Pathogenic variants in human DNA damage repair genes mostly arose in recent human history.

BACKGROUND: Genome stability is maintained by the DNA damage repair (DDR) system composed of multiple DNA repair pathways of hundreds of genes. Germline pathogenic variation (PV) in DDR genes damages function of the affected DDR genes, leading to genome instability and high risk of diseases, in particular, cancer. Knowing evolutionary origin of the PVs in human DDR genes is essential to understand the etiology of human diseases. However, answer to the issue remains largely elusive. In this study, we analyzed evolutionary origin for the PVs in human DDR genes. METHODS: We identified 169 DDR genes by referring to various databases and identified PVs in the DDR genes of modern humans from ClinVar database. We performed a phylogenetic analysis to analyze the conservation of human DDR PVs in 100 vertebrates through cross-species genomic data comparison using the phyloFit program of the PHAST package and visualized the results using the GraphPad Prism software and the ggplot module. We identified DDR PVs from over 5000 ancient humans developed a database to host the DDR PVs ( https://genemutation.fhs.um.edu.mo/dbDDR-AncientHumans ). Using the PV data, we performed a molecular archeological analysis to compare the DDR PVs between modern humans and ancient humans. We analyzed evolution selection of DDR genes across 20 vertebrates using the CodeML in PAML for phylogenetic analysis. RESULTS: Our phylogenic analysis ruled out cross-species conservation as the origin of human DDR PVs. Our archeological approach identified rich DDR PVs shared between modern and ancient humans, which were mostly dated within the last 5000 years. We also observed similar pattern of quantitative PV distribution between modern and ancient humans. We further detected a set of ATM, BRCA2 and CHEK2 PVs shared between human and Neanderthals. CONCLUSIONS: Our study reveals that human DDR PVs mostly arose in recent human history. We propose that human high cancer risk caused by DDR PVs can be a by-product of human evolution.

Evolutionary origin of germline pathogenic variants in human DNA mismatch repair genes.

BACKGROUND: Mismatch repair (MMR) system is evolutionarily conserved for genome stability maintenance. Germline pathogenic variants (PVs) in MMR genes that lead to MMR functional deficiency are associated with high cancer risk. Knowing the evolutionary origin of germline PVs in human MMR genes will facilitate understanding the biological base of MMR deficiency in cancer. However, systematic knowledge is lacking to address the issue. In this study, we performed a comprehensive analysis to know the evolutionary origin of human MMR PVs. METHODS: We retrieved MMR gene variants from the ClinVar database. The genomes of 100 vertebrates were collected from the UCSC genome browser and ancient human sequencing data were obtained through comprehensive data mining. Cross-species conservation analysis was performed based on the phylogenetic relationship among 100 vertebrates. Rescaled ancient sequencing data were used to perform variant calling for archeological analysis. RESULTS: Using the phylogenetic approach, we traced the 3369 MMR PVs identified in modern humans in 99 non-human vertebrate genomes but found no evidence for cross-species conservation as the source for human MMR PVs. Using the archeological approach, we searched the human MMR PVs in over 5000 ancient human genomes dated from 45,045 to 100 years before present and identified a group of MMR PVs shared between modern and ancient humans mostly within 10,000 years with similar quantitative patterns. CONCLUSION: Our study reveals that MMR PVs in modern humans were arisen within the recent human evolutionary history.

TP53 germline pathogenic variants in modern humans were likely originated during recent human history.

