Satomi Iwai

Use of sphingosine-1-phosphate 1 receptor agonist, KRP-203, in combination with a subtherapeutic dose of cyclosporine A for rat renal transplantation.

Background. We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. Methods. We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-1-phosphate (S1P)1 and S1P3 receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. Results. Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P1 and S1P3 receptors, KRP-203-P selectively activated S1P1, but not the S1P3 receptor (EC50: >1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P<0.05, survival time: 9.8 days (CsA) vs. >27.4 days (CsA+KRP)). Although a higher dose of CsA (3 mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P<0.05, creatinine clearance: 0.41±0.25 ml/min [CsA] vs. 1.15±0.16 ml/min [CsA+KRP]). Conclusions. The selectivity of KRP-203 for S1P1 reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.

Xenotransplanted Embryonic Kidney Provides a Niche for Endogenous Mesenchymal Stem Cell Differentiation Into Erythropoietin-Producing Tissue

Recent findings have demonstrated that stem cells can differentiate into mature tissue when supplied with a niche containing factors identical to those in the normal developmental program. A niche for the development of an organ can be provided by xenotransplantation of a similar developing organ. However, this process has many technical, safety, and ethical concerns. Here, we established xenotransplantation models that control endogenous mesenchymal stem cell (MSC) differentiation into mature erythropoietin (EPO)‐producing tissue in a niche provided by a developing xenometanephros. Transplantation of rat metanephroi into mouse omentum, and similarly pig metanephroi into cat omentum, led to the recruitment of host cells and EPO production. EPO‐expressing cells were not differentiated from integrating vessels because they did not coexpress endothelial markers (Tie‐2 and VE‐cadherin). Instead, EPO‐expressing cells were shown to be derived from circulating host cells, as shown by enhanced green fluorescent protein (EGFP) expression in the grown transplants of chimeric mice bearing bone marrow from a transgenic mouse expressing EGFP under the control of the EPO promoter. These results suggest that donor cell recruitment and differentiation in a xenotransplanted developing organ may be consistent between species. The cells responsible for EPO expression were identified as MSCs by injecting human bone marrow‐derived MSCs and endothelial progenitor cells into NOD/SCID mice. Furthermore, using metanephroi from transgenic ER‐E2F1 suicide‐inducible mice, the xenotissue component could be eliminated, leaving autologous EPO‐producing tissue. Our findings may alleviate adverse effects due to long‐lasting immunosuppression and help mitigate ethical concerns. STEM CELLS2012;30:1228–1235

Urine excretion strategy for stem cell-generated embryonic kidneys

Significance Worldwide, the number of patients with end-stage renal disease requiring renal replacement therapy is increasing because of the shortage of donor organs. We have successfully generated functional kidneys from human stem cells using the organogenic niche method. However, for these kidneys to have clinical application, a urinary excretion pathway is necessary. Using pigs, we demonstrated our stepwise peristaltic ureter system, showing that it resolves important problems regarding the construction of the urine excretion pathway and the long-term growth of the stem cell-generated embryonic kidneys. There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal’s ureter to the cloacal-developed bladder, a technique we called the “stepwise peristaltic ureter” (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.

Impact of Normothermic Preservation with Extracellular Type Solution Containing Trehalose on Rat Kidney Grafting from a Cardiac Death Donor

