INTRODUCTION

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WE HAVE SHOWN PREVIOUSLY that metanephroi from outbred rat embryos transplanted into the omentum of nonimmunosuppressed adult hosts undergo growth and differentiation, are vascularized by vessels arising from the host, and function, in that they clear inulin infused into the host's circulation (15). In contrast, developed kidneys transplanted from one rat into another undergo acute rejection within 7 days (15). Using inbred rat strains, we demonstrated identical findings after transplantation across the rat major histocompatibility complex locus RT1 and determined that a state of peripheral immune tolerance secondary to T cell ignorance is permissive, at least in part, of the survival of transplanted metanephroi in nonimmunosuppressed RT1-disparate rats (14).

Because of the state of peripheral tolerance that exists after transplantation of metanephroi (14), reduced expression of tissue antigens in transplanted metanephroi relative to adult kidneys (1), and the mounting of a T-helper 2-biased response by the host after transplantation of fetal kidneys (2), and because, unlike the case after transplantation of developed kidneys, the vasculature of transplanted developed metanephros originates, at least in part, from the host (8, 10, 13, 21), there are theoretical advantages to transplanting metanephroi relative to developed kidneys. For example, the presence of host endothelium in a transplanted organ would obviate the problem of antigen presentation by donor endothelial cells and, in the case of pig-to-human metanephric xenografts, hyperacute rejection (7).

By using a discordant model, we have carried out pig-to-rat xenotransplantation of metanephroi (8). Although our findings are preliminary, they suggest that transplantation into humans of xenograft metanephroi, for example those originating in pig embryos, could represent a novel means of xenotransplantation (7, 8).

In the case of human renal allotransplantation, there is an unavoidable delay between the time of harvest from donors and the time of implantation into recipients. The delay often results, in part, from the need to transport donor kidneys across long distances (11).

Theoretically, metanephroi could be harvested immediately before implantation into humans. However, practically it would be best if, like most human renal allografts, metanephroi could be stored in vitro for a period of time before transplantation. To determine whether transplanted metanephroi grow, differentiate, and function in hosts after preservation in vitro, we characterized the growth of embryonic day 15 (E15) rat metanephroi that had been preserved in University of Wisconsin (UW) solution for 3 days before implantation and compared their function to that of metanephroi that had been implanted directly. Our observations indicate that functional kidneys develop from metanephroi transplanted after preservation in vitro.

	METHODS

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Metanephroi were surgically dissected from E15 Sprague-Dawley rat embryos under a dissecting microscope using previously described techniques (15) and placed in ice-cold UW solution. When indicated, the following growth factors, previously shown to enhance the function of transplanted metanephroi (6, 7, 17), were added to the UW solution (UW + growth factors): 10⁷ M recombinant human insulin-like growth factor I (IGF I) (Genentech, San Francisco, CA), 10⁷ M recombinant human IGF II (Bachem, Torrance, CA), 10⁸ M recombinant human transforming growth factor- (Upstate Biotechnology, Lake Placid, NY), 10⁸ M recombinant human hepatocyte growth factor (R&D Systems, Minneapolis, MN), 5 µg/ml recombinant human vascular endothelial growth factor (Genentech), 5 µg/ml recombinant human basic fibroblast growth factor (R&D Systems), 5 µg/ml recombinant human nerve growth factor (Boehringer Mannheim, Indianapolis, IN), 10⁶ M retinoic acid (Sigma Chemicals, St. Louis, MO), 1 µg/ml corticotropin-releasing hormone (Sigma Chemicals), 1 µg/ml Tamm Horsfall protein (Biomedical Technologies, Stoughton, MA), 25 mM prostaglandin E₁, and 5 µg/ml iron-saturated transferrin. Recombinant human growth factors were used rather than rat growth factors because of their ready availability.

Some metanephroi were implanted directly in the omentum of anesthetized 6-wk-old female (host) Sprague-Dawley rats after 45 min of incubation on ice in UW solution or UW solution + growth factors. Others were implanted after 3 days of storage in a 2-ml sterile screw cap, sterile plastic microcentrifuge tube (Fisher, Houston, TX) containing 1 ml of ice-cold UW solution or UW + growth factors. During the same surgery, host rats had one kidney removed.

When indicated, 4 wk after transplantation, end-to-end ureteroureterostomy was performed using microvascular technique (interrupted 10-0 suture) between the ureter of a metanephros implanted in the omentum and the ureter of the kidney that had been removed. Eight weeks later all remaining native renal tissue (the contralateral kidney) was removed from host rats, after which inulin clearances were measured on conscious rats after placement of an indwelling bladder catheter and intravenous line exactly as in previous studies (15).

Baseline measurements for inulin were performed on urine and blood samples obtained before beginning the inulin infusions. These background values were subtracted from measurements performed after beginning the inulin infusion. Infusion was begun only after removal of all remaining native renal tissue and drainage of all urine remaining in the bladder (10-20 µl). Only the implanted metanephros remained connected to the bladder. As before, rats received no immunosuppression (15).

