Anthocyanin improves kidney function in diabetic kidney disease by regulating amino acid metabolism

Abstract

Background: Diabetic kidney disease (DKD) is among the most important causes for chronic kidney disease. Anthocyanins (ANT) are polyphenolic compounds present in various food and play an important role in ameliorating hyperglycemia and insulin sensitivity. However, the effects of ANT in DKD are still poorly understood. This study aimed to investigate the effect of ANT (cyanidin-3-O-glucoside [C3G]) on the renal function of DKD, and whether the anti-DKD effect of ANT is related to metabolic pathways.

Methods: To explore the role of ANT in DKD, we performed the examination of blood glucose, renal function, and histopathology. As for the mechanism, we designed the label-free quantification proteomics and nontargeted metabolomics analysis for kidney and serum. Subsequently, we revealed the anti-DKD effect of ANT through the bioinformatic analysis.

Results: We showed that the fasting blood glucose level (- 6.1 mmol/L, P = 0.037), perimeter of glomerular lesions (- 24.1 μm, P = 0.030), fibrosis score of glomerular (- 8.8%, P = 0.002), and kidney function (Cystatin C: - 701.4 pg/mL, P = 0.043; urine creatinine: - 701.4 mmol/L, P = 0.032) were significantly alleviated in DKD mice after ANT treatment compared to untreated in the 20th week. Further, proteins and metabolites in the kidneys of DKD mice were observed to be dramatically altered due to changes in amino acid metabolism with ANT treatment; mainly, taurine and hypotaurine metabolism pathway was upregulated (P = 0.0001, t value = 5.97). Furthermore, upregulated tryptophan metabolism (P < 0.0001, t value = 5.94) and tyrosine metabolism (P = 0.0037, t value = 2.91) pathways had effects on serum of DKD mice as responsed ANT regulating.

Conclusions: Our results suggested that prevention of the progression of DKD by ANT could be related to the regulation of amino acid metabolism. The use of dietary ANT may be one of the dietary strategies to prevent and treat DKD.

Keywords: Amino acid metabolism; Chronic kidney disease; Diabetic nephropathy; Metabolomics; Proteomics.

Fig. 1

Study design and renal histopathological characteristics. The work flow of this study (A.) The weekly body weight of mice from 8- to 20-weeks (B) and the fasting blood glucose (FBG) at 8-, 12-, 16-, and 20-weeks (C) in the control group (CT), diabetic kidney disease group (DKD), and anthocyanin (ANT)-treated group all three groups. The green, orange, and blue nodes represent the CT, DKD, and ANT group, respectively (two-way ANOVA and multiple comparison using Tukey’s honest difference test, ^#between the DKD vs. CT groups, P < 0.05; ^&between the ANT vs. DKD groups, P < 0.05; NS not significant). (D) ANT improved renal dysfunction in diabetic mice. Representative photomicrographs of the kidney sections observed with hematoxylin&eosin (H&E), periodic acid–Schiff (PAS), and Masson staining (scale bar, 20 μm). Glomeruli in the kidney sections were visualized using H&E staining. The mesangial matrix was seen in the PAS staining. The interstitial fibers were visualized with Masson staining. The glomerular perimeter (E), area (F), and fibrosis score (G) and fibrosis score of renal interstitium (H) in three groups. The cystatin-C (I), blood urea nitrogen (J), urinary creatinine (K), and urinary microalbumin (L) in the three groups. The green, orange, and blue nodes represent the CT, DKD, and ANT groups, respectively (one-way ANOVA and multiple comparison using Holm-Sidak’s multiple comparisons test; ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS not significant)

Fig. 2

Effect of ANT on the protein expression profile in the kidney of db/db mice. (A) The heatmap of the expression of proteins in the kidney in the CT, DKD, and ANT groups. The expression of each protein was normalized. Volcano plots showing the differentially expressed proteins between the DKD vs. CT (B) and ANT vs. DKD (C) groups. (D) Renal proteomic profiles after STEM analysis. STEM analysis was applied to obtain the protein expression profiles across the CT, DKD, and ANT groups. Profile ID is shown at the top left corner of the profile. Lines in each profile represent the expression pattern of proteins across the three groups (permutation test, P < 0.05). (E) Venn diagram summarizing the differential and overlapping proteins among the DKD vs. CT groups, ANT vs. DKD groups, and STEM_trend (FC > 1.5 or FC < 0.67, unpaired two-sided student’s t-tests, P < 0.05). (F) Heatmap of the expression of ANT kidney trend proteins. (G) The function analysis of ANT kidney trend proteins in Metascape platform (P < 0.01)

