Human and porcine early kidney precursors as a new source for transplantation

Kidney transplantation has been one of the major medical advances of the past 30 years. However, tissue availability remains a major obstacle. This can potentially be overcome by the use of undifferentiated or partially developed kidney precursor cells derived from early embryos and fetal tissue. Here, transplantation in mice reveals the earliest gestational time point at which kidney precursor cells, of both human and pig origin, differentiate into functional nephrons and not into other, non-renal professional cell types. Moreover, successful organogenesis is achieved when using the early kidney precursors, but not later-gestation kidneys. The formed, miniature kidneys are functional as evidenced by the dilute urine they produce. In addition, decreased immunogenicity of the transplants of early human and pig kidney precursors compared with adult kidney transplants is demonstrated in vivo. Our data pinpoint a window of human and pig kidney organogenesis that may be optimal for transplantation in humans.     

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Should We Formulate an Incentivized Model Facilitating Kidney Donation from Living Donors? A Focus on Turkey's Current System

Kidney transplantation is a lifesaving medical treatment. However, very high demand for kidneys with low kidney donation causes a black market that exploits patients’ desperation and donors’ vulnerability. The current kidney donation programs fail to produce promising results to avoid illegal and unethical kidney trafficking and commercialism. Even though the primary goal of kidney donation is to increase the number of deceased organ donations, in some countries, like Turkey, due to religious or cultural concerns, it is impossible to supply adequate deceased kidney donations. In this view, the aim of this paper is to examine kidney trafficking in the scope of Turkey's current organ donation system and propose a new model, named the Incentivized Kidney Donation Model (IKDM), to increase kidney donation from living donors. The model encompasses the following benefits offered to kidney donors; lifetime health insurance, exemptions from copayments/contribution shares, priority when receiving an organ, priority when finding a job, income tax exemptions for salaried employees, and free or discounted public utilities. This normative model has the potential to promote donors’ altruistic acts as well as the solidarity and loyalty among members of a society without violating ethical values and internationally accepted principles.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Availability of Cadaveric Kidneys for Transplantation

The availability of cadaveric donor kidneys for transplantation has been estimated from a retrospective survey of hospital deaths in Wales during 1969. Seventy-six “probable donors” and 167 “possible donors” were identified, indicating that the potential supply of donor kidneys is sufficient to meet the present demands of patients needing kidney transplantation.     

Intravenous renal cell transplantation for rats with acute and chronic renal failure

Acute kidney injury (AKI) and chronic renal failure (CKD) are the most challenging problems in nephrology. Multiple therapies have been attempted but these interventions have minimal effects on the eventual outcomes, and all too often the result is end-stage renal disease (ESRD). The only effective therapy for ESRD is renal transplantation but only a small fraction of patients receive transplants. In this work we introduce a novel approach to transplantation designed to regenerate kidneys afflicted by severe AKI or CKD: intravenous renal cell transplantation (IRCT) with adult rat primary renal cells reprogrammed to express the SAA gene localized and engrafted in kidneys of rat recipients that had severe AKI or CKD. IRCT significantly resolved renal dysfunction and limited kidney damage, inflammation, and fibrosis. Severe CKD was successfully improved by IRCT using kidney cells from donor rats or by renal cell self-donation in a form of autotransplantation. We propose that IRCT with adult primary renal cells reprogrammed to express the SAA gene can be used to effectively treat AKI and CKD.     

Current status of renal transplantation.

The success rate of renal transplantation has improved considerably during the past decade, with substantial improvements in both graft and patient survival. The quality of graft function, however, and not graft survival alone is increasingly determining the standards by which transplantation outcome is being judged. As the demand for kidney transplants continues to rise and transplants are being offered to an ever-increasing number of patients, organs are being sought from new supply pools and efforts are being made to use current resources more efficiently. Improvements in clinical management have allowed short-term complications such as infection and rejection to be better prevented or better diagnosed and treated. Fundamental advances in the understanding of the immunologic processes underlying both allograft rejection and acceptance and the introduction of new immunosuppressive agents have allowed a better use of drug therapy and have moved the goal of acquired transplant tolerance closer to attainment. With improved initial transplant success rates, the long-term transplantation outcome is becoming more important. The role of tissue matching in preventing chronic rejection is becoming more appreciated, and the long-term risks of malignancy, arteriosclerosis, and chronic rejection are being better recognized and managed.     

