Increasing renal mass improves survival in anephric rats following metanephros transplantation

Exp Physiol 92.1 pp 263–271 263

Experimental Physiology

Increasing renal mass improves survival in anephric ratsfollowing metanephros transplantation

Damian Marshall1, Mark R. Dilworth2, Marc Clancy3, Christopher A. Bravery4 and Nick Ashton2

1Intercytex Ltd, Boston, MA, USA2Faculty of Life Sciences, University of Manchester, Manchester, UK3Manchester Institute for Nephrology and Transplantation, Manchester, UK4Intercytex Ltd, Manchester, UK

Renal failure and end-stage renal disease are prevalent diseases associated with high levels ofmorbidity and mortality, the preferred treatment for which is kidney transplantation. However,the gulf between supply and demand for kidneys remains high and is growing every year. Apotential alternative to the transplantation of mature adult kidneys is the transplantation ofthe developing renal primordium, the metanephros. It has been shown previously, in rodentmodels, that transplantation of a metanephros can provide renal function capable of prolongingsurvival in anephric animals. The aim of the present study was to determine whether increasingthe mass of transplanted tissue can prolong survival further. Embryonic day 15 rat metanephroiwere transplanted into the peritoneum of anaesthetized adult rat recipients. Twenty-one dayslater, the transplanted metanephroi were anastomosed to the recipient’s urinary system, and35 days following anastomosis the animal’s native renal mass was removed. Survival times andcomposition of the excreted fluid were determined. Rats with single metanephros transplantssurvived 29 h longer than anephric controls (P < 0.001); animals with two metanephroi survived44 h longer (P < 0.001). A dilute urine was formed, with low concentrations of sodium,potassium and urea; potassium and urea concentrations were elevated in terminal serum samples,but sodium concentration and osmolality were comparable to control values. These data showthat survival time is proportional to the mass of functional renal tissue. While transplantedmetanephroi cannot currently provide life-sustaining renal function, this approach may havetherapeutic benefit in the future.

(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, OxfordRoad, Manchester M13 9PT, UK. Email: [email protected]

Renal failure and end-stage renal disease (ESRD) areprevalent diseases with high levels of morbidity andmortality. The most recent estimates of the prevalence ofrenal replacement therapy are 638 per million population(p.m.p.) for the UK (Ansell & Feest, 2005) and 1496 p.m.p.for the USA (US Renal Data System, 2005). The onlycurrent supportive treatments for renal failure andESRD are haemodialysis, peritoneal dialysis and kidneytransplantation. Dialysis is associated with morbidity andhigh costs to the health service; hence the preferredtreatment for most patients is a kidney transplant.However, because of the gulf between supply and demandfor organ transplantation, only 45% of UK and 28% of USpatients on renal replacement therapy have a functioning

transplant (Ansell & Feest, 2005; US Renal Data System,2005).

A potential therapeutic alternative to the use ofdeveloped organs for transplantation is the use ofdeveloping organ primordia. The primordium of themature kidney is the metanephros, which beginsorganogenesis during the fifth week of gestation inhumans (Moore & Persaud, 1998) and during thetwelfth day of embryonic development (E12) in therat. Metanephric development begins when the uretericbud originates as an outgrowth of the posterior end ofthe Wolffian duct and invades the adjacent metanephricmesenchyme. Metanephrogenesis then proceeds througha series of reciprocal signals between the ureteric bud and

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/expphysiol.2006.036319

264 D. Marshall and others Exp Physiol 92.1 pp 263–271

metanephric mesenchyme which causes the ureteric bud toundergo dichotomous branching to begin formation of thecollecting duct system. At the same time, the metanephricmesenchyme aggregates and begins mesenchymal-to-epithelial conversion, proceeding through the variousstages of early nephrogenesis through to the S-stage wherethe S-shaped bodies fuse with the collecting duct systemand differentiate into nephrons. The upper portion ofthe S-shaped body forms the proximal tubule, loop ofHenle and the distal tubule, while the terminal portionforms the glomerulus (Horster et al. 1999). Duringnephrogenesis, functional cell polarization is acquiredthrough cell differentiation, and changes in ion channeland transport expression occur which eventually resultin the ability to concentrate urine and regulate soluteexcretion (Huber et al. 2000).

