(F-214) Effects of Pressure Cycling on Urothelial Exosomes in vitro

Introduction:: Bladder outlet obstruction is a urological condition that affects around 18.5% of the population and leads to various lowery urinary tract symptoms (LUTs) that diminish the patients’ quality of life. 1 The obstruction is accompanied by increased bladder pressures, which are believed to cause inflammation, epithelial-mesenchymal transition (EMT) of urothelium, and subsequent tissue fibrosis.2–4 However, a tissue biopsy would be necessary to determine EMT changes in bladder tissues. Thus, there is a need for a non-invasive method to detect these alterations. Urine contains extracellular vesicles (EVs) such as exosomes, which can carry stable long-noncoding RNAs(lncRNAs). Studies have shown that stressors, including pressure, can not only influence the release of more exosomes from cells but also alter the cargo within the exosomes.5,6 Thus, we hypothesize that changes related to EMT and fibrosis in exosomal cargo may occur because of elevated pressures and could potentially be used as a non-invasive way to characterize tissue changes caused by BOO. However, the effects of elevated pressure on urothelial exosomes and their contents are unknown.In the present study, exosomes released by rat urothelial cells (MYP3) exposed to cyclic elevated pressure were characterized and their contents were examined via PCR.

Materials and Methods:: Serum starved MYP3 cells were exposed in vitro to eitherconstant atmospheric pressure or elevated pressure cycles consisting of a storage pressure of 15 cmH2O for 175 minutes and a voiding pressure of 75 cmH20 for 5 minutes for 24 hours. At the end of the experiment, supernatant culture media were collected and harvested for exosomes using three different approaches: classical ultracentrifugation, commercially available Fisher Total Exosome Isolation kit, and a novel method involving capillary-channeled polymer (C-CP) fiber spin down tips.7–9 Ultracentrifugation required around 50 mL of conditioned media to isolate EVs. Both the Fisher kit and C-CP methods allowed for EV isolation using significantly less media: 4 mL and 2 mL respectively. For the Fisher kit and C-CP isolation methods, conditioned supernatant was filtered using 0.2 µM PES syringe filters to remove debris before EV isolation.Because the EVs were eluted in a solution containing 50% glycerol with the C-CP method, EVs were filtered with 10 or 50 kDa AmiconTM Ultra centrifugal unit (Sigma) to remove theglycerol. The protein content of cell lysates and the EV solutionswas examined using Bradford and BCA assay (Fisher). Size distribution was characterized using DLS. To verify that our EVs were exosomes, markers CD81 and/or CD63 were quantified via a western or System Bioscience ExoElisa kit. Zymo or RNeasy (Qiagen) kits were used to isolate total RNA from our exosomes and quantitative reverse transcriptase (qRT-PCR) was conducted using SuperscriptTM III PlatinumTM One-Step qRT-PCR kit (Invitrogen).

Results, Conclusions, and Discussions:: When supernatant media were processed using ultracentrifugation, detectible amounts of CD81-positive EVs were observed only from MYP3 exposed to elevated pressure, but not from the cells maintained under atmospheric pressure. Subsequent attempts produced inconsistent results possibly because very small amounts of EVs were released by MYP3 cells and they tend to get lost during the washing step of UC. EVs collected using the Fisher kit from MYP3 cells were positive for CD63, but the size of these EVs was inconsistent. Some of the EVs were within exosome size range (30-150 nm) whileothers were smaller than expected 10, which may be debris generated during the procedure. In contrast, the sizes of EVs collected using C-CP method from MYP3 supernatant weremore consistent and within exosome size range, with a Z-average around 71.65 d.nm. In addition, the amount of EVs isolated with C-CP from MYP3 cells exposed to elevated pressure and control groups were similar. When comparing the three different EV isolation techniques, ultracentrifugation proved to be the least effective method as a large volume ofconditioned media was required and the yield and reproducibility was low. Although the Fisher kit was an improvement in that less media was required, the results withEVs of varying sizes make this approach unreliable and the kit can get costly for handling many samples . In conclusion, the C-CP method was the most efficient for isolating EVs because significantly less sample volume is required, a highspeed centrifuge isn’t necessary, and EV size was consistent and within the appropriate range. The results that the number of EVs collected increased when urothelial cells were exposed to elevated pressure may indicate that abnormally high bladder pressure may be detected by analyzing exosomes found in urine. Further study is needed to determine pressure-induced changes in the cargo of exosomes to help aid in the development of a non-invasive method to detect EMT and fibrotic modifications in bladder tissue through urine.

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