Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury

Abstract

MicroRNAs are endogenous, noncoding, small RNAs that regulate expression and function of genes, but little is known about regulation of microRNA in the kidneys under normal or pathologic states. Here, we generated a mouse model in which the proximal tubular cells lack Dicer, a key enzyme for microRNA production. These mice had normal renal function and histology under control conditions despite a global downregulation of microRNAs in the renal cortex; however, these animals were remarkably resistant to renal ischemia-reperfusion injury (IRI), showing significantly better renal function, less tissue damage, lower tubular apoptosis, and improved survival compared with their wild-type littermates. Microarray analysis showed altered expression of specific microRNAs during renal IRI. Taken together, these results demonstrate evidence for a pathogenic role of Dicer and associated microRNAs in renal IRI.

Figure 1.

Mouse model of targeted deletion of Dicer from renal proximal tubules. (A) Breeding protocol for the production of proximal tubule–specific Dicer knockout (PT-Dicer^−/−) mice. This study uses only male littermate mice with confirmed genotypes for experiment. (B) PCR-based genotyping to confirm Dicer deletion from renal cortical tissues. Genomic DNA is isolated from renal cortical tissues of PT-Dicer^−/− and PT-Dicer^+/+ mice. The DNA samples (50 ng per lane) are PCR-amplified using the primer pair DicerF1 and DicerDel (sequences shown in the Concise Methods section). Actin is also amplified as control. (C) Immunoblot analysis of Dicer expression in renal cortical tissues. Renal cortical tissues are dissected from kidneys of PT-Dicer^−/− and PT-Dicer^+/+ mice for homogenization to collect whole-tissue lysates for immunoblotting of Dicer and β-actin.

Figure 2.

Depletion of miRNAs from renal cortical tissues in PT-Dicer^−/− mice. Total RNA is isolated from renal cortical tissues of PT-Dicer^+/+ and PT-Dicer^−/− mice. (A) Real-time PCR analysis of miR-16, miR-192, and miR-194. Real-time PCR is performed using the Taqman miRNA assay kit as described in the Concise Methods section. The value of each miRNA is normalized by the signal of snoRNU202, an internal control. The normalized values of PT-Dicer^+/+ samples are arbitrarily set as 1; data of PT-Dicer^−/−samples are expressed as mean ± SD (n = 3). *Significantly different from the PT-Dicer^+/+ values. (B) Northern blot analysis of miR-16 and miR-194. Total RNA (10 μg per lane) is analyzed by Northern blotting as described in the Concise Methods section using a p³²-labeled probe of miR-16 or miR-194. 5s-rRNA is shown as an RNA loading control. (C) Representative heat map of microRNA microarray analysis. Total RNA samples isolated from renal cortical tissues of PT-Dicer^+/+ and PT-Dicer^−/− mice are subjected to microRNA microarray analysis. The ΔCt values of all miRNAs are used to generate the heat map. (D) Summary of microRNA microarray results. The microarray included 371 miRNA species, 173 of which were detectable in the renal cortical tissues. The value of a specific miRNA from the PT-Dicer^−/− sample is compared with that of PT-Dicer^+/+ sample to show whether the miRNA is decreased, increased, or unchanged. The results are representative of duplicate analyses.

Figure 3.

Resistance of PT-Dicer^−/− mice to renal IRI. Male PT-Dicer^+/+ and PT-Dicer^−/− mice of 8 to 10 weeks of age are subjected to sham operation (Control) or 32 minutes of bilateral renal ischemia followed by 48 hours of reperfusion (I32/48h). (A and B) Blood samples are collected to measure BUN (A) and serum creatinine (B). Data are means ± SD. (C) Renal tissues are collected for hematoxylin and eosin staining to examine histology. Histologic images of both low and high magnifications. (D) Ischemia reperfusion–induced tubular damage in PT-Dicer^+/+ and PT-Dicer^−/− mice is evaluated and scored by histology. Data are means ± SD. (E) Renal cortical and outer medulla tissues are also collected for TUNEL assay of apoptosis. (F) Representative images of TUNEL assay. TUNEL-positive cells are quantified by cell counting in comparable regions of the tissues. Data are means ± SD. n ≥ 4. *Significantly different from PT-Dicer^+/+ group.

Figure 4.

Survival of PT-Dicer^−/− and PT-Dicer^+/+ mice after severe renal IRI. PT-Dicer^−/− mice (n = 9) and their wild-type PT-Dicer^+/+ littermates (n = 10) we are subjected to 35 minutes of bilateral renal ischemia. Animal survival is monitored every day until 14 days of reperfusion. Statistical difference is determined by Kaplan-Meier survival analysis.