Uremia-Induced Gut Barrier Defect in Five-sixths Nephrectomized Mice Is Worsened By Candida Administration Through A Synergy Of Uremic Toxin, Lipopolysaccharide, And (1→3)-β-D-Glucan, But Is Attenuated By Lacticaseibacillus Rhamnosus L34

Abstract

Chronic kidney disease (CKD) causes uremic toxin accumulation and gut dysbiosis, which further induces gut leakage and worsening CKD. Lipopolysaccharides (LPS) of Gram-negative bacteria and (1→3)-β-D-glucan (BG) of fungi are the two most abundant gut microbial molecules. Due to limited data on the impact of intestinal fungi in CKD mouse models, the influences of gut fungi and Lacticaseibacillus rhamnosus L34 (L34) on CKD was investigated using oral C. albicans-administered 5/6 nephrectomy (5/6Nx) mice. At 16 weeks post-5/6Nx, Candida-5/6Nx mice demonstrated an increase in proteinuria, serum BG, serum cytokines (tumor necrotic factor-α; TNF-α and interleukin-6), alanine transaminase (ALT), and level of fecal dysbiosis (Proteobacteria on fecal microbiome) when compared to non-Candida-5/6Nx. However, serum creatinine, renal fibrosis, or gut barrier defect (FITC-dextran assay and endotoxemia) remained comparable between Candida- versus non-Candida-5/6Nx. The probiotics L34 attenuated several parameters in Candida-5/6Nx mice, including fecal dysbiosis (Proteobacteria and Bacteroides), gut leakage (fluorescein isothiocyanate (FITC)-dextran), gut-derived uremic toxin (trimethylamine-N-oxide; TMAO) and indoxyl sulfate; IS), cytokines, and ALT. In vitro, IS combined with LPS with or without BG enhanced the injury on Caco-2 enterocytes (transepithelial electrical resistance and FITC-dextran permeability) and bone marrow-derived macrophages (supernatant cytokines (TNF-α and interleukin-1 β; IL-1β) and inflammatory genes (TNF-α, IL-1β, aryl hydrocarbon receptor, and nuclear factor-κB)), compared with non-IS activation. The probiotics condition media attenuated these injuries. In conclusion, Candida administration worsens kidney damage in 5/6Nx mice through systemic inflammation, partly from gut dysbiosis-induced uremic toxins, which were attenuated by the probiotics. The additive effects on cell injury from uremic poison (IS) and microbial molecules (LPS and BG) on enterocytes and macrophages might be an important underlying mechanism.

Keywords

Lacticaseibacillus rhamnosus; Candida; gut-derived uremic toxins; gut leakage; 5/6 nephrectomy mice; chronic kidney disease.

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Introduction

Chronic kidney disease (CKD) has been recognized as an extensive worldwide burden for decades [1], causing an accumulation of various metabolic chemicals known as “uremic toxins”. Such toxins mainly derived from food components or metabolic activities in the body can contribute to various complications, such as cardiovascular diseases, pulmonary problems, and CKD progression [2]. Some circulating uremic toxins are formed in the GI tract, known as gut-derived uremic toxins [3], including trimethylamine-N-oxide (TMAO), indoxyl sulfate, p-cresol sulfate, hippuric acid, and phenylacetic acid [4]. Because of the defect of toxin elimination through kidneys in advanced CKD, the accumulated toxins are compensatorily excreted into the intestinal tract and selectively promote the overgrowth of pathogenic intestinal bacteria, so-called gut dysbiosis [5]. Dysbiosis enhances the production of gut-derived uremic toxins. The toxins (gut and non-gut derivatives) can impair intestinal epithelial tight junctions, which lead to the translocation of microbial molecules from the gut into blood circulation, so-called gut leakage or gut translocation [6]. Although the intestine is a source of gut-derived uremic toxins, the toxins distribute throughout the body (including the intestine) induce damage to several cells, including enterocytes and renal tubules [7–9]. The vicious cycle in that CKD causes uremic toxin accumulation and gut dysbiosis, which further induces gut leakage and worsens CKD, is referred to as the gut–kidney axis [10]. Gut translocation of microbial molecules and uremic toxins facilitates the inflammatory reaction and accelerates CKD progression [11].

Among microbial molecules in the gut, lipopolysaccharide (LPS) of Gram-negative bacteria and (1→3)-β-D-glucan (BG) of fungi are the two most abundant molecules in the gut [10]. However, the impact of intestinal fungi in mouse models is underappreciated, as Candida albicans are less abundant in the mouse gut than in the human intestine [12]. The amount of Candida spp. in mouse feces is insufficient to be detectable in stool culture [12], which differs from that of human feces [13]. Although gut fungi do not directly cause illness, they affect the gut microbiota and supply BG in the gut [14], contributing to the worsening of systemic inflammation following gut barrier defect (gut leakage). Candida administration in bilateral nephrectomy (acute kidney injury) mice induces more severe gut leakage and inflammatory responses [11]. However, there are still very few investigations on the impact of gut fungi on uremic disorders, and a CKD model with C. albicans presentation has never been explored. Because gut microbiota plays a major role in regulating the production of gut-derived uremic toxins and the toxins as well as endotoxemia worsen CKD progression [15,16], the use of probiotics might prevent gut dysbiosis, reduce the poison, and delay CKD progression [17–19]. Lacticaseibacillus rhamnosus L34 (L34), a strain of intestinal flora isolated from the Asian population [20], improves gut permeability integrity in several animal models of acute illnesses [21]. Due to (i) the worsening renal fibrosis and CKD progression by systemic inflammation [22], (ii) systemic inflammation-induced gut leakage in acute uremia model [11], and (iii) the anti-inflammatory properties of probiotics [21,23,24], L34 administration might also help delay the CKD progression in the Candida-administered CKD mice.

