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Objectives This study aimed to determine the diagnostic and prognostic value of urinary biomarkers of intrinsic acute kidney injury (AKI) when patients were triaged in the emergency department. Background Intrinsic AKI is associated with nephron injury and results in poor clinical outcomes. Several urinary biomarkers have been proposed to detect and measure intrinsic AKI. Methods In a multicenter prospective cohort study, 5 urinary biomarkers (urinary neutrophil gelatinase–associated lipocalin, kidney injury molecule-1, urinary liver-type fatty acid binding protein, urinary interleukin-18, and cystatin C) were measured in 1,635 unselected emergency department patients at the time of hospital admission. We determined whether the biomarkers diagnosed intrinsic AKI and predicted adverse outcomes during hospitalization. Results All biomarkers were elevated in intrinsic AKI, but urinary neutrophil gelatinase-associated lipocalin was most useful (81% specificity, 68% sensitivity at a 104-ng/ml cutoff) and predictive of the severity and duration of AKI. Intrinsic AKI was strongly associated with adverse in-hospital outcomes. Urinary neutrophil gelatinase-associated lipocalin and urinary kidney injury molecule 1 predicted a composite outcome of dialysis initiation or death during hospitalization, and both improved the net risk classification compared with conventional assessments. These biomarkers also identified a substantial subpopulation with low serum creatinine at hospital admission, but who were at risk of adverse events. Conclusion Urinary biomarkers of nephron damage enable prospective diagnostic and prognostic stratification in the emergency department.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitutes life-threatening autoimmune diseases affecting every organ, including the kidneys, where they cause necrotizing crescentic glomerulonephritis. ANCA activates neutrophils and activated neutrophils damage the endothelium, leading to vascular inflammation and necrosis. Better understanding of neutrophil-mediated AAV disease mechanisms may reveal novel treatment strategies. Here we report that ANCA induces neutrophil extracellular traps (NETs) via receptor-interacting protein kinase (RIPK) 1/3- and mixed-lineage kinase domain-like (MLKL)-dependent necroptosis. NETs from ANCA-stimulated neutrophils caused endothelial cell (EC) damage in vitro. This effect was prevented by () pharmacologic inhibition of RIPK1 or () enzymatic NET degradation. The alternative complement pathway (AP) was recently implicated in AAV, and C5a inhibition is currently being tested in clinical studies. We observed that NETs provided a scaffold for AP activation that in turn contributed to EC damage. We further established the in vivo relevance of NETs and the requirement of RIPK1/3/MLKL-dependent necroptosis, specifically in the bone marrow-derived compartment, for disease induction using murine AAV models and in human kidney biopsies. In summary, we identified a mechanistic link between ANCA-induced neutrophil activation, necroptosis, NETs, the AP, and endothelial damage. RIPK1 inhibitors are currently being evaluated in clinical trials and exhibit a novel therapeutic strategy in AAV.

In established acute kidney injury (AKI), serum creatinine poorly differentiates prerenal and intrinsic AKI. A damage-associated nephron biomarker, neutrophil gelatinase-associated lipocalin (NGAL) could be a better discriminator. We tested the hypothesis that urinary NGAL distinguishes intrinsic and prerenal AKI, and tested its performance in the prediction of a composite outcome that included progression to a higher RIFLE (“risk, injury, failure, loss of function, end stage renal disease”) severity class, dialysis, or death. We measured urinary NGAL in 161 hospitalized patients with established AKI using a standardized clinical platform. We excluded 16 patients with postrenal obstruction or insufficient clinical information. Of the remaining 145 patients, 75 patients had intrinsic AKI, 32 patients had prerenal AKI, and 38 patients could not be classified. We found that urinary NGAL levels effectively discriminated intrinsic AKI from prerenal AKI (ROC 0.87, CI 0.81-0.94). An NGAL level >104 μg/L indicated intrinsic AKI (likelihood ratio 5.97), while an NGAL level <47 μg/L made intrinsic AKI unlikely (likelihood ratio 0.2). Patients experiencing the composite outcome had higher median urinary NGAL levels on inclusion (248.2 vs. 68.3 μg/L, p<0.001). In logistic regression analysis, NGAL independently predicted the composite outcome, when corrected for demographics, co-morbidities, creatinine, and RIFLE class. Hence, urinary NGAL is useful in classifying and stratifying patients with established AKI.

The pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated necrotizing crescentic GN (NCGN) is incompletely understood. Dipeptidyl peptidase I (DPPI) is a cysteine protease required for the activation of neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase, and proteinase 3, which are enzymes that modulate inflammation. We used a mouse model of anti-myeloperoxidase (MPO) antibodyinduced NCGN to determine whether active NSPs contribute to its pathogenesis. MPO-deficient animals immunized with murine MPO, irradiated, and transplanted with wild-type bone marrow developed NCGN. In contrast, transplantation with bone marrow that lacked DPPI or lacked both neutrophil elastase and proteinase 3 protected mice from NCGN induced by anti-MPO antibody. The kidneys of mice reconstituted with DPPI-deficient bone marrow generated significantly less IL-1b than did those of mice reconstituted with wild-type bone marrow; similarly, in vitro, DPPI-deficient monocytes produced significantly less IL-1b in response to anti-MPO antibody than did wild-type monocytes. This reduction in IL-1b was NSP dependent; exogenous addition of PR3 restored IL-b production in DPPI-deficient monocytes. Last, the IL-1 receptor antagonist anakinra protected animals against anti-MPO antibody-induced NCGN (16.7%66.0% versus 2.4%61.7% crescents), suggesting that IL-1b is a critical inflammatory mediator in this model. These data suggest that the development of anti-MPO antibody-induced NCGN requires NSP-dependent IL-1b generation and that these processes may provide therapeutic targets for ANCAmediated diseases in humans.

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCAassociated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls, and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy, and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting.

Delivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-B (NF-B) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-B in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selec- IntroductionThe introduction of biologically active peptides into human polymorphonuclear neutrophils (PMNs) may help in clarifying intracellular signal transduction and ultimately could have therapeutic implications. However, all peptide delivery methods available thus far are inefficient. Previously, peptide transduction domains (PTDs) were identified that shuttle even large proteins in excess of 100 000 Da into mammalian cells in vitro and in vivo. 1 These "Trojan-horse" peptides include the homeodomain of Antennapedia (a Drosophila transcription factor), a short amino acid sequence of HIV-1, and the herpes simplex virus 1 (HSV-1) structural protein VP22. [2][3][4] We explored the use of an 11-amino acid sequence (amino acids 47-57) from the HIV TAT protein (HIV-TAT) in PMNs to target nuclear factor-B (NF-B).NF-B is a transcription factor controlling gene expression during inflammation, immunity, cell proliferation, stress response, and apoptosis. 5-8 NF-B is activated by many agents including cytokines, viral infection, UV radiation, and free radicals. 9 In unstimulated cells, NF-B is sequestered in the cytoplasm by tightly bound inhibitors (IB␣, IB␤, IB⑀). The inhibitors are phosphorylated and rapidly degraded, allowing NF-B to translocate into the nucleus and activate target genes. IB␣ is phosphorylated on serine residues by the multicomponent IB kinase (IKK) containing 2 catalytic subunits (IKK␣ and IKK␤) and one regulatory subunit (IKK␥). [10][11][12][13] In contrast to other cell types, the role of NF-B in PMNs is incompletely characterized due to rapid NF-B degradation by proteolytic enzymes, difficulties with PMN transfection, and the lack of specific NF-B inhibitors. Previous studies with pharmacologic compounds, such as pyrrolidine dithiocarbamate (PDTC), SN50, and gliotoxin, have suggested that NF-B is involved in regulating PMN apoptosis. [14][15][16] However, the specificity of these agents has been questioned. [17][18][19] We used a highly specific small peptide to block the interaction of IKK␥ with the IB kinase complex (IKK). 20 We generated a linear 2-domain peptide containing the NEMO-binding domain (NBD) to specifically block NF-B and the TAT-PTD to shuttle NBD into PMNs. Materials and methods MaterialsGranulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 8 (IL-8) were obtained from R&D Systems (Wiesbaden-Nordenstedt, Germany). Lipopolysaccharide (LPS), tumor necrosis factor ␣ (TNF-␣), dexamethasone (DEX), Ficoll-Hypaque, and pr...

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1 pos /PR3 high neutrophils, but not in the mNB1 neg /PR3 low subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/ CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1 pos /mPR3 high neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when ␤2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.