Long-term engraftment following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic rhesus macaques.

BACKGROUND: Transplantation therapy for human diabetes is limited by a shortage of donor organs, and transplant function diminished over time by cell death and limited potential for expansion of beta cells in pancreas or islets. Outcomes are complicated by immunosuppression. A way to overcome supply and expansion problems is to xenotransplant embryonic tissue. Previously, we have shown that beta cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin (STZ)-diabetic (type 1) Lewis rats or Zucker Diabetic Fatty (ZDF) diabetic (type 2) rats engraft and normalize glucose tolerance without the need for host immune-suppression. METHODS: In this study, we transplant E28 pig pancreatic primordia in the mesentery of STZ-diabetic rhesus macaques. RESULTS: Long-term engraftment of pig beta cells within liver, pancreas and mesenteric lymph nodes post-transplantation of E28 pig pancreatic primordia into STZ-diabetic rhesus macaques is demonstrated by electron microscopy, positive immune-histochemistry for insulin, and positive RT-PCR and in situ hybridization for porcine proinsulin mRNA. Insulin requirements were reduced in one macaque followed over 22 months post-transplantation and porcine insulin detected in plasma using sequential affinity chromatography, HPLC and mass spectrometry. Of potential importance for application of this transplantation technology to treatment of diabetes in humans and confirmatory of our previous findings in Lewis and ZDF rats, no host immunosuppression is required. CONCLUSIONS: Under selected circumstances, pancreatic primordia elicit a muted immune response relative to more differentiated tissue, such that engraftment occurs in non-immunosuppressed hosts. Our findings that pig pancreatic primordia engraft long-term in non-immunosuppressed STZ-diabetic rhesus macaques establishes the potential for their use in human diabetics.

Transplantation of metanephroi after preservation in vitro.

To determine whether transplanted metanephroi grow, differentiate, and function in hosts after preservation in vitro, we implanted metanephroi from embryonic day 15 (E15) Sprague-Dawley rat embryos into the omentum of nonimmunosuppressed uninephrectomized Sprague-Dawley (host) rats. Metanephroi were either implanted directly or suspended in ice-cold University of Wisconsin (UW) preservation solution with or without added growth factors for 3 days before implantation. The size and extent of tissue differentiation preimplantation of E15 metanephroi implanted directly were not distinguishable from the size and differentiation of metanephroi preserved for 3 days. In contrast, E16 metanephroi were larger than E15 metanephroi preserved for 3 days. E16 metanephroi or E13 metanephroi grown in organ culture for 3 days contained more differentiated nephron structures than those in E15 metanephroi preserved for 3 days. By 4 wk posttransplantation, metanephroi that had been preserved for 3 days had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts with normal structure had developed. At 12 wk posttransplantation, inulin clearances of preserved metanephroi were comparable to those of metanephroi that had been implanted directly. Addition of growth factors to the UW solution enhanced inulin clearances. Here we show for the first time that functional kidneys develop from metanephroi transplanted from rat embryos to adult rats after as long as 3 days of preservation in vitro.

Transplantation of rat metanephroi into mice.

To determine whether transplanted metanephroi grow and differentiate after implantation into the omentum in hosts of a different species, we implanted metanephroi from embryonic day 15 (E15) rat embryos into uninephrectomized mice (hosts). Some host mice received human CTLA4Ig (hCTLA4Ig), anti-CD45RB, and anti-CD154 (tolerance-inducing agents). E15 metanephroi contained only metanephric blastema, segments of ureteric bud, and primitive nephrons with no glomeruli. Rat metanephroi did not grow or differentiate in mice that received no tolerance-inducing agents. However, by 2 wk posttransplantation in mice that received hCTLA4Ig, anti-CD45RB, and anti-CD154, metanephroi from E15 rats had enlarged, become vascularized, and formed mature tubules and glomeruli. Rat metanephroi contained cells that stained specifically for mouse CD31, a marker for sprouting endothelial cells. Some rat glomerular capillary loops stained positively for mouse CD31. Here, we show that chimeric kidneys develop from metanephroi transplanted rat-->mouse and that glomeruli are vascularized, at least in part, by host vessels.

Growing kidneys.

