Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD

Abstract

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

Keywords: CKD; gastrointestinal medications; intestine; uremia.

Figure 1.

Effect of lubiprostone on fecal and intestinal characteristics in uremic mice. (A) Fecal shape and weight per pellet. Fecal weight per pellet was calculated as (fecal number)/(total fecal wet weight). n=6–7 for each group. (B) Residual fecal number in the colon. n=6–7. (C) Representative small intestinal images of RF and RF+Lub500 groups. (D) Intestinal transit analysis after lubiprostone administration in RF mice. The arrowheads denote the leading edge of trypan blue. *P<0.05 between the indicated groups. n=3. Scale bar, 2 cm. (E) Representative histologic images of the colon. MTS-stained cross-section of the colon containing a fecal pellet. The arrow indicates lamina propria. P<0.05 between the indicated groups. Cont, control. Scale bar, 100 µm.

Figure 2.

Effect of lubiprostone on renal fibrosis and inflammation in the uremic mouse kidney. (A) BUN concentration in mice. n=6–7 for each group. (B) Morphometric analysis of the percentage of the cortical tubular area and whole-kidney images of MTS. The cortical tubular area was calculated on the basis of the MTS image. n=6–7. Scale bar, 1 cm. *P<0.05 compared with the RF group; **P<0.01 compared with the RF group. (C) Representative histologic images of periodic acid–Schiff- (PAS-), MTS-, and picrosirius red-stained kidney. The arrowheads denote adenine-derived casts. Scale bar, 100 µm. (D) Immunostaining of α-smooth muscle actin (α-SMA) and F4/80 in kidney sections. Cont, control. Scale bar, 100 µm.

Figure 3.

Effects of lubiprostone on renal gene expression in uremic mice. Relative transcript levels of genes in (A) renal inflammation and (B) fibrosis were measured using real-time PCR. The names in parentheses indicate the coding protein: Tnfa (TNF-α), Il6 (IL-6), Pai-1 (PAI-1), Ccl2 (MCP-1), Col1a1 (type I collagen-α1), Col3a1 (type III collagen-α1), Tgfb1 (TGF-β), and Acta2 (α-smooth muscle actin). The expression levels were first normalized to those of Gapdh and then further normalized to the levels in the kidney from the control mice. n=6–7 for each group. Cont, control. *P<0.05 versus the RF group; **P<0.01 versus the RF group.

Figure 4.

Effects of lubiprostone on gut microbiota in uremic mice. (A) Unweighted unifrac distance analysis (upper panel) and diversity of bacterial species as indicated by Chao1 rarefaction measure (lower panel) of gut microbiota in the control, RF, and RF+Lub500 groups. n=6. (B) Relative abundance of microbiota on the basis of the average number of each subfamily at the order, family, and genus levels. The major subfamilies are indicated on the right. n=6. Cont, control. (C) The proportional change of each subfamily at the genus level. Genera displaying a significant change among the three groups are shown (Steel–Dwass). The y axis indicates the abundance of each microbe (percentage). C, control; RF+L, RF treated with 500 µg/kg per day lubiprostone. *P<0.05 versus the control group; ^§P<0.05 versus the RF group.

Figure 5.

Effects of lubiprostone on the plasma uremic solutes analyzed by CE-TOFMS–based metabolome analysis in uremic mice. Each of the plasma concentrations was measured by CE-TOFMS. (A) Anionic and (B) cationic solutes. The y axis indicates the micromolar concentration. n=6. C, control; GABA, γ-aminobutyric acid; RF+L, RF treated with 500 µg/kg per day lubiprostone. *P<0.05 versus the control group; ^§P<0.05 versus the RF group (ANOVA).