This part of the course will focus on the collection and processing of urine prior to the isolation of extracellular vesicles. My name is Dr. Lesley Cheng, and I am a post doctoral research fellow based at the La Trobe University in the Department of Biochemistry and Genetics in Australia. Urine has been used as a clinical specimen for many years and it is a source that can be used to measure ones health. Urine has been used to test for several disease such as glucose and diabetes or the HCG hormone to detect pregnancy. Urine is typically a sterile liquid and is a by-product of the body secreted by the kidneys. These by-products are generated by cellular metabolism which is required to be removed from the bloodstream. Urine can be the preferred clinical sample, as it is minimally invasive. 95% of urine is water, and the remaining molecules are inorganic salts such as urea and sodium. Other molecules include organic molecules such as protein and nucleotides, for example micro RNA. Cellular debris can also be found, and the pH of urine is typically between 4.6 and 8. Bacterial infections in the urethra can cause the urine to have a cloudy appearance. Extracellular vesicles can be found in urine. It is thought that most of the extracellular vesicles found in urine are secreted from the renal epithelial cells. These cells are within the renal system, which consists of the kidneys, bladder, and urethra. Upon separating urine by centrifugation, two major fractions of urine can be noticed. 1) Cell sediment, which consists of mostly renal epithelial cell debris. And 2) the remaining cell-free urine, also known as neat urine. The neat urine is where extracellular vesicles can be found. Occasionally, I may refer to extracellular vesicles as EVs. All types of EVs are found in the urine, such as microvesicles, of approximately 500 to 1,000 nanometers in diameter. And exosomes of approximately 50 to 150 nanometers in diameter, as seen here. Exosomes have been the most well-studied, and in my laboratory we have a focus on exosomes, whereby throughout this presentation I may be focusing on exosomes. More importantly, EVs are protected from RNases. And therefore miRNA contained in the EVs can be protected from the environment. These microRNAs can be found from distant sites of the body. Phenamil EVs can also contain proteins. As I alluded to before, urine has been used as a clinical specimen for many decades, and a urine test may be performed to check for the treatment of conditions such as kidney and liver related diseases. Diabetes, urinary tract infections, diseases of the urinary tract, kidney stones, and more specifically to this course, urinary exosomes or extracellular vesicles have been identified to carry biomarkers for acute kidney transplant rejection. Diabetic neuropathy, kidney diseases, renal cell carcinoma, prostate cancer, and bladder cancer. Most of these studies have been done on the smallest type of EVs, being exosomes. For example, in this published study, exosomes was isolated from urine to determine whether exosomal contents could be used as the biomarker of prostate cancer. In order to ensure processing of urine remains consistent, the collection of urine should follow standard operating procedures, depending on the final assay to be performed downstream. Clinical or biomarker information obtained from the urine is influenced by collection timing Method and handling. There are three main types of collection for urine specimens. A random specimen can be collected any time of the day, which is the easiest to obtain and readily available. However, in regards to the collection of urine for EV isolation depending on the water intake of the individual, vesicle concentration may be diluted or artificially lowered. First morning urine specimen is the first urine after sleep, and in most cases is the preferred specimen, as it is the most concentrated, thus there tends to be more vesicles in first morning urine, which helps maximizes the potential yield of exosomes for analysis. First morning urine also contains cellular debris and microbial information. Hence, these may be useful types for clinical testing. Cellular debris can be removed by low-speed centrifugation before vesicle isolation. On the other hand, midstream specimen is a collection method performed whereby patient avoids the first portion of the urine stream, which contains the most cellular contamination. This allows for the removal of potential cellular contamination. Hence, first morning or midstream urine should be used for extracellular vesicle isolation and analysis. So far I have discussed the factors that should be considered while collecting urine. Upon the collection of your urine specimen the typical differential ultracentrifugation protocol can be used to isolate the EVs. So starting with no less than 30 mls of whole urine the sample needs to be vortexed for 90 seconds. This is then followed by a low-speed centrifugation step at 2,000 Gs for 10 minutes. This is to allow the pelleting and removal of any cells in the debris. The supernatant, now being the cell-free urine is then centrifuged at 17,000 g's for 45 minutes to pallet the large micro vesicles. If you wish to analyze micro vesicles being the larger population of vesicles at approximately 500 to 1,000 nanometers in size, you can then collect and resuspend this micro vesicle pallet in P-V-S for further analysis. However, to isolate smaller vesicles such as exosomes are 50 to 150 nanometers in size. This supermatant is then centrifuged at 200,000 gs or 65 minutes. The resulting pellet of the 200,000 g spin may contain some contamination of other sized vesicles other than lysosomes. We should be characterised using western immune blood techniques will visualize underneath a transmission electron microscope. You may have noticed in this course that most particles pellet exosomes at 100,000 gs, however, here particle involves 200,000 g spin. We have found that urine samples contain a larger population of smaller vesicles at 50 nanometers compared to other sample types such as cell culture medium or serum which have more exosomes between 100 nanometers and 150 nanometers. Hence, to ensure all small, smaller size vesicles pellet