Research Paper Volume 15, Issue 20 pp 10972—10995

Figure 2. Soluble and insoluble-proteomic analyses of mouse hippocampus reveal the increase in a subset of proteins associated with BBB breakdown and Alzheimer’s disease in CKD. (A) Proteomic analyses using TBS-soluble and detergent-insoluble fractions of mouse hippocampus tissue lysates. We performed a mass spectrometry on the mixture of mice hippocampus tissue lysates in the CKD (n = 3) and control (n = 3) groups after a sequential biochemical extraction into TBS (salt buffer)-soluble fraction and sarkosyl (detergent buffer)-insoluble fraction. (B) Among the identified unique proteins or peptides over 7,000, we focused on upregulated (red) and downregulated (blue) DEGs in CKD when the fold changes were ≥3.0 compared to the controls. (C) The enrichment analysis of the DEGs in the insoluble fraction with the GO functional analysis revealed several pathways of interest, including “biological processes,” “cellular components,” and “molecular functions.” (D) Western blotting was performed to validate the proteomic analyses and showed that CKD increased expressions of soluble/insoluble IgG, and total and phosphorylated tau and MMP2 in the sarkosyl-insoluble (aggregated) fraction in the hippocampus. * Represents IgG heavy chain, and # represents the IgG light chain. The densitometry of IgG was determined with a mass of heavy and light chains. The densitometric analysis showed that CKD increased soluble IgG (n = 4 per group), insoluble IgG (n = 3 per group), insoluble phosphorylated tau (n = 14 per groups), and insoluble MMP2 (n = 3). Data are presented as mean ± standard deviation of the mean. Normality was assessed with the Shapiro–Wilk test. Statistical significance between the two groups was evaluated using an unpaired t test or Wilcoxon signed-rank test. When variables were nonparametric, we used the Wilcoxon signed-rank test. P < 0.05 was considered statistically significant. CKD, chronic kidney disease; MMP2, matrix metalloproteinase-2.