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. Author manuscript; available in PMC: 2023 Oct 14.
Published in final edited form as: Circ Res. 2022 Sep 27;131(9):e120–e134. doi: 10.1161/CIRCRESAHA.122.320184

Faecalibacterium Prausnitzii Attenuates CKD via Butyrate-Renal GPR43 Axis

Hong-Bao Li 1,*,+, Meng-Lu Xu 2,+, Xu-Dong Xu 3,+, Yu-Yan Tang 3,+, Hong-Li Jiang 4, Lu Li 2, Wen-Jie Xia 1, Nan Cui 5, Juan Bai 6, Zhi-Ming Dai 7, Bei Han 8, Ying Li 1, Bo Peng 1, Yuan-Yuan Dong 1, Sachin Aryal 9, Ishan Manandhar 9, Mahmoud Ali Eladawi 10, Rammohan Shukla 10, Yu-Ming Kang 1,*, Bina Joe 9,*, Tao Yang 9,*
PMCID: PMC9588706  NIHMSID: NIHMS1836089  PMID: 36164984

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 regarding if and how F. prausnitzii can be used as a probiotic to treat CKD remains unknown.

Methods:

We evaluated the microbial compositions in two 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. Lastly, we investigated the molecular mechanisms underlying the probiotic-induced beneficial outcomes.

Results:

We observed significant depletion of Faecalibacterium in the CKD patients 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 G protein-coupled receptor (GPR) 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 GPR43 signaling in the kidney. Our study provides the necessary foundation to harness the therapeutic potential of F. prausnitzii for ameliorating CKD.

Keywords: F. prausnitzii, chronic kidney disease, gut microbiota, butyrate, GPR43, Animal Models of Human Disease, Inflammation, Mechanisms, Nephrology and Kidney, Omics

Graphical Abstract

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Introduction

Chronic kidney disease (CKD) is one of the most important public health issues impacting approximately 10–15% of the world’s population1. The progression of CKD is commonly associated with hypertension, renal dysfunction, renal fibrosis, and ultimate chronic renal failure2,3. Because of the absence of symptoms in the early stage of the disease, timely prevention or treatment of CKD is challenging. Current strategies to manage CKD are confined to dialysis and kidney transplantation, whereby, chronic renal failure is an expensive disease to treat on a per-patient basis4. Therefore, innovative concepts for new therapeutic approaches are needed for better clinical management of CKD.

Emerging evidence suggests the important role of gut microbiota in the development and progression of CKD5. Although current evidence supports the beneficial role of rebalancing gut microbiota to manage CKD, a knowledge gap exists on whether specific gut microbiota and their metabolites can be exploited as potential therapeutics for CKD. Two classic probiotics, Lactobacillus and Bifidobacterium, have been reported to be depleted in the CKD6–8. Multiple studies have reported the beneficial effects of probiotics and prebiotics9–11. However, a recent meta-analysis of 645 participants in a total of sixteen studies by McFarlane et al. found limited evidence to support the use of traditional probiotics (including Lactobacillus and Bifidobacterium), prebiotics, or synbiotics in CKD12. Thus, our objective was to identify commensal for contemplating novel strategies and better management of CKD.

Faecalibacterium prausnitzii is a beneficial butyrate-producing bacterium with anti-inflammatory properties that promotes intestinal homeostasis13. Previous studies have demonstrated its depletion during the progression of CKD towards end-stage renal disease (ESRD)6,14. However, no studies have investigated if F. prausnitzii was a cause or effect in CKD. Thus, the current study was designed to first determine if F. prausnitzii was consistently depleted in CKD populations representing patients from both western and eastern hemispheres; next, to evaluate if supplementation of F. prausnitzii improves renal function and if so, to delineate the mechanism. Using the American Gut Project database and fecal microbial data collected from a Chinese population in Shanghai, we consistently identified reduced Faecalibacterium in the CKD patients, regardless of variations in diet preference, geographical locations and ethnicity between the two groups. These data reinforced the potentially important role of Faecalibacterium in the CKD. Oral administration of F. prausnitzii in a mouse model of CKD demonstrated beneficial effects of F. prausnitzii on intestinal homeostasis and renal function. Finally, we demonstrated the mechanism by which F. prausnitzii suppressed inflammation and improved renal function is through its metabolite butyrate and renal GPR43 receptor.

