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1 Intercytex Ltd, Boston, MA, USA 2 Faculty of Life Sciences, University of Manchester, Manchester, UK 3 Manchester Institute for Nephrology and Transplantation, Manchester, UK 4 Intercytex Ltd, Manchester, UK
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Abstract |
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(Received 25 October 2006;
accepted after revision 30 October 2006; first published online 28 September 2006)
Corresponding author N. Ashton: Faculty of Life Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, UK. Email: nick.ashton{at}manchester.ac.uk
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Introduction |
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A potential therapeutic alternative to the use of developed organs for transplantation is the use of developing organ primordia. The primordium of the mature kidney is the metanephros, which begins organogenesis during the fifth week of gestation in humans (Moore & Persaud, 1998) and during the twelfth day of embryonic development (E12) in the rat. Metanephric development begins when the ureteric bud originates as an outgrowth of the posterior end of the Wolffian duct and invades the adjacent metanephric mesenchyme. Metanephrogenesis then proceeds through a series of reciprocal signals between the ureteric bud and metanephric mesenchyme which causes the ureteric bud to undergo dichotomous branching to begin formation of the collecting duct system. At the same time, the metanephric mesenchyme aggregates and begins mesenchymal-to-epithelial conversion, proceeding through the various stages of early nephrogenesis through to the S-stage where the S-shaped bodies fuse with the collecting duct system and differentiate into nephrons. The upper portion of the S-shaped body forms the proximal tubule, loop of Henlé and the distal tubule, while the terminal portion forms the glomerulus (Horster et al. 1999). During nephrogenesis, functional cell polarization is acquired through cell differentiation, and changes in ion channel and transport expression occur which eventually result in the ability to concentrate urine and regulate solute excretion (Huber et al. 2000).
Over recent years, much research emphasis has been placed on the use of renal primordia as an alternative to transplantation of developed adult organs. A number of strategies have been employed, including the use of sectioned rodent metanephroi implanted into the renal parenchymal tissue (Woolf et al. 1990), transplantation of metanephros fragments under the renal capsule (Dekel et al. 2003), tissue engineering and therapeutic cloning (Lanza et al. 2002) and transplantation of whole metanephroi to intraperitoneal locations (Rogers et al. 1998; Dekel et al. 2002; Marshall et al. 2005). These studies have provided important insights into the ontogenetic development of transplanted renal tissue in both allogeneic (Rogers et al. 1998; Marshall et al. 2005) and xenogeneic models (Dekel et al. 2002; Rogers et al. 2003). If, as suggested in some (Dekel et al. 2003) though not all studies (Woolf & Loughna, 1998), metanephroi derive their vasculature primarily from the host, this could potentially overcome acute and hyperacute vascular rejection problems associated with xenografts of developed adult tissue. Consequently, intraperitoneal metanephros transplants have been proposed to offer immunological advantage even across xenogeneic barriers (Rogers et al. 2003).
Despite these promising advances, there has been only one published report, to date, which has described the ability of a transplanted metanephros to sustain life in an otherwise anephric animal. In this study, Rogers & Hammerman (2004) showed that rats which had received a metanephros transplant, prior to removal of their native renal tissue, were able to survive on average 58 h longer than control, anephric animals. This report suggests that transplanted metanephroi have the potential to sustain life following loss of function of native renal tissue. The major limiting factor appears to be the functional capacity of the metanephros. We (Marshall et al. 2005) and others (Rogers et al. 1998, 2001) have reported glomerular filtration rates (GFR) of the order of 30 µl min–1 (g metanephros weight)–1 for transplanted metanephroi, which equates to approximately 3% of normal GFR for an adult rat. However, despite the use of a ‘growth factor cocktail’ which has been shown to improve the growth of transplanted metanephroi (Rogers & Hammerman, 2001), the typical mass of a metanephros up to 3 months post-transplantation is 100–150 mg. Therefore, in order to improve the functional capacity of the transplanted metanephros, an increase in tissue mass is necessary. There are two possible approaches to overcome this problem: either to promote further growth of the metanephros or to increase the number of metanephroi transplanted and connected to the host's urinary system. The former approach will be difficult to achieve unless arteriogenesis and thus blood supply can be improved; hence the aim of this study was to determine whether survival in anephric rats could be increased by connecting two metanephroi to the host's ureter compared with a single connection. Since we were able to collect urine from the animals during the survival experiment, we also report, for the first time, a preliminary analysis of metanephric urine composition.
