坪井 直毅

Current studies suggest that mesenchymal stromal cells (MSCs) improve acute kidney injury (AKI) via paracrine/endocrine effects. We established human adipose tissue-derived stromal cells (hASCs) cultured in low (2%) serum (hLASCs), which have great potential of tissue regeneration. The present study was performed to investigate the therapeutic effects of hLASCs on AKI and to clarify the mechanisms involved. In low serum, hASCs proliferated well, while human bone marrow-derived stromal cells (hBMSCs) did not. hLASCs secreted higher levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) than did hASCs cultured in high (20%) serum (hHASCs) or hBMSCs cultured in high serum (hHBMSCs). AKI was induced in nude rats by folic acid, and hLASCs, hHASCs or control medium were administered into the renal subcapsules. hLASCs significantly attenuated acute renal damage, while hHASCs showed far less effect. Furthermore, interstitial fibrosis observed on day 14 was less pronounced in the hLASCs group. Cell tracking experiment showed no evidence of transdifferentiation. Intravenous injection of hLASCs or hHBMSCs or subcapsular injection of hHBMSCs did not ameliorate AKI. Concerning the mechanisms, our in vivo experiments showed that HGF knockdown by siRNA impaired the ability of hLASCs to protect the kidney from acute injury whereas VEGF knockdown did not. In conclusion, hLASCs, but not hHASCs or hHBMSCs, ameliorated AKI via paracrine effects, and HGF is one of the key mediators.

Pozzi et al. reported the effectiveness of steroid pulse therapy (Pozzi's regimen) in IgA nephropathy (IgAN). The present study was performed to clarify the predictive factors for IgAN patients treated with Pozzi's regimen. One hundred nine IgAN patients treated by Pozzi's regimen were observed for up to 112.6 (median 39.7) months, and remission of proteinuria (PR) and disappearance of urinary abnormalities [complete remission (CR)] after Pozzi's regimen were analyzed. Predictive factors for the glomerular filtration rate (GFR) slopes for up to 5 years were analyzed among 81 patients who were observed for at least 2 years. The outcome of a 50 % increase in sCr was compared between the CR and non-CR groups within 2 years. Cumulative PR and CR rates increased rapidly until 2 years (54.5 and 46.8 % at 2 years), and then slowly but steadily up to 6 years (72.8 and 66.4 % at 6 years). Baseline characteristics of the CR and non-CR groups within 2 years were similar except for proteinuria. GFR slope was steeper in the non-CR group than in the CR group (-2.44 +/- A 5.12 vs. -0.32 +/- A 3.34 ml/min/1.73 m(2)/year). On multivariate analysis, sex and CR within 2 years were associated with GFR slope. Kaplan-Meier analysis demonstrated a better survival rate in CR group patients without a 50 % increase in sCr (p = 0.024). Among IgAN patients treated with Pozzi's regimen, CR within 2 years predicts a good prognosis.

Systemic lupus erythematosus (SLE) is a chronic, multiorgan inflammatory autoimmune disorder associated with high levels of circulating autoantibodies and immune complexes. We report that passive transfer of human SLE sera into mice expressing the uniquely human Fc gamma RIIA and Fc gamma RIIIB on neutrophils induces lupus nephritis and in some cases arthritis only when the mice additionally lack the CD18 integrin, Mac-1. The prevailing view is that Mac-1 on macrophages is responsible for immune complex clearance. However, disease permitted by the absence of Mac-1 is not related to enhanced renal immune complex deposition or in situ C1q/C3 complement activation and proceeds even in the absence of macrophages. Instead, disease is associated with increased Fc gamma RIIA-induced neutrophil accumulation that is enabled by Mac-1 deficiency. Intravital microscopy in the cremasteric vasculature reveals that Mac-1 mitigates Fc gamma RIIA-dependent neutrophil recruitment in response to deposited immune complexes. Our results provide direct evidence that human SLE immune complexes are pathogenic, demonstrate that neutrophils are primary mediators of end organ damage in a novel humanized lupus mouse model, and identify Mac-1 regulation of Fc gamma RIIA-mediated neutrophil recruitment as a key step in development of target organ damage. The Journal of Immunology, 2012, 189: 3714-3723.

