SPECIAL COMMENTARY J Am Soc Nephrol 15: 1093–1095, 2004

Peter Agre, 2003 Nobel Prize Winner in Chemistry

MARK A. KNEPPER,* and SOREN NIELSEN† National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland; and †Water and Salt Research Center, University of Aarhus, Aarhus, Denmark.

Abstract. Peter C. Agre, an American Society of Nephrology somes after the incorporation of the purified protein. These member, is the recipient of the 2003 Nobel Prize in Chemistry findings sparked a veritable explosion of work that affects for his discovery of the aquaporin water channels. The function several long-standing areas of investigation such as the bio- of many cells requires that water move rapidly into and out of physics of water permeation across cell membranes, the struc- them. There was only indirect evidence that proteinaceous tural biology of integral membrane proteins, the physiology of channels provide this vital activity until Agre and colleagues fluid transport in the kidney and other organs, and the patho- purified aquaporin-1 from human erythrocytes and reported its physiological basis of inherited and acquired disorders of water cDNA sequence. They proved that aquaporin-1 is a specific balance. Agre’s discovery of the first water channel has water channel by cRNA expression studies in Xenopus oocytes spurred a revolution in animal and plant physiology and in and by functional reconstitution of transport activity in lipo- medicine.

American Society of Nephrology member, Peter Agre of ing duct, revealed that the increase in water permeability was Johns Hopkins University, received the 2003 Nobel Prize in associated with the appearance of membrane particle aggre- Chemistry for his discovery of the aquaporins, a family of gates in the apical plasma membrane. On the basis of the water channels that carry water across cell membranes in a mosaic theory of membranes, which postulated that biologic broad variety of animal and plant tissues. Since Agre’s discov- membranes are made up of proteins floating in a sea of lipid, ery of the prototypical aquaporin, now called aquaporin-1, it appeared that the particle aggregates were likely to be pro- studies by renal researchers have demonstrated that aquaporins teins and were viewed as containing putative water channels. play a central role in water reabsorption by the kidney and its With the advent of the molecular biology era in the late regulation and have implicated aquaporins in the pathophysi- 1980s and early 1990s, during which more and more cDNAs ology of several water balance disorders (1). The purpose of coding for physiologically important proteins were being this short article is to tell the story of the discovery of aqua- cloned, a number of laboratories were focused on discovering porin-1 and of the revolution in nephrology research that this the genes that code for water channels. It is interesting that the discovery sparked. success of Peter Agre in discovering the prototype water chan- Before Agre’s work, the existence of proteinaceous water nel came out of a project with an entirely different aim, the channels that mediate water transport across cell membranes identification of the protein in red cell membranes that contains was strongly suspected on the basis of studies in red blood cells the epitope responsible for Rh immunoreactivity of red cells and toad urinary bladders. Although artificial lipid membranes (4). Because of the high abundance of this contaminant, which had previously been found to be moderately permeable to could be readily purified to homogeneity, Denker et al. (5) water, studies of water permeation through the plasma mem- were spurred to characterize the contaminant biochemically. branes of red blood cells revealed exceptionally high values An antibody made by injecting rabbits with the purified holo- (2). Studies by Macey and Farmer (3) revealed that the high protein revealed that the protein was expressed not only in red water permeability of red blood cell membranes (later found to cells, but also in the kidney proximal tubule and descending contain aquaporin-1) were highly sensitive to mercurial re- limb. This localization suggested to Agre that the protein could agents, which interact with sulfhydryl groups of the amino acid play some role in transport. Gregory Preston in the Agre cysteine, a component of most proteins. Studies of toad urinary laboratory at Johns Hopkins cloned its cDNA, demonstrating bladders, which show vasopressin-stimulated increases in wa- that the protein was a polytopic integral membrane protein with ter permeability similar to that seen in the mammalian collect- six likely membrane-spanning alpha helices (6). At that time, they referred to the protein as CHIP28, for CHannel-forming Integral Protein of 28 kD. However, its function remained to be Correspondence to Dr. Mark A. Knepper, National Institutes of Health, Bldg. 10, Room 6N260, 10 Center Dr MSC 1603, Bethesda, MD 20892-1603. defined. Phone: 301-496-3064; Fax 301-402-1443; E-mail: [email protected] Agre credits John Parker, a membrane biophysicist at the 1046-6673/1504-1093 University of North Carolina, for the idea that CHIP28 could Journal of the American Society of Nephrology be the red blood cell’s water channel. To test the hypothesis, Copyright © 2004 by the American Society of Nephrology Preston and Agre teamed with Bill Guggino of the Johns DOI: 10.1097/01.ASN.0000118814.47663.7D Hopkins Department of Physiology, another ASN member, to 1094 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 1093–1095, 2004

field rapidly adopted a consistent nomenclature based on the term aquaporin to denote a member of the water channel family followed by a numeral to identify the member (9). In this nomenclature, CHIP28 was designated aquaporin-1 or AQP1. In the remainder of this article, we use this nomenclature even though many of the original papers reporting new water chan- nels did not employ the term aquaporin.

