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The last two decades have witnessed an explosion in research focused on the development and assessment of novel biomarkers for improved prognosis of diseases. As a result, best practice standards guiding biomarker research have undergone extensive development. Currently, there is great interest in the promise of biomarkers to enhance research efforts and clinical practice in the setting of chronic kidney disease, acute kidney injury, and glomerular disease. However, some have questioned whether biomarkers currently add value to the clinical practice of nephrology. The current state of the art pertaining to statistical analyses regarding the use of such measures is critical. In December 2014, the National Institute of Diabetes and Digestive and Kidney Diseases convened a meeting, "Toward Building Better Biomarker Statistical Methodology," with the goals of summarizing the current best practice recommendations and articulating new directions for methodological research. This report summarizes its conclusions and describes areas that need attention. Suggestions are made regarding metrics that should be commonly reported. We outline the methodological issues related to traditional metrics and considerations in prognostic modeling, including discrimination and case mix, calibration, validation, and cost-benefit analysis. We highlight the approach to improved risk communication and the value of graphical displays. Finally, we address some "new frontiers" in prognostic biomarker research, including the competing risk framework, the use of longitudinal biomarkers, and analyses in distributed research networks., (© 2019 John Wiley & Sons, Ltd.)

Exosomes are released by most cells and can be isolated from all biofluids including urine. Exosomes are small vesicles formed as part of the endosomal pathway that contain cellular material surrounded by a lipid bilayer that can be traced to the plasma membrane. Exosomes are potentially a more targeted source of material for biomarker discovery than unfractionated urine, and provide diagnostic and pathophysiological information without an invasive tissue biopsy. Cytoplasmic contents including protein, mRNA, miRNA, and lipids have all been studied within the exosomal fraction. Many prospective urinary exosomal biomarkers have been successfully identified for a variety of kidney or genitourinary tract conditions; detection of systemic conditions may also be possible. Isolation and analysis of exosomes can be achieved by several approaches, although many require specialized equipment or involve lengthy protocols. The need for timely analysis in the clinical setting has driven considerable innovation with several promising options recently emerging. Consensus on exosome isolation, characterization, and normalization procedures would resolve critical clinical translational bottlenecks for existing candidate exosomal biomarkers and provide a template for additional discovery studies., (2017 Published by Elsevier Inc.)

Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed when based on serum creatinine (SCr) changes, due in part, to decreased creatinine production. During experimental sepsis, we compared serum cystatin C (sCysC), SCr, and blood urea nitrogen (BUN) to inulin glomerular filtration rate (iGFR) before or 3-18 h after cecal ligation and puncture (CLP)-induced sepsis in CD-1 mice. sCysC had a faster increase and reached peak levels more rapidly than SCr in both sepsis and bilateral nephrectomy (BiNx) models. sCysC was a better surrogate of iGFR than SCr during sepsis. Combining sCysC with SCr values into a composite biomarker improved correlation with iGFR better than any biomarker alone or any other combination. We determined the renal contribution to sCysC handling with BiNx. sCysC and SCr were lower post-BiNx/CLP than post-BiNx alone, despite increased inflammatory and nonrenal organ damage biomarkers. Sepsis decreased CysC production in nephrectomized mice without changing body weight or CysC space. Sepsis decreased sCysC production and increased nonrenal clearance, similar to effects of sepsis on SCr. sCysC, SCr, and BUN were measured 6 h postsepsis to link AKI with mortality. Mice with above-median sCysC, BUN, or SCr values 6 h postsepsis died earlier than mice with below-median values, corresponding to a substantial AKI association with sepsis mortality in this model. sCysC performs similarly to SCr in classifying mice at risk for early mortality. We conclude that sCysC detects AKI early and better reflects iGFR in CLP-induced sepsis. This study shows that renal biomarkers need to be evaluated in specific contexts.

