Skip Navigation


NDT Advance Access originally published online on October 19, 2007
Nephrology Dialysis Transplantation 2008 23(2):492-496; doi:10.1093/ndt/gfm641
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/2/492    most recent
gfm641v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Mayan, H.
Right arrow Articles by Farfel, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mayan, H.
Right arrow Articles by Farfel, Z.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Increased urinary Na-Cl cotransporter protein in familial hyperkalaemia and hypertension

Haim Mayan1, Dana Attar-Herzberg1,2, Miriam Shaharabany2,3, Eliezer J Holtzman2 and Zvi Farfel1,3

1Department of Medicine E’, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Hashomer 52621, Israel, 2Nephrology Institute, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Hashomer 52621, Israel and 3Laboratory of Biochemical Pharmacology, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Hashomer 52621, Israel

Correspondence and offprint requests to: Zvi Farfel, Department of Medicine E, Sheba Medical Center, Tel Hashomer 52621 Israel. Tel: +972-35302437; Fax: +972-35302460; E-mail: farfel{at}post.tau.ac.il



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements.
 References
 
Background. Familial hyperkalaemia and hypertension (FHH), also termed pseudohypoaldosteronism type II, is a rare monogenic form of hypertension caused by mutations in the WNK1 or WNK4 kinases. In vitro expression of WNK4 reduces surface abundance and activity of coexpressed NaCl cotransporter (NCCT). This effect is lost in disease-producing WNK4 mutants. In two mice models of FHH, one expressing two extra copies of mutant WNK4 (Q562E) and another in which a mutant (D561A) WNK4 replaced wild-type WNK4, renal distal tubule hyperplasia with overexpression of NCCT was found. Currently no FHH human renal tissue is available to test for increased distal tubule surface abundance of NCCT. The availability of a unique large family with FHH and the Q565E WNK4 mutation enabled us to investigate this issue in an indirect manner.

Methods. Assuming that shedding of NCCT to the urine reflects its abundance in the distal tubule epithelium, we measured urinary NCCT protein in eight subjects of the FHH family and in eight unrelated controls by western blotting.

Results. Urinary NCCT protein was about four times higher in FHH than in controls [111.1 ± 40.5 versus 26.1 ± 16.4 densitometry units (P < 0.0001)]. No significant difference in urinary sodium and potassium concentrations was seen between FHH and controls.

Conclusions. The increased urinary NCCT in FHH most probably reflects increased NCCT abundance in the apical membrane of distal tubule cells in patients with FHH and the WNK4 mutation and points to the pathogenetic mechanism for the clinical phenotype of FHH and the WNK4 mutation, supporting results in transgenic mice with the same mutation and in knockin mice with another mutation.

Keywords: familial hyperkalaemia and hypertension; Na-Cl cotransporter; pseudohypoaldosteronism type II; urine proteomics; WNK4 kinase



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements.
 References
 
Familial hyperkalaemia and hypertension (FHH), also known as pseudohypoaldosteronism type II (PHA II), is a rare monogenic form of hypertension [1]. It was first described in a sporadic case by Paver and Pauline in 1964 [2]. The first family with FHH was described 12 years later [3]. FHH is an autosomal dominant disorder characterized by hyperkalaemia, hypertension and metabolic acidosis in the presence of normal renal function [6]. WNK1 mutations are intronic deletions and cause increased expression of the gene. WNK4 mutations are missense mutations that change the function of the gene [6]. The mechanism by which these mutations produce FHH is not clear. The efficacy of thiazides in FHH points toward the thiazide arget, the NaCl cotransporter (NCCT), as a potential molecule regulated by WNK kinase. Indeed, in in vitro experiments, expression of wild-type WNK4 with NCCT in frog oocytes caused a decrease in NCCT activity and surface abundance [9]. Additional proposed mechanisms for WNK4 in the pathogenesis of FHH include inhibition of the renal outer medullary potassium (ROMK) channel by WNK4, as found in in vitro expression studies in frog oocytes, an inhibition that is further increased using FHH-producing WNK4 mutations [10]. WNK4 was also shown to augment the paracellular Cl– transport, which was further stimulated by disease-producing mutants [17]. This is in analogy with aquaporin-2, where it was found that its amount in the urine is proportional to its amount in the whole kidney and more specifically in the apical plasma membrane of renal tubular cells [18]. Therefore, we compared the amount of urinary NCCT in subjects with FHH and the Q565E WNK4 mutation to that of healthy controls.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements.
 References
 
