Ryoichi Shiroki

Serum neutrophil gelatinase-associated lipocalin as a predictor of organ recovery from delayed graft function after kidney transplantation from donors after cardiac death.

Because of a worldwide shortage of renal grafts, kidneys procured from donors after cardiac death (DCD) have recently become an important source of renal transplants. However, DCD kidneys often have complications with delayed graft function (DGF) and recipients require hemodialysis (HD) in the early period after kidney transplantation (KTx). This study evaluated serum NGAL as a potential specific parameter to predict early functional recovery of transplanted DCD kidneys. The average serum neutrophil gelatinase-associated lipocalin (NGAL) level in normal samples was 53 ± 30 ng/ml, while that in patients with chronic renal failure requiring HD was markedly raised at 963 ± 33 ng/ml. In patients undergoing a living-related KTx from a living donor (n = 11), serum NGAL level decreased rapidly after KTx, and only in two cases, with serum NGAL levels over 400 ng/ml on postoperative day 1 (POD1), was HD required due to DGF. In contrast, all patients undergoing a KTx from a DCD (n = 5) required HD due to DGF. Even in these cases, serum NGAL levels decreased rapidly several days after a KTx prior to the recovery of urine output and preceding the decrease in serum creatinine level. The pattern of decline in serum NGAL was biphasic, the decrease after the second peak indicating a functional recovery within the next several days. These data suggest that monitoring of serum NGAL levels may allow us to predict graft recovery and the need for HD after a KTx from a DCD.

Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic

Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG1 Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG1 revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.

Increased Urinary Neutrophil Gelatinase Associated Lipocalin Levels in a Rat Model of Upper Urinary Tract Infection

PURPOSE Recurrent upper urinary tract infection is a common complication of vesicoureteral reflux that often leads to irreversible renal scarring. In our previous study of a rat model of renal bacterial infection we performed global gene expression profiling of the kidney during the onset of renal scarring. We have further investigated the product of an up-regulated gene product, NGAL, in this animal model to evaluate its potential usefulness as a biomarker of renal scarring. MATERIALS AND METHODS Renal NGAL mRNA and protein levels were examined by real-time polymerase chain reaction, Western blot and immunohistochemistry. Urinary NGAL levels were monitored by direct enzyme-linked immunosorbent assay. RESULTS Rat renal NGAL mRNA and protein levels were found to be increased soon after bacterial injection. They then decreased rapidly but subsequently persisted at high levels until the 6-week time point after injection. On histological analysis we found that NGAL protein was overproduced in macrophages and renal tubular cells 2 weeks after injection. However, renal tubular cells continued to produce NGAL 6 weeks after injection, whereas this expression was lost in infiltrating cells. Rat urinary NGAL levels were also markedly increased at the early stages of infection and they persisted at high levels throughout the latter stages of the experiment. CONCLUSIONS Urinary NGAL may be a potential noninvasive diagnostic biomarker of renal scarring.

Genomewide expression profiles of rat model renal isografts from brain dead donors

Background. It has been well documented that two factors, brain death (BD) and ischemia/reperfusion (I/R) injury, have distinct but overlapping adverse influences on the clinical outcome of renal transplantation. Method. We previously established a rat model of renal isografting from brain dead donors. In the present study, we performed genomic expression profiling with a high-density oligonucleotide microarray to identify genes that were upregulated or downregulated by BD and/or I/R injury. Results. Among a total of 20,550 genes, most of those upregulated by BD were genes for adhesion molecules and cytokines or for chemokines such as Gro1 and IP-10. When overexpression of these genes was assessed by real-time reverse transcriptase–polymerase chain reaction, it was only observed one hr after the engraftment of kidneys from BD donors and returned to baseline thereafter, indicating the presence of an acute systemic inflammatory response to BD. Analysis of biologic networks demonstrated the activation of specific pathways that were clearly different for BD and I/R injury. The p53 and NF&kgr;B pathway was involved in the acute response to BD, whereas the Myc, Jun, and c-fos pathway was involved in I/R injury. Investigation of secretory protein genes identified LCN2 and SPP1 as candidate genes for biologic markers. Conclusion. Because our experimental system is a good model of renal transplantation from brain dead or living human donors, our data may be useful for elucidating the pathologic processes involved and for identification of novel markers for graft dysfunction of renal transplantation.

