Xenotransplantation of pancreatic and kidney primordia-where do we stand?

Lack of donor availability limits the number of human donor organs. The need for host immunosuppression complicates transplantation procedures. It is possible to 'grow' new pancreatic tissue or kidneys in situ via xenotransplantation of organ primordia from animal embryos (organogenesis of the endocrine pancreas or kidney). The developing organ attracts its blood supply from the host, enabling the transplantation of pancreas or kidney in 'cellular' form obviating humoral rejection. In the case of pancreas, selective development of endocrine tissue takes place in post-transplantation. In the case of kidney, an anatomically-correct functional organ differentiates in situ. Glucose intolerance can be corrected in formerly diabetic rats and ameliorated in rhesus macaques on the basis of porcine insulin secreted in a glucose-dependent manner by beta cells originating from transplants. Primordia engraft and function after being stored in vitro prior to implantation. If obtained within a 'window' early during embryonic pancreas development, pig pancreatic primordia engraft in non immune suppressed diabetic rats or rhesus macaques. Engraftment of pig renal primordia transplanted directly into rats requires host immune suppression. However, embryonic rat kidneys into which human mesenchymal cells are incorporated into nephronic elements can be transplanted into non-immune suppressed rat hosts. Here we review recent findings germane to xenotransplantation of pancreatic or renal primordia as a novel organ replacement strategy.     

Treatment for end-stage renal disease: an organogenesis/tissue engineering odyssey

The means by which kidney function can be replaced in humans with end-stage renal disease (ESRD) include dialytic therapies and renal allotransplantation. Dialysis, is lifesaving, but often poorly tolerated. Transplantation of human kidneys is limited by the availability of donor organs. During the past decades, several different approaches have been applied towards new means to replace renal function through organogenesis and tissue engineering. These include: (1) incorporation of new nephrons into the kidney; (2) growing new kidneys in situ; (3) use of stem cells; (4) generation of histocompatible tissues using nuclear transplantation; and (5) bioengineering of an artificial kidney. The development of these approaches has depended upon understanding and integrating discoveries made in a diversity of scientific disciplines. The means by which such integration has driven advances in the treatment of ESRD provides a generic roadmap for the successful application of organogenesis and tissue engineering to organ replacement therapy.     

Tissue engineering the kidney

The means by which kidney function can be replaced in humans include dialysis and renal allotransplantation. Dialytic therapies are lifesaving, but often poorly tolerated. Transplantation of human kidneys is limited by the availability of donor organs. During the past decades, a number of different approaches have been applied toward tissue engineering the kidney as a means to replace renal function. The goals of one or another of them included the recapitulation of renal filtration, reabsorptive and secretory functions, and replacement of endocrine/metabolic activities. This review will delineate the progress to date recorded for five approaches: (1) integration of new nephrons into the kidney; (2) growing new kidneys in situ; (3) use of stem cells; (4) generation of histocompatible tissues using nuclear transplantation; and (5) bioengineering of an artificial kidney. All five approaches utilize cellular therapy. The first four employ transplantation as well, and the fifth uses dialysis.     

Cellular transplantation of nephrons

Cellular transplantation of nephrons. Embryonic renal cellular primordia transplanted into animal hosts undergo nephrogenesis in situ, become vascularized by blood vessels of host origin, exhibit excretory function, and support life in otherwise anephric hosts. Renal primordia can be transplanted across isogeneic, allogeneic, and both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. Here I review studies exploring the therapeutic potential for renal organogenesis posttransplantation of cellular kidney primordia.     

Applications of organ precursor cell therapy: can lessons from embryonic kidney transplantation be applied to the endocrine pancreas?

PURPOSE OF REVIEW: The purpose of this review is to provide an update relating to a novel approach to endocrine pancreas replacement therapy based in part on a technology developed for the transplantation of developing kidneys. The approach is to use organ primordia, and in this way transplant kidneys or pancreas in cellular form. RECENT FINDINGS: Cellular allotransplantation and xenotransplantation of both kidney and pancreatic anlagen has been successfully performed such that functioning organs develop in situ. SUMMARY: The number of human organs available for transplantation is limited. We and others have shown that it is possible to 'grow' new kidneys or endocrine pancreas from organ-specific precursor cells in situ. For the kidney, this technology takes advantage of the fact that a developing renal anlagen can attract its blood supply from an appropriate vascular bed post-transplantation, enabling the transplantation of kidneys in 'cellular' form. Techniques developed for the transplantation of embryonic kidneys can be applied to the transplantation of embryonic pancreas. Pancreatic anlagen implanted into a host peritoneum develop into a novel organ consisting of functional islets of Langerhans surrounded by stroma. The transplantation of developing pancreas could represent a novel 'cellular' treatment for diabetes mellitus, a major cause of end-stage renal disease.     

