Isolation Of Urinary Exosomes Research Articles

Quantification of urinary exosomal PSA for the diagnosis of prostate cancer: a study protocol with emphasis on application of clinical laboratory-based techniques.

Prostate cancer is the most common cancer in men and represents a major problem of public health. The current method of diagnosing/screening for prostate cancer is invasive and costly. There have been renewed and innovative studies on searching urinary biomarker for prostate cancer diagnosis, especially with the technologies of urinary exosomes. However, the technologies of urine exosomes usually need expensive machines such as ultracentrifuge and they are difficult to standardization, which hinder their application in clinical laboratory. In this study, our objective is to detect urinary exosomes from prostate cancer for the development of a test to aid in the diagnosis of prostate cancer. The exosomes from a prostate cancer cell line LNCaP was used to set up the techniques. For the analysis of urine samples of patients, the methods include the collection of first-void urine by using Colli-Pee device, the isolation of urine exosome by optimized precipitation method and the detection of exosomal PSA by Elecsys® total PSA. We have modified and optimized the isolation of urinary exosomes with precipitation method. By using the exosomes from the prostate cancer cell line, we have found that the urinary exosomal PSA can be quantified by automatic technique of Elecsys® total PSA. It will be a 2-year study. We shall start to include the patients and controls in the summer of 2024. We expect the results to be published in the last quarter of 2026. This is the first study to detect the urinary exosomal PSA by the technique of Elecsys® total PSA for the diagnosis of prostate cancer. This study emphasizes on the techniques suitable for the implementation in clinical laboratory, which will facilitate application of urinary exosomes to simplify and improve the diagnosis/screening of prostate cancer.

Urinary exosome isolation in fluorosis patients with early symptoms of nephrotoxicity

Urinary exosome isolation in fluorosis patients with early symptoms of nephrotoxicity

Urinary Exosomal MicroRNAs as Biomarkers for Obesity-Associated Chronic Kidney Disease.

The early detection of chronic kidney disease (CKD) is key to reducing the burden of disease and rising costs of care. This need has spurred interest in finding new biomarkers for CKD. Ideal bi-omarkers for CKD should be: easy to measure; stable; reliably detected, even when interfering substances are present; site-specific based on the type of injury (tubules vs. glomeruli); and its changes in concentration should correlate with disease risk or outcome. Currently, no single can-didate biomarker fulfills these criteria effectively, and the mechanisms underlying kidney fibrosis are not fully understood; however, there is growing evidence in support of microRNA-mediated pro-cesses. Specifically, urinary exosomal microRNAs may serve as biomarkers for kidney fibrosis. In-creasing incidences of obesity and the recognition of obesity-associated CKD have increased interest in the interplay of obesity and CKD. In this review, we provide: (1) an overview of the current scope of CKD biomarkers within obese individuals to elucidate the genetic pathways unique to obesi-ty-related CKD; (2) a review of microRNA expression in obese individuals with kidney fibrosis in the presence of comorbidities, such as diabetes mellitus and hypertension; (3) a review of thera-peutic processes, such as diet and exercise, that may influence miR-expression in obesity-associated CKD; (4) a review of the technical aspects of urinary exosome isolation; and (5) future areas of research.

Comparison of Human Urinary Exosomes Isolated via Ultracentrifugation Alone versus Ultracentrifugation Followed by SEC Column-Purification.

Chronic kidney disease is a progressive, incurable condition that involves a gradual loss of kidney function. While there are no non-invasive biomarkers available to determine whether individuals are susceptible to developing chronic kidney disease, small RNAs within urinary exosomes have recently emerged as a potential candidate to use for assessing renal function. Ultracentrifugation is the gold standard for urinary exosome isolation. However, extravesicular small RNA contamination can occur when isolating exosomes from biological fluids using ultracentrifugation, which may lead to misidentifying the presence of certain small RNA species in human urinary exosomes. Therefore, we characterized human urinary exosomal preparations isolated by ultracentrifugation alone, or via ultracentrifugation followed by size exclusion chromatography (SEC) column-purification. Using nanoparticle tracking analysis, we identified SEC fractions containing robust amounts of exosome-sized particles, that we further characterized using immunoblotting. When compared to exosomal preparations isolated by ultracentrifugation only, SEC fractionated exosomal preparations showed higher levels of the exosome-positive marker CD81. Moreover, while the exosome-negative marker calnexin was undetectable in SEC fractionated exosomal preparations, we did observe calnexin detection in the exosomal preparations isolated by ultracentrifugation alone, which implies contamination in these preparations. Lastly, we imaged SEC fractionated exosomal preparations using transmission electron microscopy to confirm these preparations contained human urinary exosomes. Our results indicate that combining ultracentrifugation and SEC column-purification exosome isolation strategies is a powerful approach for collecting contaminant-free human urinary exosomes and should be considered when exosomes devoid of contamination are needed for downstream applications.

