Renal Vein Levels of MicroRNA-26a Are Lower in the Poststenotic Kidney

Abstract

MicroRNA-26a (miR-26a) is a post-transcriptional regulator that inhibits cellular differentiation and apoptosis. Renal vascular disease (RVD) induces ischemic injury characterized by tubular cell apoptosis and interstitial fibrosis. We hypothesized that miR-26a levels are reduced in the poststenotic kidney and that kidney repair achieved by adipose tissue-derived mesenchymal stem cells (ad-MSCs) is associated with restored miR-26a levels. Renal function and renal miR-26a levels were assessed in pigs with RVD not treated (n=7) or 4 weeks after intrarenal infusion of ad-MSC (2.5×10(5) cells/kg; n=6), patients with RVD (n=12) or essential hypertension (n=12), and healthy volunteers (n=12). In addition, the direct effect of miR-26a on apoptosis was evaluated in a renal tubular cell culture. Compared with healthy control kidneys, swine and human poststenotic kidneys had 45.5±4.3% and 90.0±3.5% lower levels of miR-26a, respectively, which in pigs, localized to the proximal tubules. In pigs, ad-MSC delivery restored tubular miR-26a expression, attenuated tubular apoptosis and interstitial fibrosis, and improved renal function and tubular oxygen-dependent function. In vitro, miR-26a inhibition induced proximal tubular cell apoptosis and upregulated proapoptotic protein expression, which were both rescued by ad-MSC. In conclusion, decreased tubular miR-26a expression in the poststenotic kidney may be responsible for tubular cell apoptosis and renal dysfunction but can be restored using ad-MSC. Therefore, miR-26a might be a novel therapeutic target in renovascular disease.

Keywords: kidney; microRNA; renal artery stenosis; stem cell.

Copyright © 2015 by the American Society of Nephrology.

Figures

Figure 1.

Mir-26a is decreased in RVD and normalized by MSC treatment. (A) Quantitative miRs expression by plate-based hybridization. Expression was not altered in RVD, and only miR-210 slightly decreased by MSCs. (B) miR-26a was decreased in RVD and normalized in RVD+MSC. (C) Representative fluorescent in situ hybridization staining showing decreased miR-26a expression in stenotic kidney tubular cells in RVD, which was restored in RVD+MSC. (D) Dicer expression was upregulated in RVD and normalized in RVD+MSC. (E) Endogenous miR-26a expression was similar to human umbilical vein endothelial cells (HUVECs) in MSCs but elevated in kidney tubular cells (LLC-PK1). *P<0.05 versus normal or control; †P<0.05 versus RVD; ‡P<0.05 versus MSCs and HUVECs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. RLU, relative light unit.

Figure 2.

MSC treatment improved renal microvascular structure and decreased renal apoptosis in RVD. (A and B) Representative micro-CT images and quantifications showing microvascular loss that was improved by MSCs in the middle and outer cortexes of RVD kidneys. (C and D) Representative terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) images showing increased stenotic kidney apoptosis in RVD, which is attenuated in RVD+MSC. (E and F) Both AIF and caspase-3 were upregulated in RVD and attenuated in RVD+MSC. *P<0.05 versus normal; †P<0.05 versus RVD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

MSC attenuated renal inflammation and fibrosis in RVD. (A and B) Western blotting for TNF-α, NF-κB, and IL-10 in normal, RVD, and RVD+MSC pigs. TNF-α and P-NF-κB were upregulated and IL-10 was downregulated in the stenotic kidney, and MSCs improved them. MSCs attenuated renal fibrosis (trichrome staining, C and D) and glomerulosclerosis (E) in RVD. *P<0.05 versus normal; †P<0.05 versus RVD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. P-NF-κB, phospho-nuclear factor kappa B.

Figure 4.

Standard characterized MSC improved kidney function in RVD. (A) Single-kidney renal blood flow and GFR and (B) oxygen-dependent tubular function (response to furosemide by BOLD MRI) were all impaired in RVD and improved with MSC treatment. (C) Representative immunostaining of MSCs, which are positive for CD90, CD44, and CD105 and negative for CD31, CD34, and CD45. (D) MSC (red) tracking showing retention in tubular structures and interstitial space. Green shows cytokeratin-positive tubular cells. (E) Representative FACs results confirming that most MSCs are positive (FITC) for CD44, CD90, and CD105. *P<0.05 versus normal.

Figure 5.

Decreased renal vein miR-26a level in patients with RVD is associated with increased inflammatory cytokines. (A) miR-26a levels (adjusted for GFR) were significantly lower in the renal veins of patients with RVD compared with patients with EH. (B) In patients with RVD, the stenotic kidney showed greater hypoxia R2* level by BOLD MRI compared with patients with EH, whereas R2* responses to furosemide were significant in both patients with RVD and patients with EH. (C) In patients with RVD, systemic and renal vein TNF-α levels were significantly increased. (D) Patients with RVD had decreased systemic IL-10 level. (E) Compared with patients with EH, patients with RVD showed increased E-selectin levels in the renal vein. (F) Patients with RVD had increased systemic myeloperoxidase level compared with HVs. (G) Systemic granulocyte colony-stimulating factor level was only increased in patients with RVD. HV, healthy volunteers. *P<0.05 versus EH; †P<0.05 versus HV; ‡P<0.05 versus baseline.

Figure 6.

miR-26a inhibition induces kidney tubular cell apoptosis in vitro. (A) Representative FACs for apoptotic cells detected using annexin V. (B) Kidney tubular cells treated with miR-26a inhibitor increased apoptosis compared with negative controls, which were reversed by coculture with MSCs. (C) miR-26a inhibition upregulated AIF and caspase-3 expressions, which were reversed by MSC treatment. *P<0.05 versus negative control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.