Hideki Yamaya

Proteomic analysis of serum, outflow dialysate and adsorbed protein onto dialysis membranes (polysulfone and pmma) during hemodialysis treatment using SELDI-TOF-MS.

Aims: Alterations in the profiling of peptides and proteins in the serum, outflow dialysate and adsorbed protein on the dialysis membrane were investigated. Methods: Alterations in the protein profiling of routine hemodialysis using polysulfone (TS-UL) and PMMA (moderate flux membrane of polymethylmethacrylate: BK-U) in 8 patients and that of adsorption onto polysulfone and PMMA membranes in 4 patients were evaluated by SELDI-TOF-MS and ProteinChip array. Mass-to-charge ratios (m/z) between 2,000 and 120,000 were analyzed. Results: The protein with a relative intensity of m/z 11,730 measured by SELDI-TOF-MS was present in a small amount in the outflow dialysate and in a large amount in adsorption (identified as β2-microglobulin) onto PMMA membrane. Unexpectedly, 68 molecular masses of peptides that were adsorbed more onto polysulfone than onto PMMA membrane were observed. There were more peptides less than m/z 11,730 adsorbed onto polysulfone membrane than onto PMMA membrane. Dominant peaks, m/z 6,629 and 6,431 adsorbed onto polysulfone membrane were identified as apolipoprotein CI and truncated apolipoprotein CI, respectively. 37 proteins with molecular weights larger than m/z 11,730 showed greater filtration through PMMA membrane than through polysulfone membrane. 149 molecular masses that were adsorbed onto PMMA or more onto PMMA membrane than onto polysulfone membrane were observed. Conclusion: This experiment suggests that membrane adsorption is an important mechanism for the removal of middle-molecular-weight proteins by hemodialysis using not only PMMA membrane but also polysulfone membrane. Adsorption of peptide or protein onto a dialysis membrane may depend not only on the membrane material, but also on the peptide or protein.

Advances in apheresis therapy for glomerular diseases

This article is an overview of the immunomodulatory effects of apheresis in renal diseases, especially primary and secondary glomerulonephritis, and the clinical evidence for the efficacy of apheresis therapy. Permeability factor(s) derived from circulating T cells are speculated to have a crucial role in the proteinuria of nephrotic syndrome (NS). Plasma exchange (PE); immunoadsorption plasmapheresis (IAPP), using protein A sepharose cartridges; low-density lipoprotein apheresis; and lymphocytapheresis (LCAP) have been used to remove such factors or pathogenic T cells. Other glomerular diseases induced by specific antibodies such as anti-glomerular basement membrane antibodies, anti-neutrophil cytoplasmic antibodies, and immune-complexes have also been treated with PE, double-filtration plasmapheresis, IAPP, and LCAP. Recommendations, based on the evidence from recent randomized controlled studies, have been established in apheresis therapy for various glomerular diseases.

A Case Report of an Adult With Severe Hyperlipidemia During Acute Lymphocytic Leukemia Induction Therapy Successfully Treated With Plasmapheresis

Plasmapheresis for the treatment of hypertriglyceridemia has previously been performed in patients with sudden onset severe hypertriglyceridemia and acute pancreatitis; however, only a few reports of this procedure have been published. We report here on a case showing severe hypertriglyceridemia during asparaginase (Asp) treatment for acute lymphocytic leukemia (ALL), and give an overview of a lipid‐lowering apheresis therapy. To prevent the complication of pancreatitis due to hypertriglyceridemia, we performed plasma exchange (PE) three times using fresh frozen plasma. PE remarkably reduced both serum triglyceride and total cholesterol levels from 5430 mg/dL to 403 mg/dL and from 623 mg/dL to 204 mg/dL, respectively. The causes of severe hyperlipidemia in this patient were considered to include: the Asp treatment for ALL, and a genetic background with a heterozygote of familial lipoprotein lipase (LPL) defect syndrome, because the patients plasma LPL level after intravenous heparin injection was low at 137 ng/mL. Hence, PE using fresh frozen plasma may be useful not only to remove lipoproteins, but also to supply defective factors, such as LPL, in similar cases.