Development and comparative analysis of human urine exosome isolation strategies

Abstract Exosomes contain virtually every type of biomolecules that have originated from parental cells. High concentrations of exosomes are found in a variety of body fluids including urine, a body fluid that can be easily and non-invasively collected. Therefore, urinary exosomes are ideal materials for liquid biopsy. Although there are some major barriers to their use which include high variability and lack of standarization of exosome isolation methods. Therefore, it is highly recommended to systemically assess urinary exosome isolation strategies in terms of isolation concentration and purity. In this study, comparative studies among eight different strategies based on ultracentrifugation, ultrafiltration, precipitation, and size exclusion chromatography (SEC) were performed for urinary exosome isolation from healthy donors. The results indicated that SEC combined with ultrafiltration was the most compromised and balanced method for urinary exosome isolation in terms of both isolation concentration and purity. SEC combined with precipitation yielded the highest purity. Thus, the appropriate urinary exosome isolation method can be chosen depending on the downstream applications of isolated urinary exosomes. This comparative analysis of urinary exosome isolation methods contributes to the discovery of exosome-associated biomarker and the development of exosome-based diagnostics.

The Effect of Tempol on Systolic Blood Pressure in Salt‐loaded Hypertensive 129Sv Mice

The underlying mechanisms of salt‐induced hypertension are not completely understood. The purpose of this study was to determine whether oxidative stress contributes to the increase in exosomal MARCKS and ENaC and the increase in blood pressure of 129Sv mice after salt loading. Adult 129Sv mice were salt loaded to induce hypertension and the antioxidant Tempol was infused by osmotic minipump. Blood pressure was measured by tail cuff before and after Tempol infusion. Metabolic cage studies were performed to obtain 24 hour urine collections for the isolation of urinary exosomes and measurement of electrolytes in the urine. Urinary exosomes and kidneys from these mice were processed for protein biochemistry. Western blot analysis showed MARCKS and ENaC proteins are increased in urinary exosomes isolated from salt‐loaded hypertensive 129Sv mice compared to exosomes isolated from urine of the same mice before salt loading. Urinary sodium increased while potassium in the urine decreased after salt loading the 129Sv mice. Tempol infusion ameliorated the salt‐induced hypertension and decreased MARCKS and ENaC proteins in urinary exosomes of salt‐loaded hypertensive 129Sv mice. Taken together, oxidative stress may play an important role in the salt‐induced hypertension and upregulation of MARCKS and ENaC proteins in the kidneys of these animals.Support or Funding InformationThis project was funded by the U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (Grant K01 DK099617; to A.A.A.) and the University of Florida College of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A highly efficient method for isolating urinary exosomes.

In the present study, a highly efficient method, referred to as optimized ultrafiltration (OUF), was developed. This method is effective for exosome purification and also facilitates clinical work involving substantial urinary exosome isolation. In the OUF method, 0.22-µm filters along with a dialysis membrane with a molecular weight cut-off of 10,000 kDa were introduced, in order to remove extracellular microvesicles that were >200 nm and concentrate the supernatant up to 1/50 of the initial volume. The existence, purity and production of the exosomes isolated by OUF and conventional ultracentrifugation (UC) were systematically compared by transmission electron microscopy, western blotting and nanoparticle tracking analysis. In addition, colloidal Coomassie-stained gel and reverse transcription-quantitative polymerase chain reaction were used to investigate the stability and integrity of exosomes isolated by these two protocols. The time required and cost of these two methods in the process of isolating urinary exosomes were also estimated. The results indicated that OUF clearly outperforms UC in quantity, quality and biological stability, and this improved method may have extensive applications in the growing fields of clinical biomarker discovery and exosome research.

