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NDT Advance Access originally published online on October 12, 2007
Nephrology Dialysis Transplantation 2008 23(3):871-879; doi:10.1093/ndt/gfm671
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Development and functional capacity of transplanted rat metanephroi

Mark Robert Dilworth1, Marc James Clancy2, Damian Marshall3, Christopher A. Bravery3, Paul E. Brenchley2 and Nick Ashton1

1 Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK 2 Manchester Institute of Nephrology and Transplantation, Manchester Royal Infirmary, Manchester M13 9WL, UK 3 Intercytex Ltd, Innovation House, Crewe Road, Manchester M23 9QR, UK

Dr Mark R Dilworth, Faculty of Life Sciences, 1.124 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Tel: +44-161 275 5454; Fax: +44-161 275 3938; E-mail: m.r.dilworth{at}manchester.ac.uk



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Quantitative PCR
 Results
 Discussion
 References
 
Background. Transplantation of embryonic kidneys (metanephroi) offers a potential solution to the problem of kidney donor shortage. The aim of this study was to characterise the haemodynamic capacity of transplanted rat metanephroi and to determine the number and maturity of the tubules.

Methods. Metanephroi from E15 Lewis rat embryos were transplanted adjacent to the abdominal aorta of uninephrectomised adult female syngeneic Lewis rats. Twenty-one days later, a single metanephros ureter was anastomosed to the host's urinary system. Three months later animals were prepared for standard clearance measurements.

Results. Effective renal blood flow (149 ± 33 µl min–1 per g kidney weight) and glomerular filtration rate (17 ± 9 µl min–1 per g kidney weight), standardised to kidney weight, were significantly lower in transplanted metane- phroi compared with control adult kidneys (P < 0.001); renal vascular resistance (934 ± 209 mmHg ml min–1 per g kidney weight) was significantly higher (P < 0.001). Nephron number in transplanted metanephroi was significantly greater than that of E21 kidneys (P < 0.01) but lower than that of postnatal day (PND) 1 kidneys (P < 0.001). Angiotensin II type 2 receptor mRNA expression, a marker of nephrogenesis, was markedly reduced in metanephroi. Aquaporins 1 and 2, epithelial Na channel and Na-K-2Cl cotransporter type 2 mRNA and protein were expressed in transplanted metanephroi; the urea transporters-A1, 2 and 3 were absent. Vascular markers ({alpha}-smooth muscle actin and CD31) were identified in metanephroi but their expression did not differ from that of E21 and PND 1 kidneys.

Conclusions. This study shows that metanephroi continue to develop post-transplantation but only reach a stage of development equivalent to that of a normal rat kidney at birth.

Keywords: aquaporin; metanephros; nephrogenesis; renal blood flow; transplantation; urea



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Quantitative PCR
 Results
 Discussion
 References
 
In the UK alone, over 632 patients per million people have end-stage renal disease [1] and require some form of renal replacement therapy (RRT). Dialysis is the most common form of RRT but it is associated with significant comorbidity and mortality that make it less than the ideal long-term treatment. Renal transplantation using allogeneic cadaver organs or those from live donation demonstrates a better 1-year survival rate compared with dialysis [2] and for most patients this represents the optimum treatment. However, current practice is severely limited by the shortage of organs available for transplantation. Therefore, alternative strategies for transplanting renal tissue require investigation.

One promising approach is the transplantation of embryonic kidney precursors (metanephroi). The use of embryonic rather than mature kidneys has a number of immunological advantages [3]. It has been suggested by some, though not all studies, that transplanted metanephroi derive their vasculature primarily from the host [4]. As a result, hyperacute and acute rejection are diminished, which may eventually allow transplantation across the species barrier in the future [5]. Hammerman and colleagues have shown that embryonic day 15 (E15) rat metanephroi transplanted into adult rat recipients continued to grow and develop into kidney-like structures with recognisable glomeruli and tubules [3]. Glomerular filtration rates (GFR) of the order of 30 µl min–1 g metanephros weight–1 have been reported by Hammerman [6] and ourselves [7]. This falls short of GFR in the mature rat kidney (1000 µl min–1 per g kidney weight) by some margin. The growth rate of transplanted metanephroi is also diminished, with typical weights of 50–100 mg at 3 months post-transplant. This low mass suggests that fewer nephrons are present in the metanephros than in the adult kidney. In rats, nephrogenesis continues until postnatal day (PND) 11, with only 10% of nephrons present at birth [8]. Therefore, if growth rate is reduced in the transplanted metanephros, nephron number may also be reduced, contributing to the low GFR.

