湯澤 由紀夫

Capillary electrophoresis coupled with time-of-flight mass spectrometry was used to explore new serum biomarkers with high sensitivity and specificity for diabetic nephropathy (DN) diagnosis, through comprehensive analysis of serum metabolites with 78 diabetic patients. Multivariate analyses were used for identification of marker candidates and development of discriminative models. Of the 289 profiled metabolites, orthogonal partial least-squares discriminant analysis identified 19 metabolites that could distinguish between DN with macroalbuminuria and diabetic patients without albuminuria. These identified metabolites included creatinine, aspartic acid, gamma-butyrobetaine, citrulline, symmetric dimethylarginine (SDMA), kynurenine, azelaic acid, and galactaric acid. Significant correlations between all these metabolites and urinary albumin-to-creatinine ratios (p < 0.009, Spearman's rank test) were observed. When five metabolites (including gamma-butyrobetaine, SDMA, azelaic acid and two unknowns) were selected from 19 metabolites and applied for multiple logistic regression model, AUC value for diagnosing DN was 0.927 using the whole dataset, and 0.880 in a cross-validation test. In addition, when four known metabolites (aspartic acid, SDMA, azelaic acid and galactaric acid) were applied, the resulting AUC was still high at 0.844 with the whole dataset and 0.792 with cross-validation. Combination of serum metabolomics with multivariate analyses enabled accurate discrimination of DN patients. The results suggest that capillary electrophoresis-mass spectrometry based metabolome analysis could be used for DN diagnosis.

Background aims. Recent studies have demonstrated that cultured mesenchymal stromal cells derived from adipose tissue are useful for regenerative cell therapy. The stromal vascular fraction (SVF) can be obtained readily without culturing and may be clinically applicable. We investigated the therapeutic effects of SVF and used it in the treatment of acute kidney injury (AKI). Methods. Liposuction aspirates were obtained from healthy donors who had provided written informed consent. We harvested the SVF and determined the growth factor secretion and anti-apoptotic ability with conditioned medium. To investigate the effect of SVF on AKI, cisplatin was injected into rats and SVF was administrated into the subcupsula of the kidney. Results. Both human and rat SVF cells secreted vascular endothelial growth factor-A (VEGF) and hepatocyte growth factor (HGF). Human SVF-conditioned media had an anti-apoptotic effect, which was inhibited by anti-HGF antibody (Ab) but not by anti-VEGF Ab. In vivo, SVF significantly ameliorated renal function, attenuated tubular damage and increased the cortical blood flow speed. In the SVF-treated group, VEGF levels in the cortex and HGF levels in both the cortex and medulla, especially tubules in the medulla, were significantly higher. Immunostaining revealed that SVF cells expressing VEGF and HGF and remained in the subcapsule on day 14. Conclusions. The present study demonstrates that a subcapsular injection of non-expanded SVF cells ameliorates rat AKI, and that the mechanism probably involves secretion of renoprotective molecules. Administration of human SVF may be clinically applicable and useful as a novel autologous cell therapy against kidney diseases.

硫化水素（H₂S）は，有毒で時には致死的なガスであるが，一酸化窒素（NO）および一酸化炭素（CO）に加えて第3のガストランスミッターとして，その生物学的機能が近年大きく注目されている．特に神経系，心血管系，消化器系では，神経伝達物質，血圧のコントロール，インスリン分泌などに深く関与していることが確認され，さらに炎症やアポトーシス，酸化ストレスの誘導にも重要な働きをしている．さらにH₂Sの発現不全状態は，高血圧，動脈硬化，心筋梗塞，糖尿病など生活習慣病の主要な病態を引き起こす．急性腎傷害（AKI）は，直接生命予後に影響する病態として近年注目されている．虚血腎障害モデルに対して，H₂Sドナーの投与により組織傷害および腎機能が改善されたことから，H₂Sは虚血性AKIに対する重要な治療標的として期待される．また，H₂Sは内皮細胞上のアンジオテンシン変換酵素（ACE）の活性の抑制効果がある．さらに，ホモシステインは，I型アンジオテンシンII受容体（AT1R）の発現を誘導する．このため，高ホモシステイン血症に伴うH₂S産生低下は，ACE活性の上昇と血管のAT1R発現増加を介して，高血圧や腎の線維化を誘導する．一方，高ホモシステイン血症は慢性腎臓病の主要な危険因子であるが，ホモシステインはH₂Sの基質であることから，H₂S合成酵素（CBS，CSE）の発現不全に由来する内因性H₂S産生低下状態を反映した病態として理解されるようになってきた．糖尿病および糖尿病性腎症に関しては，H₂Sは直接膵でのインスリン分泌に関与し，糖尿病発症に深くかかわることが示された．高血糖に由来する糸球体でのNO発現低下は，内皮細胞障害により糖尿病性腎症における糸球体病変の発症・進展に深く関与し，CSE発現低下に伴うH₂S産生低下は，尿細管・間質での虚血障害を進行させ，腎機能低下が進行すると考えられる．

Background: Protein-energy wasting and chronic inflammation are prevalent in patients with end-stage renal disease (ESRD). We investigated the combination of serum albumin, C-reactive protein (CRP) and body mass index (BMI) at initiation of hemodialysis therapy as a predictor of all-cause and cardiovascular disease (CVD) mortality in Japanese ESRD patients. Methods: A total of 1,228 consecutive Japanese ESRD patients on hemodialysis therapy were enrolled and followed for up to 10 years. Patients were divided into quartiles according to levels of albumin, CRP and BMI. Furthermore, to clarify the joint role of these factors, albumin < 3.5 g/dl, CRP > 4.0 mg/l and BMI < 19.6 were defined as risk factors using receiver operating characteristic analysis; thereafter, patients were divided into groups according to the positive number of these factors. Results: Adjusted hazard ratios (HRs) for lower serum albumin, elevated CRP and lower BMI for 10-year all-cause mortality were 1.97, 3.13 and 2.61, respectively. Regarding the combination of these variables, adjusted HRs for mortality were 2.31, 4.28 and 8.07, respectively, in patients having any one factor, any two factors and all three factors. The C-index for an established risk model with these three positive markers was the most accurate for predicting mortality (0.768), as compared to other models with one or two markers. Similar results were seen for CVD mortality. Conclusions: Serum albumin, CRP and BMI at the start of hemodialysis therapy were able to individually stratify the risk of long-term mortality in ESRD patients. Furthermore, a combination of these variables could more accurately predict mortality. Copyright (c) 2012 S. Karger AG, Basel

Background In Japan, the population of patients on peritoneal dialysis (PD) is <4% of the total number of patients with end-stage renal disease. Few systemic analyses have examined why the number of PD patients has not increased in Japan. We organized a registry to analyze PD patients and retrospectively investigated 561 PD patients (about 5% of all Japanese PD patients) from 13 hospitals in the Tokai area for 3 years from 2005. Methods We investigated background, physical status, laboratory data, status of PD therapy, and the occurrence of PD-related complications, and analyzed reasons for withdrawal from PD. Results Nutrition did not change significantly during our observation. Urinary volume showed continued decreases after the introduction period. In contrast, PD fluid demand and ultrafiltration volume were significantly increased. For calcium metabolism, multiple phosphate binders were required after the second year of PD therapy. Early dropout within 3 years after starting PD therapy comprised 50.9% of total withdrawals, with PD-related peritonitis as the most common reason, mainly caused by Gram-positive organisms. Incidence of peritonitis was 42.8 months/patient. Culture-negative results were obtained for 32% of peritonitis cultures. Diabetes affects the prognosis of PD therapy, but not the incidence of peritonitis. Conclusion We examined clinical status over 3 years in the Tokai area. The results suggest that the incidence of peritonitis needs to be decreased to prevent early withdrawal of PD patients. Education systems to decrease the incidence of peritonitis and techniques to decrease culture-negative results might be important for improving the prognosis of peritonitis.