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein gamma (SIRP gamma) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD47(-/-) mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in a dermal air pouch model of TNF-alpha-induced inflammation. Reconstitution of CD47(-/-) mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD47(-/-) endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies showed CD47-dependent signaling required G(alpha i)-coupled pathways, this was not the case for endothelial CD47 because pertussis toxin, which inactivates G(alpha i), had no inhibitory effect, whereas G(alpha i) was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2-kinase dependent tyrosine phosphorylation of the vascular endothelial-cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent of G(alpha i). These findings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM. The Journal of Immunology, 2012, 189: 2553-2562.

腹膜透析(PD)における小・中・大分子量物質除去と腹膜機能との関連性について検討した。PD患者51例を対象とした。小分子量物質D/P ureaは、D/P Crに比し溶質透過性は高く、相関を認めた。腹膜平衡試験(PET)4時間値でD/P=1となった症倒も認めた。D/P CrとD/P phosphate、D/P β2-MGは極めて強い有意な相関を認めた。D/P Crと大分子量物質の相関については、D/P Crと比較するといずれも大分子量物質の透過性は、低かったが、D/P Alb、D/P Tf、D/P IgG、α2-MGすべてにおいて、D/P Crと有意な相関を認めた。膜透析における溶質透過牲について、PETによる腹膜機能の把握は重要で、D/P Crで中分子量物質のD/P β2-MGの予測ができる可能性が示唆された。

Background. Colonic diverticulitis is an important cause of polymicrobial peritonitis, which requires surgical treatment and cessation of peritoneal dialysis (PD). The aim of this study was to examine whether plain abdominal computed tomography (CT) is useful for evaluating colonic diverticulosis in chronic kidney disease (CKD) patients and to explore whether colonic diverticulosis is a risk factor for enteric peritonitis. Methods. The subjects consisted of 137 consecutive CKD patients (Stage 4 or 5) who were candidates for PD from February 2005 to November 2009. Abdominal CT without contrast media was performed in all PD candidates. Results. Diverticula of the colon were detected by plain CT in 57 cases (41.6%). The number of diverticula tended to increase with age. The most common site of involvement of diverticulosis was the ascending colon. In patients treated with PD, the incidence of peritonitis was higher in patients with diverticulosis than in those without diverticulosis (P = 0.004). However, only one episode of enteric peritonitis was observed among patients with diverticulosis. The presence of diverticulosis did not affect cumulative or technical survival. PD was not selected in four cases due to a high frequency of diverticula with episodes of abdominal pain. Two cases developed severe diverticulitis with peritonitis and underwent resection of the colon. Conclusions. Our study suggests that plain CT examination is useful for detecting diverticulosis in CKD patients. Silent diverticulosis is not a risk factor for enteric diverticulosis-related peritonitis. PD may be contraindicated in cases having frequent diverticulosis with episodes of lower abdominal pain.

Oxaliplatin is effective in advanced colorectal cancer and is known to have relatively few side effects, such as hemolysis and renal toxicity. We report a case of acute kidney injury (AKI) after treatment with a combination of oxaliplatin, folinic acid and 5-fluorouracil or capecitabine. The patient developed acute renal failure, hemolytic anemia and thrombocytopenia after the 34th course of chemotherapy including oxaliplatin. A positive direct antiglobulin test and detection of immunoglobulin G and complement C3b and C3d on erythrocytes suggested the diagnosis of immune-related severe intravascular hemolytic anemia. She was successfully treated and recovered using plasma exchange, corticosteroids and hemodialysis therapy. Only seven other cases of AKI associated with oxaliplatin use have been reported to date. As in this case, acute hemolysis due to autoimmune mechanisms and subsequent AKI occurred suddenly after frequent use of oxaliplatin in four of those cases. We should be aware that oxaliplatin may cause sudden life-threatening hemolysis by drug-induced antibodies and subsequent AKI, even though oxaliplatin is frequently administered without side effects. This represents the first case report of AKI-related hemolysis due to oxaliplatin in Japan.