Materials and Methods

Data availability

The raw data of 16S rRNA gene sequencing are available from NCBI Sequence Read Archive under BioProject (PRJNA797660).

Gut microbiota analysis

Illumina NovaSeq 6000 (Illumina, San Diego, CA) was used to sequence the library. The sequence reads were analyzed using QIIME 215. The reads were de-noised, merged, and chimera filtered with Divisive Amplicon Denoising Algoruthm 2 (DADA2)16, resulting in an ASVs table. The taxonomic assignments of ASVs representative sequence were performed with a naive Bayes classifier, which was trained on the SILVA database (version 138.1). Taxonomic alpha diversity (ie, Observed features, Chao1 richness and Shannon diversity) were calculated using the R package Vegan17.

Measurement of SCFAs and toxins

Plasma and fecal SCFAs levels were measured by GC-MS as reported previously with minor modification18. The levels of circulating PCS, IS, TMAO, ADMA, SDMA and GSA were determined using LC-MS as previously described19.

Microinjection of adeno-associated virus (AAV) into mouse kidney

To knockdown GPR43 expression in vivo, we designed an AAV serotype 9 vector encoding a green fluorescent protein reporter together with either short hairpin RNAs (shRNAs) targeting Gpr43 (AAV-shGpr43) or an empty vector (AAV-null) in the kidney as previously described20. AAV encoding Gpr43 (1×1012 vector genomes/mL; 10 μL) or empty vector were administered to mice at least five distributed points in the kidney as described previously21.

Results

Depletion of Faecalibacterium in the American CKD population

Gut microbial composition of 283 CKD and 294 non-CKD patients from the American Gut Project22 (obtained through Redbiom23) were compared using the DESeq2 analysis (Table S5). Two representative bacterial genera were found different between CKD and non-CKD. Faecalibacterium genus was enriched in the non-CKD (Figure 1A) and Bacteroides was enriched in the CKD (Figure 1B).

Figure 1.

Figure 1.

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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.

Microbial dysbiosis and depletion of Faecalibacterium in the Chinese CKD population

To test whether the depletion of Faecalibacterium in the CKD is a consistent feature independent of interindividual variation, we performed the microbial analysis in 105 Chinese subjects consisting of 30 healthy volunteers, 32 clinically diagnosed CKD patients without hypertension, and 43 CKD patients with hypertension (CKD-HTN). Clinical characteristics24,25 of the subjects are listed in Table S2.

The rarefaction curves of all samples based on the AVSs showed the sequencing depth of 22,000 reads (Figure S1). DESeq2 analysis was used to compare the microbial differences between groups (Table S6, Table S7). Consistently, we demonstrated that Faecalibacterium genus was enriched in the healthy controls and significantly depleted in the CKD-HTN groups (Figure 1C). We also observed an increased abundance of Bacteroides in CKD and CKD-HTN compared with controls (Figure 1D). Also, we observed a higher F/B (Firmicutes/Bacteroidetes) ratio, which is a feature identified with gut dysbiosis, in CKD-HTN patients, compared with healthy controls (Figure 1E). Parameters (i.e. observed features, Chao1 richness and Shannon diversity) of α-diversity were lower in the CKD and CKD-HTN, compared with those in the healthy controls (Figure 1F–H).

Negative correlations of Faecalibacterium with CKD-associated clinical characteristics

To investigate whether the alterations in gut microbiota was relevant to any clinical characteristics of the CKD, Spearman’s unadjusted correlations were assessed between the abundance of multiple bacterial genera and SBP, DBP, BUN, Scr, UA, eGFR, TC, TG, HDL as well as LDL. We demonstrated that Faecalibacterium was the unique bacterial genus that showed negative correlations with UA, BUN, Scr and LDL, and positive correlations with eGFR, TC and HDL (Figure 2A). These suggest its role in renal function.