Here, we explored the impact of C. albicans and L34 on renal histopathology, CKD progression, inflammatory markers, and gut leakage in the Candida-administered 5/6 nephrectomy (5/6Nx) mouse model. To understand the pathophysiologic effects of fungi on CKD, BG (the main fungal cell wall component) was used in vitro with LPS (a major component of Gram-negative bacterial cell wall) and indoxyl sulfate (a representative gut-derived uremic toxin) on enterocytes (Caco-2 cells) and macrophages (bone marrow-derived cells).

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Discussion

1. Gut Candida Altered Chronic Kidney Disease (CKD) in 5/6Nx Mice through Gut Dysbiosis, Intestinal Barrier Defect, and Systemic Inflammation

C. albicans in mouse feces is detectable only by PCR [52], not by culture [12], which differs from human conditions [13], Candida was orally administered in 5/6Nx mice to examine the impact of gut fungi in CKD. Candida-5/6Nx mice had more severe gut leakage (FITC-dextran at 8 weeks post-surgery) with higher serum BG (glycemia) that possibly worsened proteinuria and liver damage (alanine transaminase (ALT)), and increased serum cytokines compared to 5/6Nx mice. Although Candida did not alter CKD severity determined by serum creatinine and kidney fibrosis, higher serum BG in Candida-5/6Nx mice enhanced responses against endotoxemia [7,53] that induced proteinuria and high ALT [26,27]. Although fungi were nondetectable in the feces of non-Candida 5/6Nx mice (data not shown), the detectable serum BG in these mice supported the role of BG from mouse chow in gut contents [54]. While Candida gavage in acute kidney injury (AKI) enhances gut leakage-induced inflammation and mortality [11], systemic inflammation from gut-Candida in CKD is not severe enough to increase the mortality partly due to the different compensation in acute versus chronic uremia [55]. The impact of acute uremia on enterocytes might be more potent than chronic uremia with a better adaptation to the cell micro-environments [55].

Uremia-induced gut dysbiosis is a result of an increase in intestinal excretion of the accumulated uremic toxins that promote the growth of gut pathogenic bacteria, increase gut-derived uremic toxins, and enhance systemic inflammation. These effects exert injury on renal vascular endothelium [56,57] and parenchymal cells [58,59] that causes a vicious cycle of uremic toxins induced gut dysbiosis, and the dysbiosis further enhanced CKD progression through the higher toxin accumulation. Then, this vicious cycle is possibly facilitated by gut fungi due to the enhanced serum BG with the presence of Candida in the gut. Interestingly, spontaneous glycemia and endotoxemia (without systemic infection) in patients with CKD support the CKD-induced gut barrier defect [60] that might be correlated with gut dysbiosis. Indeed, gut fungi in 5/6Nx mice facilitated pathogenic Proteobacteria without alteration on Bacteroides and Firmicutes compared with non-Candida 5/6Nx. The gavage of Candida (live or heat-killed) in other mouse models facilitates fecal pathogenic bacteria [30,31,61], perhaps due to the BG fermentation properties of some bacteria [62]. Here, Candida also enhanced the growth of Helicobacter spp. and Allobaculum spp. which might be associated with uremic gastritis and mucus degradation [32,33]. Therefore, further exploration of gut fungi in CKD is interesting.

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2. Lacticaseibacillus rhamnosus L34 (L34) Attenuated Candida-Administered 5/6Nx Mice through the Anti-Inflammatory Effect on Enterocytes and Macrophages

The attenuation of uremic enteropathy (gut dysbiosis and gut leakage) and gut-derived uremic toxins by L34 has been demonstrated [11]. However, the effect of L34 on the CKD model with Candida administration has never been explored. Despite a more profound inflammation in Candida-5/6Nx than non-Candida 5/6Nx, L34 attenuated disease severity in these mice, as indicated by renal injury (improve renal fibrosis and proteinuria, but not serum creatinine), gut barrier defect (FITC-dextran, but not LPS and BG), gut-derived uremic toxins (TMAO and IS), systemic inflammation (serum cytokines), and liver damage (ALT). Notably, serum creatinine has a limitation as a CKD biomarker [63] and has a higher molecular weight (MW) than 4.4 kDa of FITC-dextran. The MW of pathogen molecules (LPS and BG) [10] also varies. Therefore, after probiotic treatment, serum creatinine, LPS, and BG might be unchanged. In parallel, L34 improved gut dysbiosis as demonstrated by a reduction of the possible pathogenic bacteria (Bacteroides, Proteobacteria, and Helicobacter spp.) [11,32] with an increase in Clostridium spp. (the possible beneficial Fermenters) [64]. However, L34 increased Cyanobacteria (toxin-producing bacteria) [41] and Allobaculum spp. (bacteria with both advantageous and disadvantageous properties [32,33]). Although L34 did not alter Firmicutes (the highly abundant bacteria in healthy conditions) in the microbiome analysis, LEfSe indicated several bacteria in the Firmicutes group in L34-treated mice. As gut dysbiosis [5], gut leakage [6,7], gut-derived uremic toxins [6,65], and inflammatory cytokines are enhanced in advanced CKD, attenuation of these mechanisms might retard the CKD progression [11]. Our findings support such effects of probiotics on the retardation of CKD progression.