Female rats were used as recipients because catheterization of the bladder, necessary to measure inulin clearances in conscious animals, is much more readily performed in female rats. E15 metanephroi transplanted into male recipients engraft, differentiate, and grow comparably to those transplanted into female rats. E15 rat embryos were used as donors because in preliminary experiments during which transplantations were carried out using E13, E14, E15, E16, or E17 rat embryos, those from E15 embryos engrafted most successfully. We have speculated previously as to why this may be the case (15).

Metanephroi were fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin exactly as in previous studies (15).

Organ culture of E13 metanephroi was carried out in a Dulbecco's modified Eagles medium-Ham's F12 solution exactly as previously described (18, 19).

Multiple comparisons of data shown in Table 1 were made using a Student-Newman-Keuls multiple comparison test.

	RESULTS

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Figure 1, a, c, e, and g, shows photomicrographs of hematoxylin- and eosin-stained sections of rat metanephroi. Higher power views are provided in Fig. 1, b, d, f, and h, for metanephroi shown in Fig. 1, a, c, e, and g, respectively.

View larger version (116K): [in this window] [in a new window]   Fig. 1.   Photomicrographs of hematoxylin- and eosin-stained midsagittal sections of metanephroi originating from an embryonic day 15 (E15) rat embryo (a and b); an E15 rat embryo after 3 days of preservation in University of Wisconsin (UW) solution (c and d); an E16 rat embryo (e and f); or an E13 metanephros grown in organ culture for 3 days (g and h). Shown are metanephric blastema (MB), branched segments of ureteric bud (UB), and developing nephron S-shaped bodies (S). Arrows delineate the nephrogenic zone. Photomicrographs are representative of >10 experiments. Bar: 150 µm (for a, c, e, and g); 375 µm (for b, d, f, and h).

Figure 1, a and b, illustrates an E15 metanephros consisting largely of undifferentiated metanephric blastema, and branches of ureteric bud. A nephrogenic zone is delineated by arrows. Figure 1, c and d, illustrates an E15 metanephros after 3 days of preservation in ice-cold UW solution. The size (midsagittal diameter) of the E15 metanephros after 3 days of preservation (Fig. 1c) is approximately the same as that of the nonpreserved E15 metanephros (Fig. 1a). As would be expected, because it originates from an embryo 1 day older, an E16 metanephros (Fig. 1e) is larger than the E15 metanephros (Fig. 1a). However, the E16 metanephros (Fig. 1e) is also larger than the E15 metanephros that had been preserved for 3 days (Fig. 1c) and has a wider nephrogenic zone (Fig. 1f, arrows) than either the E15 metanephros (Fig. 1b) or the E15 preserved metanephros (Fig. 1d).

Similar to the E15 metanephros shown in Fig. 1b, the E15 preserved metanephros shown in Fig. 1d consists largely of undifferentiated metanephric blastema, and branches of ureteric bud. In contrast, more developed nephron structures, such as an S-shaped body, can be delineated in the E16 metanephros (Fig. 1f).

Figure 1, g and h, shows photomicrographs of an E13 rat metanephros after 3 days in organ culture. While its size is approximately the same as those of metanephroi shown in Fig. 1, a and c, the state of differentiation of nephron structures, such as the S-shaped body shown in Fig. 1h, comparable to that reported in previous studies (9, 16), is more advanced than those shown in the E15 metanephros (Fig. 1b) or the E15 metanephros after 3 days of preservation in vitro (Fig. 1d).

E15 metanephroi that were preserved in UW + growth factors were histologically indistinguishable from E15 metanephroi that were preserved in UW solution (not shown).

The data shown in Fig. 1 do not exclude the possibility that some development takes place in E15 metanephroi during 3 days of preservation in UW solution on ice (chronological age 18 days). However, whatever development does occur is much less than that observed in metanephroi of lower chronological age such as E13 metanephroi after 3 days in organ culture (chronological age 16 days) (Fig. 1, g and h) or E16 metanephroi (chronological age 16 days) (Fig. 1, e and f).

Figure 2 shows photomicrographs of hematoxylin- and eosin-stained sections of a developed E15 metanephros 4 wk after implantation in the peritoneum of a host rat. The metanephros had been preserved for 3 days in UW solution (no growth factors) before implantation.

View larger version (88K): [in this window] [in a new window]   Fig. 2.   Photomicrographs of hematoxylin- and eosin-stained sections of developed E15 metanephros 4 wk after implantation in a host peritoneum. The metanephros was preserved in UW solution for 3 days before implantation. a: a ureter (u). b: a glomerulus (g), a proximal tubule (pt) with its brush border membrane delineated (arrowhead), and a distal tubule (dt). c: mature collecting ducts (cd). Photographs and photomicrographs are representative of >10 experiments. Bars: 1 mm (a) and 10 µm (b and c).