Figure 2.

Figure 2.

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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.

Decreased SCFAs and increased LPS and TMAO in the sera of CKD patients

Due to the important role of gut-kidney axis in hypertension and CKD5, we investigated the serum levels of several microbiota-derived metabolites. Trimethyl amine N-oxide (TMAO), a molecule produced from dietary phosphatidylcholine or carnitine via gut microbial metabolism, was significantly higher in the CKD-HTN than the CKD and control (Figure 2B). The level of LPS, an endotoxin of gram-negative bacteria, was the highest in the sera of CKD-HTN patients (Figure 2C). Butyrate, a major metabolite produced by Faecalibacterium was found decreased in both CKD and CKD-HTN (Figure 2D). In addition, acetate level was reduced in the CKD and CKD-HTN (Figure 2E). Propionate was found less in the CKD-HTN group compared with control group (Figure 2F).

F. prausnitzii ameliorated renal dysfunction and fibrosis in CKD mice

Next, we hypothesized that supplementation of F. prausnitzii, the only species in the Faecalibacterium genus, would ameliorate CKD-associated pathophysiological changes in the rodent model of CKD. This was because of the enrichment of Faecalibacterium in the healthy controls (Figure 1) and its strong correlation with the clinical characteristics of CKD patients (Figure 2A). Therefore, we induced CKD in mice by 5/6 Nx surgery for 4 weeks, followed by gavaging with 200μL of either 2×108 CFU of F. prausnitzii (FP)26 or vehicle (PBS) three times a week for 16 weeks (Figure 3A). The CKD group exhibited increased levels of BUN (Figure 3B) and Scr (Figure 3C) compared with the sham group. The increased BUN and Scr in the CKD mice were attenuated by F. prausnitzii treatment (Figure 3B–C). Sirius red and MTS staining results also demonstrated that F. prausnitzii treatment attenuated the pathological progression of the CKD, as demonstrated by reduced renal fibrosis in the kidney interstitium (Figure 3D–G), reduced renal lesion and tubular injury (Figure 3H–I), compared with that in the CKD group. Renal fibrosis makers COL1A1, FN and α-SMA were stained to confirm the improvement of renal fibrosis and injury by F. prausnitzii, demonstrated by significantly diminished COL1A1 (Figure 3J–K), FN (Figure 3L–M) and α-SMA (Figure 3N–O)-positive areas in the CKD+FP group, compared with the CKD.

Figure 3.

Figure 3.

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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.

Consistent with the immunohistochemical staining results, the expression levels of COL1A1, FN and α-SMA was higher in the CKD group. F. prausnitzii treatment significantly reduced the expression of the genes encoding α-SMA (Acta2) and FN (Fn1), but not COL1A1 (Col1a1) (Figure S2).

F. prausnitzii attenuated renal macrophage infiltration and inflammation in CKD mice

We next investigated the inflammatory state in the kidney. Increased renal infiltration of macrophage was observed in the CKD mice, determined by increased F4/80-positive staining. F. prausnitzii treatment ameliorated macrophage infiltration as indicated by reduced F4/80 positive staining (Figure 4A–B). In line with this, the upregulated mRNA levels of Mcpt1, Il1b and Il6 in the renal cortex of 5/6 Nx-induced CKD mice were partially rescued by F. prausnitzii treatment (Figure 4C–E).

Figure 4.

Figure 4.

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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.

F. prausnitzii reduced circulating uremic toxins in CKD mice

To demonstrate whether F. prausnitzii treatment improved circulating uremic toxins in CKD mice, we measured the levels of three microbiota-derived toxins (PCS, IS and TMAO) and three microbiota-independent uremic toxins (ADMA, SDMA and GSA) in the plasma. The CKD group exhibited increased plasma levels of all 6 toxins, compared with the sham group (Figure 5A–F). F. prausnitzii treatment markedly reduced the levels of PCS, TMAO and GSA in the plasma (Figure 5A, 5C, 5F), but not IS, ADMA and SDMA (Figure 5B, 5D, 5E).

Figure 5.

Figure 5.

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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.