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Methods |
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The survival experiments were undertaken in the US laboratories of Intercytex Ltd. Ethical approval for all animal procedures was granted through the Institutional Animal Care and Use Committee (IUCAC).
Preparation of metanephroi
Time-mated Lewis rats (embryo gestational age E15, Charles River, USA) were anaesthetized using isoflurane gaseous anaesthesia (flow rate, 1 l min–1 O2, 400 ml min–1 nitrous oxide; 3% isoflurane). Once the animals were fully anaesthetized, they were removed from the induction chamber and killed by cervical dislocation. The abdomen was dissected open and the uterus was removed and place in ice-cold phosphate-buffered saline (PBS; 0.84 mM Na2 HPo4, 0.16 mM NaH2 Po4, 0.14 M Na CL). Embryos were sequentially dissected from the uterus and placed into fresh ice-cold Dulbecco's modified Eagle's medium (DMEM). Once all the embryos were removed from the uterus, the metanephroi were dissected from the embryos under a dissecting microscope and transferred into ice-cold DMEM containing the following growth factors, which have been shown to enhance the growth of metanephroi in vivo and in vitro (Rogers et al. 1998; Rogers & Hammerman, 2001): recombinant human insulin-like growth factor I (IGF-I), 10–7
M (Upstate USA Inc., Chicago, IL, USA); recombinant human IGF-II, 10–7
M (Upstate Biotech); recombinant human vascular endothelial growth factor, 5 µg ml–1 (Upstate Biotech); recombinant human transforming growth factor 
, 10–8
M (Upstate Biotech); recombinant human nerve growth factor, 5 µg ml–1 (R&D Systems Inc., Minneapolis, MN, USA); recombinant human fibroblast growth factor, 5 µg ml–1 (R&D Systems); recombinant human hepatocyte growth factor, 10–8
M (R&D Systems); iron saturated transferrin, 5 µg ml–1 (Sigma Chemicals); corticotrophin-releasing hormone, 1 µg ml–1 (Sigma Chemicals); retinoic acid, 10–6
M (Sigma Chemicals); prostaglandin E1, 25 nM (Sigma Chemicals); and Tamm-Horsfall protein, 1 µg ml–1 (Biomedical Technologies, Stoughton, MA, USA). The metanephroi were left in this medium on ice for at least 1 h prior to transplantation into adult Lewis rat recipients.
Transplantation
Female Lewis rats (Charles River, USA) were anaesthetized using isoflurane (flow rate, 1 l min–1 O2, 400 ml min–1 nitrous oxide; 1.5% isoflurane, Vapamasta 6, Anmedic, Vallentuna, Sweden). A mid-line laparotomy was performed and the native left kidney was removed (unilateral nephrectomy). Three metanephroi, pre-incubated in the growth factor-rich medium, were then transplanted either into a pouch created in the retroperitoneal fat, adjacent to the renal vessels, close to the site of the unilateral nephrectomy, or into a pouch created adjacent to the circumflex iliac vessels of each host rat. Analgesia was administered (ketoprofen, 5 mg (kg body weight)–1, Henry Schein Inc., Indianapolis, NY, USA) subcutaneously prior to recovery from anaesthesia and 24 h later, followed by administration as required if the animals showed signs of pain (Roughan & Flecknell, 2001). All animals were housed individually with free access to chow and water.
Ureter anastomosis
Approximately 21 days after transplantation, animals were anaesthetized again using isoflurane, delivered as before (flow rate, 1 l min–1 O2, 400 ml min–1 nitrous oxide; 1.5% isoflurane), and the transplants were examined. Transplanted metanephroi that had grown sufficiently well were selected for anastomosis of the ureter with the free end of the recipient's left ureter (uretero-ureterostomy). Animals were divided into two groups at this stage: those with only a single metanephros suitable for connection (n = 5) and those with two suitable metanephroi (n = 5). Rats with only one connectable transplant underwent a single end-to-end anastomosis to connect the metanephros ureter to the host left ureter, whereas rats with two connectable transplants (one adjacent to the renal vessels and one adjacent to the circumflex iliac vessels) had both connected to the host left ureter via an end-to-end and end-to-side anastomosis, respectively. Unconnected metanephroi were left in situ, eventually becoming hydronephrotic. Analgesia was administered (ketoprofen, 5 mg (kg body weight)–1, Henry Schein Ltd) subcutaneously prior to recovery from anaesthesia and 24 h later, followed by administration as required if the animals showed signs of pain (Roughan & Flecknell, 2001). All animals were housed individually with free access to chow and water.
