Chitosan/siRNA nanoparticles targeting cyclooxygenase type 2 attenuate unilateral ureteral obstruction-induced kidney injury in mice

Abstract

Cyclooxygenase type 2 (COX-2) plays a predominant role in the progression of kidney injury in obstructive nephropathy. The aim of this study was to test the efficacy of chitosan/small interfering RNA (siRNA) nanoparticles to knockdown COX-2 specifically in macrophages to prevent kidney injury induced by unilateral ureteral obstruction (UUO). Using optical imaging techniques and confocal microscopy, we demonstrated that chitosan/siRNA nanoparticles accumulated in macrophages in the obstructed kidney. Consistent with the imaging data, the obstructed kidney contained a higher amount of siRNA and macrophages. Chitosan-formulated siRNA against COX-2 was evaluated on RAW macrophages demonstrating reduced COX-2 expression and activity after LPS stimulation. Injection of COX-2 chitosan/siRNA nanoparticles in mice subjected to three-day UUO diminished the UUO-induced COX-2 expression. Likewise, macrophages in the obstructed kidney had reduced COX-2 immunoreactivity, and histological examination showed lesser tubular damage in COX-2 siRNA-treated UUO mice. Parenchymal inflammation, assessed by tumor necrosis factor-alpha (TNF-α) and interleukin 6 mRNA expression, was attenuated by COX-2 siRNA. Furthermore, treatment with COX-2 siRNA reduced heme oxygenase-1 and cleaved caspase-3 in UUO mice, indicating lesser oxidative stress and apoptosis. Our results demonstrate a novel strategy to prevent UUO-induced kidney damage by using chitosan/siRNA nanoparticles to knockdown COX-2 specifically in macrophages.

Keywords: Cyclooxygenase type 2; chitosan; mice.; siRNA; unilateral ureteral obstruction.

Figure 1

Optical fluorescence imaging of chitosan/Cy5 siRNA nanoparticles in a murine UUO model. (A) Mice subjected to sham operation or 20-hour UUO were administered chitosan/Cy5-labelled siRNA nanoparticles or buffer i.p. Fluorescent optical imaging was performed at distinct time points before and 1, 2, 4 and 20 hours post-injection of chitosan/Cy5-siRNA. (B) Ex vivo fluorescent imaging was performed on isolated organs, including the liver, lung, spleen, heart, and kidneys 20 h after injection of chitosan/Cy5-siRNA nanoparticles on mice subjected to sham operation or UUO and showed Cy5 signal restricted to the renal region. (C) Ex vivo fluorescent imaging of Cy5 signal from the obstructed and unobstructed kidneys 20 hours after injection of chitosan/Cy5-siRNA nanoparticles. (D) Quantification of fluorescence intensity in the obstructed (LK) and unobstructed kidney (RK) 20 h after injection of chitosan/Cy5-siRNA nanoparticles.

Figure 2

siRNA distribution and macrophage accumulation in response ureteral obstruction. Mice were subjected to 3-day UUO and treated with Cy5-labeled siRNA nanoparticles i.p. at a dose of 0.5 mg/kg as described in materials and methods. (A) Total RNA was isolated from whole kidney tissue by Trizol reagent, and Northern blot was performed to analyse siRNA distribution in the right (RK) and left (LK) kidney from both sham-operated and UUO mice. Nanoparticles containing 2, 0.1, or 0.01 ng siRNA were included as control samples, Ctrl1, Ctrl 2 and Ctrl 3 respectively. (B) Quantification of Cy5-labeled siRNA nanoparticle amount in the left kidney and right kidney. (C) QPCR analysis was performed to analyse the macrophage marker CD68 mRNA levels in left and right kidney from sham-operated and 3-day UUO mice. (D) Fluorescent micrograph showing chitosan siRNA nanoparticle uptake in macrophages extracted 2 hours after i.p. administration (0.25 mg/kg chitosan/Cy5-siRNA nanoparticles). Merge pictures shows co-localization of Cy5 and the M2 macrophage marker, Mac-2, combined with DAPI. (E) Mice were subjected to 3 days UUO and treated with Cy5-flourescent labeled siRNA (0.5 mg/kg) 4 hours prior termination. Sections were stained for the macrophage marker, Mac-2. Uptake of Cy5-fluorescent labeled siRNA nanoparticles in Mac-2 positive macrophages was investigated using fluorescent confocal microscopy in UUO mice. Cy5 (red), Mac-2 (green), counterstained for DAPI (blue). Arrows represent Cy5 in Mac-2 positive cell. Original magnification: ×63. Bars = 10 µm.