Objectives: To evaluate the effects of a periurethral injection of low serum cultured adipose tissue-derived mesenchymal stromal cells (LASC) and to develop a new autologous cell therapy for stress urinary incontinence. Methods: F344 rats were divided into three groups as based on the periurethral injection of LASC, GAX collagen or vehicle (control). At 2 and 4 weeks after injection, leak point pressure (LPP) was measured before and after transection of the pelvic nerves. For cell tracking, LASC of green fluorescent protein transgenic rats were injected into nude rats. Results: At 2 weeks, both the LASC and collagen groups showed significantly higher LPP than the control group. At 4 weeks, the increase in LPP in the LASC group remained, whereas LPP in the collagen group decreased to baseline levels. In the absence of the urethral closure reflex after transection of the pelvic nerves, LPP in the LASC group was significantly higher than that in the other two groups. Histologically, the size of the urethral lumen was smaller in the LASC group than the collagen group. At 4 weeks, most of the LASC were positive for myogenic antigens including a-smooth muscle actin, desmin and calponin I. Conclusions: Periurethral injection of autologous LASC capable of myogenic differentiation made a greater contribution to the increase in urethral resistance than did the conventional collagen bulk injection. Thus, its use for treatment of stress urinary incontinence can be postulated.

Background The Committee for the Standardization of Renal Pathological Diagnosis and the Working Group for Renal Biopsy Database of the Japanese Society of Nephrology started the first nationwide, web-based, and prospective registry system, the Japan Renal Biopsy Registry (J-RBR), to record the pathological, clinical, and laboratory data of renal biopsies in 2007. Methods The patient data including age, gender, laboratory data, and clinical and pathological diagnoses were recorded on the web page of the J-RBR, which utilizes the system of the Internet Data and Information Center for Medical Research in the University Hospital Medical Information Network. We analyzed the clinical and pathological diagnoses registered on the J-RBR in 2007 and 2008. Results Data were collected from 818 patients from 18 centers in 2007 and 1582 patients from 23 centers in 2008, including the affiliated hospitals. Renal biopsies were obtained from 726 native kidneys (88.8%) and 92 renal grafts (11.2%) in 2007, and 1400 native kidneys (88.5%) and 182 renal grafts (11.5%) in 2008. The most common clinical diagnosis was chronic nephritic syndrome (47.4%), followed by nephrotic syndrome (16.8%) and renal transplantation (11.2%) in 2007. A similar frequency of the clinical diagnoses was recognized in 2008. Of the native kidneys, the most frequent pathological diagnosis as classified by pathogenesis was immunoglobulin (Ig) A nephropathy (IgAN) both in 2007 (32.9%) and 2008 (30.2%). Among the primary glomerular diseases (except IgAN), membranous nephropathy (MN) was the most common disease both in 2007 (31.4%) and 2008 (25.7%). Conclusions In a cross-sectional study, the J-RBR has shown IgAN to be the most common disease in renal biopsies in 2007 and 2008, consistent with previous Japanese studies. MN predominated in the primary glomerular diseases (except for IgAN). The frequency of the disease and the clinical and demographic correlations should be investigated in further analyses by the J-RBR.

Renal failure is a frequent complication in patients with multiple myeloma. Immunoglobulin free light chains (FLCs) form casts in the distal tubules, resulting in renal obstruction, and are also directly toxic to proximal renal tubules. Removal of FLCs contributes to renal recovery. High cut-off (HCO) membrane Theralite2100, protein leaking dialyzer PES210D alpha, plasma separator Evacure1A20 and beta(2) microglobulin adsorption column LixelleS-35 were compared in their FLC removal rate. Dialysis using Theralite2100 or Evacure1A20, diafiltration using PES210D alpha and adsorption using LixelleS-35 were performed in an in vitro circuit. The highest removal rate was obtained by Theralite2100 dialysis among the four blood purification methods. Albumin loss was also the greatest in Theralite2100 dialysis. The removal content of FLCs per 1 g albumin loss was better in PES210D alpha diafiltration. The removal rate of FLCs by Evacure EC1A-20 dialysis was the third highest. Adsorption of FLCs by the beta(2) microglobulin adsorption column Lixelle S-35 was confirmed. In conclusion, Theralite2100 dialysis was the best in removal of FLCs. PES210D alpha diafiltration can remove FLCs with smaller loss of albumin.

Background. High baseline peritoneal solute transport rate is reportedly associated with reduced patient and technique survival in continuous peritoneal dialysis (PD) patients. However, the determinants of baseline peritoneal solute transport rate remain uncertain. The aim of this study was to investigate the relationship between peritoneal local inflammation, angiogenesis and systemic inflammation and baseline peritoneal permeability. Methods. Peritoneal biopsy specimens from 42 pre-dialysis uraemic patients and 11 control individuals were investigated. Immunohistochemistry for CD68-positive macrophages, chymase- and tryptase-positive mast cells, interleukin-6 (IL-6)-positive cells, CD3-positive T cells, CD20-positive B cells, neutrophils and CD31- and pathologische anatomie Leiden-endothelium (PAL-E)-positive blood vessels in the peritoneum was performed. Baseline dialysate-to-plasma ratio for creatinine (D/P Cr) was determined within 6 months of PD induction. Clinical and laboratory parameters were measured at the time of peritoneal biopsy. Factors associated with peritoneal permeability were assessed by multiple linear regression analysis. Results. Pre-dialysis uraemic peritoneum showed infiltration by CD68-positive macrophages, and mast cells, as compared with controls. Baseline D/P Cr was correlated with density of CD68-positive macrophages (P < 0.001), IL-6-positive cells (P < 0.001), CD31-positive (P < 0.05) and PAL-E-positive blood vessels (P < 0.05) and serum albumin (P < 0.05). However, baseline peritoneal permeability was not correlated with infiltration by mast cells, B cells, T cells, neutrophils, serum C-reactive protein or other clinical factors. On multiple linear regression analysis, the number of CD68-positive macrophages in peritoneum was an independent predictor for baseline peritoneal permeability (P = 0.009). Conclusions. Peritoneal macrophage infiltration is predominant in uraemic patients and is an important factor in predicting baseline peritoneal permeability.