The number of kidney transplantations performed per year is restricted by the limited availability of donor organs. One possible solution to this shortage is the use of renal xenografts. However, the transplantation of xenografts is complicated by hyperacute and acute rejection. A second possible solution is to 'grow a kidney' from a transplanted renal anlage. It has been postulated that the host immune response might be attenuated after the transplantation of such an anlage (metanephros) instead of a developed kidney. Transplanted metanephroi become chimeric organs in that their blood supply originates, at least partly, from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one rat to another. Transplanted metanephroi grow, differentiate, become vascularized, and function in host rats. 'Growing kidneys' via the transplantation of metanephroi may hold promise as a novel therapeutic approach to the treatment of chronic renal failure.

Transplantation of metanephroi across the major histocompatibility complex in rats.

To determine whether transplanted metanephroi grow, differentiate, and function in hosts that differ in major histocompatibility complex loci (RT1 loci in rats) from donors in a defined way, we implanted metanephroi from embryonic day (E) 15 PVG (RT1(c)) rat embryos into the omentum of nonimmunosupressed uninephrectomized PVG-RT1(avl) (host) rats. By 4 wk posttransplantation, metanephroi had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts had normal structure and ultrastructure. At 12 wk posttransplantation, weights of metanephroi were 54 +/- 8 mg. Inulin clearances were 0.9 +/- 0.3 microl. min(-1). 100 g rat wt(-1). In vitro, splenocytes from PVG rats stimulated the proliferation of cells originating from both PVG-RT1(avl) rats in which a transplant had been performed and PVG-RT1(avl) rats with no transplant. Full-thickness PVG-RT1(avl) skin engrafted normally on PVG-RT1(avl) rats in which PVG metanephroi had been previously implanted and metanephroi retained a normal appearance. In contrast, skin from PVG rats sloughed, and the tubular architecture of metanephroi was obliterated by a mononuclear cell infiltrate consistent with acute rejection. Here we show for the first time that functional chimeric kidneys develop from metanephroi transplanted across the MHC into nonimmunosupressed hosts and provide evidence that a state of peripheral immune tolerance secondary to T cell "ignorance" permits their survival.

Inhibition of poly(ADP-ribose) polymerase attenuates ischemic renal injury in rats.

The enzyme, poly(ADP-ribose) polymerase (PARP), effects repair of DNA after ischemia-reperfusion (I/R) injury to cells in nerve and muscle tissue. However, its activation in severely damaged cells can lead to ATP depletion and death. We show that PARP expression is enhanced in damaged renal proximal tubules beginning at 6-12 h after I/R injury. Intraperitoneal administration of PARP inhibitors, benzamide or 3-amino benzamide, after I/R injury accelerates the recovery of normal renal function, as assessed by monitoring the levels of plasma creatinine and blood urea nitrogen during 6 days postischemia. PARP inhibition leads to increased cell proliferation at 1 day postinjury as assessed by proliferating cell nuclear antigen and improves the histopathological appearance of kidneys examined at 7 days postinjury. Furthermore, inhibition of PARP increases levels of ATP measured at 24 h postischemia compared with those in vehicle-treated animals. Our data indicate that PARP activation is a part of the cascade of molecular events that occurs after I/R injury in the kidney. Although caution is advised, transient inhibition of PARP postischemia may constitute a novel therapy for acute renal failure.

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.

Expression of CD44 in kidney after acute ischemic injury in rats.

De novo CD44 and ligand expression at wound margins accompanies cellular proliferation and migration that effect repair of injured mucosal and vascular endothelial tissues. To determine whether CD44 could play a role in recovery from acute ischemic renal injury, we characterized its renal expression and those of two of its ligands, hyaluronic acid and osteopontin. Although no expression is detectable in nonischemic kidneys, several mRNAs for CD44 are present within 1 day after injury. CD44 mRNA is expressed in proximal tubules undergoing repair. CD44 peptide is present in basal and lateral cell membranes. Hyaluronic acid is normally expressed in the interstitium of the renal papilla only. By 1 day postischemia, hyaluronic acid can be detected, in addition, in the interstitium surrounding regenerating tubules. Osteopontin, not normally expressed in the renal proximal tubule, is expressed in regenerating tubules by 3 days after induction of acute ischemic injury. Immunoreactive osteopontin peptide continues to be localized in those tubules still undergoing repair for as long as 7 days after the injury. Our data are consistent with a role for CD44-ligand interactions in the regenerating proximal tubule participating in the process of recovery after ischemic injury.

