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Acceptance of the 2001 Jean Hamburger Award
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Acceptance of the 2001 Jean Hamburger Award ANITA APERIA It is a great honor for me, a Swedish pediatrician, to be a recipient of the prestigious Jean Hamburger Award. I received my first research training at the Department of Pathology, Yale University. At that time. I was still a medical student. My mentor was Averill A. Liebow, a legendary lung pathologist, who was extremely knowledgeable in physiology. I learned from Dr. Liebow the rewards of hard work, the importance of combining morphologic and physiologic observation, and the need to address clinical questions with both patient-based and experimental work. My work at Yale concerned renal hemodynamics and oxygen consumption. I returned to Sweden and graduated from the Karolinska Institutet Medical University. A residency in pediatrics was a natural choice for me. Pediatrics is an extremely attractive specialty; its standards, both with regard to clinical practice and research, have always been very high in Sweden. Two clinical problems almost immediately caught my attention: (1) fluid and electrolyte disturbances in preterm and sick full-term babies and (2) the severe consequences of urinary tract infection in young infants. Clinical studies In the early 1970s, neonatologists made great progress in the handling of respiratory problems and the survival rate of infants born 8 to 12 weeks before term. Fluid and electrolyte disturbances were a major problem among those premature babies, indicating to me that the capacity of the kidney to regulate salt and water balance was far from mature. In a series of studies, we were able to confirm this hypothesis. During the course of these studies, I came to realize that little was known about the cellular pathways that regulate salt and water transport in the renal tubules. In 1970, many pediatric nephrologists, myself included, were studying the natural history of recurrent urinary tract infection in children. It became obvious that infants and young children were very susceptible to renal damage, even following a short course of untreated pyelonephritis, and that the resulting renal scarring was mainly located in the cortex of the kidney. The mechanisms by which this ascending infection caused scarring of cortical nephrons remained unknown. Experimental studies After 10 years of mainly patient-based studies, I realized that it was impossible to fully address my two major questions: “Which cellular pathways regulate tubular so-
dium transport and do they undergo postnatal maturation?” and “How does urinary tract infection cause scarring in the renal cortex?” without applying cell biologic methods. I decided to extend our small micropuncture laboratory and several postdoctorate students joined the group. From then on, those two questions have been addressed mainly with experimental studies, jointly named “Signal and Defense Systems in Renal Tubular Cells.” Intracellular signaling and regulation of sodium-potassium-ATPase. To study the mechanisms by which sodium-transporting proteins could be regulated, I turned first to sodium-potasssium-ATPase, the enzyme that is located in the basolateral membrane of every tubular cell. Sodium-potassium-ATPase is responsible for the active transport of sodium out of the cell. In 1985, it was generally believed that sodium-potassium-ATPase was a mechanical pump and that its activity was dependent only on the availability of it main ligands, Na⫹,K⫹, and adenosine triphosphate (ATP). By using tubular segments dissected from rat kidney, we showed that the activity of renal tubular sodium-potassium-ATPase was down-regulated by locally produced dopamine [1]. This was the first demonstration of a short-term hormonal regulation of sodium, potassium-ATPase, and it brought me into contact with Paul Greengard, the leading authority in the neurobiology of dopamine signaling. This was the beginning of a very fruitful collaboration, one in which we could demonstrate for the first time that sodium-potassium-ATPase is regulated by reversible phosphorylation [2–3], and that is could be phosphorylated by protein kinase A and C. By site-directed mutagenesis studies, we were able to demonstrate the functional importance of these phosphorylation sites [4]. By using comparative studies in neurons and nephrons, new information about the highly complex dopamine signaling pathways [5] was obtained. Intracellular signaling, receptor translocation. My work regarding dopamine regulation of sodium-potassiumATPase led me to the conclusion that dopamine plays a key role in the integrative regulation of sodium excretion [6]. This finding generated interest in what regulates the renal dopamine tonus. At least three factors have emerged as being important: (1) the availability of the dopamine precursor, l-dopa, which is taken up in the proximal tubular cells; (2) the activity of the dopamine metabolizing enzyme, catechol-O-methyl transferase; and (3) the availability of the dopamine receptors in the plasma membrane. We and others observed that, in most cells that express the dopamine type 1 receptor, the re-