Clinicopathological insights into lupus glomerulonephritis in Japanese and Asians

Lupus nephritis comprises a spectrum of glomerular, vascular, and tubulointerstitial lesions, which has significant racial variation in severity and manifestations. The current classification (ISN/RPS 2003) has been improved successfully for the categorization of lupus glomerulonephritis (LGN). On the basis of this classification, 480 Japanese cases revealed the following distribution: class I 3%, class II 16%, class III 13%, class IV-S 11%, class IV-G 41%, class V 16%, and class VI 1%. Class IV-G with chronicity tended to have the worst renal outcome. Nephrotic syndrome was a more frequent complication in class IV-S (50%), class IV-G (72%), and class V (56%), with poor renal and actuarial outcomes. With regard to therapy, treatment options including glucocorticoids alone or combined with antimetabolites (azathioprine, mizoribine, mycophenolate mofetil), calcineurin inhibitors (cyclosporine A, tacrolimus), or alkylating agents (intravenous cyclophosphamide injection) improved the outcome of LGN; however, there is no high-grade clinical evidence from Japan. Further studies are needed to resolve the clinicopathological problems of LGN, especially IV-S, IV-G, and pure membranous lupus nephritis in Japanese patients.

A patient with persistent renal AL amyloid deposition after clinical remission by HDM/SCT therapy.

A 62-year-old female patient was admitted to our hospital for evaluation of nephrotic syndrome. Monoclonal gammopathy (IgG λ type) and urinary Bence Jones proteins were detected in the serum and urine by the immunofixation method. The initial renal biopsy revealed amyloid deposition in mesangial area, glomerular capillary walls and arterioles by Congo-red staining, and amyloid fibers were detected by electron microscopy. On the bone marrow test, plasma cells accounted for 8.6%. Based on these findings, we diagnosed as AL amyloidosis associated with multiple myeloma. We treated her by high-dose intravenous injection therapy of melphalan combined with autologous peripheral blood stem cell transplantation. She achieved complete hematologic response 27 months later, however. Urine M-protein disappeared 2 months after treatment, and proteinuria slowly disappeared 17 months after treatment. On the other hand, amyloid fibers remained in renal biopsied tissues at 17 and 53 months after therapy. Electron microscopic examination also revealed the similar amyloid fibers in glomeruli. These findings suggest that, in this case, immunoglobulin light chains may cause directly and/or indirectly glomerular epithelial injury and nephrotic range proteinuria rather than via amyloid fiber formation.

Regulation of soluble Flt-1 (VEGFR-1) production by hnRNP D and protein arginine methylation.

Soluble fms-like tyrosine kinase-1 (sFlt-1) functions as a potent inhibitor of angiogenesis by trapping vascular endothelial growth factor (VEGF). However, the precise regulatory mechanism of sFlt-1 production is unknown. Here, we report that vascular sFlt-1 production is regulated by heterogeneous nuclear ribonucleoprotein D (hnRNP D) and arginine methylation. We showed that hnRNP D bound to Flt-1 pre-mRNA and that hnRNP D overexpression decreased sFlt-1 mRNA in human microvascular endothelial cells (HMVECs). In contrast, the reduction of hnRNP D levels induced an increase in sFlt-1 production. Overexpression of an hnRNP D mutant in which the arginine residue of the known arginine methylation motif (arginine-glycine-glycine; RGG) was replaced with alanine did not reduce the level of soluble-form RNA produced from the Flt-1 minigene. Moreover, we demonstrated that overexpression of arginine methyltransferase decreased the soluble-form RNA level, whereas overexpression of arginine demethylase and addition of methyltransferase inhibitors increased sFlt-1 mRNA levels. These findings indicate that hnRNP D and arginine methylation play important roles in the regulation of Flt-1 mRNA alternative polyadenylation.

