Clostridium butyricum inhibits the inflammation in children with primary nephrotic syndrome by regulating Th17/Tregs balance via gut-kidney axis

Abstract

Background: Primary nephrotic syndrome (PNS) is a common glomerular disease in children. Clostridium butyricum (C. butyricum), a probiotic producing butyric acid, exerts effective in regulating inflammation. This study was designed to elucidate the effect of C. butyricum on PNS inflammation through the gut-kidney axis.

Method: BALB/c mice were randomly divided into 4 groups: normal control group (CON), C. butyricum control group (CON+C. butyricum), PNS model group (PNS), and PNS with C. butyricum group (PNS+C. butyricum). The PNS model was established by a single injection of doxorubicin hydrochloride (DOX) through the tail vein. After 1 week of modeling, the mice were treated with C. butyricum for 6 weeks. At the end of the experiment, the mice were euthanized and associated indications were investigated.

Results: Since the successful modeling of the PNS, the 24 h urine protein, blood urea nitrogen (BUN), serum creatinine (SCr), urine urea nitrogen (UUN), urine creatinine (UCr), lipopolysaccharides (LPS), pro-inflammatory interleukin (IL)-6, IL-17A were increased, the kidney pathological damage was aggravated, while a reduction of body weights of the mice and the anti-inflammatory IL-10 significantly reduced. However, these abnormalities could be dramatically reversed by C. butyricum treatment. The crucial Th17/Tregs axis in PNS inflammation also was proved to be effectively regulated by C. butyricum treatment. This probiotic intervention notably affected the expression levels of signal transducer and activator of transcription 3 (STAT3), Heme oxygenase-1 (HO-1) protein, and retinoic acid-related orphan receptor gamma t (RORγt). 16S rRNA sequencing showed that C. butyricum could regulate the composition of the intestinal microbial community and found Proteobacteria was more abundant in urine microorganisms in mice with PNS. Short-chain fatty acids (SCFAs) were measured and showed that C. butyricum treatment increased the contents of acetic acid, propionic acid, butyric acid in feces, acetic acid, and valeric acid in urine. Correlation analysis showed that there was a closely complicated correlation among inflammatory indicators, metabolic indicators, microbiota, and associated metabolic SCFAs in the gut-kidney axis.

Conclusion: C. butyricum regulates Th17/Tregs balance via the gut-kidney axis to suppress the immune inflammatory response in mice with PNS, which may potentially contribute to a safe and inexpensive therapeutic agent for PNS.

Keywords: C. butyricum; Gut microbiota; Inflammation; PNS; Th17/Tregs; Urinary microbiota.

Fig. 1

The impacts of C. butyricum treatment on BWs, 24 h urinary protein and kidney function in DOX-induced PNS mice. Experimental design time diagram (A). BWs: Body weights (B). 24 h urine protein of mice in diverse groups (C). BUN: Blood urea nitrogen (D). SCr: Serum creatinine (E). UUN: Urine urea nitrogen (F). UCr: Urine creatinine (G). Data were expressed as mean±SD. *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. All experiments were performed in triplicate

Fig. 2

The effect of C. butyricum treatment on pathological progress of PNS mice. Representative images of kidney pathological staining including HE: hematoxylin and eosin (A), Masson's trichrome stain (B), PAS: periodic acid Schiff (C), PASM: periodic acid-silver methenamine (D)

Fig. 3

C. butyricum treatment reduced lipopolysaccharide (LPS) level in mice with PNS. Gut LPS levels (A). Kidney LPS levels .(B) Data were expressed as mean±SD. **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were performed in triplicate

Fig. 4

Regulation of Th17/Tregs balance by dietary C. butyricum treatment in mice with PNS. Flow cytometry analysis was used to separately determine the proportions of splenic Th17 cells (A), splenic Treg cells (B), peripheral blood Treg cells (C) and colon Treg cells (D) in diverse groups. Data were expressed as mean±SD. *P < 0.05, **P < 0.01. All experiments were performed in triplicate

Fig. 5

Suppression of the immune inflammatory reaction by dietary C. butyricum treatment in mice with PNS. Kidney tissue was elicited to determine the concentrations of IL-10 (A), IL-6 (B) and IL-17A (C). Data were expressed as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were performed in triplicate

Fig. 6

C. butyricum alleviated the immune inflammatory response in PNS through the HO-1/STAT3/RORγt signaling pathway. The mRNA levels of Keap1 (A), Nrf2 (B), HO-1 (C), JAK2 (D), STAT3 (E), and RORγt (F) in the kidney tissues. Representative western blot images and statistical results of HO-1 (G,H), JAK2 (G,I), STAT3 (G,J), and RORγt (G,K) expressions of protein levels in the kidney tissues. HO-1/STAT3/RORγt pathway diagram (L). Data were expressed as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All experiments were performed in triplicate

Fig. 7

The modulation of gut microbiome by probiotic C. butyricum supplementation in mice with PNS. Dilution curve (A). Alpha diversity analysis included observed species, Chao1 and Shannon (B). Beta diversity analysis includes principal coordinate (PcoA) analysis and non-metric multidimensional scaling (NMDS) analysis (C). Relative abundance of microbial species at the phylum level (D). Ratio of Firmicutes to Bacteroidetes (E). Relative abundance of microbial species at the genus level (F). Heatmap of species composition at the genus level of species clustering (G). LEfSe analysis displaying of inter-group differential taxa based on taxonomic tree H. Bacteroides (I). Parabcteroides (J). Bacillus (K). Adlercreutzia (L). Venn diagram M. Data were expressed as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 8

The changes of urinary microbiota after C. butyricum treatment in mice with PNS. Dilution curve (A). Alpha diversity analysis included observed species, Chao1 and Shannon (B). PcoA analysis (C). NMDS analysis (D). Relative abundance of microbial species at the phylum level (E). Relative abundance of microbial species at the genus level (F). Heatmap of species composition at the genus level of species clustering (G). Venn diagram (H)

Fig. 9

C. butyricum treatment increased the contents of short-chain fatty acids (SCFAs) in feces of PNS mice. Chromatogram of mouse fecal samples (A). Relative standard deviation (B). Cluster heatmap of whole metabolites of gut microbiota (C). Acetic acid (D). Propionic acid (E). Butyric acid (F). Valeric acid (G). Caproic acid (H). Isovaleric acid (I). Isobutyric acid (J). Data are expressed as mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 10

Effect of C. butyricum treatment on the contents of SCFAs in urine of PNS mice. Chromatogram of mouse urine samples (A). Relative standard deviation (B). Cluster heatmap of whole metabolites of urine microbiota (C). Acetic acid (D). Propionic acid (E). Butyric acid (F). Valeric acid (G). Caproic acid (H). Isobutyric acid (I). Isovaleric acid (J). Data were expressed as mean±SD. *P < 0.05

Fig. 11

Correlation analyses among relative abundance of gut/urine microbiota and other related indicators. Correlation of gut microbiota with inflammation, metabolic indicators, and SCFAs in PNS (A). Correlation of urine microbiota with inflammation, metabolic indicators, and SCFAs in PNS (B). *P < 0.05, **P < 0.01

Fig. 12

Patterns of effectiveness of C. butyricum for the treatment of PNS by regulating Th17/Tregs axis via the HO-1/STAT3/RORγt signaling pathway and modulating gut microbiota in mice