Analysis of altered microRNA expression profiles in the kidney tissues of ethylene glycol-induced hyperoxaluric rats

Abstract

Calcium oxalate stones account for >80% of urinary stones, however the mechanisms underlying their formation remains to be elucidated. Hyperoxaluria serves an important role in the pathophysiological process of stone formation. In the present study, differences in the miRNA expression profiles between experimental hyperoxaluric rats and normal rats were analyzed, in order to identify target genes and signaling pathways involved in the pathogenesis of hyperoxaluria. Ethylene glycol and ammonium chloride was fed to male hyperoxaluric rats (EXP) and normal age‑matched male rats (CON). The oxalate concentration in the urine of each experimental rat was collected every 24 h and measured on day 14. Three rats exhibiting the highest concentrations were selected for microarray analysis. Microarray analysis was performed to evaluate differences in the expression of microRNA (miRNA) in the kidney tissues from EXP and CON groups, and miRNAs that exhibited a >2‑fold or a <0.5‑fold alteration in expression between these groups were screened for differential expression patterns according to the threshold P‑values. Reverse transcription‑quantitative polymerase chain reaction analysis was employed to confirm the microarray results. In order to predict the potential role of miRNAs in pathophysiological processes, gene ontology (GO), pathway and target prediction analyses were conducted. A total of 28 miRNAs were observed to be differentially expressed (>2‑fold change) between EXP and CON groups. Among these miRNAs, 20 were upregulated and 8 were downregulated. GO and pathway analyses revealed that the insulin resistance and phosphatidylinositol‑bisphosphonate 3‑kinase/AKT serine threonine kinase signaling pathways were potentially associated with miRNA regulation in this setting. In conclusion, the results of the present study identified differentially expressed miRNAs in hyperoxaluric rats, and provided a novel perspective for the role of miRNAs in the formation of calcium oxalate stones.

Figure 1.

Examination of kidney tissue sections by light microscopy following von Kossa staining. Microscope images of kidney tissues derived from (A) CON and (B) EXP rats. Numerous calcium crystals (stained black-brown with argent nitrate) can be observed in Fig. 1B (magnification, ×200). CON, rats provided with water for 2 weeks; EXP, rats provided with water containing 1.0% ethylene glycol and 0.5% ammonium chloride for 2 weeks to induce hyperoxaluria.

Figure 2.

Ethylene glycol and ammonium chloride increases rat urinary oxalate levels. Urine specimens were collected (in triplicate) at 24 h-intervals from rats in the EXP and CON groups and oxalate concentrations were determined on day 14 using ion chromatography. The mean ± standard deviation urine oxalate concentration in CON and EXP groups were 24.16±1.727 and 75.91±2.885 µmol/24 h, respectively. *P<0.01 vs. CON group. miRNA, microRNA; EXP, rats provided with water containing 1.0% ethylene glycol and 0.5% ammonium chloride for 2 weeks to induce hyperoxaluria; CON, rats provided with water for 2 weeks.

Figure 3.

Hierarchical cluster heat map of differentially expressed miRNAs in rat renal tissues from EXP and CON groups. Red signals indicate downregulated expression and green signals indicate upregulated expression. EXP, rats provided with water containing 1.0% ethylene glycol and 0.5% ammonium chloride for 2 weeks to induce hyperoxaluria; CON, rats provided with water for 2 weeks.

Figure 4.

Reverse transcription-quantitative polymerase chain reaction analysis validating the expression of five differentially expressed miRNAs identified by microarray analysis of rat renal tissues from CON and EXP groups. The selected miRNAs consisted of three upregulated miRNAs and two downregulated miRNAs in rat renal tissues from the EXP group relative to the CON group. miRNA, microRNA; CON, rats provided with water for 2 weeks; EXP, rats provided with water containing 1.0% ethylene glycol and 0.5% ammonium chloride for 2 weeks to induce hyperoxaluria.

Figure 5.

Significant GO functional maps of genes targeted by differentially expressed miRNAs identified following microarray analysis of rat renal tissues from CON and EXP groups. The GO category and enrichment of GOs is presented on the vertical and horizontal axes, respectively. A higher number indicates that the function is more significant. GO, gene ontology; miRNA, microRNA; CON, rats provided with water for 2 weeks; EXP, rats provided with water containing 1.0% ethylene glycol and 0.5% ammonium chloride for 2 weeks to induce hyperoxaluria.

Figure 6.

Pathway analysis of genes targeted by differentially expressed miRNAs identified following microarray analysis of rat renal tissues from CON and EXP groups. Significant pathways targeted by (A) upregulated and (B) downregulated miRNAs. Enrichment scores of biological processes are indicated by-log (P-value). miRNA, microRNA; CON, rats provided with water for 2 weeks; EXP, rats provided with water containing 1.0% ethylene glycol and 0.5% ammonium chloride for 2 weeks to induce hyperoxaluria.