TP53 is crucial for maintaining genome stability and preventing oncogenesis. Germline pathogenic variation in TP53 damages its function, causing genome instability and increased cancer risk. Despite extensive study in TP53, the evolutionary origin of the human TP53 germline pathogenic variants remains largely unclear. In this study, we applied phylogenetic and archaeological approaches to identify the evolutionary origin of TP53 germline pathogenic variants in modern humans. In the phylogenic analysis, we searched 406 human TP53 germline pathogenic variants in 99 vertebrates distributed in eight clades of Primate, Euarchontoglires, Laurasiatheria, Afrotheria, Mammal, Aves, Sarcopterygii and Fish, but we observed no direct evidence for the cross-species conservation as the origin; in the archaeological analysis, we searched the variants in 5031 ancient human genomes dated between 45045 and 100 years before present, and identified 45 pathogenic variants in 62 ancient humans dated mostly within the last 8000 years; we also identified 6 pathogenic variants in 3 Neanderthals dated 44000 to 38515 years before present and 1 Denisovan dated 158 550 years before present. Our study reveals that TP53 germline pathogenic variants in modern humans were likely originated in recent human history and partially inherited from the extinct Neanderthals and Denisovans.

Evolutionary Origin of MUTYH Germline Pathogenic Variations in Modern Humans.

MUTYH plays an essential role in preventing oxidation-caused DNA damage. Pathogenic germline variations in MUTYH damage its function, causing intestinal polyposis and colorectal cancer. Determination of the evolutionary origin of the variation is essential to understanding the etiological relationship between MUTYH variation and cancer development. In this study, we analyzed the origins of pathogenic germline variants in human MUTYH. Using a phylogenic approach, we searched MUTYH pathogenic variants in modern humans in the MUTYH of 99 vertebrates across eight clades. We did not find pathogenic variants shared between modern humans and the non-human vertebrates following the evolutionary tree, ruling out the possibility of cross-species conservation as the origin of human pathogenic variants in MUTYH. We then searched the variants in the MUTYH of 5031 ancient humans and extinct Neanderthals and Denisovans. We identified 24 pathogenic variants in 42 ancient humans dated between 30,570 and 480 years before present (BP), and three pathogenic variants in Neanderthals dated between 65,000 and 38,310 years BP. Data from our study revealed that human MUTYH pathogenic variants mostly arose in recent human history and partially originated from Neanderthals.

Ethnic-specificity, evolution origin and deleteriousness of Asian BRCA variation revealed by over 7500 BRCA variants derived from Asian population.

Pathogenic variation in BRCA1 and BRCA2 (BRCA) causes high risk of breast and ovarian cancer, and BRCA variation data are important markers for BRCA-related clinical cancer applications. However, comprehensive BRCA variation data are lacking from the Asian population despite its large population size, heterogenous genetic background and diversified living environment across the Asia continent. We performed a systematic study on BRCA variation in Asian population including extensive data mining, standardization, annotation and characterization. We identified 7587 BRCA variants from 685 592 Asian individuals in 40 Asia countries and regions, including 1762 clinically actionable pathogenic variants and 4915 functionally unknown variants (https://genemutation.fhs.um.edu.mo/Asian-BRCA/). We observed the highly ethnic-specific nature of Asian BRCA variants between Asian and non-Asian populations and within Asian populations, highlighting that the current European descendant population-based BRCA data is inadequate to reflect BRCA variation in the Asian population. We also provided archeological evidence for the evolutionary origin and arising time of Asian BRCA variation. We further provided structural-based evidence for the deleterious variants enriched within the functionally unknown Asian BRCA variants. The data from our study provide a current view of BRCA variation in the Asian population and a rich resource to guide clinical applications of BRCA-related cancer for the Asian population.

Plasminogen Activator Inhibitor-1-Positive Platelet-Derived Extracellular Vesicles Predicts MACE and the Proinflammatory SMC Phenotype.

Patients with established coronary artery disease remain at elevated risk of major adverse cardiac events. The goal of this study was to evaluate the utility of plasminogen activator inhibitor-1-positive platelet-derived extracellular vesicles as a biomarker for major adverse cardiac events and to explore potential underlying mechanisms. Our study suggests these extracellular vesicles as a potential biomarker to identify and a therapeutic target to ameliorate neointimal formation of high-risk patients.

Elevated ß-catenin and C-myc promote malignancy, relapse, and indicate poor prognosis in patients with relapsed glioma.

