Patricia Fernández-Llama, M.D., Ph.D.
Laboratorio de Hormonologia, Hospital Clinic i Provincial, Universidad de Barcelona, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS),
Barcelona. España.
| DISCUSSION BOARD |
INTRODUCTION
Urinary concentration depends on the presence of a discrete segmental distribution of transport properties along the renal tubule. The urinary concentrating process depends on the coordinated function of the loop of Henle and the collecting ducts. The thick ascending limb of the loop of Henle powers the countercurrent multiplier process responsible for generation of a cortico-medullary osmolality gradient. The collecting ducts, under the control of vasopressin, allow variable degrees of osmotic equilibration, resulting in variable water excretion and a reciprocal relationship between urinary flow and urinary osmolality. Vasopressin modulates the collecting duct water permeability through specialized water channels protein.
Countercurrent multiplication in the renal medulla is dependent on net active NaCl absorption by the thick ascending limb of Henle's loop [1]. Urea transporters in the medullary tubules also contribute to generate the medullary osmotic gradient [1]. Therefore, several mechanisms are involved in the urinary concentration process. In this review, however, we will focus on the role of aquaporins in the urinary concentration mechanism. Complementary DNA cloning has revealed the primary structure of most of the transporters that mediate sodium, urea and water transport along the nephron, making possible a comprehensive analysis of transporter expression in different physiological and pathophysiological states. This type of approach can provide a global view of adaptation in terms of either mRNA expression ("genomics") or protein expression ("proteomics"). Because transport proteins (and not the mRNAs that code for them) mediate the transport function of the renal tubule, we have been pursuing a "proteomics" approach to the study of physiological adaptation. The strategy is to develop specific polyclonal antibodies to each transporter and channel protein expressed along the nephron and to use these antibodies for immunological techniques (immunoblotting, dot blotting and immunohistochemistry) to assess the overall pattern of adaptation to a physiological or pathophysiological stimulus [2]. This approach has been proofed to be important in understanding the molecular basis of the urinary concentration mechanism [1].
Aquaporins, water channels
The plasma membrane of all cells are known to be permeable to water. Water movement across plasma membranes occurs passively in response to osmotic gradients that are produced by primary and secondary active transport of ions and neutral solutes. Simple diffusion, however, does not account for all water movement through biological membranes.
There is now strong evidence that a family of specific water channel protein facilitate transcellular water movement in a variety of renal and extrarenal tissue in mammals [3]. The aquaporins are a family of small membrane-spanning proteins that are expressed at plasma membrane in many cells types involved in fluid transport.
Aquaporins appear to assemble in membranes as homotetramers in which each monomer contains a distinct water pore. A monomer consists in six membrane spanning domains with cytoplasmasmically oriented amino and carboxy termini [4].
Renal aquaporins.
Several aquaporins have been described in renal tubules. The abundance of individual aquaporins in the plasma membranes of renal epithelial cells is believed to be the major determinant of epithelial water permeability [1].
The renal localization of water channels is therefore, associated with water permeability in each segment of the nephron. Renal proximal tubules and descending thin limbs of Henle´s loop are known to have constitutively high water permeability allowing for the reabsorption of the majority of the water in glomerular filtrate. It is now well established that aquaporin-1 is highly abundant in the apical and basolateral membrane of these segments.
In contrast, the ascending thin limbs and thick limbs are relatively impermeable to water. Indeed no water channels have been found in these segments. Renal collecting ducts have low water permeability without vasopressin stimulation but vasopressin increases water permeability dramatically in a few seconds [5].
In the kidney collecting duct at least three aquaporins are known to be expressed, aquaporin-2, aquaporin-3 and aquaporin-4. Aquaporin-2 is expressed in the apical plasma membrane and in intracellular vesicles in principal cells of the renal collecting ducts. This water channel is the chief target for regulation of collecting duct water permeability by vasopressin. Water transport across the basolateral plasma membrane of collecting duct principal cells is thought to be mediated by aquaporin-3 and aquaporin-4.
Aquaporin-3 is most abundant in the cortical and outer medullary collecting ducts, while aquaporin-4 is predominantly found in the inner medullary collecting ducts. Aquaporin-3 protein abundance appears to be regulated by vasopressin, but there is no evidence for long-term regulation of aquaporin-4 expression in the kidney [3, 6].
Recently, a new water channel namely aquaporin-6 has been localized in intercalated cells in collecting ducts, where it is exclusively associated with intracellular vesicles. This water channel, however, contributes mainly to maintenance of acid-base homeostasis [7].
Molecular basis of urinary concentration process. Regulation of collecting duct water permeability by vasopressin.
The vasopressin receptor responsible for the regulation of water permeability in the collecting duct is the so-called V2 receptor. This receptor is an integral membrane protein coupled to adenylyl cyclase through the heterotrimeric G protein Gs. Receptor occupation increases intracellular cyclic AMP levels in collecting duct cells through activation adenylyl cyclase. Elevated cyclic AMP levels increase the water permeability of the kidney collecting duct in two separate ways [9, 10].
Clinical problems at the molecular level
Physiological studies in aquaporin knockout mice and studies in patients with mutations of the aquaporin-2 water channel have made it clear that the major fraction of water transport across renal tubule epithelia is mediated by aquaporins [8].
Clinical problems featuring impaired renal water reabsorption are associated with defect in aquaporin-2. Diabetes insipidus (DI) results from inadequate levels of vasopressin and leads to secretion of large volume of diluted urine. Nephogenic diabetes insipidus (NDI) is a disorder that occurs when vasopressin levels are not reduced, but the kidney fails to respond to the hormone.
Mutations in gene encoding renal vasopressin V2 receptor or renal aquaporin-2 have been demonstrated in patiens with NDI [11]. Acquired NDI has also been observed to be associated with decrease in the abundance of aquaporin-2. This defect has been observed with lithium treatment [12] and hypercalcemia [13].
There are other pathological situations associated with abnormal regulation of renal water excretion where a decrease in renal aquaporin-2 expression has been observed like in nephrotic syndrome [14] or liver cirrhosis [15].
In summary, aquaporins are membrane proteins that facilitate osmotic water transport across different epithelium. Renal aquaporins in the proximal tubules (aquaporin-1) and distal tubules (aquaporin-2, -3 and -4) play a central role in the renal physiological process like urine concentration.
The peptide-directed antibodies technique has been used for the detection of regulatory processes that are manifested as changes in the abundance of the target protein. Using this approach we are learning the pattern of response to vasopressin of renal aquaporin excretion.
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