iTRAQ analysis of urinary proteins: Potential use of gelsolin and osteopontin to distinguish benign thyroid goiter from papillary thyroid carcinoma.

BACKGROUND: Benign thyroid goiter (BTG) and papillary thyroid carcinoma (PTC) are often interchangeably misdiagnosed. METHODS: Pooled urine samples of patients with BTG (n=10), patients with PTC (n=9) and healthy controls (n=10) were subjected to iTRAQ analysis and immunoblotting. RESULTS: The ITRAQ analysis of the urine samples detected 646 proteins, 18 of which showed significant altered levels (p<0.01; fold-change>1.5) between patients and controls. Whilst four urinary proteins were commonly altered in both BTG and PTC patients, 14 were unique to either BTG or PTC. Amongst these, four proteins were further chosen for validation using immunoblotting, and the enhanced levels of osteopontin in BTG patients and increased levels of a truncated gelsolin fragment in PTC patients, relative to controls, appeared to corroborate the findings of the iTRAQ analysis. CONCLUSION: The data of the present study is suggestive of the potential application of urinary osteopontin and gelsolin to discriminate patients with BTG from those with PTC non-invasively. However, this needs to be further validated in studies of individual urine samples.

Cardiotrophin-1 therapy prevents gentamicin-induced nephrotoxicity in rats.

Aminoglycosides are very effective antibiotics for the treatment of severe infections, but they rank among the most frequent causes of drug-induced nephrotoxicity. Thus, prevention of aminoglycoside nephrotoxicity is an unmet therapeutic objective. Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been reported to protect the kidney against toxic and ischemic acute kidney injury (AKI). We have assessed the effect of rat CT-1 in the severity of gentamicin (G)-induced AKI. Groups of male Wistar rats received the following for 6 consecutive days: i) isotonic saline solution (group CONT), ii) G, 150mg/kg/day, i.p. (group G), iii) CT-1, 100µg/kg/day i.v. (group CT-1), or iv) G and CT-1 at the doses described above. The G group showed a manifest AKI characterized by low creatinine clearance, high plasma creatinine and urea levels, increased urinary excretion of proteins, glucose and AKI markers such as N-acetyl-glucosaminidase, neutrophil gelatinase-associated lipocalin, kidney-injury molecule-1 and T-gelsolin, increased kidney levels of CD-68, iNOS, IL-1ß and TNF-α, and markedly higher histological renal damage and leukocyte infiltration than the CONT and CT-1 groups. Administration of CT-1 together with G reduced almost all of the above-described manifestations of G-induced AKI. The results of this study have potential clinical application, as CT-1 is near to being used as a drug for organ protection.

Urinary proteome analysis of irritable bowel syndrome (IBS) symptom subgroups.

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by chronic abdominal pain associated with alterations in bowel function. Given the heterogeneity of the symptoms, multiple pathophysiologic factors are suspected to play a role. We classified women with IBS into four subgroups based on distinct symptom profiles. In-depth shotgun proteomic analysis was carried out to profile the urinary proteomes to identify possible proteins associated with these subgroups. First void urine samples with urine creatinine level≥100 mg/dL were used after excluding samples that tested positive for blood. Urine from 10 subjects representing each symptom subgroup was pooled for proteomic analysis. The urine proteome was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a data-independent method known as Precursor Acquisition Independent From Ion Count (PAcIFIC) that allowed extended detectable dynamic range. Differences in protein quantities were determined by peptide spectral counting followed by validation of select proteins with ELISA or a targeted single reaction monitoring (LC-SRM/MS) approach. Four IBS symptom subgroups were selected: (1) Constipation, (2) Diarrhea+Low Pain, (3) Diarrhea+High Pain, and (4) High Pain+High Psychological Distress. A fifth group consisted of Healthy Control subjects. From comparisons of quantitative spectral counting data among the symptom subgroups and controls, a total of 18 proteins that showed quantitative differences in relative abundance and possible physiological relevance to IBS were selected for further investigation. Three of the 18 proteins were chosen for validation by either ELISA or SRM. An elevated expression of gelsolin (GSN) was associated with the high pain groups. Trefoil Factor 3 (TFF3) levels were higher in IBS groups compared to controls. In this study, the IBS patients subclassified by predominant symptoms showed differences in urine proteome levels. Proteins showing distinctive changes are involved in homeostasis of intestinal function and inflammatory response. These findings warrant future studies with larger, independent cohorts to enable more extensive assessment and validation of urinary protein markers as a diagnostic tool in adults with IBS.

Urinary levels of regenerating islet-derived protein III ß and gelsolin differentiate gentamicin from cisplatin-induced acute kidney injury in rats.

A key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicin's nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine.

Urinary protein expression patterns in children with sleep-disordered breathing: preliminary findings.

BACKGROUND: Obstructive sleep apnea (OSA) is the most common form of sleep-disordered breathing, with almost 15 million Americans affected and many more at risk. Current diagnostic approach to OSA requires polysomnography, which is laborious, onerous, and time-consuming. There is ample evidence that inflammatory responses to the perturbations associated with OSA trigger a variety of genes and signaling cascades that ultimately lead to end-organ injury and changes in kidney function and protein expression. The aim of this study was therefore to analyze proteins in human urine and assess whether differential expression of particular proteins is associated with the presence of OSA. METHODS: Eleven OSA and 11 control children between the ages of three and 14 (males=17; females=5) underwent overnight sleep studies followed by a first-morning urine sample. Proteomic analysis of urine samples yielded a unique map of proteins, of which, five spots were selected for further analysis due to their significant intensity differences between OSA and control groups. RESULTS: Mass spectrometry followed by peptide mass fingerprinting conclusively identified four of the five spots as gelsolin, perlecan (a heparan sulfate proteoglycan), albumin, and immunoglobulin (P<0.05 and protein scores>67). CONCLUSIONS: Overall, increased expression of gelsolin and perlecan in the urinary proteome of children with OSA suggests that the episodic hypoxia associated with OSA may lead to changes in protein permeability or alternatively to increased catabolism of these proteins and their excretion in urine.