Subjects
Two groups of subjects were studied, eight control subjects and eight subjects from the large family with FHH and Q565E WNK4 mutation [16]. Their basal serum electrolytes were similar to those of all the affected family members (K 5.6 ± 0.2 mmol/l, Na 140.4 ± 2.2 mmol/l, Cl 109.0 ± 1.5 mmol/l, HCO3– 19.5 ± 1.4 mmol/l [16]. Five subjects were treated by hydrochlorothiazide when studied, since they were hypertensive. During the study all subjects were normotensive.

Detection of NCCT in Urine
Spot urine was collected and a sample was used for determination of electrolytes, creatinine and osmolarity. Fifty milliliter of urine was kept on ice with a mixture of protease inhibitors (protease inhibitor cocktail tablets, purchased from Roche). Urine was centrifuged at 17 000 g for 15 min at 4°C to remove all cell debris, and the pellet was discarded. The supernatant was centrifuged at 200 000 g for 80 min at 4°C. The resulting pellet was suspended in 500 µl of sucrose buffer [250 mM sucrose; 10 mM triethanolamine; 1 µg/ml leupeptin and 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF)] and added to Laemmli buffer with no dithiothreitol (DTT) [final concentration: Tris 50 mM pH 6.8; glycerol 10%; sodium dodecyl sulphate (SDS) 2%; bromo phenol blue 0.05%]. Samples were subjected to 8.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blot analysis. The amount of resuspended pellet loaded on each lane was calculated to represent urine that contained 3 mg of creatinine. Following SDS–PAGE, proteins were transferred to a nitrocellulose membrane. The filter was blocked with 2% bovine serum albumin (BSA) in TBST (Tris 20 mM, NaCl 150 mM, tween-20 0.05%, pH 7.4) and incubated with rabbit anti-human NCCT polyclonal antibody kindly provided by Dr MA Knepper [19]. Filter was washed with TBST and incubated with secondary horseradish peroxidase (HRP) conjugated antibody. Visualization was achieved according to the manufacturer's instructions. The intensity of the resulting bands was measured by densitometry.

Expression of results
Samples from the 16 subjects (8 controls and 8 FHH patients) were loaded on two gels, each containing both controls and FHH samples. Two of the samples (one control and one FHH) were loaded on both gels. Densitometry values were determined and the identical samples in the two gels were used for comparison of the two gels. The identical samples were very similar (not shown); thus, the gel shown in Figure 1A represents results of both gels with no normalization. Data are presented as mean ± SD. For comparison between means, Student's t-test was performed. Two tailed P-values were reported. P < 0.05 was considered significant.


Figure 1
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. (A) Western blot analysis of NCCT in the urine of subjects with FHH and controls. Each lane shows NCCT from urine that contained 3 mg of creatinine (see Methods). The arrow points to the NCCT band. (B) Densitometry values of the urinary NCCT bands in (A) for FHH and control subjects are shown and are expressed in arbitrary units. The horizontal lines show the mean value of each group.