In addition, the impact of indoxyl sulfate (IS), a representative gut-derived uremic toxin, on enterocytes and macrophages was evaluated in vitro using the extract of L34 in culture media. Indoxyl sulfate is a water-soluble form of indoxyl, a molecule converted from tryptophan amino acid by gut bacteria in the luminal side of the intestines [47] and is metabolized into IS by the liver and contacts the basolateral part of the enterocytes through blood circulation [9]. IS promotes the production of reactive oxygen species that directly induce cell damage and cell death in several organs [66,67]. Despite the resistance of Caco-2 against IS (reduced cell viability at 10 mM of IS), IS a low concentration of 0.5–1 mM could induce intestinal cell death [9] and enterocyte permeability (TEER and FITC-dextran assay) when combined with LPS or BG. These results imply an impact of IS on enterocytes during CKD-induced uremic intestine [68]. In parallel, macrophages were more susceptible to IS as the cell viability was reduced in IS at 2 mM, but the combination of IS with LPS or BG did not decrease the IS concentration. Without IS, BG alone did not induce macrophage responses, and BG with LPS demonstrated the tendency of the additive proinflammatory effect but was not different from LPS activation alone. With IS, the additive proinflammatory effect was increased as IS + LPS + BG induced a higher level of inflammatory cytokines than IS + LPS. The additive effect is possibly due to the similar downstream signaling through the NFκB transcription factor of TLR-4 and Dectin-1, the pattern recognition receptors of LPS and BG, respectively, and aryl hydrocarbon receptor (the cytosolic receptor of IS) [69] as demonstrated in the working hypothesis figure (Figure 8). Despite the profound inflammation from combined IS with LPS and BG, LCM attenuated both enterocyte permeability defect and macrophage inflammation. These findings support the benefits of probiotics on CKD [70], possibly through anti-inflammatory exopolysaccharides [71]. Although the MW of IS, at 0.23 kDa, is small enough to pass through the normal intestinal tight junction (MW lower than 0.6 kDa) [10,72], several probiotics decrease serum IS [73,74], implying a decrease in bacteria with IS production property during advanced CKD. Probiotic tests on other CKD models might be interesting because of the possible difference in the interference of gut dysbiosis and inflammatory responses in individual CKD models. For example, gut dysbiosis might be more prominent in the oral adenine-induced CKD model due to its direct effect on intestinal microbiota with inflammasome-related inflammatory mechanisms (crystal-mediated inflammation) [75–78]. With gut dysbiosis attenuation and the anti-inflammatory properties of the probiotics, further exploration in other CKD models and clinical studies for a potential application in CKD are warranted.

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Conclusions

Candida administration enhanced leaky gut and inflammatory responses in 5/6Nx CKD mice through the additive inflammatory activation of IS with LPS and BG. L. rhamnosus L34 attenuated the severity of Candida-5/6Nx mice, partly through the improved enterocyte integrity and the induced anti-inflammatory macrophages. Some probiotics would be an important adjuvant therapy in patients with CKD shortly.

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Somkanya Tungsanga 1,2 , Wimonrat Panpetch 3 , Thansita Bhunyakarnjanarat 3 , Kanyarat Udompornpitak 3 , Pisut Katavetin 1 , Wiwat Chancharoenthana 4,5 , Piraya Chatthanathon 6 , Naraporn Somboonna 6,7 , Kriang Tungsanga 1 , Somying Tumwasorn 3 and Asada Leelahavanichkul 1,3,

1. Department of Medicine, Division of Nephrology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; s.tungsanga@gmail.com (S.T.); pkatavetin@yahoo.com (P.K.); kriangtungsanga@hotmail.com (K.T.)

2. Department of Medicine, Division of General Internal Medicine-Nephrology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

3. Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; mon-med@hotmail.com (W.P.); thansitadew@gmail.com (T.B.); jubjiibb@hotmail.com (K.U.); somying.tumwasorn@gmail.com (S.T.)

4. Tropical Nephrology Research Unit, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; wiwat.cha@mahidol.ac.th

5. Tropical Immunology and Translational Research Unit, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand

6. Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; memind01@gmail.com (P.C.); naraporn.s@chula.ac.th (N.S.)

7. Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University, Bangkok 10330, Thailand