Identification of Smad3-related transcriptomes in type-2 diabetic nephropathy by whole transcriptome RNA sequencing

Abstract

Smad3 deficiency prevents the development of type 2 diabetic nephropathy; however, the underlying molecular mechanisms remain unknown. In this study, we aimed to identify Smad3-related genes involved in the pathogenesis of diabetic kidney disease. High-throughput RNA sequencing was performed to profile the whole transcriptome in the diabetic kidney of Smad3 WT-db/db, Smad3 KO-db/db, Smad3^+/- db/db and their littermate control db/m mice at 20 weeks. The gene ontology, pathways and alternative splicing of differentially expressed protein-coding genes and long non-coding RNAs related to Smad3 in diabetic kidney were analysed. Compared to Smad3 WT-db/db mice, Smad3 KO-db/db mice exhibited an alteration of genes associated with RNA splicing and metabolism, whereas heterozygosity deletion of Smad3 (Smad3^+/- db/db mice) significantly altered genes related to cell division and cell cycle. Notably, three protein-coding genes (Upk1b, Psca and Gdf15) and two lncRNAs (NONMMUG023520.2 and NONMMUG032975.2) were identified to be Smad3-dependent and to be associated with the development of diabetic nephropathy. By using whole transcriptome RNA sequencing, we identified novel Smad3 transcripts related to the development of diabetic nephropathy. Thus, targeting these transcripts may represent a novel and effective therapy for diabetic nephropathy.

Keywords: Smad3 deficiency; alternative splicing; diabetic kidney disease; whole transcriptome.

Figure 1

Overview analysis of transcriptomic RNA‐seq. A, The length of all lncRNAs, protein‐coding RNA and predict novel lncRNAs. B, The classification of lncRNAs. C, The distribution of protein‐coding and lncRNAs in all the mouse chromosomes

Figure 2

The top five significant GO terms (A‐D) and KEGG pathways (E‐H) of differentially expressed protein‐coding genes in comparisons 1‐4. BP, biological processes; CC, cellular component; MF, molecular function. When the number of significant GO terms and KEGG pathways was <5, actual number was listed in the figure

Figure 3

The top five significant GO terms (A‐D) and KEGG pathways (E‐H) of differentially expressed lncRNAs in comparisons 1‐4. BP, biological processes; MF, molecular function; CC, cellular component. When the number of GO terms and KEGG pathways was less than 5, actual number was listed in the figure

Figure 4

A, The number of alternative splicing events in each comparison. A3SS, Alternative 3′ splice site; A5SS, Alternative 5′ splice site; MXE, Mutually exclusive exon; RI, Retained intron; SE, Skipped exon. B‐E, The co‐expression network of lncRNAs and protein‐coding genes in comparisons 1‐4. The relative expression levels of protein‐coding (F) and lncRNA genes (G) in Smad3 WT‐db/db and Smad3 WT‐db/m mice kidney were validated by qPCR. Db: Smad3 WT‐db/db; dm: Smad3 WT‐db/m. *P < .05 vs dm, **P < .01 vs dm. The relative expression levels of protein‐coding genes (H‐J) and lncRNAs (K‐L) in Smad3 WT‐db/db, Smad3 KO‐db/db, Smad3 WT‐db/m or Smad3 KO‐db/m mice kidney were validated by qPCR.*P < .05 vs Smad3 WT‐db/m, ***P < .001 vs Smad3 WT‐db/m, #P < .05 vs Smad3 KO‐db/m