We report a case of peritonitis resulting from colon perforation caused by ingestion of a rare foreign body in a patient on peritoneal dialysis (PD). A 72-year-old woman on PD was hospitalized with abdominal pain and cloudy PD fluid (PDF). Although conventional antibiotic therapy was started because of a diagnosis of infectious peritonitis, low-grade fever, abdominal pain and a high number of white blood cells in PDF persisted. On day 3, anaerobic bacteria were recognized on bacterial culture of PDF, suggesting a gastrointestinal etiology. During exploratory laparotomy, sigmoidal perforation by a piece of bamboo, probably resulting from ingestion of contaminated food, was found.

Growth factor Midkine (MK), which expresses on endothelial cells and renal proximal tubules, has been implicated in inflammation-related kidney diseases such as ischemic reperfusion-induced tubulointerstitial injury and diabetic nephropathy. The biological actions of MK are elicited through its chemotactic activity and chemokine-driven inflammatory pathway. Post-infectious glomerulonephritis is caused by the deposition of immune complexes into glomeruli by infiltrating a number of inflammatory cells. Therefore, we investigated whether MK might be involved in the pathogenesis of acute glomerulonephritis. We induced endocapillary proliferative glomerulonephritis in 129/SV mice using intraperitoneal injections of a large amount of protein. In contrast to mice deficient in MK (Mdk (-/-)), Mdk (+/+) mice induced by protein overload demonstrated more diffuse cellular proliferation in the mesangial areas and capillary lumens, eventually leading to glomerular damage and tubulointerstitial injury. This pathological observation could be attributable to neutrophil infiltration through the chemotaxis and stimulation of the MK-macrophage inflammatory protein (MIP)-2 pathway, but appeared to be due to the MK-related immunoglobulin (Ig)G deposition and C3 activation. These findings are often seen in infectious-related glomerular injury. Furthermore, the profile of MK expression was strongly consistent with that of glomerular damage and tubulointersititial injury. This study might provide a new insight into understanding the deleterious role of MK in endocapillary proliferative glomerulonephritis induced by protein overload.

Background: Although the immunomodulatory effects of mesenchymal stromal cells (MSC) on T cells have been elucidated, little is known about their effects on B cells. Recently, we have established a novel culture method for adipose-derived MSC (ASC) using low (2%) serum medium containing fibroblast growth factor-2. We showed that low serum-cultured ASC (LASC) was superior to high (20%) serum cultured ASC (HASC) when used in regenerative therapy. The aim of this study was to compare the action of LASC, HASC, and bone marrow-derived MSC (BM-MSC), on xenoantibody production by B cells. Methods: Adipose-derived mesenchymal stromal cells and BM-MSC were obtained from humans or F344 rats and expanded in a low-serum or a high-serum culture medium. Proliferation of human peripheral mononuclear cells (PBMC) or rat splenocytes was induced by phytohemagglutinin (PHA) or anti-IgM-antibody. These cells were then co-cultured with LASC, HASC, or BM-MSC, and cell proliferation was studied. Porcine red blood cells (pRBC) were intraperitoneally injected into Lewis rats, and LASC, HASC, or BM-MSC obtained from F344 rats were injected intravenously or intraperitoneally. The levels of antibodies (IgM and IgG) against pRBC were examined using flow cytometry. Results: Human LASC suppressed PBMC proliferation more effectively than human HASC. Human LASC suppressed both T-cell and B-cell proliferation when incubated with PHA (a T-cell stimulus). However, human LASC did not suppress B-cell proliferation after incubation with anti-IgM-antibody (a T-cell-independent stimulus). Rat LASC suppressed PHA-stimulated splenocyte proliferation more effectively than rat HASC or rat BM-MSC. In vivo studies showed that intravenous injection of rat LASC significantly reduced the levels of IgG antibodies against pRBC, while intravenous administration of the other two types of MSC (rat HASC or rat BM-MSC) or intraperitoneal administration of rat LASC did not impede IgG production. A significant number of LASC were observed in the spleen when injected intravenously while only a few LASC were observed when given intraperitoneally. Conclusions: Administration of LASC effectively impeded xenoantibody production by B cells through the inhibition of T-cell function, while HASC or BM-MSC showed less promising effects. These results suggest that intravenous injection of LASC may be useful in attenuating antibody-mediated rejection.

