Glomerular-tubular crosstalk via cold shock Y-box binding protein-1 in the kidney.

Glomerular-tubular crosstalk within the kidney has been proposed, but the paracrine signals enabling this remain largely unknown. The cold-shock protein Y-box binding protein 1 (YBX1) is known to regulate inflammation and kidney diseases but its role in podocytes remains undetermined. Therefore, we analyzed mice with podocyte specific Ybx1 deletion (Ybx1ΔPod). Albuminuria was increased in unchallenged Ybx1ΔPod mice, which surprisingly was associated with reduced glomerular, but enhanced tubular damage. Tubular toll-like receptor 4 (TLR4) expression, node-like receptor protein 3 (NLRP3) inflammasome activation and kidney inflammatory cell infiltrates were all increased in Ybx1ΔPod mice. In vitro, extracellular YBX1 inhibited NLRP3 inflammasome activation in tubular cells. Co-immunoprecipitation, immunohistochemical analyses, microscale cell-free thermophoresis assays, and blunting of the YBX1-mediated TLR4-inhibition by a unique YBX1-derived decapeptide suggests a direct interaction of YBX1 and TLR4. Since YBX1 can be secreted upon post-translational acetylation, we hypothesized that YBX1 secreted from podocytes can inhibit TLR4 signaling in tubular cells. Indeed, mice expressing a non-secreted YBX1 variant specifically in podocytes (Ybx1PodK2A mice) phenocopied Ybx1ΔPod mice, demonstrating a tubular-protective effect of YBX1 secreted from podocytes. Lipopolysaccharide-induced tubular injury was aggravated in Ybx1ΔPod and Ybx1PodK2A mice, indicating a pathophysiological relevance of this glomerular-tubular crosstalk. Thus, our data show that YBX1 is physiologically secreted from podocytes, thereby negatively modulating sterile inflammation in the tubular compartment, apparently by binding to and inhibiting tubular TLR4 signaling. Hence, we have uncovered an YBX1-dependent molecular mechanism of glomerular-tubular crosstalk.

Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

Correction: Gupta et al. Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease. Nutrients 2022, 14, 2965.

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Single-cell profiling of vascular endothelial cells reveals progressive organ-specific vulnerabilities during obesity.

Obesity promotes diverse pathologies, including atherosclerosis and dementia, which frequently involve vascular defects and endothelial cell (EC) dysfunction. Each organ has distinct EC subtypes, but whether ECs are differentially affected by obesity is unknown. Here we use single-cell RNA sequencing to analyze transcriptomes of ~375,000 ECs from seven organs in male mice at progressive stages of obesity to identify organ-specific vulnerabilities. We find that obesity deregulates gene expression networks, including lipid handling, metabolic pathways and AP1 transcription factor and inflammatory signaling, in an organ- and EC-subtype-specific manner. The transcriptomic aberrations worsen with sustained obesity and are only partially mitigated by dietary intervention and weight loss. For example, dietary intervention substantially attenuates dysregulation of liver, but not kidney, EC transcriptomes. Through integration with human genome-wide association study data, we further identify a subset of vascular disease risk genes that are induced by obesity. Our work catalogs the impact of obesity on the endothelium, constitutes a useful resource and reveals leads for investigation as potential therapeutic targets.

Activated Protein C Ameliorates Tubular Mitochondrial Reactive Oxygen Species and Inflammation in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is an emerging pandemic, paralleling the worldwide increase in obesity and diabetes mellitus. DKD is now the most frequent cause of end-stage renal disease and is associated with an excessive risk of cardiovascular morbidity and mortality. DKD is a consequence of systemic endothelial dysfunction. The endothelial-dependent cytoprotective coagulation protease activated protein C (aPC) ameliorates glomerular damage in DKD, in part by reducing mitochondrial ROS generation in glomerular cells. Whether aPC reduces mitochondrial ROS generation in the tubular compartment remains unknown. Here, we conducted expression profiling of kidneys in diabetic mice (wild-type and mice with increased plasma levels of aPC, APChigh mice). The top induced pathways were related to metabolism and in particular to oxidoreductase activity. In tubular cells, aPC maintained the expression of genes related to the electron transport chain, PGC1-α expression, and mitochondrial mass. These effects were associated with reduced mitochondrial ROS generation. Likewise, NLRP3 inflammasome activation and sterile inflammation, which are known to be linked to excess ROS generation in DKD, were reduced in diabetic APChigh mice. Thus, aPC reduces mitochondrial ROS generation in tubular cells and dampens the associated renal sterile inflammation. These studies support approaches harnessing the cytoprotective effects of aPC in DKD.

ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis.

Diabetes mellitus is hallmarked by accelerated atherosclerosis, a major cause of mortality among patients with diabetes. Efficient therapies for diabetes-associated atherosclerosis are absent. Accelerated atherosclerosis in diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we directly compared the effects of high glucose and oxidized LDL, revealing that high glucose induced more pronounced responses in regard to maladaptive unfolded protein response (UPR), senescence, and vascular endothelial cell barrier disruption. Ex vivo, diabetic ApoE-/- mice displayed increased levels of senescence and UPR markers within atherosclerotic lesions compared with nondiabetic ApoE-/- mice. Activated protein C pretreatment maintained barrier permeability and prevented glucose-induced expression of senescence and UPR markers in vitro. These data suggest that high glucose-induced maladaptive UPR and associated senescence promote vascular endothelial cell dysfunction, which-however-can be reversed by aPC. Taken together, current data suggest that reversal of glucose-induced vascular endothelial cell dysfunction is feasible.

Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease.

Diabetes mellitus is a metabolic disease largely due to lifestyle and nutritional imbalance, resulting in insulin resistance, hyperglycemia and vascular complications. Diabetic kidney disease (DKD) is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited. Endothelial and glomerular filtration barrier (GFB) dysfunction and sterile inflammation are associated with DKD. Neutrophil extracellular traps (NETs), originally identified as an innate immune mechanism to combat infection, have been implicated in sterile inflammatory responses in non-communicable diseases. However, the contribution of NETs in DKD remains unknown. Here, we show that biomarkers of NETs are increased in diabetic mice and diabetic patients and that these changes correlate with DKD severity. Mechanistically, NETs promote NLRP3 inflammasome activation and glomerular endothelial dysfunction under high glucose stress in vitro and in vivo. Inhibition of NETs (PAD4 inhibitor) ameliorate endothelial dysfunction and renal injury in DKD. Taken together, NET-induced sterile inflammation promotes diabetes-associated endothelial dysfunction, identifying a new pathomechanism contributing to DKD. Inhibition of NETs may be a promising therapeutic strategy in DKD.

Hypercoagulability Impairs Plaque Stability in Diabetes-Induced Atherosclerosis.

Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accelerated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications are the major cause of mortality in diabetic patients. Efficient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Atherosclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we demonstrate that atherosclerotic plaque stability is reduced in hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice), which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased fibrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently, more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice. Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis.

On-a-Chip-Based Sensitive Detection of Drug-Induced Apoptosis in Polarized Gastric Epithelial Cells.

Microfluidic devices for culturing cells have been successfully utilized for biomedical applications, including drug screening. Several cell lines could be cultivated in microengineered environments with promising results, but gastric cell lines have not yet been widely used or studied. Therefore, this study focuses on establishing a polarized gastric epithelial monolayer on-a-chip and describes a general-purpose methodology applicable for bonding any porous material to PDMS through an adhesive sublayer. The fully transparent microfluidic chip consists of two microfluidic channels separated by a collagen-coated porous membrane and lined by human polarized gastric epithelial (NCI-N87) cells. We present considerations on how to ensure continuous and stable flow through the channels. The continuous flow rate was achieved using a pressure-driven pump. Media flow at a constant rate (0.5 µL/min) rapidly led the gastric epithelial cells to develop into a polarized monolayer. The barrier integrity was assessed by the FITC-dextran test. The generation of a monolayer was faster than in the static Boyden chamber. Moreover, fluorescence microscopy was used to monitor the apoptotic cell death of gastric epithelial monolayers on-a-chip in response to camptothecin, a therapeutic gastric cancer drug.

Procoagulant Extracellular Vesicles Alter Trophoblast Differentiation in Mice by a Thrombo-Inflammatory Mechanism.

Procoagulant extracellular vesicles (EV) and platelet activation have been associated with gestational vascular complications. EV-induced platelet-mediated placental inflammasome activation has been shown to cause preeclampsia-like symptoms in mice. However, the effect of EV-mediated placental thrombo-inflammation on trophoblast differentiation remains unknown. Here, we identify that the EV-induced thrombo-inflammatory pathway modulates trophoblast morphology and differentiation. EVs and platelets reduce syncytiotrophoblast differentiation while increasing giant trophoblast and spongiotrophoblast including the glycogen-rich cells. These effects are platelet-dependent and mediated by the NLRP3 inflammasome. In humans, inflammasome activation was negatively correlated with trophoblast differentiation marker GCM1 and positively correlated with blood pressure. These data identify a crucial role of EV-induced placental thrombo-inflammation on altering trophoblast differentiation and suggest platelet activation or inflammasome activation as a therapeutic target in order to achieve successful placentation.

Placental thromboinflammation impairs embryonic survival by reducing placental thrombomodulin expression.

Excess platelet activation by extracellular vesicles (EVs) results in trophoblast inflammasome activation, interleukin 1ß (IL-1ß) activation, preeclampsia (PE), and partial embryonic lethality. Embryonic thrombomodulin (TM) deficiency, which causes embryonic lethality hallmarked by impaired trophoblast proliferation, has been linked with maternal platelet activation. We hypothesized that placental TM loss, platelet activation, and embryonic lethality are mechanistically linked to trophoblast inflammasome activation. Here, we uncover unidirectional interaction of placental inflammasome activation and reduced placental TM expression: although inflammasome inhibition did not rescue TM-null embryos from lethality, the inflammasome-dependent cytokine IL-1ß reduced trophoblast TM expression and impaired pregnancy outcome. EVs, known to induce placental inflammasome activation, reduced trophoblast TM expression and proliferation. Trophoblast TM expression correlated negatively with IL-1ß expression and positively with platelet numbers and trophoblast proliferation in human PE placentae, implying translational relevance. Soluble TM treatment or placental TM restoration ameliorated the EV-induced PE-like phenotype in mice, preventing placental thromboinflammation and embryonic death. The lethality of TM-null embryos is not a consequence of placental NLRP3 inflammasome activation. Conversely, EV-induced placental inflammasome activation reduces placental TM expression, promoting placental and embryonic demise. These data identify a new function of placental TM in PE and suggest that soluble TM limits thromboinflammatory pregnancy complications.

Functional annotation of differentially expressed fetal cardiac microRNA targets: implication for microRNA-based cardiovascular therapeutics.

Gene expression pattern of a failing heart depicts remarkable similarity with developing fetal heart. Elucidating genetic as well as epigenetic mechanisms regulating the gene expression during cardiac development will improve our understanding of cardiovascular diseases. In the present study, we aimed to validate and characterize differentially expressed known microRNAs (miRNA) obtained from next generation sequencing data of two fetal cardiac developmental stages (days 4th and 14th) from chicken (G. gallus domesticus) using bioinformatic approaches. Potential mRNA targets of individual miRNA were identified and classified according to their biological, cellular, and molecular functions. Functional annotation of putative target genes was performed to predict their association with cardiovascular diseases. We identified a total of 19 differentially expressed miRNAs between 4th and 14th day sample from the data sets obtained by next generation sequencing. A total of nearly 1522 potential targets ranging from 15 to 270 for each miRNA were predicted out of which 1221 were unique, while 301 were overlapping. Gene ontology and KEGG analysis revealed that majority of these target genes regulate critical cellular and molecular processes including transcriptional regulation, protein transport, signal transduction, matrix remodeling, Ras signaling, MAPK signaling, and TGF-beta signaling pathways indicating the complex nature of microRNA-mediated gene regulation during cardiogenesis. We found a significant association between potential target genes and cardiovascular diseases validating a link between fetal cardiac miRNAs and regulation of cardiovascular disease-related genes. These important findings may lay a foundation for further understanding the regulatory mechanisms operative in gene re-programming in the failing heart.

Long- and short-term protective responses of rice seedling to combat Cr(VI) toxicity.