Urinary Neutrophil-Gelatinase Associated Lipocalin is a Potential Noninvasive Marker for Renal Scarring in Patients With Vesicoureteral Reflux

PURPOSE Renal scarring is a serious complication that often occurs with chronic pyelonephritis in the presence of vesicoureteral reflux. In a previous study we established a rat model of renal scarring in which we found the up-regulation of neutrophil-gelatinase associated lipocalin at the mRNA and protein levels. In this study we evaluated urinary neutrophil-gelatinase associated lipocalin as a potential biomarker for progression of renal scarring in patients with vesicoureteral reflux. MATERIALS AND METHODS A total of 34 patients diagnosed with vesicoureteral reflux without evidence of current urinary tract infection and 28 normal healthy children were enrolled in this study. Renal scars were evaluated by (99m)technetium dimercapto-succinic acid renal scan in 24 of the reflux cases. Urinary neutrophil-gelatinase associated lipocalin levels were monitored by ELISA. RESULTS In normal subjects urinary neutrophil-gelatinase associated lipocalin was high during infancy, decreased rapidly within the following year and reached a low stable level from age 3 years onward. Urinary neutrophil-gelatinase associated lipocalin levels, normalized to age matched standards, were significantly increased in patients with vesicoureteral reflux compared to controls. These levels did not correlate with reflux grade, but were significantly higher in patients with radiological evidence of renal scarring irrespective of reflux grade. CONCLUSIONS Estimation of urinary neutrophil-gelatinase associated lipocalin may be useful as a noninvasive diagnostic or prognostic biomarker for renal scarring.

Selection and analysis of anti-cancer antibodies for cancer therapy obtained from antibody phage library

The search for effective antibodies (Ab) for curable cancer immunotherapy has been a quest of many research groups in order to find an effective target that exists on the cancer cell surface. So far there have been no conclusive answers to shed light on the search. This study therefore aimed to bridge the gap of cancer therapy. Screening against 49 kinds of cell lines belonging to 11 kinds of solids cancers was performed. Isolation and characterization for approximately 4200 monoclonal antibodies (mAb) was also performed thereafter. Of those mAb 488 clones that turned out to bind to 29 tumor‐associated antigens (TAA) were subjected to immunohistochemical (IHC) analyses. Selection of target antigens (Ag) and a potential antibody for cancer therapy was conducted prior to clinical examinations. In order to find predictably effective targets for therapeutic Ab against solid cancers, expression of the Ag on the surface of cancer and normal cells was extensively examined by IHC analyses using fresh cancer specimens resected from patients. In this study, the tendencies of all staining patterns and distribution of the Ab are reported. While all of the TAA appeared to be involved in tumorigenesis, their expression was not restricted to some specific tumor types but rather randomly distributed among various cancers. Some kinds of Ab including anti‐epidermal growth factor receptor (EGFR) and anti‐human epidermal growth factor receptor 2 (HER2) indicated the frequency of expression in normal cells was generally low. We concluded that identification of 488 mAb and the accumulated results of IHC analyses in this study could be the key for further therapeutic Ab against cancers. The targets that showed cancer‐specific expression are expected to be better for therapeutic Ab than the other Ab. Moreover, further investigation into the growth of cancer cell lines using full human IgG form of Ab shows available efficacy in specific cases. (Cancer Sci 2011; 102: 175–181)

Robot‐assisted partial nephrectomy: Superiority over laparoscopic partial nephrectomy

