Justine M. Abais

Activation of Nod-Like Receptor Protein 3 Inflammasomes Turns on Podocyte Injury and Glomerular Sclerosis in Hyperhomocysteinemia

Inflammasome is a multiprotein complex consisting of Nod-like receptor protein 3 (NALP3), apoptosis-associated speck-like protein (ASC), and caspase 1 or 5, which functions to switch on the inflammatory process. The present study hypothesized that the formation and activation of NALP3 inflammasomes turn on podocyte injury leading to glomerulosclerosis during hyperhomocysteinemia (hHcys). RT-PCR and Western blot analysis demonstrated that murine podocytes expressed 3 essential components of the NALP3 inflammasome complex, namely, NALP3, ASC, and caspase 1. Treatment of podocytes with l-homocysteine induced the formation of NALP3 inflammasome complex, an increase in caspase 1 activity, podocyte cytoskeleton rearrangement, and decreased production of vascular endothelial growth factor from podocytes, which were all blocked by silencing the ASC gene or inhibiting caspase 1 activity. In mice with hHcys induced by feeding them a folate-free diet, NALP3 inflammasome formation and activation in glomerular podocytes were detected at an early stage, as shown by confocal microscopy, size exclusion chromatography of the assembled inflammasome complex, and increased interleukin-1&bgr; production in glomeruli. Locally silencing the ASC gene in the kidney significantly reduced NALP3 inflammasome formation and interleukin 1&bgr; production in glomeruli of mice with hHcys. Pathologically, hHcys-associated albuminuria, foot process effacement of podocytes, loss of podocyte slit diaphragm molecules, and glomerulosclerosis at the late stage were significantly improved by local ASC gene silencing or by caspase 1 inhibition. In conclusion, NALP3 inflammasome formation and activation on stimulation of homocysteine are important molecular mechanisms triggering podocyte injury and ultimately resulting in glomerulosclerosis in hHcys.

NADPH Oxidase-Mediated Triggering of Inflammasome Activation in Mouse Podocytes and Glomeruli During Hyperhomocysteinemia

AIM Our previous studies have shown that NOD-like receptor protein (NALP3) inflammasome activation is importantly involved in podocyte dysfunction and glomerular sclerosis induced by hyperhomocysteinemia (hHcys). The present study was designed to test whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling contributes to homocysteine (Hcys)-induced activation of NALP3 inflammasomes, an intracellular inflammatory machinery in podocytes in vitro and in vivo. RESULTS In vitro confocal microscopy and size-exclusion chromatography revealed that upon NADPH oxidase inhibition by gp91(phox) siRNA, gp91ds-tat peptide, diphenyleneiodonium, or apocynin, aggregation of inflammasome proteins NALP3, apoptosis-associated speck-like protein (ASC), and caspase-1 was significantly attenuated in mouse podocytes. This NADPH oxidase inhibition also resulted in diminished Hcys-induced inflammasome activation, evidenced by reduced caspase-1 activity and interleukin-1β production. Similar findings were observed in vivo where gp91(phox-/-) mice and mice receiving a gp91ds-tat treatment exhibited markedly reduced inflammasome formation and activation. Further, in vivo NADPH oxidase inhibition protected the glomeruli and podocytes from hHcys-induced injury as shown by attenuated proteinuria, albuminuria, and glomerular sclerotic changes. This might be attributed to the fact that gp91(phox-/-) and gp91ds-tat-treated mice had abolished infiltration of macrophages and T-cells into the glomeruli during hHcys. INNOVATION Our study for the first time links NADPH oxidase to the formation and activation of NALP3 inflammasomes in podocytes. CONCLUSION Hcys-induced NADPH oxidase activation is importantly involved in the switching on of NALP3 inflammasomes within podocytes, which leads to the downstream recruitment of immune cells, ultimately resulting in glomerular injury and sclerosis.

Enhancement of Autophagy by Simvastatin through Inhibition of Rac1-mTOR Signaling Pathway in Coronary Arterial Myocytes