2338 ceptor was, to a large extent, located intracellularly. This prompted us to study whether there is a regulated recruitment of dopamine type 1 receptor. Using new imaging techniques, we found that this was the case, and that dopamine type 1 receptor could be translocated to the plasma membrane both by dopamine and by atrial natriuretic factor (ANF) [7]. These studies represent novel principles for homologous and heterologous receptor sensitization and provide an explanation for the wellknown phenomenon that the natriuretic effects of ANF are attenuated in the presence of dopamine type 1 receptor antagonists. Calcium oscillations Calcium oscillation is perhaps the most versatile of all signaling systems because the cell can decode the frequency of the oscillations. The response will be very different depending on whether the frequency is in the millisecond, second, or minute range. These calcium oscillations occur as interplay between the inositol-1,4,5 triphosphate (IP3) receptor, the voltage-gated calcium channels, and the store-operated calcium channels. During the last 3 years, studies of this signaling system have been a main theme in my laboratory. We have identified two “unexpected” inducers of slow calcium oscillations that are both actually connected with my initial two research questions. Calcium oscillations induced by hemolysin Three years ago, I decided to revisit the question of why urinary tract infection in young children causes scarring of cortical nephrons. By that time, we had acquired the tools to study the interaction between Escherichia coli and proximal tubular cells. Together with a group of microbiologists, we were able to demonstrate that hemolysin excreted from uropathogenic E. coli induced calcium oscillations in proximal tubular cells in a constant, 12-minute periodicity. IP3 receptors and l-type calcium channels were both involved in this response. The oscillations resulted in the release of interleukin-6 and interleukin-8, the cytokines that are known to increase during a course of urinary tract infection with E. coli [8]. Are the calcium oscillations good or bad? We do not know yet, but we hope that future studies will show whether they will be a defense mechanism for the host, or a way by which the bacteria can cause a more favorable environment.
Calcium oscillations induced by ouabain Several recent studies have suggested that sodiumpotassium-ATPase is a multifunctional protein and that it may also act as a signal transducer. Recent studies have also convincingly shown that ouabain is an endogenous steroid hormone formed in the adrenals and the hypothalamus. The physiologic role of endogenous ouabain has been controversial. We have examined the possibility that ouabain-bound sodium-potassium-ATPase uses calcium as a signaling pathway. We found that, in rat renal proximal tubular cells, ouabain that gives only a partial inhibition of sodium-potassium-ATPase results in calcium oscillations of a very constant, 5-minute periodicity [9]. These oscillations resulted in activation of the transcriptional factors nuclear factor-kappa B (NF-B) and cyclic adenosine monophospate (cAMP)-responsive element binding protein (CREB). The downstream effect of calcium oscillation on gene activation, as well as the molecular mechanisms underlying the oscillation, are now under investigation. Scientific work is a continuous learning process. I want to thank all former and present members of my laboratory, as well as my collaborators all over the world, for having made my scientific life so rich. REFERENCES 1. Aperia A, Bertorello A, Seri I: Dopamine causes inhibition of Na⫹,K⫹ATPase activity in rat proximal convoluted tubule segments. Am J Physiol 252:F39–F45, 1987 2. Aperia A, Holtba¨ck U, Syre´n M-L, et al: Activation/deactivation of renal Na⫹,K⫹- ATPase: A final common pathway for regulation of natriuresis. FASEB J 8:436–439, 1994 3. Logvinenko NS, Dulubova I, Fedosova N, et al: Phosphorylation by protein kinase C of serine-23 of the ␣-1 subunit of rat Na⫹,K⫹ATPase affects its conformational equilibrium. Proc Natl Acad Sci USA 93:9132–9137, 1996 4. Belusa R, Wang Z, Matsubara T, et al: Mutation of the site of protein kinase C phosphorylation on rat ␣1 Na⫹,K⫹- ATPase alters regulation of intracellular Na⫹, pH and influences cell shape and adhesiveness. J Biol Chem 272:20179–20184, 1997 5. Aizman O, Brismar H, Uhle´n P, et al: Anatomical and physiological evidence for colocalization of neostriatal D1 and D2 dopamine receptors. Nat Neurosci 3:226–230, 2000 6. Aperia A: Intrarenal dopamine: A key signal in the interactive regulation of sodium metabolism. Annu Rev Physiol 62:621–647, 2000 7. Holtba¨ck U, Brismar H, Dibona GF, et al: Receptor recruitment: A mechanism for interactions between G protein-coupled receptors. Proc Natl Acad Sci USA 63:7271–7275, 1999 ˚ , Jahnukainen T, et al: ␣-Hemolysin of 8. Uhle´n P, Laestadius A uropathogenic E. coli induces Ca2⫹ oscillations in renal epithelial cells. Nature 405:694–697, 2000 9. Aizman O, Uhle´n P, Lal M, et al: Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc Natl Acad Sci USA 98:13420–13424, 2001
