Marc R. Hammerman

Regulation of canine renal vesicle Pi transport by growth hormone and parathyroid hormone.

Renal phosphate (Pi) reabsorption is increased by growth hormone (GH) and decreased by parathyroid hormone (PTH). Na+-stimulated Pi transport across the brush border membrane of the proximal tubule is the initial step in the process of Pi reabsorption. To determine whether changes in Pi reabsorption induced by GH or PTH are accompanied by changes in brush border membrane Na+-gradient-stimulated Pi transport, we examined the effect of in vivo GH and PTH administration and thyroparathyroidectomy on Pi transport by isolated brush border membrane vesicles prepared from canine kidney. In experiments in which the effect of PTH administration was examined, the same animal provided the control kidney (before PTH administration) and the experimental kidney (after PTH administration). The Na+-gradient Pi overshoot in vesicles isolated from normal, GH-treated and thyroparathyroidectomized dogs was increased after in vivo PTH administration. GH administration and thyroparathyroidectomy increased the height of the overshoot compared to normal. PTH administration decreased the apparent V value by 44% in vesicles from normal animals. The apparent V value was increased, compared to normal, by GH (34%) and thyroparathyroidectomy (57%). PTH administration decreased the apparent V in both the latter groups. GH administration to thyroparathyroidectomized dogs further increased the apparent V. Changes in the apparent V paralleled changes in Pi reabsorption in vivo induced by experimental manipulations. We conclude that changes in renal Pi reabsorption induced by GH were like those induced by PTH, accompanied by changes in the Na+-stimulated Pi transport system in the renal brush border membrane, and that the effect of PTH on vesicular Pi transport in GH-treated dogs did not differ from the effect on vesicles from normal animals.

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.

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

The renal expression of transforming growth factor-β1 (TGF-β1) is enhanced following induction of ischemic injury in rat. In cultured renal cells, TGF-β stimulates the synthesis of extracellular matrix. To link TGF-β1 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), α1(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. α1(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 α1(IV) collagen immunoreactivity was detected in thickened tubulointerstitial spaces. In contrast, TIMP-1 immunoreactivity was enhanced in distal nephron structures postischemia. Immunoneutralization of TGF-β in vivo attenuated the increases in FN-EIIIA, α1(IV) collagen, PAI-1, and TIMP-1 mRNAs by 52%, 73%, 43%, and 27%, respectively. These data are consistent with TGF-β expression postischemic injury participating in renal regeneration of extracellular matrix homeostasis in the proximal TBM.

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.

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 (IGP-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.

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.

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.

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 μg/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 μg ⋅ 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.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.

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na+-independent L-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of L-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with L-arginine, but not D-arginine, indicated that the uptake of L-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of L-arginine was demonstrated in vesicles preincubated with L-lysine and L-ornithine, but not L-alanine or L-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of L-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. L-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na+-independent uptake of L-alanine, L-glutamine, glycine or L-proline in the presence of extravesicular amino acids of similar structure. L-Arginine uptake was increased by the imposition of an H+ gradient (intravesicular pH less than extravesicular pH) and H+ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na+-independent transport of L-arginine in brush border membrane vesicles which differs from Na+-independent uptake of neutral and acidic amino acids. Na+-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na+-independent transport of dibasic amino acids across the renal brush border membrane.