Urinary Exosomes Identify Inflammatory Pathways in Vancomycin Associated Acute Kidney Injury.

BACKGROUND: Vancomycin is commonly used as a first line therapy for gram positive organisms such as methicillin resistant Staphylococcusaureus. Vancomycin-induced acute kidney injury (V-AKI) has been reported in up to 43% of patients, especially in those with higher targeted trough concentrations. The precise mechanism of injury in humans remains elusive, with recent evidence directed towards proximal tubule cell apoptosis. In this study, we investigated the protein contents of urinary exosomes in patients with V-AKI to further elucidate biomarkers of mechanisms of injury and potential responses. METHODS: Urine samples from patients with V-AKI who were enrolled in the DIRECT study and matched healthy controls from the UAB-UCSD O'Brien Center Biorepository were included in the analysis. Exosomes were extracted using solvent exclusion principle and polyethylene glycol induced precipitation. Protein identity and quantification was determined by label-free liquid chromatography mass spectrometry (LC/MS). The mean peak serum creatinine was 3.7 ± 1.4 mg/dL and time to kidney injury was 4.0 ± 3.0 days. At discharge, 90% of patients demonstrated partial recovery; 33% experienced full recovery by day 28. Proteomic analyses on five V-AKI and 7 control samples revealed 2009 proteins in all samples and 251 proteins significantly associated with V-AKI (Pi-score > 1). The top discriminatory proteins were complement C3, complement C4, galectin-3-binding protein, fibrinogen, alpha-2 macroglobulin, immunoglobulin heavy constant mu and serotransferrin. CONCLUSION: Urinary exosomes reveal up-regulation of inflammatory proteins after nephrotoxic injury in V-AKI. Further studies are necessary in a large patient sample to confirm these findings for elucidation of pathophysiologic mechanisms and validation of potential injury biomarkers.

Tamm-Horsfall glycoprotein engages human Siglec-9 to modulate neutrophil activation in the urinary tract.

Urinary tract infections are a major problem in human medicine for which better understanding of native immune defenses may reveal new pathways for therapeutic intervention. Tamm-Horsfall glycoprotein (THP), the most abundant urinary protein, interacts with bacteria including uropathogenic Escherichia coli (UPEC) as well host immune cells. In addition to its well-studied functions to antagonize bacterial colonization, we hypothesize that THP serves a critical host defense function through innate immune modulation. Using isolated human neutrophils, we found that THP binds neutrophils and that this interaction reduces reactive oxygen species generation, chemotaxis and killing of UPEC. We discovered that THP engages the inhibitory neutrophil receptor sialic acid-binding Ig-like lectin-9 (Siglec-9), and mouse functional ortholog Siglec-E, in a manner dependent on sialic acid on its N-glycan moieties. THP-null mice have significantly more neutrophils present in the urine compared with wild-type mice, both with and without the presence of inflammatory stimuli. These data support THP as an important negative regulator of neutrophil activation in the urinary tract, with dual functions to counteract bacterial colonization and suppress excessive inflammation within the urinary tract.

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis.

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.

Pirfenidone is renoprotective in diabetic kidney disease.

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-beta promoter activity, reduced TGF-beta protein secretion, and inhibited TGF-beta-induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.

Identification of Maltase Glucoamylase as a Biomarker of Acute Kidney Injury in Patients with Cirrhosis.

BACKGROUND: Acute kidney injury (AKI) is a frequent complication of decompensated cirrhosis with increased mortality. Traditional biomarkers such as serum creatinine are not sensitive for detecting injury without functional change. We hypothesize that urinary exosomes potentially carry markers that differentiate the type of kidney injury in cirrhotic patients. METHODS: This is a prospective, single-center, and observational study of adult patients with cirrhosis. The patient groups included healthy normal controls, compensated cirrhosis with normal kidney function, decompensated cirrhosis with normal kidney function, and decompensated cirrhosis with AKI. Data were extracted from the electronic health record including etiology of liver disease, MELD score, history of decompensation, Child-Turcotte-Pugh score, history of AKI, and medication exposures. Urine samples were collected at the time of consent. Urine exosome protein content was analyzed, and proteomic data were validated by immunoblotting. Statistical analysis included partial least squares-discriminant analysis coupled with variable importance in projection identification. RESULTS: Eighteen cirrhotic subjects were enrolled, and six healthy control subjects were extracted from our biorepository. Urine exosomes were isolated, and 1572 proteins were identified. Maltase-glucoamylase was the top discriminating protein confirmed by western blotting. CONCLUSIONS: Patients with cirrhosis and AKI have upregulation of renal brush border disaccharidase, MGAM, in urinary exosomes which may differentiate the type of kidney injury in cirrhosis; however, the clinical significance of this requires further validation.