Figure 3

Transfection of murine macrophage RAW 264.7 cells. Three siRNA candidates against murine COX-2 were evaluated; siCOX2-1, siCOX2-2, and siCOX2-3. siRNAs were mixed with Trans-IT-TKO^® and added at 50nM final siRNA concentration to cell cultures and incubated for 24 hours. Afterwards, transfected cells were stimulated by LPS (100 ng/mL) for 6 hours to induce COX-2. (A) RNA was extracted from cells followed by cDNA synthesis, which served as template for QPCR. COX-2 mRNA levels was investigated by QPCR. (B) Protein was extracted from cells and western blot analysis was performed to detect COX-2 protein levels. ß-actin was run in parallel to ensure equal protein loading. (C) PGE₂ levels in cell culture media evaluated by ELISA. Results are means ± SEM. WT: wildtype, LPS: lipopolysaccharide, siEGFP: negative coding control siRNA, NC: non-coding control siRNA, siCOX2: siRNA targeting murine COX-2.

Figure 4

siCOX-2 induced regulation of renal COX-2 and inflammation in mice subjected to 3-day UUO. Mice were treated with chitosan/siCOX-2 nanoparticles or chitosan/siEGFP as negative control. Following three days obstruction, whole kidney tissue was purified for QPCR and western blotting analysis. (A) QPCR analysis of COX-2 mRNA levels. (B) QPCR analysis of TNF-α and (C) IL-6 mRNA levels in response to siCOX-2 treatment. (D) QPCR analysis of CD68 mRNA level in response to 3-day UUO and siCOX-2 treatment. Each graph of statistical dot plots shows the median per cent (black bars) and p value between experimental groups (n = 6).

Figure 5

COX-2 labeling of macrophages in renal inner medullary region and effect of siCOX-2 treatment on macrophage phenotype. (A) Double staining for COX-2 and macrophage marker, Mac-2, in the renal inner medullary region. Sham, 3-day UUO siEGFP, and 3-day UUO siCOX-2 kidney sections stained for Mac-2 (red), COX-2 (green), and counterstained for TOPRO-3 (blue). 'Merge' shows co-localization of COX-2 and Mac-2. Macrophages characterized by colocalization of Mac-2 and COX-2 (arrowheads); Renal interstitial cells characterized by COX-2 positive and Mac-2 negative cells (arrows). Original magnification: ×63. Bars = 10 µm. (B, C) mRNA expression of the M1 macrophage markers, Itgax and MCP-1. (D, E) mRNA expression of the M2 macrophage markers, Arg1 and Mac-2. Each graph of statistical dot plots shows the median per cent (black bars) and p value between experimental groups (n = 6).

Figure 6

Histological evaluation of the effect of COX-2 siRNA on tubular damage in response to 3-day UUO. Following three days ureteral obstruction, renal tissue were fixed in 4% paraformaldehyde, embedded in paraffin and subsequently cut into 2µm sections. To assess the grade of tubular damage sections were stained with hematoxylin and eosin (H&E). Arrows, examples of dilated tubules. (A) Representative images of H&E staining of cortical sections. Magnification: ×40. Bars = 100 µm. (B) H&E analysis of renal tubular damage following 3-day UUO by tubular dilatation (n=20). (C, D) mRNA and protein was obtained from whole kidney tissue for QPCR and western blotting analysis. Renal tubular injury marker KIM-1 mRNA and protein levels in response to 3-day UUO and siCOX-2 treatment. Each graph of statistical dot plots shows the median per cent (black bars) and p value between experimental groups (n = 6).

Figure 7

Effect of COX-2 siRNA on apoptosis in response to 3-day UUO. Mice were subjected to 3-day UUO and treated with siCOX-2 or siEGFP for control. Protein was purified from whole kidney tissue and used for western blot analysis. (A) Immunoblots of caspase-3, and cleaved caspase-3 protein levels, with GAPDH to ensure equal protein loading. (B, C) Apoptosis markers, caspase-3 and cleaved caspase-3, protein levels in response to 3-day UUO and siCOX-2 treatment. Results are shown with total protein normalization. Each graph of statistical dot plots shows the median per cent (black bars) and p value between experimental groups (n = 6).

Figure 8

Effect of COX-2 siRNA on oxidative stress in response to 3-day UUO. Mice were subjected to 3-day UUO and treated with siCOX-2 or siEGFP for control. Protein was purified from whole kidney tissue and used for western blot analysis. (A) Immunoblots for HO-1, SOD1, and SOD2 protein levels, with GAPDH to ensure equal protein loading. (B) Protein analysis of the oxidative stress marker HO-1 protein levels in response to 3-day UUO and siCOX-2 treatment. (C, D) Regulation of antioxidant enzymes SOD1 and SOD2 protein levels in response to 3-day UUO and siCOX-2 treatment. Each graph of statistical dot plots shows the median per cent (black bars) and p value between experimental groups (n = 6).