Background. The peritoneal cavity is isolated from the outside and is usually a sterile environment. Patients on peritoneal dialysis (PD) have PD fluid (PDF) infused into the peritoneal cavity. We previously showed that unregulated complement activation could contribute to the development of peritoneal inflammation in yeast peritonitis in PD therapy. In that situation, suppression of local complement activation is essential to protect the host from further injury. The membrane complement regulators (CRegs), Crry, CD55 and CD59, are expressed in the rat peritoneum, especially along the mesothelial cell layer. Methods. We investigated CRegs' functional roles in the peritoneal cavity using blocking mAb against each CReg and complement activation in different PDFs. Results. Blockade of any single CReg did not cause spontaneous peritoneal injury in rat. Combined blockade of Crry and CD59 induced focal peritoneal tissue injury and heavy accumulation of inflammatory cells with peritoneal edema at 24 h. Deposits of C3 and C5b-9 were found on the peritoneal surface after combined blocking of Crry and CD59. Systemic complement depletion by cobra venom factor abrogated these inflammatory changes. When combined blockade of Crry and CD59 was performed with PDF of different pH and glucose concentration in rats, the peritoneal injuries were enhanced with lower pH and higher glucose concentration. These results were confirmed by in vitro experiments using primary rat mesothelial cell culture. Conclusions. Rat CRegs, Crry and CD59, specifically collaborate to control complement activation in rat peritoneum. During PD, impairment of CReg might contribute to the development of severe peritoneal inflammation.

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.

Regardless of their primary causes, progressive renal fibrosis and tubular atrophy are the main predictors of progression to end-stage renal disease. Basigin/CD147 is a multifunctional molecule-it induces matrix metalloproteinases and hyaluronan, for example-and has been implicated in organ fibrosis. However, the relationship between basigin and organ fibrosis has been poorly studied. We investigated basigin's role in renal fibrosis using a unilateral ureteral obstruction model. Basigin-deficient mice (Bsg(-/-)) demonstrated significantly less fibrosis after surgery than Bsg(+/+) mice. Fewer macrophages had infiltrated in Bsg(-/-) kidneys. Consistent with these in vivo data, primary cultured tubular epithelial cells from Bsg-/- mice produced less matrix metalloproteinase and exhibited less motility on stimulation with transforming growth factor beta. Furthermore, Bsg(-/-) embryonic fibro blasts produced less hyaluronan and a-smooth muscle actin after transforming growth factor 13 stimulation. Together, these results demonstrate for the first time that basigin is a key regulator of renal fibrosis. Basigin could be a candidate target molecule for the prevention of organ fibrosis. (Am J Pathol 2011, 178:572-579; DOI: 10.1016/j.ajpath.2010.10.009)

Background and objectives: Cardiac failure is directly affected by left ventricular (LV) dysfunction, and particularly LV systolic dysfunction is strongly associated with survival in ESRD patients. The aim of this study was to determine the prognostic value of reduced LV ejection fraction (LVEF) measured at the time of initiation of hemodialysis (HD) in incident HD patients. Design, setting, participants, & measurements: 1254 consecutive ESRD patients who electively started HD therapy were screened by echocardiography within 1 month after its inception. They were divided into five groups according to LVEF levels with a decrease of 0.1 each and were followed up for up to 7 years. Survival was examined with the Kaplan-Meier method and compared using the log-rank test. Results: Among the 1254 patients, LVEF levels >= 0.6, 0.5 to 0.6, 0.4 to 0.5, 0.3 to 0.4, and <0.3 were seen in 842 (67.1%), 247 (19.7%), 107 (8.5%), 41 (3.3%), and 17 (1.4%) patients, respectively. On Kaplan-Meier analysis, 7-year event-free rates from cardiovascular death were 84.2, 83.7, 73.6, 59.4, and 30.9% in order of groups with decreasing LVEF of 0.1 each, respectively. Seven-year event-free rates from all-cause death were 69.2, 61.7, 57.1, 45.9, and 23.1% in the respective groups. Even after adjustment for other risk factors, decreasing LVEF was a strong independent predictor for cardiovascular death. Conclusions: Reduced LVEF on starting HD therapy could stratify risk of cardiovascular and all-cause mortality in ESRD patients. Screening by echocardiography at start of HD therapy might be recommended to predict prognosis in patients with ESRD. Clin J Am Soc Nephrol 5: 1793-1798, 2010. doi: 10.2215/CJN.00050110

Aggressive removal of circulating free light chains (FLC) by blood purification accompanied by chemotherapy is a promising approach for the treatment of acute renal failure due to myeloma cast nephropathy. Plasma exchange has been performed to remove serum FLC; in order to examine an alternative strategy we performed hemodiafiltration using protein-leaking dialyzers for the treatment of dialysis-dependent acute renal failure due to myeloma cast nephropathy. In the first case with kappa-light chain cast nephropathy, the pre-treatment serum creatinine was 9.65 mg/dL, and the serum kappa-FLC was 27 100 mg/L. Plasma exchange or hemodiafiltration was performed from Monday to Friday during the first several weeks. Chemotherapy was started with high-dose dexamethasone and then switched to bortezomib plus dexamethasone. The mean removal rates of kappa-FLC were 45.8% (one plasma volume) and 66.9% (one-and-a-half plasma volumes) by plasma exchange. The removal rates of kappa-FLC by hemodiafiltration (66.9%, FB210UH beta; 71.6%, PES210D alpha; 75.2%, FXS220) were comparable to those by plasma exchange. In the second case with lambda-light chain cast nephropathy, the pre-treatment serum creatinine was 4.14 mg/dL, and the serum lambda-FLC was 4140 mg/L. The mean removal rates of lambda-FLC were 60.2% (FXS140) and 64.2% (FB210UH beta) by hemodiafiltration. Both cases became dialysis-independent. The combination of an intense blood purification regimen and bortezomib plus dexamethasone therapy appears to be an efficient approach to renal recovery. Hemodiafiltration using protein-leaking dialyzers could become an alternative to plasma exchange as a method of removing FLC.

Transforming growth factor (TGF)-β1, -β2, and -β3 are involved in control of wound repair and development of fibrosis. Connective tissue growth factor (CTGF) expression is stimulated by all TGF-β isoforms and is abundant in glomerulosclerosis and other fibrotic disorders. CTGF is hypothesized to mediate profibrotic effects of TGF-β1 or to facilitate interaction of TGF-β1 with its receptor, but its interactions with TGF-β isoforms in nonpathological conditions are unexplored so far. Tissue repair and remodeling may recapitulate gene transcription at play in organogenesis. To further delineate the relationship between CTGF and TGF-β, we compared expression patterns of CTGF and TGF-β isoforms in rat and human glomerulogenesis and in various human glomerulopathies. CTGF mRNA was present in the immediate precursors of glomerular visceral and parietal epithelial cells in the comma- and S-shaped stages, but not in earlier stages of nephron development. During the capillary loop and maturing glomerular stages and simultaneous with the presence of TGF-β1, -β2, and -β3 protein, CTGF mRNA expression was maximal and present only in differentiating glomerular epithelial cells. CTGF protein was also present on precursors of mesangium and glomerular endothelium, suggesting possible paracrine interaction. Concomitant with the presence of TGF-β2 and -β3 protein, and in the absence of TGF-β1, CTGF mRNA and protein expression was restricted to podocytes in normal adult glomeruli. However, TGF-β1 and CTGF were again coexpressed, often with TGF-β2 and -β3, in particular in podocytes in proliferative glomerulonephritis and also in mesangial cells in diabetic nephropathy and IgA nephropathy (IgA NP). Coordinated expression of TGF-β isoforms and of CTGF may be involved in normal glomerulogenesis and possibly in maintenance of glomerular structure and function at adult age. Prolonged overexpression of TGF-β1 and CTGF is associated with development of severe glomerulonephritis and glomerulosclerosis. Copyright © 2010 the American Physiological Society.

