Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Immunologic Crosstalk of Endoplasmic Reticulum Stress Signaling in Bladder Cancer.

Bladder cancer (BC) is a common malignant tumor of the urinary system. While current approaches involving adjuvant chemotherapy, radiotherapy, and immunotherapy have shown significant progress in BC treatment, challenges, such as recurrence and drug resistance, persist, especially in the case of muscle-invasive bladder cancer (MIBC). This is mainly due to the lack of pre-existing immune response cells in the tumor immune microenvironment. Micro-environmental changes (such as hypoxia and under-nutrition) can cause the aggregation of unfolded and misfolded proteins in the lumen, which induces endoplasmic reticulum (ER) stress. ER stress and its downstream signaling pathways are closely related to immunogenicity and tumor drug resistance. ER stress plays a pivotal role in a spectrum of processes within immune cells and the progression of BC cells, encompassing cell proliferation, autophagy, apoptosis, and resistance to therapies. Recent studies have increasingly recognized the potential of natural compounds to exhibit anti-BC properties through ER stress induction. Still, the efficacy of these natural compounds remains less than that of immune checkpoint inhibitors (ICIs). Currently, the ER stress-mediated immunogenic cell death (ICD) pathway is more encouraging, which can enhance ICI responses by mediating immune stemness. This article provides an overview of the recent developments in understanding how ER stress influences tumor immunity and its implications for BC. Targeting this pathway may soon emerge as a compelling therapeutic strategy for BC.

Exploring potential targets of HPV&BC based on network pharmacology and urine proteomics.

BACKGROUND: Bladder cancer (BC) caused by Human papillomavirus (HPV) infection remains a complex public health problem in developing countries. Although the HPV vaccine effectively prevents HPV infection, it does not benefit patients with BC who already have HPV. METHODS: Firstly, the differential genes of HPV-related BC patients were screened by transcriptomics, and then the prognostic and clinical characteristics of the differential genes were analyzed to screen out the valuable protein signatures. Furthermore, the compound components and targets of Astragali Radix (AR) were analyzed by network pharmacology, and the intersection targets of drug components and HPV_BC were screened out for pathway analysis. In addition, the binding ability of the compound to the Astragali-HPV_BC target was verified by molecular docking and virtual simulation. Finally, to identify potential targets in BC patients through urine proteomics and in vitro experiments. RESULTS: Eleven HPV_BC-related protein signatures were screened out, among which high expression of EGFR, CTNNB1, MYC, GSTM1, MMP9, CXCR4, NOTCH1, JUN, CXCL12, and KRT14 had a poor prognosis, while low expression of CASP3 had a poor prognosis. In the analysis of clinical characteristics, it was found that high-risk scores, EGFR, MMP9, CXCR4, JUN, and CXCL12 tended to have higher T stage, pathological stage, and grade. Pharmacological and molecular docking analysis identified a natural component of AR (Quercetin) and it corresponding core targets (EGFR). The OB of the natural component was 46.43, and the DL was 0.28, respectively. In addition, EGFR-Quercetin has high affinity. Urine proteomics and RT-PCR showed that EGFR was expressed explicitly in BC patients. Mechanism analysis revealed that AR component targets might affect HPV_BC patients through Proteoglycans in the cancer pathway. CONCLUSION: AR can target EGFR through its active component (Quercetin), and has a therapeutic effect on HPV_BC patients.

Rethinking neutrophil extracellular traps.

Neutrophils are a major subset of leukocytes in human circulating blood. In some circumstances, neutrophils release neutrophil extracellular traps (NETs). lnitially, NETs were considered to have a strong antibacterial capacity. However, currently, NETs have been shown to have a pivotal impact on various diseases. Different stimulators induce the production of different types of NETs, and their biological functions and modes of clearance do not appear to be the same. In this review, we will discuss several important issues related to NETs in order to better understand the relationship between NETs and diseases, as well as how to utilize the characteristics of NETs for disease treatment.

Performance Verification of Alternative Quality Control Materials for Urine Albumin Assessment.

