Identification of Genes Associated with Smad3-dependent Renal Injury by RNA-seq-based Transcriptome Analysis

Abstract

Transforming growth factor-β/Smad3 signaling plays a critical role in the process of chronic kidney disease (CKD), but targeting Smad3 systematically may cause autoimmune disease by impairing immunity. In this study, we used whole-transcriptome RNA-sequencing to identify the differential gene expression profile, gene ontology, pathways, and alternative splicing related to TGF-β/Smad3 in CKD. To explore common dysregulation of genes associated with Smad3-dependent renal injury, kidney tissues of Smad3 wild-type and knockout mice with immune (anti-glomerular basement membrane glomerulonephritis) and non-immune (obstructive nephropathy)-mediated CKD were used for RNA-sequencing analysis. Totally 1922 differentially expressed genes (DEGs) were commonly found in these CKD models. The up-regulated genes are inflammatory and immune response associated, while decreased genes are material or electron transportation and metabolism related. Only 9 common DEGs were found to be Smad3-dependent in two models, including 6 immunoglobulin genes (Ighg1, Ighg2c, Igkv12-41, Ighv14-3, Ighv5-6 and Ighg2b) and 3 metabolic genes (Ugt2b37, Slc22a19, and Mfsd2a). Our results identify transcriptomes associated with renal injury may represent a common mechanism for the pathogenesis of CKD and reveal novel Smad3 associated transcriptomes in the development of CKD.

Figure 1. Number of total DEGs in two CKD models of WT and Smad3 KO mice.

(a) Venn diagram has shown1922 common differentially expressed genes in two WT models. (b) Venn diagram of 84 common differentially expressed genes in two Smad3 KO models. (c) Venn diagram of 9 common Smad3-related differentially expressed genes in two models. GBM:anti-GBM GN model in wild-type or Smad3 knockout mice; UUO: UUO model in wild-type mice or Smad3 knockout mice.

Figure 2

Histogram showing the top five significant GO terms (including biological processes, molecular function and cellular component) of up-regulated genes in WT-GBM (a) or WT-UUO models (b) compared to WT-Nor.WT-UUO:UUO model in wild-type mice; WT-GBM:anti-GBM GN model in wild-type mice; WT-Nor:wild-type normal mice.

Figure 3

Histogram showing the top five significant GO terms (including biological processes, molecular function and cellular component) of down-regulated genes in WT-GBM (a) or WT-UUO models (b) compared to WT-Nor.WT-UUO:UUO model in wild-type mice; WT-GBM:anti-GBM GN model in wild-type mice; WT-Nor:wild-type normal mice.

Figure 4

Histogram showing all the significant KEGG pathways of up-regulated genes in WT-GBM (a) or WT-UUO models (b) compared to WT-Nor.WT-UUO:UUO model in wild-type mice;WT-GBM:anti-GBM GN model in wild-type mice; WT-Nor:wild-type normal mice.

Figure 5

Histogram showing all the significant KEGG pathways of down-regulated genes in WT-GBM (a) or WT-UUO models (b) compared to WT-Nor. WT-UUO:UUO model in wild-type mice; WT-GBM:anti-GBM GN model in wild-type mice; WT-Nor:wild-type normal mice.

Figure 6. Pie charts showing the number ofthe significant AS genes and events in WT and Smad3 knockout groups.

(a) WT-GBM compared toWT-Nor. (b) WT-UUOcompared to WT-Nor. (c) KO-GBM compared toKO-Nor. (d) KO-UUO compared to KO-Nor. WT-GBM:anti-GBM GN model in wild-type mice; WT-Nor:wild-type normal mice; WT-UUO:UUO model in wild-type mice; KO-GBM:anti-GBM GN model in Smad3 knockout mice; KO-UUO:UUO model in Smad3 knockout mice; KO-Nor:Smad3 knockout normal mice; SE:SkippingExon; MEX: Mutually Exclusive Exons; A5SS: Alternative 5′ splice site; A3SS: Alternative 3′ splice site; RI: Retained Intron

Figure 7. (a) Western blot analysis show protein expression levels of filamin alpha (Flna), collagen III (Col. III), collagen I (Col. I), Trem1, Pvalb.

(b) Ccl7 expression by western blot analysis. Each bar represents the mean ± SD for at least 6 animals. *p < 0.05, **p < 0.01, ***p < 0.001 vs WT-Nor or KO-Nor.WT-Nor:wild-type normal mice; WT-UUO:UUO model in wild-type mice; KO-Nor:Smad3 knockout normal mice; KO-UUO:UUO model in Smad3 knockout mice.