Context: Extensive studies have shown that ß-catenin and C-myc have been linked to a number of human cancers. However, the role of ß-catenin and C-myc in relapse glioma remains unclear. Aims: The aims of this study were to investigate the role of ß-catenin and C-myc in relapsed glioma patients and to explore the possible impact of malignancy, relapse, and prognosis. Materials and Methods: We collected surgical samples of 100 patients with primary and relapsed glioma treated at our institution. Immunohistochemistry (IHC) staining was used to evaluate the expressions of ß-catenin and C-myc. The impact of the differences on disease-free interval (DFI), initial overall survival (iOS), and overall survival from the time of glioma relapse (rOS) of the patients was analyzed. Kaplan-Meier survival functions were used to plot survival time, and a log-rank test was used for analyzing statistical significance. Cox multivariate regression analysis was used to determine independent prognostic parameters. Results: Compared to primary tumors, relapsed gliomas had higher expressions of ß-catenin and C-myc (P < 0.05). Furthermore, the expressions of ß-catenin and C-myc were significantly correlated with glioma grade (P < 0.05). These changes in expression at the time of relapse were independent of radiotherapy use. In multivariate Cox analysis, we found that ß-catenin and C-myc were independent prognostic factors for rOS (P < 0.05). Conclusions: Elevated ß-catenin and C-myc promote malignancy, relapse, and indicate poor prognosis in patients with relapsed glioma. The elevated levels of ß-catenin and c-myc in relapsed glioma were not affected by radiation therapy. The results of this study may provide a new therapeutic target for patients with relapsed glioma.

Rhoifolin Alleviates Alcoholic Liver Disease In Vivo and In Vitro via Inhibition of the TLR4/NF-κB Signaling Pathway.

Background: Alcoholic liver disease (ALD) is a common chronic liver disorder worldwide, which is detrimental to human health. A preliminary study showed that the total flavonoids within Citrus grandis "Tomentosa" exerted a remarkable effect on the treatment of experimental ALD. However, the active substances of Citrus grandis "Tomentosa" were not elucidated. Rhoifolin (ROF) is a flavonoid component present in high levels. Therefore, this research aimed to evaluate the hepatoprotective effects of ROF and its possible mechanisms. Methods: Molecular docking was performed to analyze the binding energy of ROF to the main target proteins related to ALD. Subsequently, mice were fed ethanol (ETH) for 49 days to establish the chronic alcoholic liver injury models. The liver pathological injury, serum aminotransferase levels, and oxidative stress levels in the liver tissue were measured. Human normal hepatocytes (LO2 cells) were incubated with ETH to construct the alcoholic liver cell model. The inflammatory markers and apoptosis factors were evaluated using real-time PCR and flow cytometry. Finally, the effects of ROF on the CYP2E1 and NF-κB signaling pathways were tested in vitro and in vivo. Results: Molecular docking results demonstrated that ROF was able to successfully dock with the target proteins associated with ALD. In animal studies, ROF attenuated ETH-induced liver damage in mice by decreasing the serum concentrations of AST and ALT, reducing the expression of inflammatory cytokines, and maintaining antioxidant balance in the liver tissue. The in vitro experiments demonstrated that ROF suppressed ETH-induced apoptosis in LO2 cells by promoting Bcl-2 mRNA and inhibiting Bax mRNA and caspase 3 protein expression. ROF decreased the level of LDH, ALT, AST, ROS, and MDA in the supernatant; induced the activity of GSH and SOD; and inhibited TNF-α, IL-6, and IL-1ß expression levels. Mechanistically, ROF could significantly downregulate the expression levels of CYP2E1, TLR4, and NF-κB phosphorylation. Conclusion: This study indicates that ROF is the active component within the total flavonoids, which may alleviate ETH-induced liver injury by inhibiting NF-κB phosphorylation. Therefore, ROF may serve as a promising compound for treating ALD.

Extracellular Vesicles as an Index for Endothelial Injury and Cardiac Dysfunction in a Rodent Model of GDM.

