Shinya Nagasaka

Inhibition of matrix metalloproteinases reduces ischemia-reperfusion acute kidney injury.

Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix and involved in ischemic organ injuries. The present study was designed to determine the role of MMP-2 in the development of ischemic acute kidney injury (AKI). AKI was induced in MMP-2 wild-type (MMP-2+/+) mice by 30, 60, 90, and 120 min renal ischemia and reperfusion. Renal histology, expression and activity of MMP-2 and MMP-9, and renal function were examined during the development of AKI. AKI was also induced in MMP-2-deficient (MMP-2−/−) mice and MMP-2+/+ mice treated with inhibitor of MMPs (minocycline and synthetic peptide MMP inhibitor). In MMP-2+/+ mice, MMP-2 and MMP-9 activities increased significantly at 2 to 24 h, peaked at 6 h, after reperfusion. Immunohistochemical analysis identified MMP-2 in the interstitium around tubules and peritubular capillaries in the outer medulla. Acute tubular injury (ATI), including apoptosis and necrosis, was evident in the outer medulla at 24 h, along with renal dysfunction. As ischemia period increases, MMP-2 and MMP-9 activities at 6 h and severity of AKI at 24 h increased depending on the duration of ischemia between 30 and 120 min. However, the kidneys of MMP-2−/− mice showed minimal ATI; serum creatinine 24 h after reperfusion was significantly low in these mice. Inhibitors of MMPs reduced ATI and improved renal dysfunction at 24 h. We conclude that MMPs, especially MMP-2 have a pathogenic role in ischemia-reperfusion AKI, and that inhibitors of MMPs can protect against ischemic AKI.

Statin Attenuates Experimental Anti-Glomerular Basement Membrane Glomerulonephritis Together with the Augmentation of Alternatively Activated Macrophages

Macrophages are heterogeneous and include classically activated M1 and alternatively activated M2 macrophages, characterized by pro- and anti-inflammatory functions, respectively. Macrophages that express heme oxygenase-1 also exhibit anti-inflammatory effects. We assessed the anti-inflammatory effects of statin in experimental anti-glomerular basement membrane glomerulonephritis and in vitro, focusing on the macrophage heterogeneity. Rats were induced anti-glomerular basement membrane glomerulonephritis and treated with atorvastatin (20 mg/kg/day) or vehicle (control). Control rats showed infiltration of macrophages in the glomeruli at day 3 and developed crescentic glomerulonephritis by day 7, together with increased mRNA levels of the M1 macrophage-associated cytokines, interferon-gamma, tumor necrosis factor-alpha, and interleukin-12. In contrast, statin reduced the level of proteinuria, reduced infiltration of macrophages in glomeruli with suppression of monocyte chemotactic protein-1 expression, and inhibited the formation of necrotizing and crescentic lesions. The number of glomerular ED3-positive macrophages decreased with down-regulation of M1 macrophage-associated cytokines. Furthermore, statin augmented ED2-positive M2 macrophages with up-regulation of the M2 macrophage-associated chemokines and cytokines, chemokine (C-C motif) Iigand-17 and interleukin-10. Statin also increased the glomerular interleukin-10-expressing heme oxygenase-1-positive macrophages. Statin inhibited macrophage development, and suppressed ED3-positive macrophages, but augmented ED2-positive macrophages in M2-associated cytokine environment in vitro. We conclude that the anti-inflammatory effects of statin in glomerulonephritis are mediated through inhibition of macrophage infiltration as well as augmentation of anti-inflammatory macrophages.

Inhibition of capillary repair in proliferative glomerulonephritis results in persistent glomerular inflammation with glomerular sclerosis

The pathological process of glomerulonephritis (GN) includes glomerular capillary damage, and vascular endothelial growth factor (VEGF) has an important role in glomerular capillary repair in GN. We examined the effect of inhibition of glomerular capillary repair after capillary injury in GN. Experimental Thy-1 GN was induced in rats that were divided into two groups: rats that received anti-VEGF neutralizing antibody (50 μg per 100 g body weight per day) and those treated with the vehicle from day 2 to day 9. We assessed the renal function and histopathology serially until week 6. Rats of the Thy-1 GN group showed diffuse glomerular mesangiolysis with ballooning destruction of the capillary network by day 3. VEGF164 protein levels increased in the damaged glomeruli during days 5 to 10, and endothelial-cell proliferation increased with capillary repair in the vehicle-injected group. Proliferative GN resolved subsequently with decreased mesangial hypercellularity, and recovery of most of the glomeruli to the normal structure was evident by week 6. In contrast, administration of anti-VEGF antibody significantly decreased endothelial-cell proliferation and capillary repair in glomeruli by week 2. Thereafter, glomerular mesangial-cell proliferation and activation continued with persistent infiltration of macrophages. At week 6, segmental glomerular sclerosis developed with mesangial matrix accumulation and proteinuria. Deposition of type I collagen was also noted in sclerotic lesions. We conclude that impaired capillary repair was the underlying mechanism in the prolongation of glomerular inflammation in proliferative GN and in the development of glomerular sclerosis. Capillary repair has an important role in the recovery of glomerular damage and in the resolution of proliferative GN.

