Colorectal cancer (CRC) metastasis, driven by circulating tumor cells (CTCs), remains a formidable clinical challenge, positioning CTCs' eradication as a pivotal yet underexploited therapeutic frontier for metastasis prevention. Riboflavin photochemical therapy (RPT) has emerged as a promising modality due to its dual capacity to inactivate tumor cells while inducing minimal hematological toxicity. However, the optimal parameters and mechanisms behind RPT-mediated CTCs' inactivation are still unclear. Here, we present RPT as a novel strategy to eradicate CTCs while preserving blood cell integrity. Through systematic investigations, we pioneered the application of blue light for CTCs' inactivation and established optimized RPT parameters that achieve complete CTCs' elimination with minimal impact on coagulation, immune cell functionality, and erythrocyte integrity. Mechanistically, RPT triggers mitochondrial dysfunction and oxidative stress, amplifying mitochondrial reactive oxygen species (mtROS) to induce PANoptosis, a coordinated cell death program integrating pyroptosis, apoptosis, necroptosis, and ferroptosis, in CRC cells. Transcriptomic profiling and pharmacological inhibition confirmed mtROS as the central mediator, with antioxidants rescuing PANoptotic markers and restoring cell viability. Unlike conventional therapies, RPT exploits riboflavin’s tumor-selective mitochondrial accumulation, ensuring targeted cytotoxicity without compromising blood homeostasis. Overall, this study not only deciphers the mtROS-PANoptosis axis as a therapeutic vulnerability but also establishes RPT as a promising intervention to disrupt metastatic dissemination in CRC.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder requiring improved therapeutic strategies. This study investigates the potential of ultraviolet (UV)-treated riboflavin in AD treatment. Using a MC903-induced mouse model, we demonstrate that topical UV-treated riboflavin significantly attenuates AD progression. Mechanistically, UV-treated riboflavin suppresses macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation by reducing histone H3 lysine 9 lactylation (H3K9la) on NLRP3 and apoptosis-associated speck-like protein containing a CARD (ASC) promoter, decreasing interleukin-1β (IL-1β) secretion and subsequent keratinocyte-derived thymic stromal lymphopoietin (TSLP) production. It also directly inhibits inflammatory cytokine expression in keratinocytes. NLRP3 activation in vivo partially reverses these effects, confirming the central role of NLRP3 inflammasome inhibition. Our findings reveal a novel epigenetic mechanism of UV-treated riboflavin in modulating immune responses in AD, highlighting its potential as a therapeutic strategy for inflammatory skin disorders.

In this work complex nanoparticles composed of zein and hyaluronic acid (HA), referred to as ZHPs, were fabricated through the combined effects of hydrogen bonding and electrostatic attractions. Compared to zein particles, ZHPs exhibited a larger mean particle size and a reversal in zeta potential from positive to negative. The three-phase contact angle (θo/w) of ZHPs with a 10:3 ratio was measured at 88.9°, indicating their suitability for stabilizing Pickering emulsions. Both droplet diameter and the emulsified phase volume fraction of the emulsions were significantly affected by the concentration of ZHPs and the oil phase ratio. Confocal laser scanning microscopy revealed that ZHPs adsorbed on the oil-water interface formed a compact interfacial layer. Rheological analysis showed that emulsions displayed shear-thinning behavior and exhibited characteristics of an elastic gel network. Moreover, the Pickering emulsions stabilized by ZHPs markedly suppressed curcumin degradation under UV exposure compared to both bulk oil and conventional emulsions. Results from vitro digestion indicated that the release of free fatty acids (FFAs) and the bioaccessibility of curcumin were enhanced in ZHP-stabilized emulsions relative to bulk oil. Overall, the findings support the potential of ZHP-stabilized Pickering emulsions as efficient delivery vehicles for food and pharmaceutical applications.

