Sharon A. Rogers

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.

Transplantation of pig metanephroi.

To determine whether pig metanephroi grow and differentiate after allotransplantation or xenotransplantation across a highly disparate barrier, we implanted metanephroi from embryonic day 28 (E28) pig embryos into the omentum of unilaterally nephrectomized adult pigs or C57Bl/6J mice (hosts). Some mouse hosts received anti-CD45RB, anti-CD154, and anti-CD11a (costimulatory blockade). E28 pig metanephroi were <0.2 mm in diameter and contained only metanephric blastema and segments of ureteric bud. Pig metanephroi transplanted into pigs underwent growth and differentiation of nephrons over a 2 week period without the need for costimulatory blockade of hosts. In contrast, pig metanephroi did not grow or differentiate in mice that received no costimulatory blockade. However, by 2 weeks posttransplantation in mice in which costimulation was blocked, metanephroi from E28 pigs had enlarged, become vascularized, and had formed mature tubules and glomeruli. By 3 weeks posttransplantation in mice, metanephroi had grown to the point that they were approximately half the volume of the native mouse kidney. Here we show that growth and development of pig metanephroi occurs posttransplantation across an allogeneic or highly disparate xenogeneic barrier.

Gelatinase B (MMP-9) Is Not Essential in the Normal Kidney and Does Not Influence Progression of Renal Disease in a Mouse Model of Alport Syndrome

Matrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB-/- and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB-/- embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred with Col4a3-/- mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome.

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.

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

Abstract:  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.

Growth factors and kidney development

The formation of the metanephric kidney is dependent upon the timed and sequential expression of a number of polypeptide growth factors. To shed light on the participation of members of the insulin-like growth factor (IGF) and epidermal growth factor/transforming growth factor-α (EGF/TGF-α) families, we measured the synthesis of IGF-I, IGF-II, EGF and TGF-α by developing rat metanephroi in organ culture and determined the effect of anti-growth factor antibodies on growth and development. IGF-I, IGF-II and TGF-α were produced by metanephroi and released into culture media. We could detect no EGF. Inclusion of anti-IGF-I, anti-IGF-II, anti-IGF-II receptor or anti-TGF-α antibodies in organ cultures inhibited growth and development of metanephroi. Our findings suggest that both members of the IGF family and TGF-α are produced within the developing metanephros and promote renal organogenesis.

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.

Engraftment of Cells from Porcine Islets of Langerhans and Normalization of Glucose Tolerance Following Transplantation of Pig Pancreatic Primordia in Nonimmune-Suppressed Diabetic Rats

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. However, even if toxicity can be minimalized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets may be a strategy to overcome these supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] can obviate the need for immune suppression in rats or rhesus macaques. Here, in rats transplanted previously with E28 pig pancreatic primordia in the mesentery, we show normalization of glucose tolerance in nonimmune-suppressed streptozotocin-diabetic LEW rats and insulin and porcine proinsulin mRNA-expressing cell engraftment in the kidney following implantation of porcine islets beneath the renal capsule. Donor cell engraftment was confirmed using fluorescent in situ hybridization for the porcine X chromosome and electron microscopy. In contrast, cells from islets did not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in the mesentery. This is the first report of prolonged engraftment and sustained normalization of glucose tolerance following transplantation of porcine islets in nonimmune-suppressed, immune-competent rodents. The data are consistent with tolerance induction to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.

Normalization of glucose post-transplantation into diabetic rats of pig pancreatic primordia preserved in vitro.

Embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of non-immunosuppresed steptozotocin (STZ)-diabetic Lewis rats normalize levels of circulating glucose within 2-4 weeks. Exocrine tissue does not differentiate after transplantation of pancreatic primordia. Rather individual endocrine (beta) cells engraft within the mesentery. To determine whether transplanted pig pancreatic primordia engraft, differentiate, and function in rat hosts after preservation in vitro, we implanted pig pancreatic primordia into STZ-diabetic rats either directly or after 24 hours of suspension in ice-cold University of Wisconsin (UW) preservation solution with added growth factors. Here we show engraftment in mesentery and mesenteric lymph nodes and normalization of glucose levels in STZ-diabetic rat hosts following transplantation of preserved E28 pig pancreatic primordia comparable to glucose normalization after transplantation of non-preserved E28 pancreatic primordia.