Glomerular filtration is also dependent on renal blood flow. Transplantation studies have shown that blood vessels derived from the host invade the transplanted metanephros [9], but rather than forming a single artery, several smaller arteriolar-like vessels develop [10]. This may increase vascular resistance and so reduce blood flow through the metanephros, contributing to the low GFR as reported. Blood flow has not been measured in transplanted metanephroi; hence, the aim of this study was to quantify the haemodynamic capacity of metanephroi. We also used stereological methods to quantify nephron number in order to determine whether the low GFR of transplanted metanephroi arises through a lack of glomeruli, or a reduction in blood flow. Finally, we quantified the expression of a number of transporters and channels central to the urinary concentrating process, including the urea transporters (UT)-A1, 2 and 3, the sodium:potassium:chloride cotransporter type 2 (NKCC2), the epithelial sodium channel (ENaC) and aquaporins (AQP) 1 and 2, in order to assess the maturity of tubules. We also looked for the presence of the angiotensin II type 2 receptor (AT2R) as a marker of nephrogenesis. Finally, we assessed the morphology of the vascular bed within the transplanted metanephroi by examining expression of {alpha}-smooth muscle actin ({alpha}-SMA) and platelet endothelial cell adhesion molecule-1 (CD31) by immunohistochemistry. Together, these data provide a comprehensive assessment of the development and maturation of transplanted rat metanephroi.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Quantitative PCR
 Results
 Discussion
 References
 
All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986.

Unless otherwise stated, all chemicals were purchased from Sigma–Aldrich Ltd, Dorset, UK.

Experimental animals
All animals were housed in the Biological Services Facility at the University of Manchester. Animals were maintained in a 12-h light/dark cycle with free access to water and food (Bantin and Kingman rat and mouse expanded diet, Hull, UK). E15 Lewis rat embryos were obtained from time-mated Lewis rats (Charles River, UK).

Preparation of metanephroi and transplantation
Rat metanephroi were transplanted into adult rat hosts as described previously [7]. Briefly, metanephroi were surgically dissected from E15 Lewis rat embryos and incubated in ice-cold DMEM media containing the following growth factors, shown previously to enhance the growth of metanephroi in vivo [3]: recombinant human (rh) insulin-like growth factor (IGF)-I 10–7 M, rh IGF-II 10–7 M, rh vascular endothelial growth factor 5 µg ml–1, rh transforming growth factor {alpha} 10–8 M (Upstate Biotech, Northern Ireland, UK), rh nerve growth factor 5 µg ml–1, rh fibroblast growth factor 5 µg ml–1, rh hepatocyte growth factor 10–8 M (R&D Systems, Oxon, UK), Tamm-Horsfall protein 1 µg ml–1 (Biomedical Technologies, Stoughton, MA, USA), iron saturated transferrin 5 µg ml–1, corticotrophin-releasing hormone 1 µg ml–1, retinoic acid 10–6 M and prostaglandin E1 25 nM. After 1 h, two to three metanephroi were implanted adjacent to the abdominal aorta of 6-week-old adult female Lewis (host) rats under isoflurane anaesthesia. A left nephrectomy was performed on the host rat and analgesia (0.02 mg buprenorphine, Schering-Plough Ltd, Welwyn Garden City, UK) was administered before the animals were returned to normal housing. All rats were treated with methylprednisolone (800 µg kg–1 day–1 s.c.), shown previously to improve the growth of transplanted metanephroi (M. Clancy and D. Marshall, unpublished observations), for a period of 21 days.

Three weeks following transplantation of metanephroi, animals were re-examined under isoflurane anaesthesia and assessed for the presence of urine cysts. These are formed when urine from the transplanted metanephros collects in the ureter. The free end of the metanephros ureter was then connected, via an interrupted 11–0 suture (Ethicon, Inc., TX, USA), to the free end of the host ureter (ureteroureterostomy). At this stage some metanephroi were removed for stereological or immunohistochemical analysis. Analgesia (0.02 mg buprenorphin) was administered and animals were returned to normal housing.

