Neutrophil circadian rhythm is associated with different outcomes of acute kidney injury due to cholesterol crystal embolism

Abstract

Cholesterol crystal (CC) embolism can cause acute tissue infarction and ischemic necrosis via triggering diffuse thrombotic angiopathy occluding arterioles and arteries. Neutrophils contribute to crystal-induced immunothrombosis as well as to ischemic necrosis-related necroinflammation. We speculated that CC embolism-induced acute kidney injury (AKI) would be circadian rhythm-dependent and associated with cyclic differences in neutrophil function. Injection of CC into the left kidney induced thrombotic angiopathy progressing starting as early as 3 h after CC injection followed by a progressive ischemic cortical necrosis and AKI at 24 h. In C57BL/6J mice, circulating CD11b⁺Ly6G⁺ neutrophils were higher during the day phase [Zeitgeber time (ZT) 0-12] compared to the dark phase (ZT12-24). In the time frame of thrombus formation at ZT13, more neutrophils were recruited into the injured kidney 24 h later compared to CC embolism at ZT5. This effect was associated with an increased circulating number of CXCR2⁺ neutrophils as well as an upregulated kidney adhesion molecule and chemokine expression. These findings were associated with a significant increase in kidney necrosis, and endothelial injury at ZT13. Thus, the time of day has an effect also on CC embolism-related AKI in association with the circadian rhythm of neutrophil recruitment.

Keywords: cholesterol crystal embolism (CCE); circadian rhythm; ischemic AKI; neutrophil; thrombosis.

Figure 1

Time kinetics of crystal clots formation and growth. (A) Schematic of experiment. (B) Representative images of aSMA/Fibrin staining at 3, 6, 12, and 24 h post CC injection and PBS control. (C) Partially and completely obstructed arteries in the kidney 24 h post-CC injection (n = 8–12 mice/group). (D) Representative images of PAS staining at 3, 6, 12, and 24 h post-CC injection, and kidney injury score (n = 8–12 mice/group). (E) Representative images of Ly6B2+ staining at 3, 6, 12, and 24 h post-CC injection, and neutrophils percentage in the kidney (n = 8–14 mice/group). Data are means ± SD. One-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. ****P < 0.001.

Figure 2

The circadian oscillations of neutrophils, and chemokine in mouse blood in a healthy state. (A) Flow cytometric quantification of neutrophils baseline in the blood. (B,C) MFI of surface CXCR4 (B, n = 5 mice/group) and CXCR2 (C, n = 5 mice/group) expression by neutrophils in the blood as determined by flow cytometry. (D,E) Plasma levels of CXCL12 (D, n = 5 mice/group) and CXCL1 (E, n = 5 mice/group) in healthy mice as determined by ELISA. Data are means ± SD. MFI: mean fluorescence intensity; CXCR2, CXC chemokine receptor 2; CXCR4, CXC chemokine receptor 4; CXCL12, CXC motif chemokine ligand 12.

Figure 3

The circadian oscillation of mouse GFR, kidney mRNA expression levels of adhesion molecules, and chemokines at ZT5 and ZT13 in a healthy state. (A) Circadian oscillations of mouse GFR in the healthy state (n = 8–10 mice/group). (B,C) mRNA expression of adhesion molecule Icam-1 (B, n = 7–8 mice/group) and Vcam-1 (C, n = 7–8 mice/group) in mice kidney at ZT5 and ZT13. (D) mRNA expression of neutrophil mediating chemotaxis Cxcl1 (D, n = 7–8 mice/group) and its receptor Cxcr2 (E, n = 6–7 mice/group) at ZT5 and ZT13. Icam-1, intercellular adhesion molecule 1; Vcam-1, vascular cell adhesion molecule 1; Cxcr2, CXC chemokine receptor 2; Cxcl1, CXC motif chemokine ligand 1. While background: daytime, gray background: nighttime. Data are means ± SD, unpaired Student's t-test, *P < 0.05.

Figure 4

CC embolism during the active phase leads to more kidney injury. (A) Schematic of experiment. (B) Representative images of Ly6B2+ staining, and neutrophil counts in the kidney at ZT5 and ZT13 (n = 10 mice/group). (C) Representative images of PAS staining, and mouse kidney injury score at ZT5 and ZT13 (n = 10 mice/group). (D) Representative images of CD31 staining, and endothelial injury in the kidney (n = 10 mice/group). (E) GFR reduction upon ZT5 and ZT13 24 h post-CC injection (n = 20 mice/group). While background: daytime, gray background: nighttime. Data are means ± SD, unpaired Student's t-test, *P < 0.05.

Figure 5

Enhanced kidney neutrophil recruitment is CXCR2 dependent. The intrarenal mRNA expression level of Cxcr2 (A, n = 6–8 mice/group), Cxcl1 (B, n = 7–8 mice/group), Vcam-1 (C, n = 7–8 mice/group), and Icam-1 (D, n = 7–8 mice/group) at ZT5 and ZT13 before and post-CC injection. Data are means ± SD. Icam-1, intercellular adhesion molecule 1; Vcam-1, vascular cell adhesion molecule 1; Cxcr2, CXC chemokine receptor 2; Cxcl1, CXC motif chemokine ligand 1. Unpaired Student's t-test, *P < 0.05, **P < 0.01.

Figure 6

Schematic illustration of the putative mechanism of circadian rhythm-related kidney injury. During the day, the expression of adhesion molecule (Icam-1, Vcam-1) on endothelial is lower than the night, the release of chemokine (CXCL1) from immune cells is less than the night, the expression of CXCR2 is lower, while the CXCR4 is high. Consequently, this leads to more tissue necrosis during the night.