ABSTRACT
Drug-induced kidney injury (DIKI) is a cause of drug development failure. Dogs represent a common non-rodent animal model in pre-clinical safety studies; however, biomarker assays for detecting nephrotoxicity in dogs are limited. To identify novel proteins and gain insight into the molecular mechanisms involved in DIKI, we developed an assay to evaluate proteomic changes associated with DIKI in male beagle dogs that received nephrotoxic doses of tobramycin for 10 consecutive days. Label-free quantitative discovery proteomics analysis on representative kidney cortex tissues collected on Day 11 showed that the tobramycin-induced kidney injury led to a significant differential regulation of 94 proteins mostly associated with mechanisms of nephrotoxicity such as oxidative stress and proteasome degradation. For verification of the proteomic results, we developed a multiplex peptide-centric immunoaffinity liquid chromatography tandem mass spectrometry assay (IA LC-MS/MS) to evaluate the association of eight DIKI protein biomarker candidates using kidney cortices collected on Day 11 and urine samples collected on Days -4, 1, 3, 7 and 10. The results showed that most biomarkers evaluated were detected in the kidney cortices and their expression profile in tissue aligned with the label-free data. Cystatin C was the most consistent marker regardless of the magnitude of the renal injury while fatty acid-binding protein-4 (FABP4) and kidney injury molecule-1 (KIM-1) were the most affected biomarkers in response to moderate proximal tubular injury in absence of changes in serum-based concentrations of blood urea nitrogen or creatinine. In the urine, clusterin is considered the most consistent biomarker regardless of the magnitude and time of the renal injury. To our knowledge, this is the most comprehensive multiplex assay for the quantitative analysis of mechanism-based proximal tubular injury biomarkers in dogs.
ABSTRACT
We have developed a workflow to extract, separate, and semi-quantify bioactive oxysterols from mouse colon tissues and fecal matters using solid- and liquid-phase extractions, enzymatic and chemical modifications, and stable-isotope dilution LC/MS/MS. The method was applied to a dextran sodium sulphate (DSS)-induced mouse colitis model, which revealed that one particular dihydroxycholesterol (diOHC), 7α,25-diOHC, was significantly elevated in both colon tissue and fecal matters of mice with colitis compared to that in naïve mice. The extent of 7α,25-diOHC elevation was positively correlated with colitis severity.
Subject(s)
Colitis/chemically induced , Colon/chemistry , Disease Models, Animal , Oxysterols/isolation & purification , Animals , Chromatography, Liquid , Colon/pathology , Dextran Sulfate , Female , Mice , Mice, Inbred C57BL , Oxysterols/chemistry , Tandem Mass SpectrometryABSTRACT
Amphotericin B (AmpB) nephrotoxicity was used to assess the utility of druginduced kidney injury (DIKI) biomarkers in an exploratory study in male cynomolgus monkeys. All animals had quantifiable levels of AmpB in plasma on days 1 and 4. There were no clinical signs of AmpBinduced toxicity in this study. The gold standard method used to confirm AmpBinduced DIKI was anatomic pathology which revealed microscopic lesions with varying grades of severity. Immunolocalization of alpha1 microglobulin (α1M), kidney injury molecule 1 (KIM1), osteopontin (OPN) and neutrophil gelatinaseassociated lipocalin (NGAL) proteins was evaluated in formalinfixed, paraffinembedded monkey kidney tissue sections. AmpB related immunoreactivities were identified in distinct nephron segments of treated monkeys including α1M in damaged proximal tubule epithelium, KIM1 in damaged medullary tubule epithelium, OPN mostly in the infiltrating cells of cortical tubule interstitium, and NGAL in the granular and cellular cast in dilatated cortical tubules. Variations in α1M, KIM1, OPN and NGAL immunolocalization appear as promising DIKI protein biomarkers when monitoring for AmpBinduced corticomedullary tubule injury in male cynomolgus monkeys.
Subject(s)
Amphotericin B/toxicity , Anti-Infective Agents/toxicity , Kidney Diseases/metabolism , Kidney Tubules/metabolism , Alpha-Globulins/metabolism , Amphotericin B/blood , Amphotericin B/pharmacokinetics , Animals , Anti-Infective Agents/blood , Anti-Infective Agents/pharmacokinetics , Biomarkers/metabolism , Hepatitis A Virus Cellular Receptor 1/metabolism , Immunohistochemistry , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Kidney Tubules/drug effects , Kidney Tubules/pathology , Lipocalin-2/metabolism , Macaca fascicularis , Male , Osteopontin/metabolismABSTRACT
A 28-day study was conducted to evaluate changes in urinary cytokine/chemokine expression levels in dogs with renal injury due to administration of cisplatin. Animals (n=17) were administered cisplatin at 0.75 mg/kg/day (i.v.) for five consecutive days. Urine/serum were collected at pre-dosing, 4h post-dosing and on days 2, 3, 4, 8, 10, 14, 16, 18, 21, 23, 25, 28 and unscheduled terminations. Animals were euthanized when serum creatinine (sCr) levels measured at ≥ 1.9 mg/dL, indicating significant loss of renal function (decreased glomerular filtration rate). Relevant clinical observations included lethargy and dehydration. Pre-study sCr levels ranged from 0.6 to 0.8 mg/dL; on days 1 through 4, sCr levels ranged from 0.5 and 1.1mg/dL; and terminal sCr levels ranged from 0.6 and 6.6 mg/dL. Histologically, cisplatin-related renal changes were characterized as proximal tubule dilatation, vacuolization, degeneration, regeneration, and interstitial inflammation. Increased interleukin (IL)-2, IL-8, monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GMCSF) and keratinocyte-derived chemokine (KC) occurred on days 3 through 4. Increased IL-7 occurred on day 4. This study showed for the first time that inflammatory cytokines/chemokines in urine positively identified acute renal tubular injury in dogs at time points earlier than sCr, a traditional marker of nephrotoxicity.