Background The aim of this study was to investigate factors that may improve the condition of a marginal kidney preserved with a normothermic solution following cardiac death (CD) in a model of rat kidney transplantation (RTx). Methods Post-euthanasia, Lewis (LEW) donor rats were left for 1 h in a 23°C room. These critical kidney grafts were preserved in University of Wisconsin (UW), lactate Ringers (LR), or extracellular-trehalose-Kyoto (ETK) solution, followed by intracellular-trehalose-Kyoto (ITK) solution at 4, 23, or 37°C for another 1 h, and finally transplanted into bilaterally nephrectomized LEW recipient rats (n = 4–6). Grafts of rats surviving to day 14 after RTx were evaluated by histopathological examination. The energy activity of these marginal rat kidneys was measured by high-performance liquid chromatography (HPLC; n = 4 per group) and fluorescence intensity assay (n = 6 per group) after preservation with UW or ETK solutions at each temperature. Finally, the transplanted kidney was assessed by an in vivo luciferase imaging system (n = 2). Results Using the 1-h normothermic preservation of post-CD kidneys, five out of six recipients in the ETK group survived until 14 days, in contrast to zero out of six in the UW group (p<0.01). Preservation with ITK rather than ETK at 23°C tended to have an inferior effect on recipient survival (p = 0.12). Energy activities of the fresh donor kidneys decreased in a temperature-dependent manner, while those of post-CD kidneys remained at the lower level. ETK was superior to UW in protecting against edema of the post-CD kidneys at the higher temperature. Luminescence intensity of successful grafts recovered within 1 h, while the intensity of grafts of deceased recipients did not change at 1 h post-reperfusion. Conclusions Normothermic storage with extracellular-type solution containing trehalose might prevent reperfusion injury due to temperature-dependent tissue edema.

Impact of Ex Vivo Administration of Mesenchymal Stem Cells on the Function of Kidney Grafts From Cardiac Death Donors in Rat

BACKGROUND Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). METHODS Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 μL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months. RESULTS Systemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC-injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC-non-injected group (MSC[-]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(-) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months.

Non-Invasive Magnetic Resonance Imaging in Rats for Prediction of the Fate of Grafted Kidneys from Cardiac Death Donors

The main objective of this study was to assess cardiac death (CD) kidney grafts before transplantation to determine whether blood oxygen level-dependent (BOLD) and diffusion MRI techniques can predict damage to these grafts after transplantation. We assessed CD kidney tissue by BOLD and diffusion MRI. We also examined pathological and gene expression changes in CD kidney grafts before and after transplantation. Although there was significantly more red cell congestion (RCC) in the inner stripe of the outer medulla (IS) in both 1 h after cardiac death (CD1h) and CD2h kidneys destined for grafts before transplantation compared with CD0h (p<0.05), CD2h, but not CD1h, kidney grafts had significantly different RCC in the IS 2 days after transplantation (p<0.05). Consistent with these pathological findings, tissue plasminogen activator (tPA) gene expression was increased only in the cortex and medulla of CD2h kidney grafts after transplantation. BOLD MRI successfully and non-invasively imaged and quantified RCC in the IS in both CD1h and CD2h kidney grafts (p<0.05). Diffusion MRI also non-invasively assessed increased the apparent diffusion coefficient in the IS and decreased it in the outer stripe (OS) of CD2h grafts, in concordance with interstitial edema in the IS and tubule cellular edema in the OS. These two types of edema in the outer medulla could explain the prolonged RCC in the IS only of CD2h kidney grafts, creating part of a vicious cycle inhibiting red cells coming out of capillary vessels in the IS. Perfusion with University of Wisconsin solution before MRI measurements did not diminish the difference in tissue damage between CD1h and CD2h kidney grafts. BOLD and diffusion MRI, which are readily available non-invasive tools for evaluating CD kidney grafts tissue damage, can predict prolonged organ damage, and therefore the outcome, of transplanted CD kidney grafts.

Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

Mammary tumors are the most common tumor type in both human and canine females. Mutations in the breast cancer susceptibility gene, BRCA2, have been found in most cases of inherited human breast cancer. Similarly, the canine BRCA2 gene locus has been associated with mammary tumors in female dogs. However, deleterious mutations in canine BRCA2 have not been reported, thus far. The BRCA2 protein is involved in homologous recombination repair via its interaction with RAD51 recombinase, an interaction mediated by 8 BRC repeats. These repeats are 26-amino acid, conserved motifs in mammalian BRCA2. Previous structural analyses of cancer-associated mutations affecting the BRC repeats have shown that the weakening of RAD51s affinity for even 1 repeat is sufficient to increase breast cancer susceptibility. In this study, we focused on 2 previously reported canine BRCA2 mutations (T1425P and K1435R) in BRC repeat 3 (BRC3), derived from mammary tumor samples. These mutations affected the interaction of canine BRC3 with RAD51, and were considered deleterious. Two BRC3 mutations (K1440R and K1440E), reported in human breast cancer patients, occur at amino acids corresponding to those of the K1435R mutation in dogs. These mutations affected the interaction of canine BRC3 with RAD51, and may also be considered deleterious. The two BRC3 mutations and a substitution (T1430P), corresponding to T1425P in canine BRCA2, were examined for their effects on human BRC3 function and the results were compared between species. The corresponding mutations and the substitution showed similar results in both human and canine BRC3. Therefore, canine BRCA2 may be a good model for studying human breast cancer caused by BRCA2 mutations.

