Increased gut permeability with altered gut microbiota and fecal metabolome in rats with adriamycin-induced nephropathy
Description
Primary nephrotic syndrome (PNS) is a common glomerular disease in children and adults. The most common pathological type of childhood PNS is minimal change nephrotic syndrome (MCNS). The underlying mechanism of MCNS is largely unknown. Adriamycin-induced nephropathy rat model has been also widely used for studying MCNS. Meanwhile, it has been shown that the gut-renal axis has been implicated in PNS. However, it remains un-known what is the gut-side profile of gut-renal axis in adriamycin-induced nephropathy rat model. Thus, this study aimed to investigate the gut permeability, gut microbiota and fecal metabolites in rats with adriamycin-induced nephropathy.Our data showed gut permeability increased significantly in rats with adriamycin-induced nephropathy. Accordingly, tight junction in rat ileum was dilated as shown under transmission electron microscopy. The profiles of gut micro-biota at taxonomic levels have changed in rats 6 weeks after adriamycin treatment. Particularly, the concentration of 37 fecal metabolites increased significantly, while 9 fecal metabolites decreased. Conclusions: Our findings suggested that adriamycin-induced nephropathy rat model was marked with increased gut permeability, altered gut microbiota and fecal metabolites. It might help to understand the pathogenesis of MCNS in the aspect of gut-renal axis as well as the pharmacology of Adriamycin.
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Animal experiment was approved by Ethical Committee of Shanghai Children’s Hospital, School of Medicine, Shanghai Jiao Tong University (# SHCH-IACUC-2021-XMSB-53). A total of 14 specific pathogen-free grade male Sprague-Dawley (SD) rats (Jiesijie Experimental Animal Company, Shanghai, China) at 5-weeks of age were housed under controlled temperature conditions (22-25 °C) , 50-60% relative humidity and 12:12h light/dark cycle. All rats were given food and water ad libitum. Rats were randomly and equally divided into control (CON) and adriamycin (ADR) group. Nephrotic syndrome rat model was established by a single intravenous injection of adriamycin (7.5 mg/kg, Hanhui Pharmaceutical company, China) through the tail vein according to previous reports [42]. Rats in CON group were injected intravenously with an equivalent amount of saline. At 6 weeks after ADR treatment, fecal samples, serum and tissues (Kidneys and ileums) were collected when rats were euthanized with intraperitoneal injection of overdose phenobarbital(100mg/kg).The concentration of fecal metabolites was measured in an ultra-performance liquid chromatography coupled to tandem mass spectrometry system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) . The methods of peptide mixture preparation was the same as we described before. Peptide mixture solution was put into a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100μm*2cm, nanoViper C18) binding with the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3μm resin) in buffer A (0.1% Formic acid). The condition of separation includes a gradient of 5% buffer B [acetonitrile / IPA (90:10)] (0-1 min), 5-80% buffer B (1-12 min), 80-95% buffer B (12-15 min), 95-100% buffer B (15-16 min), 100% buffer B (16-18 min ), 100-5% buffer B (18-18.1 min), 5% buffer B (18.1-20 min) at a flow rate of 300 nl/min. The differences between CON and ADR groups were evaluated by principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least square discriminant analysis (OPLS-DA) analysis. Differential metabolites were identified by using volcano plot (P<0.05) and VIP analysis (VIP >1.0, P<0.05). The quantitation of fecal metabolites and bioinformatics analysis were performed in Metabo-Profile Biotechnology Co. Ltd. (Shanghai, China).