Koji Yazawa

Analytic validation of the enzyme multiplied immunoassay technique for the determination of mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatographic assay

The analysis of mycophenolic acid (MPA) has proved a valuable adjunct to the clinical care of organ transplant recipients. The analytic validation of the enzyme multiplied immunoassay technique (EMIT) for the determination of MPA in plasma is described. The EMIT MPA standard curve was 0 to 15.0 &mgr;g/mL, and curve storage was maintained for 4 weeks. The MPA EMIT assay proved reliable and reproducible, as shown by the intra-assay and interassay coefficients of variation (1.58–3.68% and 1.23–7.57%, respectively). Excellent linear correlation (r = 0.999) was observed for dilution linearity. The sensitivity of the assay was 0.01 &mgr;g/mL. Recoveries of 99.4% to 104.2% were obtained by spiking aliquots of three controls of known MPA concentrations with MPA. No interference was observed for endogenous substances and coadministered immunosuppressant drugs, and no cross-reactivity from the major metabolite MPA glucuronide was found. The high-performance liquid chromatography (HPLC) assay used protein precipitation and C18 ion-pair chromatography with ultraviolet detection at 304 nm. Plasma concentrations of MPA were measured using EMIT and HPLC. A linear relationship was observed between EMIT and HPLC (EMIT = 1.091 × HPLC - 0.089;r 2 = 0.990, n = 129). These results indicate that EMIT is a simple, rapid, and sensitive assay method for the measurement of MPA in plasma.

Blocking of CCR5 and CXCR3 suppresses the infiltration of macrophages in acute renal allograft rejection.

Background. The chemokine receptors CCR5 and CXCR3 are expressed by T cells and macrophages. We examined effects of a CCR5/CXCR3 antagonist (TAK), with a particular focus on the role of macrophages, in a rat kidney transplant model. Methods. Dark Agouti rat kidneys were transplanted into Lewis rats. The recipients were treated daily with a 10 mg/kg TAK on posttransplant days 0 to 14 and/or 2 mg/kg of cyclosporine A (CsA) on days 0 to 5. Graft survival, histological changes, and the expression of chemokines and chemokine receptors on T cells and macrophages were studied. Results. Treatment with TAK alone suppressed CD4+T cell infiltration and slightly prolonged graft survival. The expressions of both CCR5 and CXCR3, and activated macrophage-associated cytokines and chemokines, were significantly increased on macrophages that had been separated from rejecting kidneys, compared with those from spleens. However, these upregulations were decreased in macrophages from kidneys that had been treated with TAK. Immunohistochemistry also showed that macrophages infiltrating tubules of rejecting kidney expressed both receptors. In the CsA alone group, macrophages were the dominant infiltrating cells, and all allografts were rejected within 10 days. A combined therapy involving CsA and TAK resulted in decreased macrophage infiltration, and graft survival was substantially prolonged. The levels of activated macrophage-associated cytokines and chemokines were also decreased. Conclusion. The dual blocking of CCR5/CXCR3 can be useful in decreasing rejection, with or without CsA. This mechanism acts, not only to block T-cell recruitment to a kidney graft but to suppress the infiltration of macrophages as well.

Malignant Neoplasm in Kidney Transplantation

Background: The kidney recipient is at a higher risk for cancer than is the general population, although the incidence of neoplasms in general is considered lower in Japan than in Western countries. The cause of this increased risk associated with either transplantation or geography has not yet been established.

In vivo gene transfer of hepatocyte growth factor to skeletal muscle prevents changes in rat kidneys after 5/6 nephrectomy.

Hepatocyte growth factor (HGF) has been reported to be a renal regeneration factor. We previously reported that HGF acts as a renotropic factor, inducing cell recovery from ischemic injury or drug toxicity. Gene transfer by electroporation, which uses plasmid DNA as the vector, has several advantages over the conventional gene transfer method using viral vectors, inducing the ability to perform repeated transfers without apparent immunologic responses to the DNA vector. We recently demonstrated that electroporation of the HGF gene into skeletal muscle was an effective treatment for liver injury in an animal model. We presently investigated prevention of development of chronic renal disease by repetitive HGF gene transfer in rats with 5/6 nephrectomy. Hepatocyte growth factor gene transfer‐treated rats showed better growth in body weight than untreated rats. Histologic changes such as glomerulosclerosis and interstitial fibrosis were significantly ameliorated by HGF gene transfer compared with untreated rats. Hepatocyte growth factor gene transfer by electroporation into skeletal muscle is feasible and effective against morphologic injury in subtotally nephrectomized rats.