Nitrative inactivation of thioredoxin-1 and its role in postischemic myocardial apoptosis.

BACKGROUND: Intracellular proteins involved in oxidative stress and apoptosis are nitrated in diseased tissues but not in normal tissues; definitive evidence to support a causative link between a specific protein that is nitratively modified with tissue injury in a specific disease is limited, however. The aims of the present study were to determine whether thioredoxin (Trx), a novel antioxidant and antiapoptotic molecule, is susceptible to nitrative inactivation and to establish a causative link between Trx nitration and postischemic myocardial apoptosis. METHODS AND RESULTS: In vitro exposure of human Trx-1 to 3-morpholinosydnonimine resulted in significant Trx-1 nitration and almost abolished Trx-1 activity. 3-morpholinosydnonimine-induced nitrative Trx-1 inactivation was completely blocked by MnTE-2-PyP(5+) (a superoxide dismutase mimetic) and markedly attenuated by PTIO (a nitric oxide scavenger). Administration of either reduced or oxidized Trx-1 in vivo attenuated myocardial ischemia/reperfusion injury (>50% reduction in apoptosis and infarct size, P<0.01). However, administration of nitrated Trx-1 failed to exert a cardioprotective effect. In cardiac tissues obtained from ischemic/reperfused heart, significant Trx-1 nitration was detected, Trx activity was markedly inhibited, Trx-1/ASK1 (apoptosis signal-regulating kinase-1) complex formation was abolished, and apoptosis signal-regulating kinase-1 activity was increased. Treatment with either FP15 (a peroxynitrite decomposition catalyst) or MnTE-2-PyP(5+) 10 minutes before reperfusion blocked nitrative Trx inactivation, attenuated apoptosis signal-regulating kinase-1 activation, and reduced postischemic myocardial apoptosis. CONCLUSIONS: These results strongly suggest that nitrative inactivation of Trx plays a proapoptotic role under those pathological conditions in which production of reactive nitrogen species is increased and that antinitrating treatment may have therapeutic value in those diseases, such as myocardial ischemia/reperfusion, in which pathological apoptosis is increased.

Clinical Significance of Persistent Global and Focal Computed Tomography Nephrograms After Cardiac Catheterization and Their Relationships to Urinary Biomarkers of Kidney Damage and Procedural Factors: Pilot Study.

OBJECTIVES: We evaluate the relationships between persistent computed tomography (CT) nephrograms and acute kidney injury after cardiac catheterization (CC). We compare changes in urinary biomarkers kidney injury molecule 1 (KIM-1), cystatin C, and serum creatinine to procedural factors. MATERIALS AND METHODS: From 159 eligible patients without renal insufficiency (estimated glomerular filtration rate >60 mL/min), 40 random patients (age range, 42-81 years; mean age, 64 years; 25 men, 15 women) gave written informed consent to undergo unenhanced CT limited to their kidneys 24 hours after CC. Semiquantitative assessment for global nephrograms and quantitative assessment of focal nephrograms in each kidney was performed. Computed tomography attenuation (Hounsfield units) of the renal cortex was measured. Serum creatinine, KIM-1, and cystatin C were measured before and 24 hours after CC. RESULTS: Robust linear regression showed that both relative changes in KIM-1 and cystatin C had positive relationships with kidney CT attenuation (P = 0.012 and 0.002, respectively). Spearman rank correlation coefficient showed that both absolute changes and relative changes in KIM-1 and cystatin C had positive correlations with global nephrogram grades (P = 0.025 and 0.040, respectively, for KIM-1; P = 0.013 and 0.019, respectively, for cystatin C). CONCLUSIONS: Global nephrograms on unenhanced CT in patients who have undergone CC are significantly correlated with changes in urinary biomarkers for kidney damage.