Riboflavin (RF) as a photosensitizer has been used in corneal surgery and the inactivation of blood products. However, the effect of RF on immune cells after ultraviolet (UV) light stimulation has not been investigated. This study pioneered a novel application method of RF. Firstly, UV-stimulated RF was co-cultured with human peripheral blood mononuclear cells in vitro, and the apoptosis rate of lymphocyte subsets, cell proliferation inhibition rate and concentrations of IL-1β, IL-6, IL-10, TNF-α were assessed. UV-stimulated RF was then administered intravenously to mice via the tail vein for a consecutive period of 5 days. The levels of immunoglobulin (IgG, IgM, IgA), complement (C3, C4) and cytokines (IFN-γ, IL-4, IL17, TGF-β) were detected by ELISA. Flow cytometry was employed to analyze the populations of CD3+T, CD4+T, CD8+T and CD4+T/CD8+T cells in spleen lymphocytes of mice. The data showed that UV-stimulated RF can effectively induce apoptosis in lymphocytes, and different lymphocyte subtypes exhibited varying degrees of treatment tolerance. Additionally, the proliferative capacity of lymphocytes was suppressed, while their cytokine secretion capability was augmented. The animal experiments demonstrated that UV-stimulated RF led to a significant reduction observed in serum immunoglobulin and complement levels, accompanied by an elevation in IFN-γ, IL-17 and TGF-β levels, as well as a decline in IL-4 level. In summary, the results of both in vitro and in vivo experiments have demonstrated that UV-stimulated RF, exhibits the ability to partially inhibit immune function. This novel approach utilizing RF may offer innovative perspectives for diseases requiring immunosuppressive treatment.

IgA nephropathy (IgAN) is a heterogeneous disease, which poses a series of challenges to accurate diagnosis and personalized therapy. Herein, we constructed a systematic quantitative proteome atlas from 59 IgAN and 19 normal control donors. Consensus sub-clustering of proteomic profiles divided IgAN into three subtypes (IgAN-C1, C2, and C3). IgAN-C2 had similar proteome expression patterns with normal control, while IgAN-C1/C3 exhibited higher level of complement activation, more severe mitochondrial injury, and significant extracellular matrix accumulation. Interestingly, the complement mitochondrial extracellular matrix (CME) pathway enrichment score achieved a high diagnostic power to distinguish IgAN-C2 from IgAN-C1/C3 (AUC>0.9). In addition, the proteins related to mesangial cells, endothelial cells, and tubular interstitial fibrosis were highly expressed in IgAN-C1/C3. Most critically, IgAN-C1/C3 had a worse prognosis compared to IgAN-C2 (30% eGFR decline, p = 0.02). Altogether, we proposed a molecular subtyping and prognostic system which could help to understand IgAN heterogeneity and improve the treatment in the clinic.

Introduction: Autoimmune hemolytic anemia is a potentially lethal disease characterized by autoimmune hemolysis. Although human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have been reported as a promising therapy, there is limited evidence regarding warm autoimmune hemolytic anemia (wAIHA) patients. This study aimed to investigate the potential therapeutic effects of hUC-MSCs via immune regulation in wAIHA patients. Methods: Peripheral blood mononuclear cells (PBMCs) from 10 wAIHA patients and 8 healthy controls were isolated from peripheral blood and cultured for 3 days with or without the presence of hUC-MSCs; PBMCs were co-cultured with hUC-MSCs using Transwell assays. The supernatant cytokine levels were measured after culture through AimPlex Multiple Immunoassays for Flow, including IL-2, IL-4, IL-10, IFN-γ, TNF-α, and IL-17A. The percentages of regulatory T cells, regulatory B cells, and Th1/Th2 in PBMCs were also assessed before and after culturing. Results: In the wAIHA group, hUC-MSCs could upregulate the Treg and Breg proportions after culturing for 3 days, and the Treg and Breg percentages increased after co-culturing with hUC-MSCs in the wAIHA group compared with PBMC cultured alone for 3 days (8.29 ± 8.59 vs. 6.82 ± 1.32, 3.82 ± 1.87 vs. 1.75 ± 1.20, respectively). Compared with the PBMC wAIHA group, the levels of TNF-α (2.13 ± 2.07 vs. 16.20 ± 21.13 pg/mL, p = 0.019) and IL-10 (10.51 ± 18.42 vs. 37.78 ± 44.20 pg/mL, p = 0.012) were significantly elevated in the PBMC + hUC-MSCs wAIHA group. Conclusion: The hUC-MSCs contributed to the increasing proportion of regulatory cell populations in PBMCs of wAIHA patients, thereby potentially regulating autoimmune response; thus, hUC-MSCs may be a promising approach for wAIHA treatment.