Gestational diabetes mellitus (GDM) increases risk of adverse pregnancy outcomes and maternal cardiovascular complications. It is widely believed that maternal endothelial dysfunction is a critical determinant of these risks, however, connections to maternal cardiac dysfunction and mechanisms of pathogenesis are unclear. Circulating extracellular vesicles (EVs) are emerging biomarkers that may provide insights into the pathogenesis of GDM. We examined the impact of GDM on maternal cardiac and vascular health in a rat model of diet-induced obesity-associated GDM. We observed a >3-fold increase in circulating levels of endothelial EVs (p < 0.01) and von Willebrand factor (p < 0.001) in GDM rats. A significant increase in mitochondrial DNA (mtDNA) within circulating extracellular vesicles was also observed suggesting possible mitochondrial dysfunction in the vasculature. This was supported by nicotinamide adenine dinucleotide deficiency in aortas of GDM mice. GDM was also associated with cardiac remodeling (increased LV mass) and a marked impairment in maternal diastolic function (increased isovolumetric relaxation time [IVRT], p < 0.01). Finally, we observed a strong positive correlation between endothelial EV levels and IVRT (r = 0.57, p < 0.05). In summary, we observed maternal vascular and cardiac dysfunction in rodent GDM accompanied by increased circulating endothelial EVs and EV-associated mitochondrial DNA. Our study highlights a novel method for assessment of vascular injury in GDM and highlights vascular mitochondrial injury as a possible therapeutic target.

Urinary interleukin-9 in youth with type 1 diabetes mellitus.

AIMS: Interleukin-9 (IL-9) attenuates podocyte injury in experimental kidney disease, but its role in diabetic nephropathy is unknown. We sought to relate urinary IL-9 levels to the release of podocyte-derived extracellular vesicles (EVs) in youth with type 1 diabetes. We related urinary IL-9 levels to clinical variables and studied interactions between urinary IL-9, vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) on urinary albumin/creatinine ratio (ACR) a functional measure of podocyte injury. METHODS: We performed an analysis of urine samples and clinical data from a cohort of youth with type 1 diabetes (n = 53). Cytokines were measured using a Luminex platform (Eve Technologies), and nanoscale flow cytometry was employed to quantify urinary podocyte-derived EVs. All urinary measures were normalized to urinary creatinine. RESULTS: Mean age was 14.7 ± 1.6 years, and the mean time from diagnosis was 6.7 ± 2.9 years. Mean HbA1c was 70.3 ± 13.9 mmol/mol, mean ACR was 1.3 ± 1.9 mg/mmol, and mean eGFR was 140.3 ± 32.6 ml/min/1.73 m2. IL-9 was inversely related to podocyte EVs (r = - 0.56, p = 0.003). IL-9 was also inversely related to blood glucose, HbA1C and eGFR (r = - 0.44, p = 0.002; r = - 0.41, p = 0.003; r = - 0.49, p < 0.001, respectively) and positively correlated with systolic BP (r = 0.30, p = 0.04). There was a significant interaction between IL-9, EVs and ACR (p = 0.0143), and the relationship between IL-9 and ACR depended on VEGF (p = 0.0083), TNFα (p = 0.0231) and IL-6 levels (p = 0.0178). CONCLUSIONS: IL-9 is associated with podocyte injury in early type 1 diabetes, and there are complex interactions between urinary IL-9, inflammatory cytokines and ACR.

Retraction: Overexpression of HDAC8 and down-regulation of CDKN2A is associated with worse prognosis of esophageal squamous cell carcinoma.

[This retracts the article DOI: 10.21037/tcr.2020.01.32.].

Rhoifolin Alleviates Inflammation of Acute Inflammation Animal Models and LPS-Induced RAW264.7 Cells via IKKß/NF-κB Signaling Pathway.

