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Significance: In response to infection or cellular stress, inflammasomes are assembled and activated to mediate host defense and to initiate or promote the development of different diseases, in particular, autoinflammatory diseases and chronic degenerative diseases. Understanding of inflammasomes and related physiological and pathological relevance to the cardiovascular system will open a new chapter on the pathogenesis of inflammation and related diseases and will help develop novel therapeutic strategies for prevention or treatment of cardiovascular diseases. Recent Advances: The inflammasome, in particular the nucleotide oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome, has been recently recognized as a fundamental mechanism to mediate or promote the pathogenesis of degenerative diseases. Some important mechanisms responsible for NLRP3 inflammasome activation have been proposed and many molecular targets associated with this inflammasome activation are shown to be the possible candidates of therapeutic targets for treatment of cardiovascular diseases. Critical Issues: The concepts that NLRP3 inflammasome activation occurs just in immune cells or phagocytes and that its role is only for the inflammatory progression of cardiovascular diseases are oversimplified. A large body of other cell types are capable of NLRP3 inflammasome activation, and many uncanonical effects of this inflammasome may also be implicated in the development of cardiovascular diseases, which are discussed in a great detail by this Forum. Future Directions: More mechanistic and translational studies will rapidly widen the horizon of knowledge on NLRP3 inflammasome activation and regulation, which may help develop novel effective therapeutic strategies to target this inflammasome for treatment or prevention of cardiovascular diseases. Antioxid. Redox Signal. 22, 1079-1083. [ABSTRACT FROM AUTHOR]

Membrane rafts (MRs) are specialized microdomains in the cell membrane with an altered lipid composition. Upon various stimulations, MRs can be clustered to aggregate or recruit NADPH oxidase sub-units and related proteins to form MR redox signalosomes in the membrane of cells like vascular endothelial cells (ECs). Multiple protein complexes, like MR redox signalosomes, are now considered to play a crucial role in the regulation of cell function and in the development of different cell dysfunctions. To form such redox signalosomes, ceramide will be generated from the hydrolysis of sphingomyelin by lysosomal acid sphingomyelinase that has been translocated via lysosome fusion to the MR area. In this brief review, current information is provided to help understand the occurrence and function of MR redox signalosomes. This may increase enthusiasm of the scientific community for further studies on the molecular mechanisms and the functional significance of forming such MR redox signalosomes. [ABSTRACT FROM AUTHOR]

Despite substantial evidence indicating the association of hyperhomocysteinemia (hHcys) and end-stage renal disease (ESRD), the pathogenic role of increased plasma homocysteine (Hcys) levels in the progression of ESRD remains unclear. This review will briefly summarize recent findings regarding the role of hHcys in the development of glomerulosclerosis, the association of hHcys with reduced renal transsulfuration and Hcys-induced changes of redox signaling in the development of glomerulosclerosis in rat kidneys. Based on these results, it is concluded that hHcys is implicated in glomerular sclerosis in hypertension, elevated plasma Hcys in Dahl salt-sensitive (SS) hypertensive rats is due to downregulation of cystathionine β-synthase (CBS) expression and consequent abnormality of transsulfuration in the kidney compared with normotensive rats. Hcys-induced superoxide (O2· –) production by activation of NADPH oxidase as a triggering mechanism contributes to the effects of Hcys on the homeostasis of extracellular matrix and consequent sclerosis in the glomeruli, and NADPH oxidase activation by Hcys is associated with enhanced Rac GTPase activity. Clin Chem Lab Med 2007;45:1688–93. [ABSTRACT FROM AUTHOR]

Activation of cells by receptor- and nonreceptor-mediated stimuli not only requires a change in the activity of signaling proteins but also requires a reorganization of the topology of the signalosom in the cell. The cell membrane contains distinct domains, rafts that serve the spatial organization of signaling molecules in the cell. Many receptors or stress stimuli transform rafts by the generation of ceramide. These stimuli activate the acid sphingomyelinase and induce a translocation of this enzyme onto the extracellular leaflet of the cell membrane. Surface acid sphingomyelinase generates ceramide that serves to fuse small rafts and to form large ceramide-enriched membrane platforms. These platforms cluster receptor molecules, recruit intracellular signaling molecules to aggregated receptors, and seem to exclude inhibitory signaling factors. Thus ceramide-enriched membrane platforms do not seem to be part of a specific signaling pathway but may facilitate and amplify the specific signaling elicited by the cognate stimulus. This general function may enable these membrane domains to be critically involved in the induction of apoptosis by death receptors and stress stimuli, bacterial and viral infections of mammalian cells, and the regulation of cardiovascular functions. [ABSTRACT FROM AUTHOR]

Presents information on a study which determined the role of cyclic adenosine diphosphate and adenosine-ribose in the regulation of the calcium&sup2+;-activated potassium[sup+] channel activity in coronary arterial smooth muscle cells. Materials and methods; Results and discussion.

Presents information on a study which tested the hypothesis that cyclic adenosine diphosphate-ribose increases Ca[sup 2+] release through activation of ryanodine receptors on the sarcoplasmic reticulum in coronary arterial smooth muscle cells. Materials and methods; Results; Discussion.

Presents a study which characterized the enzymatic pathway responsible for the production and metabolism of cyclic ADP-ribose in small bovine coronary arteries. Materials and methods; Results and discussion.

Focuses on the regulation of potassium calcium channel activity which is caused by cyclic adenosine diphosphate (ADP)-ribose and ADP-ribose in the smooth muscle of the coronary artery. Functions of potassium channels; Factors which influence potassium channel activities; Characteristics of arterial smooth muscles.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1−/− mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect. NEW & NOTEWORTHY: In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects. [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. Methods and Results: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1β production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. Conclusion: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response. [ABSTRACT FROM AUTHOR]

The activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been reported to importantly contribute to glomerular inflammation and injury under different pathological conditions such as obesity. However, the mechanism mediating NLRP3 inflammasome activation in podocytes and subsequent glomerular injury remains poorly understood. Given that the ceramide signaling pathway has been reported to be implicated in obesity-related glomerulopathy (ORG), the present study was designed to test whether the ceramide-producing enzyme, acid sphingomyelinase (ASM), determines NLRP3 inflammasome activation and inflammatory exosome release in podocytes leading to glomerular inflammation and injury during ORG. In Smpd1trg/Podocre mice, podocyte-specific overexpression of Smpd1 gene which encodes ASM significantly exaggerated high-fat diet (HFD)-induced NLRP3 inflammasome activation in podocytes and immune cell infiltration in glomeruli compared to WT/WT mice. Smpd1 gene deletion, however, blocked these pathological changes induced by HFD in Smpd1−/− mice. Accompanied with NLRP3 inflammasome activation and glomerular inflammation, urinary excretion of exosomes containing podocyte marker and NLRP3 inflammasome products (IL-1β and IL-18) in Smpd1trg/Podocre mice on the HFD was much higher than that in WT/WT mice. In contrast, Smpd1−/− mice on the HDF had significantly lower urinary exosome excretion than WT/WT mice. Correspondingly, HFD-induced podocyte injury, glomerular sclerosis, and proteinuria were more severe in Smpd1trg/Podocre mice, but milder in Smpd1−/− mice compared to WT/WT mice. Using podocytes isolated from these mice, we demonstrated that visfatin, a prototype pro-inflammatory adipokine, induced NLRP3 inflammasome activation and enrichment of multivesicular bodies (MVBs) containing IL-1β in podocytes, which was much stronger in podocytes from Smpd1trg/Podocre mice, but weaker in those from Smpd1−/− mice than WT/WT podocytes. By quantitative analysis of exosomes, it was found that upon visfatin stimulation, podocytes from Smpd1trg/Podocre mice released much more exosomes containing NLRP3 inflammasome products, but podocytes from Smpd1−/− mice released much less exosomes compared to WT/WT podocytes. Super-resolution microscopy demonstrated that visfatin inhibited lysosome-MVB interaction in podocytes, indicating impaired MVB degradation by lysosome. The inhibition of lysosome-MVB interaction by visfatin was amplified by Smpd1 gene overexpression but attenuated by Smpd1 gene deletion. Taken together, our results suggest that ASM in podocytes is a crucial regulator of NLRP3 inflammasome activation and inflammatory exosome release that instigate glomerular inflammation and injury during obesity. [ABSTRACT FROM AUTHOR]