BACKGROUND: Accurate detection of urine albumin is important for evaluating the progression of diabetic kidney disease. However, two levels of daily quality control may not be practically feasible in some small clinical laboratories owing to a small number of patient samples and high costs. We aimed to prepare homemade quality control material (HQM) to measure urine albumin and then verify its performance. METHODS: Normal saline solution and fresh mixed urine samples from five donors with serious kidney disease were used to prepare two levels of HQM (HQM1 and HQM2). Anhydrous ethylene glycol and sodium azide were used as antifreeze and as a preservative, respectively. RESULTS: Before being separated into Eppendorf tubes, 20 tests for HQM1 and HQM2 were performed, resulting in mean ± SD of 19.52 ± 0.91 mg/L and 105.28 ± 3.71 mg/L, respectively. After having been divided, the vial-to-vial variations of HQM1 and HQM2 were small (4.93% and 3.70%, respectively). The stability of HQM1 and HQM2 stored at 2 - 8°C was about 2 months and 80 days, respectively, and when stored at -20°C, remained stable for more than 8 months. After 1 - 8 months of cryopreservation at -20°C, once opened, the HQM in every Eppendorf tube could be kept for at least five days (CV < 6.1%). CONCLUSIONS: Our HQM stored at -20°C remained stable for a long time, and so could be considered as an alternative to standard QMs in the clinical laboratory.

Associations of urinary, glomerular, and tubular markers with the development of diabetic kidney disease in type 2 diabetes patients.

OBJECTIVES: To evaluate the associations of urinary markers (eg albumin), glomerular (eg transferrin [TRF], immunoglobulin G [IgG]), and tubular (eg α1-microglobulin [α1-MG], ß2-microglobulin [ß2-MG]) markers with the development of diabetic kidney disease (DKD) in type 2 diabetes patients, as assessed by estimated glomerular filtration rate (eGFR) and albuminuria. MATERIAL AND METHODS: A total of 252 type 2 diabetes patients and 50 nondiabetic controls from Tianjin, China, were selected. Diabetic patients were divided into three groups according to eGFR levels, including groups A, B, and C with eGFR ≥90 (n=94), 60-89 (n=94), and 30-59 (n=64) mL/min/1.73 m2 . Urine levels of glomerular and tubular markers were detected in first morning urine samples, and their associations with eGFR and albuminuria analyzed. RESULTS: Urinary levels of IgG, TRF, and ß2-MG were significantly elevated in diabetic patients with normal eGFR compared with nondiabetic control subjects. Urinary levels of all markers increased per eGFR stratum. All kidney damage markers were significantly associated with eGFR in univariate analysis (standard ß between -0.35 and -0.28; all P<.001). After adjusting for known confounders, only the tubular markers α1-MG (standard ß=-0.25; P=.013) and ß2-MG (standard ß=-0.18; P=.039) retained significant associations with eGFR. All kidney damage markers were significantly associated with albuminuria, independent of age, duration of diabetes, and eGFR (standard ß between 0.45 and 0.86; all P<.001). CONCLUSION: Only the tubular markers α1 -MG and ß2 -MG were associated with eGFR independent of albuminuria, suggesting that they may play an important role in the development of DKD.

Graphene oxide nanosheets induce DNA damage and activate the base excision repair (BER) signaling pathway both in vitro and in vivo.

Graphene oxide (GO) has widespread concerns in the fields of biological sciences and medical applications. Currently, studies have reported that excessive GO exposure can cause cellular DNA damage through reactive oxygen species (ROS) generation. However, DNA damage mediated response of the base excision repair (BER) pathway due to GO exposure is not elucidated yet. Therefore, we exposed HEK293T cells and zebrafish embryos to different concentrations of GO for 24 h, and transcriptional profiles of BER pathway genes, DNA damage, and cell viability were analyzed both in vitro and in vivo. Moreover, the deformation of HEK293T cells before and after GO exposure was also investigated using atomic force microscopy (AFM) to identify the physical changes occurred in the cells' structure. CCK-8 and Comet assay revealed the significant decrease in cell viability and increase in DNA damage in HEK293T cells at higher GO doses (25 and 50 µg/mL). Among the investigated genetic markers in HEK293T cells, BER pathway genes (APEX1, OGG1, CREB1, UNG) were significantly up-regulated upon exposure to higher GO dose (50 µg/mL), however, low exposure concentration (5, 25 µg/mL) failed to induce significant genetic induction except for CREB1 at 25 µg/mL. Additionally, the viscosity of HEK293T cells decreased upon GO exposure. In zebrafish, the results of up-regulated gene expressions (apex1, ogg1, polb, creb1) were consistent with those in the HEK293T cells. Taken all together, the exposure to elevated GO concentration could cause DNA damage to HEK293T cells and zebrafish embryos; BER pathway could be proposed as the possible inner response mechanism.

