Intestinal Dysbiosis, Barrier Dysfunction, and Bacterial Translocation Account for CKD-Related Systemic Inflammation

Abstract

CKD associates with systemic inflammation, but the underlying cause is unknown. Here, we investigated the involvement of intestinal microbiota. We report that collagen type 4 α3-deficient mice with Alport syndrome-related progressive CKD displayed systemic inflammation, including increased plasma levels of pentraxin-2 and activated antigen-presenting cells, CD4 and CD8 T cells, and Th17- or IFNγ-producing T cells in the spleen as well as regulatory T cell suppression. CKD-related systemic inflammation in these mice associated with intestinal dysbiosis of proteobacterial blooms, translocation of living bacteria across the intestinal barrier into the liver, and increased serum levels of bacterial endotoxin. Uremia did not affect secretory IgA release into the ileum lumen or mucosal leukocyte subsets. To test for causation between dysbiosis and systemic inflammation in CKD, we eradicated facultative anaerobic microbiota with antibiotics. This eradication prevented bacterial translocation, significantly reduced serum endotoxin levels, and fully reversed all markers of systemic inflammation to the level of nonuremic controls. Therefore, we conclude that uremia associates with intestinal dysbiosis, intestinal barrier dysfunction, and bacterial translocation, which trigger the state of persistent systemic inflammation in CKD. Uremic dysbiosis and intestinal barrier dysfunction may be novel therapeutic targets for intervention to suppress CKD-related systemic inflammation and its consequences.

Keywords: chronic kidney disease; inflammation; microbiota.

Figure 1.

Systemic inflammation in uremic Col4a3–deficient mice. (A) Glomerular and tubulointerstitial pathology in 9-week-old Col4a3-deficient mice is illustrated by periodic acid–Schiff staining. Representative images are shown at an original magnification of ×400. (B) CKD is illustrated by progressive decline in GFR measured by transcutaneous FITC Sinistrin clearance in conscious mice. (C) Serum levels of pentraxin-2 (PTX2)/serum amyloid P (SAP) are the murine equivalent to human PTX1/C-reactive protein and indicate systemic inflammation at 9 weeks of age. (D) Flow cytometric analysis of lymphocytes and myeloid cells in spleens of 9-week-old nonuremic wild–type and uremic Col4a3–deficient mice shows significant increased numbers of activated T cells and CD11c+ myeloid mononuclear phagocytes. Data represent means±SEMs of at least five mice in each group. *P<0.05 versus wild type; **P<0.01 versus wild type; ***P<0.001 versus wild type.

Figure 2.

Uremia affects microbiota diversity and intestinal barrier function. (A) Quantitative dysbiosis, measured in number of CFUs per gram of feces, is seen in Col4a3-deficient mice compared with wild-type animals. With increasing age, Col4a3-deficient mice developed an even more significant quantitative dysbiosis. (B) Relative abundance of different bacterial taxa detected in fecal samples. Alterations in the composition of the gut microbiota, which may be accounted for by aging, are detectable in 3- versus 9-week-old mice in both strains. Comparison of Col4a3-deficient and wild-type mice at an age of 9 weeks old depicts differences in the orders of Bacteroidales, Burholderiales (family of Alcaligenaceae), Enterobacteriales, and Verrucomicrobiales. Detailed information, including statistics, is listed in Supplemental Table 1. (C) Bacterial colonies grew from liver tissue of uremic Col4a3–deficient mice but not from nonuremic wild–type mice. (D) Sera of uremic and nonuremic mice were analyzed using limulus amebocyte lysate assay and showed significantly elevated levels of bacterial endotoxin. (E) ECIS experiments display a significant reduction for mean resistance across single-cell layers of Colon26 cells after treatment with serum of uremic Col4a3–deficient mice (time point marked by arrows) compared with cells treated with serum of wild-type mice. Data represent means±SEMs of at least five mice in each group. *P<0.05 versus wild type; ***P<0.001 versus wild type.

Figure 3.

Uremic systemic inflammation is corrected by eradication of the microbiota. All dashed lines in B and C represent values of control wild–type animals. (A) Representative pictures show a complete eradication of Gram–negative fecal flora after antibiotic treatment. The dot blot graph illustrates CFUs of feces cultures at an age of 7 weeks old. The graph depicts results of nonselective media, because a detection limit (dashed line) of 10 CFU/g feces was taken. (B) Serum endotoxin levels with and without antibiotic treatment in Col4a3-deficient mice. (C) Acute–phase protein serum amyloid P and IL-6 mRNA expression levels with and without antibiotic treatment in Col4a3-deficient mice. (D) Flow cytometry of spleen cell suspensions from uremic Col4a3–deficient mice with and without antibiotic treatment. Data represent means±SEMs of at least five mice in each group. *P<0.05 versus wild type; **P<0.01 versus wild type.