Specific Alternation of Gut Microbiota and the Role of Ruminococcus gnavus in the Development of Diabetic Nephropathy

Abstract

In this study, we aim to investigate the precise alterations in the gut microbiota during the onset and advancement of diabetic nephropathy (DN) and examine the impact of Ruminococcus gnavus (R. gnavus) on DN. Eight-week-old male KK-Ay mice were administered antibiotic cocktails for a duration of two weeks, followed by oral administration of R. gnavus for an additional eight weeks. Our study revealed significant changes in the gut microbiota during both the initiation and progression of DN. Specifically, we observed a notable increase in the abundance of Clostridia at the class level, higher levels of Lachnospirales and Oscillospirales at the order level, and a marked decrease in Clostridia_UCG-014 in DN group. Additionally, there was a significant increase in the abundance of Lachnospiraceae, Oscillospiraceae, and Ruminococcaceae at the family level. Moreover, oral administration of R. gnavus effectively aggravated kidney pathology in DN mice, accompanied by elevated levels of urea nitrogen (UN), creatinine (Cr), and urine protein. Furthermore, R. gnavus administration resulted in down-regulation of tight junction proteins such as Claudin-1, Occludin, and ZO-1, as well as increased levels of uremic toxins in urine and serum samples. Additionally, our study demonstrated that orally administered R. gnavus up-regulated the expression of inflammatory factors, including nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) and Interleukin (IL)-6. These changes indicated the involvement of the gut-kidney axis in DN, and R. gnavus may worsen diabetic nephropathy by affecting uremic toxin levels and promoting inflammation in DN.

Keywords: Gut microbiota; Ruminococcus gnavus; diabetic nephropathy; inflammation; uremic toxins.

Fig. 1. Experimental protocol for examining the effects of R. gnavus in KK-Ay mice.

Fig. 2. Gut microbiota compositions ranging from phylum to order levels in C57 and KK-Ay mice.

(A) Circos analysis providing a visual representation of the gut microbiota composition at the phylum level; (B) Community analysis pie plot presenting the relative abundance of different phyla in the gut microbiota; (C-E) Wilcoxon rank-sum test bar plots comparing the phylum, class and order level gut microbiota composition, respectively. C57, C57BL/6J group; KK, KK-Ay group; *p < 0.05, v.s. C57BL/6J group.

Fig. 3. Wilcoxon rank-sum test bar plots at the family and genus levels in C57 and KK-Ay mice.

(A) At the family level; (B) At the genus level. C57, C57BL/6J group; KK, KK-Ay group; *p < 0.05, v.s. C57BL/6J group.

Fig. 4. Gut microbiota compositions ranging from phylum to order of KK-Ay mice with different age.

(A) The Circos analysis displays the gut microbiota composition at the phylum level; (B-D) The Kruskal-Wallis H test bar plots show the statistical significance of differences at the phylum, class and order level, respectively. KK_0W, KK-Ay mice at 10 weeks old; KK_2W, KK-Ay mice at 12 weeks old; KK_4W, KK-Ay mice at 14 weeks old; KK_8W, KK-Ay mice at 18 weeks old; *p < 0.05, between groups; **p < 0.01, between groups.

Fig. 5. Gut microbiota compositions at the family and genus levels in KK-Ay mice of different ages.

(A) Circos analysis depicting the gut microbiota composition at the family level; (B, C) Kruskal-Wallis H test bar plot revealing significant differences at the family and genus level, respectively. KK_0W, KK-Ay mice at 10 weeks old; KK_2W, KK-Ay mice at 12 weeks old; KK_4W, KK-Ay mice at 14 weeks old; KK_8W, KK-Ay mice at 18 weeks old; *p < 0.05, between groups; **p < 0.01, between groups.

Fig. 6. Analysis of fecal microbiota in antibiotic-treated KK-Ay mice.

(A) Aerobic and anaerobic cultivation of intestinal contents from KK-Ay mice before and after antibiotic-treated treatment; (B) Agarose gel electrophoresis analysis of DNA abundance in KK-Ay mice with and without antibiotic-treated treatment; (C) Assessment of community diversity based on the Coverage metric; (D) Comparison of fecal microbial richness using the ACE index; (E) Calculation of fecal microbial richness using the Chao index; (F) Estimation of community diversitybased on the Sobs metric; (G) Evaluation of community diversity using the Shannon index; (H) Quantification of community diversity using the Simpson index. KK, KK-Ay group; anti: antibiotic-treated group.

Fig. 7. Impact of R. gnavus on kidney function.

(A) Electron microscopy visualization of kidney; (B) Alterations in urine UN levels; (C) Changes in urine Cr levels; (D) Fluctuations in urine protein concentrations; (E) Variation in urine KIM- 1 levels; (F) Modulation of serum KIM-1 levels. KK, KK-Ay group; anti, antibiotic-treated group; low, low dose R. gnavus group; mid, middle dose R. gnavus group; high, high dose R. gnavus group; low-res, low resolution; mid-res, mid resolution; high-res, high resolution; *p < 0.05, v.s. KK-Ay group; ^#p < 0.05, v.s. antibiotic-treated group.

Fig. 8. The effect of R. gnavus on colon.

(A) Immunohistochemistry staining of Claudin-1 in colon; (B) Immunohistochemistry staining of Occludin in colon; (C) Immunohistochemistry staining of ZO-1 in colon; (D) Alternation of TMAO in urine; (E) Alternation of TMAO in serum; (F) Alternation of pCS in urine; (G) Alternation of pCS in serum; (H) Alternation of IS in urine; (I) Alternation of IS in serum. KK, KK-Ay group; anti: antibiotic-treated group; R. gnavus: R. gnavus treatment group; low, low dose R. gnavus group; mid, middle dose R. gnavus group; high, high dose R. gnavus group; *p < 0.05, v.s. KK-Ay group; #p < 0.05, v.s. antibiotic-treated group.

Fig. 9. The effect of R. gnavus on inflammation.

(A) Alternation of NLRP3 in serum; (B) Alternation of IL-6 in serum. KK, KK-Ay group; anti: antibiotic-treated group; low, low dose R. gnavus group; mid, middle dose R. gnavus group; high, high dose R. gnavus group; *p < 0.05, v.s. KK-Ay group; ^#p < 0.05, v.s. antibiotic-treated group.