The removal or inactivation of circulating tumor cells (CTCs) can prevent distant metastasis by hematogenous route, but there is still a lack of mature and effective technical means. In the previous research, our team has initially established a method of riboflavin photosensitized treatment (RPT) for continuous treatment of peripheral blood in vitro for the inactivation of CTCs. The core of this technology is that it can selectively induce apoptosis of CTCs (HCT116 cells) without damaging immunocyte (mainly Peripheral Blood Mononuclear Cells，PBMCs) under specific parameters. To clarify the specific mechanism, firstly, the enrichment of riboflavin in HCT116 cells and PBMCs was observed under fluorescence microscope. Secondly, the apoptotic signaling pathways in HCT116 cells and PBMCs in response to RPT treatment were analyzed by transcriptomics. Finally, the mitochondrial damage in HCT116 cells and PBMCs before and after RPT treatment was observed under electron microscope. The results showed that under the same treatment conditions, HCT116 cells were significantly enriched in riboflavin compared with PBMCs. Besides, RPT treatment reduced the expression of long non﹣coding RNA (lncRNA) NEAT1, an effector gene of HCT116 cells, which further down-regulated the expression of target gene PAX2 and promoted the expression of Bax, leading to mitochondrial outer membrane permeabilization (MOMP), and consequently increased the release of pro-apoptotic factors such as cytochrome c(Cyt C), high-temperature requirement protein A2(HTRA2), apoptosis-inducing factor (AIF), endonuclease G(ENDOG), finally leading to apoptosis of HCT116 cells. In contrast, lncRNA NEAT1 remained unchanged in PBMCs before and after RPT treatment, and was unable to stimulate the PBMCs apoptotic signaling pathway. The results of the study indicated that under the specific treatment conditions, RPT technology could selectively induce apoptosis of HCT116 cells by activating the mitochondrial apoptosis pathway, which would further provide a theoretical and technical support for the effective inactivation of CTCs by RPT technology, thereby reducing the risk of recurrence of malignant tumors and improving the cure rate of malignant tumors.

BACKGROUND:Bacterial contamination still poses serious challenges to blood safety. Platelets have the highest bacterial contamination risk of all blood components. METHODS:Twenty units of manual platelets were prepared from blood donated by our hospital, which were inoculated with Staphylococcus aureus and Escherichia coli suspensions. The riboflavin sodium phosphate solution was added into platelets, adjusted to a final concentration of 160 μmol/L. Platelets added into an illumination bag and placed in the inactivation system for riboflavin photochemistry at various doses. The inactivation effect of bacteria was evaluated on a Columbia blood agar plate by the plate counting method. Meanwhile, the blood routine, blood gas analysis, platelet aggregation test, and thromboelastogram of platelets before and after treatment were detected to evaluate the changes of platelet quality after treatment. RESULTS:the inactivation effect of S. aureus and E. coli at the inactivation dose (16.9 J/cm2) could reach more than 4 logs. After treatment at 16.9 J/cm2, the blood routine results showed that the platelet count was significantly different (P<0.05), and the blood gas analysis showed that the oxygen partial pressure (pO2) and lactic acid concentration (cLac) were also significantly different (P<0.05). After 16.9 J/cm2 treatment, there was a significant difference between Arachidonic acid (AA) and Collagen (Cog) activator groups in the platelet aggregation experiment (P<0.05), but there was no significant difference in the main thrombelastogram (TEG) parameters (R value, K value, angle value, MA value) after treatment (P>0.05). CONCLUSIONS:The inactivation effect of this set of blood component pathogen inactivation system on platelet bacterial contamination could be considered to meet actual clinical needs, with the inactivation treatment having little impact on platelet function.