Selecting the optimal cell for kidney regeneration: fetal, adult or reprogrammed stem cells

Chronic kidney disease (CKD) is a progressive loss in renal function over a period of months or years. End-stage renal disease (ESRD) or stage 5 CKD ensues when renal function deteriorates to under 15% of the normal range. ESRD requires either dialysis or, preferentially, a kidney organ allograft, which is severely limited due to organ shortage for transplantation. To combat this situation, one needs to either increase supply of organs or decrease their demand. Two strategies therefore exist: for those that have completely lost their kidney function (ESRD), we will need to supply new kidneys. Taking into account the kidneys' extremely complex structure, this may prove to be impossible in the near future. In contrast, for those patients that are in the slow progression route from CKD to ESRD but still have functional kidneys, we might be able to halt progression by introducing stem cell therapy to diseased kidneys to rejuvenate or regenerate individual cell types. Multiple cell compartments that fall into three categories are likely to be worthy targets for cell repair: vessels, stroma (interstitium) and nephron epithelia. Different stem/progenitor cells can be linked to regeneration of specific cell types; hematopoietic progenitors and hemangioblastic cell types have specific effects on the vascular niche (vasculogenesis and angiogenesis). Multipotent stromal cells (MSC), whether derived from the bone marrow or isolated from the kidney's non-tubular compartment, may, in turn, heal nephron epithelia via paracrine mechanisms. Nevertheless, as we now know that all of the above lack nephrogenic potential, we should continue our quest to derive genuine nephron (epithelial) progenitors from differentiated pluripotent stem cells, from fetal and adult kidneys and from directly reprogrammed somatic cells.     

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases¹. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease². Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells³. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.     

Kidney transplantation in elderly patients

The Eurotransplant Senior Program (ESP) allocates kidneys from elderly donors to elderly recipients (> or = 65 years old). During the last 39 years, 922 kidney transplantations were performed in our transplant center. We retrospectively analysed patients included in the ESP from the our center. Eleven patients > or = 65 years old recieved kidney from donors 65 years old. Cold ischemia time was approximately 15 hours. Dual kidney transplantation was performed in one patient. Appropriate immunosuppressive protocol was given to all patients. Surgical complications were relatively common and included dissection of renal artery (1 patient), thrombosis of renal artery (1 patient), ureterovesical obstruction (1), lymphocele (1), bleeding (1), acute abdomen (2) and wound dehiscence (1). One rejection episode was registered. Delayed graft function was observed in the two patients with full recovery of kidney function. Seven patients until now have good functioning graft. Four kidneys were lost. One patient died because of pneumonia. Kidney transplantation in elderly is feasible procedure but with greater number of complications than usually.     

Experience of a Canadian multi-organ transplant service.

Organ transplantation has become the treatment of choice for selected patients with end-stage failure of the heart, liver or kidneys. The expanding role for organ transplantation, however, has led to a corresponding increase in the complexity of patient management. In response to these changes, University Hospital, London, Ont., has established an interdisciplinary multi-organ transplant service (MOTS). MOTS coordinates donor organ procurement and patient management. Donor organs have been retrieved from as far south as Dalton, Georgia, as far west as Calgary and as far east as Halifax. As of Dec. 31, 1985, 485 transplants had been performed, including 387 kidney transplants, 51 heart transplants, 3 heart/lung transplants, 43 liver transplants (in adults and children) and 1 pancreas transplant. With current immunosuppressive protocols MOTS projects 1-year patient survival rates of 95% after kidney transplantation, 88% after heart transplantation and 81% after liver transplantation. Patient rehabilitation has been excellent.     