Clearance study
Three months following ureteroureterostomy, animals with transplants (n = 6) were prepared for clearance measurements under inactin anaesthesia (thiobutabarbital sodium, 100 mg kg–1 i.p). Blood pressure was recorded via a carotid artery catheter (PowerLab, ADInstruments Ltd, Oxfordshire, UK). 3H inulin (2 µCi h–1, Amersham Biosciences, Bucks, UK) and para-aminohippuric acid (PAH, 3 mg h–1) were infused in 2.5% dextrose at 40 µl min–1 via a jugular vein catheter. To prevent mixing of urine produced by the host kidney and the transplanted metanephros, the right ureter was cannulated for collection of urine from the remaining host kidney, and the bladder was cannulated for urine collection from the transplant. After surgery, a bolus dose of 3H inulin (4 µCi) and PAH (2 mg) was injected via the venous cannula. Following a 3-h equilibration period, urine samples from both the host kidney and transplanted metanephros were allowed to drain into a vial placed below the level of the anaesthetised rat. Urine samples were collected from the host kidney every 15 min; due to the low flow rate, a single urine sample was collected from the transplanted metanephros over the 3-h experimental period. Blood samples (0.5 ml) were taken from the carotid artery at hourly intervals. A second group of intact animals with no transplanted renal tissue (n = 5) was prepared in a similar manner to act as controls. Animals were killed humanely at the end of the clearance study, and metanephroi were removed for later stereological or immunohistochemical analysis or for RNA extraction.

Analysis of urine and plasma
3H inulin was measured using a 1900CA Tri-Carb Liquid Scintillation Analyser β-counter (Canberra Industries, Meridien, CT, USA). PAH concentration was measured using a standard colorimetric assay. Mean values of 3H inulin and PAH concentrations for the native kidney and control kidneys were calculated by comparing 15 min urine collections with timed blood samples and then taking a mean over the 3-h collection period. Values for the metanephroi were taken by analysing the 3H inulin and PAH concentrations from a single 3 hour collection and comparing with a mean of the three timed blood samples obtained. Urea concentration was determined by colorimetry using a commercial kit (Enzymatic Urea Nitrogen clinical testing kit, Stanbio Laboratories, Boerne, TX, USA).

Glomerular counts
Transplanted metanephroi (n = 9) consisting of metane- phroi harvested either 3 weeks (n = 5) or 4–6 months (n = 4) following transplantation (following clearance study), and kidneys from E21 (n = 5) and PND 1 (n = 5) rats were explanted, fixed, embedded and sectioned exhaustively. Sections were stained with haematoxylin and eosin before glomerular numbers in every nth 10-µm kidney section pair (section and parallel section) were estimated using a modified version of the physical fractionator technique. Briefly, mature vascularised glomeruli were counted within a frame on one kidney section and then compared to a second ‘look-up’ section on the corresponding part of the parallel kidney section. Glomeruli that were present on both the initial section and ‘look-up’ section were discounted. The counting frame was then moved and the process repeated until the whole kidney section was counted; 8–10 section pairs were counted for each sample. The final number of glomeruli was estimated using the following formula:


Formula

where Q is the number of glomeruli counted, n is the fraction of section pairs counted, x2 implies that the estimate is doubled since glomeruli are counted two ways: initial section versus ‘look-up’ section and vice versa.

Immunohistochemical localisation
Transplanted metanephroi harvested 3 weeks to 6 months following transplantation (n = 6), E21 (n = 5), PND 1 (n = 5) and adult (n = 3) kidneys were removed, fixed and embedded. Sections (5 µm) were incubated overnight at 4°C with primary antibodies diluted in 0.1% BSA with 0.3% Triton X-100. Labelling was identified by application of either horseradish-peroxidase-conjugated swine anti-rabbit immunoglobulin (1:100, DakoCytomation Ltd, Cambridge, UK) or horseradish-peroxide-conjugated goat anti-mouse immunoglobulin (1:100, DakoCytomation Ltd). Rabbit anti-rat primary antibodies were used at the dilutions indicated: AQP1 (1:200), AQP2 (1:500, both kind gifts from Dr David Marples, University of Leeds, UK), AT2R (1:50, Abcam Ltd, Cambridge, UK), ENaC {alpha}-subunit (1:200, Sigma–Aldrich, Dorset, UK), NKCC2 (1:200, New England Biolabs, Hitchin, UK), UT-A1/2 (1:100), UT-A1/3 (1:100, both kind gifts from Dr Craig Smith, University of Manchester, UK). Mouse anti-rat primary antibodies were used at the following dilutions: CD31 (1:100 Serotec Ltd, Oxfordshire, UK), {alpha}-SMA (1:500, Sigma–Aldrich). Negative controls were carried out by the omission of either the primary or secondary antibody.