Acceptance of the 2001 Jean Hamburger Award ANITA APERIA It is a great honor for me, a Swedish pediatrician, to be a recipient of the prestigious Jean Hamburger Award. I received my first research training at the Department of Pathology, Yale University. At that time. I was still a medical student. My mentor was Averill A. Liebow, a legendary lung pathologist, who was extremely knowledgeable in physiology. I learned from Dr. Liebow the rewards of hard work, the importance of combining morphologic and physiologic observation, and the need to address clinical questions with both patient-based and experimental work. My work at Yale concerned renal hemodynamics and oxygen consumption. I returned to Sweden and graduated from the Karolinska Institutet Medical University. A residency in pediatrics was a natural choice for me. Pediatrics is an extremely attractive specialty; its standards, both with regard to clinical practice and research, have always been very high in Sweden. Two clinical problems almost immediately caught my attention: (1) fluid and electrolyte disturbances in preterm and sick full-term babies and (2) the severe consequences of urinary tract infection in young infants. Clinical studies In the early 1970s, neonatologists made great progress in the handling of respiratory problems and the survival rate of infants born 8 to 12 weeks before term. Fluid and electrolyte disturbances were a major problem among those premature babies, indicating to me that the capacity of the kidney to regulate salt and water balance was far from mature. In a series of studies, we were able to confirm this hypothesis. During the course of these studies, I came to realize that little was known about the cellular pathways that regulate salt and water transport in the renal tubules. In 1970, many pediatric nephrologists, myself included, were studying the natural history of recurrent urinary tract infection in children. It became obvious that infants and young children were very susceptible to renal damage, even following a short course of untreated pyelonephritis, and that the resulting renal scarring was mainly located in the cortex of the kidney. The mechanisms by which this ascending infection caused scarring of cortical nephrons remained unknown. Experimental studies After 10 years of mainly patient-based studies, I realized that it was impossible to fully address my two major questions: “Which cellular pathways regulate tubular so-
dium transport and do they undergo postnatal maturation?” and “How does urinary tract infection cause scarring in the renal cortex?” without applying cell biologic methods. I decided to extend our small micropuncture laboratory and several postdoctorate students joined the group. From then on, those two questions have been addressed mainly with experimental studies, jointly named “Signal and Defense Systems in Renal Tubular Cells.” Intracellular signaling and regulation of sodium-potassium-ATPase. To study the mechanisms by which sodium-transporting proteins could be regulated, I turned first to sodium-potasssium-ATPase, the enzyme that is located in the basolateral membrane of every tubular cell. Sodium-potassium-ATPase is responsible for the active transport of sodium out of the cell. In 1985, it was generally believed that sodium-potassium-ATPase was a mechanical pump and that its activity was dependent only on the availability of it main ligands, Na⫹,K⫹, and adenosine triphosphate (ATP). By using tubular segments dissected from rat kidney, we showed that the activity of renal tubular sodium-potassium-ATPase was down-regulated by locally produced dopamine [1]. This was the first demonstration of a short-term hormonal regulation of sodium, potassium-ATPase, and it brought me into contact with Paul Greengard, the leading authority in the neurobiology of dopamine signaling. This was the beginning of a very fruitful collaboration, one in which we could demonstrate for the first time that sodium-potassium-ATPase is regulated by reversible phosphorylation [2–3], and that is could be phosphorylated by protein kinase A and C. By site-directed mutagenesis studies, we were able to demonstrate the functional importance of these phosphorylation sites [4]. By using comparative studies in neurons and nephrons, new information about the highly complex dopamine signaling pathways [5] was obtained. Intracellular signaling, receptor translocation. My work regarding dopamine regulation of sodium-potassiumATPase led me to the conclusion that dopamine plays a key role in the integrative regulation of sodium excretion [6]. This finding generated interest in what regulates the renal dopamine tonus. At least three factors have emerged as being important: (1) the availability of the dopamine precursor, l-dopa, which is taken up in the proximal tubular cells; (2) the activity of the dopamine metabolizing enzyme, catechol-O-methyl transferase; and (3) the availability of the dopamine receptors in the plasma membrane. We and others observed that, in most cells that express the dopamine type 1 receptor, the re-