Rhoifolin (ROF) is a main effective component in Citrus grandis 'Tomentosa'. ROF has a potential anti-inflammatory activity, but its specific effects and mechanisms have not been studied. This study investigated the anti-inflammatory activity of ROF and searched for its possible molecular mechanisms. A mouse model of acute inflammation was induced by lipopolysaccharide, and the effects of ROF on pathological damages of the lung and liver were observed. Carrageenan-induced paw edema rat model was used to evaluate the effect of ROF on the volume of swelling paw. In LPS-induced RAW264.7 macrophages, the expression levels of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α were measured using ELISA. Real-time PCR was used to measure the mRNA levels of iNOS and CCL2. Western blot was used to detect the activation of IκBα and IKKß in NF-κB signaling pathways. The results showed that ROF accelerated the recoveries of liver and lung tissue damages in acute inflammation mice and inhibited carrageenan-induced paw edema in rats; in addition, ROF significantly suppressed the secretion of TNF-α, IL-1ß, and IL-6 in the serum of rats and mouse model. In LPS-induced RAW264.7 cells, 100 µmol/L ROF enhanced cell viability and suppressed the production of TNF-α, IL-6, and IL-1ß significantly. ROF also decreased the mRNA expression of iNOS and CCL2 and inhibited IκBα and IKKß phosphorylation. In summary, ROF had a potential therapeutic value for inflammation. Our research provided experimental basis for the further development of ROF as an anti-inflammatory drug and for clarifying the anti-inflammatory substance basis of Citrus grandis 'Tomentosa'. Graphical Abstract.

Theoretical Study of Radical-Molecule Reactions with Negative Activation Energies in Combustion: Hydroxyl Radical Addition to Alkenes.

Many of the radical-molecule reactions are nonelementary reactions with negative activation energies, which usually proceed through two steps. They exist extensively in the atmospheric chemistry and hydrocarbon fuel combustion, so they are extensively studied both theoretically and experimentally. At the same time, various models, such as a two transition state model, a steady-state model, an equilibrium-state model, and a direct elementary dynamics model are proposed to get the kinetic parameters for the overall reaction. In this paper, a conversion temperature T C1 is defined as the temperature at which the standard molar Gibbs free energy change of the formation of the reaction complex is equal to zero, and it is found that when T ≫ T C1, the direct elementary dynamics model with an inclusion of the tunneling correction of the second step reaction is applicable to calculate the overall reaction rate constants for this kind of reaction system. The reaction class of hydroxyl radical addition to alkenes is chosen as the objects of this study, five reactions are chosen as the representative for the reaction class, and their single-point energies are calculated using the method of CCSD(T)/CBS, and it is shown that the highest conversion temperature for the five reactions is 139.89 K, far below the usual initial low-temperature (550 K) oxidation chemistry of hydrocarbon fuels; therefore, the steady-state approximation method is applicable. All geometry optimizations are performed at the BH&HLYP/6-311+G(d,p) level, and the result shows that the geometric parameters in the reaction centers are conserved; hence, the isodesmic reaction method is applicable to this reaction class. To validate the accuracy of this scheme, a comparison of electronic energy difference at the BH&HLYP/6-311+G(d,p) level and the corrected electronic energy difference with the electronic energy difference at the CCSD(T)/CBS level is performed for the five representative reactions, and it is shown that the maximum absolute deviation of electronic energy difference can be reduced from 2.54 kcal·mol-1 before correction to 0.58 kcal·mol-1 after correction, indicating that the isodesmic reaction method is applicable for the accurate calculation of the kinetic parameters for large-size molecular systems with a negative activation energy reaction. The overall rate constants for 44 reactions of the reaction class of hydroxyl radical addition to alkenes are calculated using the transition-state theory in combination with the isodesmic correction scheme, and high-pressure limit rate rules for the reaction class are developed. In addition, the thermodynamic parameter is calculated and the results indicate that our dynamics model is applicable for our studied reaction class. A chemical kinetic modeling and sensitivity analysis using the calculated kinetic data is performed for the combustion of ethene, and the results indicate the studied reaction is important for the low-to-medium temperature combustion modeling of ethene.

Risk Factors for Cognitive Impairment in High-Grade Glioma Patients Treated with Postoperative Radiochemotherapy.

