Fe3O4 magnetic nanoparticles ameliorate albumin-induced tubulointerstitial fibrosis by autophagy related to Rab7.

Albuminuria is a primary feature in patients with CKD and an important contributor to tubulointerstitial fibrosis (TIF) development. Autophagy has been considered to be involved in renal tubular injury caused by albuminuria. Fe3O4 magnetic nanoparticles are related to many cellular activities, such as autophagy and inflammation. Rab7, a molecule involved in both endocytosis and autophagy, has been identified to protect renal tubular epithelial cells from albumin by regulating autophagy and MMP-2 activity in the early stage of albumin stimulation, but its role in the advanced stage is still unclear. Therefore, to investigate the effect of Fe3O4 magnetic nanoparticles on chronic renal tubular injury induced by excess albumin and to further determine the specific role of Rab7, we established a mouse model of TIF by intravenous injection of cationic bovine serum albumin (C-BSA) in Rab7-overexpressing transgenic mice. Our data revealed the decreased autophagy level, weakened MMP-2 activity and exacerbated renal tubular injury in these BSA-overloaded mice; furthermore, the degree of injury was more serious in Rab7-overexpressing transgenic mice. However, the application of Fe3O4 magnetic albumin nanoparticles (Fe3O4@BSA) enhanced MMP-2 activity and alleviated renal tubular injury, and these changes were mediated by an autophagy-dependent mechanism. Taken together, our results indicated that long-term albumin stimulation combined with overexpression of Rab7 could further decrease MMP-2 activity, exacerbate renal tubular injury and accelerate the development of TIF. Fe3O4@BSA could be a promising targeted tool for the management of CKD patients.

A multifunctional theranostic contrast agent for ultrasound/near infrared fluorescence imaging-based tumor diagnosis and ultrasound-triggered combined photothermal and gene therapy.

PURPOSE: Encapsulated microbubbles (MBs) have been reported as new theranostic carriers for simultaneous imaging and ultrasound (US)-triggered therapy. Here, we designed a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. METHODS: Gold nanorods (AuNRs) resonant at 880 nm together with the NIR797 dye were first encapsulated in lipid-shelled MBs to construct fluorescent gold microbubbles (NIR797/AuMBs) via thin film hydration and mechanical shaking in the presence of sulfur hexafluoride (SF6) gas. Then, polyethylenimine (PEI)-DNA complexes were electrostatically conjugated onto the surface of the NIR797/AuMBs, forming theranostic encapsulated MBs (PEI-DNA/NIR797/AuMBs). The potential of the PEI-DNA/NIR797/AuMBs for use as a dual-modality contrast enhancement agent was evaluated in vitro and in vivo. The antitumor effect of US/NIR laser irradiation mediating double-fusion suicide gene and photothermal therapy was also investigated using Bel-7402 cells and xenografts. RESULTS: The developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. And US + laser exposure group showed a much higher rate of cell inhibition, apoptosis and necrosis as well as a higher Bel-7402 xenograft inhibition rate than the single gene therapy or single exposure (US or laser) group. CONCLUSIONS: PEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy. STATEMENT OF SIGNIFICANCE: This is an original paper focusing on developing a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. The developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. PEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy.

Rab7 empowers renal tubular epithelial cells with autophagy-mediated protection against albumin-induced injury.

Most patients with chronic kidney disease (CKD) present with proteinuria and extracellular matrix (ECM) deposition in the interstitium. Matrix metalloproteinase-2 (MMP-2) is important for maintaining ECM metabolism and it affects the formation and development of CKD. Autophagy has been reported to be protective against renal tubular injury, but the role of autophagy related to ECM metabolism is unclear. Rab7 is a shared molecule of endocytosis and autophagy. The aim of this study is to explore the role of autophagy in regulating MMP-2 activity and to determine whether Rab7 functions in regulating MMP-2 activity and injury in albumin-overloaded TECs. In this study, abovine serum albumin (BSA)-overload rat model was first established and collagen deposition and deficient autophagic response were observed in vivo, and stimulation with albumin nanoparticles resulted in MMP-2 overactivation and obstructed autophagic flux induced by lysosomal dysfunction in vitro. Furthermore, overactivation of MMP-2 was mediated by its related regulatory molecules such as membrane-type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinase-2 (TIMP-2) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) on the membrane of TECs (HK-2 cellline). After up-regulating Rab7, albumin-induced MMP-2 overactivation was attenuated, which was reversed by chloroquine (CQ; an endocytosis inhibitor). In addition, our data indicated that up-regulation of Rab7 relieved epithelial-mesenchymal transition (EMT) and apoptosis in albumin-treated TECs. Taken together, our study demonstrated that autophagy regulates MMP-2 activity in a Rab7-dependent manner. Thus, Rab7 is a newly developed target for protecting TECs from albumin-induced injury.