The growth hormone-insulin-like growth factor axis in kidney re-revisited.

The use of renal allotransplantation to treat ESRD in the US is limited by lack of organ availability. A possible solution is the transplantation of developing kidneys (metanephric allograft or xenografts). We have conducted studies that demonstrate the feasibility of such a strategy and have shown that IGF I may be useful to accelerate the growth and development of these transplanted organs. The rationale for the use of IGF I in this setting grew from a basic understanding of the role that the growth factor plays in kidney development. ARF in humans is the most costly kidney-related disease requiring hospitalization. Its incidence is increasing. Despite many advances in dialytic therapy, the mortality rate for patients with ARF has not changed in the last several decades. Strategies for treatment of ARF are directed toward supportive care to permit renal regeneration to occur. There exists a need for new therapeutic approaches that can speed recovery and reduce mortality. Although IGF I may not prove to be the 'magic bullet' for ARF, its proposal and testing as a potential therapeutic agent has provided a paradigm for the development of treatment modalities to accelerate renal regeneration based upon a basic understanding of the injury/repair process. The basis for development of a 'growth factor' therapy for ARF will probably evolve, at least in part, out of the testing and use of IGF I in rat models and in humans. The use of GH to treat ESRD was proposed shortly after its isolation and the demonstration of its action in increasing the rate of glomerular filtration. Later, it was discovered that the actions of GH on kidney are mediated by IGF I, and the means by which IGF I enhances glomerular filtration was elucidated. We have shown that humans with ESRD are not resistant to the actions of IGF I in enhancing the GFR, establishing the potential for use of IGF I as a pharmacological agent for ESRD. There is no effective drug therapy to enhance renal function in ESRD. Although much work remains to be done, and clearly caution is advised, our observations establish the potential for the use of IGF I as a therapeutic agent in this setting and justify continued study of IGF I as a medical therapy to delay the need for dialysis.

Insulin-like growth factor I regulates renal development in rodents.

Blocking the action of insulin-like growth factor I (IGF I) impairs kidney development in vitro. However, no renal developmental abnormalities have been reported in newborn transgenic mice that do not express IGF I (Igf1-/-) mice. Ninety-five percent of Igf1-/- mice die immediately following birth. Kidney development continues following birth in rodents. To readdress the question of the participation of IGF I in the process of kidney development, we measured nephron numbers in developed kidneys from Igf1-/- mice that survived past birth, and using a second model of kidney development, characterized the effect of IGF I infused into rat hosts on the renal function of transplanted metanephroi. Igf1-/- mice were born with grossly normal kidneys. At 77 +/- 10 days after birth, Igf1-/- mice that survived were approximately 28% the weight of wild-type (WT) littermates and had proportionally smaller kidneys. The number of nephrons per kidney was reduced by approximately 20% in Igf1-/- mice. Glomerular size was also reduced in Igf1-/- mice. In untreated host rats, neither the size nor inulin clearance of transplanted metanephroi changed significantly from 12-28 weeks postimplantation. The administration of IGF I to hosts did not affect the size of transplanted metanephroi measured at 12-16 weeks following implantation. However, inulin clearances were increased significantly by the administration of IGF I to hosts. Our findings 1) indicate that IGF I plays a role in determining nephron number, 2) suggest that it enhances function in developing kidneys, and 3) establish the potential for the pharmacological use of IGF I to enhance the growth and function of transplanted metanephroi.

Insulin-like growth factor I improves renal function in patients with end-stage chronic renal failure.

There is no pharmacological treatment to increase the glomerular filtration rate in end-stage renal disease (ESRD). The administration of 100 microgram/kg of insulin-like growth factor (IGF) I twice a day to patients with ESRD increases inulin clearance. However, its effect is short-lived and IGF-I has major side effects when given this way. To assess whether the use of a lower intermittent dose of IGF-I would effect sustained improved function with tolerable side effects we performed 1) a prospective open-labeled 24-day trial in which we enrolled five patients and 2) a 31-day randomized, double-blinded, placebo-controlled trial in which we enrolled 10 patients. Patients with ESRD [creatinine clearance of <15 ml. min-1. (1.73 m2)-1] and scheduled to initiate renal replacement therapy received subcutaneous IGF-I, 50 microgram. kg-1. day-1, or vehicle. Treatment with IGF I resulted in significantly increased glomerular filtration rates (inulin clearances) during the 3rd and 4th wk of therapy in both prospective and double-blinded studies. Vehicle had no effect. No patient required discontinuation of drug secondary to side effects. We conclude that IGF-I effects sustained improvement of renal function (clearances comparable to those generally achieved by dialysis) in patients with ESRD and is well tolerated.