Life-sustaining experiments
Approximately 5 weeks after uretero-ureterostomy, experimental animals were re-anaesthetized using isoflurane, delivered as before (flow rate, 1 l min–1 O2, 400 ml min–1 nitrous oxide; 1.5% isoflurane), and the transplants were checked to ensure that the ureter anastomosis was still patent. The native right kidney was then removed (total nephrectomy) and the animals were allowed to recover. Analgesia was administered (ketoprofen, 5 mg per (kg body weight)–1, Henry Schein Ltd) subcutaneously prior to recovery from anaesthesia and 24 h later, followed by administration as required if the animals showed signs of pain (Roughan & Flecknell, 2001). Animals had unrestricted access to food and water and were monitored throughout the day and night, with health checks performed at least once per hour and a full clinical assessment at 01.00, 12.00 and 19.00. Animals were either allowed to die naturally or were killed by cervical dislocation under isoflurane anaesthesia, if they were showing signs of pain which were not alleviated by analgesia as assessed by methods devised by Roughan & Flecknell (2001), or if the animal had become moribund. The time of death or termination for each animal was recorded in hours. Urine samples (spontaneous voiding of the bladder) were taken every 24 h, and terminal serum samples were collected by cardiac puncture immediately after the death of the animals.
Two groups of control animals were also set up. Control group 1 consisted of animals that underwent all surgical procedures except uretero-ureterostomy (anephric controls, n = 6). Control group 2 consisted of animals that underwent all surgical procedures except uretero-ureterostomy and right nephrectomy (urine physiology controls, n = 5). Urine samples (spontaneous voiding of the bladder) were collected every 24 h from rats in control group 2 for analysis, and terminal serum samples were taken by cardiac puncture from both control groups.
Urine and serum analysis
Sodium and potassium concentrations in urine and serum samples were measured using a Corning 480 Flame Photometer (Ciba Corning Diagnostics Ltd, Halstead, UK). A 3 M lithium internal standard was used, and the flame photometer was standardized for urine or serum using Corning MultiCal vials. Osmolality was determined by freezing point depression using a Roebling osmometer (Camlab Ltd, Cambridge, UK).
Serum and urine urea concentrations were determined by colorimetry, using an Enzymatic Urea Nitrogen clinical testing kit (Stanbio Laboratories, Boerne, TX, USA). Briefly, 1 ml of enzyme reagent containing 120 mM phosphate buffer, 60 mM sodium salicylate, 3.2 mM sodium nitroprusside, 1 mM EDTA and 10 KU l–1 urease was added to 10 µl of either urine (diluted 1:100 in water) or serum (undiluted). The samples were mixed and incubated at room temperature for 10 min. One millilitre of colour reagent containing 130 mM sodium hydroxide and 6 mM sodium hypochloride was added; the solutions were mixed and incubated at room temperature for 10 min. The absorption of the samples was read at 600 nm using a Beckman DU-530 UV/VIS spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA), and concentrations were calculated according to the following formula:
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Statistical analysis
Survival curves were created by Kaplan-Meier survival analysis using GraphPad Prism 4 software (GraphPad Software, San Diego, CA, USA). A Pearson correlation was performed to determine the relationship between survival time and mass of transplanted tissue. The normal distribution of urine and serum composition data was confirmed by Kolmogorov–Smirnov test; differences between groups were compared by one-way ANOVA and Duncan's test. Significance was assumed at P
0.05 (SPSS for Windows, version 13.0, SPSS UK Ltd, Woking, UK).
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Results |
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At the time of transplantation, the E15 metanephros is composed mainly of undifferentiated metanephric mesenchyme with rudimentary epithelial structures (Fig. 1A). Approximately 21 days after transplantation of E15 metanephroi to the retroperitoneal fat adjacent to either the renal vessels or the circumflex iliac vessels, the transplants had developed mature renal structures, including glomeruli (Fig. 1B), proximal and distal tubules and a collecting duct system (Fig. 1C). The transplanted metanephroi had also developed a blood supply, which we have demonstrated previously (Bottomley et al. 2004) to originate from the host vasculature (Fig. 1D), and had a developed ureter which could be anastomosed end-to-end with the host ureter (Fig. 1E). Unconnected transplants became hydronephrotic and non-functional owing to urine reflux (Fig. 1F). Qualitative assessment of the metanephroi from the single and double transplant animals suggested that there were no structural differences between the groups.
