Endocrinol Metab.  2022 Jun;37(3):432-443. 10.3803/EnM.2021.1336.

Short-Chain Fatty Acids Attenuate Renal Fibrosis and Enhance Autophagy of Renal Tubular Cells in Diabetic Mice Through the HDAC2/ULK1 Axis

• Ma X
• Wang Q

• KMID: 2531387
• DOI: http://doi.org/10.3803/EnM.2021.1336

Abstract

Background
This study investigated the effect of short-chain fatty acids (SCFAs) on diabetes in a mouse model.
Methods
Autophagy in Akita mice and streptozocin (STZ)-induced diabetic C57BL/6 mice was determined by Western blots and immunohistochemistry (IHC). Western blots, IHC, hematoxylin and eosin staining, Masson staining, periodic acid-Schiff staining, and picrosirius red staining were conducted to detect whether autophagy and renal function improved in Akita mice and STZ-induced diabetic C57BL/6 mice after treatment of SCFAs. Western blots, IHC, and chromatin immunoprecipitation were performed to determine whether SCFAs affected diabetic mice via the histone deacetylase (HDAC2)/unc-51 like autophagy activating kinase 1 (ULK1) axis. Diabetic mice with kidney-specific knockout of HDAC2 were constructed, and IHC, Masson staining, and Western blots were carried out to detect whether the deletion of endogenous HDAC2 contributed to the improvement of autophagy and renal fibrosis in diabetic mice.
Results
Reduced autophagy and severe fibrosis were observed in Akita mice and STZ-induced diabetic C57BL/6 mice. Increased autophagy and reduced renal cell fibrosis were found in SCFA-treated Akita diabetic mice and STZ-induced diabetic C57BL/6 mice. Diabetic mice treated with SCFAs had lower HDAC2 expression and more enriched binding of ULK1 promoter sequences to H3K27Ac. Endogenous knockout of HDAC2 caused enhanced autophagy and decreased renal fibrosis in diabetic mice treated with SCFAs.
Conclusion
SCFAs enhanced autophagy of renal tubular cells and attenuated renal fibrosis in diabetic mice through the HDAC2/ULK1 axis.

Keyword

Diabetes; Diabetic nephropathies; Fatty acids, volatile; Autophagy; Renal fibrosis

Figure

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  Fig. 1. Decreased autophagy in renal tubular cells of diabetic mice. (A) Expression of light chain 3 (LC3)-I and LC3-II in renal cortical tissue cells of 14-week-old Akita mice detected by Western blots. (B) LC3 expression in renal tubular cells of 14-week-old Akita mice measured by immunohistochemistry (IHC). (C) Expression of LC3-I and LC3-II in renal cortical tissue cells of streptozocin (STZ)-treated C57BL/6 diabetic mice measured by Western blots. (D) LC3 expression in renal tubular cells of STZ-treated mice detected by IHC. The t test was utilized for two-group comparisons. One-way analysis of variance was used for comparisons among multiple groups, and the Tukey multiple-comparison test was used for post hoc multiple comparisons. The effect size between different experimental groups and control groups was >0.8. WT, wild-type; CT, control. aP<0.05; bP<0.01; cP<0.001 compared with WT mice (n=6).