Background/Aims: In addition to their action of lowering blood cholesterol levels, statins modulate biological characteristics and functions of arterial myocytes such as viability, proliferation, apoptosis, survival and contraction. The present study tested whether simvastatin, as a prototype statin, enhances autophagy in coronary arterial myocytes (CAMs) to thereby exert their beneficial effects in atherosclerosis. Methods and Results: Using flow cytometry, we demonstrated that simvastatin significantly increased the autophagsome formation in CAMs. Western blot analysis confirmed that simvastatin significantly increased protein expression of typical autophagy markers LC3B and Beclin1 in these CAMs. Confocal microscopy further demonstrated that simvastatin increased fusion of autophagosomes with lysosomes, which was blocked by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Simvastatin reduced mammalian target of rapamycin (mTOR) activity, which was reversed by Rac1-GTPase overexpression and the mTOR agonist phosphatidic acid. Moreover, both Rac1-GTPase overexpression and activation of mTOR by phosphatidic acid drastically blocked simvastatin-induced autophagosome formation in CAMs. Interestingly, simvastatin increased protein expression of a contractile phenotype marker calponin in CAMs, which was blocked by autophagy inhibitor 3-methyladenine. Simvastatin markedly reduced proliferation of CAMs under both control and proatherogenic stimulation. However, this inhibitory effect of simvastatin on CAM proliferation was blocked by by autophagy inhibitor 3-methyladenine or silencing of Atg7 genes. Lastly, animal experiments demonstrated that simvastatin increased protein expression of LC3B and calponin in mouse coronary arteries. Conclusion: Our results indicate that simvastatin inhibits the Rac1-mTOR pathway and thereby increases autophagy in CAMs which may stabilize CAMs in the contractile phenotype to prevent proliferation and growth of these cells.

Inflammation and Hypertension: New Understandings and Potential Therapeutic Targets

Research studying the role of inflammation in hypertension and cardiovascular disease has flourished in recent years; however, the exact mechanisms by which the activated immune cells lead to the development and maintenance of hypertension remain to be elucidated. The objectives of this brief review are to summarize and discuss the most recent findings in the field, with special emphasis on potential therapeutics to treat or prevent hypertension. This review will cover novel immune cell subtypes recently associated to the disease including the novel role of cytokines, toll-like receptors, and inflammasomes in hypertension.

Nod-like Receptor Protein 3 (NLRP3) Inflammasome Activation and Podocyte Injury via Thioredoxin-Interacting Protein (TXNIP) during Hyperhomocysteinemia *

Background: Hyperhomocysteinemia (hHcys) contributes to glomerular injury by activating NLRP3 inflammasomes in response to increased oxidative stress. Results: Thioredoxin-interacting protein (TXNIP) aggregated with NLRP3 inflammasomes, and blocking TXNIP prevented inflammasome activation during hHcys. Conclusion: TXNIP uniquely links changes in oxidative stress to hHcys-induced NLRP3 inflammasome activation. Significance: Glomerular injury related to hHcys can be prevented by TXNIP inhibition. NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1β production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1β production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.

Contribution of endogenously produced reactive oxygen species to the activation of podocyte NLRP3 inflammasomes in hyperhomocysteinemia

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.

NMDA Receptor-Mediated Activation of NADPH Oxidase and Glomerulosclerosis in Hyperhomocysteinemic Rats

This study investigated the role of NMDA receptor in hyperhomocyteinemia (hHcys)-induced NADPH oxidase (Nox) activation and glomerulosclerosis. Sprague-Dawley rats were fed a folate-free (FF) diet to produce hHcys, and a NMDA receptor antagonist, MK-801, was administrated. Rats fed the FF diet exhibited significantly increased plasma homocysteine levels, upregulated NMDA receptor expression, enhanced Nox activity and Nox-dependent O(2)(.-) production in the glomeruli, which were accompanied by remarkable glomerulosclerosis. MK-801 treatment significantly inhibited Nox-dependent O(2)(.-) production induced by hHcys and reduced glomerular damage index as compared with vehicle-treated hHcys rats. Correspondingly, glomerular deposition of extracellular matrix components in hHcys rats was ameliorated by the administration of MK-801. Additionally, hHcys induced an increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) expression and a decrease in matrix metalloproteinase (MMP)-1 and MMP-9 activities, all of which were abolished by MK-801 treatment. In vitro studies showed that homocysteine increased Nox-dependent O(2)(.-) generation in rat mesangial cells, which was blocked by MK-801. Pretreatment with MK-801 also reversed homocysteine-induced decrease in MMP-1 activity and increase in TIMP-1 expression. These results support the view that the NMDA receptor may mediate Nox activation in the kidney during hHcys and thereby play a critical role in the development of hHcys-induced glomerulosclerosis.