Vitamin D Deficiency Predicts Decline in Kidney Allograft Function: A Prospective Cohort Study

CONTEXT Vitamin D, often deficient in kidney transplant (KTx) recipients, has potential immunomodulatory effects. OBJECTIVE This study aimed to evaluate whether vitamin D status affects the rate of decline in kidney allograft function. DESIGN, SETTING, AND PATIENTS The study included a prospective cohort of 264 ambulatory KTx recipients at a single Japanese center. MAIN OUTCOME MEASURES We measured the baseline 25-hydroxyvitamin D (25D) concentration and examined its association with annual decline in estimated glomerular filtration rate (eGFR). Secondary outcome was rescue treatment with iv methylprednisolone (IV-MP) as an index of rejection episodes. RESULTS The mean serum 25D concentration was 17.1 (SD 6.5) ng/mL, and 68.4% patients had vitamin D inadequacy or deficiency. Time after KTx was a significant effect modifier for the association of serum 25D concentration with annual eGFR change and need for IV-MP (P for interaction < .1). We divided patients according to the median time after KTx (10 y) and found that low vitamin D was significantly associated with a rapid eGFR decline at less than 10 years after KTx but not at 10 or more years after KTx. The same was true for rescue treatment with IV-MP. Overall, propensity score matching showed independent associations of low vitamin D with both outcomes. Stratified matching confirmed pronounced associations at less than 10 years after KTx. CONCLUSIONS Vitamin D deficiency predicts a rapid decline in eGFR and need for IV-MP at less than 10 years after KTx. Future studies are warranted to evaluate the clinical efficacy of vitamin D supplementation.

Hydrogen-rich University of Wisconsin solution attenuates renal cold ischemia-reperfusion injury.

Background Renal ischemia-reperfusion (I/R) injury is unavoidable in kidney transplantation and frequently influences both short- and long-term allograft survival rates. One of the major events in I/R injury is the generation of cytotoxic oxygen radicals. Recently, hydrogen gas has been reported to display antioxidant properties and protective effects against organ dysfunction induced by various I/R injuries. We investigated whether hydrogen-rich University of Wisconsin (HRUW) solution attenuates renal cold I/R injury. Methods We prepared HRUW solution by a novel method involving immersion of centrifuge tubes containing UW solution into hydrogen-saturated water. Hydrogen readily permeates through the centrifuge tubes, and thus, the hydrogen concentration of the UW solution gradually increases in a time-dependent manner. Syngeneic rat kidney transplantation was performed, and the animals were divided into three groups: recipients with nonpreserved grafts (control group), recipients with grafts preserved in UW solution for 24 to 48 hr (UW group), and recipients with grafts preserved in HRUW solution for 24 to 48 hr (HRUW group). Results In the early phases, HRUW solution decreased oxidative stress, tubular apoptosis, and interstitial macrophage infiltration in the kidney grafts. Consequently, HRUW solution improved renal function and prolonged recipient survival rate compared with simple cold storage using UW solution. Histopathologically, HRUW treatment alleviated tubular injury and suppressed development of interstitial fibrosis. Conclusions HRUW solution improved graft function and prolonged graft survival compared with simple cold storage using UW solution by protecting tubular epithelial cells from inflammation and apoptosis. Our new method of organ preservation is a groundbreaking, safe, and simple strategy that may be applied in the clinical setting.

Epigallocatechin-3-gallate protects kidneys from ischemia reperfusion injury by HO-1 upregulation and inhibition of macrophage infiltration

Epigallocatechin‐3‐gallate (EGCG) shows diverse chemical and biological activities. We investigated the effects of EGCG in a rat renal ischemia reperfusion (I/R) injury model. Sprague–Dawley rats received intraperitoneal injection of 50 mg/kg EGCG 48 h, 24 h, and 30 min prior to I/R injury. The animals were subjected to left renal occlusion for 45 min. EGCG treatment suppressed the peak in serum creatinine. EGCG‐treated kidneys showed significantly less tubular damage and a decreased number of apoptotic cells. The I/R‐induced elevation in the renal MDA level was significantly decreased in the EGCG group. Reverse‐transcriptase polymerase chain reaction showed that EGCG significantly decreased the expression of MHC class II, TLR2, TLR4, MCP‐1, IL‐18, TGF‐β1, procollagen Ia1, TIMP‐1, and Kim‐1. ED‐1 staining showed reduced macrophage infiltration and α‐SMA staining revealed less interstitial expression. Heme oxygenase‐1 (HO‐1) expression in I/R kidneys was upregulated in the EGCG group based on the results of both RT‐PCR and Western blotting analysis. Blockade of HO‐1 gene induction by SnPP increased renal tubular damage and macrophage infiltration. These findings suggest that EGCG protects the kidneys against I/R injury by reducing macrophage infiltration and decreasing renal fibrosis. These beneficial effects may be mediated, in part, by augmentation of the HO‐1 gene.

