Identification of key metabolic changes in renal interstitial fibrosis rats using metabonomics and pharmacology

Abstract

Renal fibrosis is one of the important pathways involved in end-stage renal failure. Investigating the metabolic changes in the progression of disease may enhance the understanding of its pathogenesis and therapeutic information. In this study, (1)H-nuclear magnetic resonance (NMR)-based metabonomics was firstly used to screen the metabolic changes in urine and kidney tissues of renal interstitial fibrotic rats induced by unilateral ureteral obstruction (UUO), at 7, 14, 21, and 28 days after operation, respectively. The results revealed that reduced levels of bioenergy synthesis and branched chain amino acids (BCAAs), as well as elevated levels of indoxyl sulfate (IS) are involved in metabolic alterations of renal fibrosis rats. Next, by pharmacological treatment we found that reduction of IS levels could prevent the renal fibrotic symptoms. Therefore, we suggested that urinary IS may be used as a potential biomarker for the diagnosis of renal fibrosis, and a therapeutic target for drugs. Novel attempt combining metabonomics and pharmacology was established that have ability to provide more systematic diagnostic and therapeutic information of diseases.

Figure 1

Changes in renal morphology in sham (A) and UUO-treated (B) for day 7, (C) for day 14 and (D) for day 28) kidneys as revealed by HE staining (100-fold).

Figure 2

Representative ¹H-NMR spectra of the urine samples obtained from a UUO rat (A) and a sham rat (B). Keys: 1, isoleucine/leucine; 2, lactate; 3, acetate; 4, acetamide; 5, N-acetylglycine; 6, succinate; 7, 2-ketoglutarate; 8, citrate; 9, methylamine; 10, dimethylamine; 11, trimethylamine; 12, creatinine/creatine; 13, cis-aconitate; 14, N-nitrosodimethylamine; 15, taurine; 16, trimethylamine-N-oxide; 17, trans-aconitate; 18, glycine; 19, hippurate; 20, trigonelline; 21, 1-methylnicotinamide (1-MNA); 22, allantoin; 23, urea; 24, indoxyl sulfate; 25, formate; 26, niacinamide; 27, creatinine.

Figure 3

OPLS-DA scores (left) and coefficient-coded loading plots (right) for the models discriminating the UUO-treated groups (red dots) and time-matched control rats (blank squares) for data obtained from urine samples in 7 days (A), 14 days (B), 21 days (C), and 28 days (D) after UUO or sham operation. Peaks in the positive direction indicate metabolites that are more abundant in the control groups than UUO group (↑CON). Consequently, metabolites that are more abundant in the UUO group are presented as peaks in the negative direction (↓UUO). Metabolite keys are the same as in Fig. 2.

Figure 4

Representative ¹H-NMR spectra of kidney extracts from rats in the UUO (A) and control (B) groups. Keys: 1, Isoleucine/Leucine; 2, Valine; 3, 3-Hydroxybutyrate; 4, Lactate; 5, Alanine; 6, Acetate; 7, Proline; 8, Glutamate; 9, Pyruvate; 10, Succinate; 11, Carnitine; 12, Methylamine; 13, Citrate; 14, Dimethylamine; 15, Trimethylamine (TMA); 16, Creatinine/Creatine; 17, Choline; 18, Phosphocholine/Glycerophosphosphorylcholine (PC/GPC); 19, Taurine; 20, Betaine; 21, Trimethylamine-N-oxide (TMAO); 22, 1, 3-Dimethylurate; 23, Methanol; 24, Glucose; 25, myo-Inositol; 26, Glycine; 27, Hippurate; 28, myo-Inosine; 29, Trigonelline; 30, Allantion; 31, Uracil; 32, cis-Aconitate; 33, Adenosine; 34, Fumarate; 35, Tyrosine; 36, Histidine; 37, Phenylalanine; 38, Niacinamide; 39, Histamine; 40, Xanthine; 41, 3-Methylxanthine; 42, Hypoxanthine; 43, Adenine; 44, Formate; 45, AMP/ATP.

Figure 5. Multivariate pattern recognition analysis of kidney extracts from UUO-treated and control rats at different time points.

OPLS-DA scores (left) and coefficient-coded loading plots (right) for the models discriminating the UUO-treated group (red dots) and time-matched control group (blank squares) for data obtained from kidney extracts in 7 days (A), 14 days (B), 21 days (C), and 28 days (D) after UUO or sham surgery. Peaks in the positive direction indicate metabolites that are more abundant in the control groups than UUO group (↑CON). Consequently, metabolites that are more abundant in the UUO group are presented as peaks in the negative direction (↓UUO). Metabolite keys are the same as in Fig. 4.

Figure 6. Serum clinical chemistry parameters at different days after administration of BCAAs, ATP and meclofenamate, respectively.

Keys: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, compared with UUO rats, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared with sham operation rats.

Figure 7

Representative HE-stained sections (100-fold) of kidneys from UUO (A), and BCAAs-treated UUO rats (B), ATP-treated UUO rats (C), and meclofenamate-treated UUO rats (D) for 28 days, respectively.

Figure 8. Altered metabolic pathways related to renal fibrosis induced by UUO.

The pathways referenced to the KEGG database and Roche Biochemical Pathways Part 1 (website: http://biochemical-pathways.com/#/map/1) show the interrelationship of the identified metabolic pathways involved in UUO rats. The metabolites with the color blue or red are representative of decline and elevation in levels, respectively, compared to controls. Keys: ^adetected in kidney; ^bdetected in urine; ^cdetected in both kidney and urine.

Figure 9. Linear regression analysis of urinary IS levels in UUO rats.

The y-axis is fold changes of IS in urine [(UUO-control)/control]. IS levels were increased at all the studied time points (r² = 0.6475, P < 0.0001).