Investigation of urinary components in rat model of ketamine-induced bladder fibrosis based on metabolomics

Abstract

Background: Ketamine abuse has been linked to the system's damage, presenting with lower urinary tract symptoms (LUTS). While the pathogenesis of ketamine-induced urinary damage is not fully understood, fibrosis is believed to be a potential mechanism. A metabolomic investigation of the urinary metabolites in ketamine abuse was conducted to gain insights into its pathogenesis.

Methods: A rat model of ketamine induced bladder fibrosis was established through tail vein injection of ketamine hydrochloride and control group was established through tail vein injection of the equivalent normal saline. Hematoxylin and eosin (H&E) staining and Masson trichrome staining were performed to evaluated bladder pathology. Urinary components were detected based on a metabolomic approach using ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOFMS platform). Orthogonal projections analyzed the data to latent structures discriminant analysis (OPLS-DA) and bioinformatics analysis.

Results: The rat model of ketamine induced bladder fibrosis was confirmed through H&E and Masson trichrome staining. There were marked differences in the urinary metabolites between the experimental group and the control group. Compared to the control group, 16 kinds of differential metabolites were up-regulated and 102 differential metabolites were down-regulated in the urine samples of the ketamine group. Bioinformatics analysis revealed the related metabolic pathways.

Conclusions: Using a ketamine-induced bladder fibrosis rat model, this study identified the differential urinary metabolites expressed following ketamine treatment. These results provide vital clues for exploring the pathogenesis of ketamine-induced LUTS and may further contribute to the disease's diagnosis and treatment.

Keywords: Ketamine; bladder fibrosis; metabolomics.

Figure 1

Histopathological changes of bladder specimen induced by 8-week ketamine injection in rat. Comparison between the control group (A) and ketamine group (B) through HE staining showed urothelium desquamation (red arrow), inflammatory cell infiltration (blue arrow), vascular distension and congestion (yellow arrow) in ketamine group. Bladder fibrosis was confirmed via Masson trichrome staining in control (C) and ketamine group (D). It showed collagen deposition (green arrow). Original magnification ×100.

Figure 2

Score scatter plot of OPLS-DA model for ketamine group vs. control group (A for negative ion and B for positive ion mode) and permutation test of OPLS-DA model for ketamine group vs. control group (C for negative ion and D for positive ion mode). OPLS-DA, orthogonal projections to latent structures-discriminant analysis.

Figure 3

Comparison of all differentially expressed metabolites levels in the ketamine and control groups. Heatmaps showed 118 significantly altered metabolites between these two groups (A). The colors correspond to the abundance value of each metabolite. Volcano map showed differentially expressed metabolites screened by VIP value >1 and P<0.05 (B). VIP, variable importance in the projection.

Figure 4

ROC curves (A,B,C,D,E,F,G,H,I,J) and box plots (K,L,M,N,O,P,Q,R) for differential metabolites screened with AUC >0.9. (A,J) Methamphetamine; (B,K) 1-deoxy-D-xylulose-5-phosphate; (C,L) 7-methylguanosine; (D,M) allocystathionine; (E,N) clofibrate; (F,O) epsilon-caprolactone; (G,P) myristoleic acid; (H,Q) N-acetyl-L-aspartic acid; (I,R) Tyr-Glu. AUC, area under the curve of ROC.

Figure 5

Heatmap of correlation analysis for differential metabolites (A). Pathway analysis bubble plot for model group vs. control group (B for negative ion and C for positive ion mode). Regulatory network map for model group vs. control group (D for negative ion and E for positive ion mode).