Compartmentalization of neutrophils in the kidney and lung following acute ischemic kidney injury

Abstract

During renal ischemia-reperfusion, local and distant tissue injury is caused by an influx of neutrophils into the affected tissues. Here we measured the kinetics of margination and transmigration of neutrophils in vivo in the kidney and lungs following renal ischemia-reperfusion. After bilateral renal injury, kidney neutrophil content increased threefold at 24 h. The neutrophils were found primarily in the interstitium and to a lesser degree marginated to the vascular endothelium. These interstitial neutrophils had significantly lower levels of intracellular IFN-gamma, IL-4, IL-6, and IL-10 a tendency for decreased amounts of IL-4 and TNF-alpha compared to the marginated neutrophils. Localization of the neutrophils to the kidney interstitium was confirmed by high resolution microscopy and these sites of transmigration were directly associated with areas of increased vascular permeability. Activation of the adenosine 2A receptor significantly decreased both kidney neutrophil transmigration by about half and vascular permeability by about a third. After unilateral renal ischemia-reperfusion, the unclipped kidney and lungs did not accumulate interstitial neutrophils or have increased vascular permeability despite a marked increase of neutrophil margination in the lungs. Our findings suggest there is a sequential recruitment and transmigration of neutrophils from the vasculature into the kidney interstitium at the site of tissue injury following renal ischemia-reperfusion.

Figure 1. Total kidney neutrophil content

Mouse kidneys were subjected to sham or 32 min ischemia and reperfusion for 2, 24, 48, or 72 h. Kidney suspensions were prepared for flow cytometry. Neutrophils were identified by (1) their typical appearance in the forward-side scatter, (2) their CD45⁺ expression, and (3) two independent neutrophil markers, GR-1 APC and 7/4 FITC. N = 3–8 for all groups. *P<0.05, **P<0.01, ***P<0.005 compared with sham.

Figure 2. Schematic diagram for analysis of kidney and lung neutrophils using flow cytometry

Twenty-four hours following renal ischemia-reperfusion injury (IRI) (1), mice were injected with APC-conjugated GR-1 anti-mouse Ly-6G antibody or isotype control through tail vein (2). Five minutes later, mice were anesthetized and perfused (3), and the kidneys and lungs were excised and processed for FACS analysis (4). CD45 mAb was used to identify total leukocyte population and 7-AAD was used to distinguish live from dead cells. Neutrophil populations were sorted according to either 7/4⁺ GR-1⁺ (marginated neutrophils) or 7/4⁺ GR-1⁻ (neutrophils from the interstitium).

Figure 3. Compartmentalization of neutrophils following kidney ischemia-reperfusion injury

We harvested kidneys and lungs 5 min after injection of anti-GR-1 antibody or isotype control in sham or 24 h renal ischemia-reperfusion injury (IRI) mice. Marginated neutrophil content was identified as 7/4⁺GR-1⁺ and interstitial neutrophil content was identified as 7/4⁺GR-1⁻. Graphs show representative dot plots after 24 h of isotype control, sham, or IRI.

Figure 4. Histological and immunofluorescence localization of neutrophils to interstitial and marginated compartments in the kidney and lung

Histological staining of kidney outer medulla of mice subjected to 24 h renal ischemia-reperfusion injury (IRI) showing peritubular capillary neutrophils (a), interstitial neutrophils (b and c: arrows), and intraluminal neutrophils (b: arrowhead). Immunofluorescence staining of kidney outer medulla (d and e) and lung (f) using antibodies to 7/4 (green; for neutrophils) and CD31 (red; for vascular endothelium). Nuclei are depicted by DAPI labeling (blue). Neutrophils in both sides of the vascular endothelial wall, and circle shows a neutrophil transmigrating through the vascular wall of the kidney (d: arrowhead). Z-stack image (7.0 mm) of 12 optical slices of the kidney at 0.6 mm intervals (e). Kidney interstitial neutrophils (arrows) are shown in inset (e). Lung tissue shows all neutrophils in the vasculature (f).

Figure 5. Differential analysis of intracellular cytokine content between marginated and interstitial neutrophils

Twenty-four hours following renal ischemia-reperfusion injury (IRI), mice were injected with APC-conjugated GR-1 anti-mouse Ly-6G antibody through tail vein. Five minutes later, mice were anesthetized and perfused, and kidneys were excised and processed for FACS analysis for intracellular cytokine content. Values are mean ± s.e.m.; n = 5 all groups. *P<0.05, **P<0.005 compared with interstitial neutrophils. Open bars, interstitial neutrophils; filled bars, marginated neutrophils.

Figure 6. Time course of in vivo neutrophil trafficking following renal IRI

Representative dot plot graphs for both the kidney and lung (a) and quantitative analysis of neutrophil transmigration following renal ischemia-reperfusion injury (IRI) in the kidney (b) and lung (c). Values are mean ± s.e.m.; n = 6–10 all groups. *P<0.05, **P<0.005 compared with sham. Filled squares, interstitial neutrophils; filled diamonds, marginated neutrophils.

Figure 7. Effect of ATL313 on kidney and lung interstitial neutrophil content after renal IRI

Mouse kidneys were subjected to 32 min ischemia and 24 h reperfusion and treated with vehicle or ATL313 (1 ng/kg per min) through osmotic pump. The kidney (a) and lung (b) neutrophil trafficking was determined using flow cytometry. Values are mean ± s.e.m.; n = 6–9 all groups. *P<0.005 compared with sham and ^#P<0.05 compared with 24 h ischemia-reperfusion injury (IRI) groups. Open bars, sham; filled bars, 24 h renal IRI; light gray bars, 24 h renal IRI + ATL313.

Figure 8. Compartmentation of neutrophils following bilateral vs unilateral renal ischemia-reperfusion injury (IRI)

Representative dot plot graphs of sham, unilateral (only left kidney was clipped), or bilateral (both kidneys were clipped) kidney and lung after renal IRI (n = 4 each groups). Neutrophils were labeled 5 min after injection of anti-GR-1 antibody. Marginated neutrophil content was identified as 7/4⁺ GR-1⁺ and interstitial neutrophil content was identified as 7/4⁺ GR-1⁻

Figure 9. Functional analysis following renal IRI

(a) Mouse kidneys were subjected to 32 min ischemia and 24 h reperfusion. Two percent Evans Blue Dye (EBD) was administered through tail vein 1 h before harvesting the kidneys. EBD was extracted in formamide and the amount of extravasated EBD in each kidney and the lung was calculated against a standard curve. Values are mean ± s.e.m.; n = 4 all groups. *P<0.005 compared with sham, ^#P<0.005 compared with bilateral renal ischemia-reperfusion injury (IRI). (b) Blood was obtained following 24 h of renal IRI and plasma creatinine was assayed. Values are mean ± s.e.m.; n = 4 all groups. **P<0.005 compared with sham; ^#P<0.005 compared with bilateral renal IRI. Open bars, sham; filled bars, 24 h bilateral renal IRI; light gray bars, 24 h bilateral renal IRI + ATL313; dark gray bars, 24 h unilateral renal IRI.