BPI Fold-Containing Family A Member 2/Parotid Secretory Protein Is an Early Biomarker of AKI.

AKI is a major cause of morbidity and mortality and an important contributor to the development and progression of CKD. Molecular biomarkers that improve the detection and prognostication of AKI are therefore required. We assessed the utility as such of BPI fold-containing family A member 2 (BPIFA2), also known as parotid secretory protein, which we identified via a multiplex quantitative proteomics screen of acutely injured murine kidneys. In physiologic conditions, BPIFA2 is expressed specifically in the parotid glands and is abundant in salivary secretions. In our study, AKI induced Bpifa2 expression in the kidneys of mice within 3 hours. Furthermore, we detected BPIFA2 protein in plasma and urine in these models as early as 6 hours after injury. However, renal injury did not induce the expression of Bpifa2 in mice lacking Nur77, an immediate early gene expressed in the kidneys during AKI. Notably, patients with AKI had higher blood and urine levels of BPIFA2 than did healthy individuals. Together, our results reveal that BPIFA2 is a potential early biomarker of AKI.

Combined serum and EPS-urine proteomic analysis using iTRAQ technology for discovery of potential prostate cancer biomarkers.

Prostate cancer (PCa) is one of the most common malignant tumors and a major cause of cancer-related death for men worldwide. The aim of our study was to identify potential non-invasive serum and expressed prostatic secretion (EPS)-urine biomarkers for accurate diagnosis of PCa. Here, we performed a combined isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to compare protein profiles using pooled serum and EPS-urine samples from 4 groups of patients: benign prostate hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), localized PCa and metastatic PCa. The differentially expressed proteins were rigorously selected and further validated in a large and independent cohort using classical ELISA and Western blot assays. Finally, we established a multiplex biomarker panel consisting of 3 proteins (serum PF4V1, PSA, and urinary CRISP3) with an excellent diagnostic capacity to differentiate PCa from BPH [area under the receiver operating characteristic curve (AUC) of 0.941], which showed an evidently greater discriminatory ability than PSA alone (AUC, 0.757) (P<0.001). Importantly, even when PSA level was in the gray zone (4-10 ng/mL), a combination of PF4V1 and CRISP3 could achieve a relatively high diagnostic efficacy (AUC, 0.895). Furthermore, their combination also had the potential to distinguish PCa from HGPIN (AUC, 0.934). Our results demonstrated that the combined application of serum and EPS-urine biomarkers can improve the diagnosis of PCa and provide a new prospect for non-invasive PCa detection.

Opiorphin analysis in equine plasma and urine using hydrophilic interaction LC-MS.

BACKGROUND: Due to opiorphin's analgesic and antidepressant functions, its illicit use is rumored in some racing jurisdictions. Opiorphin is very difficult to detect due to its hydrophilic nature and rapid degradation in plasma and urine samples. METHODOLOGY & RESULTS: We have developed a sensitive, reliable method for opiorphin detection and confirmation in equine samples, using EDTA to inhibit analyte degradation between the time of collection and analysis. Opiorphin was extracted by weak cation exchange followed by analysis using HILIC-MS/MS. The method was validated and the LOD was determined to be 50 pg/ml in equine plasma and urine. CONCLUSION: The method has good selectivity and precision and is the first reported method for the detection of opiorphin in equine plasma and urine.

Copious urinary excretion of a male Syrian hamster (Mesocricetus auratus) salivary gland protein after its endocrine-like release upon ß-adrenergic stimulation.

Salivary glands, although widely considered as typically exocrine, may also release specific proteins in an endocrine manner. However, endocrine release of salivary gland proteins is not generally acknowledged since the evidences are not easily demonstrable. Submandibular salivary glands (SMG) of male Syrian hamsters express male-specific secretory proteins (MSP; which are lipocalins) visible in SDS-PAGE of SMG extracts, as major bands and also detectable in immunoblots of whole-saliva and urine as low MSP crossreactions. We report here that MSP is localized in acinar cells of SMG and acute treatment with isoproterenol (IPR; non-specific ß1/ß2-adrenergic agonist) results in considerable release of MSP in SMG-saliva. Moreover, acute IPR treatment markedly depletes SMG-MSP in a dose- and time-dependent manner. However, MSP depleted from SMG, far exceeds that recovered in SMG-saliva. Blood, submandibular lymph nodes and kidney of IPR-treated males showed MSP crossreactions and SDS-PAGE of their urine revealed profuse MSP excretion; this was undetectable in IPR-treated-SMG-ablated males, confirming that a substantial amount of MSP depleted from SMG after IPR treatment enters circulation and is excreted in urine. Treatments with specific ß1- or ß2-adrenergic agonists also reduced SMG-MSP levels and resulted in copious urinary excretion of MSP. Co-treatments with specific ß1/ß2-blockers indicated that above effects of IPR, ß1- and even ß2-agonists are very likely mediated by ß1-adrenoceptors. MSP's detection by SDS-PAGE in urine after ß-agonist treatment is a compelling and easily demonstrable evidence of release into circulation of a salivary gland protein. The possible means (endocrine-like or otherwise) of MSP's release into circulation and significance of its presence in saliva, blood and urine of male hamsters are discussed.

Mucin 7 and cytokeratin 20 as new diagnostic urinary markers for bladder tumor.

PURPOSE: We determine the sensitivity and specificity of cytokeratin 20 (CK-20) and mucin 7 (MUC7) gene expression in voided urine samples taken from patients with bladder tumor and from control groups to investigate putative, noninvasive urinary markers for bladder tumor detection and monitoring. MATERIALS AND METHODS: Voided urine samples were collected from 50 patients with histologically proven bladder neoplasms (pTaN0M0G1-3 in 19 and pTisN0M0G3-pT4pN1M1G3 in 31), 20 patients with urolithiasis, 20 patients with urinary tract infection, 20 patients with other urological neoplasms and 20 healthy volunteers. Total RNA was extracted from exfoliated cells collected from 200 ml. voided urine. All RNA samples were investigated by a specific CK-20 and MUC7 nested reverse transcriptase polymerase chain reaction. RESULTS: The overall sensitivity of CK-20 gene expression in voided urine samples for the detection of bladder neoplasms was 78%. In contrast, voided urine samples from control patients and healthy volunteers showed a high rate of false-positive CK-20 detection resulting in a low specificity of 36%. The overall sensitivity of the MUC7 test for all bladder tumor cases was 66%. The sensitivity for papillary urothelial neoplasms (pTaN0M0G1-3) was 42% whereas analysis of the carcinoma in situ and invasive bladder cancer group (pTisN0M0G3-pT4pN1M1G3) yielded a sensitivity of 81%. The overall specificity of the MUC7 nested reverse transcriptase polymerase chain reaction method in the control groups was 80%. CONCLUSIONS: A high positive CK-20 detection rate was found not only in voided urine samples from patients with bladder tumor, but also in urine specimens from control groups. Therefore, CK-20 is not a reliable urinary tumor marker for bladder neoplasms. In contrast to CK-20, analysis of MUC7 demonstrated a high sensitivity and high specificity for carcinoma in situ and invasive bladder cancer, thus fulfilling the criteria of a urinary tumor marker.