Dysbiosis of Gram-negative gut microbiota and the associated serum lipopolysaccharide exacerbates inflammation in type 2 diabetic patients with chronic kidney disease

Abstract

Lipopolysaccharide (LPS), a potent endotoxin present in the outer membrane of Gram-negative bacteria, causes chronic immune responses associated with inflammation. In the present study, the association between LPS and the dysbiosis of Gram-negative bacteria in the gut microbiome was determined in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (T2DM-CKD; stages 4 and 5, not on dialysis) compared with healthy individuals. Microbiome diversity was analyzed in patients with T2DM-CKD and healthy controls by sequencing the hypervariable sub-regions of the 16S ribosomal RNA gene from stool samples. Serum samples were assayed by ELISA for LPS, C-reactive protein (CRP), tumor necrosis factor-α (TNFα), interleukin-6 (IL6) and endothelin-1. A total of four gut Gram-negative phyla (Bacteroidetes, Proteobacteria, Fusobacteria and Verrucomicrobia) were identified in the gut microbiome of the T2DM-CKD and control groups. Proteobacteria, Verrucomicrobia and Fusobacteria exhibited significantly increased relative abundance in patients with T2DM-CKD when compared with controls (P<0.05). The levels of serum LPS were significantly increased in patients with T2DM-CKD compared with controls (P<0.05). Elevated serum LPS was significantly correlated with increased levels of TNFα, IL6 and CRP. The dysbiosis of Gram-negative bacteria in the gut microbiome of patients with T2DM-CKD may contribute to the elevated serum levels of LPS and the consequential effects on the inflammatory biomarkers in these patients. The association between the dysbiosis of Gram-negative bacteria in the gut microbiome of patients with T2DM-CKD, increased LPS levels and the effects on inflammatory biomarkers may provide insight into potential diagnostic and therapeutic approaches in the treatment of T2DM-CKD.

Keywords: Gram-negative bacteria; chronic kidney disease; gut microbiome; inflammatory markers; lipopolysaccharide; type 2 diabetes mellitus.

Figure 1.

Identified gut microbiota phyla in patients with T2DM-CKD and healthy individuals. The relative abundances were expressed as mean percentages. T2DM, type 2 diabetes mellitus.

Figure 2.

Relative abundance of four gut Gram-negative phyla in the T2DM-CKD and control groups. No significant difference was observed between the relative abundance of Bacteroidetes. Significant differences were observed in the relative abundance of the three Gram-negative phyla Proteobacteria, Verrucomicrobia and Fusobacteria between the two groups. P<0.05. T2DM-CKD, type 2 diabetes mellitus and chronic kidney disease.

Figure 3.

Identified families and genera in the Gram-negative phylum Bacteroidetes in the type 2 diabetes mellitus-chronic kidney disease and control groups with high relative abundances.

Figure 4.

Members of the gut microbiota identified in three Gram-negative phyla with low relative abundances within patients with type 2 diabetes mellitus and controls. (A) The phylum Proteobacteria comprised of 5 families and 17 genera. (B) A single genus was identified in the phylum, Verrucomicrobia; (C) 2 genera were identified in the phylum, Fusobacteria.

Figure 5.

Multiple comparison analysis with adjusted P-values of the relative abundances of Proteobacteria and Fusobacteria in patients with T2DM and controls. The two phyla demonstrated a significant difference for the specified source of variations. P<0.05. T2DM-CKD, type 2 diabetes mellitus and chronic kidney disease.

Figure 6.

Correlation between elevated serum LPS and inflammatory biomarkers in the T2DM-CKD and control groups. (A) Serum LPS (ng/ml) levels in T2DM-CKD patients compared with the controls. (B-D) Elevated serum levels of LPS revealed a significant positive correlation with (B) CRP, (C) IL6 and (D) TNFα in the blood of patients with T2DM-CKD. P<0.05. CRP, C-reactive protein; IL6, interleukin-6; LPS, lipopolysaccharide; T2DM-CKD, type 2 diabetes mellitus and chronic kidney disease; TNFα, tumor necrosis factor α.

Figure 7.

Multifactorial effects of host genotype, diet, drugs and the potential influence of T2DM on alterations in the gut microbiome, which is followed by a cascade of events that promote systemic inflammation and reduces the clearance of endotoxins associated with chronic kidney disease. T2DM, T2DM type 2 diabetes mellitus.