Background/Aim: Plasma trimethylamine-N-oxide (TMAO), a product of intestinal microbial metabolism of dietary phosphatidylcholine has been recently associated with atherosclerosis and increased risk of cardiovascular diseases (CVD) in rodents and humans. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. The present study tested whether TMAO induces NLRP3 inflammasome formation and activation and thereby contributes to endothelial injury initiating atherogenesis. Methods: Inflammasome formation and activation was determined by confocal microscopy, caspase-1 activity was measured by colorimetric assay, IL-1ß production was measured using ELISA, cell permeability was determined by microplate reader and ZO-1 expression was determined by western blot analysis and confocal microscopy. In in vivo experiments, TMAO was infused by osmotic pump implantation. Results: TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase-1, caspase-1 activity, IL-1ß production, cell permeability in carotid artery endothelial cells (CAECs) compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD or Nlrp3 siRNA abolished the TMAO-induced inflammasome formation, activation and cell permeability in these cells. In addition, we explored the mechanisms by which TMAO activates NLRP3 inflammasomes. TMAO-induced the activation of NLRP3 inflammasomes was associated with both redox regulation and lysosomal dysfunction. In animal experiments, direct infusion of TMAO in mice with partially ligated carotid artery were found to have increased NLRP3 inflammasome formation and IL-1ß production in the intima of wild type mice. Conclusion: The formation and activation of NLRP3 inflammasomes by TMAO may be an important initiating mechanism to turn on the endothelial inflammatory response leading to endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Background: Proteinuria-induced epithelial-mesenchymal transition (EMT) plays an important role in progressive renal tubulointerstitial fibrosis in chronic renal disease. Stem cell therapy has been used for different diseases. Stem cell conditioned culture media (SCM) exhibits similar beneficial effects as stem cell therapy. The present study tested the hypothesis that SCM inhibits albumin-induced EMT in cultured renal tubular cells. Methods: Rat renal tubular cells were treated with/without albumin (20 µmg/ml) plus SCM or control cell media (CCM). EMT markers and inflammatory factors were measured by Western blot and fluorescent images. Results: Albumin induced EMT as shown by significant decreases in levels of epithelial marker E-cadherin, increases in mesenchymal markers fibroblast-specific protein 1 and α-smooth muscle actin, and elevations in collagen I. SCM inhibited all these changes. Meanwhile, albumin induced NF-κB translocation from cytosol into nucleus and that SCM blocked the nuclear translocation of NF-κB. Albumin also increased the levels of pro-inflammatory factor monocyte chemoattractant protein-1 (MCP)-1 by nearly 30 fold compared with control. SCM almost abolished albumin-induced increase of MCP-1. Conclusion: These results suggest that SCM attenuated albumin-induced EMT in renal tubular cells via inhibiting activation of inflammatory factors, which may serve as a new therapeutic approach for chronic kidney diseases. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

R5175 --> 839.9 --> Circulatory exosomes have been reported to induce vascular endothelial injury and neointima proliferation in mice with liver‐specific deletion of acid ceramidase gene (Asah1fl/fl/Albcre, Asah1 is mouse code of this gene). However, the precise mechanism mediating this exosome‐induced endothelial injury remains unknown. The present study tested a hypothesis that plasma exosomes from Asah1fl/fl/Albcre mice on the high fat diet (HFD) activate endothelial NLRP3 inflammasome activation leading to endothelial dysfunction. To test this hypothesis, wild type (WT/WT) mice and Asah1fl/fl/Albcre mice were fed with normal diet (ND) or HFD for 2 months. Using immunofluorescence confocal microscopy, we confirmed that ceramide levels significantly increased in the liver of Asah1fl/fl/Albcre mice compared to its WT/WT littermates and that HFD further enhanced lysosome ceramide levels in the liver as shown by the colocalization of ceramide with lysosome marker, lamp‐1. Then, isolated exosomes from the plasma of these mice were used to treat the primary cultured WT/WT endothelial cells from the carotid artery. It was found that exosomes isolated from HFD‐treated mice (HFD‐Exo) even from WT/WT mice significantly stimulated NLRP3 inflammasome formation and activation as shown by the significant increases in the colocalization of NLRP3 with Caspase‐1 or ASC, caspase‐1 activity and interleukin 1 beta (IL‐1β) level in the endothelial cells compared to exosomes from ND treated mice (ND‐Exo). This inflammasome formation and activation were remarkably enhanced if endothelial cells were treated by HFD‐Exo isolated from Asah1fl/fl/Albcre mice. To confirm whether lysosomal ceramide contributes to HFD‐Exo‐induced endothelial NLRP3 inflammasome activation, we further treated endothelial cells with C(2)‐ceramide, which was shown similar effects comparable to that induced by HFD‐Exo. Using dibutyryl‐cAMP, a blocker of ceramide‐induced action significantly attenuated C(2)‐ceramide‐ or HDF‐Exo‐induced enhancement of endothelial NLRP3 inflammmasome activation. Together, these results suggest that liver‐specific deletion of lysosomal Asah1 gene promotes release of HFD‐Exos that may contain high ceramide, activating endothelial NLRP3 inflammasome and thereby resulting in endothelial dysfunction during HFD‐induced liver steatosis. [ABSTRACT FROM AUTHOR]

Abstract: Cathepsin L is a cysteine protease that can generate endogenous endostatin in vascular and epithelial basement membranes and importantly participates in a variety of pathophysiological processes. The present study was designed to determine whether this cathepsin L-derived endogenous endostatin alters endothelium-dependent vasodilator responses in coronary arteries via NAD(P)H oxidase activation. In isolated and perfused small bovine coronary arteries, administration of cathepsin L (200 ng/ml) markedly attenuated endothelium-dependent vasodilator responses to bradykinin or A23187 by 56.16±9.58% and 68.95±10.32%, respectively. This inhibitory effect of cathepsin L on endothelium-dependent vasodilator responses could be significantly reversed by pre-incubation of the arteries with O2 − scavenger, Tiron, or neutralizing anti-endostatin antibody. By fluorescent ELISA assay, cathepsin L dose-dependently increased endostatin production in coronary arteries. In situ high-speed dual wavelength switching fluorescent microscopic imaging showed that cathepsin L decreased bradykinin- and A23187-induced NO levels in the intact endothelium, but it had no effect on Ca2+ response to these vasodilators. This cathepsin L-induced reduction of NO was restored by the pretreatment of an anti-endostatin antibody. Electron spin resonance (ESR) analysis demonstrated that cathepsin L increased O2 − production which could be markedly attenuated by the NAD(P)H oxidase inhibitors, apocynin or anti-endostatin antibody. It is concluded that endostatin could be endogenously produced in coronary arteries when cathepsin L is increased and that this cathepsin L-derived endostatin, if excessive, may result in endothelial dysfunction through enhanced production of O2 − due to NAD(P)H oxidase activation. [Copyright &y& Elsevier]

Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N‐terminal nucleophile hydrolase family. This enzyme is located in the lysosome, which mediates conversion of ceramide (CER) into sphingosine and free fatty acids at acidic pH. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation. The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LEs)/lysosomes may control cargo selection, membrane biogenesis, and exosome secretion, which may be fine controlled by lysosomal sphingolipids such as CER. This lysosomal‐CER‐mTOR signaling may be a crucial molecular mechanism responsible for development of arterial medial calcification (AMC). Torin‐1 (5 mg/kg/day), an mTOR inhibitor, significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin (OSP) and runt‐related transcription factor 2 (RUNX2) and upregulation of smooth muscle 22α (SM22‐α) in mice receiving high dose of Vitamin D (500 000 IU/kg/day). Asah1fl/fl/SMCre mice had markedly increased co‐localization of mTORC1 with lysosome‐associated membrane protein‐1 (Lamp‐1) (lysosome marker) and decreased co‐localization of vacuolar protein sorting‐associated protein 16 (VPS16) (a multivesicular bodies [MVBs] marker) with Lamp‐1, suggesting mTOR activation caused reduced MVBs interaction with lysosomes. Torin‐1 significantly reduced the co‐localization of mTOR vs Lamp‐1, increased lysosome–MVB interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using coronary artery smooth muscle cells (CASMCs), Pi‐stimulation significantly increased p‐mTOR expression in Asah1fl/fl/SMCre CASMCs as compared to WT/WT cells associated with increased calcium deposition and mineralization. Torin‐1 blocked Pi‐induced calcium deposition and mineralization. siRNA mTOR and Torin‐1 significantly reduce co‐localization of mTORC1 with Lamp‐1, increased VPS16 vs Lamp‐1 co‐localization in Pi‐stimulated CASMCs, associated with decreased exosome release. Functionally, Torin‐1 significantly reduces arterial stiffening as shown by restoration from increased pulse wave velocity and decreased elastin breaks. These results suggest that lysosomal CER‐mTOR signaling may play a critical role for the control of lysosome–MVB interaction, exosome secretion and arterial stiffening during AMC. [ABSTRACT FROM AUTHOR]