F. prausnitzii altered gut microbial composition in CKD mice

Next, we tested if the beneficial effects of F. prausnitzii administration were through alterations in CKD-associated gut microbiota. Rarefaction of samples to 45,000 reads showed consistent and plateauing diversity metrics across samples (Figure S3A). Decreased Shannon diversity was observed in the CKD mice when compared with sham control, although changes in the Observed features and Shannon diversity were not significant (Figure S3B–D). F. prausnitzii treatment significantly increased observed features, Chao 1 richness and Shannon diversity (Figure S3B–D). In the principal coordinate analysis (PCoA), four clusters formed, each representing one group. CKD group showed significant separation from sham control group (ANOSIM, R= 0.802; P= 0.003, Figure S3E). Treatment with FP in the CKD (CKD+FP) significantly altered the microbiota and separated it from the CKD group (ANOSIM, R= 0.704; P= 0.002, Figure S3E). At the genus level, we observed that Desulfovibrio and Collinsella were more abundant in the CKD group as compared to control group, while Faecalibacterium and Roseburia were less abundant. 16 weeks of F. prausnitzii treatment decreased the average abundance of Desulfovibrio and Collinsella genera and increased the average abundance of Roseburia and Faecalibacterium genera in the CKD mouse (Figure S3F–I). This suggested that F. prausnitzii altered gut microbiota towards a balance of diversity and homeostasis.

F. prausnitzii ameliorated intestinal inflammation and permeability in CKD mice

Increased intestinal inflammation and barrier injury contributes to the pathogenesis of CKD27,28. Therefore, we investigated the effects of F. prausnitzii treatment on intestinal inflammation and permeability. CKD induced increases in the gene expression of Mcpt1, Il6, Il1b, Tlr2 and Tlr4 in proximal colon of CKD group, compared with those in the sham group (Figure S4A–E). F. prausnitzii treatment reduced Mcpt1, Il6 and Tlr2 mRNA levels in the proximal colon of CKD+FP group (Figure S4A, B, E). For the intestinal permeability, we performed (i) real-time RT-PCR29 for tight junction proteins; (ii) oral gavage of FITC-dextran30, followed by the evaluation of its level in plasma; (iii) ELISA for the plasma level of intestinal fatty acid binding protein 2 (I-FABP, a surrogate marker shows damage to enterocytes). The mRNA levels of Tjp1 (tight junction protein 1) and Cldn4 (claudin 4) in proximal colon were significantly lower in the CKD group than those in the control group. F. prausnitzii treatment restored the expression of Tjp1 and Cldn4 (Figure S4F–G). FITC-dextran and I-FABP levels were increased in plasma of the CKD mice, both of which were rescued by the treatment of F. prausnitzii (Figure S4H–I).

Effects of F. prausnitzii on plasma and fecal SCFAs production in CKD mice

F. prausnitzii is a well-recognized butyrate-producer. To elucidate the underlying mechanism in the renoprotective effect by F. prausnitzii, we analyzed the levels of SCFAs in all four groups of mice. As shown in Table S4, less butyrate was observed in the feces and plasma of the CKD mice, compared with sham mice. F. prausnitzii increased butyrate levels in both feces and plasma. No effects of F. prausnitzii on acetate or propionate were found. These results along with the changes of microbiota suggest that increased butyrate production may be the underlined mechanisms responsible for the renoprotective effect by F. prausnitzii.

F. prausnitzii increased the expression of GPR43 in CKD mice

To further investigate if the F. prausnitzii-mediated renoprotection was through butyrate, we assessed the expression of butyrate receptors, G-protein–coupled receptor (GPR) 43 (Figure S5A), GPR109A (Figure S5B), and GPR41 (Figure S5C). The gene expression of Gpr43, which was lower in CKD, was rescued in the kidney tissues by F. prausnitzii treatment of CKD mouse (Figure S5A), suggesting that the renoprotection by the butyrate producing F. prausnitzii is likely via the excess availability of butyrate to bind to its receptor GPR43. Immunohistochemical staining of GPR43 illustrated consistent expression of GPR43 in the tubular epithelial cells and renal podocytes in the CKD mice (Figure S5D, S5E) and CKD patients (Figure S5F). Immunofluorescent staining also demonstrated that AQP3 (a marker for tubular epithelial cells)-positive and WT-1 (a marker for podocytes)-positive cells expressed GPR43 (Figure S6A–S6D).