   Quantitative PCR
Top
 Abstract
 Introduction
 Subjects and methods
 Quantitative PCR
Results
 Discussion
 References
 
Total RNA was extracted from transplanted metanephroi harvested 3 weeks to 6 months following transplantation (n = 6), E21 (n = 7), PND 1 (n = 7) and adult (n = 3) kidneys by trizol extraction and reverse-transcribed using SuperScript II RT (200 U, Invitrogen, UK). All primers and TaqMan probes (Table 1) were designed using Primer Express (ABI Instruments, Foster City, CA, USA). Reverse transcriptase–polymerase chain reaction was performed using the ABI Prism 7000 system (ABI Prism, Foster City, CA, USA). PCR amplification had shown that the gene efficiencies of all genes tested were comparable with the gene efficiency of β-actin, allowing it to be used as a reference gene for relative quantification. Relative gene expression was calculated using the 2–{Delta}{Delta}CT method [11].


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  		Table 1 Taqman probe and primer sequences (5' to 3') used for qPCR

 
Statistical analysis
Data were analysed by t-test and one way one-way analysis of variance (ANOVA) following log10 transformation and Tukey's post hoc test, where appropriate (SPSS 13.0 for Windows, SPSS UK Ltd, Surrey, UK). Significance was attributed where P < 0.05.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Quantitative PCR
 Results
Discussion
 References
 
Clearance study
Table 2 summarises baseline data for animals with a transplanted metanephros and control rats with two intact kidneys. Transplanted metanephroi, 4–6 months post-transplant, weighed 50 ± 10 mg, which was significantly less than the remaining native kidney (P < 0.001) and the kidneys of control animals (P < 0.001). Mean arterial pressure was constant throughout the experiment and did not differ between animals with transplants and controls. Haematocrit was similar in both groups.


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  		Table 2 Baseline data in animals with transplanted metanephroi and controlsa

 
Effective renal blood flow (ERBF, Figure 1a) in transplanted metanephroi was 149 ± 33 µl min–1 per g kidney weight, approximately 5% of that in the remaining native kidney (P < 0.001) and in the kidneys of the control animals (P < 0.001). Consequently, the calculated renal vascular resistance (RVR, Figure 1b) of transplanted metanephroi (933 ± 209 mmHg ml min–1 per g kidney weight) was significantly higher (P < 0.001) compared with all other kidneys. Uninephrectomy of the host animals resulted in a compensatory increase in ERBF in the contralateral kidney by comparison with the left kidney of control rats (P < 0.05). GFR (Figure 1c) was significantly lower (P < 0.001) in metanephroi compared with all adult kidney tissue. Urine flow rate from the metanephroi, when standardised to kidney weight, tended to be lower than that of the native adult kidney and control kidneys, but this difference was not statistically significant. Urine flow rates were comparable between the host kidney and control kidneys.


Figure 1
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  		Fig. 1 (a) ERBF, (b) RVR, (c) GFR and (d) urine flow rate (UV) in transplanted metanephroi and controls. All parameters were measured in transplanted metanephroi and the remaining right native kidney of animals with transplants, and left and right kidneys of control animals. All data are presented as mean ± SEM. Statistical analysis was by one-way ANOVA, following log10 transformation of data, and Tukey's post hoc test. ***P < 0.001 compared with metanephroi, {dagger}P < 0.05 compared with left kidney of controls.

 
Urea analysis
Urea concentrations (Table 3) reflected the maturity of the renal tissue. The concentration of urea in cyst fluid was significantly lower than that of urine produced by the metanephros (P < 0.005), which in turn was significantly lower than that of urine produced by the native kidney (P < 0.001). Despite the comparatively low urea concentrations of cyst fluid and metanephric urine, these were significantly higher than the serum urea concentration (P < 0.001).


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  		Table 3 Urea concentrations in transplanted metanephroi and controlsa

 
Glomerular counts
Transplanted metanephroi had 4399 ± 216 nephrons, which was significantly more than E21 kidneys (2578 ± 358, P < 0.01) but significantly fewer than PND 1 kidneys (7023 ± 587, P < 0.001).