BACKGROUND:The various pathogenesis between Clear cell renal carcinoma (CCRCC) and Chromophobe renal carcinoma (CHRCC) contributes to the different tumor growth rate and metastasis. In this study, we explored the distinct proteomic profiles between these two cancers and found different expression of glycogen phosphorylases in two cancers. METHODS:We explored novel targets by proteomics. Five CCRCC cases and five CHRCC cases were selected for tandem mass tag-labeling liquid chromatography-mass spectroscopy (LC-MS). Gene ontology and KEGG pathway were applied for bioinformatic analysis. Glycogen phosphorylases were detected by Western blotting. RESULTS:CHRCC were younger, more commonly female, and had larger tumors compared to those with CCRCC. 101 differentially expressed proteins (DEPs) in CCRCC and 235 DEPs in CHRCC were detected by LC-MS. It was found that disruption of metabolic pathways, epithelial cell differentiation, and cell response were the common characters for two tumor types. Activation of cell-cell adhesion and oxidation-reduction process stimulate CCRCC growth and epithelial cell differentiation and transferrin transport was involved in CHRCC growth, We also found that oxidative phosphorylation is activated in CHRCC and inhibited in CCRCC. More importantly, we found and confirmed that upregulation of glycogen phosphorylase liver type in CCRCC and glycogen phosphorylase brain type in CHRCC mediated differential glycogenolysis in the two tumor types, which could serve as potential therapeutic targets. CONCLUSION:We found different expression of glycogen phosphorylases in CCRCC and CHRCC by quantitative proteomics, which provides potential therapeutic targets in the future.

OBJECTIVE:To evaluate the characteristics of autoimmune hemolytic anemia caused by salvianolate by antibody detection and clinical index monitoring.METHODS:Micro-column gel anti-human globulin method was used for irregular antibody screening and antibody identification. Salvianolate, sodium creatine phosphate and levocarnitine were used to sensitize red blood cells that were compatible with the patient's plasma, and the RBCs were used to test drug antibody in patient plasma respectively. The patient's clinical examination of hemolysis index and blood transfusion effect were analyed retrospectively.RESULTS:The patients were positive for irregular antibody screening, and there were antoanti-Ce antibodies in serum. The erythrocytes sensitized with salvianolate in the patient's serum were positive, while those sensitized with sodium creatine phosphate and levocarnitine were negative.CONCLUSION:Salvianolate causes drug-induced autoimmune hemolytic anemia in this patient.

Background: Platelet antibodies can lead to clinical diseases such as platelet transfusion refractoriness (PTR), fetal/neonatal alloimmune thrombocytopenia (FNAIT), etc. This study is aimed at understanding CD36 expression, platelet alloantibody distribution in different populations in Northern China, and effects of platelet alloantibodies on pregnancy. Study design and methods: Whole blood samples of 612 subjects including hematological patients, pregnant women, and blood donors were collected at a single center, then CD36 expressions were determined, followed by platelet antibody screening and characterization of platelet antibody specificity. A retrospective analysis was performed in 1552 pregnant women admitted to Department of Obstetrics, in order to investigate FNAIT occurrence. Results: Rate of CD36 deficiency expression was 2.12% (13/612), all cases exhibited type II deficiency without type I deficiency being detected, and such rate is lower than that in Southern China (3.43%), Japanese (4.87%) and in the black people (4.18%), and higher than that in the White people (0.09%). Positive rates of platelet antibody screening in hematological patient group (6.86%, 14/204) and in pregnant women group (6.31%, 13/206) are higher than that in blood donor group (0.49%, 1/202), P < .01. Out of 1552 pregnant women, there were not children with FNAIT. Conclusion: The frequency of CD36 deficiency in northern China was low, all of them were type II deficiency, and no CD36 antibody was detected. It is speculated that the risk of immune-related thrombocytopenia caused by CD36 deficiency in this population is very low. Platelet antibodies should be monitored early in patients with hematological and multiple miscarriages pregnant.