Ito Y, Goldschmeding R, Kasuga H, Claessen N, Nakayama M, Yuzawa Y, Sawai A, Matsuo S, Weening JJ, Aten J. Expression patterns of connective tissue growth factor and of TGF-beta isoforms during glomerular injury recapitulate glomerulogenesis. Am J Physiol Renal Physiol 299: F545-F558, 2010. First published June 24, 2010; doi:10.1152/ajprenal.00120.2009.-Transforming growth factor (TGF)-beta(1),-beta(2), and -beta(3) are involved in control of wound repair and development of fibrosis. Connective tissue growth factor (CTGF) expression is stimulated by all TGF-beta isoforms and is abundant in glomerulosclerosis and other fibrotic disorders. CTGF is hypothesized to mediate profibrotic effects of TGF-beta(1) or to facilitate interaction of TGF-beta(1) with its receptor, but its interactions with TGF-beta isoforms in nonpathological conditions are unexplored so far. Tissue repair and remodeling may recapitulate gene transcription at play in organogenesis. To further delineate the relationship between CTGF and TGF-beta, we compared expression patterns of CTGF and TGF-beta isoforms in rat and human glomerulogenesis and in various human glomerulopathies. CTGF mRNA was present in the immediate precursors of glomerular visceral and parietal epithelial cells in the comma-and S-shaped stages, but not in earlier stages of nephron development. During the capillary loop and maturing glomerular stages and simultaneous with the presence of TGF-beta(1), -beta(2), and -beta(3) protein, CTGF mRNA expression was maximal and present only in differentiating glomerular epithelial cells. CTGF protein was also present on precursors of mesangium and glomerular endothelium, suggesting possible paracrine interaction. Concomitant with the presence of TGF-beta(2) and -beta(3) protein, and in the absence of TGF-beta(1), CTGF mRNA and protein expression was restricted to podocytes in normal adult glomeruli. However, TGF-beta(1) and CTGF were again coexpressed, often with TGF-beta(2) and -beta(3), in particular in podocytes in proliferative glomerulonephritis and also in mesangial cells in diabetic nephropathy and IgA nephropathy (IgA NP). Coordinated expression of TGF-beta isoforms and of CTGF may be involved in normal glomerulogenesis and possibly in maintenance of glomerular structure and function at adult age. Prolonged overexpression of TGF-beta(1) and CTGF is associated with development of severe glomerulonephritis and glomerulosclerosis.

We report the pathological findings of the peritoneum in a patient with chronic eosinophilic peritonitis. Peripheral blood eosinophilia was confirmed before insertion of Tenckhoff catheter. Eosinophilic peritonitis continued from the second day after initiation of peritoneal dialysis for 18 months. Pathological findings showed numerous eosinophils in peritoneal blood vessels. Mast cells were also detected in the peritoneum, while neoangiogenesis was not prominent. The highly permeable state of the peritoneal membrane may be due to inflammatory mediators, such as tryptase. Mast cells may be involved in high peritoneal permeability in such patients. © 2010 The Author.

Darbepoetin alfa, which has a much longer half-life than recombinant human erythropoietin, is used to treat renal anemia. However, there are few reports investigating the efficacy of darbepoetin alfa administered every 2 weeks (Q2W). We performed the single-center, prospective, and randomized study in chronic hemodialysis patients. Clinically stable patients on hemodialysis were recruited, and darbepoetin alfa 15-40 mu g/week was administered intravenously once a week (QW) to achieve a hemoglobin (Hb) level of 10.5-12.0 g/dl for 8 weeks prior to randomization. The patients were randomly assigned to 2 different dosing frequency groups: once a week (QW) or every 2 weeks (Q2W). We switched to a double dose in the Q2W group. We measured Hb level every 2 weeks and administered darbepoetin alfa to achieve an Hb level of 10.5-11.5 g/dl. The primary endpoints were the weekly dose of darbepoetin alfa administered at week 24. We assigned 19 and 20 patients into QW and Q2W, respectively. There were no significant differences between the groups in Hb, transferrin saturation, ferritin, and weekly dose of darbepoetin alfa at end of the baseline period. There was no significant difference in Hb level at week 24, at which time the weekly dose requirement and dose per dry body weight were much higher in the Q2W than in the QW group. Administration of darbepoetin alfa Q2W could maintain Hb level similarly to to that obtained QW, but we did not confirm efficiency at a higher dose requirement or blood pressure elevation.

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis (PD) therapy that often occurs in association with peritoneal high transport rate and ultrafiltration failure (UFF). To study the possible pathogenic role of connective tissue growth factor (CTGF) in the relationship of PF and UFF, dialysate CTGF contents (n = 178) and tissue CTGF expression (n = 61) were investigated by ELISA, real-time PCR, immunohistochemistry, and in situ hybridization. CTGF production with and without TGF-β1 stimulation in human peritoneal mesothelial cells (HPMC) from the spent patients' peritoneal dialysate (n = 32) was studied in vitro. The dialysate-to-plasma ratio for creatinine (D/P Cr) was positively correlated to dialysate CTGF concentration and estimated local peritoneal production of CTGF. CTGF mRNA expression was 11.4-fold higher in peritoneal membranes with UFF than in pre-PD renal failure peritoneum and was correlated with thickness of the peritoneum. CTGF protein and mRNA were detected in mesothelium and in fibroblast-like cells. In cultured HPMC, TGF-β1-induced expression of CTGF mRNA was increased at 12 and 24 h and was correlated with D/P Cr. In contrast, bone morphogenic protein-4 mRNA expression was inversely correlated with D/P Cr. Our results suggest that high peritoneal transport state is associated with fibrosis and increased peritoneal CTGF expression and production by mesothelial cells, which can be stimulated by TGF- β1. Dialysate CTGF concentration could be a biomarker for both peritoneal fibrosis and membrane function. Functional alteration of mesothelial cells may be involved in progression of peritoneal fibrosis in high transport state. Copyright © 2010 American Physiological Society.