Over recent years, much research emphasis has beenplaced on the use of renal primordia as an alternativeto transplantation of developed adult organs. A numberof strategies have been employed, including the use ofsectioned rodent metanephroi implanted into the renalparenchymal tissue (Woolf et al. 1990), transplantationof metanephros fragments under the renal capsule(Dekel et al. 2003), tissue engineering and therapeuticcloning (Lanza et al. 2002) and transplantation ofwhole metanephroi to intraperitoneal locations (Rogerset al. 1998; Dekel et al. 2002; Marshall et al. 2005).These studies have provided important insights into theontogenetic development of transplanted renal tissuein both allogeneic (Rogers et al. 1998; Marshall et al.2005) and xenogeneic models (Dekel et al. 2002; Rogerset al. 2003). If, as suggested in some (Dekel et al.2003) though not all studies (Woolf & Loughna, 1998),metanephroi derive their vasculature primarily from thehost, this could potentially overcome acute and hyperacutevascular rejection problems associated with xenograftsof developed adult tissue. Consequently, intraperitonealmetanephros transplants have been proposed to offerimmunological advantage even across xenogeneic barriers(Rogers et al. 2003).

Despite these promising advances, there has been onlyone published report, to date, which has described theability of a transplanted metanephros to sustain life inan otherwise anephric animal. In this study, Rogers &Hammerman (2004) showed that rats which had receiveda metanephros transplant, prior to removal of their nativerenal tissue, were able to survive on average 58 h longerthan control, anephric animals. This report suggests thattransplanted metanephroi have the potential to sustain lifefollowing loss of function of native renal tissue. The majorlimiting factor appears to be the functional capacity of themetanephros. We (Marshall et al. 2005) and others (Rogerset al. 1998, 2001) have reported glomerular filtrationrates (GFR) of the order of 30 µl min−1 (g metanephrosweight)−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 oftransplanted metanephroi (Rogers & Hammerman, 2001),the typical mass of a metanephros up to 3 monthspost-transplantation is 100–150 mg. Therefore, in orderto improve the functional capacity of the transplantedmetanephros, an increase in tissue mass is necessary. Thereare two possible approaches to overcome this problem:either to promote further growth of the metanephrosor to increase the number of metanephroi transplantedand connected to the host’s urinary system. The formerapproach will be difficult to achieve unless arteriogenesisand thus blood supply can be improved; hence the aim ofthis study was to determine whether survival in anephricrats could be increased by connecting two metanephroi tothe host’s ureter compared with a single connection. Sincewe were able to collect urine from the animals during thesurvival experiment, we also report, for the first time, apreliminary analysis of metanephric urine composition.

Methods

Ethical approval

The survival experiments were undertaken in the USlaboratories of Intercytex Ltd. Ethical approval for allanimal procedures was granted through the InstitutionalAnimal Care and Use Committee (IUCAC).

Preparation of metanephroi

Time-mated Lewis rats (embryo gestational age E15,Charles River, USA) were anaesthetized using isofluranegaseous anaesthesia (flow rate, 1 l min−1 O2, 400 ml min−1

nitrous oxide; 3% isoflurane). Once the animals werefully anaesthetized, they were removed from the inductionchamber and killed by cervical dislocation. The abdomenwas dissected open and the uterus was removed andplace in ice-cold phosphate-buffered saline (PBS; 0.84 mmNa2 HPo4, 0.16 mm NaH2 Po4, 0.14 m Na CL). Embryoswere sequentially dissected from the uterus and placedinto fresh ice-cold Dulbecco’s modified Eagle’s medium(DMEM). Once all the embryos were removed fromthe uterus, the metanephroi were dissected from theembryos under a dissecting microscope and transferredinto ice-cold DMEM containing the following growthfactors, which have been shown to enhance the growthof metanephroi in vivo and in vitro (Rogers et al.1998; Rogers & Hammerman, 2001): recombinant humaninsulin-like growth factor I (IGF-I), 10−7 m (UpstateUSA Inc., Chicago, IL, USA); recombinant humanIGF-II, 10−7 m (Upstate Biotech); recombinant humanvascular endothelial growth factor, 5 µg ml−1 (UpstateBiotech); recombinant human transforming growthfactor α, 10−8 m (Upstate Biotech); recombinant human

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society

Exp Physiol 92.1 pp 263–271 Survival following metanephros transplantation 265

nerve growth factor, 5 µg ml−1 (R&D Systems Inc.,Minneapolis, MN, USA); recombinant human fibroblastgrowth factor, 5 µg ml−1 (R&D Systems); recombinanthuman 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 (SigmaChemicals); 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 thismedium on ice for at least 1 h prior to transplantation intoadult Lewis rat recipients.