Nephron‐sparing surgery has been proven to positively impact the postoperative quality of life for the treatment of small renal tumors, possibly leading to functional improvements. Laparoscopic partial nephrectomy is still one of the most demanding procedures in urological surgery. Laparoscopic partial nephrectomy sometimes results in extended warm ischemic time and severe complications, such as open conversion, postoperative hemorrhage and urine leakage. Robot‐assisted partial nephrectomy exploits the advantages offered by the da Vinci Surgical System to laparoscopic partial nephrectomy, equipped with 3‐D vision and a better degree in the freedom of surgical instruments. The introduction of the da Vinci Surgical System made nephron‐sparing surgery, specifically robot‐assisted partial nephrectomy, safe with promising results, leading to the shortening of warm ischemic time and a reduction in perioperative complications. Even for complex and challenging tumors, robotic assistance is expected to provide the benefit of minimally‐invasive surgery with safe and satisfactory renal function. Warm ischemic time is the modifiable factor during robot‐assisted partial nephrectomy to affect postoperative kidney function. We analyzed the predictive factors for extended warm ischemic time from our robot‐assisted partial nephrectomy series. The surface area of the tumor attached to the kidney parenchyma was shown to significantly affect the extended warm ischemic time during robot‐assisted partial nephrectomy. In cases with tumor‐attached surface area more than 15 cm2, we should consider switching robot‐assisted partial nephrectomy to open partial nephrectomy under cold ischemia if it is imperative. In Japan, a nationwide prospective study has been carried out to show the superiority of robot‐assisted partial nephrectomy to laparoscopic partial nephrectomy in improving warm ischemic time and complications. By facilitating robotic technology, robot‐assisted partial nephrectomy will be more frequently carried out as a safe, effective and minimally‐invasive nephron‐sparing surgery procedure.

Tyrosine kinase inhibitor-induced vasculopathy in clear cell renal cell carcinoma: an unrecognized antitumour mechanism

To evaluate the pathological features of clear cell renal cell carcinoma (CCRCC) treated with tyrosine kinase inhibitors (TKIs), and to elucidate the mechanism of action of TKIs.

Serum Neutrophil Gelatinase Associated Lipocalin During the Early Postoperative Period Predicts the Recovery of Graft Function After Kidney Transplantation From Donors After Cardiac Death

PURPOSE Kidneys procured from donors after cardiac death hold great potential to expand the donor pool. However, they have not yet been fully used, in part due to the high incidence of delayed graft function. Although urine neutrophil gelatinase-associated lipocalin is a well-known early biomarker for renal injury after kidney transplantation, its usefulness is limited in cases with delayed graft function because of the unavailability of a urine sample. We evaluated serum neutrophil gelatinase-associated lipocalin as a potential biomarker to predict the functional recovery of kidneys transplanted from donors after cardiac death. MATERIALS AND METHODS Consecutive patients transplanted with a kidney from a living related (39), brain dead (1) or post-cardiac death (27) donor were retrospectively enrolled in the study. Serum samples were collected serially before and after kidney transplantation. Serum neutrophil gelatinase-associated lipocalin was measured using the ARCHITECT® assay. RESULTS Average serum neutrophil gelatinase-associated lipocalin was markedly high during the pre transplantation period. It decreased rapidly after transplantation. The slope of the decrease correlated well with the recovery period. By analyzing ROC curves we determined cutoffs to predict immediate, slow or delayed graft function requiring hemodialysis for longer than 1 week with high sensitivity and specificity. CONCLUSIONS These data suggest that serial monitoring of serum neutrophil gelatinase-associated lipocalin may allow us to predict graft recovery and the need for hemodialysis after kidney transplantation from a donor after cardiac death.

Global expression profiles in 1-hour biopsy specimens of human kidney transplantation from donors after cardiac death.

Because of the worldwide shortage of renal grafts, kidney transplantation (KTx) from donors after cardiac death (DCD) is an alternative way to obtain KTx from brain-dead donors. Although the prognosis of DCD KTx is gradually improving, the graft often undergoes delayed graft function (DGF), rendering the control of DGF essential for post-KTx patient care. In an attempt to characterize etiology of DGF, genome-wide gene expression profiling was performed using renal biopsy samples performed at 1 h after KTx from DCD and the data were compared with those of KTx from living donors (LD). A total of 526 genes were differentially expressed between them. Genes involved in acute inflammation were activated, while metabolic pathways were consistently downregulated in DCD. These findings imply the inferior performance of the DCD grafts relative to LD grafts. Several genes were identified where the expression levels were correlated well with parameters indicating short- and long-term prognosis of the DCD patients. In addition, several genes encoding secretory proteins were identified that might reflect the performance of the graft and be potential noninvasive biomarkers. These data provide a good source for candidates of biomarkers that are potentially useful for the control of DGF.