Endothelial cells co-cultured with renal carcinoma cells significantly reduce RECK expression under chemical hypoxia.

Renal cell carcinoma (RCC) is characterized by excessive angiogenesis, while chronic kidney disease (CKD) suffers from the opposite problem-failure of reparative angiogenesis. It can be due to their different responses to hypoxic environment. But the specific molecular regulators are still unclear. This study is aimed to explore the influence of human renal cell cancer cells (786-0) and human renal tubular epithelial cells (HK-2) on RECK expression, proliferation, and angiogenesis of adjacent microvascular endothelial cells (HMEC-1) under chemical hypoxia. Cobalt chloride (CoCl2 ) treatment was used to simulate the hypoxia environment in RCC and CKD. Co-culture, cell proliferation assay, and tube formation assay were used to evaluate the influence of 786-0 or HK-2 cells on proliferation and angiogenesis of adjacent HMEC-1 cells. Effects of different environments on RECK expressions in 786-0, HK2, or HMEC-1 cells were determined by Western blot. We found that both 786-0 cells and HK2 cells can upregulate RECK expression of adjacent HMEC-1 cells in normoxic conditions. However, under hypoxia, the HMEC-1 cells co-cultured with 786-0 significantly reduced RECK expression and there was no significant change in HMEC-1 cells co-cultured with HK2 cells. We also found that 786-0 significantly enhanced the proliferation and angiogenesis of adjacent HMEC-1 cells. Our results suggested that some paracrine substances produced by 786-0 cells may reduce RECK expression of adjacent HMEC-1 cells and enhance their proliferation and in vitro angiogenic capacity.

Circulating tumor cell detection: A direct comparison between negative and unbiased enrichment in lung cancer.

Circulating tumor cells (CTCs), isolated as a 'liquid biopsy', may provide important diagnostic and prognostic information. Therefore, rapid, reliable and unbiased detection of CTCs are required for routine clinical analyses. It was demonstrated that negative enrichment, an epithelial marker-independent technique for isolating CTCs, exhibits a better efficiency in the detection of CTCs compared with positive enrichment techniques that only use specific anti-epithelial cell adhesion molecules. However, negative enrichment techniques incur significant cell loss during the isolation procedure, and as it is a method that uses only one type of antibody, it is inherently biased. The detection procedure and identification of cell types also relies on skilled and experienced technicians. In the present study, the detection sensitivity of using negative enrichment and a previously described unbiased detection method was compared. The results revealed that unbiased detection methods may efficiently detect >90% of cancer cells in blood samples containing CTCs. By contrast, only 40-60% of CTCs were detected by negative enrichment. Additionally, CTCs were identified in >65% of patients with stage I/II lung cancer. This simple yet efficient approach may achieve a high level of sensitivity. It demonstrates a potential for the large-scale clinical implementation of CTC-based diagnostic and prognostic strategies.

Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in Hepatocytes.

