Renal Proximal Tubular Cells Injury Induced By Gold Nanoparticles In Kidney Disease

Background:Gold nanoparticles (GNPs) have been widely used in biomedical fields ranging from therapeutics to diagnostics in the recent past, particularly for biosensors, biomarkers, molecular imaging, photothermal therapy, and targeted delivery of drugs and antigens, not only concerning the wide variety of biological molecules that can be applied, and but also concerning their characterization including simple synthetic protocol simple synthetic protocol, physical and chemical properties, optical properties, and stability. Due to the rapid development of GNPs applications, human safety concerns are gaining increased attention, which makes it necessary to better understand the biodistribution of GNPs in the body, their elimination from the body. However, little attention has been paid to kidneys, especially in pathological states.Chronic kidney disease (CKD) is increasingly recognized as a worldwide public health problem, and nephrotic syndrome is a common clinical kidney disease. The injury of renal tubular epithelial cells plays an important role in the progression of real fibrosis. To the best of our knowledge, renal tubules, particularly proximal tubules, are susceptible to different types of injuries, such as environmental toxins, nephrotoxic drugs, ischemia/reperfusion and hypoxia. It has been known for some time that CKD are often accompanied by hypoxia, which may play an important pathogenic role in the development of renal damage. Based on these critical questions, we put forward the hypothesis of this research:glomerular filtration membrane damage in CKD and GNPs were increased excretion into the urine, then heavily reabsorbed by renal proximal tubular epithelial cell, causing nephrotoxicity; when in hypoxic conditions, GNPs were preferentially accumulated in the kidneys, further to induce renal injury and renal fibrosis. In order to verify the above hypothesis, we employed the AN mice, the human renal proximal tubular epithelial cell line HK-2 and macrophages to evaluate the renal toxicity of GNPs and explore the relative mechanisms of toxicity to determine the biosafety of using gold or gold-derived nanoparticles in patients with CKD.Methods:1. GNPs of 5 nm were produced via NaBH4 reduction of a starting solution of sodium citrate and HAuCl4 in water.13 nm GNPs synthesis was performed using the classical citrate reduction route. Transmission electron microscope (TEM), dynamic light scattering (DLS) and UV-Vis Spectrophotometry were used to determine the size, shape, and aggregation state of the nanoparticles as previously described.2. We assessed the biodistribution and acute toxicity of 5 nm and 13 nm solid GNPs in AN mice 24 hours after intravenous injection. GNPs suspensions were administered intravenously via tail vein to the AN mice. The quantitative amount of the particles in blood, urine, and several organs was measured after 24 h by inductively coupled plasma mass spectrometry (ICP-MS). Pathology, transmission electron microscopy (TEM) and TUNEL staining were used to observe the general toxicity to kidney of the AN mice.3. In vitro studies have explored the impact of GNPs to hypoxia human renal tubular epithelial cells including the proliferation of cells as well as its related cell autophagy, apoptosis and oxidative stress.4. The indirect influence of GNPs-exposed hypoxic macrophages to renal tubular epithelial cells was explored. The influence of GNPs on the activation of NLRP3 inflammasome and production of its downstream inflammatory cytokines such as IL-1β in GNPs-exposed hypoxic macrophages was also explored. Moreover, the relationship between GNP-induced inflammatory response and renal tubular epithelium intercellular epithelial mesenchymal transdifferentiation (epithelial mesenchymal transition, EMT) was examined.Results:1. Both 5 nm and 13 nm GNPs were synthesized and characterized using various biophysical methods, including TEM, DLS and UV-vis spectrophotometry.2. AN murine model was successfully established by intravenous injection with a single dose of adriamycin (10 mg/kg) to the male BALB/c mice. The ICP-MS analysis show that the concentration of GNPs in the kidneys increased after 5 nm GNPs administration to the AN mice compared with normal mice. The histological alterations were mainly seen in the proximal renal tubules. In comparison with control mice, exposure to GNPs the AN mice has produced the following renal tubular alterations:expansion or contraction, cloudy swelling and renal tubular epithelial cell injury. The TUNEL staining further confirmed 5nm GNPs can effectively induce apoptosis in renal tubular epithelial cells in AN mice.3. We detected the cytotoxicity of 5 and 13 nm GNPs to renal proximal tubular cells HK-2 by Cell Counting Kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay, but we just found the toxic effect in the 5 nm GNP-treated cells at 50 nM dose under hypoxic condition. Furthermore, TEM images revealed that GNPs were either localized in vesicles or free in the lysosomes in 5 nm GNPs-treated HK-2 cells, and the cellular uptake of the GNPs in the hypoxic cells was significantly higher than that in normoxic cells. The result of Annexin V-FITC/PI double staining indicated that the 5-nm GNPs were more efficient to induce cellular apoptosis in hypoxic environments. TEM, immunofluorescence and Western blotting results strongly suggested that the 5 nm GNPs (50 nM) could cause autophagy in normoxic conditions, while hypoxic treatment could exaggerate the effect. Moreover, we found in normoxic HK-2 cells,5 nm GNPs (50 nM) treatment could cause autophagy and cell survival, while in hypoxic conditions, GNP exposure at the same condition led to the production of reactive oxygen species (ROS), the loss of mitochondria! membrane potential (AΨM), and an increase in apoptosis and autophagic cell death.4. The cellular uptake of the GNPs-treated hypoxic macrophages was significantly higher than that in normoxic cells, and the aggregation of GNPs induced more loss of cell viability or cell membrane integrity in hypoxic macrophages. In hypoxic macrophages, GNPs-treatment significantly activated the NLRP3 inflammasome and increased the expression of its downstream inflammatory cytokines such as IL-1β.At the meantime, ROS scavenger N- acetyl cysteine acid (NAC) could significantly inhibit the expression of NLRP3, Caspase-1 and IL-1β. Furthermore, we found GNPs-induced secretion of IL-1β in hypoxic macrophages can significantly improve the expression of a-SMA, while reduce the expression of E-cad in hypoxic HK-2 cells. Besides, z-VAD-fmk (a well-known caspase-1 inhibitor) significantly inhibited the production of Caspase-1 and IL-1β in hypoxic macrophages, meanwhile inhibit the expression of a-SMA and induce the expression of E-cad in hypoxic HK-2 cells.Conclusion:1. This aggravation of renal injury in GNPs-treated AN mice is associated with an increase cellular uptake of GNPs, and this effect may be through promotion of edema and apoptosis in renal proximal tubular cells.2. GNPs treatment could cause autophagy and cell survival in normoxic conditions, while cell apoptosis and autophagic cell death in hypoxic conditions. Increase in ROS generation and loss of AΨM may play important roles in the induction of GNPs-mediated apoptosis.3. In hypoxic conditions, GNP can cause cytotoxicity and induce activation of the NLRP3 inflammasome and expression of IL-1β in macrophages, while the increased secretion of IL-1β in macrophage can promote EMT in renal tubular epithelial cells.