F. prausnitzii reduced renal inflammation through butyrate-GPR43 pathway

Next, we established in vitro models with LPS-induced inflammation in the tubular epithelial cells TCMK-131,32 and renal podocytes MPC-533 to test if F. prausnitzii harbors any renal anti-inflammatory effects (Figure 6A), because renal inflammation is a hallmark throughout different stages of CKD. In both cell lines, we confirmed that the LPS-induced overexpression of proinflammatory Il1b and Il6 was suppressed by culture supernatant of F. prausnitzii and sodium butyrate (Figure 6A). To demonstrate the role of GPR43 in F. prausnitzii and butyrate-mediated suppression of renal inflammation, GPR43 was knocked down using siRNA34,35 and confirmed by western blot36 in TCMK-1 and MPC-5 (Figure 6B–C). GPR43 knockdown blocked the F. prausnitzii-mediated downregulation of Il1b and Il6 mRNA levels in LPS-treated TCMK-1 and MPC-5 cells (Figure 6D–E).

Figure 6.

Figure 6.

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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.

F. prausnitzii improved renal dysfunction and inflammation via GPR43 in CKD mice

To determine if the F. prausnitzii-mediated renoprotection was through GPR43, we knocked down GPR43 in mouse kidneys using adeno-associated virus that carries short hairpin RNA targeting mouse Gpr43 coding sequence (Figure 7A). The knockdown of GPR43 in the kidney was confirmed by immunofluorescent staining (Figure S6A–D). As shown in Figure 7B and 7C, GPR43 knockdown blocked the reduced BUN and Scr by F. prausnitzii treatment in the CKD mice. As determined by MTS and PAS staining37, GPR43 knockdown inhibited the alleviation of fibrosis (Figure 7D, 7E) and tubular injury (Figure 7D, 7F) in the kidney by F. prausnitzii treatment. Knockdown of GPR43 in the kidney blocked the reduced positive staining of F4/80 by F. prausnitzii treatment (Figure 7D, 7G). The reduced mRNA expression of several inflammatory genes, including Mcpt1 (Figure 7H), Il1b (Figure 7I) and Il6 (Figure 7J), in the kidney by F. prausnitzii treatment was also inhibited by GPR43 knockdown.

Figure 7.

Figure 7.

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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.

Butyrate ameliorated renal dysfunction and inflammation via GPR43 in CKD mice

To further determine if the beneficial effects of F. prausnitzii are due to increased butyrate and GPR43 signaling in the kidney, we knocked down GPR43 expression using adeno-associated virus and given sodium butyrate treatment38 in the CKD mice (Figure 8A). Renal GPR43 expression was knocked down by transduction with the AAV vector after four weeks (Figure 8B–C). GPR43 knockdown blocked the butyrate-mediated amelioration of BUN (Figure 8D) and Scr (Figure 8E) in the CKD mice. MTS and PAS staining results also showed that GPR43 knockdown inhibited the alleviation of fibrosis (Figure 8F, 8G) and tubular injury (Figure 8F, 8H) in the kidney by butyrate treatment. We also found that GPR43 knockdown blocked the reduced F4/80 positive staining (Figure 8F, 8I) and mRNA levels of Mcpt1 (Figure 8J), Il1b (Figure 8K) and Il6 (Figure 8L) in the kidney by butyrate treatment. Taken together, our results demonstrated that protection conferred by F. prausnitzii on renal inflammation and dysfunction operates through the butyrate-GPR43 pathway.

Figure 8.

Figure 8.

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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.