Proliferative glomerulonephritis with monoclonal immunoglobulin G3κ deposits in association with parvovirus B19 infection

Proliferative glomerulonephritis with monoclonal immunoglobulin G deposits is a recently described disease entity, characterized by nonorganized electron-dense deposits in glomeruli and immunofluorescence findings indicating monoclonal immunoglobulin G deposits. The pathogenesis of many cases of proliferative glomerulonephritis with monoclonal immunoglobulin G deposits remains unknown. We herein report 2 patients with parvovirus B19 infection who developed acute nephritic syndrome with hypocomplementemia (patient 1) or persistent proteinuria and congestive heart failure (patient 2); however, neither patient had detectable levels of serum monoclonal immunoglobulin G. Renal biopsy in both patients showed diffuse endocapillary proliferative glomerulonephritis with monoclonal immunoglobulin G3κ deposits, and electron microscopy showed nonorganized electron-dense deposits mainly in the subendothelial and mesangial areas. Clinical symptoms, abnormal laboratory findings, and urinary abnormalities recovered spontaneously in both cases within 4 weeks. Our 2 cases may be the first reported patients with proliferative glomerulonephritis with monoclonal immunoglobulin G deposits possibly associated with parvovirus B19 infection. Virus infection-associated immune disorders could be implicated in the pathogenesis of proliferative glomerulonephritis with monoclonal immunoglobulin G deposits.

Lymphangiogenesis Associated With Acute Cellular Rejection in Rat Liver Transplantation

Lymphangiogenesis may be important for the cellular immune response in liver transplantation. In the present study, we examined lymphangiogenesis in liver allografts displaying acute cellular rejection (ACR), or long-term acceptance, or severe ACR plus antibody-mediated rejection (AMR). ACR and subsequent long-term graft acceptance developed in liver transplantations from DA to PVG rats without immunosuppression (mean survival time more than 90 days). Severe ACR and AMR developed in liver transplantations from DA to Lewis rats without immunosuppression (mean survival = 11 days). Normal DA donor livers before transplantation showed a small number of lymphatic vessels around portal veins. DA liver grafts in PVG showed ACR with lymphangiogenesis in portal areas and portal-portal bridging areas with cellular infiltration. Newly formed lymphatic vessels in ACR were characterized by proliferating endothelial cells with expression of the homeobox transcription factor PROX-1 and surrounded by discontinuous basement membranes. Thereafter, the infiltrates spontaneously disappeared, and the grafts survived more than 90 days. During the resolution of the cellular infiltration, expanded lymphatic vessels were packed with many lymphocytes. Thereafter, the number of lymphatic vessels decreased. In contrast, severe ACR and AMR in DA-to-Lewis transplantations showed lymphatic vessels disappeared with edema in the portal areas at day 11. In conclusion, lymphangiogenesis occurred during ACR. It may be involved in the resolution of ACR and reduction of inflammation. In severe ACR and AMR, lymphatic vessels were destroyed, which may be involved in persistent severe inflammation.