Expression of urea, sodium and water transporters and channels and vascular markers
The expression patterns of UT-A1, 2 and 3, AQP1 and 2, ENaC, NKCC2 and AT2R are summarised in Table 4. Positive immunostaining for UT-A1, 2 and 3 was visible in adult kidney (Figure 2a) within the inner medullary collecting duct (IMCD) and thin descending limbs but not in transplanted metanephroi (Figure 2b), E21 or PND 1 kidneys. AQP1 was localised to the proximal tubules and thin descending limbs in transplanted metanephroi (Figure 2c), with more intense staining visible in E21 (Figure 2d), PND 1 and adult kidneys. AQP2 was localised to the IMCD in all kidneys examined including transplanted metanephroi (Figure 2e) and PND 1 kidneys (Figure 2f). Positive staining for ENaC (data not shown) was observed in the collecting ducts and that for NKCC2 (Figure 2g and h) was localised to the thick ascending limbs in all groups; there were no differences between transplanted metane- phroi and either E21 or PND 1 kidneys. The developmental marker AT2R showed localised staining of developing tubules in the cortex of the metanephroi, E21 and PND 1 kidneys but not in adult kidneys (data not shown). Expression of CD31 (Figure 2i) and {alpha}-SMA (Figure 2j) was also observed in the transplanted metanephroi; {alpha}-SMA was particularly abundant, whereas CD31 staining was less widespread.


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  		Table 4 A summary of immunohistochemical staining

 

Figure 2
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  		Fig. 2 Immunohistochemical localisation of (a and b) UT-A1/3, (c and d) AQP1 and (e and f) AQP2, (g and h) NKCC2, (i) CD31 and (j) {alpha}SMA. Intense UT-A1/3 staining was observed in the collecting ducts of adult rat controls (a) but not in transplanted metanephroi (b). AQP1 was localised to the proximal tubules (PT) of transplanted metanephroi (c) and embryonic day 21 rats (arrowed) close to the glomeruli (g) in (d). Staining for AQP2 was present in the collecting ducts of transplanted metanephroi (arrowed) (e) and also in the collecting ducts of PND 1 kidney (f). Staining for NKCC2 was visible in the thick ascending limbs of metanephroi (g, arrowed) and PND 1 kidneys (h). CD31 was present in endothelial cells (arrowed) of transplanted metanephroi (i) and abundant staining for {alpha}SMA was also observed within the transplants. Original magnification for all images was x100. Scale bars for all images as for (a).

 
Analysis by qPCR confirmed these observations. Combined UT-A1 and 2 expression in metanephroi was approximately 10% of adult levels, but not significantly different from E21 and PND 1 kidneys (Figure 3a). Combined UT-A1 and 3 mRNA expression in metanephroi was significantly lower than that in both E21 and PND 1 kidneys (Figure 3b, P < 0.001). UT-A1 and 3 expression was significantly higher in PND 1 compared with E21 kidneys (P < 0.05). Messenger RNA expression of AQP1 and 2 in metanephroi was approximately 10% of adult levels. Although mRNA expression tended to be greater in E21 and PND 1 kidneys by comparison with metanephroi, this difference was not statistically significant (Figure 3c and d). The mRNA expression of ENaC and NKCC2 in metanephroi was between 5 and 10% of adult expression levels. This was not statistically different compared with E21 and PND 1 kidneys (Figure 3e and f). AT2R mRNA expression in metanephroi, despite being 13-fold higher than that in adult samples (13.2 ± 2.0 arbitrary units), was markedly reduced compared with both E21 (1048.5 ± 229.2, P < 0.001) and PND 1 kidneys (899.3 ± 97.5, P < 0.001).


Figure 3
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  		Fig. 3 Expression of transporters and channels involved in the urinary concentrating process. mRNA expression of (a) UT-A1/2, (b) UT-A1/3, (c) AQP1, (D) AQP2, (e) NKCC2 and (f) ENaC is shown for transplanted metanephroi, embryonic day 21 animals (E21) and PND 1 animals. All values were compared with adult expression levels, assigned an arbitrary value of 1. Statistical analysis was by one-way ANOVA. ***P < 0.001 compared with transplanted metanephroi, {dagger}P < 0.05 compared with E21 kidney.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Quantitative PCR
 Results
 Discussion
References
 
This study has shown that E15 rat metanephroi transplanted into syngeneic host animals continue to grow and develop for a short period of time. On the basis of nephron number and the expression levels of transporters and channels involved in the urinary concentrating process, the transplanted metanephroi appear to be at a stage of development equivalent to that of the normal rat kidney at the time of birth. This observation is supported by the clearance measurements, which showed that the GFR (17 µl min–1 per g kidney weight) was broadly comparable with those observed in neonatal rats in the first week of life (90 µl min–1 per g kidney weight) [12]. Thus, the development of the metanephroi has been arrested around the E21/PND 1 stage.