 
The study was approved by the Ethics Committee of the Sheba Medical Center, adhering to the Helsinki Declaration. Informed consent was obtained from all subjects studied.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements.
 References
 
The characteristics of the subjects studied are shown in Table 1. The gender distribution in FHH and control subjects was identical. The FHH subjects and control subjects did not differ significantly in their age and BMI. Measurement of urinary electrolytes revealed that FHH subjects had lower urinary sodium content than the control subjects (8.6 ± 6.0 versus 13.6 ± 4.0 mmol/mmol creatinine), but this was statistically insignificant (P = 0.07). FHH subjects had also lower urinary potassium concentration than controls (4.4 ± 1.3 versus 6.5 ± 2.6 mmol/mmol creatinine), which is not statistically significant (P = 0.06). In our previous studies of the family [15] or in first morning urinary samples [16]. In addition, in these studies no subject was on thiazide treatment [19]. The bands are easily seen in the lanes of the FHH subjects. In the controls, they are clearly seen in subjects 2 and 8. The upper bands that are seen in control subjects 4 and 5 are irrelevant since they also appear while using the secondary antibody only (not shown). The amount of NCCT in the urine of FHH subjects is strikingly higher than that found in controls (Figure 1A and B). Densitometry shows that the mean urinary abundance of NCCT in FHH is more than four times higher than the mean abundance of NCCT of controls (11.1 ± 40.5 versus 26.1 ± 16.4 arbitrary densitometry units, P < 0.0001). There is almost no overlap of the individual values in the two groups (Figure 1B). There was no correlation between individual urinary NCCT values and urinary sodium in FHH or controls (FHH: r = 0.01, P = 0.99, control: r = 0.45, P = 0.27). Also, no correlation was found between individual urinary NCCT values and urinary potassium in FHH or controls (FHH: r = –0.46, P = 0.25, control: r = 0.23, P = 0.58). Five of the FHH subjects were treated by thiazides since they were hypertensive. Although the groups were small, it seems that thiazide therapy did not affect urinary NCCT [(100.8 ± 48.2 versus 128.1 ± 20.2 densitometry units, P = 0.40) in FHH subjects, on or off thiazide therapy, respectively].


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics and urinary NCCT data of the groups of subjects studied

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements.
 References
 
The progress in urine proteomics with its capacity to detect very low concentrations of urinary proteins enabled advances in the study of physiological as well as pathological states [17]. An important example was the finding that urinary aquaporin-2 can be measured in the urine. Since it is shed from principal cell membranes into the urine, changes in the urinary concentration of aquaporin-2 reflect the action of vasopressin on the kidney, which increases insertion and consequently the number of aquaporin-2 molecules in the apical membrane of the cortical collecting duct [20]. One of the proteins that can be determined in the urine is NCCT which, like aquaporin-2, is an apical membrane protein and has a crucial role in FHH. Indeed in rats urine, NCCT as well as other Na+ transporters can be detected [21]. NCCT was also detected in human urine [19]. It is inserted in urinary exosomes that are supposed to represent vesicles released from the apical plasma membrane of renal tubule epithelial cells [17]. Determination of NCCT in the urine can be utilized to perform quantitative measurement of NCCT in FHH subjects in comparison to controls.