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Figure 2A shows Kaplan-Meier survival curves for animals with one or two transplanted metanephroi compared with anephric control animals. Control animals (anephric controls n = 6) with no native renal mass lived for 76.6 ± 9.3 h (range, 67–89 h), which compares well with published data from other groups (67 ± 2.7 h; Rogers & Hammerman, 2004). Animals that had a single transplant connected to the host ureter (n = 5) lived significantly longer than the control animals (P < 0.001), surviving for 105.6 ± 13.1 h (range, 96–120 h). Life was prolonged further in animals with two transplants connected to the host ureter (n = 5); this group had an average lifespan of 121.2 ± 25.6 h (range, 97–150 h), which was significantly greater than that of both the control animals (P < 0.001) and the single transplant group (P < 0.05).
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Excretory function of transplanted metanephroi
Urine (Fig. 3) and serum (Table 2) osmolality and the concentrations of sodium, potassium and urea were determined as markers of excretory function by transplanted metanephroi. The concentrations of sodium and potassium in the urine produced by metanephroi was significantly lower (P < 0.01) than that of the unilateral nephrectomy control group (urine physiology controls). The urinary sodium to potassium concentration ratio tended to be greater in both transplant groups compared with the controls; however, this difference failed to reach statistical significance (control, n = 5, 0.6 ± 0.1; single transplant, n = 5, 1.2 ± 0.3; double transplant, n = 5, 1.5 ± 0.3, one-way ANOVA F2,24 = 2.9, P = 0.076). The decrease in electrolyte concentration was associated with a reduction in urine osmolality in both transplant groups (P < 0.001). The urinary concentration of urea in rats with a single connected metanephros was significantly lower (P < 0.001) than that from control animals. Surprisingly, the urea concentration in urine collected from rats with two connected metanephroi did not differ from that of control animals.
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Discussion |
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A number of earlier studies have shown that transplanted metanephroi have the ability to perform at least some of the functions of the mature kidney, including filtration of plasma (Rogers et al. 1998, 2001; Dekel et al. 2002; Marshall et al. 2005). Reported glomerular filtration rates have been of the order of 30 µl min–1 (g metanephros weight)–1 (Rogers et al. 1998, 2001; Marshall et al. 2005), which equates to around 3% of the GFR of a normal adult rat kidney. This degree of filtration capacity is too low to sustain life in the long term, but Rogers & Hammerman (2004) have reported recently that the survival of anephric rats was prolonged by 58 h following the transplantation and connection of a single metanephros. These experiments were performed 20 weeks after initial transplantation of the metanephros. Here, we show that survival was prolonged by 29 h in an anephric rat just 8 weeks postmetanephros transplantation; this was increased to 45 h in rats with two connected metanephric transplants. These data show that, by increasing the amount of renal tissue and therefore filtration capacity, life can be extended. Rogers & Hammerman (2004) did not report the mass of their metanephroi at 20 weeks post-transplantation; however, they indicated that the cross-sectional diameter was similar to that of an adult rat kidney. The metanephroi in the present study were of the order of 180 mg, which is considerably smaller than an adult kidney (typically 1–1.5 g), which again suggests that if the mass of tissue were increased, survival would also be improved.
Tissue size is not the only important feature that is likely to influence long-term survival. Maturation of the renal tubules is also essential if the metanephros is going to be able to regulate extracellular fluid volume and composition adequately. Nephrogenesis continues after birth in the rat until postnatal day 8–11 (Kavlock & Gray, 1982); however, full tubular function does not develop until up to 6 weeks postnatally (Rane & Aperia, 1985). The osmolality of spontaneously voided urine samples collected from rats surviving with one or two metanephroi (350–380 mosmol kg–1) is comparable to that reported for a 10-day-old rat (Gray & Kavlock, 1991), suggesting that the urinary concentrating mechanism is far from fully developed in the metanephroi. Hence, it is not surprising that the concentrations of sodium, potassium and urea measured in urine samples from both experimental groups were lower than those of control animals. The urinary sodium to potassium ratios provide further evidence that the metanephric nephrons were still immature. The ratios for both experimental groups were greater than unity, reflecting proportionately lower potassium excretion by the tubules.