This is the first study to report nephron number in transplanted metanephroi. Using non-biased stereology, we have shown that the number of nephrons present in the metanephroi is intermediate between that seen in E21 and PND 1 kidneys, which suggests that nephrogenesis was arrested at a stage equivalent to that at the time of birth. The nephron counts were performed on two groups of transplanted metanephroi, some of which were harvested at 3 weeks post-transplant (5/9) and some at 4–6 months post-transplant (4/9). Despite a difference of 13 weeks between the groups, there was no significant difference in the number of nephrons present. This suggests that the period of nephrogenesis post-transplantation was limited to the first 3 weeks.

The AT2R expression levels observed also suggest that nephrogenesis had ceased in transplanted metanephroi. AT2R plays a role in renal morphogenesis and in the regulation of fetal kidney function [13]. It is highly expressed in fetal rat kidneys from E14 to PND 7. There is debate over its expression level in the mature rat, with some suggesting that AT2R is undetectable by PND 28 [14]. We did not detect AT2R mRNA or protein in adult kidneys, but both were highly expressed in E21 and PND 1 kidneys that were undergoing nephrogenesis. AT2R mRNA expression was markedly reduced in metanephroi and positive immunostaining was only observed in 2/6 samples. These observations suggest that nephron formation had ceased in the metanephroi.

Functionally, the metanephroi were at a stage of development comparable with the early post partum kidney. The ERBF reported herein is the first estimation of blood flow through transplanted metanephroi. Metanephric blood flow was much lower than that through the remaining native kidney of the host animal or the kidneys of controls. Even when corrected for mass, ERBF in the metanephroi remains very low. This contrasts with previous reports on normal kidney development that have shown that ERBFs, per gram of tissue, at 3–4 weeks of age are comparable with values in adult rats [15]. The transplanted metanephroi in this study were approximately 16 weeks of age at the time of clearance, so clearly they do not have an ERBF consistent with their chronological age.

One reason for the decrease in ERBF is the marked increase in RVR observed in transplanted metanephroi. The elevated RVR may reflect abnormal development of the vascular tree in the metanephroi following transplantation. Recent work has shown that vascularisation of transplanted metanephroi occurs primarily as a result of angiogenesis from the host rather than vasculogenesis from the transplant [9]. However, this does not appear to proceed normally as, rather than developing a single renal artery, a number of smaller arteriolar-type vessels develop [10]. This, coupled with a lack of arteriogenesis, means that the vascular tree consists of a series of small, high-resistance vessels. Consequently, it is not surprising that RVR is elevated and blood flow is reduced. CD31 and {alpha}-SMA were detected in transplanted metanephroi at levels comparable with E21 and PND 1 kidneys. This observation suggests that a poor vascular network within the transplanted metanephros is unlikely to be the major cause of increased RVR; rather it seems likely that the lack of a single renal artery is the main reason for the low blood flow rate measured in the current study.

The elevated RVR and low ERBF measured in this study were associated with a GFR equivalent to 2% in an adult rat kidney. Although this rate of filtration is very low, it may not be abnormal for a kidney of the transplanted metanephros's apparent developmental age. Measurements made at PND 4 revealed a GFR of 90 µl min–1 per g kidney weight [12], which is within the same range of GFR as observed in the metanephroi in the current study (17 µl min–1 per g kidney weight). Similarly, urine flow rates, while very low (3 µl min–1 per g kidney weight), are comparable with those observed in neonatal rats [12]. Hence, the metanephroi may actually be performing within the expected physiological range for their size and stage of development.

