(A) Compared to sham inhalation, P. aeruginosa inhalation leads to impaired oxygenation, as demonstrated by reduced S_pO₂. (B) P. aeruginosa CFUs can be detected in lung homogenates after inhalation of bacteria but not of NaCl 0.9%. (C) P. aeruginosa inhalation gives rise to pulmonary recruitment of neutrophils, as measured by an increase in lung myeloperoxidase activity. (D) Antibody (α-GR-1)-mediated neutrophil-depletion prior to P. aeruginosa inhalation further impairs oxygenation when compared to control injections. (E) 24h after inhalation, neutrophil-depleted (α-GR-1-treated) mice reveal larger numbers of P. aeruginosa CFUs in lung homogenates than mice treated with a control antibody. (F) I.p. injection of FA induces severe AKI, as evidenced by more than 3-fold higher plasma creatinine concentrations than control mice. * and # indicate p<0.05 vs. sham/control experiments.

(A) Unremarkable lung histology in mice after control injection and sham inhalation (group 1). (B) Normal kidney histology in mice after control injection and sham inhalation (group 1). (C) I.p. injection of FA with sham inhalation does not induce histological damage in the lung (group 2). (D) Severe histological damage occurrs in kidneys after i.p. injection of FA (and sham inhalation, group 2), in particular flattening of tubule epithelia (acute tubular necrosis). (E) Inhalation of P. aeruginosa (group 3: control+pneumonia) leads to severe histological damage, including diffuse alveolar damage, neutrophil influx, and interstitial edema. (F) Inhalation of P. aeruginosa (group 3) only causes mild histological changes in the kidneys, characterized by hyperemia and tubular edema. (G) Combined i.p. injection of FA and inhalation of P. aeruginosa (group 4) gives rise to alveolar damage consistent with bacterial pneumonia but milder than that observed in ‘simple pneumonia’ (see E for comparison). Here, cellular infiltrates and interstitial edema seem markedly decreased. (H) Histological damage in kidneys after combined i.p. injection of FA and inhalation of P. aeruginosa (group 4) is characterized by both tubular damage typical for FA-toxicity and pneumonia-related hyperemia.

(A) Inhalation of P. aeruginosa but not sham inhalation gives rises to a significant elevation in plasma creatinine. (B) Compared to sham inhalation, inhalation of P. aeruginosa also increases plasma cystatin C concentrations in mice. (C) Significant increases in urinary NGAL concentrations after inhalation of P. aeruginosa indicate significant tubular injury in these mice compared to mice with sham inhalation. (D) Similar pulse distention in mice with sham inhalation and mice with prior P. aeruginosa inhalation suggests comparable systemic hemodynamics between these groups. (E) Inhalation of P. aeruginosa does not induce neutrophil recruitment into the kidneys, as evidenced by unchanged renal MPO levels between mice with sham inhalation and mice with prior P. aeruginosa inhalation.

Compared to control treatment, platelet-depletion prior to inhalation of P. aeruginosa worsens (A) oxygenation and (B) bacterial load in lung homogenates. (C) Despite worsening pneumonia, platelet-depleted mice display better kidney function than mice treated with control serum, as shown by significantly lower plasma creatinine concentrations.

(A) At t=0h, mice receive i.p injections of FA or NaHCO₃. 24h later, mice are placed in an air-tight chamber to inhale aerosolized P. aeruginosa or NaCl 0.9%. After 48h, vital signs are obtained, and mice are then anesthetized to collect blood samples and harvest organs. This design results in 4 different experimental groups (Fig. 1): Mice with control injection and sham inhalation (group 1), mice with AKI and sham inhalation (group 2), mice with control injection and pneumonia (group 3), and mice with AKI and pneumonia (group 4). (B) Both i.p. Injection of folic acid (FA) and i.m. injection of glycerol induced significant acute kidney injury, RIFLE-R (FA) and RIFLE-I/F (glycerol), respectively. Relative changes in plasma creatinine and cystatin C (C) were very similar. (D) Inhalation of P. aeruginosa leads to a significant increase in plasma IL-6 levels. (E) FA-induced AKI itself only leads to a small, non-significant rise in plasma IL-6 levels. Pre-existing AKI does not affect plasma IL-6 levels during pneumonia. * indicates p<0.05 vs. sham/control experiments.

(A) Compared to mice without AKI (group 1: AKI+sham), FA-induced AKI (group 2: AKI+sham) itself does not affect oxygenation in mice with sham inhalation. However, pre-existing AKI worsens oxygenation in mice with P. aeruginosa inhalation (group 3: control+pneumonia vs. group 4: AKI+pneumonia). (C) Mice with pre-existing AKI display a significantly greater number of P. aeruginosa CFUs in lung homogenates after inhalation (group 4: AKI+pneumonia) than mice without AKI (group 3: pneumonia+control). (E) Pre-existing AKI itself (group 2: AKI+sham vs. group 1: control+sham) does not cause significant increase in lung lung MPO, indicating no substantial influx of neutrophils into lungs. Pre-existing AKI, however, significantly decreases lung MPO in the setting of bacterial pneumonia (group 3: control+pneumonia vs. group 4: AKI+pneumonia), indicating attenuated recruitment of neutrophils into the lung. Compared to FA-induced AKI, glycerol-induced myohemoglobinuric AKI had similar effects on oxygenation (B), bacterial load in the lung (D), and pulmonary neutrophil recruitment (F). * and # indicate p<0.05 vs. sham and control experiments, respectively.

(A) Neutrophils from mice with FA-induced AKI show significantly reduced transmigration towards fMLP. (B) F-actin structures in Z series of fluorescence images: Neutrophils from vehicle-treated mice exhibited one short and narrow lamellipodia of F-actin at one pole of the cell. In contrast, FA-treated neutrophils were characterized by a prominent and large protrusion of F-actin. (C) The significant increased in surface area of F-actin in neutrophils from mice with FA-induced AKI suggest that these neutrophils cannot limit F-actin formation. The black-and-white pictures represent phase-contrast images of stimulated cells. The images are one x-y view of the z-series analyzed by deconvolution in Volocity. Histograms reflect surface area of lamellipodia (n=3 mice, 40 cells per mouse). Arrows indicate F-actin protrusions. Scale bar represents 5 μm.* indicate p<0.05 vs. sham/control experiments

(A) PAS-staining reveals brush-border defects in renal tubular epithelial cells after inhalation of P. aeruginosa as well as after (B) FA injections. (C) TUNEL staining only reveals very few and weak apoptotic changes in the kidney after inhalation of P. aeruginosa. (D) FA injections lead to massive apoptosis in renal tubular epithelial cells, as demonstrated by positive TUNEL staining.