In India, rice is the principal crop and is the staple diet of majority of the population. Widespread use of hexavalent chromium [Cr(VI)] in leather processing, wood preservatives, stainless-steel manufacture, and electroplating industries has resulted in contamination of paddy fields and poses a great challenge to the society be it crops, animals, or human beings. Cr(VI) toxicity results in growth inhibition and leading to changes in components of antioxidant systems as well as secondary metabolites. We evaluated the comparative short and long term effects of Cr(VI) stress on rice plants to explore the plant defense responses against Cr stress. Different assays including the phenolic and flavonoid content evaluation, malondialdehyde (MDA), proline, antioxidant enzyme analysis, and DPPH assay were performed to understand the plant response against the Cr(VI) stress. Total phenols and flavonoids were significantly higher in Cr stressed plants as compared to control groups. Under Cr(VI) exposure, significant higher accumulation of proline was observed. Similarly, high levels of MDA content were also observed after 7 days of Cr stress. In addition, the antioxidant activities such as GST, APX, and SOD including DPPH radical scavenging were also markedly increased during Cr(VI) stress. Further identification and quantification of phenols were done spectrophotometrically to view the whole spectrum of phenolics. HPLC analysis showed gallic acid as the main contributor to abiotic defense response. Our study showed that Cr stress imposes serious toxic effects and plant phenolics have a protective role against metal stress.

Response to inhaled corticosteroids on serum CD28, quality of life, and peak expiratory flow rate in bronchial asthma.

BACKGROUND: CD28 is a 44 kDa glycoprotein that is important in initiating T-cell responses and that results in increased T-cell proliferation and interleukin-2 production. This study estimated the serum CD28 levels in patients with asthma and evaluated the effect of inhaled corticosteroids (ICS) on the levels of CD28, the peak expiratory flow rate (PEFR), and quality of life (QOL). METHODS: This prospective, open-label, observational study enrolled 40 adult patients with asthma of mild-to-moderate severity who were started on ICS and 40 healthy controls. Patients with bronchial asthma were evaluated for their serum CD28 level and QOL by using Mini Asthma Quality of Life Questionnaire scores, severity of symptoms, and PEFR at baseline and after 4 weeks. RESULTS: The mean (standard deviation [SD]) serum CD28 concentration in patients with asthma was 107 ± 4.98 ng/mL, which was significantly elevated (p < 0.01) compared with the healthy individuals (37.67 ± 18.28 ng/mL). ICS treatment for 4 weeks reduced the mean (SD) serum CD28 levels to 40.9 ± 2.82 ng/mL. PEFR increased significantly, from 190.75 ± 10.55 to 263 ± 10.69 L/min (p < 0.01) after 4 weeks. Similarly, the mean (SD) Mini Asthma Quality of Life Questionnaire scores significantly increased, from 36.90 ± 10.31 on day 0 to 70.63 ± 11.56 on day 28 after ICS therapy (p < 0.01). A negative correlation between the elevations of serum CD28 levels was seen with QOL. CONCLUSION: The serum CD28 concentration, a marker of inflammation, is increased in bronchial asthma and reduced by ICS therapy. ICS also improved QOL scores and objective clinical outcomes in patients with asthma.

An Evaluation of Indian Consumers' Reporting of Suspected Adverse Drug Reactions with a Designated Reporting Form.

BACKGROUND: The Pharmacovigilance Program of India recently initiated a process for direct patient reporting of Adverse Drug Reactions (ADRs) with a designated form. PATIENTS AND METHODS: A survey of 200 patients reporting ADRs filling the form. Forms were analysed for patient data, the suspected medication(s), ADRs and possible causality. RESULTS: 54.3% of respondents provided their contact information; the implicated medicine was mentioned in 60% and the description of ADRs in 80% although 46.2% were not interpretable. The severity of ADRs was mentioned in 73.5%. No responder filled the expiry date component of the implicated modification and a causality assessment from most forms was unclassifiable (57%) or unclassified (26%). Details of concomitant drugs were missing. CONCLUSION: Missing information was a deterrent in analysing the consumer ADR reports for signal detection. It is recommended that the following fields are highlighted in the form: consumer's initials, address, date suspected reaction started, description of event, name, dose, and the reason for the use the medication as well as its expiry date. These should be mandatory in the existing form and new fields added for weight and height, batch number for vaccines and biological products, de challenge and rechallenge results to the suspected medicine and concomitantly used medicines. To improve the quality of information in the consumer reporting form an awareness campaign is also suggested.