Candida Administration in Bilateral Nephrectomy Mice Elevates Serum (1→3)-β-D-glucan That Enhances Systemic Inflammation Through Energy Augmentation in Macrophages

Abstract

Systemic inflammation, from gut translocation of organismal molecules, might worsen uremic complications in acute kidney injury (AKI). The monitoring of gut permeability integrity and/or organismal molecules in AKI might be clinically beneficial. Due to the less prominence of Candida albicans in human intestine compared with mouse gut, C. albicans were orally administered in bilateral nephrectomy (BiN) mice. Gut dysbiosis, using microbiome analysis, and gut permeability defect (gut leakage), which was determined by fluorescein isothiocyanate-dextran and intestinal tight-junction immunofluorescent staining, in mice with BiN-Candida was more severe than BiN without Candida. Additionally, profound gut leakage in BiN-Candida also resulted in gut translocation of lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG), the organismal components from gut contents, that induced more severe systemic inflammation than BiN without Candida. The co-presentation of LPS and BG in mouse serum enhanced inflammatory responses. As such, LPS with Whole Glucan Particle (WGP, a representative BG) induced more severe macrophage responses than LPS alone as determined by supernatant cytokines and gene expression of downstream signals (NFκB, Malt-1 and Syk). Meanwhile, WGP alone did not induced the responses. In parallel, WGP (with or without LPS), but not LPS alone, accelerated macrophage ATP production (extracellular flux analysis) through the upregulation of genes in mitochondria and glycolysis pathway (using RNA sequencing analysis), without the induction of cell activities. These data indicated a WGP pre-conditioning effect on cell energy augmentation. In conclusion, Candida in BiN mice accelerated gut translocation of BG that augmented cell energy status and enhanced pro-inflammatory macrophage responses. Hence, gut fungi and BG were associated with the enhanced systemic inflammation in acute uremia.

Keywords: (1→3)-β-D-glucan; Candida; bilateral nephrectomy; endotoxin; uremia mice.

Figure 1

Characteristics of mice with sham, bilateral nephrectomy (BiN) and BiN with Candida administration (BiN+Cand) at 48 h after surgery, as indicated by fungal abundance in feces (culture method) (A), serum creatinine (B), serum cytokines (C–E), gut leakage; FITC-dextran, endotoxemia and serum (1→3)-β-D-glucan (BG) and the abundance of tight junction molecules (Claudin-1 and Occludin) as evaluated by immunofluorescent staining and scored in percentage of the detected-area with the representative pictures (F–K) are demonstrated (n = 7–9/group). Notably, data of sham with Candida administration (Sham+Cand) are not demonstrated due to the similarity to the sham group without Candida.

Figure 2

Fecal microbiome analysis from mice with sham, bilateral nephrectomy (BiN) and BiN with Candida administration (BiN+Cand) at 48 h after surgery as indicated by relative abundance of bacterial diversity at phylum level, genus level, and the average of both levels (A–C), the heterogeneity of fecal bacteria by operational taxonomic units (OTUs) and alpha-diversity indices (Chao and Shannon) (D), the comparison of bacterial diversity in phylum level in graphs (E) and the non-metric multidimensional scaling (NMDS) based on Thetayc beta diversity matrices at the genus level (F) are demonstrated (n = 5–6/group). Notably, data of sham with Candida administration (Sham+Cand) are not demonstrated due to the similarity to the sham group without Candida.

Figure 3

Characteristics of macrophage responses after 6 h incubation of media control (control), Whole Glucan Particle (WPG) with or without lipopolysaccharide (LPS) as indicated by supernatant cytokines (A–C), expression of the downstream signaling molecules (NFκB, Syk and Malt-1) (D–F), protein abundance of AMPK, p-AMPK and a ratio between AMPK/p-AMPK (a sensor of cell energy status) with the representative figures of Western blot analysis (G,H), phagocytosis and bactericidal activity (I) are demonstrated (independent triplicate experiments were performed).

