Published papers

Rhabdomyolysis is a serious syndrome caused by skeletal muscle injury and the subsequent release of breakdown products from damaged muscle cells into systemic circulation. The muscle damage most often results from strenuous exercise, muscle hypoxia, medications, or drug abuse and can lead to life-threatening complications, such as acute kidney injury (AKI). Rhabdomyolysis and the AKI complication can also occur during crush syndrome, an emergency condition that commonly occurs in victims of natural disasters, such as earthquakes, and man-made disasters, such as wars and terrorism. Myoglobin released from damaged muscle is believed to trigger renal dysfunction in this form of AKI. Recently, macrophages were implicated in the disease pathogenesis of rhabdomyolysis-induced AKI, but the precise molecular mechanism remains unclear. In the present study, we show that macrophages released extracellular traps (ETs) comprising DNA fibers and granule proteins in a mouse model of rhabdomyolysis. Heme-activated platelets released from necrotic muscle cells during rhabdomyolysis enhanced the production of macrophage extracellular traps (METs) through increasing intracellular reactive oxygen species generation and histone citrullination. Here we report, for the first time to our knowledge, this unanticipated role for METs and platelets as a sensor of myoglobin-derived heme in rhabdomyolysis-induced AKI. This previously unknown mechanism might be targeted for treatment of the disease. Finally, we found a new therapeutic tool for prevention of AKI after rhabdomyolysis, which might rescue some sufferers of this pathology.

CD18 integrins promote neutrophil recruitment, and their engagement activates tyrosine kinases, leading to neutrophil activation. However, the significance of integrin-dependent leukocyte activation in vivo has been difficult to prove. Here, in a model of thrombohemorrhagic vasculitis, the CD18 integrin Mac-1 on neutrophils recognized complement C3 deposited within vessel walls and triggered a signaling pathway involving the Src-family kinase Hck and the Syk tyrosine kinase. This led to neutrophil elastase release, causing hemorrhage, fibrin deposition, and thrombosis. Mice genetically deficient in any of these components (C3, Mac-1, Hck, Syk, or elastase) were resistant to disease despite normal tissue neutrophil accumulation. Disease was restored in Mac-1-deficient mice infused with wild-type, but not kinase- or elastase-deficient, neutrophils. Elastase release in the inflamed tissue was reduced in Mac-1-deficient mice, and a deficiency of Mac-1 or the kinases blocked neutrophil elastase release in vitro. These data suggest that Mac-1 engagement of complement activates tyrosine kinases to promote elastase-dependent blood vessel injury in vivo. © 2006 Elsevier Inc. All rights reserved.

BACKGROUND-: Inflammation and thrombosis coexist in several disorders. Although it is recognized that leukocytes may induce a procoagulant state at sites of inflammation, the critical molecular determinants of this process remain largely unknown. METHODS AND RESULTS-: To examine mechanisms of inflammation-induced thrombosis, we developed a murine model of thrombotic glomerulonephritis (TGN), a known cause of acute renal failure in patients. This model, induced by lipopolysaccharide and antibody to the glomerular basement membrane, led to rapid glomerular neutrophil recruitment, thrombotic glomerular lesions with endothelial cell injury, and renal dysfunction. In mice immunodepleted of neutrophils or lacking the leukocyte-specific integrin Mac-1, neutrophil recruitment, endothelial injury, glomerular thrombosis, and acute renal failure were markedly attenuated despite the robust generation of renal cytokines. Neutrophil elastase is a likely effector of Mac-1 because its activity was reduced in Mac-1-deficient mice and the phenotype in mice deficient in Mac-1 or neutrophil elastase was similar. Platelets accumulated in glomerular capillaries within 4 hours of TGN before evidence of thrombosis. Platelet immunodepletion before TGN markedly exacerbated hematuria (hemorrhage), inflammation, and injury, whereas thrombocytopenic Mac-1-deficient mice remained resistant to disease, indicating that initial glomerular platelet deposition protects the vessel wall from neutrophil-mediated sequelae. The subsequent thrombosis relied on the interaction of Mac-1 on recruited neutrophils with glycoprotein Ibα on platelets as antibody-mediated disruption of this interaction attenuated TGN without affecting renal neutrophil accumulation. CONCLUSIONS-: These observations establish Mac-1 on neutrophils as a critical molecular link between inflammation and thrombosis and suggest it as an attractive target for antithrombotic therapy.