Discussion

In this study, we verified that depletion of Faecalibacterium is a key signature in both Western and Eastern CKD patients, independent of ethnicity. Moreover, we uncovered an unrecognized role of F. prausnitzii against CKD progression and further demonstrated the mechanism that F. prausnitzii-derived butyrate interacts with GPR43 to exhibit its beneficial effects against CDK. Taken together, our study illustrated a mechanistic role of F. prausnitzii in restoration of renal function and expanded the prospect for the use of F. prausnitzii as a probiotic for CKD treatment.

The association of gut dysbiosis with CKD has been determined in both rodent models and human patients5,7. The dysbiotic hallmark in CKD is an imbalanced gut microbiota with the depletion of short-chain fatty acid-producing bacteria and the bloom of uremic toxin-producing pathobionts6,39,40. Accumulation of uremic toxins deteriorates multiple organs including the kidney. On the other hand, quantitative reduction in SCFAs, especially butyrate, also contributed to the progression of CKD18. Provided with the evidence, we aim to identify a bacterial species that could be used as a probiotic for CKD. In this study, we verified that CKD patients suffer from dysbiosis with a significant reduction in butyrate-producing bacteria F. prausnitzii and resultant fecal butyrate levels.

To further dissect the role of gut microbiota, Faecalibacterium was selected because of (1) its substantial link with various clinical characteristics of CKD patients; (2) its health-promoting characteristics as an emerging probiotic; (3) its consistent depletion in CKD. F. prausnitzii, the genus’s only known species, is a potent butyrate producer. It constitutes more than 5% of the human gut microbiome, making it a highly abundant microbe among all the bacterial species in the gut41. Depletion of F. prausnitzii has been considered a biomarker for inflammatory bowel disease, especially in the context of the F. prausnitzii – Escherichia coli ratio42. However, the role of F. prausnitzii in CKD remains poorly understood. Taking the advantage of successful culture of strictly anaerobic F. prausnitzii41, we showed that the renoprotective effects of F. prausnitzii are associated with the altered gut microbial composition, remodeled intestinal homeostasis and the anti-inflammatory butyrate-GPR43 signaling, all of which qualify F. prausnitzii as a probiotic.

Butyrate exerts anti-inflammatory effects, maintains gut homeostasis and also has an impact on tissues and organs beyond the gut when absorbed into circulation43,44. In the diabetic nephropathy mice, Li et al. found that the SCFA-mediated protection was reliant on GPR43 and/or GPR109A45. We found a feedback mechanism that elevates GPR43 expression in response to F. prausnitzii oral gavage in the mouse 5/6 Nx CKD model. Additionally, since the expression of GPR43 was mainly found in tubular epithelial cells and renal podocytes, we investigated the role of GPR43 in renal inflammation, a common feature in CKD. We observed that knock down of GPR43 in the tubular epithelial cells and renal podocytes in vitro diminished the anti-inflammatory effects of butyrate as well as cultured medium of F. prausnitzii. These strongly support that F. prausnitzii-derived butyrate exhibits anti-inflammation through GPR43 receptor. Multiple lines of evidence have shown that inflammation has a direct pathogenic role in the pathogenesis of CKD46. Pro-inflammatory cytokines such as IL-1β47 and IL-648 have been shown to positively correlate with the severity of chronic kidney disease. Numerous studies showed the potential of treating CKD using anti-inflammatory agents49. Therefore, our data suggest that inflammation suppression through renal butyrate-GPR43 signaling may be one of the mechanisms underlying F. prausnitzii’s favorable effects in CKD therapy. Additionally, the butyrate-GPR43 axis was examined in vivo for its involvement in renal function. In CKD mice, we demonstrated that butyrate supplementation in drinking water decreased BUN and Scr levels in the urine, attenuated renal pathology and macrophage infiltration, and reduced kidney inflammation. The lack of kidney specific Gpr43 deletion model precludes us from further examining the specificity of these observations. Instead, in a new group of mice, we used multiple-site injections of AAV-shGpr43 to knock down GPR43 in the kidney. Protection from butyrate was diminished in CKD+AAV-shGpr43 mice compared to CKD mice injected with AAV-null. Taken together, our data support the hypothesis that gut commensal F. prausnitzii ameliorated chronic kidney disease in mice through the butyrate-Gpr43 axis.