Proteomic analyses of Urine Exosomes reveal New Biomarkers of Diabetes in Pregnancy.

OBJECTIVE: To evaluate 24 hour urine exosome protein content changes among pregnant US subjects with diabetes and obesity during early pregnancy. METHODS: The exosome proteome content from 24 hour urine samples of pregnant subjects with gestational diabetes mellitus (GDM, N=8) and pre-gestational Type 2 diabetes (PGD, N = 10) were compared with control samples (CTRL, N = 10) obtained at week 20 of pregnancy. Differences in exosome protein load between groups was identified by liquid chromatography/mass spectrometry, analyzed by linear regression in negative binomial distribution, visualized in MetaboAnalyst (version 3.0), and validated by western immunoblotting. RESULTS: At the 20th week of pregnancy, we identified 646, 734 and 856 proteins in exosomes from 24 hour urine samples of patients from the CTRL, GDM and PGD groups, respectively. S100 calcium binding protein A9, damage associated molecular pattern (DAMP) signal, was found to be significantly increased in both GDM and PGD subjects. In GDM subjects the peptide counts for S100A9 protein independently correlated with maternal obesity and macrosomia of the newborn infants. Early to late pregnancy developmental changes in the GDM group were shown to utilize pathways and protein expression levels differently from those in PGD or CTRL groups. CONCLUSIONS: Urinary exosome proteomic analysis non-invasively provides insights into maternal changes during diabetic pregnancy. Exosome biomarkers early in pregnancy can be potentially used to better understand pathophysiologic mechanisms of diabetes at a cellular level, and to distinguish between gestational and pre-gestational diabetes at the pathway level. This information can aid intervention efforts to improve pregnancy outcomes in women with diabetes.

Proteomic analysis of urine exosomes reveals renal tubule response to leptospiral colonization in experimentally infected rats.

BACKGROUND: Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. CONCLUSIONS: We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.

Novel systems biology insights using antifibrotic approaches for diabetic kidney disease.

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. To explore the therapeutic potential of PFD, we studied the PFD-treated db/db diabetic mouse kidney by liquid chromatography-tandem mass spectrometry proteomics. A total of 21 proteins unique to PFD-treated diabetic kidneys were identified. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA translation. Two key proteins involved in mRNA translation initiation and elongation were further evaluated and found to be regulated by PFD at the level of phosphorylation. In conclusion, insights from combining proteomics and bioinformatics improve the likelihood of rapid advancement of novel clinical therapies focused on reducing inflammation and fibrosis for diabetic complications.

Thioredoxin glycation: A novel posttranslational modification that inhibits its antioxidant and organ protective actions.

Thioredoxin (Trx) is an antioxidant and antiapoptotic molecule, and its activity is regulated by posttranslational modifications. Trx-1 has recently been reported to exert potent protective action against endotoxic liver injury. However, whether Trx-1 activity is affected by endotoxin has never been previously investigated. The aim of the present study was to determine endotoxic regulation of Trx-1, and the potential mechanism involved. In vitro coincubation of Trx-1 with lipopolysaccharide (LPS) inhibited Trx-1 activity in a dose- and time-dependent fashion. The core (polysaccharide containing) region of LPS had a greater inhibitory effect on Trx-1 activity than its Lipid A fragment, suggesting the involvement of sugar groups. Periodic acid-Schiff staining and fructosamine assay demonstrated that Trx-1 was rapidly glycated by LPS. Aminoguanidine, a competitive glycation-inhibitor, completely blocked the inhibitory effect of LPS on Trx-1. Moreover, Trx-1 activity was also significantly inhibited by in vitro ribose incubation. Finally, in vivo administration of Trx-1, but not glycated Trx-1, reduced LPS-induced hepatic injury. Taken together, these results demonstrated for the first time that Trx-1 is susceptible to glycative inactivation. This novel posttranslational Trx-1 modification contributes to LPS cytotoxicity, suggesting that blockading protein glycation might be a new therapeutic strategy against endotoxic organ injury.

Role of reactive oxygen species in hyperadrenergic hypertension: biochemical, physiological, and pharmacological evidence from targeted ablation of the chromogranin a (Chga) gene.