The use of a supercooling refrigerator improves the preservation of organ grafts

Current medical transplantation confronts major problems such as the shortage of donors and geographical restrictions that inhibit efficient utilization of finite donor organs within their storage lives. To overcome these issues, expanding organ preservation time has become a major concern. We investigated whether a strategy which best preserves organ grafts can be achieved by the use of a newly developed refrigerating chamber, which is capable of establishing a supercooled and unfrozen state stably by generating an electrostatic field in its inside. When adult rat organs such as heart, liver, and kidneys were stored in the supercooled conditions, the levels of major biochemical markers leaked from the preserved organs were significantly lower than in the ordinary hypothermic storage. No apparent tissue damages were observed histologically after the supercooled preservation. Our results suggest that the use of this supercooling refrigerator improves organ preservation and may provide an innovative technique for human organ transplantation.     

IFN-gamma alters the pathology of graft rejection: protection from early necrosis

We studied the effect of host IFN-gamma on the pathology of acute rejection of vascularized mouse heart and kidney allografts. Organs from CBA donors (H-2k) were transplanted into BALB/c (H-2d) hosts with wild-type (WT) or disrupted (GKO, BALB/c mice with disrupted IFN-gamma genes) IFN-gamma genes. In WT hosts, rejecting hearts and kidneys showed mononuclear cell infiltration, intense induction of donor MHC products, but little parenchymal necrosis at day 7. Rejecting allografts in GKO recipients showed infiltrate but little or no induction of donor MHC and developed extensive necrosis despite patent large vessels. The necrosis was immunologically mediated, since it developed during rejection, was absent in isografts, and was prevented by immunosuppressing the recipient with cyclosporine or mycophenolate mofetil. Rejecting kidneys in GKO hosts showed increased mRNA for heme oxygenase 1, and decreased mRNA for NO synthase 2 and monokine inducible by IFN-gamma (MIG). The mRNA levels for CTL genes (perforin, granzyme B, and Fas ligand) were similar in rejecting kidneys in WT and GKO hosts, and the host Ab responses were similar. The administration of recombinant IFN-gamma to GKO hosts reduced but did not fully prevent the effects of IFN-gamma deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-gamma has unique effects in vascularized allografts, including induction of MHC and MIG, and protection against parenchymal necrosis, probably at the level of the microcirculation. This is probably a local action of IFN-gamma produced in large quantities in the allograft.     

Comparative survival and economic benefits of deceased donor kidney transplantation and dialysis in people with varying ages and co-morbidities

Background

Deceased donor kidneys for transplantation are in most countries allocated preferentially to recipients who have limited co-morbidities. Little is known about the incremental health and economic gain from transplanting those with co-morbidities compared to remaining on dialysis. The aim of our study is to estimate the average and incremental survival benefits and health care costs of listing and transplantation compared to dialysis among individuals with varying co-morbidities.

Methods

A probabilistic Markov model was constructed, using current outcomes for patients with defined co-morbidities treated with either dialysis or transplantation, to compare the health and economic benefits of listing and transplantation with dialysis.

Findings

Using the current waiting time for deceased donor transplantation, transplanting a potential recipient, with or without co-morbidities achieves survival gains of between 6 months and more than three life years compared to remaining on dialysis, with an average incremental cost-effectiveness ratio (ICER) of less than $50,000/LYS, even among those with advanced age. Age at listing and the waiting time for transplantation are the most influential variables within the model. If there were an unlimited supply of organs and no waiting time, transplanting the younger and healthier individuals saves the most number of life years and is cost-saving, whereas transplanting the middle-age to older patients still achieves substantial incremental gains in life expectancy compared to being on dialysis.

Conclusions

Our modelled analyses suggest transplanting the younger and healthier individuals with end-stage kidney disease maximises survival gains and saves money. Listing and transplanting those with considerable co-morbidities is also cost-effective and achieves substantial survival gains compared with the dialysis alternative. Preferentially excluding the older and sicker individuals cannot be justified on utilitarian grounds.     

