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ABSTRACT: 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 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 gp91phox siRNA, gp91ds-tat peptide, diphenyleneiodonium (DPI) 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 (IL-1β) production. Similar findings were observed in vivo where gp91phox-/- mice and mice receiving a gp91ds-tat treatment exhibited markedly reduced inflammasome formation and activation. Furthermore, 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 gp91phox-/- 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.
Antioxidants & Redox Signaling 10/2012; · 8.20 Impact Factor
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ABSTRACT: 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-1β production in glomeruli. Locally silencing the ASC gene in the kidney significantly reduced NALP3 inflammasome formation and interleukin 1β 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.
Hypertension 05/2012; 60(1):154-62. · 6.21 Impact Factor
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ABSTRACT: The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91(phox), and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function.
AJP Heart and Circulatory Physiology 09/2011; 301(5):H2028-37. · 3.71 Impact Factor
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ABSTRACT: 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.
American Journal Of Pathology 09/2011; 179(5):2210-9. · 4.89 Impact Factor
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ABSTRACT: The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1), in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide (O⁻₂) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91( phox ) (gp91(-/-)), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91(+/+)) mice, hHcys induced by a folate-free diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91(-/-) mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91(+/+) mice with hHcys were all significantly attenuated in gp91(-/-) mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury.
Pflügers Archiv - European Journal of Physiology 06/2011; 462(3):455-67. · 4.46 Impact Factor
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ABSTRACT: The adipokine visfatin, produced during obesity, has been reported to participate in the development of cardiovascular disease associated with metabolic syndrome. The present study was designed to test a hypothesis that visfatin causes coronary endothelial dysfunction through lysosome trafficking and fusion to cell membranes, membrane raft (MR) clustering, and formation of redox signalosomes.
By using confocal microscopy, it was found that visfatin, but not adiponectin, stimulated NADPH oxidase (NOX) subunits, gp91(phox) aggregation in MR clusters and p47(phox) translocation to these MR clusters in bovine coronary arterial endothelial cells (CAECs), leading to activation of NOX with a 2.5-fold increase in O(2)(·-) production. A signalling lipid, ceramide, was found to be enriched in such membrane MR-NOX complexes of CAECs. Lysosomal fluorescent dye (FM1-43) quenching and de-quenching revealed that visfatin induced the fusion of lysosomes to cell membranes and incorporation of acid sphingomyelinase and its product, ceramide, in such MR-NOX signalling platforms. Functionally, visfatin significantly attenuated endothelium-dependent vasodilation in small coronary arteries (by 80%), which was blocked by lysosomal function inhibitor and MR disruptors.
These results suggest that lysosome-associated molecular trafficking and consequent ceramide accumulation in cell membrane may mediate the assembly of NOX subunits and their activation in response to adipokine visfatin in CAECs, thereby producing endothelial dysfunction in coronary circulation.
Cardiovascular research 02/2011; 89(2):401-9. · 5.80 Impact Factor
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ABSTRACT: Epithelial-to-Mesenchymal Transition (EMT) is an important pathogenic mechanism mediating glomerular injury or sclerosis in a variety of renal and systemic diseases such as hyperhomocysteinemia (hHcys). The present study was designed to test whether Hcys-induced EMT in podocytes is reversed by growth hormone (GH), a hormone regulating cell differentiation and growth and to explore the cellular and molecular mechanism mediating its action. It was found that Hcys induced significant EMT in podocytes, as shown by marked decreases in slit diaphragm-associated protein P-cadherin and zonula occludens-1 as epithelial markers and by dramatic increases in the expression of mesenchymal markers, fibroblast specific protein-1 and α-smooth muscle actin, which were detected by all examinations via immunocytochemistry, real time RT-PCR and Western blot analysis. When podocytes were treated with GH at 25 ng/mL, however, Hcys failed to induce podocyte EMT. Using electromagnetic spin resonance spectrometry, Hcys-induced superoxide (O(2).(-)) production via NADPH oxidase was found to be significantly inhibited by GH (66%). Functionally, GH was shown to substantially inhibit Hcys-induced increases in the permeability of podocyte monolayers and to block the decrease in podocin expression in these cells. In addition, NADPH oxidase subunit, gp91(phox) and GH receptors aggregated in membrane raft clusters, which produced O(2).(-) in response to Hcys and could be blocked by GH, membrane raft disruptors filipin and MCD or NADPH oxidase inhibitor, apocynin. It is concluded that Hcys-induced podocyte EMT is associated with transmembrane membrane raft-redox signaling and that GH reverses this Hcys-induced EMT protecting podocytes from functional disturbance.