In line with the improved renal function in the CKD mice treated with F. prausnitzii, we also demonstrated that F. prausnitzii altered gut microbial composition and restored intestinal homeostasis determined by ameliorated intestinal inflammation and permeability. For the gut microbiota, inhibition of the toxins-producing bacteria (e.g. Desulfovibrio and Collinsella) and promotion of the butyrate-producing bacteria (e.g. Roseburia and Faecalibacterium) were observed in the mice treated with F. prausnitzii. Desulfovibrio and Prevotella are TMAO producing bacteria and have a strong positive correlation with the risk of adverse cardiovascular events50–52. Furthermore, increased TMAO production has been directly linked to the progressive of renal fibrosis and the poorer long-term survival of CKD patients53,54. Consistently, an increase in TMAO in circulation was noted in the CKD mice. Furthermore, F. prausnitzii treatment markedly reduced the elevated TMAO level. On the other hand, increased abundances of Roseburia and Faecalibacterium were observed in F. prausnitzii treated-CKD mice. Along with the reduced gut inflammation and permeability by F. prausnitzii treatment, these positive changes resulted in the restoration of gut homeostasis and ecology, which we interpret as further contributions to the re-balancing of gut microbiota-derived metabolites (i.e. SCFAs, uremic toxins) in the circulation. These findings underscored the importance of gut-kidney communication in the management of CKD.

The importance of mutual communication between kidney and gut is widely recognized. Dysbiotic microbiota increased the production of gut-derived uremic toxins and altered the intestinal barrier. These changes in turn contribute to the acceleration of renal injury5. Multiple strategies have been attempted to target gut microbiota or its metabolites for therapy of CKD. In the Synbiotics Easing Renal Failure by Improving Gut Microbiology (SYNERGY) trial, supplementation of synbiotics (i.e. high–molecular weight inulin, fructo-oligosaccharides, and galacto-oligosaccharides as prebiotics and nine different strains across the Lactobacillus, Bifidobacteria, and Streptococcus genera as probiotics) reduced p-cresyl sulfate, but not indoxyl sulfate in the serum, though the gut microbiome was favorably changed11. Resistant starch55,56 have been used as prebiotics to slow the progression of CKD, reduced plasma levels of indoxyl sulfate, improve gut homeostasis. In a recent meta-analysis, McFarlane et al. found that probiotics in Lactobacillus, Bifidobacterium, and Streptococcus genera have not shown positive benefits in CKD. Thus, there is a need to identify beneficial commensal potentially for CKD. In our study, we utilized the well-established rodent 5/6 nephrotomy CKD model, which represents a simple model of progressive renal disease by reduced nephron number57. This minimizes the influences of other factors (e.g. hypertension and diabetes) contributing to CKD pathogenesis. We verified the depletion of F. prausnitzii in CKD patients and further validated its probiotic property to attenuate CKD progression in a mouse model. In addition, F. prausnitzii was able to restore the gut environment favorable for its colonization and butyrate production. This is important for the successful colonization of introduced probiotics, since deteriorated uremic milieu in the gut was found in CKD58. It is also important to note that the identification of the beneficial effects of F. prausnitzii-butyrate-GPR43 axis in the 5/6 nephrotomy CKD model presents with the novel idea to expand the composition of this commensal in the gut to treat CKD. It does not rule out other possible pathways as CKD treatment targets, especially because the etiology of CKD is multifactorial.

In summary, the most significant findings in our current study include: (1) verification of the depletion of F. prausnitzii as a marker in two independent populations; (2) for the first-time demonstration that F. prausnitzii attenuated renal inflammation, reduced uremic toxins, and delayed the progression of CKD in a mouse model. (3) demonstration that F. prausnitzii corrected gut dysbiosis, restored gut homeostasis in mouse CKD model. (4) illustration that F. prausnitzii-derived butyrate exerted renal protective effect through GPR43, which contributes to its beneficial effect in CKD. Given the global growth of CKD incidence and associated mortality coupled with the challenges for prevention and limited therapeutic solutions for CKD, our study in mice proposes a novel means of exploiting a commensal, F. prausnitzii, as a probiotic for the treatment of CKD.