Donor derived malignancy following transplantation: a review

Organ and tissue transplant is now the treatment of choice for many end stage diseases. In the recent years, there has been an increasing demand for organs but not a similar increase in the supply leading to a severe shortage of organs for transplant resulted in increasing wait times for recipients. This has resulted in expanded donor criteria to include older donors and donors with mild disease. In spite of implementation of more stringent criteria for donor selection, there continues to be some risk of donor derived malignancy. Malignancy after transplantation can occur in three different ways: (a) de-novo occurrence, (b) recurrence of malignancy, and (c) donor-related malignancy. Donor related malignancy can be either due to direct transmission of tumor or due to tumor arising in cells of donor origin. We will review donor related malignancies following solid organ transplantation and hematopoeitic progenitor cell transplantation. Further, we will briefly review the methods for detection and management of these donor related malignancies.     

Prolongation of Life in Anephric Rats Following De Novo Renal Organogenesis

One solution to the shortage of human organs available for transplantation envisions growing new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. Allotransplantation of embryonic day 15 metanephroi into the omentum of adult hosts is followed by differentiation, growth, vascularization and function of the implants. Here we show that survival of rats with all native renal mass removed can be increased by prior metanephros transplantation and ureteroureterostomy. Excretion of urine formed by metanephroi is prerequisite for enhanced survival. This is the first demonstration that life can be extended following de novo renal organogenesis.Key Words: cell therapy, end-stage renal disease, kidney, metanephros, transplantation     

In Vivo imaging of differences in early donor cell proliferation in graft-versus-host disease hosts with different pre-conditioning doses

Graft-versus-host disease (GVHD) results from immunemediated attacks on recipient tissues by donor-originated cells through the recognition of incompatible antigens expressed on host cells. The pre-conditioning irradiation dose is a risk factor influencing GVHD severity. In this study, using newly generated luciferase transgenic mice on a B6 background (B6.Luc^Tg) as bone marrow and splenocyte donors, we explored the effects of irradiation doses on donor cell dynamics in major histocompatibility complex (MHC)-matched allogeneic GVHD hosts via bioluminescence imaging (BLI). Results from BLI of GVHD hosts showed higher emission intensities of luminescence signals from hosts irradiated with 900 cGy as compared with those irradiated with 400 cGy. In particular, BLI signals from target organs, such as the spleen, liver, and lung, and several different lymph nodes fluctuated with similar time kinetics soon after transplantation, reflecting the synchronous proliferation of donor cells in the different organs in hosts irradiated with 900 cGy. The kinetic curves of the BLI signals were not synchronized between the target organs and the secondary organs in hosts irradiated with 400 cGy. These results demonstrate that pre-conditioning doses influence the kinetics and degree of proliferation in the target organs soon after transplantation. The results from this study are the first describing donor cell dynamics in MHC-matched allogeneic GVHD hosts and the influence of irradiation doses on proliferation dynamics, and will provide spatiotemporal information to help understand GVHD pathophysiology.     

Organogenesis forum lecture

Renal replacement therapy (i.e., kidney transplantation) represents the optimal treatment for end-stage renal disease (a condition which is expected to increase over the next decade or so). However, the demand for transplantable kidneys currently outpaces the availability of donor kidneys, a situation not expected to improve in the foreseeable future. An alternative route to cadaveric or living-related donors would be to engineer kidneys for allograft transplantation from cells based on concepts derived from our current understanding of normal kidney development. Although the use of cells for this purpose remains hypothetical, recent research from our laboratory has provided strong evidence that implantation of kidney-like tissue bio-engineered from the recombination of in vitro culture systems which model discrete aspects of kidney development (i.e., cell culture, isolated WD, isolated UB, and isolated MM) is possible. These recent findings are discussed here. Pathway based system biology approaches to understanding the mechanism(s) of kidney development are also discussed, particularly in the setting of this novel and seemingly powerful xeno-based tissue engineering strategy.     

Contribution of renal secreted complement C3 to the circulating pool in humans

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.