R4017 --> The nucleotide‐binding oligomerization domain‐like receptor containing pyrin domain 3 (NLRP3) inflammasome in podocytes has been implicated in the initiation of glomerular inflammation during hyperhomocysteinemia (hHcy). However, the mechanism by which NLRP3 inflammasome products are released from podocytes remains unknown. The present study tested whether exosome secretion from podocytes is enhanced by NADPH oxidase‐produced reactive oxygen species (ROS), which may serve as a pathogenic mechanism mediating the release of products by NLRP3 inflammasome activation in podocytes. We first demonstrated the remarkable elevation of endogenously produced ROS in podocytes treated with homocysteine (Hcy) compared with control podocytes, which was abolished by pre‐treatment with catalase, a hydrogen peroxide (H2O2) scavenger or gp91 ds‐tat peptide, a NADPH oxidase inhibitor. Hcy also induced activation of NLRP3 inflammasome and formation of MVBs containing the products of NLRP3 inflammasome activation such as IL‐1β in podocytes, which were prevented by catalase and gp91 ds‐tat peptide. By super‐resolution microscopy and nanoparticle tracking analysis (NTA), we found that ML‐SA1, a TRPML1 channel agonist that increases lysosome trafficking by calcium release, significantly enhanced lysosome‐MVB interaction and reduced exosome release in podocytes, which attenuated Hcy effects. In the presence of catalase or gp91 ds‐tat peptide, moreover, Hcy‐induced reduction of lysosome‐MVB interaction and exosome secretion was restored. We also tested whether exogenous ROS can mimic the effects of Hcy on exosome release in podocytes. It was found that H2O2 activated NLRP3 inflammasome and induced formation of MVBs containing inflammatory cytokines in a dose‐dependent manner. Furthermore, we confirmed that H2O2 decreased lysosome‐MVB interaction and increased exosome release in podocytes. Based on these results, we conclude that endogenously produced ROS importantly contributes to inflammatory exosome secretion from podocytes during activation of NLRP3 inflammasome in response to high Hcy due to inhibition of lysosome‐MVB interaction. This redox regulation of Hcy‐induced inflammatory exosome release may be a critical molecular mechanism triggering glomerular inflammatory response during hHcy (supported by NIH grant DK120491). [ABSTRACT FROM AUTHOR]

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum -infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo , pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources. [ABSTRACT FROM AUTHOR]

Podocytes are visceral epithelial cells covering the outer surface of glomerular capillaries in the kidney. Blood is filtered through the slit diaphragm of podocytes to form urine. The functional and structural integrity of podocytes is essential for the normal function of the kidney. As a membrane-bound organelle, lysosomes are responsible for the degradation of molecules via hydrolytic enzymes. In addition to its degradative properties, recent studies have revealed that lysosomes may serve as a platform mediating cellular signaling in different types of cells. In the last decade, increasing evidence has revealed that the normal function of the lysosome is important for the maintenance of podocyte homeostasis. Podocytes have no ability to proliferate under most pathological conditions; therefore, lysosome-dependent autophagic flux is critical for podocyte survival. In addition, new insights into the pathogenic role of lysosome and associated signaling in podocyte injury and chronic kidney disease have recently emerged. Targeting lysosomal functions or signaling pathways are considered potential therapeutic strategies for some chronic glomerular diseases. This review briefly summarizes current evidence demonstrating the regulation of lysosomal function and signaling mechanisms as well as the canonical and noncanonical roles of podocyte lysosome dysfunction in the development of chronic glomerular diseases and associated therapeutic strategies. [ABSTRACT FROM AUTHOR]

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC. [ABSTRACT FROM AUTHOR]

Elevated homocysteine (Hcy) levels have been shown to activate nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome leading to podocyte dysfunction and glomerular injury. However, it remains unclear how this inflammasome activation in podocytes is a therapeutic target for reversal of glomerular injury and ultimate sclerosis. The present study tested whether inhibition of Rac1 GTPase activity suppresses NLRP3 inflammation activation and thereby blocks podocyte injury induced by elevated Hcy. In cultured podocytes, we found that L-Hcy (the active Hcy form) stimulated the NLRP3 inflammasome formation, as shown by increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1, which was accompanied by increased interleukin-1β production and caspase-1 activity, indicating NLRP3 inflammasome activation. Rac1 activator, uridine triphosphate (UTP), mimicked L-Hcy-induced NLRP3 inflammasome activation, while Rac1 inhibitor NSC23766 blocked it. This Rac1 inhibition also prevented L-Hcy-induced podocyte dysfunction. All these effects were shown to be mediated via lipid raft redox signaling platforms with nicotinamide adenine dinucleotide phosphate oxidase subunits and consequent O2– production. In animal studies, hyperhomocysteinemia (hHcy) induced by folate-free diet was shown to induce NLRP3 inflammasome formation and activation in glomeruli, which was also mimicked by UTP and inhibited by NSC23766 to a comparable level seen in Nlrp3 gene knockout mice. These results together suggest that Rac1 inhibition protects the kidney from hHcy-induced podocyte injury and glomerular sclerosis due to its action to suppress NLRP3 inflammasome activation in podocytes. [ABSTRACT FROM AUTHOR]

Abstract The NLRP3 inflammasome is activated in the cytoplasm of cells and its products such as IL-1β are exported through a non-classical ER-Golgi pathway. Several mechanistically distinct models including exocytosis of secretory lysosomes, microvesicles (MVs) and extracellular vehicles (EVs) have been proposed for their release. In this study, we hypothesized that the NLRP3 inflammasome product, IL-1β in response to exogenously administrated and endogenously produced d -ribose stimulation is released via extracellular vesicles including EVs via a sphingolipid-mediated molecular mechanisms controlling lysosome and multivesicular body (MVB) interaction. First, we demonstrated that both endogenous and exogenous d -ribose induced NLRP3 inflammasome activation to produce IL-1β, which was released via EVs in podocytes. Then, we found that colocalization of marker MVB marker VPS16 with IL-1β within podocytes increased upon d -ribose stimulation, which was accompanied by decreased colocalization of lysosome marker Lamp-1 and VPS16, suggesting decrease in MVB inclusion of IL-1β due to reduced lysosome and MVB interaction. All these changes were mimicked and accelerated by lysosome v-ATPase inhibitor, bafilomycin. Moreover, ceramide in podocytes was found elevated upon d -ribose stimulation, and prior treatments of podocyte with acid sphingomyelinase (Asm) inhibitor, amitriptyline, acid ceramidase (AC) inducer, genistein, or AC CRISPR/cas9 activation plasmids were found to decrease d -ribose-induced ceramide accumulation, EVs release and IL-1β secretion due to reduced interactions of lysosome with MVBs. These results suggest that inflammasome-derived products such as IL-1β during d -ribose stimulation are released via EVs, in which lysosomal sphingolipid-mediated regulation of lysosome function plays an important role. Highlights • Exogenous and endogenous d -ribose induced NLRP3 inflammasome activation in podocytes. • IL-1β secretion through extracellular vehicles releases in d -ribose-induced podocytes. • Lysosomal-ceramide sphingolipid pathway regulates inflammasome-dependent IL-1β release through extracellular vehicles. [ABSTRACT FROM AUTHOR]

