miR-335 and miR-34a Promote renal senescence by suppressing mitochondrial antioxidative enzymes

Abstract

The molecular basis for aging of the kidney is not well understood. MicroRNAs (miRNAs) contribute to processes such as development, differentiation, and apoptosis, but their contribution to the aging process is unknown. Here, we analyzed the miRNA expression profile of young (3-month) and old (24-month) rat kidneys and identified the biologic pathways and genes regulated by differentially expressed miRNAs. We observed upregulation of 18 miRNAs with aging, mainly regulating the genes associated with energy metabolism, cell proliferation, antioxidative defense, and extracellular matrix degradation; in contrast, we observed downregulation of 7 miRNAs with aging, principally targeting the genes associated with the immune inflammatory response and cell-cycle arrest. Bioinformatics analysis suggested that superoxide dismutase 2 (SOD2) and thioredoxin reductase 2 (Txnrd2), located in the mitochondria, are potential targets of miR-335 and miR-34a, respectively. Aging mesangial cells exhibited significant upregulation of miR-335 and miR-34a and marked downregulation of SOD2 and Txnrd2. miR-335 and miR-34a inhibited expression of SOD2 and Txnrd2 by binding to the 3'-untranslated regions of each gene, respectively. Overexpression of miR-335 and miR-34a induced premature senescence of young mesangial cells via suppression of SOD2 and Txnrd2 with a concomitant increase in reactive oxygen species (ROS). Conversely, antisense miR-335 and miR-34a inhibited senescence of old mesangial cells via upregulation of SOD2 and Txnrd2 with a concomitant decrease in ROS. In conclusion, these results suggest that miRNAs may contribute to renal aging by inhibiting intracellular pathways such as those involving the mitochondrial antioxidative enzymes SOD2 and Txnrd2.

Figure 1.

The lesions in the aged renal tissues were significantly increased. (A) Semiquantitative analyses of the renal lesions in aged rats. Glomerular scoring: *P < 0.01 versus young. Interstitial scoring: #P < 0.01 versus young. (B) Histologic analysis of 3- and 24-month rat renal tissues by PAS staining. In some glomeruli, focal segmental glomerular sclerosis may exist occasionally.

Figure 2.

miR-184, miR-335 and miR-347 exhibited differential expressions in (A) aged rats and (B) mice. (A) Fold differences between old and young rat kidneys measured by chip and qRT-PCR. (B) Expression levels of mmu-miR-184, mmu-miR-335, and mmu-miR-347 in the aged mouse kidneys detected by qRT-PCR. n = 5 per miRNA. *P < 0.01 versus young.

Figure 3.

The purity of the primary renal cells was identified by immunofluorescence staining. (A and B) The glomerular mesangial cells were stained with antibodies against desmin and vimentin. (C and D) The glomerular endothelial and epithelial cells were stained with antibodies against CD31 and nephrin, respectively. (E and F) The tubular epithelial cells and interstitial fibroblasts were stained with antibodies against cytokeratin 18 and FSP-1, respectively. Green color: fluorescein isothiocyanate-labeled secondary antibodies were used; red color: tetramethyl rhodamine isothiocyanate-labeled secondary antibodies were used.

Figure 4.

Expressions of miR-335 and miR-34a in aging mesangial cells, endothelial cells, tubular epithelial cells and interstitial fibroblasts were significantly upregulated and expressions of SOD2 and Txnrd2 in aging mesangial cells were downregulated. (A) Expression levels of miR-335 and miR-34a in aging renal mesangial cells were detected by qRT-PCR. n = 5 per miRNA. *P < 0.01 versus young. (B) Levels of SOD2 and Txnrd2 proteins were analyzed by Western blot in aging renal mesangial cells. The graph is representative of three separate experiments. (C) Expression levels of miR-335 and miR-34a were detected by qRT-PCR in the aging glomerular epithelial cells, endothelial cells, tubular epithelial cells, and fibroblasts. n = 5 per miRNA. *P < 0.01 versus young, #P < 0.05 versus young.

Figure 5.

SOD2 and Txnrd2 were potential target genes of miR-335 and miR-34a, respectively. (A) Construction of luciferase-wild-type UTR vectors (Luc-SOD2-UTR and Luc-Txnrd2-UTR) and the corresponding luciferase-mutated UTR vectors (Luc-SOD2-mutant UTR and Luc-Txnrd2-mutant UTR). (B) The effect of miR-335 on luciferase activity in mesangial cells cotransfected with miR-335 mimic (premiR-335) and Luc-SOD2-UTR vector or Luc-SOD2-mutant UTR. *P < 0.05 versus premiR control. (C) The effect of miR-34a on luciferase activity in mesangial cells cotransfected with miR-34a mimic (premiR-34a) and Luc-Txnrd2-UTR vector or Luc-Txnrd2-mutant UTR. *P < 0.05 versus premiR control.

Figure 6.

(A) miR-335 and miR-34a mimics inhibit SOD2 and Txnrd2 expressions in young mesangial cells, and (B) antisense miR-335 and miR-34a increase SOD2 and Txnrd2 expressions in aging mesangial cells. Co, miRNA control; CA, antisense miRNA control; miR335 and miR34a, miR-335 and miR-34a mimics, respectively; AmiR335 and AmiR34a, antisense miR-335 and miR-34a inhibitors, respectively. (C) Analysis of expression levels of SOD2 and Txnrd2 by Western blot in aged renal tissues. The graph is representative of three separate experiments.

Figure 7.

miR-335 and miR-34a mimics induce premature senescent phenotypes in young mesangial cells, and antisense miR-335 and miR-34a relieve senescent phenotypes in old mesangial cells. (A) SA-β-gal staining in young mesangial cells transfected with miR-335 and miR-34a mimics. Blue precipitation in the cytoplasm was observed in the senescent cells. (B) Analysis of SAHF formation in young mesangial cells transfected with miR-335 and miR-34a mimics. The cells were stained with DAPI, and heterochromatin foci were observed in the senescent cells. (C) SA-β-gal staining in aging mesangial cells transfected with antisense miR-335 and miR-34a inhibitors. (D) Analysis of SAHF formation in aging mesangial cells transfected with antisense miR-335 and miR-34a inhibitors. Co, miRNA control; CA, antisense miRNA control. The above results of SA-β-gal staining and SAHF analysis are representative images of three experiments.

Figure 8.

miR-335 and miR-34a mimics significantly elevated ROS levels in young mesangial cells and miR-335 and miR-34a inhibitors significantly decreased ROS levels in aging mesangial cells. (A) Level of ROS was determined in young mesangial cells transfected with miR-335 and miR-34a mimics. *P < 0.01 versus control. (B) Level of ROS was determined in old mesangial cells transfected with antisense miR-335 and miR-34a inhibitors. #P < 0.05 versus control.