Distinguished Scientists Lecture Series. New developments in kidney development.

BACKGROUND/AIMS: The number of kidney transplantations performed per year is limited due to the 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 rejection. METHODS: It has been postulated that the host immune response might be attenuated following the transplantation of embryonic kidneys (metanephroi) rather than developed (adult kidneys). Transplanted metanephroi become chimeric organs in that their blood supply originates from the host. It is possible to transplant a developing metanephros, without the use of immunosuppression, from one outbred rat to another. RESULTS: Transplanted metanephroi grow, develop, become vascularized, and function in host rats. In contrast, developed adult kidneys transplanted from one rat to another undergo rejection within 7 days after transplantation. CONCLUSIONS: These observations suggest that metanephric tissue may be less immunogenic than adult kidney. Transplantation of metanephroi represents a new development that could lead to a novel therapeutic approach to the treatment of chronic renal failure.

Expression of CD27 and ischemia/reperfusion-induced expression of its ligand Siva in rat kidneys.

BACKGROUND: Studies identifying genes that are differentially expressed following induction of acute ischemic injury have been useful in delineating the pathophysiology of acute renal failure. METHODS: A differential cDNA library screening technique was used to identify genes that are differentially expressed in rat kidney following induction of acute ischemic renal injury. RESULTS: Levels of mRNA with a high homology to that coding for Siva, a human proapoptotic protein, were increased approximately 4.5-fold in kidneys obtained from rats within 12 hours following ischemia, compared to kidneys from sham-operated rats. A partial cDNA sequence for the rat protein (rat Siva) was determined that overlaps 92% of the human open reading frame. The cDNA sequence predicts a protein 177 amino acids in length with 76% homology to human Siva. Levels of rat Siva in kidneys were elevated at one, five and seven days post-ischemia were not different from those in kidneys from sham-operated controls. In situ hybridization demonstrated that rat Siva mRNA was expressed in cells lining damaged sections in the S3 segment of the proximal tubule at 12 hours and one day post-ischemia. At five and seven days, Siva mRNA was located in epithelial cells of regenerating tubules including in papillary proliferations. TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells colocalized with cells containing Siva mRNA. CD27, the receptor for Siva was localized by immunohistochemistry to sloughed cells in the lumens of damaged S3 segments at 12 hours post-ischemia and to cells within papillary proliferations at five days post-injury. CONCLUSIONS: Siva that is produced within the kidney could be a mediator of apoptosis post-ischemia via an interaction with CD27.

Extracellular matrix-related genes in kidney after ischemic injury: potential role for TGF-beta in repair.

The renal expression of transforming growth factor-beta1 (TGF-beta1) is enhanced following induction of ischemic injury in rat. In cultured renal cells, TGF-beta stimulates the synthesis of extracellular matrix. To link TGF-beta1 expression with the regulation of extracellular matrix postischemia, we characterized the expression of several genes known to regulate extracellular matrix synthesis at various times during recovery from acute ischemic renal injury in rat. Levels of mRNA for plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloprotease-1 (TIMP-1), alpha1(IV) collagen, and fibronectin-EIIIA (FN-EIIIA) mRNAs were significantly enhanced in kidneys within 12 h to 3 days after injury and remained elevated at 7-28 days postischemia relative to levels in kidneys of sham-operated controls. PAI-1 mRNA and peptide were localized in regenerating proximal tubules at 3 and 7 days postischemic injury. alpha1(IV) Collagen and FN-EIIIA mRNAs were expressed primarily in regenerating proximal tubule cells. Immunoreactivity for FN-EIIIA was enhanced in the tubular basement membrane (TBM) of regenerating proximal tubules, and alpha1(IV) collagen immunoreactivity was detected in thickened tubulointerstitial spaces. In contrast, TIMP-1 immunoreactivity was enhanced in distal nephron structures postischemia. Immunoneutralization of TGF-beta in vivo attenuated the increases in FN-EIIIA, alpha1(IV) collagen, PAI-1, and TIMP-1 mRNAs by 52%, 73%, 43%, and 27%, respectively. These data are consistent with TGF-beta expression postischemic injury participating in renal regeneration of extracellular matrix homeostasis in the proximal TBM.