Alanine aminotransferase is more sensitive to the decrease in hepatitis B virus-DNA load than other liver markers in chronic hepatitis B patients.

BACKGROUND: A direct correlation between hepatitis B virus DNA (HBV-DNA) and liver markers has not been identified in chronic hepatitis B (CHB) patients. However, the effect of HBV-DNA changes on liver markers remains unclear. We explored the association between decreased HBV-DNA and liver makers in CHB patients. METHODS: Chronic hepatitis B patients who visited Jinhua Central Hospital twice were selected for analysis. Finally, 171 participants with a 1-log reduction in HBV-DNA between the two visits were enrolled as the case group, and 158 participants with no significant changes in HBV-DNA were enrolled as the control group. RESULTS: There was no significant correlation between HBV-DNA and liver markers (P>.05). However, in longitudinal analysis, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT) were significantly different between the two tests (P<.05) in the case group. Conversely, there was no significant difference in the control group. When HBV-DNA decreased >26 times, ALT was reduced by half or more. A similar trend was observed with a decrease of >63 times for AST and a decrease of >76 times for GGT. CONCLUSIONS: A large change in HBV-DNA can lead to a significant variation in liver markers. In particular, ALT was more sensitive than other liver markers to a reduction in HBV-DNA.

Second Morning ACR Could Be the Alternative to First Morning ACR to Assess Albuminuria in Elderly Population.

BACKGROUND: Albumin/creatinine ratio (ACR) from a first morning urine is recommended as a early indicator for diabetic nephropathy. However, it is not always feasible to collect the first morning urine for outpatients. We aimed to explore whether ACR from a second morning urine had a good consistency with that from a first morning urine to predict albuminuria in Chinese elderly citizens. METHOD: One hundred and ninety-one elderly citizens (≧60 years old) from Junliangcheng community, Dongli district, Tianjin, China were included. A first and second morning urine was collected from each participants, successfully and detected the urinary albumin and creatinine of each urine sample. Albumin to creatinine ratio from a first morning urine (ACR1) was compared with that from a second morning urine (ACR2), and the ability of ACR1 and ACR2 to predict albuminuria was assessed. RESULT: ACR1 and ACR2 were highly correlated (r = 0.901), especially in male and hypertension group (r = 0.938 and 0.904). The slope and intercept were 0.93 and 0.11 after log-transformed. And there was no statistical difference between values of ACR1 and ACR2 (P = 0.271). Overall, 26.2% participants were detected with albuminuria when judged by ACR1 and 28.3% by ACR2. A good concordance of ACR category (normal or albuminuria) was found between ACR1 and ACR2 (Kappa value = 0.815 in overall; in male and hypertension group were 0.900 and 0.850). CONCLUSION: A second morning urine ACR could be the alternative to a first morning urine ACR for albuminuria detection in elderly population.

Tubular proteinuria is the dominant type of proteinuria in an elderly community population in China.

PURPOSE: To explore the types of proteinuria in the elderly population in China. METHODS: Seven hundred and fourteen elderly people (≥ 60 years old) from Tianjin, China, were selected for the study. The albumin-to-creatinine ratio and α1-microglobulin-to-creatinine ratio from morning urine samples were used as indicators of proteinuria. The prevalence of proteinuria was evaluated and the proportion of three different types of proteinuria (mixed, glomerular, and tubular) was assessed in the subjects by analyzing these indicators. RESULTS: Of the 714 subjects, 29.13 % had elevated ACR and 46.36 % had elevated MCR. The proportion of subjects with either elevated ACR or MCR was 53.78 %. The correlation between MCR and ACR was moderate (r = 0.58, R (2) = 0.34, P < 0.001). Overall, tubular proteinuria was dominant (45.83 %), followed by mixed glomerular and tubular proteinuria (35.68 %), and significantly higher than glomerular proteinuria. A diet high in salt was the independent risk factor for tubular proteinuria; physical activity was the independent risk factor for glomerular proteinuria. The risk of glomerular proteinuria was lower in males than in females, but the risk of tubular proteinuria was higher in males. CONCLUSIONS: The prevalence of tubular proteinuria was higher than that of glomerular proteinuria, and the risk factors are different, in the elderly in China; therefore, tubular damage markers should get more attention in the overall population.