Inhibition of microbiota-dependent TMAO production attenuates chronic kidney disease in mice

Abstract

Patients with chronic kidney disease (CKD) have elevated circulating levels of trimethylamine N-oxide (TMAO), a metabolite derived from gut microbes and associated with cardiovascular diseases. High circulating levels of TMAO and its dietary precursor, choline, predict increased risk for development of CKD in apparently healthy subjects, and studies in mice fed TMAO or choline suggest that TMAO can contribute to kidney impairment and renal fibrosis. Here we examined the interactions between TMAO, kidney disease, and cardiovascular disease in mouse models. We observed that while female hyperlipidemic apoE KO mice fed a 0.2% adenine diet for 14 weeks developed CKD with elevated plasma levels of TMAO, provision of a non-lethal inhibitor of gut microbial trimethylamine (TMA) production, iodomethylcholine (IMC), significantly reduced multiple markers of renal injury (plasma creatinine, cystatin C, FGF23, and TMAO), reduced histopathologic evidence of fibrosis, and markedly attenuated development of microalbuminuria. In addition, while the adenine-induced CKD model significantly increased heart weight, a surrogate marker for myocardial hypertrophy, this was largely prevented by IMC supplementation. Surprisingly, adenine feeding did not increase atherosclerosis and significantly decreased the expression of inflammatory genes in the aorta compared to the control groups, effects unrelated to TMAO levels. Our data demonstrate that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO reduction may be a novel strategy in treating CKD and its cardiovascular disease complications.

Figure 1

Inhibition of TMAO production attenuates chronic kidney development induced by adenine feeding. Female apoE KO mice (n = 10 per group) were fed control (Con), control + IMC (Con + IMC), adenine (Ade), or adenine + IMC (Ade + IMC) diets for 14 weeks before measurements of traits. Plasma levels of (a) TMAO, (b) creatinine, (c) Urea, (d) Indoxyl sulfate, (e) Cystatin C, and (f) c-terminal FGF23 (cFGF23) are shown. (g) Urine albumin (Alb) to creatinine (Cre) ratios are shown. Representative kidney sections stained with Masson’s trichrome are shown in (h–k). Quantification of kidney pathology, (l) cortical scar, and (m) collagen deposition, in the kidney sections of all 4 groups are shown. Individual values, means, and standard errors are shown. Sample sizes were n = 10/group for (a–f), n = 8–10/group for (g), and n = 8 kidneys/group for (l,m). For (a–f,l,m), post hoc multiple comparisons analysis was performed after significant two-way ANOVA. Symbols: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 2

Inhibition of TMAO generation decreases inflammation and fibrosis in the kidneys of adenine diet-induced CKD. Gene expression analysis of kidney samples from the same mice described in Fig. 1 were performed using qPCR (a), n = 8 to 9 per group, and RNA-seq (b,c). Based on the RNA-seq data, pathway enrichment analysis was performed to identify down-regulated (b), and up-regulated (c) functional clusters in the kidneys of Ade + IMC group compared to the Ade group (n = 6 per group). Gene expression (d, n = 4–5/group) and immunoblotting (e,f, n = 3/group) analysis of MDCK II kidney epithelial cells treated with control, and various doses of TMAO (d) or 200 μM of TMAO (e,f) for 48 h. (g) MDCKII cells were treated with control media, 100 nM NF-κB inhibitor IV (Inh), 200 μM TMAO (TMAO), or 200 μM TMAO + 100 nM NF-κB inhibitor IV (TMAO + Inh) for 48 h before gene expression analysis (n = 9 for each group). For (a,f), two tailed Student’s t-test was performed. Symbols: *p < 0.05, and **p < 0.01. For (d) Post hoc Tukey’s multiple comparisons test was performed after significant one-way ANOVA. Symbols: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the control group. For (g) Post hoc analysis was performed after significant two-way ANOVA. Symbols: **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 3

IMC supplementation protects against myocardial hypertrophy and dyslipidemia associated with adenine diet-induced CKD. Data obtained from tissue samples of the same animals described in Fig. 1 are presented in this figure, n = 10 per group. (a) Heart weight/body weight ratios, (b) Plasma levels of triglyceride (TG), total cholesterol (TC), HDL cholesterol (HDL), and VLDL/IDL/LDL cholesterol (VLDL/IDL/LDL), (c) mean atherosclerotic lesion area at the aortic root, and (d) aortic gene expression analysis by qPCR are shown. For data presented in all panels, post hoc multiple comparisons analysis was performed after significant two-way ANOVA. Symbols: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.