Neutrophils are central players in the innate immune system. They generate neutrophil extracellular traps (NETs), which protect against invading pathogens but are also associated with the development of autoimmune and/or inflammatory diseases and thrombosis. Here, we report that lactoferrin, one of the components of NETs, translocated from the cytoplasm to the plasma membrane and markedly suppressed NETs release. Furthermore, exogenous lactoferrin shrunk the chromatin fibers found in released NETs, without affecting the generation of oxygen radicals, but this failed after chemical removal of the positive charge of lactoferrin, suggesting that charge-charge interactions between lactoferrin and NETs were required for this function. In a model of immune complex-induced NET formation in vivo, intravenous lactoferrin injection markedly reduced the extent of NET formation. These observations suggest that lactoferrin serves as an intrinsic inhibitor of NETs release into the circulation. Thus, lactoferrin may represent a therapeutic lead for controlling NETs release in autoimmune and/or inflammatory diseases.

IL-15, a T cell growth factor, has been linked to exacerbating autoimmune diseases and allograft rejection. To test the hypothesis that IL-15-deficient (IL-15-/-) mice would be protected from T cell-dependent nephritis, we induced nephrotoxic serum nephritis (NSN) in IL-15-/- and wild-type (IL-15+/+) C57BL/6 mice. Contrary to our expectations, IL-15 protects the kidney during this T cell-dependent immunologic insult. Tubular, interstitial, and glomerular pathology and renal function are worse in IL-15-/- mice during NSN. We detected a substantial increase in tubular apoptosis in IL-15-/- kidneys. Moreover, macrophages and CD4 T cells are more abundant in the interstitia and glomeruli in IL-15-/- mice. This led us to identify several mechanisms responsible for heightened renal injury in the absence of IL-15. We now report that IL-15 and the IL-15 receptor (α,β,γ chains) are constitutively expressed in normal tubular epithelial cells (TECs). IL-15 is an autocrine survival factor for TECs. TEC apoptosis induced with anti-Fas or actinomycin D is substantially greater in IL-15-/- than in wild-type TECs. Moreover, IL-15 decreases the induction of a nephritogenic chemokine, MCP-1, that attracts leukocytes into the kidney during NSN. Taken together, we suggest that IL-15 is a therapeutic for tubulointerstitial and glomerular kidney diseases.

Endothelial cell-cell junctional proteins and cortical actin are of central importance for regulating vascular permeability. Rap1, a member of the Ras family of GTPases, is enriched at endothelial cell-cell contacts and activated by cyclic AMP (cAMP) through a PKA-independent pathway. Activation of a cAMP-inducible gua nine-exchange factor for Rap, Epac, results in markedly enhanced basal endothelial barrier function by increasing cortical actin and subsequent redistribution of adherens and tight junctional molecules to cell-cell contacts. Activation of Epac also counteracts thrombin-induced hyperpermeability through down-regula tion of Rho GTPase activation, suggesting cross-talk between Rap and Rho GTPases. Thus, Epac/Rap activation represents a new pathway for regulating endothelial cell barrier function.

Background. Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-κB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-κB affects TNF-α- induced apoptosis in rat mesangial cells (MCs). Methods. To specifically inhibit NF-κB activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κB. Electrophoretic mobility shift assay was performed to evaluate NF-κB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results. TNF-α-induced NF-κB activation was blocked by overexpression of IκBΔN. Overexpression of IκBΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α- induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion. Overexpression of IκBΔN potentiates TNF-α- induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκBΔN to induce apoptosis in MCs under inflammatory conditions.

