Sodium butyrate ameliorates insulin resistance and renal failure in CKD rats by modulating intestinal permeability and mucin expression

Abstract

Background: The associated increase in the lipopolysaccharide (LPS) levels and uremic toxins in chronic kidney disease (CKD) has shifted the way we focus on intestinal microbiota. This study shows that a disruption of the intestinal barrier in CKD promotes leakage of LPS from the gut, subsequently decreasing insulin sensitivity. Butyrate treatment improved the intestinal barrier function by increasing colonic mucin and tight junction (TJ) proteins. This modulation further ameliorated metabolic functions such as insulin intolerance and improved renal function.

Methods: Renal failure was induced by 5/6th nephrectomy (Nx) in rats. A group of Nx and control rats received sodium butyrate in drinking water. The Nx groups were compared with sham-operated controls.

Results: The Nx rats had significant increases in serum creatinine, urea and proteinuria. These animals had impaired glucose and insulin tolerance and increased gluconeogenesis, which corresponded with decreased glucagon-like peptide-1 (GLP-1) secretion. The Nx animals suffered significant loss of intestinal TJ proteins, colonic mucin and mucin 2 protein. This was associated with a significant increase in circulating LPS, suggesting a leaky gut phenomenon. 5'adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, known to modulate epithelial TJs and glucose metabolism, was significantly reduced in the intestine of the Nx group. Anti-inflammatory cytokine, interleukin 10, anti-bacterial peptide and cathelicidin-related antimicrobial peptide were also lowered in the Nx cohort. Butyrate treatment increased AMPK phosphorylation, improved renal function and controlled hyperglycemia.

Conclusions: Butyrate improves AMPK phosphorylation, increases GLP-1 secretion and promotes colonic mucin and TJ proteins, which strengthen the gut wall. This decreases LPS leakage and inflammation. Taken together, butyrate improves metabolic parameters such as insulin resistance and markers of renal failure in CKD animals.

Keywords: chronic renal failure; diabetic kidney disease; gut; nephrectomy; short chain fatty acid.

FIGURE 1

Butyrate ameliorates hyperglycemia in 5/6th Nx rats. Butyrate treatment was started 2 weeks after surgery and continued for 6 weeks. (A) Oral GTT: Plasma glucose concentration in response to oral glucose administration. (B) AUC of glucose after oral glucose administration. (C) ITT: The animals were given insulin i.p. and plasma glucose measured by glucometer. (D) AUC of glucose after insulin administration. (E) PTT: Plasma glucose concentration in response to i.p. pyruvate administration. (F) AUC of glucose after pyruvate administration. Data are expressed as mean ± SD (n = 6). Butyrate control consisted of five animals. Statistically significant differences between the groups are given as follows: ^aP < 0.05 compared with control; ^bP < 0.01 compared with control; ^cP < 0.05 compared with butyrate control; ^dP < 0.01 compared with butyrate control; ^eP < 0.05 compared with Nx + butyrate; ^fP < 0.01 compared with Nx + butyrate.

FIGURE 2

Butyrate increases GLP-1 secretion and reduces plasma LPS levels of 5/6th Nx rats. Butyrate was given for 6 weeks. (A) Blood was collected 8 weeks after surgery. Plasma GLP was measured by enzyme immunoassay 30 min after oral glucose administration (2 g/kg). GLP levels of the Nx group were significantly lower than the control and butyrate control groups. Butyrate treatment significantly abated the decrease. There was no significant difference in the GLP levels of the control, butyrate control and Nx + butyrate groups. (B) Plasma LPS was measured by a Lonza kit. The LPS levels of the Nx group were significantly higher than the control and butyrate control groups. Although butyrate treatment (Nx + butyrate) significantly decreased the LPS levels, it was still significantly higher than the control (P = 0.005) and butyrate control (P = 0.006) groups. Data are mean ± standard deviation of five to six animals per group. Butyrate control consisted of five animals.

FIGURE 3

Decreased abundance of colonic TJ proteins of the 5/6th Nx group was reversed by butyrate. Representative western blot of ZO-1 and claudin 1. (A) Cytosolic ZO-1 but not claudin 1 was significantly reduced in the Nx group. Butyrate treatment significantly improved ZO-1 expression. (B) Reduction of membrane claudin 1 in the Nx group was abrogated by butyrate treatment. ZO-1 protein could not be detected in the membrane fraction. Data are mean ± standard deviation of six animals per group. Cytosolic ZO-1 and membrane claudin 1 of the control and Nx + butyrate groups were not significantly different from each other.

FIGURE 4

Butyrate treatment reverted the decrease in AMPK phosphorylation and CAMKKβ in the colon of Nx animals. (A) The representative western blot of phosphorylated AMPK (pAMPK) and AMPK of the colon. A significant decrease in the pAMPK:AMPK ratio in the Nx group was reversed by butyrate treatment. The pAMPK:AMPK ratio between the control and Nx + butyrate groups was not significantly different from each other. (B) The representative western blot of CAMKKβ and β-actin of the colon. Reduced CAMKKβ in the Nx colon was reversed by butyrate. Data are mean ± standard deviation of six animals per group. The CAMKKβ between the control and Nx + butyrate groups was not significantly different from each other.

FIGURE 5

Reduced expression of colonic mucin stained by Alcian blue in Nx animals was corrected by butyrate treatment. Representative sections of colonic mucin of two animals from each group (control, Nx and butyrate-treated Nx animals) are shown. The proximal part of the colon (0.5 cm) was collected and stored in Carnoy’s solution. Alcian blue stained the mucin in the colon, imparting a blue color under a light microscope (×10 magnification). Please note the decrease in mucin expression of CKD animals (shown by the arrows) and its resurgence following butyrate treatment. Compared with the controls, the blue-stained areas representing mucin expression were significantly less in the Nx groups. Butyrate treatment reversed this effect. A total of six animals per group were used to assess mucin expression.

FIGURE 6

Muc2 expression in the colon analyzed immunohistochemically revealed decreased expression of Muc2 in the Nx group was abrogated by butyrate. Fluorescent microscopy of Muc2, stained red, merged with DNA-capturing 4′-6-diamidino-2-phenylindole, stained blue. Representative sections of colonic mucin of two animals from each group (control, Nx and butyrate-treated Nx) are shown. A total of six animals per group were used to assess Muc2 expression. The proximal part of the colon (0.5 cm) was collected and stored in Carnoy’s solution. Muc2 expression was determined by immunohistochemistry using Muc2 antibody. The yellow arrow points to the Muc2 (red) expression. Compared with controls, Muc2 expression was significantly lower in the Nx groups. The butyrate-treated group had a significant increase in mucin expression.

FIGURE 7

Butyrate treatment reversed the decrease of colonic IL-10 and duodenal CRAMP of Nx animals. (A) Representative western blot of IL-10 normalized to β-actin. Significant reduction of IL-10 in the colon of Nx animals reversed with butyrate; however, Nx + butyrate was significantly higher than in the control group (<0.001). (B) Representative western blot of colon CRAMP normalized to β-actin. (C) Representative western blot of duodenal CRAMP normalized to β-actin. Compared with the controls there was reduced expression of CRAMP in the colon and duodenum of Nx animals that was reversed with butyrate. Although butyrate treatment (Nx + butyrate) increased CRAMP expression in the colon, it was still significantly lower (P < 0.05) than in the control group. Only 50% of the colon from the Nx animals had detectable CRAMP. The expression of duodenal CRAMP in the Nx + butyrate and control groups was not significantly different. Data are mean ± standard deviation of six animals per group.