Transplantation

Female Lewis rats (Charles River, USA) were anaesthetizedusing 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 wasperformed and the native left kidney was removed(unilateral nephrectomy). Three metanephroi, pre-incubated in the growth factor-rich medium, werethen transplanted either into a pouch created in theretroperitoneal fat, adjacent to the renal vessels, close to thesite of the unilateral nephrectomy, or into a pouch createdadjacent to the circumflex iliac vessels of each host rat.Analgesia was administered (ketoprofen, 5 mg (kg bodyweight)−1, Henry Schein Inc., Indianapolis, NY, USA)subcutaneously prior to recovery from anaesthesia and24 h later, followed by administration as required if theanimals showed signs of pain (Roughan & Flecknell, 2001).All animals were housed individually with free access tochow and water.

Ureter anastomosis

Approximately 21 days after transplantation, animals wereanaesthetized 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 sufficientlywell were selected for anastomosis of the ureter withthe free end of the recipient’s left ureter (uretero-ureterostomy). Animals were divided into two groupsat this stage: those with only a single metanephrossuitable for connection (n = 5) and those with two suitablemetanephroi (n = 5). Rats with only one connectabletransplant underwent a single end-to-end anastomosisto connect the metanephros ureter to the host leftureter, whereas rats with two connectable transplants(one adjacent to the renal vessels and one adjacentto the circumflex iliac vessels) had both connected tothe host left ureter via an end-to-end and end-to-sideanastomosis, respectively. Unconnected metanephroi wereleft in situ, eventually becoming hydronephrotic. Analgesiawas administered (ketoprofen, 5 mg (kg body weight)−1,

Henry Schein Ltd) subcutaneously prior to recovery fromanaesthesia and 24 h later, followed by administration asrequired if the animals showed signs of pain (Roughan& Flecknell, 2001). All animals were housed individuallywith free access to chow and water.

Life-sustaining experiments

Approximately 5 weeks after uretero-ureterostomy,experimental animals were re-anaesthetized usingisoflurane, delivered as before (flow rate, 1 l min−1

O2, 400 ml min−1 nitrous oxide; 1.5% isoflurane), andthe transplants were checked to ensure that the ureteranastomosis was still patent. The native right kidneywas then removed (total nephrectomy) and the animalswere allowed to recover. Analgesia was administered(ketoprofen, 5 mg per (kg body weight)−1, Henry ScheinLtd) subcutaneously prior to recovery from anaesthesiaand 24 h later, followed by administration as required ifthe animals showed signs of pain (Roughan & Flecknell,2001). Animals had unrestricted access to food and waterand were monitored throughout the day and night, withhealth checks performed at least once per hour and a fullclinical assessment at 01.00, 12.00 and 19.00. Animalswere either allowed to die naturally or were killed bycervical dislocation under isoflurane anaesthesia, if theywere showing signs of pain which were not alleviated byanalgesia 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 wasrecorded in hours. Urine samples (spontaneous voidingof the bladder) were taken every 24 h, and terminal serumsamples were collected by cardiac puncture immediatelyafter the death of the animals.

Two groups of control animals were also set up.Control group 1 consisted of animals that underwentall surgical procedures except uretero-ureterostomy(anephric controls, n = 6). Control group 2 consisted ofanimals that underwent all surgical procedures excepturetero-ureterostomy and right nephrectomy (urinephysiology controls, n = 5). Urine samples (spontaneousvoiding of the bladder) were collected every 24 h from ratsin control group 2 for analysis, and terminal serum sampleswere taken by cardiac puncture from both control groups.

Urine and serum analysis

Sodium and potassium concentrations in urine andserum samples were measured using a Corning 480 FlamePhotometer (Ciba Corning Diagnostics Ltd, Halstead,UK). A 3 m lithium internal standard was used, and theflame photometer was standardized for urine or serumusing Corning MultiCal vials. Osmolality was determinedby freezing point depression using a Roebling osmometer(Camlab Ltd, Cambridge, UK).



