A specific immunoassay for detection of feline kidney injury molecule 1.

OBJECTIVES: The aim of this study was to design and carry out a preliminary evaluation of a urine point-of-care test for kidney injury molecule 1 (KIM-1) in healthy and diseased cats. METHODS: Part of the feline KIM-1 gene was amplified, ligated into a plasmid with a signal peptide and monomeric human IgGFc, and transfected into a mammalian cell line. Supernatant was purified and tested for the fusion protein by gel electrophoresis and Western blot. Mice were immunized three times with purified proteins, and hybridomas were generated from splenocytes. Antibodies were tested by ELISA for detection of recombinant KIM-1 and naturally occurring KIM-1 in disease-state urine. Next, a lateral flow assay (LFA) with capture and detection antibodies was constructed, and tested with 34 urine samples from healthy and diseased cats. Antibodies were also tested for reactivity with formalin-fixed paraffin-embedded kidney tissue. RESULTS: Three antibodies were assessed. Antibodies detected between 0.4 and 60 ng/ml feline KIM-1 fusion protein in the LFA. Urine samples from healthy cats yielded faint bands in the LFA corresponding to optical density (OD) values of 4.8-8.8. Samples from cats with suspected or confirmed acute kidney injury (AKI) had OD values ranging from 1.6-20.5. Urine KIM-1 varied over multiple days in cats with sepsis or urethral obstruction despite normalizing serum creatinine concentration. In tissue sections, KIM-1 antibodies labeled tubular cells with morphological features of injury. CONCLUSIONS AND RELEVANCE: A practical patient-side assay for detection of KIM-1 in feline urine has been developed. Preliminary results show marked though transient increases in cats with sepsis and urethral obstruction-associated AKI, and expression in injured tubules. Although initial data indicating that the LFA is sensitive and specific for KIM-1 in cats with AKI are promising, values associated with different types of injury, urine collection, urine storage and specific gravity need to be investigated.

Benzo(a)pyrene suppresses tracheal antimicrobial peptide gene expression in bovine tracheal epithelial cells.

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). ß-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of ß-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 µM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 µM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP-a toxic pollutant found in crude oil-on this respiratory innate immune response.

Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle.

ß-defensins are an important element of the mucosal innate immune response against bacterial pathogens. Tracheal antimicrobial peptide (TAP) has microbicidal activity against the bacteria that cause bovine respiratory disease, and its expression in tracheal epithelial cells is upregulated by bacterial products including lipopolysaccharide (LPS, a TLR4 agonist), Pam3CSK4 (an agonist of Toll-like receptor 2/1), and interleukin (IL)-17A. The objectives of this study were to identify the signalling pathway by which LPS, Pam3CSK4 and IL-17A induce TAP gene expression, and to determine the effect of glucocorticoid as a model of stress on this epithelial innate immune response. In primary cultures of bovine tracheal epithelial cells (bTEC), LPS, Pam3CSK4 and IL-17A each stimulated TAP gene expression. This effect was abrogated by caffeic acid phenylester (CAPE), an inhibitor of NF-κB. Similarly, western analysis showed that LPS, Pam3CSK4 and IL-17A each induced translocation of NF-κB p65 from the cytoplasm to the nucleus, but pre-treatment with CAPE inhibited this response. Finally, pre-treatment of bTEC with the glucocorticoid dexamethasone abolished the stimulatory effect of LPS, Pam3CSK4 and IL-17A on upregulation of TAP gene expression. These findings indicate that NF-κB activation is necessary for induction of TAP gene expression by LPS (a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist), or IL-17A. Furthermore, this stimulatory response is inhibited by glucocorticoid, suggesting this as one mechanism by which stress increases the risk of bacterial pneumonia. These findings have implications for understanding the pathogenesis of stress-associated bacterial pneumonia, and for developing methods to stimulate innate immune responses in the respiratory tract of cattle.

Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle.

Bovine respiratory disease is a complex of bacterial and viral infections of economic and welfare importance to the beef industry. Although tracheal antimicrobial peptide (TAP) has microbicidal activity against bacterial pathogens causing bovine respiratory disease, risk factors for bovine respiratory disease including BVDV and stress (glucocorticoids) have been shown to inhibit the induced expression of this gene. Lipopolysaccharide is known to stimulate TAP gene expression, but the maximum effect is only observed after 16 h of stimulation. The present study investigated other agonists of TAP gene expression in primary cultures of bovine tracheal epithelial cells. PCR analysis of unstimulated tracheal epithelial cells, tracheal tissue and lung tissue each showed mRNA expression for Toll-like receptors (TLRs) 1-10. Quantitative RT-PCR analysis showed that Pam3CSK4 (an agonist of TLR1/2) and interleukin (IL)-17A significantly induced TAP gene expression in tracheal epithelial cells after only 4-8 h of stimulation. Flagellin (a TLR5 agonist), lipopolysaccharide and interferon-α also had stimulatory effects, but little or no response was found with class B CpG ODN 2007 (TLR9 agonist) or lipoteichoic acid (TLR2 agonist). The use of combined agonists had little or no enhancing effect above that of single agonists. Thus, Pam3CSK4, IL-17A and lipopolysaccharide rapidly and significantly induce TAP gene expression, suggesting that these stimulatory pathways may be of value for enhancing innate immunity in feedlot cattle at times of susceptibility to disease.

Effect of corticosteroids and neuropeptides on the expression of defensins in bovine tracheal epithelial cells.

Susceptibility to bacterial pneumonia in cattle is enhanced by stressors such as transportation, weaning, and commingling, which trigger a physiologic stress response resulting in elevated levels of endogenous corticosteroids and catecholamines. To determine the effect of neuroendocrine mediators on the expression of innate defense peptides in the lung, bovine tracheal epithelial cells were exposed to dexamethasone, catecholamines, acetylcholine, or substance P, and then beta-defensin expression was quantified using real-time reverse transcription-PCR. Basal expression of tracheal antimicrobial peptide (TAP) mRNA was not affected by any of the mediators tested. However, induction of TAP expression by lipopolysaccharide was significantly inhibited by pretreatment with dexamethasone. Bronchial biopsy specimens from dexamethasone-treated calves had significantly lower expression of TAP and lingual antimicrobial peptide (LAP) mRNA than saline-treated controls following 48 h of treatment. Lipopolysaccharide-elicited neutrophil recruitment was enhanced in the lungs of dexamethasone-treated calves compared to saline-treated controls. These findings indicate that modulation of epithelial antimicrobial peptide expression is one mechanism through which corticosteroids and stress may impair innate pulmonary defenses.