Integrative microRNA and mRNA expression profiling in acute aristolochic acid nephropathy in mice

Abstract

In acute aristolochic acid nephropathy (AAN), aristolochic acid (AA) induces renal injury and tubulointerstitial fibrosis. However, the roles of microRNAs (miRNAs/miRs) and mRNAs involved in AAN are not clearly understood. The aim of the present study was to examine AA‑induced genome‑wide differentially expressed (DE) miRNAs and DE mRNAs using deep sequencing in mouse kidneys, and to analyze their regulatory networks. In the present self‑controlled study, mice were treated with 5 mg/kg/day AA for 5 days, following unilateral nephrectomy. AA‑induced renal injury and tubulointerstitial fibrosis were detected using hematoxylin and eosin staining and Masson's trichrome staining in the mouse kidneys. A total of 82 DE miRNAs and 4,605 DE mRNAs were identified between the AA‑treated group and the self‑control group. Of these DE miRNAs and mRNAs, some were validated using reverse transcription‑quantitative PCR. Expression levels of the profibrotic miR‑21, miR‑433 and miR‑132 families were significantly increased, whereas expression levels of the anti‑fibrotic miR‑122‑5p and let‑7a‑1‑3p were significantly decreased. Functions and signaling pathways associated with the DE miRNAs and mRNAs were analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 767 DE pairs (in opposing directions) of miRNAs and their mRNA targets were identified. Among these, regulatory networks of miRNAs and mRNAs were analyzed using KEGG to identify enriched signaling pathways and extracellular matrix‑associated pathways. In conclusion, the present study identified genome‑wide DE miRNAs and mRNAs in the kidneys of AA‑treated mice, as well as their regulatory pairs and signaling networks. The present results may improve the understanding of the role of DE miRNAs and their mRNA targets in the pathophysiology of acute AAN.

Figure 1.

Flow chart of the experimental design. AA, aristolochic acid; H&E, hematoxylin and eosin; miRNA, microRNA; DE, differentially expressed; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 2.

Histopathological changes induced by AA treatment analyzed by H&E and Masson's trichrome staining in kidney samples. Magnification, ×400. Compared with (A) the self-control group, notably widened tubular lumens and flattened tubular cells were observed in (B) the AA-treated group by H&E staining. The arrows pointed out to the representative widened tubular lumens or flattened tubular cells Compared with (C) the self-control group, a notable increase in collagen, as shown by an increase in Masson's trichrome staining, was observed in (D) the AA-treated group. AA, aristolochic acid; H&E, hematoxylin and eosin; CON, control.

Figure 3.

HCA and PCA of mRNA expression patterns. HCA and PCA were used to separate all 8 samples into different groups or clusters. Each group or cluster had similar mRNA expression patterns. (A) All 8 samples were separated into two groups, consistent with the treatment groups and the self-control groups, by HCA of mRNA expression levels. Each column represents an individual sample. Each row represents a differentially expressed mRNA. Different colors represent different expression levels of mRNA. The intensity of the red color is associated with higher expression levels. By contrast, the intensity of the blue color is associated with lower expression levels. (B) PCA separated all 8 samples into the AA-treated and the self-control clusters. PCA decreased a large amount of mRNA expression information in samples to a few independent variables (principal components) to compare the samples and identify clusters with high similarities in mRNA expression levels. The separate points represent separate samples. The x- and y-axes represent the contributor rate of the first component and second component, respectively. HCA, hierarchical clustering analysis; PCA, principal component analysis; AA, aristolochic acid.

Figure 4.

Analysis of GO and KEGG pathways of differentially expressed mRNAs. (A) GO analysis. (B) KEGG analysis of pathway enrichment. (C) Enriched KEGG pathways. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5.

Expression levels of DE miRNAs and DE mRNAs validated by RT-qPCR. Expression levels of 4 DE miRNAs and 4 DE mRNAs were measured using RT-qPCR and normalized to U6 or GAPDH, respectively. Relative expression levels of (A) miR-124-3p (P=0.0398), (B) miR-21a-5p (P=0.0391) and (C) miR-132-3p (P=0.0399) were significantly increased in the AA group compared with in the self-control group. Expression levels of (D) miR-1968-5p (P=0.0014) were significantly decreased in the AA group compared with in the self-control group. Relative mRNA levels of (E) Timp1 (P=0.0416), (F) Serpine1 (P=0.0475), (G) IL11 (P=0.0038) and (H) TGF-β1 (P=0.0146) were significantly increased in the AA group compared with in the self-control group. *P<0.05. AA, aristolochic acid; DE, differentially expressed; miRNA/miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; AA, aristolochic acid; IL11, interleukin 11; TGF-β1, transforming growth factor β1; mmu, Mus musculus.

Figure 6.

GO and KEGG pathway analysis of DE miRNAs and concurrently oppositely regulated DE mRNAs. (A) GO analysis. (B) KEGG analysis of pathway enrichment. (C) Enriched KEGG pathways. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; DE, differentially expressed; miRNA, microRNA.

Figure 7.

Networks of DE miRNAs and concurrently oppositely regulated DE mRNAs involved in signaling pathways and ECM-associated pathways following AA treatment. (A) Networks of decreased DE miRNAs and concurrently increased target DE mRNAs. (B) Networks of increased DE miRNAs and concurrently decreased target DE mRNAs. DE miRNAs are indicated by an oval. DE mRNAs are indicated by a rectangle. The increased DE miRNAs are marked in yellow and the decreased DE miRNAs are marked in blue. The increased DE mRNAs are marked in red and the decreased DE mRNAs are marked in green. The magnitude of the differential expression changes in DE miRNAs and DE mRNAs following AA treatment is indicated by the size of the node. AA, aristolochic acid; ECM, extracellular matrix; DE, differentially expressed; miRNA/miR, microRNA.