Cellular Physiology and Biochemistry 01/2011; 27(6):691-702. · 2.86 Impact Factor
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ABSTRACT: Adipokines have been reported to contribute to glomerular injury during obesity or diabetes mellitus. However, the mechanisms mediating the actions of various adipokines on the kidney remained elusive. The present study was performed to determine whether acid sphingomyelinase (ASM)-ceramide associated lipid raft (LR) clustering is involved in local oxidative stress in glomerular endothelial cells (GECs) induced by adipokines such as visfatin and adiponectin. Using confocal microscopy, visfatin but not adiponectin was found to increase LRs clustering in the membrane of GECs in a dose and time dependent manner. Upon visfatin stimulation ASMase activity was increased, and an aggregation of ASMase product, ceramide and NADPH oxidase subunits, gp91(phox) and p47(phox) was observed in the LR clusters, forming a LR redox signaling platform. The formation of this signaling platform was blocked by prior treatment with LR disruptor filipin, ASMase inhibitor amitriptyline, ASMase siRNA, gp91(phox) siRNA and adiponectin. Corresponding to LR clustering and aggregation of NADPH subunits, superoxide (O(2)(-)) production was significantly increased (2.7 folds) upon visfatin stimulation, as measured by electron spin resonance (ESR) spectrometry. Functionally, visfatin significantly increased the permeability of GEC layer in culture and disrupted microtubular networks, which were blocked by inhibition of LR redox signaling platform formation. In conclusion, the injurious effect of visfatin, but not adiponectin on the glomerular endothelium is associated with the formation of LR redox signaling platforms via LR clustering, which produces local oxidative stress resulting in the disruption of microtubular networks in GECs and increases the glomerular permeability.
Biochimica et Biophysica Acta 12/2010; 1801(12):1294-304. · 4.66 Impact Factor
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ABSTRACT: 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.
Antioxidants & Redox Signaling 10/2010; 13(7):975-86. · 8.20 Impact Factor
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ABSTRACT: The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD). Treatment of mice with the acid sphingomyelinase (ASMase) inhibitor amitriptyline significantly attenuated the HFD-induced plasma ceramide levels. Corresponding to increase in plasma ceramide, the HFD significantly increased the body weight gain, plasma leptin concentration, urinary total protein and albumin excretion, glomerular damage index, and adipose tissue ASMase activity compared with the LFD-fed mice. These HFD-induced changes were also significantly attenuated by treatment of mice with amitriptyline. In addition, the decline of plasma glucose concentration after an intraperitoneal injection of insulin (0.15 U/kg b.wt.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg b.wt.) resulted in a slow increase followed by a rapid decrease in the plasma glucose concentration in LFD and HFD plus amitriptyline-treated mice, but such blood glucose response was not observed in HFD-fed mice. Immunofluorescence analysis demonstrated a decrease in the podocin and an increase in the desmin in the glomeruli of HFD-fed mice compared with the LFD and HFD plus amitriptyline-treated mice. In conclusion, our results reveal a pivotal role for ceramide biosynthesis in obesity, metabolic syndrome, and associated kidney damage.
Journal of Pharmacology and Experimental Therapeutics 09/2010; 334(3):839-46. · 3.83 Impact Factor
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ABSTRACT: 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.
