Identification of an antibiotic-treated mouse model to study the gut-kidney axis

Abstract Background and Aims Microbiota and its modulation could influence several physiological functions, and there is growing interest in it as a cutting-edge research topic in the field of renal diseases. According to the literature, germ-free (GF) mice are the gold-standard animal model for studying the correlation between the changing microbiota and the effects in the host. Unfortunately, their generation and maintenance are often unfeasible and costly, and the lack of microbiota could influence the circulating levels of amino acids, thus affecting kidney growth. This study aims to identify a more advantageous mouse model, specifically an antibiotic-treated one, that exhibits a strong reduction of the gut microbiota without compromising the kidney functionality. Method Five-six weeks old C57/BL6 male mice were randomly divided into 5 groups: Group 1 (Tr1) received a cocktail containing ampicillin, gentamicin, metronidazole, neomycin (1mg/mL each) and vancomycin (0.5 mg/mL) by oral gavage for 10 days; Group 2 (Tr2) received drinking water (DW) supplemented with ampicillin (1 mg/mL) neomycin (1 mg/mL), metronidazole (1mg/mL) and vancomycin (0,5 mg/mL) for 14 days; Group 3 (Comb3) was treated with amphotericin-B (0.2 mg/mL) by oral gavage for 3 days followed by an antibiotic cocktail composed by vancomycin (10 mg/mL), neomycin (20 mg/mL), metronidazole (20 mg/mL) and amphotericin-B (1 mg/mL) administrated by oral gavage and ampicillin (1 mg/mL) provided in DW for 7 days; Group 4 (Comb4) was treated with a combination of ampicillin, neomycin, metronidazole and vancomycin (40mg/mL each) both by oral gavage and DW; Control Group (Ctr) received milliQ water only. Fecal DNA was used to assess the reduction of the intestinal microbiota by the 16S gene rRNA sequencing, and intestinal permeability was determined both in vivo by FITC-dextran and by measuring the expression of Zonulin-1 (ZO-1) on intestine sections. To verify the kidney integrity after treatment, renal morphology and fibrosis were evaluated by multiphoton microscopy, while transdermal measurement of GFR occurred by using MediBeacon device. Results Apart from Tr1, with the less percentage of microbiota depletion, the rest of antibiotics cocktails exhibited a significant microbiota reduction. Data regarding intestinal permeability in vivo by FITC-dextran were consistent with hematoxylin-eosin staining and revealed a reduced expression of ZO-1 in all antibiotics-treated groups versus Ctr, confirming that microbiota has been depleted as expected. In all the experimental groups, particularly Tr2, urine/serum parameters reestablished at Ctr levels after recovery and only Comb4 showed signs of fibrosis. The low intragroup variability and the homogeneous decrease of the most abundant phyla, as well as renal physiology evaluation, point to Tr2 as the most promising antibiotics cocktail among the tested ones. Conclusion The potential of our study lies in the feasibility to have a manageable mouse model to perform studies on the gut-kidney axis. In particular, it will be specifically used for the evaluation of fecal microbiota transplant (FMT) with feces of patients affected by kidney diseases, enabling us to determine whether intestinal dysbiosis may impair the kidney functionality. However, the characterized mouse model might be employed to assess the effect of the FMT from patients with renal pathologies not only on kidneys but also at systemic level.