Phagocytosis of complement-opsonized targets is a primary function of neutrophils at sites of inflammation, and the clearance of neutrophils that have phagocytosed microbes is important for the resolution of inflammation. Our previous work suggests that phagocytosis leads to rapid neutrophil apoptosis that is inhibited by antibody to the β2 integrin, Mac-1, and requires NADPH oxidase-derived reactive oxygen species (ROS) generated during phagocytosis. Here we report that phagocytosis-induced cell death (PICD) does not occur in Mac-1-deficient murine neutrophils, suggesting that PICD proceeds through a bona fide Mac-1-dependent pathway. A sustained, intracellular oxidative burst is associated with PICD. Furthermore, PICD does not require traditional death receptors, Fas, or tumor necrosis factor (TNF) receptor. TNF but not Fas synergizes with phagocytosis to enhance significantly PICD by increasing the oxidative burst, and this is Mac-1-dependent. Phagocytosis-induced ROS promote cleavage/activation of caspases 8 and 3, key players in most extrinsic ("death receptor") mediated pathways of apoptosis, and caspases 8 and 3 but not caspase 9/mitochondria, are required for PICD. This suggests that ROS target the extrinsic versus the intrinsic ("stress stimulus") apoptotic pathway. Phagocytosis also triggers a competing MAPK/ERK-dependent survival pathway that provides resistance to PICD likely by down-regulating caspase 8 activation. The anti-apoptotic factor granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhances ROS generation associated with phagocytosis. Despite this, it completely suppresses PICD by sustaining ERK activation and inhibiting caspase 8 activation in phagocytosing neutrophils. Together, these studies suggest that Mac-1-mediated phagocytosis promotes apoptosis through a caspase 8/3-dependent pathway that is modulated by NADPH oxidase-generated ROS and MAPK/ERK. Moreover, TNF and GM-CSF, likely encountered by phagocytosing neutrophils at inflammatory sites, exploit pro-(ROS) and anti-apoptotic (ERK) signals triggered by phagocytosis to promote or suppress PICD, respectively, and thus modulate the fate of phagocytosing neutrophils.

Background. Severe proteinuria not only indicates the presence of progressive glomerular disease, but also causes tubular epithelial cells to produce inflammatory mediators leading to tubulointerstitial (TI) injury. We investigated the role of nuclear factor-κB (NF-κB) in tubular epithelial cells in the development of proteinuria-induced TI injury. Methods. To specifically inhibit NF-κB activation, a recombinant adenovirus vector expressing a truncated form of IκBα (AdexIκBδN) was injected into renal arteries of protein-overloaded rats, a model of TI injury characterized by infiltration of mononuclear cells and fibrosis. Results. Activation of NF-κB in the renal cortex, observed in protein-overloaded rats treated with a control vector, recombinant lacZ adenovirus, was prevented in AdexIκBΔN-injected rats. Microscopic examination revealed AdexIκBΔN treatment to markedly attenuate proteinuria-induced TI injury. Increased immunostaining of vascular cell adhesion molecule-1, transforming growth factor-β, and fibronectin in TI lesions also was suppressed by AdexIκBΔN injection. Conclusions. These findings provide evidence of the critical role of NF-κB activation in TI injury and suggest the therapeutic potential of adenovirus-mediated IκBΔN gene transfer into the kidney as a means of interrupting the process of TI damage.

The signals linking neutrophil opsonic receptors, FcγRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcγRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcγRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcγR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcγR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions. Copyright © 2008 by The American Association of Immunologists, Inc.

Small-vessel vasculitis is a life-threatening autoimmune disease that is frequently associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Conventional immunotherapy including steroids and cyclophosphamide can cause serious adverse events, limiting the efficacy and safety of treatment. Eicosapentaenoic acid (EPA), a key component of fish oil, is an omega-3 polyunsaturated fatty acid widely known to be cardioprotective and beneficial for vascular function. We report two elderly patients with systemic ANCA-associated vasculitis (AAV) in whom the administration of EPA in concert with steroids safely induced and maintained remission, without the use of additioal immunosuppressants. To explore the mechanisms by which EPA enhances the treatment of AAV, we employed SCG/Kj mice as a spontaneous murine model of AAV. Dietary enrichment with EPA significantly delayed the onset of crescentic glomerulonephritis and prolonged the overall survival. EPA-derived anti-inflammatory lipid mediators and their precursors were present in the kidney, plasma, spleen, and lungs in the EPA-treated mice. Furthermore, a decrease in ANCA production and CD4/CD8-double negative T cells, and an increase in Foxp3+ regulatory T cells in the lymph nodes of the kidney were observed in the EPA-treated mice. These clinical and experimental observations suggest that EPA can safely support and augment conventional therapy for treating autoimmune small-vessel vasculitis.

