δ-Opioid receptor activation modified microRNA expression in the rat kidney under prolonged hypoxia

Abstract

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.

Figure 1. (A) Body weight measurement.

Data points represent the mean ± SE with a minimum n = 9. C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. **p<0.01 (C vs. H; C+DOR vs. H+DOR) for all time points encompassed by the bar. (B) Kidney weight/body weight ratios for each study group. *p<0.05, **p<0.01 with student’s t-test.

Figure 2. Dendrogram of aberrant miRNA expression in the kidney after hypoxia.

Unsupervised hierarchical cluster analysis of 22 miRNAs differentially expressed in the kidney after hypoxia separates hypoxic kidney from control samples based on miRNA profiling. Upregulated miRNAs are labeled red and downregulated miRs are colored green.

Figure 3. Relative miRNA expression levels of miR-363*, miR-let-7f, and miR-370 in the kidney following 10 days of hypoxia as determined by quantitative RT-PCR.

C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. **p<0.01.

Figure 4. Relative miRNA expression levels of miR-466b and miR-511 in the kidney following 10 days of hypoxia as determined by quantitative RT-PCR.

C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Relative miRNA expression levels of miR-298, miR-324-3p, and miR-20b-5p in the kidney following either 5 or 10 days of hypoxia as determined by quantitative RT-PCR.

C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. *p<0.05, **p<0.01, ***p<0.001.

Figure 6. Relative miRNA expression levels of miR-347 and miR-212 in the kidney following either 1, 5, or 10 days of hypoxia as determined by quantitative RT-PCR.

C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. *p<0.05, **p<0.01, ***p<0.001.

Figure 7. Relative miRNA expression levels of miR-351 and miR-29a in the kidney following either 1, 5, or 10 days of hypoxia as determined by quantitative RT-PCR.

C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. *p<0.05, **p<0.01.

Figure 8. Relative miRNA expression levels of miR-21 and miR-29b in the kidney following either 1, 5, or 10 days of hypoxia as determined by quantitative RT-PCR.

C, control; C+DOR, control+UFP-512; H, hypoxia; H+DOR, hypoxia+UFP-512. *p<0.05, **p<0.01, ***p<0.001.