Serum and urine urea concentrations were determinedby colorimetry, using an Enzymatic Urea Nitrogen clinicaltesting kit (Stanbio Laboratories, Boerne, TX, USA).Briefly, 1 ml of enzyme reagent containing 120 mmphosphate buffer, 60 mm sodium salicylate, 3.2 mmsodium nitroprusside, 1 mm EDTA and 10 KU l−1 ureasewas added to 10 µl of either urine (diluted 1:100 inwater) or serum (undiluted). The samples were mixed andincubated at room temperature for 10 min. One millilitreof colour reagent containing 130 mm sodium hydroxideand 6 mm sodium hypochloride was added; the solutionswere mixed and incubated at room temperature for 10 min.The absorption of the samples was read at 600 nm using aBeckman DU-530 UV/VIS spectrophotometer (BeckmanInstruments Inc., Fullerton, CA, USA), and concentrationswere calculated according to the following formula:

Urea nitrogen (mg dl−1)

= Absorption of unknown sample

Absorption of standard× 30(mg dl−1)

Statistical analysis

Survival curves were created by Kaplan-Meier survivalanalysis using GraphPad Prism 4 software (GraphPad

Figure 1. Growth and development of metanephroi following transplantationA, a Haematoxylin and Eosin (H&E) stained section of an E15 metanephros showing undifferentiated metanephricmesenchyme (Mm) and rudimentary epithelial structures (∗). B, H&E stained section of a metanephros 21 dayspost-transplantation showing development of numerous glomeruli (G) and tubules (T). C, H&E stained section of atransplant following life-sustaining experiments showing mature adult structure including glomeruli (G), collectingtubules (CT), proximal tubules (PT) and distal tubules (DT). D, photograph of a metanephros transplant (Tx) 21 dayspost-transplantation; a blood vessel originating from the host vasculature can be delineated (arrow). E, photographof a metanephros transplant (Tx) showing end-to-end anastomosis between host and transplant ureters (arrow).F, photograph of a non-connected transplant (Tx) that has become hydronephrotic owing to urine reflux.

Software, San Diego, CA, USA). A Pearson correlationwas performed to determine the relationship betweensurvival time and mass of transplanted tissue. Thenormal distribution of urine and serum composition datawas confirmed by Kolmogorov–Smirnov test; differencesbetween groups were compared by one-way ANOVA andDuncan’s test. Significance was assumed at P ≤ 0.05 (SPSSfor Windows, version 13.0, SPSS UK Ltd, Woking, UK).

Results

Morphology and growth of transplantedmetanephroi

At the time of transplantation, the E15 metanephrosis composed mainly of undifferentiated metanephricmesenchyme with rudimentary epithelial structures(Fig. 1A). Approximately 21 days after transplantation ofE15 metanephroi to the retroperitoneal fat adjacent toeither the renal vessels or the circumflex iliac vessels,the transplants had developed mature renal structures,including glomeruli (Fig. 1B), proximal and distal tubulesand a collecting duct system (Fig. 1C). The transplantedmetanephroi had also developed a blood supply, whichwe have demonstrated previously (Bottomley et al. 2004)to originate from the host vasculature (Fig. 1D), and



Table 1. Analysis of transplant growth, cyst development and successful ureteranastomosis

Published data∗ Experimental data

Number of animals 26 17Metanephroi transplanted 79 52Number of transplants grown 66 (83.5%) 44 (84.6%)Number of transplants with urine cyst 50 (75.8%) 36 (81.8%)Number of uretero-ureterostomies 20 (76.9%) 12 (70.6%)

∗ Marshall et al. (2005).

had a developed ureter which could be anastomosedend-to-end with the host ureter (Fig. 1E). Unconnectedtransplants became hydronephrotic and non-functionalowing to urine reflux (Fig. 1F). Qualitative assessment ofthe metanephroi from the single and double transplantanimals suggested that there were no structural differencesbetween the groups.

In a previous study, we have shown that transplantationof metanephroi into pouches in the retroperitoneal fatadjacent to the renal vessels and/or circumflex iliac vesselsresults in high growth success rates. The transplants arealso more likely to develop a urine cyst 21 days post-transplantation (Marshall et al. 2005), a feature whichnot only demonstrates the start of urine productionby the transplants but also aids in the connection ofthe transplants to the recipient urinary system. In thisstudy, we again show this to be the case (Table 1);84.6% of transplants grew and 81.8% of the transplantshad a urine cyst at 21 days post-transplantation. Of thetransplanted animals, 70.6% subsequently had at leastone transplant connected to the host ureter. There wasno difference in the success rate of single and doubleconnections.