Mizutani M, Ito Y, Mizuno M, Nishimura H, Suzuki Y, Hattori R, Matsukawa Y, Imai M, Oliver N, Goldschmeding R, Aten J, Krediet RT, Yuzawa Y, Matsuo S. Connective tissue growth factor (CTGF/CCN2) is increased in peritoneal dialysis patients with high peritoneal solute transport rate. Am J Physiol Renal Physiol 298: F721-F733, 2010. First published December 23, 2009; doi:10.1152/ajprenal.00368.2009.-Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis (PD) therapy that often occurs in association with peritoneal high transport rate and ultrafiltration failure (UFF). To study the possible pathogenic role of connective tissue growth factor (CTGF) in the relationship of PF and UFF, dialysate CTGF contents (n = 178) and tissue CTGF expression (n = 61) were investigated by ELISA, real-time PCR, immunohistochemistry, and in situ hybridization. CTGF production with and without TGF-beta(1) stimulation in human peritoneal mesothelial cells (HPMC) from the spent patients' peritoneal dialysate (n = 32) was studied in vitro. The dialysate-to-plasma ratio for creatinine (D/P Cr) was positively correlated to dialysate CTGF concentration and estimated local peritoneal production of CTGF. CTGF mRNA expression was 11.4-fold higher in peritoneal membranes with UFF than in pre-PD renal failure peritoneum and was correlated with thickness of the peritoneum. CTGF protein and mRNA were detected in mesothelium and in fibroblast-like cells. In cultured HPMC, TGF-beta(1)-induced expression of CTGF mRNA was increased at 12 and 24 h and was correlated with D/P Cr. In contrast, bone morphogenic protein-4 mRNA expression was inversely correlated with D/P Cr. Our results suggest that high peritoneal transport state is associated with fibrosis and increased peritoneal CTGF expression and production by mesothelial cells, which can be stimulated by TGF-beta(1). Dialysate CTGF concentration could be a biomarker for both peritoneal fibrosis and membrane function. Functional alteration of mesothelial cells may be involved in progression of peritoneal fibrosis in high transport state.

Besides its effect on calcium metabolism, vitamin D may play a part in preventing the onset and progression of cardiovascular disease (CVD) events. Only a few reports on the studies relating to whether vitamin D may reduce CVD events in patients with predialysis chronic kidney disease (CKD) are available, and many ambiguities remain. We conducted a retrospective cohort study of 665 patients with predialysis CKD. With log-rank test using the Kaplan-Meyer survival curve, comparison of incidences of CVD events, CVD-related mortality, and all-cause mortality were made between patients in the alfacalcidol treatment group (107 patients) in the predialysis stage to whom alfacalcidol 0.25-0.5 mu g/day was orally administered for at least 24 weeks, and patients in the nontreatment group (558 patients) who received no administration of alfacalcidol or other type of activated vitamin D and its analogues. Patients to whom alfacalcidol administration was discontinued within 24 weeks as well as initiation of dialysis of < 24 weeks were excluded for this study. Factors relating to CVD events were examined using Cox's proportional hazards analysis. The mean follow-up period was 55.1 +/- A 38.9 months in the alfacalcidol treatment group and 41.9 +/- A 38.4 months in the nontreatment group. CVD events occurred in 172 patients during the follow-up period, and 74 of those occurred during the predialysis period. In the alfacalcidol treatment group, the incidence of cumulative CVD events was significantly lower. In relation to all-cause deaths and CVD-related deaths, the cumulative mortality rate was significantly lower in the alfacalcidol treatment group during the follow-up period. Throughout the follow-up period, the association between CVD events and alfacalcidol use was detected when adjusted for age, sex, diabetes, hypertension, use of renin-angiotensin system inhibitors, estimated glomerular filtration rate, and albumin and parathyroid hormone. These data showed that oral administration of alfacalcidol for predialysis CKD patients was associated with reduced risk for CVD.

Background: Percutaneous transluminal angioplasty (PTA) for femoropopliteal lesions in peripheral artery disease has been performed in patients undergoing hemodialysis as well as in the general population. Cilostazol, a selective inhibitor of phosphodiesterase 3, has been reported to reduce target lesion revascularization after PTA for femoropopliteal lesions in the general population. Objective: This study investigated the effects of cilostazol use on long-term patency after PTA in patients with femoropopliteal disease undergoing hemodialysis. Methods: fit this retrospective study, data from patients undergoing hemodialysis who underwent successful PTA for femoropopliteal disease, defined as a final luminal diameter stenosis <30%, without angiographically Visual arterial dissection and no in-hospital complications, were included. One Study group received long-term treatment with oral cilostazol 100 mg BID after PTA; the control group did not. The duration of follow-tip was ! 6 years. The primary outcome of interest Was cumulative patency, as measured by the event-free rate 6 years after PTA, with event defined as restenosis of >50% of the vessel diameter in femoropophreal lesions. Data on baseline characteristics, patency, and covariates (diabetes, hypertension, hyperlipidemia, smoking, coronary artery disease, critical limb ischemia, TransAtlantic Inter-Society Consensus classification, and stenting) were obtained from electronic medical records and tele-phone interviews with patients. To minimize the effects of selection bias for cilostazol administration, a propensity-matched analysis using Cox univariate and multivariate models including the previously mentioned covariates was conducted. The propensity, scores of the 2 groups were matched 1:1 (AUC = 0.69 [receiving operating characteristics analysis]). Data were obtained from electronic medical records and telephone interviews with patients by trained personnel who were blinded to treatment assignment. Results: A total of 358 consecutive lesions of 1,74 patients undergoing hemodialysis were Included (103 men, 7.1 women; mean [SD] age, 66 [11] years; cilostazol group, 61. patients, 121 lesions; control group, I 13 patients, 237 lesions). The mean duration of follow-Lip was 37 (27) months. The 6-year event-free rate of restenosis of >50% of the vessel diameter was significantly higher in the cilostazol group than in the control group (72/121 [59.5%] vs 120/237 [50.6%,]; P = 0.005 [log-rank test]; hazard ratio [HR] = 0.63; 95% CI, 0.45-0.88; P = 0.008 [Cox univariate analysis]). Also, event-free rates of target lesion revascularization and limb amputation were significantly higher in the cilostazol group than in the control group (40/61 [65.6%] vs 57/113 [50.4%]; P = 0.013 [log-rank test] and 54161 [88.5%] vs 90/113 [79.6%]; P = 0.047 [log-rank test], respectively). On propensity score matching (105 lesions), the baseline characteristics were comparable between the 2 groups. The 6-year event-free rate of restenosis was significantly higher in the cilostazol group than in the control group (66/105 [62.9%] vs 52/105 [49.5%]; HR = 0.58; 95% CI, 0.38-0.88; P = 0.012 [Cox univariate analysis]). On propensity-matched (COX multivariate) analysis, cilostazol (HR = 0.51; 95% CI, 0.27-0.84; P = 0.008), age (HR = 1.01; 95% CI, 1.01-1.04; P = 0.031), and critical limb ischemia (HR = 2.21; 95% CI, 1.39-3.53; P = 0.001) were independent predictors of restenosis. None of the patients in the cilostazol group discontinued cilostazol treatment during the follow-up period. Four patients (6.6%) experienced mild headache. Conclusion: This study found that ill these patients with femoropopliteal lesions in peripheral artery disease who were undergoing hemodialysis, those treated with cilostazol 100 mg BID after PTA had a higher mean rate of cumulative patency after PTA than those in the control group. (Clin Ther. 2010;32:24-33) (C) 2010 Excerpta Medica Inc.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease that originates in abnormal pluripotent bone marrow stem cells and it is consistently associated with the Philadelphia chromosome and/or BCR/ABL fusion gene. Renal infiltration of leukemic cells is relatively rare in CML and is associated with renal impairment. We describe a patient who developed acute renal failure by tubulointerstitial nephropathy during treatment with imatinib mesylate for CML. The acute kidney injury was subsequently found to be due to direct leukemic infiltration. Treatment with hydroxycarbamide and prednisolone resulted in stabilization of the renal function for approximately 4 months. Leukemic infiltration into the kidney should always be considered when a patient with CML presents with renal impairment, regardless of the clinical stage, as the renal failure often responds well to chemotherapy.

