An ultrasmall ruthenium-based nanoparticle to protect acute kidney injury and promote renal repair

Acute kidney injury (AKI) is common, with high morbidity and mortality. However,  treatment options are limited. Many studies have shown a strong link between AKI and the development of chronic kidney disease (CKD), with AKI greatly increasing the risks of CKD and end-stage renal disease (ESRD). Recently, nanotechnology-based antioxidative therapy has emerged as a promising approach to alleviating oxidative stress caused by excessive reactive oxygen species (ROS). This study reports an ultrasmall ruthenium-based nanoparticle as a ROS scavenger to mitigate AKI and promote renal repair in C57 mice. 

The ultrasmall ruthenium-based nanoparticles, designated as Ru@TiO2, were synthesized by a facile one-pot solvothermal method. First, 10 ml of N, N'-dimethylformamide and 30 ml of isopropyl alcohol were mixed as the organic solvent. Subsequently, tetrabutyl orthotitanate and RuCl3 were added. We analyzed the morphology and chemical structure using transmission electron microscopy (TEM). We also assessed its enzyme-mimicking abilities in vitro, biocompatibility of Ru@TiO2, and the toxicity of Ru@TiO2 on HK2 cells and C57 mice. For AKI experiment, AKI was induced by intramuscular injection of 50% glycerin (6 ml/kg) into both hind thighs. Mice in the treatment group received Ru@TiO2 via intravenous injection into the tail following AKI induction. 24 hours later, the mice were sacrificed. For post-AKI experiment, AKI mice were treated with Ru@TiO2 every three days. 14 days later, the mice were sacrificed, and serum and renal tissue were collected for analysis. 

1. Characterizations of Ru@TiO2 nanozyme

The low-magnification TEM images of Ru@TiO2 confirm the morphology of the nanoparticles, with a specific size of approximately 50 nm (Figure 1a-c). High-resolution TEM (HR-TEM) of the surface region showed Ru clusters measuring approximately 1.7 nm (Figure 1b). Figure 1d-f confirmed that Ru@TiO2 had excellent antioxidant property, SOD-like and CAT-like capabilities.

2. Biocompatibility of Ru@TiO2 nanozyme

As shown in Figure 2a, the hemolysis rate of Ru@TiO2 was under 5% even at a high dose of 200 µg/mL, meeting the ASTM standard. It has negligible effect on routine blood tests, coagulation, or liver and kidney function (Figure 2b-e), and did not alter the histopathology of major organs in normal mice (Figure 2f). The cell counting kit-8 assay and Calcein AM/PI double stain also showed no cytotoxicity to HK2 cells (Figures 2g-h). These results confirm the excellent biocompatibility of Ru@TiO2.

3. Protective effect of Ru@TiO2 nanozyme in AKI mice

Figure 3a shows that Ru@TiO2 reduced the serum creatinine and urea levels in glycerin-induced AKI mice. Hematoxylin and eosin (H&E) staining results showed many casts (red arrow) in the kidney tissue of AKI mice, which were significantly reduced after treatment with Ru@TiO2 (Figure 3b). Additionally, Ru@TiO2 treatment effectively downregulated the expression of NGAL and KIM-1 proteins (Figure 3c-d). Furthermore, treatment with Ru@TiO2 reduced IL-6 expression levels, indicating a reduction in kidney inflammation (Figure 3e-f). We also observed that Ru@TiO2 treatment increased the activity of SOD, CAT, and GPX in renal tissue (Figure 3g). These results suggest that Ru@TiO2 nanozymes enhance renal antioxidant capacity, protects kidney function and ameliorates kidney damage in AKI mice.

After 14 days, mice in glycerin-induced AKI group exhibited weight loss, whereas mice in the Ru@TiO2 treatment group gained weight (Figure 4a). Ru@TiO2 also reduced serum creatinine and urea levels, even below those in the control group(Figure 4b). H&E staining revealed tubular damage in AKI mice, which was alleviated following Ru@TiO2 treatment. Additionally, Masson staining showed collagen fibers formation after severe AKI, while repeated Ru@TiO2 treatment slowed collagen buildup in the kidneys and alleviated the progression of renal fibrosis (Figure 4c). 

Our findings demonstrate that the ultrasmall Ru@TiO2 nanoparticle has excellent anti-ROS enzyme-mimicking capabilities. In AKI mice, Ru@TiO2 treatment effectively enhance renal antioxidant capacity, mitigate AKI and alleviate renal fibrosis progression.