As we have shown previously for E15 metanephroi that are not preserved in vitro before transplantation (15), transplanted developed preserved metanephroi are kidney shaped (Fig. 2a). A ureter is present. Cortices contain mature-appearing glomeruli and proximal and distal tubules (Fig. 2b). The medulla contains mature collecting ducts (Fig. 2c).

Weights, urine volumes, and inulin clearances were measured at 12 wk posttransplantation in metanephroi that had been transplanted directly or transplanted after 3 days of preservation in UW solution with or without growth factors.

The addition of growth factors to the UW solution (compared with no growth factors) increased the weights of metanephroi transplanted directly, but not after 3 days of preservation (Table 1). Urine volumes measured at the time of inulin clearances were significantly increased compared with all other groups by the addition of growth factors to the UW solution of preserved metanephroi.

Inulin clearances were expressed as microliters per minute per 100 g rat weight. Rat weights did not vary between groups at the time inulin clearances were measured (Table 1). Clearances of developed metanephroi transplanted directly (0.43 ± 0.06 µl · min¹ · 100 g¹) or after 3 days of preservation in UW solution without growth factors (0.38 ± 0.08 µl · min¹ · 100 g¹) were comparable to clearances previously measured in Sprague-Dawley to Sprague-Dawley transplants (14, 15, 17). Addition of growth factors to the UW solution increased inulin clearances measured in developed metanephroi that had been implanted directly (1.1 ± 0.2 µl · min¹ · 100 g¹) or after 3 days of preservation in vitro (1.2 ± 0.2 µl · min¹ · 100 g¹) compared with clearances measured in either group of metanephroi that were not exposed to growth factors (Table 1).

	DISCUSSION

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A shortage exists of human kidneys available for transplantation. It has been suggested that the transplantation of animal kidneys into humans could substitute for allotransplantation of human kidneys (20).

In many ways, pigs represent the ideal renal organ donor for humans. The reason is that, relative to more closely related nonhuman primates, pigs are plentiful. In addition, their size and digestive, circulatory, respiratory, and renal physiologies are very similar to those of humans. Unfortunately, the transplantation of porcine vascularized organs such as kidneys into humans is rendered problematic, in part because of the reaction of preformed antibodies against antigens present on the vascular endothelium of the pig (hyperacute rejection) (20).

Hyperacute rejection of xenografts should be circumvented to the extent that the transplanted organ is supplied by host vessels. An example of host vascularization of a transplanted tissue (4, 12) permitting pig-to-human transplantation is provided by the demonstration that fetal porcine islets of Langerhans can be transplanted to humans without eliciting hyperacute rejection (3).

Insight into the origin of the renal blood supply is provided by experiments in which developing kidneys are transplanted to ectopic sites. In the case of 11-day-old mouse or chick metanephroi grafted onto the chorioallantoic membrane of the quail, the vasculature is derived entirely from the host (21). In the case of 11- to 12-day-old mouse metanephroi grafted into the anterior chamber of the eye, the glomerular microvascular endothelium derives from both donor and host (10). In either case, large external vessels derive from the host (10, 21). In the case of rat metanephroi transplanted into the peritoneal cavity of mice, most of the glomerular capillary loops are of host origin (13). In that at least a portion of their vasculature originates from the host, transplanted metanephroi are similar to transplanted islets.

Transplanted metanephroi elicit a muted host immune response relative to transplanted developed kidneys (1, 2, 14, 15). This probably results, at least in part, from reduced expression of tissue antigens including human major histocompatibility complex (HLA) class I and II in developing fetal kidneys relative to developed kidneys (1), the mounting of a T-helper 2-biased response by the host after transplantation of fetal kidneys in contrast to a T-helper 1-biased response after developed-kidney transplantation (2), and the relative absence of dendritic cells in a transplanted metanephros relative to a mature kidney (14).

To the extent that transplanted metanephroi are more vascularized by the host and elicit a muted immune response relative to transplanted developed kidneys, the xenotransplantation of the former is advantageous relative to the xenotransplantation of the latter. Therefore, should the use of metanephroi become an alternative to the use of human renal allografts, it would be useful to be able to preserve these renal anlage in vitro for a time before their use, similar to the way in which renal allografts are preserved (11).

Perspectives

No direct analogy is possible between storage of metanephroi and kidneys. Nonetheless, comparisons can be made. The duration of warm ischemia for metanephroi, i.e., the time between removal from embryos from rats, dissection of metanephroi, and placement in ice-cold UW solution, is ~15 min. For the preserved metanephroi used to generate data shown in Figs. 1 and 2 and Table 1, the duration of cold ischemia is 3 days. Here we show that under these conditions of preservation in vitro, functional chimeric kidneys develop from metanephroi transplanted into adult rats. Our findings indicate that, as is the case with developed kidneys, preservation of metanephroi in vitro before transplantation is a feasible option, in that metanephroi transplanted directly or after preservation develop into an organ that clears insulin from the host's circulation.