Indeed, the tubules themselves may be more mature than the nephron counts and clearance data suggest. Urea concentrations were measured in cyst fluid and urine produced by the metanephros. Cyst fluid sampled at 3 weeks after initial transplantation had a significantly lower urea concentration than that in urine from the metanephros at 4 months after transplantation. This is consistent with previous reports in rat transplants [3], and suggests that between 3 weeks and 4 months following transplantation, the metanephros transplants developed a greater concentrating ability. Furthermore, by comparing transplanted metanephroi with kidneys from 4-day-old animals [12], it is apparent that the transplants have a greater concentrating ability than their mass might suggest. Urea concentration in metanephric urine was 30-fold greater than that of serum, compared with a 20-fold difference between urine and serum in 4-day-old animals. This concentration of urea in metanephric urine is unlikely to have arisen through facilitated transport. No immunohistochemical staining of UT-A1, 2 or 3 was observed in metanephroi and only very low levels of UT-A1 and 2 or UT-A1 and 3 mRNA were observed. Therefore, the concentration of urea observed in metanephric urine implies that water has been reabsorbed from the tubular fluid.

Transplanted metanephroi expressed both AQP1 and 2 protein and mRNA, which is consistent with reports of AQP1 expression in the renal vasculature as early as E16 [16] and AQP2 from E18 [17]. AQP1 and 2 expressions tended to be lower in metanephroi by comparison with E21 and PND 1 kidneys, although this difference was not statistically significant, suggesting that the transplants should have been capable of transcellular water transport. Sodium transport could have acted as the driving force for the movement of water as metanephroi expressed both ENaC and NKCC2. ENaC mRNA is expressed in the normal fetal kidney from E16, with expression increasing rapidly until 3 days after birth [18]. NKCC2 is expressed in the thin ascending limbs at birth [19], although expression of NKCC2 protein and mRNA were detected as early as E21 in the current study. These observations suggest that transplanted metanephroi are able to concentrate urine to a limited extent, consistent with their stage of arrested development.

Developmental expression of NKCC2 has been linked to the appearance of UT-A transporters in fetal mice [20]. Expression of NKCC2 at E14 was followed by that of the tonicity-responsive binding protein (TonEBP) at E15 which in turn stimulated UT-A expression at E16. UT-A expression was reduced by the administration of the NKCC2 inhibitor furosemide, leading to the conclusion that the hypertonicity generated by NKCC2 activates TonEBP and thus UT-A expression [20]. This pathway appears to be disrupted in the transplanted metanephroi as NKCC2 expression was not associated with UT-A expression in the current study. One possible explanation is that methylprednisolone treatment, which was used to promote metanephric growth, may have delayed medullary development in the transplanted tissue as exposure to supranormal concentrations of glucocorticoids has been shown to impair growth of the outer medulla [21]. This study also showed that glucocorticoid treatment accelerated the development of the TAL epithelium, by up-regulating NKCC2 expression to adult levels. We observed no such increase in NKCC2 in methylprednisolone-treated metanephroi; indeed, NKCC2 mRNA expression was only 5% of adult levels suggesting that brief glucocorticoid exposure may not have had a marked impact on the loop of Henle in transplanted metanephroi.

In conclusion, this study has demonstrated that transplanted E15 rat metanephroi continue to grow and develop for a short period of time in the host. Nephrogenesis had ceased by 3 weeks post-transplantation, having progressed to a stage comparable with a normal rat kidney at birth, which amounts to 15% of adult nephron number [22]. We have shown previously that increasing nephron number by transplanting and connecting two metanephroi to an otherwise anephric rat prolongs life for 20 h more than a rat with a single metanephros, but life is still only sustained for 120 h [23]. This suggests that increasing nephron number alone is not sufficient; the maturity of the tubules must also be increased if life is to be prolonged indefinitely. The current study shows that the expression levels of urea, water and sodium transporters and channels in transplanted metanephroi were also comparable with those seen in the rat kidney at or shortly after birth. The immature tubules are able to concentrate urine to a degree, but are not able to perform all of the functions of the normal adult kidney to an extent compatible with the long-term maintenance of life. This study suggests that, if transplantation of embryonic kidneys is to become a viable approach to RRT, the period of growth and development of post-transplantation must be prolonged.



   Acknowledgments
 
M.R.D. was supported by an MRC Collaborative Studentship. M.J.C. was supported by a fellowship grant from Intercytex Ltd. The authors thank Dr Jens Nyengaard, University of Aarhus, for assistance with the stereological measurements, Dr David Marples, University of Leeds, for the AQP1 and AQP2 antibodies and Dr Craig Smith, University of Manchester, for the UT-A1/2 and UT-A1/3 antibodies.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Quantitative PCR
 Results
 Discussion
 References
 

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Received for publication: 8. 6.07
Accepted in revised form: 3. 9.07


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