PURPOSE: Fractionated radiotherapy as well as concomitant and adjuvant chemotherapy such as temozolomide for postoperative high-grade glioma (HGG) patients improves progression-free survival and overall survival. Multiple factors such as chemotherapy, radiotherapy, tumor grade, residual tumor volume, and genetic modifications might play a role in the formation of cognitive impairment. The risk factors of cognitive impairment in postoperative patients with HGG receiving radiotherapy and chemotherapy remains a concern in this population. The purpose of this study was to identify risk factors for cognitive impairment in patients of postoperative HGG. MATERIALS AND METHODS: A total of 229 patients with HGG who underwent surgery were analyzed. Cognitive impairment was defined as a decrease of Cognitive Assessment Montreal (MoCA)'s score in at least two cognitive domains or any MoCA's score of less than 26 points at the time of study compared with baseline level. Multiple potential risk factors including methylated status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter, glioma World Health Organization (WHO) grade, residual tumor volume, education, and sex were analyzed. Cox univariate and multivariate regression analysis was used to detect the significant risk factors for cognitive impairment. RESULTS: At the end of follow-up among the 229 patients, 147 patients (67%) developed cognitive impairment. 82 patients (36%) remained in normal cognitive condition. In multivariate analysis, unmethylated MGMT promoter (hazard ratio [HR], 1.679; 95% confidence interval [CI], 1.212 to 2.326; p=0.002), glioblastoma (HR, 1.550; 95% CI, 1.117 to 2.149; p=0.009), and residual tumor volume > 5.58 cm3 (HR, 1.454; 95% CI, 1.047 to 2.020; p=0.026) were independent risk factors for cognitive impairment. CONCLUSION: Methylated status of the MGMT promoter, glioma WHO grade, and residual tumor volume might be risk factors for the cognitive impairment in postoperative patients with HGG.

Over-expression of HDAC8 down-regulate CDKN2A is associated with worse prognosis of esophageal squamous cell carcinoma.

BACKGROUND: The epigenetic alteration has an impact on cancer cell cycle regulation. Expression of histone deacetylase 8 (HDAC8) ruled out the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A) and this is linked with the prognosis of Esophageal cancer (EC) patients. METHODS: By the immunohistochemical staining, we examined the expression status of HDAC8 and CDKN2A protein of 110 esophageal squamous cell carcinoma (ESCC) patients in the tissue microarray. The nuclear staining intensity was counted by immunoreactivity scoring ranging from 0 to 12 and grouped them into two; weak or non-expression and over-expression. RESULTS: The median follow-up duration of our study was 71 months postoperatively. Up-regulation of HDAC8 expression and down-regulation of CDKN2A (p16) indicate decreased 5-year overall survival (OS) (P=0.003) and (P=0.001) respectively and progression-free survival (PFS) (P=0.005, P=0.001) respectively, which are statistically significant and determined by Kaplan-Meier estimates using log-rank test. Consequently, HDAC8 and CDKN2A act as an independent prognostic biomarker, for OS and PFS that analyzed by multivariate cox-regression analysis, HR 1.173 with 95% CI: 0.215-6.416 and P=0.003 and HR 1.217 with 95% CI: 0.224-6.600 and P=0.005 respectively. Spearman rank test establish the negative correlation between HDAC8 and CDKN2A. CONCLUSIONS: Over-expression of HDAC8 and weak or no expression of CDKN2A have a worse impact on EC patients and indicate poor survival and progression of the disease and act as a promising prognostic parameter.

Prostaglandin E2 receptor EP1 (PGE2/EP1) deletion promotes glomerular podocyte and endothelial cell injury in hypertensive TTRhRen mice.