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis therapy. The present study was performed to examine the mechanisms of PF in view of the plasminogen activator (PA)/plasmin/matrix metalloproteinase (MMP) cascade. PF was induced in tissue-type PA (tPA) deficient mice and wild-type mice by intraperitoneal injection of chlorhexidine gluconate. Mice were killed on day 21, and tissue samples were taken. Histopathological studies were performed. Plasmin activity, gelatinases activity, and the levels of tPA, transforming growth factor-β1 (TGF-β1), and MMP-2 mRNA were determined. Protein levels of MMP-3, tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -3, phospho-Smad3, membrane-type 1 (MT1)-MMP, and MT3-MMP were also studied. On day 21, tPA +/+ mice showed severe PF, whereas tPA -/- mice showed milder change. Submesothelial basement membranes were dissolved in tPA +/+ mice while they were relatively preserved in tPA -/- mice. The levels of macrophage infiltration, staining for α-smooth muscle actin (α-SMA) and collagen type III, and vascular density were all significantly lower in tPA -/- mice than in tPA +/+ mice. The levels of plasmin activity, pro- and active MMP-2, mRNA expression of tPA and TGF-β1, and phospho-Smad3 protein were also lower in tPA -/- mice. No difference was observed between the two groups concerning the protein levels of MMP-3, TIMP-1, TIMP-2, TIMP-3, MT1-MMP, or MT3-MMP. These results indicate that the presence of tPA enhances inflammation, angiogenesis, and fibrogenesis in the peritoneum of the PF model mice. Activation of the PA/plasmin/MMP cascade may play a pivotal role in the pathogenesis of PF. Copyright © 2009 the American Physiological Society.

Kurata K, Maruyama S, Kato S, Sato W, Yamamoto J, Ozaki T, Nitta A, Nabeshima T, Morita Y, Mizuno M, Ito Y, Yuzawa Y, Matsuo S. Tissue-type plasminogen activator deficiency attenuates peritoneal fibrosis in mice. Am J Physiol Renal Physiol 297: F1510-F1517, 2009. doi:10.1152/ajprenal.90330.2008.-Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis therapy. The present study was performed to examine the mechanisms of PF in view of the plasminogen activator (PA)/plasmin/matrix metalloproteinase (MMP) cascade. PF was induced in tissue-type PA (tPA) deficient mice and wild-type mice by intraperitoneal injection of chlorhexidine gluconate. Mice were killed on day 21, and tissue samples were taken. Histopathological studies were performed. Plasmin activity, gelatinases activity, and the levels of tPA, transforming growth factor-beta 1 (TGF-(sic)1), and MMP-2 mRNA were determined. Protein levels of MMP- 3, tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -3, phospho-Smad3, membrane-type 1 (MT1)-MMP, and MT3-MMP were also studied. On day 21, tPA +/+ mice showed severe PF, whereas tPA -/- mice showed milder change. Submesothelial basement membranes were dissolved in tPA +/+ mice while they were relatively preserved in tPA -/- mice. The levels of macrophage infiltration, staining for alpha-smooth muscle actin (alpha-SMA) and collagen type III, and vascular density were all significantly lower in tPA -/- mice than in tPA +/+ mice. The levels of plasmin activity, pro- and active MMP- 2, mRNA expression of tPA and TGF-beta 1, and phospho-Smad3 protein were also lower in tPA -/- mice. No difference was observed between the two groups concerning the protein levels of MMP-3, TIMP-1, TIMP-2, TIMP-3, MT1-MMP, or MT3-MMP. These results indicate that the presence of tPA enhances inflammation, angiogenesis, and fibrogenesis in the peritoneum of the PF model mice. Activation of the PA/plasmin/MMP cascade may play a pivotal role in the pathogenesis of PF.

Background-Percutaneous coronary intervention (PCI) using drug-eluting stents significantly reduces the risk of restenosis in the general population. However, in patients on hemodialysis, adverse cardiac events are frequently seen even if treated with drug-eluting stents. Recent studies suggest that C-reactive protein (CRP) reflects vascular wall inflammation and can predict adverse cardiac events. We evaluated possible prognostic values of CRP on outcomes in patients on hemodialysis undergoing PCI with drug-eluting stents. Methods and Results-A total of 167 patients undergoing PCI with sirolimus-eluting stents for stable angina (322 lesions) were enrolled. They were divided into tertiles according to serum CRP levels. We analyzed the incidence of major adverse cardiovascular events including cardiovascular death, nonfatal myocardial infarction, and target lesion revascularization after PCI as well as quantitative coronary angiographic data. The mean follow-up was 31 months (SD, 14). Major adverse cardiac events occurred in 11 patients (19.6%) of the lowest tertile, in 22 patients (39.3%) of the middle tertile, and in 28 patients (50.9%) of the highest tertile during follow-up period (P=0.0009). There was a progressive increase in neointimal growth after sirolimus-eluting stent implantation during follow-up because preprocedural CRP levels were higher, despite similar angiographic data just after PCI. Angiographic restenosis at 6 to 8 months after PCI was seen in 10.6% in the lowest tertile, 17.9% in the middle tertile, and 32.0% in the highest tertile (P=0.0007). Conclusions-Increased preprocedural serum CRP levels would predict higher major adverse cardiac events and restenosis rates after sirolimus-eluting stents implantation in patients on hemodialysis. (Circ Cardiovasc Interv. 2009; 2: 513-518.)

Hyperuricemia has recently been recognized to be a risk factor for nephropathy in the diabetic subject. We tested the hypothesis that lowering uric acid with a xanthine oxidase inhibitor might reduce renal injury in the diabetic mouse. Diabetic (db/db) mice were treated with allopurinol or no treatment for 8 wk. Serum uric acid, renal function, and histology were assessed at death. The direct effect of uric acid in human proximal tubular epithelial cells was also evaluated under normal or high glucose condition. We found that db/db mice developed hyperuricemia, albuminuria, mesangial matrix expansion, and mild tubulointerstitial disease. Allopurinol treatment significantly lowered uric acid levels, reduced albuminuria, and ameliorated tubulointerstitial injury, but it did not prevent mesangial expansion. The mechanism for protection was shown to be due to a reduction in inflammatory cells mediated by a reduction in ICAM-1 expression by tubular epithelial cells. Interestingly, allopurinol did not reduce oxidative stress in the kidney. An inflammatory role of uric acid on tubular cells was also confirmed by our in vitro evidence that uric acid directly induced ICAM-1 expression in the human proximal tubular cell. In conclusion, hyperuricemia has a pathogenic role in the mild tubulointerstitial injury associated with diabetic nephropathy but not glomerular damage in db/db mice. Lowering uric acid may reduce tubulointerstitial injury in diabetes. Copyright © 2009 the American Physiological Society.

