Candida Administration in 5/6 Nephrectomized Mice Enhanced Fibrosis in Internal Organs: An Impact of Lipopolysaccharide and (1→3)-β-D-Glucan from Leaky Gut

Abstract

Uremic toxins and gut dysbiosis in advanced chronic kidney disease (CKD) can induce gut leakage, causing the translocation of gut microbial molecules into the systemic circulation. Lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG) are the major gut microbial molecules of Gram-negative bacteria and fungi, respectively, and can induce inflammation in several organs. Here, the fibrosis in the kidney, liver, and heart was investigated in oral C. albicans-administered 5/6 nephrectomized (Candida-5/6 Nx) mice. At 20 weeks post 5/6 Nx, Candida-5/6 Nx mice demonstrated increased 24 h proteinuria, liver enzymes, and serum cytokines (TNF-α, IL-6, and IL-10), but not weight loss, systolic blood pressure, hematocrit, serum creatinine, or gut-derived uremic toxins (TMAO and indoxyl sulfate), compared to in 5/6 Nx alone. The gut leakage in Candida-5/6 Nx was more severe, as indicated by FITC-dextran assay, endotoxemia, and serum BG. The areas of fibrosis from histopathology, along with the upregulated gene expression of Toll-like receptor 4 (TLR-4) and Dectin-1, the receptors for LPS and BG, respectively, were higher in the kidney, liver, and heart. In vitro, LPS combined with BG increased the supernatant IL-6 and TNF-α, upregulated the genes of pro-inflammation and pro-fibrotic processes, Dectin-1, and TLR-4 in renal tubular (HK-2) cells and hepatocytes (HepG2), when compared with LPS or BG alone. This supported the pro-inflammation-induced fibrosis and the possible LPS-BG additive effects on kidney and liver fibrosis. In conclusion, uremia-induced leaky gut causes the translocation of gut LPS and BG into circulation, which activates the pro-inflammatory and pro-fibrotic pathways, causing internal organ fibrosis. Our results support the crosstalk among several organs in CKD through a leaky gut.

Keywords: 5/6 nephrectomized mice; Candida; chronic kidney disease; fibrosis; gut leakage; gut-derived uremic toxins.

Figure 1

The characteristics of Sham control and 5/6 nephrectomized (5/6 Nx) mice with phosphate buffer solution (PBS) or Candida administration (Candida) at 20 weeks (wk) after right nephrectomy (20 week post Nx) (see method), as indicated by body weight (A), mean arterial pressure (B), hematocrit (C), renal function (24 h urine protein and serum creatinine) (D,E), serum gut-derived uremic toxins, including trimethylamine N-oxide (TMAO) and indoxyl sulfate (F,G), liver enzyme (alanine transaminase) (H), and serum cytokines (TNF-α, IL-6, and IL-10) (I–K) are demonstrated (n = 11/group).

Figure 2

The characteristics of Sham control and 5/6 nephrectomized (5/6 Nx) mice with phosphate buffer solution (PBS) or Candida administration (Candida) at 20 weeks (wk) after right nephrectomy (20 week post Nx) (see method), as indicated by the gut barrier defect; FITC-dextran assay, endotoxemia, and serum (1→3)-β-D-glucan (BG) (A–C), area of organ fibrosis with the gene expression of Toll-like receptor 4 (TLR-4) and Dectin-1 in the kidney (D–F), liver (G–I), and heart (J–L) are demonstrated (n = 11/group).

Figure 3

The representative kidney histopathological pictures stained by hematoxylin and eosin (H&E) (upper part) or Masson’s trichrome color (lower part) (original magnification 40–200×) of Sham control, 5/6 nephrectomized mice with phosphate buffer solution (5/6 Nx PBS) or Candida administration (5/6 Nx Candida) are demonstrated.

Figure 4

The representative liver histopathological pictures stained by hematoxylin and eosin (H&E) (upper part) or Masson’s trichrome color (lower part) (original magnification 40–200×) of Sham control, 5/6 nephrectomized mice with phosphate buffer solution (5/6 Nx PBS) or Candida administration (5/6 Nx Candida) are demonstrated.

Figure 5

The representative heart histopathological pictures stained by hematoxylin and eosin (H&E) (upper part) or Masson’s trichrome color (lower part) (original magnification 40–200×) of Sham control, 5/6 nephrectomized mice with phosphate buffer solution (5/6 Nx PBS) or Candida administration (5/6 Nx Candida) are demonstrated.

Figure 6

The characteristics of renal tubular cells (HK-2 cells) after 24 h incubation in the culture medium (Control) or lipopolysaccharide (LPS) with or without (1→3)-B-D-glucan (BG), as indicated by supernatant cytokines (TNF-α and IL-8) (A,B), and the gene expression of cytokines (TGF-β and IL-8) (C,D), hypoxia-inducible factor-1α (HIF-1α) (E), fibrosis-associated genes, including fibronectin, alpha-smooth muscle actin (α-SMA), collagen (type I, III, and IV) (F–J), and inflammatory mediators (Dectin-1 and Toll-like-receptor 4; TLR-4) (K,L) are demonstrated. Independent triplicate experiments were performed for all in vitro experiments.

Figure 7

The characteristics of hepatocyte (HepG2 cells) after 1 and 72 h incubation in the culture medium (Control) or lipopolysaccharide (LPS) with or without (1→3)-B-D-glucan (BG), as indicated by supernatant cytokines (TNF-α and IL-8) (A,B), and the gene expression of cytokines (TGF-β and IL-8) (C,D), hypoxia-inducible factor-1α (HIF-1α) (E), fibrosis-associated genes, including fibronectin, alpha-smooth muscle actin (α-SMA), collagen (type I, III and IV) (F–J), and inflammatory mediators (Dectin-1 and Toll-like-receptor 4; TLR-4) (K,L) are demonstrated. Independent triplicate experiments were performed for all in vitro experiments.

Figure 8

The proposed working hypothesis of the enhanced fibrosis from chronic kidney disease through leaky-gut-induced pro-inflammation that is activated by lipopolysaccharide (LPS) together with (1→3)-β-D-glucan (BG). Firstly (1), the increased uremic toxins in the intestine, and the substitution of kidney excretion, affects gut organisms and causes the intestinal barrier defect. Secondly (2), the LPS and BG from the gut translocate into the blood (leaky gut). Thirdly (3), LPS and BG additively activate pro-inflammation in several organs, partly through the activation of Toll-like-receptor 4 (TLR-4; LPS receptor) and Dectin-1 (BG receptor). The enhanced inflammatory responses from the leaky gut during uremia possibly worsen several uremic complications. The diagram was created using Biorender.com.