C-C Motif Chemokine 5 Attenuates Angiotensin II-Dependent Kidney Injury by Limiting Renal Macrophage Infiltration

Abstract

Inappropriate activation of the renin angiotensin system (RAS) is a key contributor to the pathogenesis of essential hypertension. During RAS activation, infiltration of immune cells into the kidney exacerbates hypertension and renal injury. However, the mechanisms underpinning the accumulation of mononuclear cells in the kidney after RAS stimulation remain unclear. C-C motif chemokine 5 (CCL5) drives recruitment of macrophages and T lymphocytes into injured tissues, and we have found that RAS activation induces CCL5 expression in the kidney during the pathogenesis of hypertension and renal fibrosis. We therefore evaluated the contribution of CCL5 to renal damage and fibrosis in hypertensive and normotensive models of RAS stimulation. Surprisingly, during angiotensin II-induced hypertension, CCL5-deficient (knockout, KO) mice exhibited markedly augmented kidney damage, macrophage infiltration, and expression of proinflammatory macrophage cytokines compared with wild-type controls. When subjected to the normotensive unilateral ureteral obstruction model of endogenous RAS activation, CCL5 KO mice similarly developed more severe renal fibrosis and greater accumulation of macrophages in the kidney, congruent with enhanced renal expression of the macrophage chemokine CCL2. In turn, pharmacologic inhibition of CCL2 abrogated the differences between CCL5 KO and wild-type mice in kidney fibrosis and macrophage infiltration after unilateral ureteral obstruction. These data indicate that CCL5 paradoxically limits macrophage accumulation in the injured kidney during RAS activation by constraining the proinflammatory actions of CCL2.

Figure 1

CCL5-deficient mice have a preserved chronic hypertensive response to Ang II. A: MAPs of WT and CCL5 KO mice were measured by radiotelemetry before and during Ang II-dependent hypertension B and C: Heart weight-to-body weight (B) and albumin-to-creatinine ratios (C) were determined after 4 weeks of chronic saline or Ang II infusion in WT and CCL5 KO mice. n = 22 mice per group. *P < 0.05 versus WT. Ang, angiotensin; CCL5, C-C motif chemokine 5; KO, knockout; MAP, mean arterial pressure; WT, wild-type.

Figure 2

CCL5 mitigates Ang II-mediated glomerular injury. After 28 days of chronic saline or Ang II infusion, kidney sections of WT and CCL5 KO mice were blindly assessed (A.F.B.) for glomerular injury. A and B: Representative kidney sections from Ang II-infused WT (A) and CCL5 KO (B) mice. Injured glomeruli were scored based on the combined levels of glomerulosclerosis and epithelial cell reactivity (C). CCL5 KO mice had augmented glomerular injury compared with WT controls. ^∗P < 0.05 versus WT. Scale bar = 100 μm (A and B). Ang, angiotensin; AU, arbitrary units; CCL5, C-C motif chemokine 5; KO, knockout; WT, wild-type.

Figure 3

CCL5 protects from renal interstitial damage by RAS activation. After 4 weeks of saline control or Ang II-induced hypertension, sections of WT and CCL5 KO kidneys were blindly analyzed (A.F.B.) using a semiquantitative scoring method of interstitial fibrosis ranging from no injury (0) to severe fibrosis (4). A and B: Representative kidney sections from Ang II-infused WT (A) and CCL5 KO (B) mice. C: Fibrosis scores. Ang II-infused CCL5 KO mice were significantly more susceptible to kidney fibrosis. ^∗P < 0.05 versus WT. Scale bar = 100 μm (A and B). Ang, angiotensin; AU, arbitrary units; CCL5, C-C motif chemokine 5; KO, knockout; RAS, renin angiotensin system; WT, wild-type.

Figure 4

mRNA expression of markers and mediators of hypertensive kidney injury. Whole kidney mRNA expression of Lcn2 (A), Col1a1 (B), Serpine1 (Pai1), and Tgfb1 (C) was determined in WT and CCL5 KO mice after 28 days of chronic Ang II infusion. Expression of these injury markers was elevated in CCL5 KO mice compared with WT controls. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 versus WT. Ang, angiotensin; CCL5, C-C motif chemokine 5; KO, knockout; WT, wild-type.

Figure 5

CCL5 deficiency permits augmented macrophage infiltration and inflammation in the hypertensive kidney. A and B: WT (A) and CCL5 KO (B) kidney sections were stained for the macrophage marker F4/80 after 28 days of Ang II. C: The density of macrophage staining was digitally scored into quintiles and subjected to χ² analysis (P < 0.0001). D and E: mRNA levels of the proinflammatory mediators Tnf, Il1b, and Il6 (D) and the alternative CCL5 ligands Ccl3 and Ccl4 and the CCL5 receptors Ccr1 and Ccr5 (E) were measured in WT and CCL5 KO kidneys. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus WT. Scale bar = 100 μm (A and B). Ang, angiotensin; CCL5, C-C motif chemokine; CCR, C-C chemokine receptor; KO, knockout; Tnf, tumor necrosis factor; WT, wild-type.

Figure 6

CCL5 deficiency exaggerates fibrosis and infiltration of macrophages in the obstructed kidney. A and B: Seven days after UUO, sections of WT (A) and CCL5 KO (B) kidneys were stained with picrosirius red to visualize collagen fibrils. C: The percentage of picrosirius red stain per section was digitally quantified in obstructed and contralateral, unobstructed kidneys. D–F: Sections of WT (D) and CCL5 KO (E) obstructed kidneys were stained with anti-F4/80, and the percentage of positive stain per section was computed (F). G: mRNA expression of Ccl2, a strong chemoattractant for macrophages, was up-regulated in obstructed CCL5 KO compared with WT kidneys. Scale bars = 100 μm (A, B, D, and E). ^∗P < 0.05 versus WT. CCL5, C-C motif chemokine 5; KO, knockout; UUO, unilateral ureteral obstruction; WT, wild-type.

Figure 7

CCL5 limits the infiltration of inflammatory Ly6C^hi myeloid cells into the obstructed kidney. Seven days after UUO, renal myeloid cells were evaluated via flow cytometry for their expression of Ly6C. A: The gating strategy for analyzing viable myeloid cells. B and C: Within the myeloid cell population, Ly6C^hi expression was significantly enhanced in the CCL5 KO obstructed kidney compared with WT. ^∗∗P < 0.01 versus WT. CCL5, C-C motif chemokine 5; FSC, forward scatter; KO, knockout; Ly6C, lymphocyte antigen 6 complex; SSC, side scatter; UUO, unilateral ureteral obstruction; WT, wild-type.

Figure 8

CCL2 antagonism reduces UUO-induced fibrosis and macrophage infiltration in the CCL5 KO kidneys to WT levels. Mice were treated with the CCL2 inhibitor propagermanium after UUO. A–C: CCL2 inhibition yields similar levels of picrosirius red staining for collagen fibrils in obstructed WT (A) and CCL5 KO (B) kidneys, quantified by morphometric analysis (C). D–F: CCL2 antagonist reduces and renders macrophage infiltration comparable in obstructed WT (D) and CCL5 KO (E) kidneys, quantified in panel F. Scale bars = 100 μm (A, B, D, and E). CCL2, C-C motif chemokine 2; CCL5, C-C motif chemokine 5; KO, knockout; UUO, unilateral ureteral obstruction; WT, wild-type.