Survival of animals with metanephroi transplants

Figure 2A shows Kaplan-Meier survival curves for animalswith one or two transplanted metanephroi comparedwith anephric control animals. Control animals (anephriccontrols n = 6) with no native renal mass lived for76.6 ± 9.3 h (range, 67–89 h), which compares well withpublished data from other groups (67 ± 2.7 h; Rogers &Hammerman, 2004). Animals that had a single transplantconnected to the host ureter (n = 5) lived significantlylonger than the control animals (P < 0.001), survivingfor 105.6 ± 13.1 h (range, 96–120 h). Life was prolongedfurther in animals with two transplants connected to thehost ureter (n = 5); this group had an average lifespan of121.2 ± 25.6 h (range, 97–150 h), which was significantlygreater than that of both the control animals (P < 0.001)and the single transplant group (P < 0.05).

At time of killing, the total mass of transplantedtissue was significantly greater in those animals thathad received two metanephroi compared with those that

had a single transplant (one metanephros transplant,n = 5, 187 ± 11 mg versus two metanephroi transplant,n = 5, 259 ± 33 mg, P = 0.002). However, there wasno significant difference in the weights of individualmetanephros transplants from animals with single(187 ± 11 mg) or double connections (174 ± 16 mg).

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Figure 2. Survival of animals with metanephroi transplantsA, Kaplan-Meier survival curve for animals with two metanephroitransplants connected (double connection, continuous line, n = 5),one metanephros transplant connected (single connection, dashedline, n = 5) and anephric control animals (controls, dotted line, n = 6).B, plot of metanephros weight against lifespan for anephric rats witheither one or two transplanted metanephroi. There was a significantcorrelation between the mass of transplanted tissue and survival(Pearson r = 0.71, P = 0.021)



Table 2. Analysis of terminal serum samples for sodium, potassium and urea concentrations and for osmolality

Unilateral nephrectomy controls Anephric controls Single transplant Double transplant

Sodium (mmol l−1) 137.4 ± 8.3 126.0 ± 2.0 122.4 ± 6.3 133.2 ± 6.7Potassium (mmol l−1) 6.1 ± 0.8 16.0 ± 2.4∗∗ 20.6 ± 1.8∗∗∗ 18.5 ± 3.2∗∗Urea (mg dl−1) 40.6 ± 4.7 156.6 ± 0.4∗∗∗ 151.7 ± 6.5∗∗∗ 158.7 ± 4.8∗∗∗Osmolality (mosmol kg−1) 427.0 ± 48.4 474.5 ± 16.5 534.3 ± 27.1 509.3 ± 19.1

n = 5 for each experimental group. ‘Single transplant’ represents animals with one metanephros connected to host ureter; ‘doubletransplant’ represents animals with two metanephroi connected to host ureter. ‘Unilateral nephrectomy controls’ represent animalsthat had undergone all surgical procedures except uretero-ureterostomy and right nephrectomy; ‘anephric controls’ represent animalsthat underwent all surgical procedures except uretero-ureterostomy. Statistical comparisons were by one-way ANOVA and Duncan’stest: ∗∗P < 0.01, ∗∗∗P < 0.001 versus unilateral nephrectomy control.

Combining data from both experimental groups, therewas a significant positive correlation (r = 0.71, P = 0.02)between survival time and the total mass of transplantedtissue (Fig. 2B).

Excretory function of transplanted metanephroi

Urine (Fig. 3) and serum (Table 2) osmolality andthe concentrations of sodium, potassium and urea

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Figure 3. Analysis of daily urine samples for sodium (A), potassium (B) and urea concentrations (C) andosmolality (D)‘Single’ represents animals with 1 metanephros connected to host ureter (n = 5); ‘double’ represents animals withtwo metanephroi connected to host ureter (n = 5); and ‘control’ represents animals with unilateral nephrectomy(n = 5). Statistical comparisons were by one-way ANOVA and Duncan’s test: ∗∗P < 0.01, ∗∗∗P < 0.001 versuscontrol.