A 68-year-old man was admitted with acute renal failure caused by cholesterol embolization after undergoing carotid artery stenting. Hemodialysis therapy (HD) was immediately required because of uremia, using nafamostat mesilate as an anticoagulant for HD. However, blue toes and gangrene of the feet worsened. To prevent use of anticoagulants and stabilize BP, HD was changed to peritoneal dialysis (PD). After starting PD, blue toes and gangrene improved markedly. Residual renal function also partially recovered. Although BP was unstable during HD, stability of BP and avoidance of anticoagulants during PD therapy might have contributed to the good results.

A 54-year-old woman on peritoneal dialysis (PD) was hospitalized with peritonitis with a high body temperature, abdominal pain and cloudy peritoneal fluid. She progressively fell into septic-like shock within only 6 hours after onset. The causative bacteria were Streptococcus mitis (S. mitis), part of the normal flora of oral cavity, intestine, female genial tract and upper respiratory tract. S. mitis shows pathogenicity for diseases such as endocarditis, brain abscesses and sepsis in children with malignancy or transplantation. However, S. mitis rarely shows severe pathogenic responses in adults. We report herein a case of fulminant peritonitis caused by S. mitis in an adult PD patient.

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 Ib alpha 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. (Circulation. 2009;120:1255-1265.)

Aim: Cot/Tpl2, a serine/threonine (Ser/Thr) protein kinase, has been classified as a member of the mitogen-activated protein kinase (MAPK) family, and is known to have a pleiotropic role. Many studies have reported the involvement of Cot/Tpl2, mainly as a member of the Toll-like receptor (TLR) 4 signalling pathway in lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) production. At the same time, it is also related to the caspase-dependent apoptotic pathway. Thus, the role of Cot/Tpl2 in ischaemia/reperfusion injury (IRI) in which TNF-alpha and apoptosis are the major pathogenetic factors was studied. Methods: IRI was induced in wild type (Cot/Tpl2(+/+)) mice and in Cot/Tpl2-deficient (Cot/Tpl2(-/-)) mice. The extent of tubular injury and renal function were studied. TNF-alpha production, neutrophil infiltration and apoptosis were also compared between the two groups. Results: Cot/Tpl2(-/-) mice had preserved renal function compared with wild type mice in IRI. Although Cot/Tpl2 was phosphorylated in IRI and in the cultured tubular epithelial cells (TEC) after stimulation with LPS and hydrogen peroxide, there were no significant differences in terms of TNF-alpha production, neutrophil infiltration or MAPK activation between Cot/Tpl2(+/+) and Cot/Tpl2(-/-) mice. In contrast, Cot/Tpl2(-/-) mice showed obviously reduced terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling positive cells and cleaved caspase-3 positive cells. Furthermore, Cot/Tpl2-deficient TECs demonstrated significantly less caspase-3 activation after hydrogen peroxide stimulation with comparable caspase-9 activation to wild type TEC. Conclusion: Tpl2 did not function as a member of MAPK family, but as a promoter of apoptosis in IRI. These results suggest that Cot/Tpl2 could be a possible therapeutic target in IRI.