AQP1 Mutations in Humans Agre’s previous work with red blood cell antigens led him to the recognition that the polymorphism responsible for the discrimination of Colton blood groups occurs in the coding sequence of AQP1 (10). He and his colleagues recently pub- lished a paper in the New England Journal of Medicine de- scribing patients that are Colton-null due to mutations in the AQP1 gene (11). These patients fail to express functional AQP1 and fail to concentrate their urine, demonstrating the importance of AQP1 in normal renal function.

Cloning and Characterization of Other Members of the Aquaporin Family Peter Courtland Agre On the basis of the primary structure of the AQP1 gene and its mRNA, investigators worldwide have succeeded in identi- express CHIP28 in Xenopus oocytes. A few years earlier, fying many other aquaporins by homology cloning, many of Ernest Wright, from UCLA, had demonstrated the utility of the demonstrated clinical importance. From the perspective of Xenopus oocyte as an expression system for functional cloning renal medicine, none is more important than aquaporin-2 of cDNAs, and Guggino had imported this method into his (AQP2), the vasopressin-regulated water channel of the col- laboratory. In addition, Guggino had set up a state-of-the-art lecting duct, cloned by Fushimi et al. (12). Deen, van Os, and video microscopy system that facilitated quantification of the colleagues demonstrated that human individuals with muta- swelling rates of oocytes when submersed in hypotonic solu- tions in AQP2 suffer from severe nephrogenic diabetes insip- tions. When Preston injected cRNA made from his CHIP28 idus (NDI), underscoring that aquaporin-2 is essential for uri- cDNA clone into Xenopus oocytes, he found a marked increase nary concentration. Soon after the discovery of AQP2, we were in the rate of osmotic swelling (7). Thus, expression of CHIP28 able to demonstrate that vasopressin regulates collecting duct conveyed a high water permeability to Xenopus oocyte plasma water permeability, and hence body water balance, by regulat- membranes, providing the first direct evidence for the exis- ing trafficking of AQP2-laden vesicles to and from the apical tence of water channel proteins. Nevertheless, it could be plasma membrane (13), validating the shuttle hypothesis of argued that CHIP28 might have been an accessory protein that Wade et al. (14). Further studies identified another mode of activated endogenous water channels native to the oocyte. To regulation of AQP2 that was unanticipated; long-term eleva- confirm that CHIP28 was indeed a water channel itself, Agre tions of circulating vasopressin was shown to markedly in- teamed with Mark Zeidel, another ASN member, to reconsti- crease the abundance of AQP2 in the collecting duct (15,16). tute purified CHIP28 protein into artificial lipid membranes Extensive studies of the pathophysiology of water balance (8). Zeidel measured water permeability in these vesicles using disorders in rats (too extensive to review here) have revealed a a stop-flow light-scattering method that he had set up in his central role of AQP2 dysregulation in many clinical important laboratory and found that vesicles in which CHIP28 was in- syndromes, including congestive heart failure, cirrhosis, ne- corporated manifested a dramatic increase in water permeabil- phrotic syndrome, and acquired NDI due to lithium adminis- ity over vesicles containing only lipid. The water permeability tration (1). As referred to above, mutations in AQP2 have been was demonstrated to be sensitive to mercurials just as seen in also shown to be cause of 20% of the rare heritable NDI with the native red blood cells. Thus, with the incontrovertible autosomal recessive inheritance. Most of the remaining cases demonstration that CHIP28 is a molecular water channel, the are caused by mutations in the vasopressin-V2 receptor, lead- door was opened for other investigators to clone other water ing to inability to regulate AQP2 and hence severe NDI. Thus, channels on the basis of homology (i.e., structural similarity to Peter Agre’s discovery of the aquaporins has led to an under- CHIP28). standing of the pathophysiology of water balance abnormalities that affect millions of patients, and it is leading us toward Nomenclature better therapies for these disorders. To deal with confusion stemming from differing naming Peter Agre and co-workers also cloned an additional mem- systems for water channels offered by various laboratories, the ber of the water channel family, AQP4, which has been found J Am Soc Nephrol 15: 1093–1095, 2004 1095 to be expressed in a polarized fashion in astrocytes in the brain, 4. Agre P, Saboori AM, Asimos A, Smith BL: Purification and on the aspect of the cells that face the vasculature (17). Func- partial characterization of the Mr 30,000 integral membrane tional studies have provided evidence that AQP4 is an impor- protein associated with the erythrocyte Rh(D) antigen. J Biol tant element of the blood brain barrier and may be an important Chem 262: 17497–17503, 1987 molecular target in treatment of post-traumatic brain edema (18). 