To investigate the interaction between endothelin (ET) and the nitric oxide system, we examined the effects of ET-1 and ET-3 on the induction of inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GTP:CHI), the rate-limiting enzyme of de novo synthesis of the cofactor tetrahydrobiopterin (BH4), in rat mesangial eels. ET-1 inhibited the nitrite accumulation induced by a combination of interleukin-1β, tumor necrosis factor-α, and lipopolysaccharide in a concentration-dependent manner. The inhibitory effect of ET-3 was less potent than that of ET-1. A selective ET(A) antagonist, BQ-485, and an ET(A) and ET(B) antagonist, TAK-044, abolished the inhibitory effects of ET-1, whereas the selective ET(B) antagonist BQ-788 had no effect on the inhibition produced by ET-1. These observations indicate that ET-1 inhibits cytokine-stimulated nitrite accumulation through the ETA receptor. Western blot analysis showed that the suppression of nitrite accumulation was accompanied by a decrease in iNOS protein. Northern blot analysis showed that ET-1 inhibited the expression of both iNOS and GTP:CHI mRNA. In conclusion, ET-1 inhibits cytokine-stimulated nitric oxide production through the ETA receptor by suppressing the expression of iNOS and GTP:CHI mRNA in rat mesangial cells.

Cytokines and cytokine-induced nitric oxide (NO) play important roles in inflammatory glomerular diseases, and both platelet-derived growth factor and transforming growth factor-β inhibit cytokine-induced NO production. In this study, we demonstrated that a selective endothelin ET(A) receptor antagonist, BQ-485 (Hexahydro-1H-azepinylcarbonyl-Leu-d-Trp-d-Trp-OH), reversed the inhibitory effect of platelet-derived growth factor on cytokine-induced NO production, but not that of transforming growth factor-β. Our findings suggest a difference between the inhibitory mechanisms of platelet-derived growth factor and transforming growth factor-β on cytokine-induced NO production. Copyright (C) 1999 Elsevier Science B.V.

A 65-year-old-woman presented with edema, ascites, proteinuria and abnormal liver function tests. A small amount of mixed cryoglobulin was detected in her serum. Liver biopsy revealed mild chronic active hepatitis, but tests for hepatotropic viral infection were negative. Electron microscopy of the renal biopsy revealed glomerular electrondense deposits that contained numerous tubular structures. Renal amyloidosis and light chain deposition disease were ruled out by appropriate histological techniques. The ultrastructural findings of renal biopsy suggested either cryoglobulinemic glomerulonephritis or immunotactoid glomerulopathy. Although the exact interrelationship among the peculiar glomerulopathy, cryoglobulinemia and chronic active hepatitis in the present case remains undetermined, this report enlarges the spectrum of glomerulopathy characterized by extracellular deposition of microtubules.

The acute onset of idiopathic nephrotic syndrome (NS) is often accompanied by acute kidney injury, which can lead to congestive heart failure and lung edema. In this report, we present two cases of NS-induced acute kidney injury successfully treated with a low dose of carperitide, a human atrial natriuretic peptide. In combination with standard diuretic therapy and immunotherapy, carperitide retained the renal function and spared the need for renal replacement therapy, including hemodialysis. Although further investigation in clinical trials is required to validate these findings, carperitide may be useful for maintaining the renal function in cases of NS-induced acute kidney injury.

Immunoglobulin (Ig) A nephropathy is a prevalent form of primary glomerulonephritis, which leads to end-stage renal failure in a significant proportion of patients. Immunotherapy, including steroid use, is widely used to induce disease remission; however, it can cause serious side effects. We herein report 3 cases of progressive IgA nephropathy and their successful treatment with a combination of aspirin and eicosapentaenoic acid (EPA) without the use of steroids. The precise mechanism responsible for the combination therapy is still unknown; however, aspirin may potentiate the production of anti-inflammatory lipid mediators derived from EPA. Further clinical trials are required to substantiate this treatment regimen.