Characterization of Urinary Exosomes from Diabetic Hypertensive db/db Mice

Hypertension can occur before or after the onset of type 2 diabetes mellitus. The db/db mouse model is commonly used to study type 2 diabetes mellitus and its comorbidities including obesity and hypertension. Diabetic db/db mice develop salt‐sensitive hypertension after salt loading. Urinary exosomes are nano‐sized vesicles derived from cell types within the kidney that contain lipids, proteins, and nucleic acids. Recent studies showed urinary exosomes are useful biomarkers for kidney associated diseases including hypertension. We hypothesized the production of urinary exosomes is augmented and that their cargo is altered during the development of salt‐sensitive hypertension. Metabolic cage studies were performed to collect urine for the isolation of urinary exosomes from diabetic db/db mice and wild‐type littermate mice before and after salt‐loading. Western blot analysis was performed using validated antibodies to probe for the exosomal markers flotillin‐1, Hsp70, and annexin V in urinary exosomal lysates. NanoSight analysis showed an increase in the concentration of urinary exosomes from salt‐loaded, diabetic, hypertensive db/db mice and Western blot analysis revealed the enrichment of ENaC and MARCKS in these exosomes compared to non‐salt loaded diabetic db/db mice or healthy wild‐type littermate mice after salt‐loading. Future studies will investigate the physiological role of exosomal MARCKS in the development of diabetes associated hypertension.Support or Funding InformationThis research was supported by a National Institutes of Health grant K01 DK099617 (to A. A. Alli).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A PEG-based method for the isolation of urinary exosomes and its application in renal fibrosis diagnostics using cargo miR-29c and miR-21 analysis.

To assess a new and highly specific, but low-cost, easily performed and suitable for large-scale applications method for renal fibrosis (RF) diagnostics. Thirty-five RF and twenty non-RF patients were enrolled in the study. An appropriate polyethylene glycol (PEG) was used to isolate urinary exosomes. The efficiency of isolation process was evaluated by the morphology and size observation, as well as the detection of specific markers (CD63, CD9). The expression level of exosomal miR-29c, miR-21 and the endogenous control snRNA-U6 were detected by qRT-PCR. The diagnostic potency of urinary exosomal miR-29c and miR-21 was estimated by the ROC method. Spearman's rank-order correlations analysis was used to assess the correlation between the miRNAs and clinical parameters, including pathological index. PEG-based method for isolation urinary exosome was effective and could be completed with a relatively low-speed centrifugal machine. Exosomal miR-29c and miR-21 were detected in all samples. The analysis of miRNAs in urinary exosomes revealed significant dys-regulation of miR-29c and miR-21 associated with RF. Exosomal miR-29c and miR-21 could predict degree of RF with AUC of 0.8333 and 0.7639 (P<0.05). Correlation analysis showed that the level of miR-29c had a significant negative relationship with eGFR and the interstitial relative area. The PEG-based method for isolation urinary exosome is an inexpensive and easily performed approach. The application for cargo miRNA analysis is feasible. Urinary exosomal miR-29c may present a promising diagnostic approach.

Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients.

Chronic kidney disease (CKD) is a severe disorder with an increasing incidence worldwide. An early detection may help to prevent its progression and to minimize the risk of cardiovascular diseases as one of the major comorbidities. Recently, extracellular miRNAs like urinary exosomal miRNAs became of great interest as non-invasive biomarkers which can be determined by RT-qPCR. But until now, there is no consensus regarding the normalization of miRNAs isolated from body fluids. The present study analyzed the miRNAs miR-16, miR-92a, miR-21, miR-124a and the small nuclear RNA RNU6B for their applicability as an endogenous reference gene in expression studies of exosomal miRNAs isolated from CKD patients. For this purpose, miRNA expression levels were determined by RT-qPCR after the isolation of urinary exosomes from 33 CKD patients and from 5 healthy controls. Expression data was analyzed with the normalization determination software NormFinder, BestKeeper, GeNorm and DeltaCt. Our results revealed an abundant expression of the four candidate miRNAs in urinary exosomes and no detectable expression of RNU6B. We identified miR-16 as the most stable endogenous reference gene in our data set, making it a suitable endogenous reference gene for miRNA studies of urinary exosomes derived from CKD patients.