Fine-needle aspiration biopsy sampling in renal transplantation: interstitial cellular infiltrates and major histocompatibility complex class-II antigen expression in renal tubular cells

The dynamics of major histocompatibility complex (MHC) class-II (DR) antigen products in renal tubular cells and cellular infiltrations in the interstitium of the kidney following renal transplantation without immunosuppressive therapy are presented. DR antigen in 27 normal kidneys and in 18 of 19 kidneys transplanted as autograft showed no DR-antigen expression on their tubular cells. These DR-antigen-negative tubular cells could be induced to express the antigen during courses of untreated rejection both in primary (n = 6) and repeat (n = 4) allografts. Parallel to the increasing DR-antigen expression in the allograft, the autologous kidney expressed this antigen with the same time dependency and with the same intensity. Graft infiltrating cells were evaluated by daily fine-needle aspiration biopsy and core biopsy. Induction of DR antigen was associated with a significant infiltration of blastogenic cells and monocytes/macrophages in the allograft. During rejection of the allograft no cellular infiltrate was noted in the autograft, but during an initial time period after allograft removal cellular infiltrates were seen. Following both courses of rejection DR antigen returns to negative by 6-8 days as did blastogenic cells and monocytes/macrophages in the autograft interstitium. Inducible antigen expression in normally DR-antigen-negative allograft parenchymal cells during rejection is shown to depend on inflammatory cells in the graft. The antigen expression is not restricted to the tissue transplanted, but also affects autologous tissue of the host organism.     

Renal organogenesis from transplanted metanephric primordia

One novel solution to the shortage of human organs available for transplantation envisions growing new organs in situ via xenotransplantation of developing primordia from animal embryos. It has been shown that renal primordia (metanephroi) transplanted into animal hosts undergo organogenesis in situ, become vascularized by blood vessels of host origin, and exhibit excretory function. Metanephroi can be stored for up to 3 d in vitro before transplantation with no impairment in growth or function post-implantation. Metanephroi can be transplanted across both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. This is a review of studies exploring the therapeutic potential for renal organogenesis posttransplantation of kidney primordia.     

Endothelial cell chimerism after renal transplantation in a rat model

BACKGROUND: Endothelial chimerism, that is, the replacement of damaged donor endothelium by recipient precursors, has been proposed to reduce the allogeneic stimulus and graft rejection. However, both its mechanism and consequence are poorly understood. In this study, we set up a rat model of renal transplantation to investigate the phenomenon of endothelial chimerism and the relationship among the factors involved in its induction, such as preischemic injury, severity of graft rejections, and the role of cyclosporine A toxicity. METHODS: Female Lewis rats received renal transplants from male Brown Norway kidneys. Groups were divided according to ischemia or nonischemia of the donor kidney and cyclosporine treatment or nonimmunosuppression. To investigate the endothelial chimerism, an in situ hybridization by an X-chromosome-specific DNA probe was performed. The severity of allograft rejection was scored according to the Banff '97 classification. RESULTS: In grafts without preischemic injury or without cyclosporine A treatment, a low degree of endothelial chimerism was detected, although severe vascular rejection and tissue necrosis developed. More chimerism was found in recipients receiving an ischemic kidney followed by immunosuppressive treatment, although less severe rejection developed. In recipients receiving an ischemic kidney without cyclosporine A treatment, the highest degree of endothelial chimerism occurred. CONCLUSIONS: Endothelial chimerism demonstrated in rats after renal transplantation may be caused by endothelial damage induced by vascular rejection or ischemia. Ischemia had the strongest association with the induction of chimerism, which may function as a synergistic promoter. A low-dose cyclosporine A treatment was shown to inhibit endothelial replacement.     

The use of double renal transplants from paediatric cadaver donors.