Supplementary Material

Table S5
Table S6
Table S7
Table S8
Table S9
Table S10
320184 ARRIVE Guidelines
320184 Data Supplement
320184 Major Resources Table

Novelty and Significance.

What is known?

  • Supplementation using “traditional” probiotics in chronic kidney disease (CKD) has shown inconsistent outcomes.

  • Depletion of butyrate producing Faecalibacterium prausnitzii is found in chronic kidney disease.

What new information does this article contribute?

  • Levels of F. prausnitzii are decreased in both the US and Chinese CKD population.

  • Supplementation of F. prausnitzii or butyrate attenuated CKD and partially restored renal function.

  • Knock down of GPR43 in the kidney abolished the beneficial effects of F. prausnitzii or butyrate in the CKD models.

Lack of symptoms in the early stage makes the diagnosis and treatment of CKD a significant public health issue. Limited available treatments for late stage disease impacts quality of life.. Gut dysbiosis was reported in CKD, but clinical trials testing probiotic supplementation have been disappointing. Our current study identified a novel commensal bacterium F. prausnitzii that attenuated the progression of CKD and restored renal functions in murine CKD models. Furthermore, we demonstrated that the beneficial effects of F. prausnitzii on the kidney was through the metabolite butyrate and renal GPR43. This study provides evidence for the potential use of F. prausnitzii as a probiotic in CKD.

Acknowledgments

The authors would like to convey heartfelt gratitude to the participants for their contribution.

Sources of funding

This study is supported by the grants (No. 82170443, No.82200831, No. 81774060, No. 82004083, and No. 81770426) from the National Natural Science Foundation of China, the grant (No. 2021SF-153) from Key Research and Development Program of Shaanxi Province, Fundamental Research Funds for the Central Universities (Xi’an Jiaotong University, xzy012020087), Natural Science Foundation of Shaanxi (No. 2022JQ-840), Scientific Research Program Funded by Shaanxi Provincial Education Department (Program No. 22JK0551), NIH R01HL143082 to BJ, American Heart Association Career Development Award 852969 and NIH R21AG079357 to TY.

Non-standard Abbreviations and Acronyms

FP

F. prausnitzii

CKD

chronic kidney disease

BUN

blood urea nitrogen

Scr

serum creatinine

FDR

false discovery rate

Egfr

estimated glomerular filtration rate

SCFAs

short-chain fatty acids

GPR

G protein-coupled receptor

BP

blood pressure

HTN

hypertension

TMAO

trimethylamine N-oxide

LPS

lipopolysaccharide

ADMA

asymmetric dimethylarginine

SDMA

symmetric dimethylarginine

GSA

guanidinosuccinic acid

Footnotes

Disclosures

None.

Supplemental Material

Expanded Materials & Methods

References 15–20, 22–26, 29, 30, 34–38

Online Figure S1–S6

Online Tables S1–S4

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S5
NIHMS1836089-supplement-Table_S5.xlsx (800.1KB, xlsx)
Table S6
NIHMS1836089-supplement-Table_S6.xlsx (96.9KB, xlsx)
Table S7
NIHMS1836089-supplement-Table_S7.xlsx (98.9KB, xlsx)
Table S8
NIHMS1836089-supplement-Table_S8.xlsx (38.4KB, xlsx)
Table S9
NIHMS1836089-supplement-Table_S9.xlsx (38.7KB, xlsx)
Table S10
NIHMS1836089-supplement-Table_S10.xlsx (43.9KB, xlsx)
320184 ARRIVE Guidelines
NIHMS1836089-supplement-320184_ARRIVE_Guidelines.pdf (21.4KB, pdf)
320184 Data Supplement
NIHMS1836089-supplement-320184_Data_Supplement.pdf (2MB, pdf)
320184 Major Resources Table
NIHMS1836089-supplement-320184_Major_Resources_Table.pdf (147.7KB, pdf)

Data Availability Statement

The raw data of 16S rRNA gene sequencing are available from NCBI Sequence Read Archive under BioProject (PRJNA797660).

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