Using an immunoblot assay, we have found a marked increase in the abundance of urinary NCCT in FHH subjects compared to controls. FHH subjects had a mean of about four times higher abundance of urinary NCCT than controls with almost no overlap between individual data in the two groups. Although FHH subjects had lower (statistically insignificant) urinary sodium and potassium concentrations, it does not seem that this was the cause for the marked increase in urinary NCCT in FHH. In no group of subjects was there a correlation between individual urinary NCCT values and urinary sodium or potassium. The difference in urinary NCCT is apparently the result of the WNK4 mutation that causes the clinical phenotype. It probably reflects increased shedding of NCCT into the urine. In analogy with the finding in the study of aquaporin-2 [18,20], we may assume that distal tubule apical cell membranes of FHH subjects have an increased abundance of NCCT. In support of such a mechanism are the findings in the recent report that transgenic mice which have two extra copies of the FHH-producing mutant Q562E WNK4 gene (the same mutant, Q565E, affecting the currently reported FHH subjects), have in addition to hyperkalaemia, hypercalciuria and hypertension, also marked hyperplasia of the distal convoluted tubule [13]. Using immunohistochemical techniques, it was found that hyperplasia is confined to the distal tubule cells and does not occur in other nephron segments. In this segment, distal convoluted tubule surface area and total NCCT expression are increased. Very recently, Yang et al. produced knockin mice expressing the FHH-producing D561A WNK4 mutant [14]. Like Lalioti et al. [13], Yang et al. found increased expression of NCCT in the renal distal nephron in their animal model [14]. In addition, they have shown increased phosphorylation of the Ste20-related kinases, SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress response kinase [1]) in the mutant mice, establishing a role for the WNK4-SPAK/OSR1-NCCT cascade [14] is different than that proposed by Lifton and colleagues [13], which was based on the findings in frog oocytes, both groups find overexpression of NCCT in the distal convoluted tubule. Our findings of increased urinary NCCT in FHH support the notion that in the human disease, NCCT is overexpressed in this nephron segment.

These findings support the central role of the increased NCCT membrane abundance in the pathophysiology of FHH and diminish the importance of the role of paracellular chloride transport in the production of FHH [25]. Similarly in the transgenic mice model, homozygous knockout of the NCCT gene reversed the phenotypic abnormalities, indicating that the effect of WNK4 on NCCT only is sufficient to produce hyperkalaemia, hypercalciuria and hypertension [13]. In addition, in both mice models [14].

It is interesting to note that in Gitelman's syndrome, which features hypokalaemia, metabolic alkalosis and hypotension, is a mirror image of FHH and is caused by inactivating mutations of NCCT, immunobloting of the urine showed that patients had reciprocal decreased amounts of urinary NCCT [26].

Our finding may shed light on additional pathophysiological mechanisms of action of WNK kinases. For example, the question whether the effect of WNK1 in the pathogenesis of FHH is mediated solely by the kidney or whether extrarenal effects are involved has not been settled [27]. Similar to WNK4, WNK1 stimulates and phosphorylates SPAK and OSR1 [28).

Finally, the mechanism by which the presumed increased abundance of renal cell membrane NCCT is associated with increased sensitivity to thiazides is not known. In the current study, thiazide therapy did not change urinary and presumably renal abundance of NCCT. A major limitation of the study is that only three of the eight FHH subjects were off thiazides. Further studies are necessary to confirm these results.



   Acknowledgements.
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements.
References
 
This work was supported by a grant from the Israel Science Foundation to Z.F. and E.J.H. Z.F. holds The Dr Boris (Dov) Quartin Chair in Chemical Pathology of the Tel Aviv University. We thank Dr MA Knepper for providing us the anti-human NCCT antibody.

Conflict of interest statement. No author has a conflict of interest to declare.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements.
 References
 