were determined as markers of excretory function bytransplanted metanephroi. The concentrations of sodiumand potassium in the urine produced by metanephroi wassignificantly lower (P < 0.01) than that of the unilateralnephrectomy control group (urine physiology controls).The urinary sodium to potassium concentration ratiotended to be greater in both transplant groups comparedwith the controls; however, this difference failed to reachstatistical 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). Thedecrease in electrolyte concentration was associated witha reduction in urine osmolality in both transplant groups(P < 0.001). The urinary concentration of urea in rats witha single connected metanephros was significantly lower(P < 0.001) than that from control animals. Surprisingly,the urea concentration in urine collected from rats withtwo connected metanephroi did not differ from that ofcontrol animals.

Analysis of the terminal serum samples revealed asomewhat different pattern. Compared with the unilateralnephrectomy control group (urine physiology controls),the serum concentrations of potassium and urea wereelevated significantly (P < 0.01) in both the single anddouble transplant groups; serum sodium concentrationsdid not differ between groups (Table 2). Serum osmolalitytended to be higher in both transplant groups comparedwith the unilateral nephrectomy controls, but thisdifference did not reach statistical significance. Theanephric controls resembled the transplant groups,having elevated (P < 0.01) serum potassium and ureaconcentrations compared with the unilateral nephrectomycontrols. Serum sodium concentrations and osmolalitieswere not statistically different between the two controlgroups.

Discussion

In this study, we have shown that the life expectancyof anephric animals was prolonged significantly by thetransplantation and subsequent connection to the urinarysystem of a metanephros. Furthermore, we have extendedthe observations of Rogers & Hammerman (2004) to showthat increasing the mass of renal tissue by connectingtwo metanephroi further increased survival. There wasa positive correlation between survival time and totalmetanephric mass, which suggests that if the mass oftransplanted renal tissue is increased sufficiently, anephricanimals may be able to survive for an extended periodof time. This represents an important step towards theultimate goal of developing a renal replacement therapy, orat the very least, supplementation of limited renal functionin ESRD patients.

A number of earlier studies have shown thattransplanted metanephroi have the ability to performat 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). Reportedglomerular filtration rates have been of the order of30 µl min−1 (g metanephros weight)−1 (Rogers et al. 1998,2001; Marshall et al. 2005), which equates to around3% of the GFR of a normal adult rat kidney. Thisdegree of filtration capacity is too low to sustain lifein the long term, but Rogers & Hammerman (2004)

have reported recently that the survival of anephric ratswas prolonged by 58 h following the transplantation andconnection of a single metanephros. These experimentswere performed 20 weeks after initial transplantationof the metanephros. Here, we show that survival wasprolonged by 29 h in an anephric rat just 8 weekspostmetanephros transplantation; this was increased to45 h in rats with two connected metanephric transplants.These data show that, by increasing the amount of renaltissue and therefore filtration capacity, life can be extended.Rogers & Hammerman (2004) did not report the massof their metanephroi at 20 weeks post-transplantation;however, they indicated that the cross-sectional diameterwas similar to that of an adult rat kidney. The metanephroiin the present study were of the order of 180 mg, which isconsiderably smaller than an adult kidney (typically 1–1.5 g), which again suggests that if the mass of tissue wereincreased, survival would also be improved.

Tissue size is not the only important feature that islikely to influence long-term survival. Maturation of therenal tubules is also essential if the metanephros is goingto be able to regulate extracellular fluid volume andcomposition adequately. Nephrogenesis continues afterbirth in the rat until postnatal day 8–11 (Kavlock & Gray,1982); however, full tubular function does not developuntil up to 6 weeks postnatally (Rane & Aperia, 1985).The osmolality of spontaneously voided urine samplescollected from rats surviving with one or two metanephroi(350–380 mosmol kg−1) is comparable to that reportedfor a 10-day-old rat (Gray & Kavlock, 1991), suggestingthat the urinary concentrating mechanism is far from fullydeveloped in the metanephroi. Hence, it is not surprisingthat the concentrations of sodium, potassium and ureameasured in urine samples from both experimental groupswere lower than those of control animals. The urinarysodium to potassium ratios provide further evidence thatthe metanephric nephrons were still immature. The ratiosfor both experimental groups were greater than unity,reflecting proportionately lower potassium excretion bythe tubules.