2338 ceptor was, to a large extent, located intracellularly. This prompted us to study whether there is a regulated recruitment of dopamine type 1 receptor. Using new imaging techniques, we found that this was the case, and that dopamine type 1 receptor could be translocated to the plasma membrane both by dopamine and by atrial natriuretic factor (ANF) [7]. These studies represent novel principles for homologous and heterologous receptor sensitization and provide an explanation for the wellknown phenomenon that the natriuretic effects of ANF are attenuated in the presence of dopamine type 1 receptor antagonists. Calcium oscillations Calcium oscillation is perhaps the most versatile of all signaling systems because the cell can decode the frequency of the oscillations. The response will be very different depending on whether the frequency is in the millisecond, second, or minute range. These calcium oscillations occur as interplay between the inositol-1,4,5 triphosphate (IP3) receptor, the voltage-gated calcium channels, and the store-operated calcium channels. During the last 3 years, studies of this signaling system have been a main theme in my laboratory. We have identified two “unexpected” inducers of slow calcium oscillations that are both actually connected with my initial two research questions. Calcium oscillations induced by hemolysin Three years ago, I decided to revisit the question of why urinary tract infection in young children causes scarring of cortical nephrons. By that time, we had acquired the tools to study the interaction between Escherichia coli and proximal tubular cells. Together with a group of microbiologists, we were able to demonstrate that hemolysin excreted from uropathogenic E. coli induced calcium oscillations in proximal tubular cells in a constant, 12-minute periodicity. IP3 receptors and l-type calcium channels were both involved in this response. The oscillations resulted in the release of interleukin-6 and interleukin-8, the cytokines that are known to increase during a course of urinary tract infection with E. coli [8]. Are the calcium oscillations good or bad? We do not know yet, but we hope that future studies will show whether they will be a defense mechanism for the host, or a way by which the bacteria can cause a more favorable environment.
Calcium oscillations induced by ouabain Several recent studies have suggested that sodiumpotassium-ATPase is a multifunctional protein and that it may also act as a signal transducer. Recent studies have also convincingly shown that ouabain is an endogenous steroid hormone formed in the adrenals and the hypothalamus. The physiologic role of endogenous ouabain has been controversial. We have examined the possibility that ouabain-bound sodium-potassium-ATPase uses calcium as a signaling pathway. We found that, in rat renal proximal tubular cells, ouabain that gives only a partial inhibition of sodium-potassium-ATPase results in calcium oscillations of a very constant, 5-minute periodicity [9]. These oscillations resulted in activation of the transcriptional factors nuclear factor-kappa B (NF-B) and cyclic adenosine monophospate (cAMP)-responsive element binding protein (CREB). The downstream effect of calcium oscillation on gene activation, as well as the molecular mechanisms underlying the oscillation, are now under investigation. Scientific work is a continuous learning process. I want to thank all former and present members of my laboratory, as well as my collaborators all over the world, for having made my scientific life so rich. REFERENCES 1. Aperia A, Bertorello A, Seri I: Dopamine causes inhibition of Na⫹,K⫹ATPase activity in rat proximal convoluted tubule segments. Am J Physiol 252:F39–F45, 1987 2. Aperia A, Holtba¨ck U, Syre´n M-L, et al: Activation/deactivation of renal Na⫹,K⫹- ATPase: A final common pathway for regulation of natriuresis. FASEB J 8:436–439, 1994 3. Logvinenko NS, Dulubova I, Fedosova N, et al: Phosphorylation by protein kinase C of serine-23 of the ␣-1 subunit of rat Na⫹,K⫹ATPase affects its conformational equilibrium. Proc Natl Acad Sci USA 93:9132–9137, 1996 4. Belusa R, Wang Z, Matsubara T, et al: Mutation of the site of protein kinase C phosphorylation on rat ␣1 Na⫹,K⫹- ATPase alters regulation of intracellular Na⫹, pH and influences cell shape and adhesiveness. J Biol Chem 272:20179–20184, 1997 5. Aizman O, Brismar H, Uhle´n P, et al: Anatomical and physiological evidence for colocalization of neostriatal D1 and D2 dopamine receptors. Nat Neurosci 3:226–230, 2000 6. Aperia A: Intrarenal dopamine: A key signal in the interactive regulation of sodium metabolism. Annu Rev Physiol 62:621–647, 2000 7. Holtba¨ck U, Brismar H, Dibona GF, et al: Receptor recruitment: A mechanism for interactions between G protein-coupled receptors. Proc Natl Acad Sci USA 63:7271–7275, 1999 ˚ , Jahnukainen T, et al: ␣-Hemolysin of 8. Uhle´n P, Laestadius A uropathogenic E. coli induces Ca2⫹ oscillations in renal epithelial cells. Nature 405:694–697, 2000 9. Aizman O, Uhle´n P, Lal M, et al: Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc Natl Acad Sci USA 98:13420–13424, 2001