We have also shown, in a preliminary study, that metanephroi at a similar post-transplant stage did not express the tubular urea transporters UT-A1 or UT-A3 (Dilworth et al. 2005). In the animals with two metanephric transplants connected, the urinary urea concentration was comparable to that of control animals, suggesting that there was either greater urea excretion or increased water reabsorption by this particular group of metanephroi. The reason for this is unclear. Rates of metanephros development do vary; we have observed different levels of aquaporin 1 and 2 expression in similar groups of metanephroi (Dilworth et al. 2005). However, it seems unlikely that those in the two metanephroi connection group would develop more rapidly by chance than those in the single connection group, unless the presence of two functioning metanephroi enhances growth and tubular maturation in some way. Nonetheless, the serum urea concentration was still fivefold higher than that of the unilateral nephrectomy control group, suggesting that the urinary excretion rate of urea was insufficient to reduce the serum urea concentration and thus serum osmolality. Indeed, the serum profile of both experimental groups was identical to that of the anephric controls at death. This implies that while transplanted metanephroi are able to regulate serum composition to a degree compatible with life for a limited time, eventually their excretory capacity is overwhelmed, leading to death.
Clearly, the collection of a timed urine sample would have been preferable to spontaneously voided samples, since this would have allowed the calculation of urinary excretion rates and thus a more detailed assessment of excretory function. However, the very low flow rates exhibited by metanephroi (typically of the order of 0.16 µl min–1 compared with host kidney flow rates of 14 µl min–1) mean that evaporation would have had a significant impact on sample collection in a standard metabolism cage. In order to overcome this limitation, acute clearance experiments are now required in order to assess tubular handling of solutes by metanephroi. We have made measurements of GFR (Marshall et al. 2005), but the small volumes of fluid collected precluded further analysis of urine composition. Therefore, in the meantime, the measurements of urinary sodium, potassium and urea concentrations reported herein provide the best indication of excretory function by transplanted metanephroi to date.
Nonetheless, it appears from our results that metanephros transplants have some ability to excrete products filtered from the blood, although their overall ability to sustain life is limited at present. However, several important considerations have to be made relating to the potential use of metanephros transplants as a therapy for end-stage renal disease. Firstly, in the model described here, the animals were devoid of all native renal mass, and so were totally reliant on the transplants for renal function. This is unlikely to be the case in the therapeutic setting, where the metanephros transplants would probably be used to supplement any remaining functional capacity of the native kidneys. Secondly, in previous studies (Marshall et al. 2005), we have demonstrated that transplantation of metanephroi to the sites used in this study can result in a glomerular filtration rate equivalent to 10% of normal renal function at 12 weeks post-transplantation. This is a longer time frame than the one used in the present study and suggests that the transplants may develop to a stage where they could provide life-sustaining renal function. Rogers et al. (1998) have shown previously that metanephroi transplanted onto the omentum cease to grow after 12 weeks inside the host, which compares well with our previous renal function data (Marshall et al. 2005). This suggests that maximum transplant development (in a rat model) has occurred after 12 weeks, and at this point transplants to the sites used in this report may be capable of sustaining life. However, the recent report by Rogers & Hammerman (2004) described a prolongation of life in anephric rats 20 weeks post-transplantation of similar magnitude to that described here after just 8 weeks. Whether this is linked to the site of transplantation, the potential deleterious effect of intrarenal fluid build-up prior to uretero-ureterostomy or the metabolic and excretory function of the transplants remains to be examined. Finally, in a larger animal model or in a human patient, it may be possible to connect numerous metanephros transplants to the host urinary system. Survival time was positively correlated with the total weight of transplanted tissue, so increasing renal mass should have an additive effect for each subsequent transplant anastomosed.
In summary, this study has demonstrated that renal primordia have the potential to extend survival in the absence of functional mature renal tissue in the rat, albeit in the short term. The challenge for the future is to improve this capacity if metanephros transplantation is to become a viable clinical treatment in end-stage renal disease.
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References |
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Bottomley M, Marshall D, Clancy M, Symmonds K, Brenchley PE & Bravery CA (2004). Xenotransplanted porcine metanephroi overcome acute vascular rejection by deriving their blood supply solely from the recipient. In 7th International Congress of the Cell Transplant Society, P015, Boston.
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