Bovine serum albumin aggravates macrophage M1 activation and kidney injury in heterozygous Klotho-deficient mice via the gut microbiota-immune axis

Abstract

Klotho expression abnormalities induces kidney injury and chronic kidney disease, however, the underlying mechanism remains unclear. Here, Klotho^+/- mice and wild-type mice were treated with low-dose bovine serum albumin (BSA). Pathological examination demonstrated that the area of glomerular collagen deposition and fibrosis in BSA-Kl^-/+ mice was significantly larger than that in BSA-WT mice. The serum levels of superoxide dismutase, malondialdehyde, creatinine, and urea in BSA-Kl^-/+ mice were significantly increased. Sequencing of gut microbiota 16S rRNA v3-v4 region indicated that BSA-Kl^-/+ mice showed a significantly higher relative abundance of the genera Dubosiella, Akkermansia, Alloprevotella, and Lachnospiraceae and a significantly lower relative abundance of the genera Allobaculum and Muribaculaceae than BSA-WT mice. KEGG analysis revealed that the metabolic pathways of signal transduction, xenobiotic biodegradation and metabolism, and lipid metabolism increased significantly in BSA-Kl^-/+ mice. Flow cytometry showed that the proportion of CD68⁺/CD11b⁺ cells in the peripheral blood was significantly higher in BSA-KL^-/+ mice than that in BSA-WT mice. qPCR and western blot suggested that Klotho and Nrf2 expression in MΦ1 cells of BSA-KL^-/+ mice was significantly decreased. Thus, the findings suggest during the immune activation and chronic inflammation induced by the gut microbiota imbalance in Klotho-deficient mice treated to BSA, disrupted expression of proteins in the Nrf2/NF-κB signaling pathway in monocyte-derived macrophage M1 cells leads to the aggravation of inflammation and kidney injury.

Keywords: Klotho; Nrf2/NF-κB pathway; gut microbiota; kidney injury; macrophage M1.

Figure 1

Bovine serum albumin (BSA)-induced kidney injury in mice. (A) Hematoxylin and eosin staining. Magnification 200×. (B) Masson staining. Magnification 200×. The arrow indicated the collagen deposition sites. (C) Comparisons of mesangial matrix area and collagen area fraction of kidney tissues in BSA-treated Klotho-deficient and wild-type mice. **p < 0.01 (t test); n = 4. (D) Periodic acid-Schiff staining results. Magnification 200×. The arrow indicated the mesangial hyperplasia sites. (E) Results of serum superoxide dismutase and malondialdehyde assays of BSA-treated Klotho-deficient and wild-type mice. **p < 0.01 (t test); n = 4. (F) Creatinine and urea in serum and urea in urine in BSA-treated Klotho-deficient and wild-type mice. **p < 0.01 (t test); n = 4. (G) Western blot of Klotho in the kidney tissue of BSA-treated Klotho-deficient and wild-type mice.

Figure 2

Analysis of operational taxonomic units. (A) Number of operational taxonomic units. (B) Venn diagram of operational taxonomic units. (C) Gut microbiota clustering and species distribution. (D) Species phylogenetic tree.

Figure 3

Heat map of species richness clustering at the level of: (A) Phylum; (B) Species.

Figure 4

Alpha diversity analysis. (A) Rank abundance curve; (B) Rarefaction curve; (C) Shannon index curve; (D) Alpha diversity analysis.

Figure 5

Beta diversity analysis. (A) Results of principal component analysis, principal coordinate analysis, and non-metric multidimensional scaling analysis. (B) Sample unweighted pair-group method with arithmetic mean clustering tree. (C) Combined drawing of clustering tree and histogram. (D) Heat map showing sample abundance.

Figure 6

Significant difference analysis between groups. (A) Value distribution histogram of line discriminant analysis effect size. (B) Results of species annotation were visualized using KRONA. (C) Species network at the genus level.

Figure 7

Metabolic signaling pathways and protein differences in gut microbiota. (A) Results of Kyoto Encyclopedia of Genes and Genomes metabolic pathway analysis. (B) Clusters of orthologous groups of proteins analysis of distribution and abundance of homologous protein clusters in gut microbiota.

Figure 8

Bovine serum albumin (BSA) increased the number and activity of MΦ1. (A) Analysis of the proportion of monocyte-derived macrophage subsets using flow cytometry in BSA-treated Klotho-deficient and wild-type mice. **p < 0.01 (t test); n = 4. (B) qPCR results **p < 0.01 vs WT+BSA; t test; n = 4. (C) Western blot results.

Figure 9

BSA aggravates macrophage M1 activation and kidney injury in heterozygous Klotho-deficient mice via the gut microbiota-immune axis.