Fig. 3

Effect of ANT on the protein expression profile in the serum of db/db mice. (A) The heatmap of the expression of serum proteins in the CT, DKD, and ANT groups. The expression of each protein was normalized. Volcano plots showing the differentially expressed proteins between the DKD vs. CT (B) and ANT vs. DKD (C) groups. (D) Serum proteomic profiles after STEM analysis. STEM analysis was applied to obtain the protein expression profiles across the CT, DKD, and ANT groups. Profile ID is shown at the top left corner of the profile. Lines in each profile represent the expression pattern of proteins across the three groups (permutation test, P < 0.05). (E) Venn diagram summarizing the differential and overlapping proteins among the DKD vs. CT groups, ANT vs. DKD groups, and STEM_trend (FC > 1.5 or FC < 0.67, unpaired two-sided student’s t-tests, P < 0.05). (F) The heatmap of the expression of ANT serum trend proteins in the three groups. (G) The function analysis of ANT serum trend proteins in Metascape platform (P < 0.01)

Fig. 4

Metabolic profiles in the kidney and serum in DKD. The score plot of Orthogonal projections to latent structures discriminant analysis (OPLS-DA) for distinguishing metabolites in the kidney (A) and serum (D) between the DKD and CT groups. The OPLS-DA model for distinguishing metabolites in the kidney (B) and serum (E) between the DKD and CT groups. The upset plot for differentially expressed metabolites in the kidney (C) and serum (F) between the DKD and CT groups. Bubble plot for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in the kidney (G) and serum (H) (P < 0.05). The differentially activated KEGG pathways between the DKD and CT groups in the kidney (I) and serum (J). The red and blue bands represent the upregulated and downregulated pathways, respectively (moderated t-test, P < 0.05, |t value|> 2)

Fig. 5

Effect of ANT on the metabolite expression profile in the kidney and serum of db/db mice. The score plot of OPLS-DA for distinguishing the metabolites in the kidney (A) and serum (D) between the ANT and DKD groups. The OPLS-DA model for distinguishing the metabolites in the kidney (B) and serum (E) between the ANT and DKD groups. The upset plot for differentially expressed metabolites among the DKD vs. CT groups, ANT vs. DKD groups, and STEM_trend in the kidney (C) and serum (F). The heatmap for the expression of ANT kidney trend metabolites (G) and ANT serum trend metabolites (H) in the three groups. Bar plot showing the percentage of differentially expressed metabolites among the DKD vs. CT groups, ANT vs. DKD groups, and STEM_trend in the kidney (I) and serum (J)

Fig. 6

GSVA analysis of KEGG pathways for ANT trend metabolites and metabolic pathways altered by ANT in the kidney and serum. The enrichment score for nine KEGG pathways by GSVA analysis in the kidney (A) and serum (C) among the CT, DKD, and ANT three groups. The red and blue nodes represent enrichment score of KEGG pathways in each sample for three groups, respectively. The gray and purple nodes represent P value of KEGG pathways (moderated t-test, P < 0.05). The differentially activated KEGG pathways between the DKD vs. CT and ANT vs. DKD groups in the kidney (B) and serum (D). The blue and orange bands represent the activated KEGG pathways of the DKD vs. CT and ANT vs. DKD groups, respectively, and the gray bands represent the nonactivated KEGG pathways. (E) The diagram of a part of the taurine and hypotaurine metabolism pathway. The expression of taurine, 5-L-glutamyl-taurine, and acetyl-CoA is shown in (E). The enzymatic expression of Ado, Cdo1, Fmo5, and Ggt1 is shown in (E). Other metabolites, including hypotaurine, cysteamine, L-cysteine, and 3-sulfino-L-alanine, were not identified in the current study. (F) The diagram of a part of the tryptophan and tyrosine metabolism pathway. The expression of L-tryptophan, L-kynurenine, 5-methoxyindoleacetate, indoleacetaldehyde, maleic acid, fumaric acid, and indole-5,6-quinone is shown in (F). The expression of Aldh1a7, Acat1, Cat, Hgd, Comt, and Fah is shown in (F). Other metabolites, including 3-hydroxyanthranilate, cinnavalininate, Acetyl-CoA, tyrosine, L-DOPA, L-metanephrine, and 4-maleylacetoacetate, were not identified in the current study