We have also shown, in a preliminary study, thatmetanephroi at a similar post-transplant stage did notexpress the tubular urea transporters UT-A1 or UT-A3 (Dilworth et al. 2005). In the animals with twometanephric transplants connected, the urinary ureaconcentration was comparable to that of control animals,suggesting that there was either greater urea excretionor increased water reabsorption by this particular groupof metanephroi. The reason for this is unclear. Rates ofmetanephros development do vary; we have observeddifferent levels of aquaporin 1 and 2 expression in similargroups of metanephroi (Dilworth et al. 2005). However,it seems unlikely that those in the two metanephroiconnection group would develop more rapidly by chancethan those in the single connection group, unless the



presence of two functioning metanephroi enhances growthand tubular maturation in some way. Nonetheless, theserum urea concentration was still fivefold higher than thatof the unilateral nephrectomy control group, suggestingthat the urinary excretion rate of urea was insufficientto reduce the serum urea concentration and thus serumosmolality. Indeed, the serum profile of both experimentalgroups was identical to that of the anephric controls atdeath. This implies that while transplanted metanephroiare able to regulate serum composition to a degreecompatible with life for a limited time, eventually theirexcretory capacity is overwhelmed, leading to death.

Clearly, the collection of a timed urine sample wouldhave been preferable to spontaneously voided samples,since this would have allowed the calculation of urinaryexcretion rates and thus a more detailed assessment ofexcretory function. However, the very low flow ratesexhibited by metanephroi (typically of the order of0.16 µl min−1 compared with host kidney flow rates of14 µl min−1) mean that evaporation would have had asignificant impact on sample collection in a standardmetabolism cage. In order to overcome this limitation,acute clearance experiments are now required in orderto assess tubular handling of solutes by metanephroi. Wehave made measurements of GFR (Marshall et al. 2005),but the small volumes of fluid collected precluded furtheranalysis of urine composition. Therefore, in the meantime,the measurements of urinary sodium, potassium andurea concentrations reported herein provide the bestindication of excretory function by transplantedmetanephroi to date.

Nonetheless, it appears from our results thatmetanephros transplants have some ability to excreteproducts filtered from the blood, although their overallability to sustain life is limited at present. However, severalimportant considerations have to be made relating to thepotential use of metanephros transplants as a therapy forend-stage renal disease. Firstly, in the model describedhere, the animals were devoid of all native renal mass, andso 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 beused to supplement any remaining functional capacity ofthe native kidneys. Secondly, in previous studies (Marshallet al. 2005), we have demonstrated that transplantation ofmetanephroi to the sites used in this study can result ina glomerular filtration rate equivalent to 10% of normalrenal function at 12 weeks post-transplantation. This isa longer time frame than the one used in the presentstudy and suggests that the transplants may develop toa stage where they could provide life-sustaining renalfunction. Rogers et al. (1998) have shown previouslythat metanephroi transplanted onto the omentum ceaseto grow after 12 weeks inside the host, which compareswell with our previous renal function data (Marshall

et al. 2005). This suggests that maximum transplantdevelopment (in a rat model) has occurred after 12 weeks,and at this point transplants to the sites used in thisreport may be capable of sustaining life. However, therecent report by Rogers & Hammerman (2004) describeda prolongation of life in anephric rats 20 weeks post-transplantation of similar magnitude to that describedhere after just 8 weeks. Whether this is linked to thesite of transplantation, the potential deleterious effect ofintrarenal fluid build-up prior to uretero-ureterostomy orthe metabolic and excretory function of the transplantsremains to be examined. Finally, in a larger animal modelor in a human patient, it may be possible to connectnumerous metanephros transplants to the host urinarysystem. Survival time was positively correlated with thetotal weight of transplanted tissue, so increasing renalmass should have an additive effect for each subsequenttransplant anastomosed.

In summary, this study has demonstrated that renalprimordia have the potential to extend survival in theabsence of functional mature renal tissue in the rat, albeitin the short term. The challenge for the future is to improvethis capacity if metanephros transplantation is to becomea viable clinical treatment in end-stage renal disease.

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Acknowledgements

The authors wish to thank Kirsten Symmonds and MartynBottomley (Intercytex Ltd, Manchester, UK) for help withhistology and Erica Philips (Intercytex Ltd, Boston, MA, USA)for help collecting urine samples.


Increasing renal mass improves survival in anephric rats following metanephros transplantation

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