Kosugi T, Nakayama T, Heinig M, Zhang L, Yuzawa Y, Sanchez-Lozada LG, Roncal C, Johnson RJ, Nakagawa T. Effect of lowering uric acid on renal disease in the type 2 diabetic db/db mice. Am J Physiol Renal Physiol 297: F481-F488, 2009. First published May 20, 2009; doi:10.1152/ajprenal.00092.2009.-Hyperuricemia has recently been recognized to be a risk factor for nephropathy in the diabetic subject. We tested the hypothesis that lowering uric acid with a xanthine oxidase inhibitor might reduce renal injury in the diabetic mouse. Diabetic (db/db) mice were treated with allopurinol or no treatment for 8 wk. Serum uric acid, renal function, and histology were assessed at death. The direct effect of uric acid in human proximal tubular epithelial cells was also evaluated under normal or high glucose condition. We found that db/db mice developed hyperuricemia, albuminuria, mesangial matrix expansion, and mild tubulointerstitial disease. Allopurinol treatment significantly lowered uric acid levels, reduced albuminuria, and ameliorated tubulointerstitial injury, but it did not prevent mesangial expansion. The mechanism for protection was shown to be due to a reduction in inflammatory cells mediated by a reduction in ICAM-1 expression by tubular epithelial cells. Interestingly, allopurinol did not reduce oxidative stress in the kidney. An inflammatory role of uric acid on tubular cells was also confirmed by our in vitro evidence that uric acid directly induced ICAM-1 expression in the human proximal tubular cell. In conclusion, hyperuricemia has a pathogenic role in the mild tubulointerstitial injury associated with diabetic nephropathy but not glomerular damage in db/db mice. Lowering uric acid may reduce tubulointerstitial injury in diabetes.

E-selectin and its ligands are essential for extravasation of leukocytes in inflammation. Here, we report that basigin (Bsg)/CD147 is a ligand for E-selectin that promotes renal inflammation in ischemia/reperfusion. Compared with wild-type mice, Bsg-deficient (Bsg(-/-)) mice demonstrated striking suppression of neutrophil infiltration in the kidney after renal ischemia/reperfusion. Although E-selectin expression increased similarly between the two genotypes, Bsg(-/-) mice exhibited less renal damage, suggesting that Bsg on neutrophils contribute to renal injury in this model. Neutrophils expressed Bsg with Winked polylactosamine chains and Bsg(-/-) neutrophils showed reduced binding to E-selectin. Bsg isolated from HL-60 cells bound to E-selectin, and tunicamycin treatment to abolish Winked glycans from Bsg abrogated this binding. Furthermore, Bsg(-/-) neutrophils exhibited reduced E-selectin-dependent adherence to human umbilical vein endothelial cells in vitro. Injection of labeled neutrophils into mice showed that Bsg(-/-) neutrophils were less readily recruited to the kidney after renal ischemia/reperfusion than Bsg(+/+) neutrophils, regardless of the recipient's genotype. Taken together, these results indicate that Bsg is a physiologic ligand for E-selectin that plays a critical role in the renal damage induced by ischemia/reperfusion.

Fungal peritonitis is an important complication in peritoneal dialysis patients; either continuous or recurrent peritonitis may enhance peritoneal damage. Even when the peritoneal dialysis catheter is removed in patients with fungal peritonitis, peritoneal fibrosis can progress and evolve into encapsular peritoneal sclerosis. It is unclear why fungal infections are worse than bacterial in these respects. Zymosan is a cell wall component of yeast that strongly activates the complement system. In this study, we compared the effects of zymosan and bacterial LPS on peritoneal inflammation in a rat peritoneal injury model induced by mechanical scraping. Intraperitoneal administration of zymosan, but not LPS or vehicle, caused markedly enhanced peritonitis with massive infiltration of cells and deposition of complement activation products Cab and membrane attack complex on day 5. In rats administered zymosan and sacrificed on days 18 or 36, peritoneal inflammation persisted with accumulation of ED-1-positive cells, small deposits of Cab and membrane attack complex, exudation of fibrinogen, and capillary proliferation in subperitoneal tissues. When zymosan was administered daily for 5 days after peritoneal scrape, there was even greater peritoneal inflammation with peritoneal thickening, inflammatory cell accumulation, and complement deposition. Inhibition of systemic complement by pretreatment with cobra venom factor or local inhibition by i.p. administration of the recombinant complement regulator Crry-Ig reduced peritoneal inflammation in zymosan-treated rats. Our results show that yeast components augment inflammation in the injured peritoneum by causing complement activation within the peritoneal cavity. Local anticomplement therapy may therefore protect from peritoneal damage during fungal infection of the peritoneum. The Journal of Immunology, 2009, 183: 1403-14.12.

The renin-angiotensin system plays a pivotal role in regulating blood pressure and is involved in the pathogenesis of kidney disorders and other diseases. Here, we report that the growth factor midkine is what we believe to be a novel regulator of the renin-angiotensin system. The hypertension induced in mice by 5/6 nephrectomy was accompanied by renal damage and elevated plasma angiotensin II levels and was ameliorated by an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin receptor blocker. Notably, ACE activity in the lung, midkine expression in the lung, and midkine levels in the plasma were all increased after 5/6 nephrectomy. Exposure to midkine protein enhanced ACE expression in primary cultured human lung microvascular endothelial cells. Furthermore, hypertension was not induced and renal damage was less severe in midkine-deficient mice. Supplemental administration of midkine protein to midkine-deficient mice restored ACE expression in the lung and hypertension after 5/6 nephrectomy. Oxidative stress might be involved in midkine expression, since expression of NADH/NADPH oxidase-1, -2, and -4 was induced in the lung after 5/6 nephrectomy. Indeed, the antioxidative reagent tempol reduced midkine expression and plasma angiotensin II levels and consequently ameliorated hypertension. These results suggest that midkine regulates the renin-angiotensin system and mediates the kidney-lung interaction after 5/6 nephrectomy.

Accumulating evidence suggests that the delivery of human adipose tissue-derived stromal cells (hASCs) has great potential as regenerative therapy. This was performed to develop a method for expanding hASCs by reducing the amount of serum required. We demonstrate that hASCs were able to expand efficiently in media containing 2% serum and fibroblast growth factor-2. These cells, or low serum cultured hASCs (hLASCs), expressed cell surface markers similar to those on bone marrow-derived mesenchymal stem cells, and could be differentiated into cells of mesenchymal lineage. Of interest, hLASCs secreted higher levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) than hASCs cultured in 20% serum (hHASCs). Moreover, hLASC-conditioned media significantly increased endothelial cell (EC) proliferation and decreased EC apoptosis compared to that obtained from hHASCs or control media only. Antibodies against VEGF and HGF virtually negated these effects. When hASCs were administered into the ischemic hindlimbs of nude rats, hLASCs improved blood flow, increased capillary density, and raised the levels of VEGF and HGF in the muscles as compared with hHASCs. In conclusion, we demonstrate a novel low serum culture system for hASCs, which may have great potential in regenerative cell therapy for damaged organs in the clinical setting.