Role of survivin in acute lung injury: epithelial cells of mice and humans

Survivin, an inhibitor of apoptosis, regulates cell division and is a potential target for anticancer drugs because many cancers express high survivin levels. However, whether survivin would be toxic to human lung cells and tissues has not been determined. This report clarified the involvement of survivin in acute lung injury. We used immunohistochemical analysis, immunoelectron microscopy, and real-time reverse transcription-quantitative polymerase chain reaction to study survivin expression and localization in injured mouse and human lungs. We also used cultured human lung epithelial cells (BEAS-2B and A549) to study survivin cytoprotection. Nuclei and cytoplasm of epithelial cells in day 3 and day 7 models of bleomycin-injured lung showed survivin-positive results, which is consistent with upregulated survivin mRNA expression. These nuclei also evidenced double positive findings for proliferating cell nuclear antigen and survivin. Day 7 models had similar Smac/DIABLO-positive and survivin-positive cell distributions. The cytoplasm and nuclei of epithelial cells in lesions with diffuse alveolar damage manifested strong survivin-positive findings. Bleomycin stimulation in both epithelial cell lines upregulated expression of survivin and apoptosis-related molecules. Suppression of survivin expression with small interfering RNA rendered human lung epithelial cells susceptible to bleomycin-induced damage, with markedly upregulated activation of caspase-3, caspase-7, poly (ADP-ribose) polymerase, and lactate dehydrogenase activity and an increased number of dead cells compared with mock small interfering RNA-treated cells. Overexpression of survivin via transfection resulted in these epithelial cells being resistant to bleomycin-induced cell damage, with reduced activation of apoptosis-related molecules and lactate dehydrogenase activity and fewer dead cells compared with results for mock-transfected cells. Survivin, acting at the epithelial cell level that depends partly on apoptosis inhibition, is therefore a key mediator of cytoprotection in acute lung injury. Understanding the precise role of survivin in normal lung cells is required for the development of therapeutic survivin.

Acute Graft-Versus-Host Disease of the Kidney in Allogeneic Rat Bone Marrow Transplantation

Allogeneic hematopoietic cell or bone marrow transplantation (BMT) causes graft-versus-host-disease (GVHD). However, the involvement of the kidney in acute GVHD is not well-understood. Acute GVHD was induced in Lewis rats (RT1l) by transplantation of Dark Agouti (DA) rat (RT1a) bone marrow cells (6.0×107 cells) without immunosuppression after lethal irradiation (10 Gy). We examined the impact of acute GVHD on the kidney in allogeneic BMT rats and compared them with those in Lewis-to-Lewis syngeneic BMT control and non-BMT control rats. In syngeneic BMT and non-BMT control rats, acute GVHD did not develop by day 28. In allogeneic BMT rats, severe acute GVHD developed at 21–28 days after BMT in the skin, intestine, and liver with decreased body weight (>20%), skin rush, diarrhea, and liver dysfunction. In the kidney, infiltration of donor-type leukocytes was by day 28. Mild inflammation characterized by infiltration of CD3+ T-cells, including CD8+ T-cells and CD4+ T-cells, and CD68+ macrophages to the interstitium around the small arteries was noted. During moderate to severe inflammation, these infiltrating cells expanded into the peritubular interstitium with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis. Renal dysfunction also developed, and the serum blood urea nitrogen (33.9±4.7 mg/dL) and urinary N-acetyl-β-D-glucosaminidase (NAG: 31.5±15.5 U/L) levels increased. No immunoglobulin and complement deposition was detected in the kidney. In conclusion, the kidney was a primary target organ of acute GVHD after BMT. Acute GVHD of the kidney was characterized by increased levels of urinary NAG and cell-mediated injury to the renal microvasculature and renal tubules.

Hepatic artery reconstruction prevents ischemic graft injury, inhibits graft rejection, and mediates long-term graft acceptance in rat liver transplantation.

BACKGROUND Hepatic artery (HA) reconstruction is performed in the clinical liver transplantation. METHODS We assessed the importance of HA reconstruction in the success of liver transplantation. Orthotopic liver transplantation was performed without immunosspression from Lewis (RT1l) to Lewis rats (syngeneic transplantation) as well as Lewis to BN (RT1n) rats (allogeneic transplantation) with or without HA reconstruction. We examined graft function, pathology, and mRNA levels using DNA arrays in both arterialized and nonarterialized liver grafts. RESULTS In Lewis-to-Lewis syngeneic grafts, both the arterialized and nonarterialized grafts survived >120 days with normal graft function. lnfiltration of CD3(+) T cells and CD68(+) macrophages, marked bile duct proliferation with apoptotic epithelial cells, and expansion and increasing fibrosis of portal areas were evident in the nonarterialized grafts at day 120, although preservation of architecture was noted in the arterialized grafts. DNA array analysis of nonarterialized syngeneic grafts demonstrated the upregulation of mRNA of cell death-related proteins, cell cycle-related proteins, and inflammation-related proteins than those in arterialized grafts. Moreover, the arterialized Lewis-to-BN allogeneic grafts could survive for a long time with less severe graft dysfunction than those in non-arterialized allogeneic grafts. CONCLUSIONS HA reconstruction in liver transplantation inhibited hypoxic injury and subsequent inflammation and bile duct proliferation, prevented the augmentation of T-cell-and antibody-mediated rejection, and mediated long-term graft acceptance. HA reconstruction is essential factor in the success of liver transplantation.