BACKGROUND: Radiation and filtration have achieved satisfactory results in inactivation or removal of tumor cells mixed in salvage blood, but some drawbacks remain. This study evaluated the inactivation on HCT116 cells mixed in simulative salvage blood by riboflavin photochemical treatment. METHODSHCT: 116 cells were added to the whole blood to simulate contaminated salvaged blood. The mixed blood was added with riboflavin of 50 mu mol/L final concentration and illuminated by ultraviolet light. The samples were divided into control group and Experimental Groups 1 (18 J/cm(2)), 2 (23.4 J/cm(2)), and 3 (28.8 J/cm(2)). An autotransfusion system (Cell Saver Elite, Haemonetics) was used to simulate the intraoperative blood salvage procedure to deal with whole blood. The apoptosis rate and tumorigenicity of HCT116 cells and the superimposed damage to red blood cells (RBCs) were evaluated. RESULTS: The apoptosis rates of HCT116 in Experimental Groups 1, 2, and 3 were much higher than that in the control group. Tumor growth was found in the control group, but no tumor growth was found in the three experimental groups. The hemolysis rates in the three experimental groups were significantly higher than that in the control group, but much lower than the quality standard of RBCs at the end of preservation. The concentration of adenosine triphosphate in RBCs was comparable in the control and experimental groups. CONCLUSION: Riboflavin at a 50 mu mol/L final concentration and 18 J/cm(2) ultraviolet illumination can effectively inactivate HCT116 cells in salvaged blood, with minimum damage to the structure and function of RBCs, and the main quality indexes of salvaged RBCs were within the standard range.

Surgical resection is one of the most common radical treatments for cancers. However, tumors may be compressed or the local intravascular pressure may be increased during surgical manipulation, causing the shedding and entry of tumor cells into the blood circulation and hence distant recurrence and metastasis of tumors. We have preliminarily established a method of riboflavin photosensitization treatment (RPT) for inactivation of circulating tumor cells. This technology promises to solve the problems of shedding and entry of solid tumor cells into blood circulation before surgical manipulation, and almost unavoidable hematogenous dissemination of tumor cells during surgical resection. In the present study, apoptosis detection and tumorigenicity experiment in immunodeficient mice were conducted to evaluate the effect of RPT for inactivation of circulating tumor cells respectively. Next, functional evaluation was carried out for the immune cells through detecting apoptosis rate and cytokine secretion of lymphocyte. Finally, thromboelastography (TEG) and free hemoglobin were detected to assess peripheral blood coagulation and red blood cell damage. The results showed that RPT (50 μmol/L riboflavin, 10.8 J/cm2 UV) could effectively make tumor cell lose the ability of proliferation in the peripheral blood. In the meantime, the damage caused to peripheral blood coagulation, immune cell function and red blood cells was generally acceptable. The results of the study showed that RPT had huge potential in addressing the problems of shedding and entry of solid tumor cells into blood circulation before surgical manipulation, and almost unavoidable hematogenous dissemination of tumor cells during surgical resection. This therapy is expected to be an auxiliary and supportive method to reduce the risk of hematogenous metastasis and recurrence of cancers, and to increase the surgical success rate of malignant solid tumors.

Antibody reagents have been remained as a standard approach to characterize blood group (BG) antigens in clinic. The specificity and cross-reactivity of these BG antibodies are routine detected using the gel microcolumn assay (GMA). However, the GMA is neither specific nor sensitive, thus increasing the risk of improperly matched RBC transfusions. In this work, we describe a bead-based RBC membrane antigen array to detect BG antibody antigen binding with similar to 700-fold higher sensitivity and dynamic range than the GMA. RBC membrane antigen arrays were fabricated using fragmented RBC membranes highly enriched in BG panel antigens. The arrays were then used to screen the interactions of 15 BG reagents to three antigen panels. The majority of the antibody reactions (i.e., 86.7%; 39/45) aligned with those obtained with the GMA. The six cross reactive, nonspecific antibody reactions identified only by our arrays (i.e., 13.3%; 6/45) were confirmed by agglutination inhibition and genotyping assays. These results demonstrate that our RBC membrane antigen array has great potential in screening BG antibodies and improving the safety of RBC transfusions.

Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the nature of disease progression and its regulation remain unclear. In the present study, we monitored the course of anti-Thy1 nephritis from days 1 to 5 and established gene expression profiles at each time point using microarrays to explore the development of inflammation. According to the gene expression profiles, macrophage infiltration (triggered by CCL2 activation) was evident on day 1 and enhanced inflammation over the next few days. We screened for genes with expression levels similar to CCL2 and found that the upregulation of the circadian gene albumin D-site-binding protein (DBP) was involved in CCL2 activation in mesangial cells. More importantly, CCL2 expression showed oscillatory changes similar to DBP, and DBP induced peak CCL2 expression at 16:00 a clock on day 1 in the anti-Thy1 nephritis model. We knocked down DBP through transfection with a small interfering RNA (siRNA) and used RNA sequencing to identify the DBP-regulated TNF-α-CCL2 pathway. We performed chromatin immunoprecipitation sequencing (ChIP-Seq) and the dual luciferase assay to show that DBP bound to the TRIM55 promoter, regulating gene expression and in turn controlling the TNF-α-CCL2 pathway. In conclusion, DBP-regulated circadian CCL2 expression by the TRIM55-TNF pathway in injured mesangial cells at an early stage, which promoted macrophage recruitment and in turn triggered infiltration and inflammation in a model of anti-Thy1 nephritis.

The purpose of the present study was to identify the differential proteins that synchronously change in urine and glomeruli and could be used to monitor glomerular lesions of IgA nephropathy (IgAN). The proteomes of urine and glomeruli from four IgAN patients who were graded III/IV according to the grading system of Lee et al. were compared to the urine proteomes of four healthy volunteers and the glomeruli proteomes of adjacent normal tissue from four patients with renal tumors, respectively. Western blot, enzyme-linked immunosorbent assay and immunofluorescence assay were applied to verify the results of the proteomic analysis. In the proteomic analysis of urine from IgAN patients and healthy volunteers, 714 proteins were identified, with 246 proteins identified as differential proteins. In the proteomic analysis of glomeruli from renal biopsy tissue of IgAN patients and from adjacent normal tissue of patients with renal tumors, 161 proteins were identified altogether, and 20 proteins of these were recognized as differential proteins. After comparatively analyzing the differential proteins identified in the urine and glomeruli, five synchronously changed differential proteins were found: complement C9, Ig kappa chain C region and three cytoskeleton proteins. In summary, our findings indicate that certain immunological indices in urine appear to be associated with glomerular lesions of IgAN.

Membranous nephropathy (MN) is an autoimmune inflammatory disease in which proteins related with plenty of biological processes play an important role. However, the role of these proteins in the pathogenesis of MN is still unclear. This study aimed to screen differential proteins in kidney tissue samples from MN patients by using protein arrays and determine the pathways involved in the pathogenesis of MN. This study first tested a quantitative protein array (QAH-INF-3) and two semiquantitative protein arrays (L-493 and L-507) with normal renal tissue and identified L-493 as the most appropriate assay to compare protein levels between MN tissues and normal control tissues. The L-493 array identified 66 differentially expressed proteins (DEPs) that may be associated with MN. The gene oncology (GO) and protein-protein interaction (PPI) analyses revealed several processes potentially involved in MN, including extracellular matrix disassembly and organization, cell adhesion, cell-cell signaling, cellular protein metabolic process, and immune response (P < 0.05). We suggest that these different pathways work together via protein signaling and result in the pathogenesis and progression of MN.