BACKGROUND: Oxidative stress, an excessive production of reactive oxygen species (ROS) outstripping antioxidant defense mechanisms, occurs in cardiovascular pathologies, including hypertension. In the present study, we used biochemical, physiological, and pharmacological approaches to explore the role of derangements of catecholamines, ROS, and the endothelium-derived relaxing factor nitric oxide (NO(•)) in the development of a hyperadrenergic model of hereditary hypertension: targeted ablation (knockout [KO]) of chromogranin A (Chga) in the mouse. METHODS AND RESULTS: Homozygous ⁻(/)⁻ Chga gene knockout (KO) mice were compared with wild-type (WT, +/+) control mice. In the KO mouse, elevations of systolic and diastolic blood pressure were accompanied by not only elevated catecholamine (norepinephrine and epinephrine) concentrations but also increased ROS (H2O2) and isoprostane (an index of lipid peroxidation), as well as depletion of NO(•). Renal transcript analyses implicated changes in Nox1/2, Xo/Xdh, and Sod1,2 mRNAs in ROS elevation by the KO state. KO alterations in blood pressure, catecholamines, H2O2, isoprostane, and NO(•) could be abrogated or even normalized (rescued) by either sympathetic outflow inhibition (with clonidine) or NADPH oxidase inhibition (with apocynin). In cultured renal podocytes, H2O2 production was substantially augmented by epinephrine (probably through ß2-adrenergic receptors) and modestly diminished by norepinephrine (probably through α1-adrenergic receptors). CONCLUSIONS: ROS appear to play a necessary role in the development of hyperadrenergic hypertension in this model, in a process mechanistically linking elevated blood pressure with catecholamine excess, renal transcriptional responses, ROS elevation, lipid peroxidation, and NO(•) depletion. Some of the changes appear to be dependent on transcription, whereas others are immediate. The cycle could be disrupted by inhibition of either sympathetic outflow or NADPH oxidase. Because common genetic variation at the human CHGA locus alters BP, the results have implications for antihypertensive treatment as well as prevention of target-organ consequences of the disease. The results document novel pathophysiological links between the adrenergic system and oxidative stress and suggest new strategies to probe the role and actions of ROS within this setting.

Reactive oxygen species production via NADPH oxidase mediates TGF-beta-induced cytoskeletal alterations in endothelial cells.

Cytoskeletal alterations in endothelial cells have been linked to nitric oxide generation and cell-cell interactions. Transforming growth factor (TGF)-beta has been described to affect cytoskeletal rearrangement in numerous cell types; however, the underlying pathway is unclear. In the present study, we found that human umbilical vein endothelial cells (HUVEC) have marked cytoskeletal alterations with short-term TGF-beta treatment resulting in filipodia formation and F-actin assembly. The cytoskeletal alterations were blocked by the novel TGF-beta type I receptor/ALK5 kinase inhibitor (SB-505124) but not by the p38 kinase inhibitor (SB-203580). TGF-beta also induced marked stimulation of reactive oxygen species (ROS) within 5 min of TGF-beta exposure. TGF-beta stimulation of ROS was mediated by the NAPDH oxidase homolog Nox4 as DPI, an inhibitor of NADPH oxidase, and dominant-negative Nox4 adenovirus blocked ROS production. Finally, inhibition of ROS with ROS scavengers or dominant-negative Nox4 blocked the TGF-beta effect on cytoskeleton changes in endothelial cells. In conclusion, our studies show for the first time that TGF-beta-induced ROS production in human endothelial cells is via Nox4 and that TGF-beta alteration of cytoskeleton in HUVEC is mediated via a Nox4-dependent pathway.

Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy.

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5-6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.

Paradigm shifts in malaria parasite biochemistry and anti-malarial chemotherapy.

A fatty acid synthesis (FAS) pathway was recently discovered and established in the obligate human parasite Plasmodium falciparum. Its inhibition by triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) leads to its classification as a type II FAS. Humans, the vertebrate host for the malarial parasite utilize type I FAS, which is not inhibited by triclosan. This discovery thus paves the way for novel approaches to the treatment of malaria. In direct contrast to the delayed-death phenotype associated with poisoning of the apicoplast using certain other drugs, the rapid and striking action of triclosan suggests the possibility of developing new drug(s) for the treatment of malaria.