A folic acid-induced rat model of renal injury to identify biomarkers of tubulointerstitial fibrosis from urinary exosomes

Abstract

Background: Chronic tubulointerstitial (TI) fibrosis is difficult to diagnose. Urinary exosomes contain many potential biomarkers, and may be useful to identify biomarkers of TI fibrosis. However, a suitable animal model to investigate urinary exosome biomarkers for TI fibrosis has not yet been specifically identified. Objective: To test the suitability of a folic-acid induced rat model of renal injury for identifying potential biomarkers of fibrosis from urinary exosomes. Methods: We employed a rat model of renal injury using a single intraperitoneal injection of folic acid. Urinary exosomes were isolated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: We demonstrated severe acute kidney injury after folic acid injection by a 6-fold decrease in creatinine clearance at day 2, severe inflammatory cell infiltration in the TI area at day 7, and renal fibrosis at day 14. We collected 24 h urine samples from folic acid-injected rats and vehicle-injected control rats at days 7 and 14, then isolated urinary exosomes using a differential centrifugation method. We found that levels of exosomal marker proteins ALG-2-interacting protein 1 (ALIX; PDCD6IP) and tumor susceptibility gene 101 (TSG101) were higher in folic acid-injected rats than in control rats. LC-MS/MS analysis of urinary exosomal proteins from the folic acid-injected and control rats identified 372 proteins, including various potential TI fibrosis biomarkers. Conclusion: We demonstrated the feasibility of using a folic acid-induced rat model of TI fibrosis for urinary proteomic analysis. These rats excreted sufficient urinary exosomes permitting the large-scale analysis and discovery of potential biomarkers of renal fibrosis. Keywords: Biomarkers, chronic kidney disease, folic acid, interstitial fibrosis, proteomics, urine exosomes