0.02 vs. WT, ^†P = 0.02 vs. WT, ^‡P = 0.01 vs. WT, ^#P = 0.03, ^§P < 0.02. At day 7, *P < 0.03 vs. WT, ^†P = 0.02 vs. WT, ^#P = 0.05, ^§P < 0.001.

Figure 2. AT1A receptor activation on macrophages suppresses their M1 proinflammatory polarization.

(A) Heat map showing hierarchical cluster analysis of ΔCT values of CD11b⁺Ly6C^hi macrophages isolated from obstructed WT and Macro KO kidneys. (B) mRNA expression of IL-1β measured by real-time RT-PCR in Ly6C^hi and Ly6C^lo macrophages isolated from obstructed kidneys. (C) mRNA levels of proinflammatory cytokines Il1b, Il1a, Ccl5, Ccl2, Il12a, and Il12b in WT and Macro KO peritoneal macrophages following M1 stimulation. (D) ColI and Ngal gene expression in WT (Il1r1 WT) or IL-1R–deficient (Il1r1 KO) renal tubular epithelial cells cocultured with M0- or M1-activated macrophages from WT or Macro KO (MKO) animals.

Figure 3. Activation of the kidney IL-1R mediates exaggerated renal damage induced by AT1A receptor–deficient macrophages.

(A) Right panel shows section of a WT kidney transplanted into a LysM-Cre⁺ mT/mG recipient and subjected to ureteral obstruction (KT-UO). Green fluorescence marks infiltrating LysM⁺ myeloid cells from recipient. Left panel shows section of an unobstructed native contralateral kidney from the same LysM-Cre⁺ mT/mG recipient. (B) Right panel shows section of a LysM-Cre⁺ mT/mG kidney transplanted into a non-Cre mT/mG recipient and subjected to ureteral obstruction. Green fluorescence marks LysM⁺ macrophages resident in KT-UO kidney. Left panel shows section of an unobstructed native contralateral kidney from the same non-Cre mT/mG recipient. Blue fluorescence is a nuclear DAPI stain. Original magnification, ×20. (C–E) Use of KT-UO model to dissect the contribution of WT vs. AT1 receptor–deficient macrophages to kidney damage mediated through renal IL-1R activation. WT (Macro AT1A WT) or Macro KO (KO) recipients were transplanted with an IL-1R WT or KO kidney subjected to UUO (groups I–IV as in Supplemental Table 2; n = 6–7 per group). (C) Representative polarized images of obstructed kidneys stained with picrosirius red at day 7 KT-UO. Original magnification, ×20. Quantitation is shown on the right. Excessive fibrosis due to AT1 receptor deficiency on macrophages in Macro KO recipient is abrogated in IL-1R–deficient donor kidneys. mRNA expression of (D) Col I and (E) NGAL in obstructed kidneys 7 days after KT-UO.