In 224 consecutive kidney transplantations, 3 patients, aged 44, 37 and 13 years received double renal transplants from paediatric cadaver donors aged 1 day, 23 and 19 months respectively. Both adult recipients developed severe hypertension during postoperative rejection crises leading to multiple graft ruptures in the recipient of the neonatal kidneys. 1 patient died from massive gastrointestinal bleeding 4 weeks after transplantation while kidney function was satisfactory. Excellent graft function was achieved in the 3rd patient. The techniques of removal, preservation and transplantation of double renal transplants are described. The significance of increasing the potential number of donor kidneys by the use of paediatric cadaver donors is stressed.     

Long-term outcome of ABO-incompatible renal transplantation.

Based on the long-term experience with ABO-incompatible kidney transplantation, the following can be concluded: 1. Renal transplantation across ABO incompatibility is an acceptable treatment for patients with end-stage renal failure. [table: see text] 2. Long-term patient and graft survival in ABO-incompatible kidney transplantation is influenced primarily by acute rejection episodes occurring within 1 year. 3. Despite the removal of anti-ABO natural antibodies before transplantation, hyperacute rejection crises may occur in some cases. 4. Humoral rejection is the most prominent type of rejection in ABO-incompatible renal transplantation. Even though most of this rejection is controllable with anti-rejection therapy, the prognosis for a graft that undergoes humoral rejection is significantly poor. 5. The maximum IgG titers of anti-A/B antibody before transplantation may have a harmful effect on graft acceptance in ABO-incompatible kidney transplantation. 6. Renal transplantation across ABO incompatibility is principally the most significant risk factor to affect long-term allograft function in ABO-incompatible living kidney transplantation.     

Technology insight: Innovative options for end-stage renal disease--from kidney refurbishment to artificial kidney

The steadily growing number of patients with chronic kidney disease who will eventually develop end-stage renal disease, together with the qualitative limitations of currently available renal replacement therapies, have triggered the exploration of innovative strategies for renal replacement therapy and for salvage of renal function. Currently, new hemodialysis modalities and membranes are being used with the aim of increasing clearance of uremic toxins to afford better metabolic control. In addition to these conventional approaches, there are four innovative potential solutions to the problem of replacing renal function when kidneys fail. The first is a small, implantable device with the potential to be supplemented with human cells ('artificial kidney'). The second involves restoration of the damaged kidney by harnessing recent advances in stem-cell technology and knowledge of developmental programing ('refurbished kidney'). The third is (partially) growing a kidney in vitro with the use of therapeutic cloning ('cultured kidney'). The fourth innovative solution involves the use of other organs to replace various renal functions ('distributed kidney'). In this article we review the efforts that have been made to improve renal replacement therapies, and explore innovative approaches. We will not cover all potential solutions in detail. Rather, we aim to indicate directions of future endeavor and arouse enthusiasm in clinicians and scientists for exploration of these exciting avenues.     

Organogenesis of kidneys following transplantation of renal progenitor cells

One novel solution to the shortage of human organs available for transplantation envisions 'growing' new organs in situ via xenotransplantation of developing anlagen from animal embryos. We and others have shown that renal progenitor cells (metanephroi) transplanted into animal hosts undergo organogenesis (differentiation and growth), become vascularized by blood vessels of host origin and exhibit excretory function. Metanephroi can be stored for up to 3 days in vitro prior to transplantation with no impairment in growth or function post-implantation. Metanephroi can be transplanted across both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. Here we review studies exploring the potential therapeutic use of embryonic kidney transplantation as a means to achieve renal organogenesis.     

Chronic rejection in renal transplantation

With renal transplantation, chronic rejection is currently the most prevalent cause of late transplant failure. Clinically, chronic rejection presents as chronic transplant dysfunction, characterized by a slow loss of function, often in combination with hypertension and proteinuria. Transplant glomerulopathy and multilayering of the peritubular capillaries are highly characteristic for chronic rejection. Risk factors have been identified and include young recipient age, black race, presensitization, histoincompatibility, and acute rejection episodes, especially vascular rejection episodes and rejections that occur late after transplantation. Chronic rejection develops in grafts that undergo intermittent or persistent damage from cellular and humoral immune responses resulting from indirect recognition of alloantigens. Progression factors such as advanced donor age, renal dysfunction, hypertension, proteinuria, hyperlipidemia, and smoking play an important role. At the tissue level, senescence conditioned by ischemia/reperfusion may contribute to the development of chronic rejection. The most effective option to prevent renal failure from chronic rejection is to avoid graft injury from both immune and nonimmune mechanism together with nonnephrotoxic maintenance immunosuppression.     