Amitriptyline is a tricyclic antidepressant and an inhibitor of lysosomal acid sphingomyelinase (ASM). Amitriptyline is well known for its cardiovascular side effects and toxicity in psychiatric patients. However, the mechanisms underlying the cardiovascular side effects of amitriptyline remain largely undefined. This study aimed to determine the effects of amitriptyline on angiogenic capability of vascular endothelial cells in physiological settings and identify its mechanism of action. The ex vivo aortic ring angiogenesis and in vitro‐cultured endothelial cell tube formation assay were used to assess the effects of amitriptyline on endothelial angiogenic capability. It was demonstrated that amitriptyline impaired the angiogenesis of aortic rings, which was similar to that found in aortic rings with haploinsufficiency of the ASM gene. In cultured mouse microvascular endothelial cells (MVECs), amitriptyline impaired the proliferation and tube formation under basal condition, which were accompanied by attenuated angiogenic signalling pathways such as endothelial nitric oxide synthase, Akt and Erk1/2 pathways. Mechanistically, amitriptyline inhibited autophagic flux without affecting autophagosome biogenesis at basal condition. ASM gene silencing or autophagy inhibition mimics the inhibitory effects of amitriptyline on endothelial cell proliferation and tube formation. Collectively, our data suggest that amitriptyline inhibits endothelial cell proliferation and angiogenesis via blockade of ASM‐autophagic flux axis. It is implicated that the cardiovascular side effects of amitriptyline may be associated with its inhibitory action on physiological angiogenesis. [ABSTRACT FROM AUTHOR]

Abstract The pannexin-1 (Panx1) channel has been reported to mediate the release of ATP that is involved in local tissue inflammation, obesity, and many chronic degenerative diseases. It remains unknown whether Panx1 is present in podocytes and whether this channel in podocytes mediates ATP release leading to glomerular inflammation or fibrosis. To answer these questions, we first characterized the expression of Panx channels in podocytes. Among the three known pannexins, Panx1 was the most enriched in podocytes, either cultured or native in mouse glomeruli. Using a Port-a-Patch planar patch-clamp system, we recorded a large voltage-gated outward current through podocyte membrane under the Cs+in/Na+out gradient. Substitution of gluconate or aspartate for chloride in the bath solution blocked voltage-gated outward currents and shifted the reversal potential of Panx1 currents to the right, indicating the anion permeability of this channel. Pharmacologically, the recorded voltage-gated outward currents were substantially attenuated by specific Panx1 channel inhibitors. Given the anti-inflammatory and intracellular ATP restorative effects of adiponectin, we tested whether this adipokine inhibits Panx1 channel activity to block ATP release. Adiponectin blocked Panx1 channel activity in podocytes. Mechanistically, inhibition of acid ceramidase (AC) remarkably enhanced Panx1 channel activity under control conditions and prevented the inhibition of Panx1 channel by adiponectin. Correspondingly, intracellular addition of AC products, sphingosine or sphingosine-1-phosphate (S1P), blocked Panx1 channel activity, while elevation of intracellular ceramide had no effect on Panx1 channel activity. These results suggest that adiponectin inhibits Panx1 channel activity in podocytes through activation of AC and associated elevation of intracellular S1P. Highlights • The Panx1 channel is highly enriched in murine podocytes and it conducts anion currents. • Adiponectin inhibits Panx1 channel activity. • Inhibition of the Panx1 channel by adiponectin is dependent on activation of AC. • Enhanced production of S1P due to activation of AC contributes to inhibition of Panx1 channel by adiponectin. [ABSTRACT FROM AUTHOR]

Background: Our previous study has detected a stem cell deficiency in the renal medulla in Dahl salt-sensitive (S) rats. This study determined whether infusion of valproic acid (VA), an agent known to stimulate the stem cell function, attenuated salt-sensitive hypertension in Dahl S rats. Methods: Uninephrectomized Dahl S rats were infused with vehicle or VA (50mg/kg/d) into the renal medulla and fed with a low (LS) or high salt diet (HS). Stem cell marker and number were analyzed by immunohistochemistry, Real-time RTPCR and Western blot. Sodium excretion and blood pressure were measured. Results: VA significantly increased the mRNA and protein levels of FGF2, a stem cell niche factor, and CD133, a stem cell marker. The number of CD133+ cells was significantly increased in the renal medulla in VA-treated rats. Meanwhile, high salt-induced increases in the mRNA level of proinflammatory factors interleukin-1β and interleukin-6 were blocked in VA-treated rats. Functionally, sodium excretion in response to the blood pressure increase and acute sodium loading was significantly enhanced, sodium retention attenuated, high salt-induced increase of blood pressure reduced in VA-treated rats. Conclusion: Activation of stem cell function by VA inhibits the activation of proinflammatory factors and attenuates salt-sensitive hypertension in Dahl S rats. [ABSTRACT FROM AUTHOR]

Background: Autophagy disorder contributes to dedifferentiation of arterial smooth muscle cells, but the mechanisms are poorly understood. Here, we sought to investigate the role of scaffolding adaptor p62/SQSTM1 (p62) in phenotype switching of mouse coronary arterial myocytes (CAMs) induced by CD38 gene deficiency or lysosomal dysfunction which blocks autophagic flux in the cells. Methods: Protein expression was measured by western blot analysis and immunofluorescent staining. Cell cycle and proliferation rate were analyzed by flow cytometry and MTS assay respectively. mRNA abundance was tested by qRT-PCR. Results: CD38 gene deficiency or bafilomycin A1 (baf), a selective lysosomal inhibitor treatment increased proliferation rate and vimentin expression in CAMs which was prevented by p62 gene silencing. Cell percentage in G2/M and G0/G1 phase was decreased and increased by CD38 deficiency or baf treatment, respectively which was accompanied by accrual of cyclin-dependent kinase 1 (CDK1) protein. Although free ubiquitin content was increased, the colocalization of it to CDK1 was markedly decreased in CD38-/- or baf treated CAMs. Furthermore, the changes in both cell cycle and CDK1 ubiquitinylation could be restored by p62 gene silencing. Conclusion: The results suggest in CD38-/- or baf treated CAMs, p62 accumulation promotes phenotype transition and proliferation by accelerating cell cycle progress through G2/M which might relate to the compromised ubiquitinylation and degradation of CDK1. [ABSTRACT FROM AUTHOR]

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) have been demonstrated to mediate the activation of NOD-like receptor protein 3 (NLRP3) inflammasomes in podocytes in response to elevated levels of homocysteine (Hcys). However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The present study tested whether the guanine nucleotide exchange factor Vav2 mediates Rac1-mediated NOX activation in response to elevated Hcys leading to NLRP3 inflammasome activation in podocytes and consequent glomerular injury. In a mouse model of hyperhomocysteinemia (hHcys), we found that mice with hHcys (on the FF diet) or oncoVav2 (a constitutively active form of Vav2) transfection in the kidney exhibited increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated IL-1β levels in glomeruli, indicating the formation and activation of the NLRP3 inflammasome. This glomerular NLRP3 inflammasome activation was accompanied by podocyte dysfunction and glomerular injury, even sclerosis. Local transfection of Vav2 shRNA plasmids significantly attenuated hHcys-induced NLRP3 inflammasome activation, podocyte injury, and glomerular sclerosis. In cultured podocytes, Hcys treatment and oncoVav2 transfection were also found to increase NLRP3 inflammasome formation and activation, which were all inhibited by Vav2 shRNA. Furthermore, Vav2 shRNA prevented Hcys-induced podocyte damage as shown by restoring Hcys-impaired VEGF secretion and podocin production. This inhibitory action of Vav2 shRNA on Hcys-induced podocyte injury was associated with reduction of Rac1 activity and ROS production. These results suggest that elevated Hcys levels activate Vav2 and thereby increase NOX activity leading to ROS production, which triggers NLRP3 inflammasome activation, podocyte dysfunction and glomerular injury. [ABSTRACT FROM AUTHOR]

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1fl/fl/Podocre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1fl/fl/Podocre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca2+ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome. • Asah1 gene deletion impairs autophagic flux and thereby induces autophagosome accumulation in podocytes. • Asah1 gene knockout inhibits lysosome trafficking through blockade of TRPML1 channel. • Asah1 gene deletion induces podocyte dedifferentiation. • Enhancement of TRPML1 channel activity attenuates autophagosome accumulation and dedifferentiation in podocytes lacking Asah1 gene. [ABSTRACT FROM AUTHOR]

