Faecalibacterium prausnitzii Attenuates CKD via Butyrate-Renal GPR43 Axis

Abstract

Background: Despite available clinical management strategies, chronic kidney disease (CKD) is associated with severe morbidity and mortality worldwide, which beckons new solutions. Host-microbial interactions with a depletion of Faecalibacterium prausnitzii in CKD are reported. However, the mechanisms about if and how F prausnitzii can be used as a probiotic to treat CKD remains unknown.

Methods: We evaluated the microbial compositions in 2 independent CKD populations for any potential probiotic. Next, we investigated if supplementation of such probiotic in a mouse CKD model can restore gut-renal homeostasis as monitored by its effects on suppression on renal inflammation, improvement in gut permeability and renal function. Last, we investigated the molecular mechanisms underlying the probiotic-induced beneficial outcomes.

Results: We observed significant depletion of Faecalibacterium in the patients with CKD in both Western (n=283) and Eastern populations (n=75). Supplementation of F prausnitzii to CKD mice reduced renal dysfunction, renal inflammation, and lowered the serum levels of various uremic toxins. These are coupled with improved gut microbial ecology and intestinal integrity. Moreover, we demonstrated that the beneficial effects in kidney induced by F prausnitzii-derived butyrate were through the GPR (G protein-coupled receptor)-43.

Conclusions: Using a mouse CKD model, we uncovered a novel beneficial role of F prausnitzii in the restoration of renal function in CKD, which is, at least in part, attributed to the butyrate-mediated GPR-43 signaling in the kidney. Our study provides the necessary foundation to harness the therapeutic potential of F prausnitzii for ameliorating CKD.

Keywords: Faecalibacterium; butyrates; kidney diseases; microbiota; receptors, G-protein-coupled.

Figure 1.

Depletion of Faecalibacterium in the database of American Gut Project and Chinese CKD population. Comparison of Faecalibacterium (A) and Bacteroides (B) abundances between non-CKD group (n=294) and CKD group (n=283) of American Gut Project Complete analysis of taxonomical genera with false discovery rate (FDR) adjusted P value was included in Table 5 The adjusted P values were determined byDESeq2 analysis. Comparison of Faecalibacterium (C) and Bacteroides (D) abundances between Chinese human fecal microbiota: Control (n=30), CKD (n=32) and CKD-HTN (n=43). Complete analysis of taxonomical genera with false discovery rate (FDR) adjusted P value was included in Table 6 and Table 7. The adjusted P values were determined by DESeq2 analysis. (E) Increased ratio of Firmicutes to Bacteroidetes (F/B), (F) decreased Observed features, (G) Chao1 richness, and (H) Shannon diversity scores in Control, CKD and CKD-HTN subjects. Data are presented as mean ± SD in Figure 1E–H. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons in Figure 1E–H.

Figure 2.

Correlation analysis of differential genera with clinical characteristics in all three groups. (A) The abundance of differential genera enriched in Control (n=30), CKD (n=32) and CKD-HTN (n=43) groups was analyzed for conversation with differential clinical characteristics (ie, SBP, DBP, BUN, Scr, UA, eGFR, TC, TG, HDL and LDL) using Spearman’s correlation analysis. The horizontal axis represents different renal function related indexes. The vertical axis represents the abundance of differential genera enriched in all three groups. The correlation coefficient is indicated by a color gradient from orange (positive correlation) to blue (negative correlation). * indicates P< 0.05, ** indicates P< 0.01. Higher serum levels of TMAO (B) and LPS (C) in the CKD-HTN (n=30) than those in control (n=20) and CKD (n=22) subjects. Lower serum levels of butyrate (D), acetate (E) and propionate (F) in the CKD-HTN than those in control subjects. Data are presented as mean ± SD. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons. FP, F. prausnitzii; SBP, systolic blood pressure; DBP, diastolic blood pressure; BUN, blood urea nitrogen; Scr, serum creatinine; UA, uric acid; eGFR, estimated glomerular filtration rate. TG, triglyceride; TC, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LPS, lipopolysaccharide; TMAO, trimethylamine N-oxide.

Figure 3.

F. prausnitzii ameliorated renal dysfunction and fibrosis in CKD mice. (A) Schematic protocol for F. prausnitzii treatment on 5/6 Nx-induced CKD mice. The levels of BUN (B) and Scr (C) were significantly decreased upon treatment with F. prausnitzii compared with those in the CKD group. (D) Representative images showing Masson’s trichrome staining of the renal interstitium. (E) Quantitative analysis of the fibrotic area in different groups. (F) Representative drawings of Sirius red staining show the fibrosis in the renal interstitium. (G) Quantitative analysis of the Sirius red staining area in different groups. (H) Representative drawings of periodic acid-Schiff (PAS) staining show the renal injury. (I) Evaluation of tubular injury score in different groups. (J) Representative immunohistochemical drawings of COL1A1 in different groups (scale bar=100 μm). (K) Quantitative analysis of COL1A1 positive staining area in different groups. (L) Representative immunohistochemical drawings of FN in different groups (scale bar=100 μm). (M) Quantitative analysis of FN positive staining area in different groups. (N) Representative immunohistochemical drawings of α-SMA in different groups (scale bar=100 μm). (O) Quantitative analysis of α-SMA positive staining area in different groups. Data are presented as mean ± SD. n=6 per group. P values were determined by two-way ANOVA followed by Tukey’s multiple comparisons. FP, F. prausnitzii; SBP, systolic blood pressure; BUN, blood urea nitrogen; Scr, serum creatinine; COL1A1, collagen type I alpha 1; FN, fibronectin; α-SMA, α-smooth muscle actin.