Subject(s)
Acute Kidney Injury/chemically induced , Antineoplastic Agents/toxicity , Cisplatin/toxicity , Acute Kidney Injury/physiopathology , Acute Kidney Injury/urine , Animals , Cytokines/blood , Cytokines/urine , Dogs , Glomerular Filtration Rate , MaleABSTRACT
The objective of this study was to characterize acute coronary artery injury evoked by the endothelin A receptor (ETAR) antagonist, CI-1034. Male dogs (n = 5) were intravenously administered CI-1034 at 120 mg/kg for 4 d. Control animals (n = 3) received vehicle. Macroscopically, drug-related hemorrhage was observed in the right coronary groove and atrium. Histologically, drugrelated coronary changes were characterized as medial hemorrhage and necrosis, with mixed inflammatory-cell infiltrates in the adventitia and media. Immunohistochemistry staining indicated increased expression of inducible nitric oxide synthase (iNOS), cleaved caspase-3, and S100A8/A9 (within in monocytes and neutrophils) proteins in coronary arteries of CI-1034-treated animals. However, there were similar expression levels of endothelial nitric oxide synthase (eNOS) among control and CI-1034-treated animals. Significant drug-related nitric oxide (NO) accumulation occurred on days 1 through 4 in serum. Increased interleukin (IL)-6 and fibrinogen in plasma and serum amyloid A (SAA) occurred on days 2 through 5 in CI-1034-treated animals. Increased levels of NO accumulation in serum; increased IL-6 and fibrinogen levels in plasma; increased SAA levels; and increased expressions of iNOS, cleaved caspase-3, and S100A8/A9 complex appear to be characteristic of CI-1034-induced acute vascular injury in dogs.
Subject(s)
Coronary Vessels/drug effects , Endothelin A Receptor Antagonists , Thiazines/toxicity , Acute Disease , Animals , Biomarkers/metabolism , Calgranulin A/metabolism , Calgranulin B/metabolism , Caspase 3 , Caspases/metabolism , Coronary Vessels/metabolism , Coronary Vessels/pathology , Dogs , Fibrinogen/analysis , Heart Atria/drug effects , Heart Atria/pathology , Hemorrhage/chemically induced , Hemorrhage/pathology , Injections, Intravenous , Interleukin-6/blood , Male , Necrosis , Nitric Oxide/blood , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide Synthase Type III/metabolism , Tunica Media/drug effects , Tunica Media/metabolism , Tunica Media/pathologyABSTRACT
Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNgamma and TNFalpha at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1beta, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1beta, IL-5, IL-10, TNFalpha and IFNgamma at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.
Subject(s)
Cytokines/blood , Endotoxemia/blood , Lipopolysaccharides , Protein Array Analysis , Animals , Antibodies/chemistry , Antibodies/metabolism , Blotting, Western , Disease Models, Animal , Disease Progression , Endotoxemia/chemically induced , Female , MiceABSTRACT
Methods for the stepwise isolation of endothelial cells and smooth muscle cells from individual canine coronary arteries are described. Both cell types can be isolated in pure culture with high yields. Dogs are a common species used in the study of atherosclerosis and coronary artery disease. Capacity to isolate endothelial cells and smooth muscle cells from individual canine coronary arteries should prove useful in the study of coronary artery disease.
Subject(s)
Coronary Vessels/cytology , Endothelium, Vascular/cytology , Muscle, Smooth, Vascular/cytology , Animals , Cell Separation/methods , Coloring Agents , Dogs , Microscopy, Phase-Contrast/methodsABSTRACT
The mechanisms by which nitric oxide (NO) exerts its protective effect in the ischemia/reperfusion (I/R) injury of the kidney have not been fully determined. The hypothesis of this study was based on the assumption that I/R upregulates some chemokines (MIP-2 and MIP-1alpha) as well as certain protein kinases (MAPK p44/42), and therefore we aimed in this work at recognizing if an exogenous NO donor would downregulate these effects in rat ischemic kidneys at the same time that it would offer functional protection as measured by serum creatinine. Sprague-Dawley rats were subjected to renal warm ischemia (75 min) and contralateral nephrectomy. Animals were divided into 3 groups (n = 8 per group): sham, ischemic control, and ischemic group treated with sodium nitroprusside (NaNP 5 mg/kg) given 15 min prior to reperfusion. Serum creatinine (SCr), serum chemokines (MIP-2 and MIP-1alpha), kidney tissue MAPK p44/42, kidney neutrophil infiltration determined by myeloperoxidase (MPO), and light histology were evaluated 4 h after reperfusion began. There were significant improvements in SCr and better histopathological features in the I/R-NaNP group compared with the I/R group. Similarly, the I/R-NaNP kidneys exhibited a downregulating effect of serum chemokines (MIP-2 and MIP-1alpha) and kidney tissue MAPK p44/42 that was not observed in the I/R group alone. The MPO levels were lower in the I/R-NaNP group compared with the I/R untreated group. We can conclude from these experiments that I/R of the rat kidney upregulated the production of MIP-2 and MIP-1alpha chemokines and the activation of MAPKp44/42. It also had a detrimental effect on the function and structure of the ischemic kidney. Exogenous NO had a temporal protective effect in organ function and histology and exerted a downregulating response in the production of MIP-2 and MIP-1alpha chemokines and the activation of MAPK p44/42 following I/R.