Objective: The aim of this study was to examine the presence of microRNAs (miRNAs) within exosomes isolated from human saliva and to optimize and test methods for successful downstream applications., Design: Exosomes isolated from fresh and frozen glandular and whole human saliva were used as a source of miRNAs. The presence of miRNAs was validated with TaqMan quantitative PCR and miRNA microarrays., Results: We successfully isolated exosomes from human saliva from healthy controls and a patient with Sjögren's syndrome. microRNAs extracted from the exosomal fraction were sufficient for quantitative PCR and microarray profiling., Conclusions: The isolation of miRNAs from easily and non-invasively obtained salivary exosomes with subsequent characterization of the miRNA expression patterns is promising for the development of future biomarkers of the diagnosis and prognosis of various salivary gland pathologies.

Urinary exosomes are excreted from all nephron segments and may serve as biomarkers for classifying renal diseases. Isolation of urinary exosomes by the established ultracentrifugation method has some limitations for use in a clinical laboratory. We sought a rapid and simple way to obtain urinary exosomes. We used a commercially available nanomembrane concentrator to enrich exosomes from urine by centrifugation at 3,000 g for 10-30 min. Urinary exosomal markers tumor susceptibility gene 101, aquaporin-2, neuron-specific enolase, annexin V, angiotensin-converting enzyme, and podocalyxin (PODXL) were recovered from the nanomembrane concentrator and detected by Western blotting, and typical features of urinary vesicles were found by electron microscopy. Exosomal markers were detected in as little as 0.5 ml of urine. By the nanomembrane method, exosomal proteins could be recovered from urine samples frozen at -80 degrees C or refrigerated overnight at 4 degrees C then stored at -80 degrees C. By enriching exosomes we could detect PODXL, a podocyte marker, which decreased by 71% in five male patients with focal segmental glomerulosclerosis and abundant proteinuria. We conclude that 1) use of a nanomembrane concentrator simplifies and accelerates the enrichment of urinary exosomes; and 2) the nanomembrane concentrator can concentrate exosomal proteins from clinical urine samples. This enhanced method may accelerate the translation of urinary exosomal biomarkers from bench to bedside for the diagnosis, classification, and prognostication of renal diseases.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Recent progress in biotechnology offers the promise of better medical care at lower costs. Among the techniques that show the greatest promise is mass spectrometry of proteins, which can identify proteins present in body fluids and tissue specimens at a large scale. Because urine can be collected in large amounts in a non-invasive fashion, the potential exists to use mass spectrometry to discover urinary biomarkers that are early predictors of renal disease, or useful in making therapeutic choices. Recently, the authors demonstrated that both membrane proteins and cytosolic proteins from renal epithelia are highly enriched in low-density urinary structures identified as exosomes. Exosomes were found to contain many disease-associated proteins including aquaporin-2, polycystin-1, podocin, non-muscle myosin II, angiotensin-converting enzyme, Na+ K+ 2Cl- cotransporter (NKCC2), thiazide-sensitive Na-Cl cotransporter (NCC), and epithelial sodium channel (ENaC). Potentially, other disease biomarkers could be discovered by mass spectrometry-based proteomic studies in well-defined patient populations. Herein is described the advantages of using urinary exosomes as a starting material for biomarker discovery. In addition, the purpose of this review is to present an overall strategy for biomarker discovery in urine using exosomes and for developing cost-effective clinical assays for these biomarkers, which can potentially be used for early detection of disease, as a means of differential diagnosis, or as a means of guiding therapy. Finally, potential barriers that need to be overcome before urinary proteomics can be applied clinically, are emphasized.

Animal models and human studies have been useful in dissecting the molecular mechanisms of renal disease and finding new disease targets; however, translation of these findings to new clinical therapeutics remains challenging. Difficulties with detecting early disease, measuring drug effectiveness, and the daunting cost of clinical trials hampers the development of new therapeutics for renal diseases. Many existing laboratory tests were discovered because of inspired recognition that a particular protein might prove useful in clinical practice. New unbiased genomic and proteomic techniques identify many constituents present in biologic samples and thus may greatly accelerate biomarker research. This review focuses on the steps needed to develop new biomarkers that are useful in laboratory and clinical investigations, with particular focus on new proteomic screening technologies. New biomarkers will speed the laboratory and clinical development of new treatments for renal diseases through mechanistic insights, diagnoses that are more refined, early detection, and enhanced proof of concept testing.