5. Denker BM, Smith BL, Kuhajda FP, Agre P: Identification, purification, and partial characterization of a novel M 28,000 Aside from progress in the physiology and pathophysiology r integral membrane protein from erythrocytes and renal tubules. of water transport in the animal tissues, the discovery of J Biol Chem 263: 15634–15642, 1988 aquaporins by Agre has spurred important new work in plant 6. Preston GM, Agre P: Isolation of the cDNA for erythrocyte physiology (19). Many aquaporin genes are expressed in dif- integral membrane protein of 28 kilodaltons: Member of an ancient ferent parts of plants and are responsible for water transport in channel family. Proc Natl Acad SciUSA88: 11110–11114, 1991 plant tissues. For example, when a fruit grows, it accumulates 7. Preston GM, Carroll TP, Guggino WP, Agre P: Appearance of large amounts of water that are transported through aquaporins water channels in Xenopus oocytes expressing red cell CHIP28 in the stem. Given the importance of aquaporins in plant protein. Science 256: 385–387, 1992 physiology, it appears likely that plants can be engineered for 8. Zeidel ML, Ambudkar SV, Smith BL, Agre P: Reconstitution of better growth in arid environments through manipulation of functional water channels in liposomes containing purified red aquaporin gene expression. cell CHIP28 protein. Biochemistry 31: 7436–7440, 1992 9. Agre P, Sasaki S, Chrispeels MJ: Aquaporins: A family of water Biochemical Characterization and Structural channel proteins. Am J Physiol 265: F461, 1993 10. Smith BL, Preston GM, Spring FA, Anstee DJ, Agre P: Human Analysis of AQP1 red cell aquaporin CHIP. I. Molecular characterization of ABH Aside from his initial discovery of the aquaporins and the and Colton blood group antigens. J Clin Invest 94: 1043–1049, 1994 cloning of many of the important animal aquaporins, Agre has 11. King LS, Choi M, Fernandez PC, Cartron JP, Agre P: Defective played a central role in understanding the relationship between urinary-concentrating ability due to a complete deficiency of structure and function of the aquaporins. His initial work aquaporin-1. N. Engl J Med 345: 175–179, 2001 demonstrated that AQP1 assemble as homotretramers. By use 12. Fushimi K, Uchida S, Hara Y, Hirata Y, Marumo F, Sasaki S: of a variety of biochemical techniques, studies of recombinants Cloning and expression of apical membrane water channel of rat led Agre and coworkers to predict that AQP1 and homologous kidney collecting tubule. Nature 361: 549–552, 1993 proteins would have a unique fold referred to as the “hour- 13. Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, glass,” an aqueous channel formed by the intersection of Knepper MA: Vasopressin increases water permeability of kid- hemichannels dipping into the bilayer from the extracellular ney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad SciUSA and intracellular surfaces of the lipid bilayer (20). 92: 1013–1017, 1995 The ability of the Agre laboratory to purify human red cell 14. Wade JB, Stetson DL, Lewis SA: ADH action: evidence for a AQP1 protein in milligram amounts made it a candidate for membrane shuttle mechanism. Ann NY Acad Sci 372: 106–117, 1981 structural determination. Collaborating with Andreas Engel 15. DiGiovanni SR, Nielsen S, Christensen EI, Knepper MA: Reg- and his group in Basel, purified aquaporin-1 protein was re- ulation of collecting duct water channel expression by vasopressin constituted into membrane crystals and structure was deter- in Brattleboro rat. Proc Natl Acad SciUSA91: 8984–8988, 1994 mined at 16-Angstrom resolution in a biologically active form 16. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, Harris (21,22). This structure was advanced to 3.8-Angstrom resolu- HW: Cellular and subcellular immunolocalization of vasopres- tion in collaboration with Yoshi Fujiyoshi and his group in sin-regulated water channel in rat kidney. Proc Natl Acad Sci U Kyoto (23). This work confirmed the hourglass model and SA90: 11663–11667, 1993 explained how AQP1 is freely permeated by water molecules 17. Nielsen S, Nagelhus EA, Amiry-Moghaddam M, Bourque C, Agre P, Ottersen OP: Specialized membrane domains for water but is impermeable to protons, an important issue because our transport in glial cells: High resolution immunogold cytochem- kidneys reabsorb up to 180 L of fluid each day without reab- istry of aquaporin-4 in rat brain. J Neurosci 17: 171–180, 1997 sorbing excreted acid. Moreover, this revealed for the first time 18. Amiry-Moghaddam M, Ottersen OP: The molecular basis of the structure of a human channel protein solved from human water transport in the brain. Nat Rev Neurosci 4: 991–1001, 2003 tissue and sparked the interest of other research groups who 19. Maurel C, Chrispeels MJ: Aquaporins. A molecular entry into further resolved the structure by electron crystallography and plant water relations. Plant Physiol 125: 135–138, 2001 x-ray crystallography. 20. 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