Recent studies indicate that inflammasomes serve as intracellular machinery to initiate classical cytokine-mediated inflammatory responses and play a crucial role in the pathogenesis of cardiovascular diseases. However, whether or not the activation of endothelial inflammasomes directly causes cell dysfunction or tissue injury without recruitment of inflammatory cells is unknown. We explored the role of endothelial cell inflammasome activation in mediating tight junction disruption, a hallmark event of endothelial barrier dysfunction leading to endothelial hyperpermeability in diabetes. We used confocal microscopy to study the formation and activation of NOD-like receptor family pyrin domain containing-3 (Nlrp3) inflammasomes and expression of tight junction proteins in coronary arteries of streptozotocin-treated diabetic wild type and Nlrp3 gene-deleted mice. We found that Nlrp3 ablation prevented inflammasome activation and tight junction disassembly in the coronary arterial endothelium of diabetic mice. Similarly, Nlrp3 gene silencing prevented high glucose-induced down-regulation of tight junction proteins in cultured mouse vascular endothelial cells (MVECs). The high glucose-induced tight junction disruption and consequent endothelial permeability were attributed to increased release of the high mobility group box protein-1 (HMGB1), which is dependent on enhanced Nlrp3 inflammasome activity. Mechanistically, reducing reactive oxygen species (ROS) production abolished high glucose-induced inflammasome activation, tight junction disruption, and endothelial hyperpermeability in MVECs. Collectively, the ROS-dependent activation of endothelial Nlrp3 inflammasomes by hyperglycemia may be an important initiating mechanism to cause endothelial dysfunction. These effects could contribute to the early onset of endothelial injury in diabetes. Key message: [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. Methods and Results: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1β production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. Conclusion: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response. [ABSTRACT FROM AUTHOR]

[Display omitted] The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure. [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies have indicated that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle cells upon atherogenic stimulations. However, the molecular mechanisms mediating this vascular smooth muscle (SMC) phenotypic switching remain unknown. Methods & Results: In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs) from CD38-/- mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin, SM22α and α-SMA but increased the expression of SMC dedifferentiation marker, vimentin, which was accompanied by enhanced cell proliferation. This phenotypic change in CD38-/- CAMs was enhanced by 7-ketocholesterol (7-Ket), an atherogenic stimulus. We further found that the CD38 deficiency decreased the expression and activity of nuclear factor E2-related factor 2 (Nrf2), a basic leucine zipper (bZIP) transcription factor sensitive to redox regulation. Similar to CD38 deletion, Nrf2 gene silencing increased CAM dedifferentiation upon 7-Ket stimulation. In contrast, the overexpression of Nrf2 gene abolished 7-Ket-induced dedifferentiation in CD38-/- CAMs. Given the sensitivity of Nrf2 to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity associated with CD38 effect in CAM phenotype changes. It was demonstrated that in CD38-/- CAMs, 7-Ket failed to stimulate the production of O2-., while in CD38+/+ CAMs 7-Ket induced marked O2-. production and enhancement of Nrf2 activity, which was substantially attenuated by NOX4 gene silencing. Finally, we demonstrated that 7-Ket-induced and NOX4-dependent O2-. production was inhibited by 8-Br-cADPR, an antagonist of cADPR or NED-19, an antagonist of NAADP as product of CD38 ADP-ribosylcyclase, which significantly inhibited the level of cytosolic Ca2+ and the activation of Nrf2 under 7-Ket. Conclusion: Taken together, these results suggest that CD38 activity is required for 7-Ket-induced Ca2+ and consequently O2-. production in CAMs, which increases Nrf2 activity to maintain their differentiated status. When CD38 gene expression and function are deficient, the Nrf2 activity is suppressed, thereby leading to phenotypic switching of CAMs. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol buildup in atherosclerosis, contradictory results have been generated, indicating that free cholesterol produces both pro- and anti-atherosclerosis effects in macrophages. This inconsistency might stem from the examination of only select concentrations of free cholesterol. In the present study, we sought to investigate the implication of excess free cholesterol loading in the pathophysiology of atherosclerosis across a broad concentration range from (in µg/ml) 0 to 60. Methods: Macrophage viability was determined by measuring formazan formation and flow cytometry viable cell counting. The polarization of M1 and M2 macrophages was differentiated by FACS (Fluorescence-Activated Cell Sorting) assay. The secretion of IL-1β in macrophage culture medium was measured by ELISA kit. Macrophage apoptosis was detected by flow cytometry using a TUNEL kit. Results: Macrophage viability was increased at the treatment of lower concentrations of free cholesterol from (in µg/ml) 0 to 20, but gradually decreased at higher concentrations from 20 to 60. Lower free cholesterol loading induced anti-inflammatory M2 macrophage polarization. The activation of the PPARγ (Peroxisome Proliferator-Activated Receptor gamma) nuclear factor underscored the stimulation of this M2 phenotype. Nevertheless, higher levels of free cholesterol resulted in pro-inflammatory M1 activation. Moreover, with the application of higher free cholesterol concentrations, macrophage apoptosis and secretion of the inflammatory cytokine IL-1β increased significantly. Conclusion: These results for the first time demonstrate that free cholesterol could render concentration-dependent diversification effects on macrophage viability, polarization, apoptosis and inflammatory cytokine secretions, thereby reconciling the pros and cons of free cholesterol buildup in macrophages to the pathophysiology of atherosclerosis. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Pulmonary edema associated with increased vascular permeability is a severe complication of Staphylococcus aureus-induced sepsis and an important cause of human pathology and death. We investigated the role of the mammalian acid sphingomyelinase (Asm)/ceramide system in the development of lung edema caused by S. aureus. Our findings demonstrate that genetic deficiency or pharmacologic inhibition of Asm reduced lung edema in mice infected with S. aureus. The Asm/ceramide system triggered the formation of superoxide, resulting in degradation of tight junction proteins followed by lung edema. Treatment of infected mice with amitriptyline, a potent inhibitor of Asm, protected mice from lung edema caused by S. aureus, but did not reduce systemic bacterial numbers. In turn, treatment with antibiotics reduced bacterial numbers but did not protect mice from lung edema. In contrast, only the combination of antibiotics and amitriptyline inhibited both pulmonary edema and bacteremia protecting mice from lethal sepsis and lung dysfunction suggesting the combination of both drugs as novel treatment option for sepsis. Key messages: [ABSTRACT FROM AUTHOR]

Aims: This study hypothesized that activation of endothelial nucleotide oligomerization domain-like receptor protein with pyrin domain containing 3 (Nlrp3) inflammasomes directly produces endothelial dysfunction during hypercholesterolemia, which is distinct from its canonical roles in inflammation. Results: Acute hypercholesterolemia in mice was induced by intraperitoneal administration of poloxamer 407 (0.5 g/kg) for 24 h. Endothelial dysfunction was assessed by evaluating endothelium-dependent vasodilation in isolated, perfused, and pressurized coronary arteries in response to bradykinin (10−10-10−6 M) and acetylcholine (10−9-10−5 M). Impaired endothelium-dependent vasodilation was observed in Nlrp3+/+ mice with acute hypercholesterolemia, which was markedly ameliorated in Nlrp3−/− mice. Treatment of mice with inhibitors for caspase-1 or high mobility group box 1 (HMGB1) significantly restored endothelium-dependent vasodilation in Nlrp3+/+ mice with acute hypercholesterolemia. Confocal microscopic analysis demonstrated that hypercholesterolemia markedly increased caspase-1 activity and HMGB1 expression in coronary arterial endothelium of Nlrp3+/+ mice, which was absent in Nlrp3-deficient mice. Further, recombinant HMGB1 directly induced endothelial dysfunction in normal Nlrp3+/+ coronary arteries. In vitro, Nlrp3 inflammasome formation and its activity were instigated in cultured endothelial cells by cholesterol crystal, a danger factor associated with hypercholesterolemia. Moreover, cholesterol crystals directly induced endothelial dysfunction in coronary arteries from Nlrp3+/+ mice, which was attenuated in Nlrp3−/− arteries. Such cholesterol crystal-induced impairment was associated with enhanced superoxide production, downregulation of endothelial nitric oxide synthase activity, and pyroptosis. Innovation and Conclusion: Our data provide the first evidence that activation of endothelial Nlrp3 inflammasome directly impairs endothelial function beyond its canonical inflammatory actions. This novel non-canonical action of Nlrp3 inflammasomes may initiate or exacerbate vascular injury during hypercholesterolemia. Antioxid. Redox Signal. 22, 1084-1096. [ABSTRACT FROM AUTHOR]

