Lacticaseibacillus rhamnosus attenuates uremic toxins in patients with nondialysis chronic kidney disease through the anti-inflammatory molecules

Abstract

Because of the strain-dependent effect and the lack of simultaneous in vitro test with limited clinical data on Lacticaseibacillus rhamnosus L34 (L34) isolated from the Thai population, L34 was tested and compared with L. rhamnosus GG (LGG). The before and after test using L34 and a randomized placebo-controlled trial using placebo, L34, and LGG, for 4 weeks in patients with non-dialysis chronic kidney disease stage 3-5 (CKD) together with the in vitro experiments using indoxyl sulfate (IS, a representative uremic toxin) were performed. In comparison with the baseline, 4-week-L34 administration reduced gut-derived uremic toxins (GDUTs), except total IS, and attenuated several biomarkers, including i) systemic inflammation, as measured by cytokines and neutrophil extracellular traps using citrullinated histone 3, cell-free DNA, and fluorescent-stained nuclear morphology; ii) gut permeability defect (beta-D-glucan but not by endotoxemia); and iii) gut dysbiosis (fecal microbiome analysis). Additionally, L34-conditioned media attenuated IS-induced injuries on Caco-2 enterocytes, THP-1-derived-macrophages, and isolated neutrophils. Despite the possible different active compounds, both probiotics similarly attenuated IS-induced inflammation in vitro and in patients when compared with the placebo. In conclusion, L34 and LGG similarly attenuated systemic inflammation in patients with CKD, through the improved gut dysbiosis and anti-inflammation.

Keywords: Lacticaseibacillus rhamnosus; Chronic kidney disease; Gut leakage; Gut-derived uremic toxins; Probiotics.

Fig. 1

The characteristics of patients with chronic kidney disease (CKD) at before and after 4 week of L. rhamnosus L34 (L34) administration, as indicated by hemoglobin (A), serum creatinine (B), estimated glomerular filtration rate (eGFR) (C), C-reactive protein (CRP) (D), white blood cell count (WBC) (E), serum cytokines (TNF-α, IL-6, and IL-10) (F–H), gut-derived uremic toxins (free indoxyl sulfate, total indoxyl sulfate, and p-cresol) (I-K), endotoxemia (L), serum (1→3)-beta-d-glucan (BG) (M), neutrophil extracellular traps (NETs), as determined by nuclear morphology with representative fluorescent pictures (N, O), cell-free DNA (P), citrullinated histone 3 (CitH3) (Q), the selected graph presentation of fecal bacterial abundance (phylum and genus levels) from fecal microbiome analysis (R-X), and fecal fungal abundance (expressing in cycle threshold or Ct) (Y), are demonstrated (n = 10/group). *, p < 0.05.

Fig. 2

The fecal microbiome analysis of patients with chronic kidney disease (CKD) at before and after 4 weeks of L. rhamnosus L34 (L34) administration as indicated by the abundance of bacteria in phylum, class, order, family, genus, and species is demonstrated (n = 10/group).

Fig. 3

The fecal microbiome analysis of patients with chronic kidney disease (CKD) at before and after 4 week of L. rhamnosus L34 (L34) administration as indicated by the Cladogram (a branching diagram showing the cladistic relationship among species) (A), the Linear discriminant analysis Effect Size (LEfSe) (the identified bacteria characterizing the differences between groups) (B), and the principal coordinate analysis (PCoA) (the visualization indicating the differences between microbial communities by projecting data onto a 2 dimensional plot) on Bray–Curtis (C) are demonstrated.

Fig. 4

The characteristics of different cells after the 24 h incubation by the control culture media (Control) or Lacticaseibacillus condition media from L. rhamnosus L34 (LCM) or indoxyl sulfate (IS) (1 mM) or IS with LCM (IS + LCM) are demonstrated. For enterocytes (Caco-2 cells), supernatant IL-8 (A), the expression of several genes, including IL-8 (CXCL8), NF-κB, MUC-2, and occludin (OCLN) (B-E), and transepithelial electrical resistance (TEER) (F) are indicated. For THP-1-derived-macrophages (THP-1), supernatant cytokines (TNF-α, IL-6, and IL-10) (G-I) and the expression of several genes, including NF-κB and NOS2 (J, K), are used. For neutrophils, the neutrophil extracellular traps (NETs), as determined by nuclear morphology with representative fluorescent pictures (L, M), cell-free DNA (N), citrullinated histone 3 (CitH3) (O), are shown. The results were retrieved from the isolated triplicated experiments. #, p < 0.05 vs. Control, *, p < 0.05.

Fig. 5

The characteristics of enterocytes (Caco-2 cells), THP-1-derived-macrophages (THP-1), and neutrophils after 24 h incubation with indoxyl sulfate (IS) (1 mM) together with the Lacticaseibacillus condition media (LCM) from L. rhamnosus L34 (LCM from L34) or from L. rhamnosus GG (LCM from LGG) after neutralizing by several enzymes, including α-amylase (Amy), lipase (Lip), protease (Pro), and lysozyme (Lyz), or no enzyme (non) in comparison with the control media (no LCM), as demonstrated by supernatant IL-8 for Caco-2 (A), supernatant IL-6 for THP-1 (B), and citrullinated histone 3 (CitH3) for neutrophils (C), are demonstrated. The results were retrieved from the isolated triplicated experiments. #, p < 0.05 vs. No LCM, *, p < 0.05 vs. Non.

Fig. 6

Schema of the cross-sectional analysis and the characteristics of patients with chronic kidney disease (CKD) after 4 week administration of L. rhamnosus L34 (L34) or L. rhamnosus GG (LGG) or placebo control (placebo) is demonstrated.

Fig. 7

The characteristics of participants after the randomized control trial and the characteristics of patients with chronic kidney disease (CKD) after 4 week administration of L. rhamnosus L34 (L34) or L. rhamnosus GG (LGG) or placebo control (placebo), as indicated by hemoglobin, blood urea nitrogen (BUN), serum creatinine, estimated glomerular filtration rate (eGFR), urine protein creatinine ratio (UPCI) (A-E), serum cytokines (TNF-α, IL-6, and IL-10) (F–H), gut-derived uremic toxins (free indoxyl sulfate, total indoxyl sulfate, and p-cresol) (I-K), endotoxemia (L), serum (1→3)-beta-D-glucan (BG) (M), cell-free DNA (N), and citrullinated histone 3 (CitH3) (O) are demonstrated (n = 25/group). *, p < 0.05 vs. placebo.