Background. Even in the drug-eluting stent (DES) era, the restenosis rate of the follow-up period after percutaneous coronary intervention (PCI) is higher in haemodialysis (HD) patients than in non-HD patients. Therefore, higher restenosis remains a clinical limitation in HD patients, and a simple clinical method to predict patients likely to have restenosis after stent implantation is attractive. The present study investigated the potential relationship between aortic valvular calcification (AVC) and angiographical restenosis at follow-up after DES implantation in patients on maintenance HD. Methods. In the study, 97 patients were enrolled. We prospectively performed echocardiography before elective PCI with DES implantation. Angiographic follow-up was scheduled between 6 and 8 months after PCI. Restenosis at follow-up was defined as a diameter stenosis of >= 50% by measuring quantitative coronary angiography. Results. Of the enrolled patients, 59 patients (60.8%) had AVC. Complete angiographical follow-up was obtained in 86 patients (88.7%). The angiographical restenosis rate during the follow-up period was 24.7% in patients with AVC and 8.9% in patients without AVC [hazard ratio (HR) 3.36; 95% confidence interval (CI) 1.18-9.56, P = 0.023]. Even after multivariate adjustment including covariates related to atherogenecity, AVC remained an independent predictor of restenosis after implanting DES (HR 3.83; 95% CI 1.14-12.9, P = 0.029). Late lumen loss suggesting neointimal growth after DES implantation was 0.28 +/- 0.70 mm in the non-AVC group and 0.64 +/- 0.90 mm in the AVC group (P = 0.013). Conclusions. AVC provides predictive information regarding DES implantation in patients on maintenance HD.

Bleomycin (BLM) induces cellular apoptosis or necrosis by producing reactive oxygen species, and has been used to induce scleroderma in adult mice. We wondered whether BLM induces the same pathological phenotype in newborn mice as in adult mice. BLM was subcutaneously administrated to newborn BALB/c mice. At 1 month of age, BLM-treated mice showed severe destruction of salivary glands with enlargement of nearby lymph nodes. These nodes contained CD4(+) T cells and B220(+) cells with high expression of MHC class II molecules. In addition, autoantibodies were detected by HEp-2 staining and western blotting. The cell transfer experiments were performed to evaluate the role of autoimmune phenomena in these pathological changes. Following the transfer of enriched CD4(+) T cells to 1-month-old BALB/c nude mice, the salivary glands were severely damaged with CD4(+) T cell and B220(+) cells infiltrations. The number of T-cell antigen receptor V beta 8.3(+) CD4(+) T cells was significantly increased in BLM-treated murine spleen. These findings will provide new insights into the causal factors of environment in autoimmunity and the relationship between autoreactive CD4(+) T cells and autoantibodies. Immunology and Cell Biology ( 2009) 87, 351-358; doi:10.1038/icb.2009.1; published online 10 February 2009

We report a case of peripheral neuropathy and skin ulcer in a patient with rheumatoid arthritis (RA) who received tocilizumab. A 65-year-old woman with a 20-year history of RA participated in a tocilizumab clinical trial. She received a single dose of 8 mg/kg tocilizumab intravenously. The following day the patient started to experience numbness and purpura in all four extremities. The purpura of her left lower limb became necrotic, and a skin ulcer appeared 3 weeks later. Steroid-pulse treatment was initiated 12 weeks after tocilizumab administration, with the result that the numbness improved, and the skin ulcers showed complete epithelialization.

Recent progress with specific markers of lymphatic vessel endothelium allowed recognition of lymphangiogenic events in various disease states; however, there is little information concerning this process in human chronic renal diseases. To determine this we measured expression of the lymphatic marker D2-40 and vascular endothelial growth factor-C (VEGF-C), a major growth factor in lymphangiogenesis, in 124 human renal biopsy specimens. In the kidneys of control subjects and in uninjured areas of pathologic specimens, lymphatic vessels were detected only around the arcuate and interlobular arteries. An increase in the number of lymphatic vessels was found at the site of tubulointerstitial lesions correlating with the degree of tissue damage and more strongly correlating with areas of fibrosis than inflammation. On serial sections, lymphatic vessel proliferation was found in the tubulointerstitial area at the site of tuft adhesions to Bowman's capsule. Lymphatic growth was associated with VEGF-C expression in inflammatory mononuclear cells and tubular epithelial cells, mainly of proximal tubules. Lymphangiogenesis and VEGF-C expression was elevated in diabetic nephropathy in comparison to other renal diseases. Our results indicate that lymphangiogenesis is a common feature in the progression of the tubulointerstitial fibrosis.

Recently, we and others reported that diabetic endothelial nitric oxide synthase knockout (eNOSKO) mice develop advanced glomerular lesions that include mesangiolysis and nodular lesions. Interestingly, insulin treatment lowered blood pressure and prevented renal lesions, raising the question as to whether these beneficial effects of insulin were due to its ability to lower either high glucose levels or high blood pressure. We, therefore, examined the effect of lowering blood pressure using hydralazine in this diabetic eNOSKO mouse model. Hydralazine treatment significantly blocked the development of mesangiolysis and microaneurysms, whereas tubulointerstitial injury was not prevented in these mice. Additionally, hydralazine did not reduce expression levels of either tubulointerstitial thrombospondin-1 or transforming growth factor-beta despite controlling blood pressure. On the other hand, the critical role of high glucose levels on the development of tubulointerstitial injury was suggested by the observation that serum glucose levels were correlated with tubulointerstitial injury, as well as with the expression levels of both transforming growth factor-beta and thrombospondin-1. Importantly, controlling blood glucose with insulin completely blocked tubulointerstitial injury in diabetic eNOSKO mice. These data suggest that glomerular injury is dependent on systemic blood pressure, whereas hyperglycemia may have a more important role in tubulointerstitial injury, possibly due to the stimulation of the thrombospondin-l-transforming growth factor-beta pathway in diabetic eNOSKO mice. This study could provide insights into the pathogenesis of advanced diabetic nephropathy in the presence of endothelial dysfunction. (Am J Pathol 2009, 174:1221 1229; DOI: 10.2353/ajpath.2009.080605)

Background. Hemodialysis (HD) patients are susceptible to atypical tuberculosis (TB), especially among patients presenting with fever of unknown origin (FUO), because of their impaired cellular immunity. Diagnostic trials of anti-TB drugs are therefore recommended in some TB endemic countries, including Japan, though clinical evidence for this therapy is scarce. Methods. We prospectively collected data for incident cases of clinical FUO for two years in 78 of 169 dialysis facilities in Aichi prefecture, located in central Japan. Clinical FUO was defined as sustained fever without any localizing signs and no infiltration on chest x-rays after a one-week antibiotic trial. The baseline characteristics, subsequent body temperatures on the days of HD therapy, and names of antibiotics including anti-TB drugs with the durations of medication were reported until fever alleviation or fever sustainment for over eight weeks. Results. We identified 15 newly developed clinical FUO patients among 8,125 HD patients. The incidence rate was estimated to be 92 (95% CI, 26-158) per 100,000 person-years. This corresponds to 244 cases per year among 264,473 HD patients in Japan. Anti-TB drugs were secondarily prescribed in 8 of 15 clinical FUO patients (53%). No improved fever alleviation was observed when anti-TB drugs were secondarily prescribed compared with cases in which other antibiotics were preferred. Conclusion. We investigated the incidence of FUO in HD patients and found that the rate was not very high, whereas anti-TB drugs were frequently used for FUO cases. The efficacy of this diagnostic therapy should be elucidated in large-scale studies.