Reversal of Acute Kidney Injury-Induced Neutrophil Dysfunction: A Critical Role for Resistin

Abstract

Objectives: To assess the reversibility of acute kidney injury-induced neutrophil dysfunction and to identify involved mechanisms.

Design: Controlled laboratory experiment and prospective observational clinical study.

Setting: University laboratory and hospital.

Subjects: C57BL/6 wild-type mice.

Patients: Patients with septic shock with or without acute kidney injury.

Interventions: Murine acute kidney injury was induced by intraperitoneal injections of folic acid (nephrotoxic acute kidney injury) or by IM injections of glycerol (rhabdomyolysis-induced acute kidney injury). After 24 hours, we incubated isolated neutrophils for 3 hours in normal mouse serum or minimum essential medium buffer. We further studied the effects of plasma samples from 13 patients with septic shock (with or without severe acute kidney injury) on neutrophilic-differentiated NB4 cells.

Measurements and main results: Experimental acute kidney injury significantly inhibited neutrophil migration and intracellular actin polymerization. Plasma levels of resistin, a proinflammatory cytokine and uremic toxin, were significantly elevated during both forms of acute kidney injury. Incubation in serum or minimum essential medium buffer restored normal neutrophil function. Resistin by itself was able to induce acute kidney injury-like neutrophil dysfunction in vitro. Plasma resistin was significantly higher in patients with septic shock with acute kidney injury compared with patients with septic shock alone. Compared with plasma from patients with septic shock, plasma from patients with septic shock and acute kidney injury inhibited neutrophilic-differentiated NB4 cell migration. Even after 4 days of renal replacement therapy, plasma from patients with septic shock plus acute kidney injury still showed elevated resistin levels and inhibited neutrophilic-differentiated NB4 cell migration. Resistin inhibited neutrophilic-differentiated NB4 cell migration and intracellular actin polymerization at concentrations seen during acute kidney injury, but not at normal physiologic concentrations.

Conclusions: Acute kidney injury-induced neutrophil dysfunction is reversible in vitro. However, standard renal replacement therapy does not correct this defect in patients with septic shock and acute kidney injury. Resistin is greatly elevated during acute kidney injury, even with ongoing renal replacement therapy, and is sufficient to cause acute kidney injury-like neutrophil dysfunction by itself.

Figures

Figure 1. Renal function in murine models of nephrotoxic and rhabdomyolysis-induced AKI

Plasma creatinine levels are elevated as early as 4 hours after injection in mice with rhabdomyolysis-induced AKI (A, n=8) and are even further increased after 24 hours (B, n=7–9). In nephrotoxic AKI (n=8), elevated plasma creatinine levels are only seen after 24 hours (B). Similar changes are seen with plasma concentrations of cystatin C (B + C).

Figure 2. Neutrophil transwell migration during AKI

Rhabdomyolysis-induced AKI causes impaired neutrophil transwell migration as early as 4h after induction (A, n=7–8), lasting at least as long as 24h (B, n=6–9). Nephrotoxic AKI (n=6) significantly inhibits neutrophil transwell migration 24h after induction (B). Incubation in normal serum completely restores normal neutrophil transwell migration in rhabdomyolysis-induced AKI (n=8–10) but only incompletely and nephrotoxic AKI (n=8) (C). Normal neutrophil transwell migration is completely restored in both nephrotoxic AKI (n=6) and rhabdomyolysis-induced AKI (n=8–10) after incubation in MEM buffer (D).

Figure 3. The effect of restoration of renal function on different neutrophil subpopulations in vitro

After stratifying neutrophils from all mice (n=37) into quartiles according to their transwell migration rates (A), the effects of incubating in normal mouse serum (B) or MEM buffer (C) on each quartile of neutrophils were analyzed. Neutrophils with the highest transwell migration rates, i.e. the 4th quartile neutrophils, are not significantly affected by either incubation. The ratios of transwell migration rates after and before incubation are close to 1 (B+C). Contrary, neutrophils with the lowest transwell migration rates, i.e. the 1st quartile neutrophils, show great improvement in transwell migration. The ratios of transwell migration rates after and before incubation are significantly increased (B+C). Although a small difference, incubating poorly migrating neutrophils in MEM buffer provides significantly better transwell migration rates than incubating in normal serum (D).

Figure 4. Intracellular actin polymerization during AKI

Intracellular actin polymerization in neutrophils was stimulated by in vitro incubation with fMLP and subsequently measured using flow cytometry. As seen in (A), intracellular actin, under normal conditions (control), polymerizes quickly within 30 seconds after stimulation (F-actin spike) and then depolymerizes over a longer period of time. Quantitative analysis of F-actin formation (n=7), expressed as AUC0–30″, shows that rhabdomyolysis-induced AKI significantly diminishes actin polymerization (B). Incubating neutrophils in either normal mouse serum (C) or in MEM buffer (E) restores a normal actin polymerization pattern and results in AUC0–30″ that are statistically not different from control neutrophils (D & F).

Figure 5. Resistin in murine models of nephrotoxic and rhabdomyolysis-induced AKI

Plasma resistin levels are elevated as early as 4h after injection in mice with rhabdomyolysis-induced AKI (A) and remain elevated at 24h after induction (B). In nephrotoxic AKI, elevated plasma resistin levels are seen after 24 hours (B). (C) In vitro pretreatment with resistin significantly decreases neutrophil transwell migration rates, similar to that seen with either nephrotoxic or rhabdomyolysis-induced AKI.

Figure 6. Immunofluorescence staining for intracellular F-actin

Intracellular F-actin shows a normal pattern of distribution in neutrophils from control mice (A) or neutrophils undergoing control pretreatment (C). F-actin is primarily localized to the leading edge of the migrating cell, i.e. neutrophils exhibit one short and narrow lamellipodia of F-actin at one pole of the cell (arrow). Contrary to that, F-actin localization is nearly lost in neutrophils from mice with AKI (B) or neutrophils pretreated with resistin (D), i.e. neutrophils are characterized by a lack of distinctly localized F-actin formation (arrow). No formation of a leading edge can be seen in either cell type, suggesting impaired migration of neutrophils during AKI or after pretreatment with resistin.

Figure 7. The effects of AKI in septic shock on plasma resistin and cell transwell migration

(A) As to be expected, patients with AKI during septic shock show significantly elevated plasma creatinine concentrations when compared to those without AKI. RRT significantly reduces plasma creatinine levels by day 4. (B) Plasma resistin levels are also significantly elevated in patients with septic shock and AKI. Contrary to serum creatinine levels, our modes of RRT do not significantly lower plasma resistin levels. (C) NB4PMN transwell migration is significantly reduced in patients with septic shock and AKI. RRT fails to restore normal NB4PMN transwell migration.

Figure 8. The effects of different resistin concentrations of NB4PMN transwell migration and intracellular F-actin formation

(A) Resistin at a physiological concentration has no effect on NB4PMN transwell migration when compared to a resistin-free control. However, resistin at concentrations seen during (severe) AKI significantly inhibits transwell migration (n=8). (B) Resistin at a physiological concentration also has no effect on intracellular F-actin formation in NB4PMN cells. By contrast, resistin at concentrations seen during (severe) AKI significantly reduces intracellular F-actin formation in NB4PMN cells (n=8).