Figure 4.

F. prausnitzii attenuated renal macrophage infiltration and inflammation in CKD mice. (A) Representative immunohistochemical drawings of F4/80 in different groups (scale bar=100 μm). 5/6Nx-induced CKD mice had a significant increase in the positive expression of F4/80 compared with the sham mice, which ameliorated by F. prausnitzii treatment. (B) Quantitative analysis of F4/80 positive staining area in different groups. Effects of F. prausnitzii treatment on the gene expression levels of Mcpt1 (C), Il1b (D) and Il6 (E) in different groups by real-time RT-PCR. n=6 per group. Data are presented as mean ± SD. *P<0.05; ^**P<0.01; ^***P<0.001; ^****P<0.0001 in two-way ANOVA followed by Tukey’s multiple comparisons. FP, F. prausnitzii; Mcpt1, monocyte chemotactic protein 1; Il1b, interleukin-1β; Il6, interleukin 6.

Figure 5.

F. prausnitzii reduced circulating uremic toxins in CKD mice. Comparison of microbiota-derived uremic toxins levels in the serum: p-cresyl sulfate (A), indoxyl sulfate (B) and TMAO (C). Comparison of plasma microbiota-independent uremic toxins levels: ADMA (D), SDMA (E) and GSA (F). n=6 per group. Data are presented as mean ± SD. P values were determined by two-way ANOVA followed by Tukey’s multiple comparisons. FP, F. prausnitzii; TMAO, trimethylamine N-oxide; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; GSA, guanidinosuccinic acid.

Figure 6.

F. prausnitzii reduced renal inflammation through butyrate-GPR43 pathway. (A) LPS-induced increases in expression of Il1b and Il6 were suppressed by F. prausnitzii supernatant and sodium butyrate in renal podocytes (MPC-5) and tubular epithelial (TCMK-1) cells. GPR43 was knocked down by siRNA in TCMK-1 (B) and MPC-5 (C) cells. One-way ANOVA was performed at the end of the experiment. GPR43 knockdown blocked the F. prausnitzii and sodium butyrate-mediated suppression of Il1b (D) and Il6 (E) expression induced by LPS in the TCMK-1 and MPC-5 cells. Two-way ANOVA was performed at the end of the experiment. FP, F. prausnitzii; NaBu, sodium butyrate.

Figure 7.

F. prausnitzii ameliorated renal dysfunction and inflammation via GPR43 in CKD mice. (A) A schematic diagram for the GPR43 knockdown and F. prausnitzii treatment experiment on 5/6 Nx-induced CKD mice. GPR43 knockdown blocked the butyrate-mediated amelioration of BUN (B) and Scr (C) in the CKD mice. (D) Representative images of MTS, PAS and F4/80 staining in different groups. Quantitative analysis of MTS (E), PAS (F) and F4/80 (G) staining in different groups. Effects of GPR43 knockdown and F. prausnitzii treatment on the gene expression levels of Mcpt1 (H), Il1b (I) and Il6 (J) in different groups by Real-time RT-PCR. n=6 per group. Data are presented as mean ± SD. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons. BUN, blood urea nitrogen; Scr, serum creatinine; MTS, Masson’s trichrome staining; PAS, periodic acid-Schiff staining; Mcpt1, monocyte chemotactic protein 1; Il1b, interleukin-1β; Il6, interleukin 6.

Figure 8.

Butyrate ameliorated renal dysfunction and inflammation via GPR43 in CKD mice. (A) A schematic diagram for the GPR43 knockdown and butyrate treatment experiment on 5/6 Nx-induced CKD mice. (B) Western blotting was used to detect GPR43 expression on protein level. (C) Quantitative analysis of GPR43 protein expression in different groups. GPR43 knockdown blocked the butyrate-mediated amelioration of BUN (D) and Scr (E) in the CKD mice. (F) Representative images of MTS, PAS and F4/80 staining in different groups. Quantitative analysis of MTS (G), PAS (H) and F4/80 (I) staining in different groups. Effects of GPR43 knockdown and butyrate treatment on the gene expression levels of Mcpt1 (J), Il1b (K) and Il6 (L) in different groups by Real-time RT-PCR. n=6 per group. Data are presented as mean ± SD. P values were determined by one-way ANOVA followed by Tukey’s multiple comparisons. BUN, blood urea nitrogen; Scr, serum creatinine; MTS, Masson’s trichrome staining; PAS, periodic acid-Schiff staining; Mcpt1, monocyte chemotactic protein 1; Il1b, interleukin-1β; Il6, interleukin 6.