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  Fig. 2. Short-chain fatty acids (SCFAs) enhanced autophagy in renal tubular cells of diabetic mice. Diabetic Akita mice were randomly divided into four groups. Healthy wild-type mice were fed distilled water, and diabetic Akita mice were fed with distilled water, sodium propionate, sodium butyrate, and sodium valproate respectively, per the standard of 300 mg/kg/day for 14 weeks; these were recorded as the wildtype (WT), diabetes, diabetes (D)+sodium propionate (SP), D+sodium butyrate (SB), and D+sodium valproate (SV) groups. (A) Expression of light chain 3 (LC3)-I and LC3-II in renal cortical tissues of WT and Akita mice detected by Western blots. (B) LC3 expression in renal tubular cells of WT and Akita mice measured by immunohistochemistry (IHC). C57BL/6 mice were randomly divided into five groups; after diabetes was induced by streptozocin (STZ) treatment, mice were fed with distilled water, sodium propionate, sodium butyrate, or sodium valproate for 14 weeks. These groups were recorded as control (CT; fed with distilled water without diabetes induction), STZ, STZ+SP, STZ+SB, and STZ+SV. (C) Expressions of LC3-I and LC3-II in renal cortical tissues of C57BL/6 control mice and diabetic mice measured by Western blots. (D) Expression of LC3 in renal tubular cells of C57BL/6 control mice and diabetic mice detected by IHC. One-way analysis of variance was used for comparisons among multiple groups, and the Tukey multiple-comparison test was used for post hoc multiple comparisons. The effect size between different experimental groups and control groups was >0.8. aP<0.01 compared with WT mice fed with distilled water; bP<0.05 compared with diabetic Akita mice fed with distilled water; cP<0.01 compared with WT mice; dP<0.05 compared with STZ-treated mice fed with distilled water (n=6).

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  Fig. 3. Short-chain fatty acids (SCFAs) reduced renal fibrosis in diabetic mice. hematoxylin-eosin (H&E), Masson, periodic acid-Schiff (PAS), and picrosirius red staining were performed on renal medulla tissue sections in each experimental group and the control group. (A) Staining results of Akita diabetic mice. (B) Staining results of streptozocin (STZ)-induced C57BL/6 mice. One-way analysis of variance was used for comparisons among multiple groups, and the Tukey multiple-comparison test was used for post hoc multiple comparisons. The effect size between different experimental groups and control groups was >0.8. WT, wild type; D, diabetes; SP, sodium propionate; SB, sodium butyrate; SV, sodium valproate; CT, control. aP<0.01 compared with the WT group; bP<0.05 compared with diabetic mice; cP<0.01 compared with the CT group; dP<0.05 compared with STZ mice (n=6).

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  Fig. 4. Effects of short-chain fatty acids (SCFAs) on histone deacetylase (HDAC) and histone acetylation in diabetic mice. (A) HDAC2 expression in Akita mice in the five groups detected by immunohistochemistry (IHC). (B) H3 histone acetylation levels in the five groups of Akita mice detected by IHC. One-way analysis of variance was used for comparisons among multiple groups, and the Tukey multiple-comparison test was used for post hoc multiple comparisons. The effect size between different experimental groups and control groups was >0.8. WT, wild type; D, diabetes; SP, sodium propionate; SB, sodium butyrate; SV, sodium valproate. aP<0.01 compared with the WT group; bP<0.05 compared with the diabetes group; cP<0.05 compared with the WT group; dP<0.01 compared with the diabetes group (n=6).

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  Fig. 5. Short-chain fatty acids (SCFAs) affected autophagy and renal fibrosis in diabetic mice through the histone deacetylase (HDAC2)/unc51 like autophagy activating kinase 1 (ULK1) axis. (A) The ULK1 promoter region in the University of California Santa Cruz (UCSC) database had a peak for H3K27Ac. (B) ULK1 expression in the wild type (WT), diabetes (D)+sodium propionate (SP), D+sodium butyrate (SB), and D+sodium valproate (SV) mice renal medulla tissues and cells obtained from the renal cortex or medulla was measured by Western blots. (C) ULK1 promoter sequences bound to H3K27Ac in mice renal tissues and cells obtained from the renal cortex or medulla from the above five groups detected by chromatin immunoprecipitation (ChIP). (D) The kidneys from 14-week-old mice of four genotypes were weighed, with the ratio of kidney to body weight calculated. (E) The degree of renal medulla fibrosis in mice of four genotypes detected by Masson staining. (F) Light chain 3 (LC3) expression measured by immunochemistry (IHC) in mice of the four genotypes. (G) ULK1 expression in mice of the four genotypes detected by Western blots. One-way analysis of variance was used for comparisons among multiple groups, and the Tukey multiple-comparison test was used for post hoc multiple comparisons. The effect size between different experimental groups and control groups was >0.8. aP<0.05 compared with the WT group; bP<0.05 compared with the diabetes group; cP<0.05; dP<0.01; eP<0.01 (n=6).

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