Endothelial NLRP3 Inflammasome Activation and Enhanced Neointima Formation in Mice by Adipokine Visfatin

Inflammasomes serve as an intracellular machinery to initiate inflammatory response to various danger signals. The present study tested whether an inflammasome centered on nucleotide oligomerization domain-like receptor protein 3 (NLRP3) triggers endothelial inflammatory response to adipokine visfatin, a major injurious adipokine during obesity. NLRP3 inflammasome components were abundantly expressed in cultured mouse microvascular endothelial cells, including NLRP3, apoptosis-associated speck-like protein, and caspase-1. These NLRP3 inflammasome molecules could be aggregated to form an inflammasome complex on stimulation of visfatin, as shown by fluorescence confocal microscopy and size exclusion chromatography. Correspondingly, visfatin significantly increased caspase-1 activity and IL-1β release in microvascular endothelial cells, indicating an activation of NLRP3 inflammasomes. In animal experiments, direct infusion of visfatin in mice with partially ligated left carotid artery were found to have significantly increased neointimal formation, which was correlated with increased NLRP3 inflammasome formation and IL-1β production in the intima. Further, visfatin-induced neointimal formation, endothelial inflammasome formation, and IL-1β production in mouse partially ligated left carotid artery were abolished by caspase-1 inhibition, local delivery of apoptosis-associated speck-like protein shRNA or deletion of the ASC gene. In conclusion, the formation and activation of NLRP3 inflammasomes by adipokine visfatin may be an important initiating mechanism to turn on the endothelial inflammatory response leading to arterial inflammation and endothelial dysfunction in mice during early stage obesity.

Acid Sphingomyelinase Gene Deficiency Ameliorates the Hyperhomocysteinemia-Induced Glomerular Injury in Mice

Hyperhomocysteinemia (hHcys) enhances ceramide production, leading to the activation of NADPH oxidase and consequent glomerular oxidative stress and sclerosis. The present study was performed to determine whether acid sphingomyelinase (Asm), a ceramide-producing enzyme, is implicated in the development of hHcys-induced glomerular oxidative stress and injury. Uninephrectomized Asm-knockout (Asm(-/-)) and wild-type (Asm(+/+)) mice, with or without Asm short hairpin RNA (shRNA) transfection, were fed a folate-free (FF) diet for 8 weeks, which significantly elevated the plasma Hcys level compared with mice fed normal chow. By using in vivo molecular imaging, we found that transfected shRNAs were expressed in the renal cortex starting on day 3 and continued for 24 days. The FF diet significantly increased renal ceramide production, Asm mRNA and activity, urinary total protein and albumin excretion, glomerular damage index, and NADPH-dependent superoxide production in the renal cortex from Asm(+/+) mice compared with that from Asm(-/-) or Asm shRNA-transfected wild-type mice. Immunofluorescence analysis showed that the FF diet decreased the expression of podocin but increased desmin and ceramide levels in glomeruli from Asm(+/+) mice but not in those from Asm(-/-) and Asm shRNA-transfected wild-type mice. In conclusion, our observations reveal that Asm plays a pivotal role in mediating podocyte injury and glomerular sclerosis associated with NADPH oxidase-associated local oxidative stress during hHcys.

Attenuation by Statins of Membrane Raft-Redox Signaling in Coronary Arterial Endothelium

Membrane raft (MR)–redox signaling platforms associated with NADPH oxidase are involved in coronary endothelial dysfunction. Here, we studied whether statins interfere with the formation of MR-redox signaling platforms to protect the coronary arterial endothelium from oxidized low-density lipoprotein (OxLDL)–induced injury and from acute hypercholesterolemia. In cultured human coronary arterial endothelial cells, confocal microscopy detected the formation of an MRs clustering when they were exposed to OxLDL, and such MR platform formation was inhibited markedly by statins, including pravastatin and simvastatin. In these MR clusters, NADPH oxidase subunits gp91phox and p47phox were aggregated and were markedly blocked by both statins. In addition, colocalization of acid sphingomyelinase (ASM) and ceramide was induced by OxLDL, which was blocked by statins. Electron spin resonance spectrometry showed that OxLDL-induced superoxide (O2.−) production in the MR fractions was substantially reduced by statins. In coronary artery intima of mice with acute hypercholesterolemia, confocal microscopy revealed a colocalization of gp91phox, p47phox, ASM, or ceramide in MR clusters. Such colocalization was rarely observed in the arteries of normal mice or significantly reduced by pretreatment of hypercholesterolemic mice with statins. Furthermore, O2.− production in situ was 3-fold higher in the coronary arteries from hypercholesterolemic mice than in those from normal mice, and such increase was inhibited by statins. Our results indicate that blockade of MR-redox signaling platform formation in endothelial cell membrane may be another important therapeutic mechanism of statins in preventing endothelial injury and atherosclerosis and may be associated with their direct action on membrane cholesterol structure and function.