Background and Aims: Hypoxia and oxidative stress contribute toward liver fibrosis. In this experiment, we used small hairpin RNA (shRNA) to interfere with the intracellular oxygen sensor-prolyl hydroxylase 1 (PHD1) and the intracellular oxidative stress sensor-kelch-like ECH associated protein 1 (Keap1) in the hypoxic hepatocytes in order to investigate the function of PHD1and Keap1. Methods: We first established the CCl4-induced liver fibrosis model, subsequently, the levels of the PHD1, hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), Keap1, and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) were detected in liver tissues. Simultaneously, AML12 cells co-transfected with PHD1 and Keap1shRNAs were constructed in vitro, then the intracellular oxidative stress, the proportion of cells undergoing apoptosis, and cell viability were measured. The expression of pro-fibrogenic molecules were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The level of alpha-1 type I collagen (COL1A1) was determined using an enzyme-linked immunosorbent assay (ELISA). Finally, serum-free "conditioned medium" (CM) from the supernatant of hypoxic AML12 hepatocytes was used to culture rat hepatic stellate cells (HSC-T6), and the levels of fibrosis-related molecules, apoptosis, and cell proliferation were determined. Results: The marker of hypoxia-HIF-1α and HIF-2α in the livers with fibrosis were upregulated, however, the increase in PHD1 expression was not statistically significant in comparison to the control group. Sign of oxidative stress-Keap1 was increased, while the expression of Nrf2, one of the Keap1 main downstream molecules, was reduced in the hepatocytes. And in vitro, the double-knockdown of PHD1 and Keap1 in AML12 hepatocytes presented with decreased hypoxia-induced oxidative stress and apoptosis, furthermore, these hypoxic AML12 cells showed the increased cell viability and the doweregulated expression of pro-fibrogenic molecules. In addition, HSC-T6 cells cultured in the hypoxic double-knockdown CM demonstrated the downregulation of fibrosis-related molecules, diminished cell proliferation, and enhanced apoptosis. Conclusions: Our study demonstrated that double-knockdown of PHD1 and Keap1 attenuated hypoxia and oxidative stress induced injury in the hepatocytes, and subsequently inhibited HSC activation, which offers a novel therapeutic strategy in the prophylaxis and treatment of liver fibrosis.

Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK­2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK­2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK­2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma.

Inhibition of liver fibrosis using vitamin A-coupled liposomes to deliver matrix metalloproteinase-2 siRNA in vitro.

Hepatic fibrosis is a common form of wound healing in response to chronic liver injuries and can lead to more serious complications, including mortality. It is well­established that hepatic stellate cells (HSCs) are central mediators of hepatic fibrosis, and matrix metalloproteinase­2 (MMP­2) is important in the formation of liver fibrosis. In addition, HSCs are the primary cells secreting MMP­2 and extracellular matrix, therefore, there has been increasing interest in developing agents with high selectivity towards HSCs. However, no clinical drugs based on MMP­2, directed against HSCs, have been used to prevent fibrosis. Following consideration of the abundant vitamin A (VitA) receptors expressed on the cellular membrane of HSCs, the present study constructed VitA­coupled liposomes (VitA­lips) using dicyclohexylcarbodiimide­1, 3­diaminopentane condensation, rotatory film processing and ultrasonic oscillation. The results revealed that the liposomes exhibited low cytotoxicity and a suitable binding ability to MMP­2 small interference (si)RNA. Furthermore, the liposomes effectively delivered MMP­2 siRNA to the HSC­T6 cells. When HSCs were treated with the liposomes carrying MMP­2 siRNA (VitA­lip­MMP­2 siRNA), the mRNA expression and activity of MMP­2, and the protein expression levels of α­smooth muscle actin and type I collagen were significantly reduced. These results suggested that inhibition of the expression of MMP­2 in HSC­T6 cells may contribute to preventing hepatic fibrosis, and provided experimental support to the development of specific drugs against MMP­2 to prevent fibrogenesis in chronic liver disease.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells.

BACKGROUND: Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225)-conjugated, gemcitabine (GEM)-containing magnetic albumin nanospheres (C225-GEM/MANs) were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI), and double-targeted thermochemotherapy against pancreatic cancer cells. METHODS: Fe3O4 nanoparticles (NPs) and GEM co-loaded albumin nanospheres (GEM/MANs) were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2) and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry (FCM) assay. RESULTS: When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal intensity was significantly lower when magnetic and C225 targeting were combined, rather than used alone. The inhibitory and apoptotic rates of each thermochemotherapy group were significantly higher than those of the chemotherapy-alone groups. Additionally, both MTT and FCM analysis verified that double-targeted thermochemotherapy had the highest targeted killing efficiency among all groups. CONCLUSION: The C225-GEM/MANs can distinguish various EGFR-expressing live pancreatic cancer cells, monitor diverse cellular targeting effects using targeted MRI imaging, and efficiently mediate double-targeted thermochemotherapy against pancreatic cancer cells.