The miRNAs in urinary sediment are easy to obtain, which provides a new approach to searching for non-invasive biomarkers of IgA nephropathy (IgAN). Compared with normal controls (n = 3), 214 different miRNAs in the urinary sediment of IgAN (n = 9) were found by miRNA chip assay. By quantitative PCR analysis, miR-25-3p, miR-144-3p and miR-486-5p were confirmed to be significantly higher in IgAN (n = 93) than in the normal group (n = 82) or disease control (n = 40). These three miRNAs had good specificity and sensitivity for the diagnosis of IgAN by receiver operating characteristic curve analysis, in which the AUC value of miR-486-5p was the largest at 0.935. Urinary sediment miR-25-3p, miR-144-3p and miR-486-5p were demonstrated to be mainly derived from urinary erythrocytes, which were separated by CD235a magnetic beads. The increased expression of urinary erythrocyte miRNAs in IgAN patients was not associated with those in the blood erythrocytes. In addition, urinary supernatant microvesicles of miR-144-3p and miR-486-5p in the IgAN group were also significantly increased. This study showed that the miR-25-3p, miR-144-3p and miR-486-5p in urinary sediment were mainly derived from urinary erythrocytes, which could be non-invasive candidate biomarkers for IgA nephropathy.

Kidney aging together with related renal disease had become a major clinical problem. Understanding the mechanisms of aging was important for suspending senescence and decreasing the incidence of aging-related diseases. In the present work, 24-month-old F344 rats were used as aging rats and 3-month-old rats were used as young controls. Senescence-associated-β-galactosidase staining results showed that the degree of senescence in renal tubules was more severe than that in glomeruli. We performed quantitative LC-MS to assess the differential protein expression profiles of senescent glomeruli and tubules. Bioinformatics analysis showed that aging, response to oxidative stress, nucleotide metabolism, amine acid metabolism, and inflammatory response were common mechanisms of glomerulus and tubule senescence. Differentially expressed proteins network mediated Golgi vesicle transport, actin filament based process, and regulation of cell death were associated with tubule senescence. More importantly, we found that the changes of four and a half LIM protein 2 (FHL2) were opposite in senescent glomeruli and tubules, and FHL2 could regulate p16 by suppressing T-box 3, which was involved in regulation of senescence in glomeruli and tubules. In conclusion, we assessed the mechanisms of senescence in aging glomeruli and tubules, and the results yielded new insight into kidney senescence.

BACKGROUND:Dysfunction of renal tubule epithelial cells is associated with renal tubulointerstitial fibrosis. Exploration of the proteomic profiles of senesced tubule epithelial cells is essential to elucidate the mechanism of tubulointerstitium development.METHODS:Primary human proximal tubule epithelial cells from passage 3 (P3) and passage 6 (P6) were selected for evaluation. EdU and SA-β-galactosidase staining were used to detect cell senescence. p53, p21, and p16 were detected by Western blot analysis. Liquid chromatography mass spectrometry (LC-MS) was used to examine differentially expressed proteins (DEPs) between P6 and P3 cells. The expression of DEPs was examined by Western blot analysis. Bioinformatics analysis was performed by protein-protein interaction and gene ontology analyses.RESULTS:The majority of tubule cells from passage 6 (P6) stained positive for SA-β-galactosidase, whereas passage 3 (P3) cells were negative. Senescence biomarkers, including p53, p21, and p16, were upregulated in P6 cells relative to P3 cells. EdU staining results showed a lower rate of EdU positive cells in P6 cells than in P3 cells. LC-MS was used to examine DEPs between P6 and P3 cells. These DEPs are involved in glycolysis, response to stress, cytoskeleton regulation, oxidative reduction, ATP binding, and oxidative stress. Using Western blot analysis, we validated the down-regulation of AKR1B1, EEF2, EEF1A1, and HSP90 and the up-regulation of VIM in P6 cells seen in the LC-MS data. More importantly, we built the molecular network based on biological functions and protein-protein interactions and found that the DEPs are involved in translation elongation, stress, and glycolysis, and that they are all associated with cytoskeleton regulation, which regulates senescent cell activities such as apoptosis and EMT in tubule epithelial cells.CONCLUSIONS:We explored proteomic profile changes in cell culture-induced senescent cells and built senescence-associated molecular networks, which will help to elucidate the mechanisms of senescence in human proximal tubule epithelial cells.