The Pathological Characteristics of Acute Antibody-Mediated Rejection in DA-to-Lewis Rat Orthotopic Liver Transplantation

The category of acute antibody-mediated rejection (AMR) is not included in the Banff classification of liver transplantation pathology. We investigated the pathology of acute AMR using an orthotopic rat liver transplantation from DA-to-Lewis rats without immunosuppression. We studied liver graft samples at days 5, 7, and 9 to 11, focusing on the pathological characteristics of acute AMR. Progressive acute cellular rejection and AMR led to irreversible graft failure by day 11 ± 2. At day 5 immunoglobulin G (IgG) was deposited on endothelial cells in the portal veins and small arteries. Thereafter, at day 7 to day 11 the IgG deposition expanded on endothelial cells in portal veins and hepatic arteries, epithelial cells in bile ducts, sinusoids and hepatic cells in lobules. Light microscopic studies during the development of acute AMR showed interstitial edema in portal areas with neutrophilic infiltration. Rejecting grafts revealed congestion and/or thrombi in portal veins and hepatic arteries with neutrophil infiltration and fibrinogen deposition, severe degeneration of epithelial cells in bile ducts with periductal edema, intralobular edema, and hemorrhage with neutrophil infiltration and fibrinogen deposition, as well as hepatic cell degeneration and necrosis. In conclusion, acute AMR that developed in liver transplantation was characterized by endothelial cell injuries in microvasculature of portal veins, hepatic arteries, and sinusoids, accompanied by congestion, hemorrhage, thrombus formation, and neutophilic infiltration, as well as by bile duct and hepatic cell degeneration and necrosis.

Impact of anti-glomerular basement membrane antibodies and glomerular neutrophil activation on glomerulonephritis in experimental myeloperoxidase-antineutrophil cytoplasmic antibody vasculitis

BACKGROUND Antineutrophil cytoplasmic antibody (ANCA) and neutrophil interactions play important roles in ANCA-associated vasculitis (AAV) pathogenesis. However, mechanisms underlying the pathogenesis of crescent formation in ANCA-associated vasculitis have not been completely elucidated. To ascertain the involvement of these interactions in necrotizing crescentic glomerulonephritis (NCGN), we used an AAV rat model and investigated the effects of the anti-myeloperoxidase (MPO) antibody (Ab) titer, tumor necrosis factor α (TNF-α), granulocyte colony-stimulating factor (G-CSF) and subnephritogenic anti-glomerular basement membrane (GBM) Abs, as proinflammatory stimuli. METHODS NCGN was induced in Wistar Kyoto rats by human MPO (hMPO) immunization. Renal function, pathology, and glomerular cytokine and chemokine expression were evaluated in hMPO-immunized rats with/without several co-treatments (TNF-α, G-CSF or subnephritogenic anti-GBM Abs). Rat neutrophils activation by IgG purified from rat serum in each group was examined in vitro. RESULTS The hMPO-immunized rats had significantly higher level of anti-hMPO Ab production. The induced anti-hMPO Abs cross-reacted with TNF-α- or G-CSF-primed rat neutrophils secreting TNF-α and interleukin-1β in vitro. The reactivity of anti-MPO Abs against rat MPO, crescent formation with neutrophil extracellular traps and glomerular-activated neutrophil infiltration in the rat model were significantly enhanced by subnephritogenic anti-GBM Ab but not by TNF-α or G-CSF administration. The model rats injected with the subnephritogenic anti-GBM Abs showed increased urinary albumin excretion and serum TNF-α, chemokine (C-X-C) ligand 1 (CXCL1) and CXCL2 levels. TNF-α, CXCL1, CXCL2 and CXCL8 increased in the glomeruli with significant amounts of crescent formation. In addition, in vitro activated neutrophils decreased CXC chemokine receptor 1 (CXCR1) and CXCR2 expressions. CONCLUSIONS The coexistence of subnephritogenic anti-GBM Abs leads to the inflammatory environment in glomeruli that is amplified by the interaction of ANCA and neutrophils. Development of NCGN in MPO-AAV may be necessary for not only the accumulation of neutrophils in glomeruli, but also the aberrant neutrophil activation on glomerulonephritis.