Macrophage cell counts.

<p>(A) Differential counts for macrophages were assessed ex vivo using cytospin smears of BAL samples. 150 µL of cell suspension was centrifuged in a Shandon Single Cytofunnel for 3 minutes. The slides were processed and stained using 3 Hema 3 step stain set and cells were scored. Results were expressed as total cell number/mL. (B) Macrophage Inflammatory responses assessed by flow cytometry on mouse lung tissue-recovered cells. For each mouse experimental group, 50,000–100,000 events were collected on an LSRII flow cytometer and analyzed for the presence of resident macrophages (CD11c+Siglec F+) (C) and recruited macrophages (CD11b+Siglec F−). (One-way ANOVA test, significant at *p<0.05, **p<0.01).</p

Neutrophil cell counts.

<p>(A) Differential counts for neutrophils were assessed ex vivo using cytospin smears of BAL samples. Results were expressed as total cell number/mL. (B) Neutrophil Inflammatory responses assessed ex vivo by flow cytometry on mouse lung tissue-recovered cells. For each mouse experimental group, 50,000–100,000 events were collected on an LSRII flow cytometer and analyzed for the presence of neutrophils (Ly6G+CD11b+). (One-way ANOVA test, significant at *p<0.05, **p<0.01 and ***p<0.001).</p

Demographic and clinical characteristics of the study population.

<p>Abbreviations: G = GOLD stage; na = not available; F = Female, M = Male; 1 = Not Hispanic, 2 = Hispanic; FEV1 = forced expiratory volume in 1 second, FVC = Forced Vital Capacity.</p><p>Demographic and clinical characteristics of the study population.</p

<i>Novosphingobium</i> mouse challenge model (A).

<p>C57BL/6 mice were challenged once every seven days with 5×10⁷CFU total of <i>Novosphingobium panipatense</i>. Mice were then exposed to room air conditions or to cigarette smoke from 3R4F reference cigarettes for six weeks. Control mice, challenged with sterile PBS, were exposed to room air or to cigarette smoke for six weeks. At the end of the six weeks of exposure, BAL and lung tissue were harvested, processed and analyzed for markers of inflammation and levels of bacteria. (B) Body weight of C57BL/6 mice after six weeks of weekly challenge with either 5×10⁷CFU total of <i>Novosphingobium panipatense</i> or sterile PBS. Mice were exposed to room air conditions or to cigarette smoke from 3R4F reference cigarettes during the six weeks of challenge. (C) Total BAL cell counts. Bronchoalveolar lavage (BAL) fluid was collected from mouse lungs by aspiration with phosphate buffered saline which was repeated three times. Total viable cell counts were determined in a hemocytometer using trypan blue exclusion. (One-way ANOVA test, **significant at p<0.01).</p

Evaluation of the presence and levels of total bacteria by qPCR on BAL.

<p>BAL samples were harvested from mice, seven days or six weeks post IT challenge with either sterile PBS or with 5×10⁷CFU total of <i>Novosphingobium panipatense</i>. BAL was analyzed by an all-bacteria qPCR. (One-way ANOVA test, significant at *p<0.05).</p

Bacterial detection in mouse lung tissue.

<p>(A) Evaluation of the presence and levels of total bacteria by qPCR on mouse lung tissue. DNA was extracted from mouse lung tissue and analyzed for levels of total bacteria in samples harvested six weeks post weekly IT challenge with either sterile PBS or with 5×10⁷CFU total of <i>Novosphingobium panipatense</i>. (B) Evaluation of the presence and levels of <i>Novosphingobium</i> by a two-step <i>Novosphingobium</i>-specific qPCR on mouse lung tissue. DNA was extracted from mouse lung tissue and analyzed for levels of <i>Novosphingobium</i> in samples harvested six weeks post weekly IT challenge with either sterile PBS or with 5×10⁷CFU total of <i>Novosphingobium panipatense</i>. The <i>Novosphingobium</i>-specific qPCR performed on these tissue samples resulted in undetectable levels of <i>Novosphingobium</i> in all tissue samples.</p

Reconstitution of <i>Ntn1^+/−</i> mice with exogenous netrin-1.

<p>Renal function and inflammation in mice with partial deficiency for netrin-1 (<i>Ntn1^+/−</i>) treated with exogenous netrin (5 µg/mouse I.V.) or vehicle prior to 30 minutes of renal ischemia. (A) Glomerular filtration rate (as measured by FITC-inulin clearance) was measured after 1 hour of reperfusion. (B) Quantification of neutrophil tissue accumulation by measurement of myeloperoxidase (MPO) (mean ± SD; n = 6–8).</p

<i>In vitro</i> expression of netrin-1 in HK-2 cells.

<p>(A) Expression of netrin-1 in human renal epithelial cells (HK-2 cells) following exposure to hypoxia (1% O₂) for indicated time periods. One representative blot of three is displayed. (B) Quantification or netrin-1 protein in HK-2 cells relative to β-actin.</p

Histological tissue insure induced by AKI in mice with partial netrin-1 deficiency (<i>Ntn1^+/−</i>) or control mice (<i>Ntn1^+/+</i>).

<p>Renal histology in <i>Ntn1^+/−</i> mice exposed to renal ischemia or age-, weight-, and gender-matched littermate controls (<i>Ntn1^+/+</i>) were subjected to 30 minutes of left renal artery ischemia. Renal histology was obtained after 24 hours of reperfusion. (A–D) Representative H&E staining (400×). Arrow marks destructed tubules. (E) Quantification of histological tissue damage assessed by Jablonski index.</p

Renal inflammatory changes in <i>Ntn1^+/−</i> mice following ischemia.

<p><i>Ntn1^+/−</i> mice and their respective age-, weight-, and gender-matched littermate controls (<i>Ntn1^+/+</i>) were subjected to 30 minutes of left renal artery ischemia. (A–D) Neutrophil staining. Arrows indicate neutrophils (magnification 400×). (E) Quantification of neutrophil tissue accumulation by measurement of myeloperoxidase (MPO). (F) TNF-α and (G) interleukin-6 (IL-6) and (H) interleukin-10 (IL-10) were assessed by real-time RT-PCR from renal tissues. Data were calculated relative to ß-actin and are expressed as fold change compared to sham-operated animals without ischemia (−I). Data are representative of four to six independent experiments for each experimental condition (mean ± SD).</p