Hematopoietic microRNA-126 protects against renal ischemia/reperfusion injury by promoting vascular integrity

Abstract

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.

Figure 1.

Validation of overexpression of miR-126 and BM transplantation in vivo. (A) Microscopic images (×200) of PB and BM smears show dsRed⁺ cells in animals transplanted with LV-C or LC-126 compared with cells of nontransplanted animals (donor). (B) Representative FACS plots demonstrate successful chimerism in PB and BM by CD45.1 (donor) and CD45.2 (acceptor) levels. Numbers represent percentage of cells in corresponding quadrant. Quantitative RT-PCR demonstrates significant upregulation of miR-126 levels in BM (C) and PB (D) of LV-126 mice compared with LV-C mice.

Figure 2.

Overexpression of miR-126 in BM cells leads to increased neovascularization in subcutaneously implanted Matrigel plugs. (A) Representative microscopic images of both sides of Matrigel plugs 7 days after implantation in LV-C mice (left micrographs) and LV-126 mice (right micrographs). Arrowheads point to neovasculature. (B) A movie still displaying the presence of red blood cells in the neovasculature of the Matrigel plugs. (C) Quantification of the number of vessels per plug shows increased vascularization in animals transplanted with LV-126 BM with borderline significance (P=0.05). (D) Vessels were “digitalized” and the total length (in pixel area) was quantified. LV-126 animals demonstrated a significantly higher vessel surface area compared with LV-C animals. (E) Total surface area of plug-vessels correlates significantly with the expression levels of miR-126 in the BM (P=0.05). (F and G) Representative confocal images (×400) of a vessel and corresponding cross-sectional profiles of fluorescent labels (colors correspond to images) stained for MECA32⁺/dsRed⁺ EC in 2 different planes (F) and stained for PDGFRβ⁺/dsRed⁺ pericytes (G). *P<0.01; ^#P<0.10.

Figure 3.

miR-126 protects against renal IRI. (A) miR-126 overexpression results in decreased blood urea levels after IRI. (B) miR-126 overexpression results in less weight loss 3 days after IRI. Quantitative RT-PCR analysis normalized on glyceraldehyde 3-phosphate dehydrogenase mRNA levels of renal gene expression in LV-126 and LV-C mice of (C) KIM-1, neutrophil gelatinase-associated lipocalin, chemokine (C-C motif) ligand 2, IL-6, and IL-10 3 days after injury and (D) α-smooth muscle actin, Col1α1, Col3α1, and TGF-β 3 weeks after injury. Increase or decrease is relative to LV-C group. (E) Representative images (×200) of hematoxylin and eosin staining on LV-C and LV-126 kidney sections. Graphs show quantification of atubular necrosis and cast score. (F–I) Representative images (×100) of staining for tubular damage (E, KIM-1), leukocytes (F, CD45), macrophages (G, F4/80) and neutrophils (H, Gr-1) 3 days after IRI. Graphs below images show quantification of staining results for all mice (n=10 per group).

Figure 4.

miR-126 preserves capillary density by increasing incorporation of BM-derived EC. (A) Representative microscopic images (×100) and (G) quantification of MECA32 staining in corticomedullary junctions show higher capillary density in mice that overexpress miR-126 3 days after IRI. (B) Representative microscopic images (×100) and (H) quantification of dsRed staining in the kidney show increased dsRed signal in LV-126 mice. (C) Representative images (×400) showing CD31⁺/dsRed⁺ cells as indicated by arrows and (D) quantification showing higher numbers of BM-derived EC in the miR-126 group. (E and F) Representative microscopic images (×100) and (G and H) quantification of MECA32 and dsRed staining in corticomedullary junctions of nonischemic kidney shows no differences as a result of miR-126 overexpression. (I) Confocal images (×400) confirm dsRed⁺ ECs by MECA staining. (J) Zoomed image of dsRed⁺ EC and corresponding crosssectional profile of fluorescent labels (K, colors correspond to images) show overlap of MECA32 and dsRed.

Figure 5.

Overexpression of miR-126 results in increased hematopoietic stem/progenitor cell levels in PB and altered CXCR4/SDF-1 signaling. (A) FACS analysis of LSK and (B) LSF cells in PB and BM shows an increase of these circulating cells after injury after miR-126 overexpression. (C) FACS analysis on dsRed⁺/Lin⁺ cells shows an increased mean fluorescence intensity (MFI) for CXCR4 in LV-126 group, while (D) the dsRed⁺ LSK cells shows a decreased MFI for CXCR4 in LV-126 group. (E) Quantitative RT-PCR in kidneys shows increased SDF-1 mRNA levels in response to miR-126 overexpression in BM, while in nonischemic kidneys this increase is absent. (F) Quantification and (G) representative microscopic images (×200; 3 days after IRI) of SDF-1 staining in kidney sections show increased SDF-1 expression in miR-126–overexpressing mice.