A modified precipitation method to isolate urinary exosomes.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

Perturbations in the urinary exosome in transplant rejection.

Background: Urine exosomes are small vesicles exocytosed into the urine by all renal epithelial cell types under normal physiologic and disease states. Urine exosomal proteins may mirror disease specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery.Methods: Urine exosomes were isolated by centrifugal filtration of urine samples collected from kidney transplant patients with and without acute rejection (AR), which were biopsy matched. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Ue) underwent mass spectroscopy-based quantitative proteomics analysis. The proteome data were analyzed for significant differential protein abundances in AR.Results: A total of 1018 proteins were identified in Uw and 349 proteins in Ue. Two hundred seventy-nine overlapped between the two urinary compartments and 70 proteins were unique to the Ue compartment. Of 349 exosomal proteins identified from transplant patients, 220 had not been previously identified in the normal Ue fraction. Eleven Ue proteins, functionally involved in an inflammatory and stress response, were more abundant in urine samples from patients with AR, three of which are exclusive to the Ue fraction. Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw.Conclusion: A rapid urinary exosome isolation method and quantitative measurement of enriched Ue proteins was applied. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Ue fraction from normal healthy subjects. Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

Use and isolation of urinary exosomes as biomarkers for diabetic nephropathy.

Diabetes represents a major threat to public health and the number of patients is increasing alarmingly in the global scale. Particularly, the diabetic kidney disease (nephropathy, DN) together with its cardiovascular complications cause immense human suffering, highly increased risk of premature deaths, and lead to huge societal costs. DN is first detected when protein appears in urine (microalbuminuria). As in other persisting proteinuric diseases (like vasculitis) it heralds irreversible damage of kidney functions up to non-functional (end-stage) kidney and ultimately calls for kidney replacement therapy (dialysis or kidney transplantation). While remarkable progress has been made in understanding the genetic and molecular factors associating with chronic kidney diseases, breakthroughs are still missing to provide comprehensive understanding of events and mechanisms associated. Non-invasive diagnostic tools for early diagnostics of kidney damage are badly needed. Exosomes – small vesicular structures present in urine are released by all cell types along kidney structures to present with distinct surface assembly. Furthermore, exosomes carry a load of special proteins and nucleic acids. This “cargo” faithfully reflects the physiological state of their respective cells of origin and appears to serve as a new pathway for downstream signaling to target cells. Accordingly, exosome vesicles are emerging as a valuable source for disease stage-specific information and as fingerprints of disease progression. Unfortunately, technical issues of exosome isolation are challenging and, thus, their full potential remains untapped. Here, we review the molecular basis of exosome secretion as well as their use to reveal events along the nephron. In addition to novel molecular information, the new methods provide the needed accurate, personalized, non-invasive, and inexpensive future diagnostics.

Isolation of urinary exosomes for RNA biomarker discovery using a simple, fast, and highly scalable method.

Urinary exosomes are nanovesicles (40-100 nm) of endocytic origin that are secreted into the urine when a multivesicular body fuses with the membrane of cells from all nephron segments. Interest in urinary exosomes intensified after the discovery that they contain not only protein and mRNA but also microRNA (miRNA) markers of renal dysfunction and structural injury. Currently, the most widely used protocol for the isolation of urinary exosomes is based on ultracentrifugation, a method that is time consuming, requires expensive equipment, and has low scalability, which limits its applicability in the clinical practice. In this chapter, a simple, fast, and highly scalable step-by-step method for isolation of urinary exosomes is described. This method starts with a 10-min centrifugation of 10 ml urine, then the supernatant is saved (SN1), and the pellet is treated with dithiothreitol and heat to release and recover those exosomes entrapped by polymeric Tamm-Horsfall protein. The treated pellet is then resuspended and centrifuged, and the supernatant obtained (SN2) is combined with the first supernatant, SN1. Next, 3.3 ml of ExoQuick-TC, a commercial exosome precipitation reagent, is added to the total supernatant (SN1 + SN2), mixed well, and saved for at least 12 h at 4 °C. Finally, a pellet of exosomes is obtained after a 30-min centrifugation of the supernatant/ExoQuick-TC mix. We previously compared this method with five others used to isolate urinary exosomes and found that this is the simplest, fastest, and most effective alternative to ultracentrifugation-based protocols if the goal of the study is RNA profiling. A method for isolation and quantification of miRNAs and mRNAs from urinary exosomes is also described here. In addition, we provide a step-by-step description of exosomal miRNA profiling using universal reverse transcription and SYBR qPCR.

Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis

Diabetic nephropathy (DN) is a major complication of diabetes mellitus (DM), the most frequent cause of end-stage renal disease (ESRD). Exosomes isolated from urine are considered a rich non-invasive source of markers for renal events. Proteinuria associated with DN patients at advanced stages may result in "contamination" of exosomal fraction by co-precipitation of high abundance urine proteins, making it enormously difficult to obtain a reliable comparison of healthy individuals and DN patients and to detect minor proteins. We evaluated different protocols for urinary exosome isolation (ultracentrifugation-based and Exoquick® reagent-based) in combination with an easy and quick depletion procedure of contaminating high abundance proteins (albumin). The optimal methodology was then applied to investigate the proteome of human urinary exosomes in DN and controls using spectral counting LC-MS/MS analysis followed by selected reaction monitoring (SRM) confirmation. A panel of 3 proteins (AMBP, MLL3, and VDAC1) is differentially present in urinary exosomes from DN patients, opening a new field of research focused on improving diagnosis and follow-up of this pathology. Diabetic nephropathy (DN) is a progressive proteinuric kidney disease, a major complication of diabetes mellitus, and the most frequent cause of end-stage renal disease. Current markers of disease (i.e. creatinine and urinary albumin excretion) have proven limitations (i.e. some patients regress to normoalbuminuria, kidney damage may be already present in recently diagnoses microalbuminuric patients and renal function may decrease in the absence of significant albuminuria). We show here the first study on human DN proteome of urinary exosomes. Proteinuria associated to DN patients resulting in contamination of exosomal fraction and the associated difficulty to reliably compare healthy and disease conditions, are here overcome. A combined methodology pointed to increase exosomal proteome recovery and depletion of high-abundance proteome was here set-up. A total of 352 proteins were here identified for the first time associated to human urinary exosomes. Label-free quantitative comparison of DN urinary exosomes vs control group and SRM further validation, resulted in the discovery of a panel of three proteins (AMBP, MLL3 and VDAC1) which changes in DN, opening a new field of research focused to improve diagnosis and follow-up of this pathology.

Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers

Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine.

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases

Exosomes are 40–100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.

TAMM-HORSFALL PROTEIN AND URINARY EXOSOME ISOLATION: PP.9.380

Urinary exosomes have been proposed as a starting material for discovery of protein biomarkers of disease. Currently, standard protocols for urinary exosome isolation involve a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the 17,000 Xg supernatant and to sediment only when the sample is spun at 200,000 Xg. Here we show, using immunoblotting and electron microscopy, that urinary exosomes are also present in the 17,000 Xg pellet as a result of entrainment by polymeric Tamm-Horsfall protein (THP). This diminishes the reproducibility of isolation. We show, by electron microscopy, that the addition of DTT to the 17,000 Xg pellet results in disruption of the THP polymeric network presumably by reduction of disulfide bonds that link the monomers. This procedure was shown to deplete the exosomal proteins Alix, TSG101, and CD9 from the 17,000 Xg pellet. Also, by shifting the THP to the 200,000 Xg pellet, the use of DTT makes it theoretically feasible to use THP in this fraction to normalize excretion rates of other proteins in spot urines. Immunoblotting to test this idea showed a high degree of correlation between exosomal proteins and THP. We conclude that 1) THP polymeric networks entrain urinary exosomes; 2) the yield of exosomes by differential centrifugation can be increased by chemical reduction of the sample; and 3) THP may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical. Optimize urinary exosome isolation is a key point in the field of biomarker discovery

Tamm-Horsfall protein and urinary exosome isolation

Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical.