Table 1.
The sequence for qPCR primers and shRNA.
Fig 1.
Bioinformatics analysis identifies differentially expressed genes associated with ribosome biogenesis in renal fibrosis.
(A) Differential gene expression analysis was performed on GEO datasets GSE42303 (including 3 samples of UUO-induced renal fibrosis samples and 3 controls) and GSE121190 (containing 3 UUO-induced renal fibrosis samples and 3 control samples) using the limma package in R studio. (B) Venn diagram showing overlapping differentially expressed genes with consistent expression trends in both datasets. (C-D) Metascape was used for the Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment annotation of the differentially expressed genes.
Fig 2.
Elevated ribosome biogenesis in a unilateral ureteral obstruction (UUO)-induced renal fibrosis mouse model.
Mice were randomly allocated into UUO and control groups. UUO was conducted as described. (A) Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured from serum samples. (B) Gross morphology of kidneys. (C) Hematoxylin and eosin (H&E) staining was performed to assess pathological damage in the renal cortex. (D) MASSON staining was performed to detect collagen production in the renal cortex. (E) Immunoblotting was applied to assess the expression levels of KIM-1 and NGAL, both markers of tubular injury, in kidney tissues. (G) The levels of TNF-α, IL-1β, and IL-6 in mouse renal tissues were measured by ELISA. (F) Immunoblotting was performed to detect the protein levels of E-CADHERIN, α-SMA, FIBRONECTIN, and COLLAGEN I. (H) qRT-PCR was performed to measure rRNA levels in the kidneys. Data are analyzed using unpaired Student’s t-test and presented as the mean ± SD. N = 6 (biological replicates) for A, C, D, G and H. N = 3 (biological replicates) for E and F. ** P < 0.01 compared to control group.
Fig 3.
Renal injury is accompanied by significant upregulation of Isg20 and ribosome biogenesis increase.
(A) Intersection analysis identifying 1708 overlapping differentially expressed genes related to 331 ribosome biogenesis and 23 overlapping genes were identified. (B) Among these 23 overlapping genes, DEGs with the top 10 log2|FC| values were compared and 3 were overlapping. (C) Expression levels of Rps19, Rps9, and Isg20 in renal fibrosis samples from datasets GSE42303 and GSE121190. (D) The mRNA expression levels of Rps19, Rps9, and Isg20 were determined in mice kidneys using qRT-PCR. N = 6 (biological replicates). (E) ISG20 protein level was determined in mice kidneys using Immunoblotting. N = 3 (biological replicates). (F) IHC quantitative analysis was performed to evaluate the cytoplasm and nuclei distribution of ISG20. N = 3 (biological replicates). Data are analyzed using unpaired Student’s t-test and presented as the mean ± SD. ** P < 0.01 compared to control group.
Fig 4.
Knockdown of Isg20 ameliorates renal fibrosis and reduces ribosome biogenesis.
Isg20 knockdown was achieved in UUO-induced CKD mice models by introducing short hairpin RNA against Isg20. (A-B) Isg20 knockdown was confirmed in mice kidneys using qRT-PCR and Immunoblotting. (C) SCr and BUN levels measured from serum samples obtained via abdominal aorta blood collection and centrifugation. (D) Gross morphology of kidneys. (E) H&E staining was performed to assess pathological damage in the renal cortex. (F) MASSON staining was performed to detect collagen production in the renal cortex. (G) Immunoblotting was used to determine the protein levels of KIM-1 and NGAL in kidney tissues. (H) Immunoblotting was performed to detect the protein levels of E-CADHERIN, α-SMA, FIBRONECTIN, and COLLAGEN I in kidney tissues. (I) The expression levels of TNF-α, IL-1β, and IL-6 in mice renal tissues were measured by ELISA. (J) qRT-PCR was performed to detect rRNA levels in kidney tissues. Data are analyzed using unpaired Student’s t-test and presented as the mean ± SD. N = 6 (biological replicates) for A, C, D, E, I and J. N = 3 (biological replicates) for B, G and H. ** P < 0.01 compared to UUO + sh-NC group.
Fig 5.
Knockdown of Isg20 potentially ameliorates endoplasmic reticulum (ER) stress and cell death by inhibiting ribosome biogenesis.
Mice were randomly allocated into four groups: Control group, UUO group, UUO + sh-NC group, and UUO + sh-Isg20 group and received corresponding operation and treatments. (A-B) The protein levels of PERK, IRE1, and GRP78 in kidney tissues were determined using Immunoblotting. N = 3 (biological replicates). (C) TUNEL staining was performed to detect cell apoptosis in the renal cortex of mice. N = 6 (biological replicates). (D) Quantification of apoptosis rate in kidney tissues from TUNEL staining. (E-F) The protein levels of apoptosis-related factors BCL-2 and BAX in kidney tissues were determined using Immunoblotting. N = 3 (biological replicates). Data are analyzed using one-way ANOVA followed LSD test for (B, D and F). ** P < 0.01 compared to control group; ## P < 0.01 compared to UUO + sh-NC group.