Figure 4

Graphic pictures indicate the action of mitochondrial inhibitory agents, including Rotenone and Antimycin A (the blockage of protein complex I and complex III, respectively) and oligomycin (a blockage of protein complex IV), and the activity of Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial augmented-agent through mitochondrial uncoupling (a process that transfers proton back to mitochondrial matrix to increase mitochondrial ATP synthesis) (A) and area of the graph from extracellular flux analysis, including several parameters of mitochondrial respiration based on oxygen consumption rate (OCR) (B). Characteristics of macrophage responses after 3 h incubation of media control (control), Whole Glucan Particle (WGP) with or without lipopolysaccharide (LPS) (the representative cell wall molecules of fungi and bacteria in gut) as evaluated by OCR mitochondrial respiration (C) with the better visualization on graph presentation of basal respiration and maximal respiration (D,E) and the heat-map of mitochondria-associated genes from RNA sequencing analysis (F) are demonstrated (independent triplicate experiments were performed). Notably, other OCR parameters, including proton leak, ATP production, and non-mitochondrial respiration are not shown due to the similar patterns as basal respiration and maximal respiration.

Figure 5

Graphic pictures indicate the action of a glycolysis inhibitory agents, 2-Deoxy-d-glucose (2-DG) on hexokinase (A) and area of the graph from extracellular flux analysis, including several parameters of glycolysis activity based on extracellular acidification rate (ECAR) (B). Characteristics of macrophage responses after 3 h incubation of media control (control), Whole Glucan Particle (WPG) with or without lipopolysaccharide (LPS) (the representative cell wall molecules of fungi and bacteria in gut) as evaluated by ECAR glycolysis activity (C) with the better visualization on graph presentation of glycolysis and glycolysis capacity (D,E) and the heat-map of glycolysis-associated genes from RNA sequencing analysis (F) are demonstrated (independent triplicate experiments were performed).

Figure 6

A graphic picture indicates the ATP analysis (A) that separates ATP from mitochondria and glycolysis through oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, by the extracellular flux analysis machine using mitochondrial inhibitory agents, Oligomycin and Rotenone with Antimycin A (R&A) is demonstrated. The measurement is based on the basic knowledge that a blockage of mitochondrial ATP accelerates ATP production from the glycolysis pathway. Additionally, characteristics of macrophage responses after 3 h incubation of media control (control), Whole Glucan Particle (WPG) with or without lipopolysaccharide (LPS) (the representative cell wall molecules of fungi and bacteria in the gut) as evaluated by ATP production from either mitochondria (by OCR) or glycolysis (by ECAR) (B,C), the cell energy phenotype profile (by OCR versus ECAR) (D), percentage of ATP that derived from mitochondria (oxidative phosphorylation) (E) and from glycolysis (F), ATP production rate from mitochondria and glycolysis (G) and the heat-map of genes for ATP production from RNA sequencing analysis (H) are demonstrated (independent triplicate experiments were performed).

Figure 7

The biological process gene ontology terms (Go term) enrichment analysis of differential gene expression (DEGs) in macrophages with various conditions. The up- and downregulated genes (−1 to 1-fold change cut-off value) were identified using ontology enrichment analysis and the top ten significantly Go term with their –log10 p value in comparison between macrophages with control condition (Control) versus the activation by Whole Glucan Particle (WPG) with or without lipopolysaccharide (LPS) (the representative cell wall molecules of fungi and bacteria in gut) (A–C) and between WGP plus LPS (WGP+LPS) versus the activation by WGP or LPS alone (D,E) are demonstrated.

Figure 8

The proposed hypothesis demonstrates uremia-induced fecal dysbiosis that is more severe in bilateral nephrectomy (BiN) mice with Candida (BiN-Candida) than BiN alone (non-Candida BiN). There is a lesser translocation of (1→3)-β-D-glucan (BG) in non-Candida BiN mice than BiN-Candida (because of the increased BG level in feces of BiN-Candida mice), with a similar LPS translocation in both models. Then, LPS+BG (LPS with BG) activates TLR-4 and Dectin-1, respectively, and upregulates NFκB, a transcriptional factor for cytokine production, through Syk and Malt-1. In parallel, BG also enhances the important enzymes in glycolysis pathway (in cell cytoplasm) and in OXPHOS (oxidative phosphorylation) of mitochondria that increase cell energy status (ATP production) and enhance LPS responses.