Inflammation mediated by anti body-antigen complexes contributes to autoimmune diseases. Mice deficient in the common Fc gamma-chain are protected from IgG-mediated glomerulonephritis and the reverse passive Arthus (RPA) reaction and FcR-bearing macrophages, and mast cells have been assigned primary roles in these processes. Here we demonstrate that neutrophil-selective transgenic expression of the two uniquely human neutrophil Fc gamma receptors (Fc gamma Rs), Fc gamma RIIA and Fc gamma RIIIB, in Fc gamma-chain-deficient mice restored susceptibility to progressive glomerulonephritis and the cutaneous RPA reaction. Fc gamma RIIIB and Fc gamma RIIA mediated neutrophil accumulation, whereas Fc gamma RIIA alone promoted organ injury. In a model of soluble immune complexes deposited within the vasculature, Fc gamma RIIIB was responsible for neutrophil slow rolling and adhesion whereas in the cremaster RPA, induced by both vascular and tissue soluble immune complexes, Fc gamma RIIA predominated. Thus, human Fc gamma Rs on neutrophils serve as molecular links between antibody and immunological disease, with Fc gamma RIIA promoting tissue injury and Fc gamma RIIIB and Fc gamma RIIA displaying specialized context-dependent functions in neutrophil recruitment.

Tissue-specific silencing of genes may be used for genetic engineering in mice and has possible therapeutic applications in humans. Current strategies in mice rely on Cre/IoxP technology requiring the generation of multiple transgenic lines and breeding strategies. Here, we describe the selective silencing of CD18, a leukocyte-specific integrin in neutrophils using a micro RNA(miRNA) strategy that requires the generation of one transgenic line. CD18-specific miRNA hairpin driven by the myeloid specific human MRP8 promoter resulted in the generation of transgenic lines with 75% to 95% reduction in CD18 protein levels in neutrophils and monocytes. Minimal decreases in T cells and a partial diminution in macrophages were observed. Neutrophil CD18 silencing resulted in neutrophilia, splenomegaly, and significant defects in neutrophil trafficking with the degree of alterations correlating with the extent of CD18 silencing. Thus, our data demonstrate the utility of using miRNA approaches to silence genes in neutrophils, which are terminally differentiated cells with a short half-life that largely pre- cludes their genetic manipulation in vitro. Furthermore, the mouse models provide a valuable tool to examine the contribution of CD18 on neutrophils to leukocyte adhesion deficiency type 1 (LAD-1), a complex inherited disorder in which reduced or absent CD18 expression in multiple leukocyte subsets leads to impaired innate and adaptive immune responses.

The programmed death 1/programmed death 1 ligand (PD-L) pathway is instrumental in peripheral tolerance. Blocking this pathway exacerbates experimental autoimmune diseases, but its role in autoimmune kidney disease has not been explored. Therefore, we tested the hypothesis that the programmed death 1 ligands (PD-L1 and PD-L2), provide a protective barrier during T cell-and macrophage (M phi)-dependent autoimmune kidney disease. For this purpose, we compared nephrotoxic serum nephritis (NSN) in mice lacking PD-L1 (PD-L1(-/-)), PD-L2 (PD-L2(-/-)), or both (PD-L1/L2(-/-)) to wild-type (WT) C57BL/6 mice. Kidney pathology, loss of renal function, and intrarenal leukocyte infiltrates were increased in each PD-L(-/-) strain as compared with WT mice. Although the magnitude of renal pathology was similar in PD-L1(-/-) and PD-L2-/- mice, our findings suggest that kidney disease in each strain is regulated by distinct mechanisms. Specifically, we detected increased CD68(+) cells along with elevated circulating IgG and IgG deposits in glomeruli in PD-L2(-/-) mice, but not PD-L1(-/-) mice. In contrast, we detected a rise in activated CD8(+) T cells in PD-L1(-/-) mice, but not PD-L2-/- mice. Furthermore, since PD-L1 is expressed by parenchymal and hemopoietic cells in WT kidneys, we explored the differential impact of PD-L1 expression on these cell types by inducing NSN in bone marrow chimeric mice. Our results indicate that PD-L1 expression on hemopoietic cells, and not parenchymal cells, is primarily responsible for limiting leukocyte infiltration during NSN. Taken together, our findings indicate that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints poised to suppress autoimmune renal disease.