at 200,000g spin is recommended to ensure a large yield of exosomal vesicles for analysis in urine samples. Now to also ensure the your urine sample is prepared and optimised for vesicle isolation, additional steps can be performed before the procedure to increase the final pellet of microvesicles or exosomes. As mentioned before, it is recommended that protease inhibitors are added to the urine sample to inhibit protein degradation. Protease inhibitors typically come as a tablet form, which can then be added to the urine just after collection. And therefore the urine is preserved before analysis or storage. A study by Zhou et al shows the differences and benefits of adding protease inhibitors. Here, eight fresh urine samples from healthy volunteers were collected without inhibitors and with the addition of protease inhibitors. So B1 to B8 are from eight different volunteers, and D1 to D8 another eight different volunteers. Exosome fractions were prepared and analyzed by Western immunoblotting for the sodium-potassium-calcium cotransporter isoform 2, which is found in exosomes. You can see that there were no successful detection of the cotransporter protein in most of the samples without protease treatment, indicating the degradation of protein in these samples. Whereas, the protein cotransporter protein was detected in every sample that was treated with protease inhibitor. Hence protease inhibitors should be used for proteomic analysis such as with standard immunoblotting. The effects of vortexing and storage of whole urine before the ultracentrifugation procedure was also studied by Zhou et al. This morning urine was collected from three healthy volunteers and subjected to different storage protocols with or without vortexing prior to vesicle isolation. Lane one is a sample stored at 4 degrees and processed within one hour with no prior vortexing. Lane two and three were stored at -20 or -80 for one week without vortexing before use. Lane four and five were stored at -20 or -80 for one week and subjected to intensive vortexing for 90 seconds after complete thawing of the sample. Following vesicle isolation by ultracentrifugation, the final vesicle pellets were loaded on an SDS-PAGE, with was then stained to visualize total protein. Proteins were also transferred to a membrane to propol various exosome associated protein such as NAG-3, TSG101, ALIX, and AQP2. In lane one, you can see the high presence of protein extracted from the fresh samples which were stored 4 degrees and processed within one hour. This protein profile is also seen in the sample stored at -80 degrees. There's also a high recovery of many exosomal proteins. So vortexing is not required if you were to process fresh samples, or flash-freezed samples at -80 degrees. However, less protein is obtained when samples are stored at -20 degrees if vortexing was not employed, compared to a vesicle yield if all urine was vortexed prior to ultracentrifugation. This study indicates that vortexing the sample before vesicle isolation helps to potentially release exosomes into the supernatant to increase vesicle yield. Zhou et al also measured the amount of protein obtained from the exosome pellet under different storage and vortexing conditions. Here, maximal amounts of protein and consistent amounts was obtained if samples were stored at -20 or -80 degrees with vortexing. Thus, storage at -20 and -80 does not affect vesicle isolation so long that samples are vortexed before proceeding to differential ultracentrifugation. Also, the study investigated that long-term storage up to 12 months is found to be fine. Upon isolating your vesicles, you can visualize their morphology under transmission electron microscopy, commonly referred to as. You may find that the samples contain many rope-like structures, not only vesicles. These are the rope-like structures, and inside them you can see vesicles trapped within these networks. These rope-like structures are typically known as the Tamm-Horsfall protein, abbreviated as THP. They form long polymeric filaments in urine samples. It is the most abundant protein found in urine. THP can form a network of these filaments and trap small vesicles such as exosomes. It has been shown that dithiothreitol, DTT, treatment can assist with releasing these vesicles entrapped in the THP network, thereby releasing the vesicles into the supernatant and allowing for increased exosome recovery. DTT treatment can be performed by treating the 200,000g pellet with DTT before diluting with PVS4, an additional 200,000g spin to recover otherwise entrapped exosomes. I have investigated the use of DTT treatment to increase exosomal yields. In this Western blot you can see that there is no significant change of exosomal markers between exosomes treated with or without DTT. SH-SY5Y cell lines and exosomes were used as a Western immuno-bonding positive control here. Upon extracting the RNA content from these exosomes, DTT treatment did not seem to increase or improve RNA yields. However, upon visualizing the exosome preparations on the TEM, you can see that the exosomes treated with DTT have less THP contamination. There is a lack of rope-like structures in these TEM pictures. Thus, the treatment of the resulting vesicle pellet with DTT at that 200,000g spin may assist with removing THP contamination for certain downstream applications such as protein analysis. To further characterize your 200,000g pellet and to also determine the purity of vesicles or exosomes in your preparation, a particle diameter counter can be used such as the key nano lemanocyte instruments. Here the dark grey shows the vesicle population obtained by simply passing through cell free urine which contains vesicles and comparing them to a sample that has undergone ultracentrifugation to isolate a potential pure exosome pellet, which is in light grey. Approximately the same population of vesicles are found in both sample types, and the vesicles are approximately the same size, at about 70 nanometers in size. This indicates that the majority of vesicles found in the urine are of exosomal size, which is between 50 to 150 nanometers. Exosome pallets along with the controls can also be run on a Western blot. And exosome