Overendocytosis of gold nanoparticles increases autophagy and apoptosis in hypoxic human renal proximal tubular cells.

BACKGROUND: Gold nanoparticles (GNPs) can potentially be used in biomedical fields ranging from therapeutics to diagnostics, and their use will result in increased human exposure. Many studies have demonstrated that GNPs can be deposited in the kidneys, particularly in renal tubular epithelial cells. Chronic hypoxic is inevitable in chronic kidney diseases, and it results in renal tubular epithelial cells that are susceptible to different types of injuries. However, the understanding of the interactions between GNPs and hypoxic renal tubular epithelial cells is still rudimentary. In the present study, we characterized the cytotoxic effects of GNPs in hypoxic renal tubular epithelial cells. RESULTS: Both 5 nm and 13 nm GNPs were synthesized and characterized using various biophysical methods, including transmission electron microscopy, dynamic light scattering, and ultraviolet-visible spectrophotometry. We detected the cytotoxicity of 5 and 13 nm GNPs (0, 1, 25, and 50 nM) to human renal proximal tubular cells (HK-2) by Cell Counting Kit-8 assay and lactate dehydrogenase release assay, but we just found the toxic effect in the 5 nm GNP-treated cells at 50 nM dose under hypoxic condition. Furthermore, the transmission electron microscopy 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. In normoxic HK-2 cells, 5 nm GNPs (50 nM) treatment could cause autophagy and cell survival. However, in hypoxic conditions, the GNP exposure at the same condition led to the production of reactive oxygen species, the loss of mitochondrial membrane potential (ΔΨM), and an increase in apoptosis and autophagic cell death. CONCLUSION/SIGNIFICANCE: Our results demonstrate that renal tubular epithelial cells presented different responses under normoxic and hypoxic environments, which provide an important basis for understanding the risks associated with GNP use-especially for the potential GNP-related therapies in chronic kidney disease patients.

[Prognostic significance of a newly proposed grading and scoring system in stage I pulmonary adenocarcinoma].

OBJECTIVE: To evaluate the prognostic significance of a new grading and scoring system (based on the new IASLC/ATS/ERS classification) in stage I pulmonary adenocarcinoma, as compared with the WHO grading system. METHODS: The clinicopathologic characteristics of 125 patients with stage I pulmonary adenocarcinoma primarily treated by surgical resection were reviewed retrospectively. All cases were classified according to the new IASLC/ATS/ERS classification and graded into three prognostic groups based on the new classification, the Sica scoring system and the WHO grading system, respectively. The differences in prognosis of the three groups were analyzed. RESULTS: There was a statistically significant correlation between the new grading system and the WHO grading system (P = 0.000). Both of them showed negative correlation with overall survival. The new scoring system however better correlated with disease recurrence and/or metastasis (P = 0.855, P = 0.073 versus P = 0.011). According to univariate Log-rank test, the prognosis correlated with tumor size (P = 0.004), clinical stage (P = 0.000), the WHO grading (P = 0.020), the new grading system (P = 0.000), the new scoring system (P = 0.000), vascular invasion (P = 0.021), and recurrence and/or metastasis (P = 0.000). The Cox regression analysis demonstrated that clinical stage (P = 0.014), the new grading system (P = 0.047), the new scoring system (P = 0.043), and recurrence and/or metastasis (P = 0.018) were significantly independent poor prognostic factors. CONCLUSIONS: The new grading and scoring system shows good correlation with the WHO grading system. Compared with the WHO grading system, the new scoring system based on the new IASLC/ATS/ERS classification provides valuable information in categorizing stage I pulmonary adenocarcinoma cases with different risks of disease recurrence, tumor metastasis and prognosis.