Prostaglandin E2 receptor EP1 (PGE2/EP1) promotes diabetic renal injury, and EP1 receptor deletion improves hyperfiltration, albuminuria, and fibrosis. The role of EP1 receptors in hypertensive kidney disease (HKD) remains controversial. We examined the contribution of EP1 receptors to HKD. EP1 null (EP1-/-) mice were bred with hypertensive TTRhRen mice (Htn) to evaluate kidney function and injury at 24 weeks. EP1 deletion had no effect on elevation of systolic blood pressure in Htn mice (HtnEP1-/-) but resulted in pronounced albuminuria and reduced FITC-inulin clearance, compared with Htn or wild-type (WT) mice. Ultrastructural injury to podocytes and glomerular endothelium was prominent in HtnEP1-/- mice; including widened subendothelial space, subendothelial lucent zones and focal lifting of endothelium from basement membrane, with focal subendothelial cell debris. Cortex COX2 mRNA was increased by EP1 deletion. Glomerular EP3 mRNA was reduced by EP1 deletion, and EP4 by Htn and EP1 deletion. In WT mice, PGE2 increased chloride reabsorption via EP1 in isolated perfused thick ascending limb (TAL), but PGE2 or EP1 deletion did not affect vasopressin-mediated chloride reabsorption. In WT and Htn mouse inner medullary collecting duct (IMCD), PGE2 inhibited vasopressin-water transport, but not in EP1-/- or HtnEP1-/- mice. Overall, EP1 mediated TAL and IMCD transport in response to PGE2 is unaltered in Htn, and EP1 is protective in HKD.

Effect of hemodialysis on extracellular vesicles and circulating submicron particles.

BACKGROUND: Although hemodialysis is a highly effective treatment for diffusive clearance of low molecular weight uremic toxins, its effect on circulating extracellular vesicles and submicron particles is less clear. The purpose of this study was to examine the impact of hemodialysis on circulating levels of submicron particles. METHODS: Plasma samples from patients were collected immediately before and after the mid-week hemodialysis session. Total submicron particles were assessed by nanoparticle tracking analysis and levels of endothelial (CD144+), platelet (CD41+), leukocyte (CD45+), and total (Annexin V+) membrane microparticles (MPs) were assessed by flow cytometry. RESULTS: Total submicron particle number was significantly lower post-dialysis with reductions in particles < 40 nm, 40-100 nm, and 100-1000 nm in size. Circulating annexin V+ MPs, platelet MPs, leukocyte MPs, and endothelial MPs were all reduced following dialysis. Assessment of protein markers suggested that extracellular vesicles were not present in the dialysate, but rather adsorbed to the dialysis membrane. CONCLUSIONS: In summary, hemodialysis is associated with reductions in circulating submicron particles including membrane MPs. Accordingly, there may be significant interdialytic variation in circulating submicron particles. Investigators interested in measuring extracellular vesicles in patients undergoing hemodialysis should therefore carefully consider the timing of biosampling.

Podocyte-derived microparticles promote proximal tubule fibrotic signaling via p38 MAPK and CD36.

Tubulointerstitial fibrosis is a hallmark of advanced diabetic kidney disease that is linked to a decline in renal function, however the pathogenic mechanisms are poorly understood. Microparticles (MPs) are 100-1000 nm vesicles shed from injured cells that are implicated in intercellular signalling. Our lab recently observed the formation of MPs from podocytes and their release into urine of animal models of type 1 and 2 diabetes and in humans with type 1 diabetes. The purpose of the present study was to examine the role of podocyte MPs in tubular epithelial cell fibrotic responses. MPs were isolated from the media of differentiated, untreated human podocytes (hPODs) and administered to cultured human proximal tubule epithelial cells (PTECs). Treatment with podocyte MPs increased p38 and Smad3 phosphorylation and expression of the extracellular matrix (ECM) proteins fibronectin and collagen type IV. MP-induced responses were attenuated by co-treatment with the p38 inhibitor SB202190. A transforming growth factor beta (TGF-ß) receptor inhibitor (LY2109761) blocked MP-induced Smad3 phosphorylation and ECM protein expression but not p38 phosphorylation suggesting that these responses occurred downstream of p38. Finally, blockade of the class B scavenger receptor CD36 completely abrogated MP-mediated p38 phosphorylation, downstream Smad3 activation and fibronectin/collagen type IV induction. Taken together our results suggest that podocyte MPs interact with proximal tubule cells and induce pro-fibrotic responses. Such interactions may contribute to the development of tubular fibrosis in glomerular disease.