Abstract: FR-PO524
Urinary Stone Forming Crystals Activate Renal Medullary Epithelial Cells to Shed Extracellular Vesicles Containing Specific MicroRNAs and Proteins
Session Information
- Bone and Mineral Metabolism: Basic
October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bone and Mineral Metabolism
- 401 Bone and Mineral Metabolism: Basic
Authors
- Chen, Guotao, Department of Nephrology, The Bishan Hospital, Chongqing City, P.R. China, Chongqing city, China
- Jayachandran, Muthuvel, Mayo Clinic, Rochester, Minnesota, United States
- Han, Jing jing, Mayo Clinic College of Medicine (Rochester), Rochester, Minnesota, United States
- Haskic, Zejfa, Mayo clinic , Rochester, Minnesota, United States
- Lieske, John C., Mayo Clinic, Rochester, Minnesota, United States
Background
The majority (~80%) of urinary stones are comprised of calcium oxalate (CaOx) and/or hydroxyapatite (HA). Our previous studies suggest that populations of urinary extracellular vesicles (EVs) and their contents differ in stone formers. This study was designed to examined whether CaOx or HA crystals activate human renal medullary epithelial cells (HRMdEC) to shed EVs containing specific microRNAs (miRNAs) and their target proteins.
Methods
HRMdEC grown in 6-well tissue culture plates were synchronized by incubation in RenaLife Basal Medium without fetal bovine serum for one hour before exposure to CaOx monohydrate (COM) or HA crystals (100µg/mL). Aliquots of medium (250µL) were collected at 0, 0.5, 1, 3, 6, 12, and 24 hour (h). Populations of EVs in medium were analyzed by digital flow cytometer. Cells were harvested at 24h to determine expression of miRNAs identified previously in the urinary EVs of stone formers and their target proteins using qPCR and Western blotting, respectively. Data were analyzed by Student’s t-test or Wilcoxon rank sum test.
Results
The total number of EVs (exosomes and microvesicles) in the medium was significantly increased (P<0.05) by 3h and progressively increased out to 24h after addition of COM or HA crystals. Cellular expression of preselected miR-1299, miR-146b-5p and miR-483-5p were increased whereas miR-532-5p and 664a-3p were decreased significantly (P<0.05) in response to COM crystals. Expression of miR-146b-5p was increased whereas miR-532-3p was decreased significantly (P<0.05) in response to HA crystals. Cellular expression of miR-146-5p and miR-532-3p targeted matrix matalloproteinase-16 was increased significantly (P<0.05) following COM crystal treatment.
Conclusion
Exposure of cultured renal medullary epithelial cells to urinary stone forming crystals activates them to shed EVs and express specific miRNAs and target proteins. The response to COM crystals was more robust that to HA. Further analysis of intra- and inter-cellular signaling pathways via EVs and miRNAs could elucidate novel cellular mechanisms in early urinary stone pathogenesis.
Funding
- NIDDK Support