Bioartificial kidney for full renal replacement therapy

The rapid understanding of the cellular and molecular basis of organ function and disease processes will be translated in the next millennium into new therapeutic approaches to a wide range of clinical disorders, including acute and chronic renal failure. Central to these new therapies are the developing fields of gene therapy, cell therapy, and tissue engineering. These new technologies are based on the ability to expand stem or progenitor cells in tissue culture to perform differentiated tasks and to introduce these cells into the patient either in extracorporeal circuits or as implantable constructs. Cell therapy devices are currently being developed to replace the filtrative, metabolic, and endocrinologic functions of the kidney lost in both acute and chronic renal failure. This article summarizes the current state of device development for a renal tubule assist device, a bioartificial hemofilter, and a regulatable erythropoietin cell therapy device. These individual devices have the promise to be combined to produce a wearable or implantable bioartificial kidney for full renal replacement therapy. These new approaches may result in therapeutic modalities that significantly diminish the morbidity and mortality in patients with acute renal failure or end-stage renal disease.     

Bilateral native nephrectomy improves renal isograft function in rats

Bilateral native nephrectomy has been suggested to improve renal allograft survival in man. This effect may be most prominent in patients experiencing acute tubular necrosis following transplantation. Thus, native kidneys may alter the course of ischemic acute tubular necrosis in the transplanted kidney. In the present studies, we utilized an experimental model of syngeneic transplantation in which rejection does not occur. We studied Lewis rat renal isografts transplanted into littermates following sham, unilateral or bilateral native nephrectomy. In a fourth group of rats, we evaluated the importance of native kidney excretory function by studying isografts transplanted into littermates with bilaterally obstructed native kidneys. Renal blood flow and excretory function were measured in vivo, eight days following transplantation. Renal excretory function of isografts transplanted into animals following bilateral native nephrectomy was similar to normal nontransplanted Lewis kidneys. The presence of either one or both functioning native kidneys significantly reduced isograft inulin clearance, PAH clearance, and blood flow. However, when isografts were transplanted into Lewis rats with bilaterally obstructed native kidneys, renal isograft inulin clearance and blood flow were not significantly impaired. Nontransplanted kidneys demonstrated "functional hypertrophy" following contralateral nephrectomy, with glomerular filtration rate and renal blood flow increasing by approximately 50%. In contrast, isograft glomerular filtration rate in animals following bilateral native nephrectomy was equivalent to that of single kidneys from normal animals with both kidneys in situ. However, renal blood flow of isografts from these animals increased to the same level as nontransplanted Lewis kidneys following contralateral nephrectomy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute Humoral Rejection in an ABO Compatible Combined Liver–Kidney Transplant—The Kidney Is Not Always Protected

Combined liver–kidney transplantation has become a common practice for the treatment of patients with concurrent end-stage renal disease and end-stage liver disease. Liver transplantation in the setting of multiorgan transplantation is thought to have a protective effect against humoral rejection even when a positive crossmatch is obtained prior to surgery. In most centers, a pre liver–kidney transplant crossmatch is rarely performed because of the known immunoprotective effect of the liver allograft. In this report, a case of acute humoral rejection in the kidney allograft after a combined liver–kidney transplant is described. Although humoral rejection was treated using plasmapheresis, intravenous immunoglobulin and rituximab, the kidney required 3 months to recover function and finally progressed to chronic allograft nephropathy. A heightened index of suspicion for acute humoral rejection of the renal allograft is necessary when performing combined liver–kidney transplants to highly sensitized patients due to previous organ transplants.     