associated markers and non-exosomal associated markers. Here you can see that the vesicles isolated by the ultracentrifugation protocol are positive and enriched for TSG101, flotillin and CD63. These are not seen enriched or present in the cell free urine or the exosome depleted supernatant of the 200,000 G spin. The urinal exosomes are negative for non-exosomal markers such as nucleoporin that would indicate contamination of nuclear bodies, GM 130 for golgi apparatus and BCRT bodies. That's the Western blot and particle diameter results here suggest that the vesicles isolated are likely to contain a high population of exosomes. Again the SHSY5Y cells and the exosomes were used here as a positive control. As mentioned earlier, exosomes can contain protein and RNA which can be used as biomarkers. One can extract the RNA contents from vesicles such as exosomes using a commercial RNA kit, such as the miRNA Easy Kit from Qiagen. This key is able to extract all RNA species including micro RNA at 22 nucleotides long. It is important to note that most RNA kits on the market only extract RNA of 200 nucleotides in one go. Here on this slide, RNA was extracted from urinary exosomes as well as other fractions of urine for comparison using the miRNA easy kit. Upon RNA extraction, the samples were run on the Agilent Bioanalyser small RNA assay. This chip provides a read-out of small RNA quality of your sample. The x-axis here shows the length of the nucleotides detected. Here, RNA extracted from the cellular debris of urine is observed to contain a large amount of RNA species at 20 to 40 nucleotides in length, and some other RNA species from 40 to 200 nucleotides in length. Most of this cellular RNA is degraded as there is no ribosome or peak, such as A10S or 28S. This was determined by running the sample on a total RNA nano 6000 assay chip. In the original urine you can see that the original unprocessed urine contains mostly RNA characteristic of micro-RNA at 20 to 40 nucleotides long and other small RNA graded in 40 nucleotides long. This profile is deemed similar to the one found from cell-free urine. On the other hand, exosomes are enriched with microRNA as you can see from this bioanalyzer profile. However, to determine whether these bioanalyzer traces are indeed RNA species one can perform several molecular base experiments. Upon extracting the RNA contents a number of RNA downstream experiments can be performed to investigate and profile the RNA contents in your sample, using techniques such as quantitative RTPCR or next generation deep sequencing. Quantitative RTPCR requires prior knowledge of the RNA species that may be contained in your sample so that specific primers are designed for amplification and detection. However next generation DNA sequencing can provide an unbiased profile of RNA species without prior knowledge of the sample. Here on this slide, next-generation date sequencing was performed to profile the RNA species in urinary exosomes, and it was found that exosomes were indeed enriched with micro RNA at 35%. The urinary exosomes contain 3% coding RNA and less than 1% small nuclear RNA, small nucleolar RNA, long intergenic noncoding RNAs, and long non-coding RNAs. Other RNA species found were un-annotated, non-coding species which had not been identified or otherwise known as junk RNA. In regards to the number of micro RNA detected, only 12 micro RNA species were found in the cell free urine while 184 microRNA species were found in exosomes isolated by the ultracentrifugation method. Using a commercial urine exosomal kit, 66 microRNA species were detected. From this data obtained from the next generation date sequencing of exosomes it can be observed that urine exosome samples indeed contain mostly micro RNA. Apart from RNA, urinary exosomes have also been found to carry protein cargo. This study by Pisitkun et al, performed a proteonic study to profile protein-containing urinary exosomes. They found that the majority of proteins were cytoplasmic. Furthermore, the majority of proteins were also associated with plasma membrane and extracellular proteins. Other proteins included those form the peripheral part of the plasma membrane, lysosomal, G P Adling's plasma proteins, nuclei, golgi, and mitochondrial proteins. Further analysis by Western immunoblotting performed by Pisitkun et al prepared the presence of several proteins initially identified in their proteomic profiling in exosomes isolated from urine and compared them to tissues of the kidney. You can see that a number of these proteins, such as angiotensin converting enzyme ACE and ALEX, which is a exosomal associated protein enriched in the urine exosomes. Compared to different tissues isolated from several regions of the kidneys such as the cortex and the outer and inner medulla. This completes my presentation and I would like to end with this summary slide. The collection of urine prior to the isolation of extracellular vesicles is highly important. Here are some elements that are important to ensure optimal recovery of urinary exosomes or vesicles. Protease inhibitors are necessary for the preservation of vesicular proteins. It is not necessary to remove cellular debris before storage. Freezing urine samples at -80 degrees will preserve urine for vesicular isolation. Intensive vortexing after thawing improves the recovery of urinary vesicles. Ideally more than 20-30 ml of urine is required for vesicular isolation. First and second morning urine can be used for vesicle isolation. DTT treatment during ultracentrifugation procedure is ideal for downstream protein experiments but not necessary for RNA experiments. The use urinary extracellular vesicles can be applied to many types of studies such as micro RNA analysis. This is highly useful as there is an enrichment of RNA species in urinary vesicles. Several proteins derived from the kidneys are enriched in urinary vesicles. These could be used as protein biomarkers or to understand diseases of the renal system. Enriching for urinary vesicles may allow the identification of protein and RNA biomarkers, which may have been diluted in cell-free urine. Please feel free to look up these references using this course material for further details.