Significance: Inflammasomes are multiprotein complexes localized within the cytoplasm of the cell that are responsible for the maturation of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18, and the activation of a highly inflammatory form of cell death, pyroptosis. In response to infection or cellular stress, inflammasomes are assembled, activated, and involved in host defense and pathophysiology of diseases. Clarification of the molecular mechanisms leading to the activation of this intracellular inflammatory machinery may provide new insights into the concept of inflammation as the root of and route to human diseases. Recent Advances: The activation of inflammasomes, specifically the most fully characterized inflammasome-the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome, is now emerging as a critical molecular mechanism for many degenerative diseases. Several models have been developed to describe how NLRP3 inflammasomes are activated, including K+ efflux, lysosome function, endoplasmic reticulum (ER) stress, intracellular calcium, ubiquitination, microRNAs, and, in particular, reactive oxygen species (ROS). Critical Issues: ROS may serve as a 'kindling' or triggering factor to activate NLRP3 inflammasomes as well as 'bonfire' or 'effector' molecules, resulting in pathological processes. Increasing evidence seeks to understand how this spatiotemporal action of ROS occurs during NLRP3 inflammasome activation, which will be a major focus of this review. Future Directions: It is imperative to know how this dual action of ROS works during NLRP3 inflammation activation on different stimuli and what relevance such spatiotemporal redox regulation of NLRP3 inflammasomes has in cell or organ functions and possible human diseases. Antioxid. Redox Signal. 22, 1111-1129. [ABSTRACT FROM AUTHOR]

Background: Autophagy is of importance in the regulation of cell differentiation and senescence in podocytes. It is possible that derangement of autophagy under different pathological conditions activates or enhances Epithelial-to-Mesenchymal Transition (EMT) in podocytes, resulting in glomerular sclerosis. To test this hypothesis, the present study produced lysosome dysfunction by inhibition of the vacuolar H+-ATPase (V-ATPase) to test whether deficiency of autophagic flux leads to enhancement of EMT in podocytes. Methods and Results: By Western blot and confocal analysis, lysosome inhibition using a V-ATPase inhibitor or its siRNA was found to markedly decreases the epithelial markers (P-cadherin and ZO-1) and increases the mesenchymal markers (FSP-1 and α-SMA). This enhancement was accompanied by deficient autophagic flux, as demonstrated by marked increases in LC3B-II and p62/Sequestosome 1. However, inhibition of autophagosome formation using spaudin-1 significantly attenuated both enhancement of EMT and deficiency of autophagic flux. To explore the mechanisms by which deficient autophagic flux enhances EMT, we tested the role of accumulated p62 as a signal hub in this process. Neither the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear kappa-light-chain-enhancer pathways of p62 contributed to enhanced EMT. However, inhibition of cyclin-dependent kinase 1 (CDK1) activity reduced the phosphorylation of p62 and enhanced EMT in podocytes similar to lysosome dysfunction. Conclusion: The lack of phosphorylated p62 leads to a faster exit from cell mitosis, enhanced EMT associated with lysosome dysfunction may be attributed to accumulation of p62 and associated reduction of p62 phosphorylation. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Adult stem cell deficiency has been implicated in the pathogenic mechanism for various diseases. Renal medullary dysfunction is one of the major mechanisms for the development of hypertension in Dahl salt-sensitive (S) rats. The present study first detected a stem cell deficiency in the renal medulla in Dahl S rats and then tested the hypothesis that transplantation of mesenchymal stem cells (MSCs) into the renal medulla improves salt-sensitive hypertension in Dahl S rats. Immunohistochemistry and flowcytometry analyses showed a significantly reduced number of stem cell marker CD133+ cells in the renal medulla from Dahl S rats compared with controls, suggesting a stem cell deficiency. Rat MSCs or control cells were transplanted into the renal medulla in uninephrectomized Dahl S rats, which were then treated with a low- or high-salt diet for 20 days. High-salt-induced sodium retention and hypertension was significantly attenuated in MSC-treated rats compared with control cell-treated rats. Meanwhile, high-salt-induced increases of proinflammatory factors, monocyte chemoattractant protein-1, and interleukin-1β, in the renal medulla were blocked by MSC treatment. Furthermore, immunostaining showed that high-salt-induced immune cell infiltration into the renal medulla was substantially inhibited by MSC treatment. These results suggested that stem cell defect in the renal medulla may contribute to the hypertension in Dahl S rats and that correction of this stem cell defect by MSCs attenuated hypertension in Dahl S rats through anti-inflammation. Key message: [ABSTRACT FROM AUTHOR]

Background/Aims: Hyperhomocysteinemia (hHcys) has been reported to initiate Nod-like receptor protein 3 (NLRP3) inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the formation and activation of inflammasomes in glomeruli of hHcys mice. Methods: Plasma homocysteine concentration was estimated utilizing HPLC, inflammasome formation and immunofluorescence expression from confocal microscopy, IL-1β production from ELISA. Results: Uninephrectomized Nlrp3 knockout (Nlrp3-/-) and wild type (Nlrp3+/+) and intra renal Nlrp3 shRNA-transfected wild type mice (Nlrp3 shRNA) were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. The plasma Hcys levels were significantly elevated in both Nlrp3-/- and Nlrp3+/+ mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with apoptosis-associated speck-like protein (ASC) or caspase-1, caspase-1 activity and IL-1β production in glomeruli of Nlrp3+/+, but not in Nlrp3-/- mice and local Nlrp3 shRNA transfected mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nlrp3+/+ mice compared to ND fed mice. However, the hHcys-induced increase in GDI and proteinuria were significantly lower in Nlrp3-/- and local Nlrp3 shRNA transfected mice than in Nlrp3+/+ mice. Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin expression in glomeruli of Nlrp3+/+ mice compared to Nlrp3-/- mice. Conclusion: Nlrp3 gene is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys-induced injury. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2 −) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O2 − production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2 − production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF. [Copyright &y& Elsevier]

Recent studies have indicated a protective role of autophagy in regulating vascular smooth muscle cells homeostasis in atherogenesis, but the mechanisms controlling autophagy, particularly autophagy maturation, are poorly understood. Here, we investigated whether acid sphingomyelinase (ASM)-regulated lysosome function is involved in autophagy maturation in coronary arterial smooth muscle cells (CASMCs) in the pathogenesis of atherosclerosis. In coronary arterial wall of ASM-deficient ( Smpd1) mice on Western diet, there were high expression levels of both LC3B, a robust marker of autophagosomes (APs), and p62, a selective autophagy substrate, compared with those in wild-type ( Smpd1) mice. By Western blotting and flow cytometry, atherogenic stimulation of Smpd1 CASMCs with 7-ketocholesterol was found to significantly enhance LC3B expression and increase the content of both APs and autophagolysosomes (APLs). In Smpd1 CASMCs, such 7-ketocholesterol-induced increases in LC3B and p62 expression and APs were further augmented, but APLs formation was abolished. Analysis of fluorescence resonance energy transfer between fluorescence-labeled LC3B and Lamp1 (lysosome marker) showed that 7-ketocholesterol markedly induced fusion of APs with lysosomes in Smpd1 CASMCs, which was abolished in Smpd1 CASMCs. Moreover, 7-ketocholesterol-induced expression of cell dedifferentiation marker vimentin and proliferation was enhanced in Smpd1 CASMCs compared with those in Smpd1 CASMCs. Lastly, overexpression of ASM further increased APLs formation in Smpd1 CASMCs and restored APLs formation in Smpd1 CASMCs indicating that increased ASM expression is highly correlated with enhanced APLs formation. Taken together, our data suggest that the control of lysosome trafficking and fusion by ASM is essential to a normal autophagic flux in CASMCs, which implicates that the deficiency of ASM-mediated regulation of autophagy maturation may result in imbalance of arterial smooth muscle cell homeostasis and thus serve as an important atherogenic mechanism in coronary arteries. Key messages: • Acid sphingomyelinase (ASM) controls autophagy maturation in smooth muscle cells. • ASM maintains smooth muscle cell homeostasis and its contractile phenotype. • ASM plays a protective role in smooth muscle dysfunction and atherosclerosis. [ABSTRACT FROM AUTHOR]