Generation of a Felinized Swine Endothelial Cell Line by Expression of Feline Decay-Accelerating Factor

Embryonic stem cell research has facilitated the generation of many cell types for the production of tissues and organs for both humans and companion animals. Because ≥30% of pet cats suffer from chronic kidney disease (CKD), xenotransplantation between pigs and cats has been studied. For a successful pig to cat xenotransplant, the immune reaction must be overcome, especially hyperacute rejection. In this study, we isolated the gene for feline decay-accelerating factor (fDAF), an inhibitor of complement proteins, and transfected a swine endothelial cell line with fDAF to “felinize” the pig cells. These fDAF-expressing cells were resistant to feline serum containing anti-pig antibodies, suggesting that felinized pig cells were resistant to hyperacute rejection. Our results suggest that a “felinized” pig kidney can be generated for the treatment of CKD in cats in the future.

Transcatheter arterial embolization for treatment of hepatocellular carcinoma in a cat

CASE DESCRIPTION A 16-year-old 6.8-kg (15-lb) castrated male domestic shorthair cat was evaluated because of a 3 × 6-cm mass in the right medial lobe of the liver. CLINICAL FINDINGS The cat had a history of frequent vomiting and anorexia along with 10% weight loss over the past year. TREATMENT AND OUTCOME Transcatheter arterial embolization was selected because surgery (standard first-line treatment) was declined and only 1 vessel feeding the tumor was apparent on contrast-enhanced CT. A 4F sheath was placed in the left carotid artery, and a 3.3F guide catheter was advanced into the celiac artery. A 0.014-inch guidewire and 1.7F microcatheter were inserted into the hepatic artery through the guiding catheter and advanced into the feeding vessel. A mixture of polyvinyl alcohol particles and contrast agent was injected for embolization. A hypoechoic area in the tumor was identified on ultrasonography on posttreatment day 6, and necrotic and degenerated cells in the area were identified cytologically. By posttreatment day 71, vomiting had resolved and CT revealed decreased tumor size, but altered attenuation suggested a more solid mass on day 205. No feeding vessel for embolization was found on contrast-enhanced CT, so ultrasonic emulsification to remove the tumor was performed on day 231. No recurrence was seen on contrast-enhanced CT on day 420 or day 721. CLINICAL RELEVANCE Findings suggested that transcatheter arterial embolization may be suitable for treating hepatic tumors in cats, but alternative approaches are needed in cats, compared with dogs, owing to anatomic differences.

Effect of Propofol Continuous-Rate Infusion on Intravenous Glucose Tolerance Test in Dogs

Hyperglycemia causes perioperative complications and many anesthetics impair glucose metabolism and cause hyperglycemia. We evaluated the effects of propofol on blood glucose metabolism and insulin secretion during an intravenous glucose tolerance test (IVGTT) in dogs. Blood glucose, insulin, triglyceride, cholesterol, and free fatty acid (FFA) levels were measured in dogs during IVGTT in a conscious state and under the effect of 2.0% isoflurane, low-concentration propofol (0.2 mg/kg/min), and high-concentration propofol (0.4 mg/kg/min) anesthesia. Plasma glucose levels significantly increased in all of the treatment groups when compared with those in the conscious group. The prolonged half-life period of plasma glucose suggested that isoflurane and propofol attenuated glucose metabolism in dogs. Plasma insulin levels were significantly lower in the isoflurane group when compared with those in the other groups, whereas blood FFA levels were increased in the propofol groups when compared with the other groups. These results suggest that propofol itself does not directly raise plasma glucose levels, but attenuates glucose metabolism by accumulating FFA.