Regulation of cell survival during renal development.

Apoptosis occurs in an orchestrated fashion during kidney development. In contrast, it is a relatively rare event in normal developed (adult) kidney. However, a predictable pattern of apoptosis is observed in adult kidney in two settings, during which parts of the developmental pattern are recapitulated. These are regeneration following acute ischemic injury and the process of cystogenesis in polycystic kidney disease. Apoptosis in kidney is regulated by agents both intrinsic and extrinsic to the renal cell. The protooncogene b-cell lymphoma/leukemia gene product-2 (bcl-2) is an important intrinsic factor. The growth factor epidermal growth factor is an important extrinsic regulator. A thorough understanding of the control of renal apoptosis during development and recovery from ischemic injury and in cystogenesis may lead to new therapies to prevent or ameliorate abnormalities of kidney formation, to enhance regeneration following acute ischemic injury, and to slow the progression of renal failure in polycystic kidney disease.

Growth factors and apoptosis in acute renal injury.

The precisely orchestrated pattern of growth factor expression within the kidney following acute renal injury indicates that growth factors regulate the process of repair. The use of growth factors as therapeutic agents to accelerate renal regeneration in this setting stems from this observation. In animal models of acute renal injury, administration of epidermal growth factor (EGF), insulin-like growth factor I (IGF-I) or hepatocyte growth factor (HGF) accelerates restoration of kidney function and normalization of histology post-acute renal injury and reduces mortality. IGF-I has been safely administered to humans and protects against post-surgical renal dysfunction. Renal cellular apoptosis occurs in a predictable pattern during recovery from acute ischemic injury. Renal apoptosis is regulated by agents both intrinsic and extrinsic to the kidney cell. The protooncogene, B-cell lymphoma/leukemia gene product-2 (bcl-2), is an important intrinsic factor. The growth factor, EGF, is an important extrinsic regulator. A thorough understanding of the control of renal apoptosis during recovery from ischemic injury coupled with an increased understanding of the roles that growth factors play in this process, is likely to result in the development of new therapies to enhance kidney regeneration.

Transplantation of developing metanephroi into adult rats.

BACKGROUND: Transplantation of developing metanephroi into adult hosts has been proposed as a means to augment host renal function. METHODS: We implanted whole metanephroi from embryonic day 15 (E15) rats subcapsularly in kidneys or into the omentum of non-immunosupressed adult rat hosts. At the time of implantation, some host rats underwent unilateral nephrectomy (UNX) or unilateral nephrectomy and partial contralateral renal infarction (1 1/2 NX). E15 metanephroi contained only metanephric blastema, segments of ureteric bud, and primitive nephrons with no glomeruli. RESULTS: Four to six weeks post-implantation, metanephroi from E15 rats had enlarged, become vascularized, and had formed mature tubules and glomeruli. Ureters of metanephroi transplanted into the omentum were anastomosed to hosts' ureters that remained after UNX. Four weeks following ureteroureterostomy, the contralateral kidney was removed. Inulin clearances of seven metanephroi implanted into UNX hosts averaged 0.11 +/- 0.02 microliters/min/100 g (2.42 +/- 0.70 microliters/min/g kidney wt) and the creatinine clearances averaged 0.65 +/- 0.18 microliters/min/100 g. Metanephroi weighed 71 +/- 15 mg (approximately 4% of the contralateral native kidney). The transplanted metanephroi were vascularized by arteries originating from the omentum. Both weights of transplanted metanephroi (145 +/- 24 mg) and inulin clearances of transplanted metanephroi (30.1 +/- 8.7 microliters/min/g kidney weight) were significantly increased in rats that underwent 1 1/2 NX compared to UNX. In contrast, transplantation of developed kidneys resulted in rejection. CONCLUSIONS: Our findings establish that functional chimeric kidneys develop from metanephroi transplanted in adult hosts.