Acute graft-versus-host-disease in kidney transplantation: case report and review of literature.

Graft-versus-host-disease (GVHD) is a complication of solid organ transplantation, most commonly of the small bowel or liver. Herein, we have presented a case of GVHD in a 27-year-old man who underwent an human leukocyte antigen (HLA) minor mismatch renal transplantation from his father. After the procedure, the patient presented with a fever, skin rash, and watery diarrhea. An allograft kidney biopsy demonstrated no sign of rejection; however, anti-A antibody was detected in plasma and progressive anemia was attributed to hemolytic anemia owing to a passenger lymphocyte syndrome. From those findings, we suspected that the clinical symptoms were caused by acute GVHD. An endoscopic biopsy of the colon revealed apoptotic cells consistent with the disease. We found reports of only 5 other GVHD cases after kidney transplantation. Several risk factors are associated with GVHD, such as transfer of graft lymphocytes, donor HLA homozygosity, and a relationship between recipient immunogenicity and immunosuppression. In this case, detection led to early diagnosis of donor-derived GVHD due to passenger lymphocyte syndrome. It is important keep GVHD in mind and to understand its risk factors as the mortality rate is high.

Adipose tissue-derived stem cells suppress acute cellular rejection by TSG-6 and CD44 interaction in rat kidney transplantation.

Background In addition to its abundance and easy accessibility, adipose tissue yields more potent immunoregulatory stem cells (adipose tissue-derived stem cells, ADSCs) than does bone marrow. However, the beneficial effects of ADSCs on alloreactivity are scarcely known. This study evaluated the beneficial effects of ADSCs in rat kidney transplantation and analyzed the underlying molecular mechanism. Methods Dark Agouti rat kidneys were transplanted into Lewis rats. Autologous ADSCs (2×106) were injected through the left renal artery of the donors before the nephrectomy (ADSCs group). Graft survival, histologic changes, and the expression of several cytokines and proteins were assessed. In an in vitro experiment, the immunosuppressive capacity of ADSCs was tested in a mixed lymphocyte reaction. Results Histologic findings of the ADSCs group revealed a reduced rejection grade, whereas the number of infiltrated CD4+/CD8+ T cells was also significantly decreased as compared to the control. Relative to these findings, injection of ADSCs led to a significantly prolonged mean graft survival compared with the control. In vitro, autologous ADSCs dose-dependently suppressed alloreactive lymphocytes. Moreover, ADSCs increased the level of tumor necrosis factor-inducible gene 6 protein (TSG-6) in mixed lymphocyte reaction, which has an anti-inflammatory capacity. Recombinant TSG-6 markedly suppressed alloreactive T cells through downregulating CD44, which may lead to the suppression of T-cell activation and infiltration into allografts. Conclusion Our findings clearly showed that ADSCs attenuated acute rejection by secreting TSG-6 as well as through direct cell interaction. These findings contribute to the clinical application of these cells in solid organ transplantation.

Transplantation of allogenic fetal membrane-derived mesenchymal stem cells protects against ischemia/reperfusion-induced acute kidney injury.

Mesenchymal stem cells (MSCs) are an attractive therapeutic cell source for treating renal diseases. MSC administration has been shown to improve renal function, although the underlying mechanisms are not completely understood. We recently showed that allogenic fetal membrane-derived MSCs (FM-MSCs), which are available noninvasively in large amounts, had a renoprotective effect in an experimental glomerulonephritis model. Here we investigated whether allogenic FM-MSC administration could protect kidneys from ischemia/ reperfusion (I/R) injury. Lewis rats were subjected to right nephrectomy and left renal I/R injury by clamping the left renal artery as an acute kidney injury (AKI) model. After declamping, FM-MSCs (5 × 105 cells) obtained from major histocompatibility complex (MHC)-mismatched ACI rats were intravenously administered. I/R-injured rats exhibited increased serum creatinine and BUN, whereas FM-MSC administration significantly ameliorated renal function. Histological analysis revealed that FM-MSC administration significantly suppressed tubular apoptosis and infiltration of macrophages and T-cells. Administration of FM-MSCs mainly homed into the lung, but increased serum IL-10 levels. Of interest is that renoprotective effects of FM-MSCs were abolished by using anti-IL-10 neutralization antibody, suggesting that IL-10 would be one of the candidate factors to protect rat kidney from I/R injury in this model. We concluded that allogenic FM-MSC transplantation is a potent therapeutic strategy for the treatment of AKI.