Abstract: Inflammasome, an intracellular inflammatory machinery, has been reported to be involved in a variety of chronic degenerative diseases such as atherosclerosis, autoinflammatory diseases and Alzheimer's disease. The present study hypothesized that the formation and activation of inflammasomes associated with apoptosis associated speck-like protein (ASC) are an important initiating mechanism resulting in obesity-associated podocyte injury and consequent glomerular sclerosis. To test this hypothesis, Asc gene knockout (Asc−/− ), wild type (Asc+/+ ) and intrarenal Asc shRNA-transfected wild type (Asc shRNA) mice were fed a high fat diet (HFD) or normal diet (ND) for 12weeks to produce obesity and associated glomerular injury. Western blot and RT-PCR analyses demonstrated that renal tissue Asc expression was lacking in Asc −/− mice or substantially reduced in Asc shRNA transfected mice compared to Asc +/+ mice. Confocal microscopic and co-immunoprecipitation analysis showed that the HFD enhanced the formation of inflammasome associated with Asc in podocytes as shown by colocalization of Asc with Nod-like receptor protein 3 (Nalp3). This inflammasome complex aggregation was not observed in Asc−/− and local Asc shRNA-transfected mice. The caspase-1 activity, IL-1β production and glomerular damage index (GDI) were also significantly attenuated in Asc−/− and Asc shRNA-transfected mice fed the HFD. This decreased GDI in Asc −/− and Asc shRNA transfected mice on the HFD was accompanied by attenuated proteinuria, albuminuria, foot process effacement of podocytes and loss of podocyte slit diaphragm molecules. In conclusion, activation and formation of inflammasomes in podocytes are importantly implicated in the development of obesity-associated glomerular injury. [Copyright &y& Elsevier]

Aim Autophagic flux is an important process during autophagy maturation in smooth muscle cells. However, the molecular mechanisms underlying autophagic flux in these cells are largely unknown. Here, we revealed a previously undefined role of CD38, an enzyme that metabolizes NADP+ into NAADP, in the regulation of autophagic flux in coronary arterial myocytes (CAMs). Methods and results In vivo CD38 gene knockout mice (CD38−/−) fed the high-fat Western diet showed increased accumulation of autophagosomes in coronary arterial media compared with that in wild-type (CD38+/+) mice, suggesting that CD38 gene deletion results in a defective autophagic process in CAMs of coronary arteries. In primary cultured CAMs, CD38 gene deletion markedly enhanced 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer)-induced accumulation of autophagosomes and increased expression of an autophagic marker, LC3B. However, no difference in autophagosome formation was observed between CD38+/+ and CD38−/− CAMs when autophagic flux was blocked, which indicates that CD38 regulates autophagic flux rather than induction of autophagosome formation. Further, 7-Ket-induced formation of autophagolysosomes was markedly attenuated in CD38−/− CAMs compared with CD38+/+ CAMs. Mechanistically, CD38 gene deletion markedly inhibited 7-Ket-induced dynein activation and autophagosome trafficking, which were associated with attenuated lysosomal Ca2+ release. Importantly, coronary arterial smooth muscle from CD38−/− mice fed the Western diet exhibited phenotypic changes towards a more dedifferentiated state with abnormal extracellular matrix metabolism. Conclusion Taken together, these results suggest that CD38 plays a critical role in autophagosome trafficking and fusion with lysosomes, thus controlling autophagic flux in CAMs under atherogenic stimulation. [ABSTRACT FROM PUBLISHER]

Abstract: Hyperhomocysteinemia (hHcys) is an important pathogenic factor contributing to the progression of end-stage renal disease. Recent studies have demonstrated the implication of nicotinamide adenine dinucleotide phosphate oxidase-mediated NLRP3 inflammasome activation in the development of podocyte injury and glomerular sclerosis during hHcys. However, it remains unknown which reactive oxygen species (ROS) are responsible for this activation of NLRP3 inflammasomes and how such action of ROS is controlled. This study tested the contribution of common endogenous ROS including superoxide (O2 −), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical ( OH) to the activation of NLRP3 inflammasomes in mouse podocytes and glomeruli. In vitro, confocal microscopy and size-exclusion chromatography demonstrated that dismutation of O2 − by 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (Tempol) and decomposition of H2O2 by catalase prevented Hcys-induced aggregation of NLRP3 inflammasome proteins and inhibited Hcys-induced caspase-1 activation and IL-1β production in mouse podocytes. However, scavenging of ONOO− or OH had no significant effect on either Hcys-induced NLRP3 inflammasome formation or activation. In vivo, scavenging of O2 − by Tempol and removal of H2O2 by catalase substantially inhibited NLRP3 inflammasome formation and activation in glomeruli of hHcys mice as shown by reduced colocalization of NLRP3 with ASC or caspase-1 and inhibition of caspase-1 activation and IL-1β production. Furthermore, Tempol and catalase significantly attenuated hHcys-induced glomerular injury. In conclusion, endogenously produced O2 − and H2O2 primarily contribute to NLRP3 inflammasome formation and activation in mouse glomeruli resulting in glomerular injury or consequent sclerosis during hHcys. [Copyright &y& Elsevier]

Although Nlrp3 inflammasome activation in macrophages has been shown to be critical for the development of atherosclerosis upon atherogenic stimuli, it remains unknown whether activated Nlrp3 inflammasomes by other non-atherogenic stimuli induce alterations in macrophages that may contribute in the concert with other factors to atherogenesis. Thus, the present study tested the hypothesis that activation of Nlrp3 inflammasomes by ATP, which is a classical non-lipid danger stimulus, enhances the migration of macrophage and increases lipids deposition in macrophages accelerating foam cell formation. We first demonstrated that extracellular ATP (2.5 mM) markedly increased the formation and activation of Nlrp3 inflammasomes in bone marrow macrophages (BMMs) from wild type (Asc+/+) mice resulting in activation of caspase-1 and IL-1β production. In these Asc+/+ macrophages, such stimulation of inflammasomes by non-lipid ATP was similar to those induced by atherogenic stimuli such as cholesterol crystals or 7-ketocholesterol. Both non-lipid and lipid forms of stimuli induced formation and activation of Nlrp3 inflammasomes, which were prevented by Asc gene deletion. Interestingly, Asc+/+ BMMs had dramatic lipids accumulation after stimulation with ATP. Further, we demonstrated that large amount of cholesterol was accumulated in lysosomes of Asc+/+ BMMs when inflammasomes were activated by ATP. Such intracellular and lysosomal lipids deposition was not observed in Asc−/− BMMs and also prevented by caspase-1 inhibitor WEHD. In addition, in vitro and in vivo experiments revealed that migration of Asc+/+ BMMs increased due to stimulation of Nlrp3 inflammasomes, which was markedly attenuated in Asc−/− BMMs. Together, these results suggest that activation of Nlrp3 inflammasomes remarkably increases the susceptibility of macrophages to lipid deposition and their migration ability. Such novel action of inflammasomes may facilitate entry or retention of macrophages into the arterial wall, where they form foam cells and ultimately induce atherosclerosis. [ABSTRACT FROM AUTHOR]