Free radical research 08/2010; 44(8):831-42. · 2.22 Impact Factor
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ABSTRACT: In this study, mice lacking the gp91(phox) gene were used to address the role of NADPH oxidase in hyperhomocysteinemia-induced podocyte injury. It was found that a folate-free diet increased plasma homocysteine levels, but failed to increase O(2)(-) production in the glomeruli from gp91(phox) gene knockout (gp91(-/-)) mice, compared with wild-type (gp91(+/+)) mice. Proteinuria and glomerular damage index (GDI) were significantly lower, whereas the glomerular filtration rate (GFR) was higher in gp91(-/-) than in gp91(+/+) mice when they were on the folate-free diet (urine albumin excretion, 21.23+/-1.88 vs 32.86+/-4.03 microg/24 h; GDI, 1.17+/-0.18 vs 2.59+/-0.49; and GFR, 53.01+/-4.69 vs 40.98+/-1.44 microl/min). Hyperhomocysteinemia-induced decrease in nephrin expression and increase in desmin expression in gp91(+/+) mice were not observed in gp91(-/-) mice. Morphologically, foot process effacement and podocyte loss due to hyperhomocysteinemia were significantly attenuated in gp91(-/-) mice. In in vitro studies of podocytes, homocysteine was found to increase gp91(phox) expression and O2(*)(-) generation, which was substantially inhibited by gp91(phox) siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A production was abolished by gp91(phox) siRNA or diphenyleneiodonium, a NADPH oxidase inhibitor. These results suggest that the functional integrity of NADPH oxidase is essential for hyperhomocysteinemia-induced podocyte injury and glomerulosclerosis.
Free radical biology & medicine 04/2010; 48(8):1109-17. · 5.42 Impact Factor
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ABSTRACT: Our recent studies have indicated that hyperhomocysteinemia (hHcys) may induce podocyte damage, resulting in glomerulosclerosis. However, the molecular mechanisms mediating hHcys-induced podocyte injury are still poorly understood. In the present study, we first demonstrated that an intact NADPH oxidase system is present in podocytes as shown by detection of its membrane subunit (gp91(phox)) and cytosolic subunit (p47(phox)). Then, confocal microscopy showed that gp91(phox) and p47(phox) could be aggregated in lipid raft (LR) clusters in podocytes treated with homocysteine (Hcys), which were illustrated by their colocalization with cholera toxin B, a common LR marker. Different mechanistic LR disruptors, either methyl-beta-cyclodextrin (MCD) or filipin abolished such Hcys-induced formation of LR-gp91(phox) or LR-p47(phox) transmembrane signaling complexes. By flotation of detergent-resistant membrane fractions we found that gp91(phox) and p47(phox) were enriched in LR fractions upon Hcys stimulation, and such enrichment of NADPH oxidase subunits and increase in its enzyme activity were blocked by MCD or filipin. Functionally, disruption of LR clustering significantly attenuated Hcys-induced podocyte injury, as shown by their inhibitory effects on Hcys-decreased expression of slit diaphragm molecules such as nephrin and podocin. Similarly, Hcys-increased expression of desmin was also reduced by disruption of LR clustering. In addition, inhibition of such LR-associated redox signaling prevented cytoskeleton disarrangement and apoptosis induced by Hcys. It is concluded that NADPH oxidase subunits aggregation and consequent activation of this enzyme through LR clustering is an important molecular mechanism triggering oxidative injury of podocytes induced by Hcys.
Biochimica et Biophysica Acta 04/2010; 1803(4):482-91. · 4.66 Impact Factor
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ABSTRACT: We currently reported that Vav2, a member of the guanine nucleotide exchange factor-Vav subfamily, participates in homocysteine-induced increases in Rac1 activity and consequent activation of NADPH oxidase in rat mesangial cells. However, the physiological relevance of this cellular action of Vav2 remains unknown. The present study tested a hypothesis that Vav2 importantly mediates the injurious action of homocysteine on glomeruli and thereby contributes to the development of glomerulosclerosis during hyperhomocysteinemia. We found that, among Vav members, Vav2 was abundantly expressed in glomeruli. When Vav2 short hairpin RNA was transfected into the kidneys of Sprague-Dawley rats, hyperhomocysteinemia induced by folate-free diet failed to significantly enhance Rac1 activity and increase NADPH-dependent superoxide production. In these rats with silenced renal Vav2 gene, glomerular injury during hyperhomocysteinemia was markedly attenuated compared with those rats only receiving mock vector transfection, as shown by ameliorated albuminuria and extracellular matrix metabolism. In the rat kidneys with transfection of a dominant-active Vav2 variant (onco-Vav2), we found that overexpression of Vav2 led to significant increases in Rac1 activity, superoxide production, and glomerular injury, which was similar to that induced by hyperhomocysteinemia. However, this Vav2 overexpression was unable to further enhance homocysteine-induced glomerular injury. We concluded that Vav2-mediated activation of NADPH oxidase is an important initiating mechanism resulting in hyperhomocysteinemic glomerular injury through enhanced local oxidative stress.
Hypertension 12/2008; 53(1):90-6. · 6.21 Impact Factor