Stem cells and progenitor cells in renal disease

Stem cells and progenitor cells are necessary for repair and regeneration of injured renal tissue. Infiltrating or resident stem cells can contribute to the replacement of lost or damaged tissue. However, the regulation of circulating progenitor cells is not well understood. We have analyzed the effects of erythropoietin on circulating progenitor cells and found that low levels of erythropoietin induce mobilization and differentiation of endothelial progenitor cells. In an animal model of 5/6 nephrectomy we could demonstrate that erythropoietin ameliorates tissue injury. Full regeneration of renal tissue demands the existence of stem cells and an adequate local "milieu," a so-called stem cell niche. We have previously described a stem cell niche in the kidneys of the dogfish, Squalus acanthus. Further analysis revealed that in the regenerating zone of the shark kidney, stem cells exist that can be induced by loss of renal tissue to form new glomeruli. Such animal models improve our understanding of stem cell behavior in the kidney and may eventually contribute to novel therapies.     

Pathologic characteristics of transplanted kidney xenografts

For xenotransplantation to become a clinical reality, we need to better understand the mechanisms of graft rejection or acceptance. We examined pathologic changes in α1,3-galactosyltransferase gene-knockout pig kidneys transplanted into baboons that were treated with a protocol designed to induce immunotolerance through thymic transplantation (n=4) or were treated with long-term immunosuppressants (n=3). Hyperacute rejection did not occur in α1,3-galactosyltransferase gene-knockout kidney xenografts. By 34 days, acute humoral rejection led to xenograft loss in all three xenografts in the long-term immunosuppression group. The failing grafts exhibited thrombotic microangiopathic glomerulopathy with multiple platelet-fibrin microthrombi, focal interstitial hemorrhage, and acute cellular xenograft rejection. Damaged glomeruli showed IgM, IgG, C4d, and C5b-9 deposition. They also demonstrated endothelial cell death, diffuse endothelial procoagulant activation with high expression of tissue factor and vWF, and low expression of the ectonucleotidase CD39. In contrast, in the immunotolerance group, two of four grafts had normal graft function and no pathologic findings of acute or chronic rejection at 56 and 83 days. One of the remaining kidneys had mild but transient graft dysfunction with reversible, mild microangiopathic glomerulopathy, probably associated with preformed antibodies. The other kidney in the immunotolerance group developed unstable graft function at 81 days and developed chronic xenograft glomerulopathy. In summary, the success of pig-to-primate xenotransplantation may necessitate immune tolerance to inhibit acute humoral and cellular xenograft rejection.     

Transplantation of metanephroi to sites within the abdominal cavity

A novel approach to circumventing the shortage in transplantable donor organs is the use of embryonic primordia that develop inside the host. Previously published work has shown that transplantation of rat fetal kidney primordia (metanephroi) onto the omentum of adult rat hosts results in growth and development of the metanephroi into functioning kidney units capable of providing a measurable renal function. However, for anatomical and physiological reasons the omentum may not provide the ideal site for transplantation and may limit the maximum renal function that the transplants can achieve. We postulate that it may be possible to increase the renal function of the transplants by transplantation to sites with increased blood flow. To test this we transplanted rat embryonic day 15 metanephroi into the retroperitoneal fat adjacent to major blood vessels in the peritoneum of unilaterally nephrectomized rats; 21 days later the transplants were examined and suitable transplants connected to the host urinary system. Approximately 130 days later the glomerular filtration rate of the connected transplants was analyzed. Our results show that transplantation of metanephroi to the regions highlighted in this study results in an increased presence of urinary cysts, suggesting increased early renal function in the transplants compared to metanephroi transplanted onto the omentum, but most importantly we show that we can increase the renal function of the transplants to a level comparable with other renal therapies such as dialysis. This work suggests life-sustaining renal function could be achieved through transplantation of renal primordia.     

Transplantation of renal precursor cells: a new therapeutic approach

The number of kidney transplantations performed per year is limited due to availability of donor organs. One possible solution to the organ shortage is the use of renal xenografts. However, the transplantation of xenografts is complicated by hyperacute and acute rejection. It has been postulated that the host immune response might be attenuated following the transplantation of renal precursor cells or embryonic kidneys (metanephroi) instead of developed (adult) kidneys. Transplanted metanephroi become chimeric organs in that their blood supply originates, at least in part, from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one rat to another. Transplanted metanephroi grow, develop, become vascularized, and function in host rats. Transplantation of metanephroi may be a promising novel therapeutic approach for the treatment of chronic renal failure. Received: 29 December 1999 / Revised: 13 March 2000 / Accepted: 13 March 2000