R3995 --> 98.4 --> Acid Sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)‐induced NLRP3 inflammasome activation in podocytes and glomerular sclerosis. The present study was designed to test whether somatic podocyte‐specific silencing of ASM gene (its mouse code is Smpd1) attenuates hHcy‐induced NLRP3 inflammasome activation and associated exosome release in podocytes and thereby suppresses glomerular inflammatory response and injury. We first constructed a floxed Smpd1 shRNA construct (Vigene Bioscience, Cat# IS100001) that has both RFP and GFP as reporter genes, where RFP is expressed if there is no cre excision. If cre excision occurs, GFP is expressed. This floxed Smpd1 shRNA was transfected into podocytes isolated from WT/Podocre mice, which decreased ASM mRNA and protein levels by 90% and 60%, respectively. This suggests efficient gene silencing in podocytes with cre gene. When this floxed Smpd1 shRNA was transfected into mouse kidney by intravenous infusion and ultrasound‐microbubble directed introduction, ASM expression decreased by 65% in WT/Podocre mice and 51% in Smpd1trg/Podocre mice after 8 weeks of shRNA transfection. It was also found that transfection of floxed Smpd1 shRNA substantially attenuated hHcy‐induced increase in NLRP3 inflammasome product, IL‐1β. By nanoparticle tracking analysis, floxed Smpd1 shRNA transfection was shown to significantly block hHcy‐induced elevation of urinary exosome excretion, an indicative of NLRP3 inflammasome product release from the cells. In Smpd1trg/Podocre mice, hHcy was found to induce exaggerated NLRP3 inflammasome activation, exosome excretion and glomerular inflammatory response such as immune cell infiltration compared to WT/WT mice. This exaggerated glomerular pathological response to hHcy was also significantly suppressed by podocyte‐specific Smpd1 gene silencing. Besides, podocyte‐specific Smpd1 gene deletion substantially attenuated proteinuria and albuminuria. Taken together, our results suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and associated exosome release and that podocyte‐specific silencing of ASM gene may be an important therapeutic strategy for treatment of glomerular injury and ultimate sclerosis during hHcy. [ABSTRACT FROM AUTHOR]

R4087 --> 98.5 --> The nucleotide‐binding oligomerization domain‐like receptor containing pyrin domain 3 (NLRP3) inflammasome products such as IL‐1β, IL‐18, and HMGB1 are critical factors to trigger glomerular inflammatory responses under different pathological conditions. The present study was designed to test a hypothesis that during obesity exosome release serves as the molecular mechanism mediating the release and action of NLRP3 inflammasome products from podocytes thereby leading to glomerular inflammation and sclerosis. By confocal microscopy and immunohistochemistry, NLRP3 inflammasome activation and associated T cell infiltration were found significantly enhanced in glomeruli of obese mice receiving high‐fat diet (HFD). Nanoparticle tracking analysis showed that podocyte‐derived exosomes containing NLRP3 inflammasome products remarkably increased in urine of these mice. By structured illumination microscopy, we observed the inhibition of lysosome‐MVB interaction by visfatin, a pro‐inflammatory adipokine, in podocytes isolated from mice on the normal diet, which was accompanied by enhanced exosome release. The HFD‐induced NLRP3 inflammasome activation and exosome release in mouse glomeruli and visfatin‐induced impairment of lysosome‐MVB interaction in primary cultures of podocytes were attenuated by Smpd1 gene knockout but amplified by podocyte‐specific Smpd1 gene overexpression (Smpd1 is the gene code of mouse acid sphingomyelinase). Correspondingly, HFD‐induced podocyte injury, glomerulosclerosis, and proteinuria were enhanced in mice with podocyte‐specific Smpd1 gene overexpression but reduced in Smpd1 gene knockout mice. Taken together, our results suggest that the release of exosomes containing NLRP3 inflammasome products from podocytes is critically implicated in the triggering of glomerular inflammatory response leading to glomerular sclerosis during obesity. The acid sphingomyelinase‐ceramide signaling pathway plays a key role in this exosome‐mediated release of NLRP3 inflammasome products. [ABSTRACT FROM AUTHOR]

R4100 --> 845.6 --> Obesity has been reported to induce NLRP3 inflammasome activation in podocytes, leading to podocyte injury and glomerular dysfunction. The present study tested whether exosome biogenesis and lysosome function are involved in the regulation of exosome release from podocytes during obesity and whether obesity still induces glomerular inflammatory response without robust release of exosomes. By nanoparticle tracking analysis, increased urinary exosome excretion was found in WT/WT mice fed with high‐fat diet (HFD), which was exaggerated by podocyte‐specific overexpression of Smpd1 gene (Smpd1 is the gene code of mouse acid sphingomyelinase) in Smpd1trg/Podocre mice. Exosome biogenesis inhibitor‐GW4869, lysosome function enhancer‐rapamycin, and acid sphingomyelinase inhibitor‐amitriptyline were found to inhibit the elevation of urinary exosome excretion during obesity in both WT/WT and Smpd1trg/Podocre mice. We also isolated podocytes from WT/WT and Smpd1trg/Podocre mice for in vitro studies. Visfatin as a pro‐inflammatory adipokine enhanced exosome release from podocytes, which was exaggerated by Smpd1 gene overexpression. Correspondingly, pre‐treatment of cells with GW4869, rapamycin, and amitriptyline blocked the increase in exosome release induced by visfatin. By confocal microscopy, we observed HFD‐induced NLRP3 inflammasome activation in glomeruli, which was enhanced by podocyte‐specific Smpd1 gene overexpression but attenuated by amitriptyline. Moreover, HFD‐induced immune cell infiltration into glomeruli and glomerular sclerotic changes were worsen by podocyte‐specific Smpd1 gene overexpression but inhibited by all 3 treatments. However, GW4869 and rapamycin had no effects on NLRP3 inflammasome in podocytes, but only amitriptyline suppressed NLRP3 inflammasome activation. It is concluded that exosome release from podocytes during obesity contributes to the inflammatory response in glomeruli, which is regulated by exosome biogenesis and lysosome function. Without robust release of exosomes, NLRP3 inflammasome activation may not induce glomerular inflammatory response during obesity. [ABSTRACT FROM AUTHOR]

R2622 --> 844.4 --> The renal medulla has been long known to exert antihypertensive action by releasing different bioactive substances. However, so far it remains unknown what is the identity of such renal medullary antihypertensive substances. In the present study, we hypothesized that exosomes, but not a single molecule released from the renal medulla are importantly implicated in the renal medullary antihypertensive action. To test this hypothesis, we first produced the 2 kidney‐one clip (2K1C) hypertension mouse model by clipping the right renal artery (127.9 ± 1.40 mmHg vs. 94.8 ± 1.75 mmHg of sham mice). After 1 week, we unclipped the right renal artery and monitored the mean arterial blood pressure (MAP) for 1 hour and at the same time collected blood samples for analysis of exosomes. It was found that unclipping the right renal artery of 2K1C mice significanty reduced MAP by 21.2 ± 2.65 mmHg but increased the plasma exosome concentrations by more than 5 folds (before unclipping 2.9 ± 0.7 vs. 3.3±0.5, N.S., after unclipping 4±0.5 vs. 17±5× 108particles/mL, p<0.05). Immunohistochemical (IHC) assessments showed that the clipped kidneys had dramatic increase in exosome markers (CD63 and Annexin II) in the renal medulla of 2K1C mice compared to contralateral kidneys or sham mouse kidneys. These functional and IHC changes in 2K1C mice were almost completely blocked by pretreatment of mice with an exosome biogenesis inhibitor, GW‐4869 (1 mg/kg/day, i.p., n=8). We also found that rapamycin, a compound that increases autophagy and lysosome trafficking (6 mg/kg/day, i.p., n=8) substantially attenuated exosome release from the renal medulla and associated antihypertensive action in 2K1C with unclipping the right renal artery, even though it had some effects to reduce MAP and increase exosome release before the unclipping. In another series of studies using 2K1C hypertensive mice, we extracted and purified exosomes from the blood of mice with unclipping of the right renal artery and then infused these exosomes intravenously to another group of 2K1C mice. It was found that plasma exosomes from 2K1C mice with unlipping of the right renal artery signicantly reduced MAP when they are infused into recipient 2K1C mice (at a concentration of 3 x 108 exosomes) compard to exosomes from sham mice. Urinary flow and sodium excretion of recipient mice also increased slightly by infusion of exosomes from 2K1C mice. These results suggest that exosomes are enriched in the renal medulla of the clipped kidney in 2K1C mice and they can be released into the blood if the renal artery clip is released. These exosomes have strong effects to lower arterial blood pressure. [ABSTRACT FROM AUTHOR]