Progressive multifocal leukoencephalopathy developed 26 years after renal transplantation

Department of Neurology, Kanazawa Medical University, Ishikawa, Japan Division of Nephrology, Kanazawa Medical University, Ishikawa, Japan Department of Virology I, National Institute of Infectious Disease, Tokyo, Japan Department of Neurosurgery, Brain Research Institute, University of Niigata, Niigata, Japan Department of Pathology, Brain Research Institute, University of Niigata, Niigata, Japan

Circulating fibrocytes in ischemia-reperfusion injury and chronic renal allograft fibrosis.

Background: Interstitial fibrosis in chronic allograft injury has been suggested as a major cause of the loss of allograft. Methods: To clarify the involvement of circulating fibrocytes (CF) and α-smooth muscle actin (SMA)-positive cells in renal allograft injury, we investigated 36 renal transplanted cases at 0 h, 1 h, 2–4 weeks, 4–8 weeks, and 1 year, and 5 normal controls. Double immunofluorescence analysis for both COL1 and CD45 indicating CF (/mm2), and the positive area (%) of α-SMA and Masson trichrome (MT) stain were detected by an image analyzing system. Results: The mean number of CF was 0 in controls and 4.0 in total transplanted specimens (p < 0.05). CF correlated with the α-SMA-positive area in the graft (R2 = 0.39, p < 0.01), but not with Banff 2005 scores. The number of CF increased in 2–4 weeks; however, decreased 1 year after transplantation. α-SMA-positive area gradually increased at 1 year concomitant with the increase of MT-positive area. A similar phenomenon was observed in a case of primary nonfunction kidney from 0 h to 6 weeks after transplantation. The electron microscopy score of fibrosis around peritubular capillaries was correlated positively with COL1-positive area (R2 = 0.72, p < 0.01), but negatively with infiltrated CF (R2 = 0.25, p < 0.05). Conclusion: CF were transiently induced, probably due to ischemia-reperfusion injury, but fibrosis only slightly progressed in this process. The α-SMA-positive myofibroblasts may accelerate the expansion of fibrosis around peritubular capillaries in chronic allograft injury.

A case of second renal transplantation with acute antibody-mediated rejection complicated with BK virus nephropathy

Atsumi H, Asaka M, Kimura S, Imura J, Fujimoto K, Chikazawa Y, Nakagawa M, Okuyama H, Yamaya H, Moriyama M, Tanaka T, Suzuki K, Yokoyama H. A case of second renal transplantation with acute antibody‐mediated rejection complicated with BK virus nephropathy.
Clin Transplant 2010: 24 (Suppl. 22): 35–38.

Ischemia–reperfusion injury as the leading cause of primary non‐function in renal transplantation using donors with prolonged warm ischemic time

Abstract:  Renal transplantation (RTx) from marginal donors has been performed in most Japanese transplant centers because of severe donor organ shortage. RTx using marginal donors with prolonged warm ischemic time (WIT) has sometimes resulted in primary non‐function (PNF). Among 53 cadaveric RTx performed at Kanazawa Medical University between October 1986 and May 2006, five cases using non‐heart‐beating donors resulted in PNF. In these five patients, histologic examination of one h RBx (graft biopsy one h after revascularization) revealed extensive congestion in glomerular and peritubular capillaries. Glomerular subendothelial swelling and mesangiolysis were also detected, and electron microscopic examination showed widening of the subendothelial spaces of glomerular capillaries. Such findings of endothelial damage were not detected in the other 48 cases without PNF. In contrast, no distinct abnormalities other than acute tubular necrosis were found in zero h RBx (graft biopsy before implantation) obtained from three patients with PNF. Prolonged WIT was the prominent feature of the five patients with PNF, and multiple logistic analysis showed that prolonged WIT was a significant risk factor for the development of endothelial damage on reperfusion. Paired allografts from the same donors of these five cases also ended in PNF. It is conceivable that the endothelial damage was caused by ischemia–reperfusion injury and extensive congestion followed as a result of endothelial injury. Ischemia–reperfusion injury is the leading cause of PNF in RTx using donors with prolonged WIT. Guidelines to assess the viability of marginal grafts should be established in the future to prevent transplantation of non‐functioning grafts.