  1. Lifton RP, Ghaharavi DS, Geller DS. Molecular mechanisms of human hypertension. Cell (2001) 104:545–556.[CrossRef][Web of Science][Medline]
  2. Paver WKA, Pauline GJ. Hypertension and hyperpotassemia without renal disease in a young male. Med J Aust (1964) 2:305–306.[Medline]
  3. Farfel Z, Rosenthal T, Shibolet S, et al. Familial hyperkalaemia and hypertension. Harefuah (1976) 90:468–470.[Medline]
  4. Gordon RD, Klemm SA, Tunny TJ, et al. Gordon's Syndrome: a sodium-volume dependent form of hypertension with a genetic basis. In: Hypertension: pathophysiology, diagnosis, and management.—Laragh JH, Brenner BM, eds. (1995) 2nd edn. New York: Raven Press. 2111–2123.
  5. Farfel Z, Iaina A, Rosenthal T, et al. Familial hyperpotassemia and hypertension accompanied by normal plasma aldosterone levels: possible cell membrane defect. Arch Intern Med (1978) 138:1828–1832.[Abstract/Free Full Text]
  6. Wilson F, Disse-Nicodeme S, Choate K, et al. Human hypertension caused by mutations in WNK kinases. Science (2001) 293:1107–1112.[Abstract/Free Full Text]
  7. Wilson FH, Kahle KT, Sabath E, et al. Molecular pathogenesis of inherited hypertension with hyperkalaemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4. Proc Natl Acad Sci USA (2003) 100:680–684.[Abstract/Free Full Text]
  8. Yang CL, Angell J, Mitchell R, et al. WNK kinases regulate thiazide-sensitive Na-Cl cotransport. J Clin Invest (2003) 111:1039–1045.[CrossRef][Web of Science][Medline]
  9. Cai H, Cebotaru V, Wang YH, et al. WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells. Kidney Int (2006) 69:2162–2170.[CrossRef][Web of Science][Medline]
  10. Kahle KT, Wilson FH, Leng Q, et al. WNK4 regulates the balance between renal NaCl reabsorption and K+ secretion. Nature Genet (2003) 35:372–376.[CrossRef][Web of Science][Medline]
  11. Yamauchi K, Rai T, Kobayashi K, et al. paracellularCl-permeability and phosphorylates claudins. Proc Natl Acad Sci USA (2004) 101:4690–4694.[Abstract/Free Full Text]
  12. Kahle KT, Macgregor GG, Wilson FH, et al. Paracellular Cl– permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension. Proc Natl Acad Sci USA (2004) 101:14877–14882.[Abstract/Free Full Text]
  13. Lalioti MD, Zhang J, Volkman HM, et al. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nat Genet (2006) 38:1124–1132.[CrossRef][Web of Science][Medline]
  14. Yang SS, Morimoto T, Rai T, et al. Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4D561A/+ knockin mouse model. Cell Metabolism (2007) 5:331–344.[CrossRef][Web of Science][Medline]
  15. Mayan H, Vered I, Mouallem M, et al. Pseudohypoaldosteronism type II: marked sensitivity to thiazides, hypercalciuria, normomagnesemia, and low bone mineral density. J Clin Endocrinol Metab (2002) 87:3248–3254.[Abstract/Free Full Text]
  16. Mayan H, Munter G, Shaharabany M, et al. Hypercalciuria in familial hyperkalaemia and hypertension accompanies hyperkalaemia and precedes hypertension: description of a large family with the Q565E WNK4 mutation. J Clin Endocrinol Metab (2004) 89:4025–30.[Abstract/Free Full Text]
  17. Hoorn EJ, Pisitkun T, Zieste R, et al. Prospects for urinary proteomics: exosomes as a source of urinary biomarkers. Nephrology (2005) 10:283–290.[CrossRef][Medline]
  18. Wen H, Frokiaer J, Kwon TH, et al. Urinary excretion of Aquaporin-2 in rat is mediated by a vasopressin-dependent apical pathway. J Am Soc Nephrol (1999) 10:1416–1429.[Abstract/Free Full Text]
  19. Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci USA (2004) 101:13368–13373.[Abstract/Free Full Text]
  20. Kanno K, Sasaki S, Hirata Y, et al. Urinary excretion of Aquaporine-2 in patients with diabetes insipidus. N Engl J Med (1995) 332:1540–1545.[Abstract/Free Full Text]
  21. McKee JA, Kumar S, Ecelbarger CA, et al. Detection of Na+ transporter proteins in urine. J Am Soc Nephrol (2000) 11:2128–2132.[Abstract/Free Full Text]
  22. Vitari AC, Deak M, Morrice NA, et al. The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases. Biochem J (2005) 391:17–24.[CrossRef][Web of Science][Medline]
  23. Moriguchi T, Urushiyama S, Hisamoto N, et al. WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE-related kinases, SPAK and OSR1. J Biol Chem (2005) 280:42685–42693.[Abstract/Free Full Text]
  24. Gagnon KBE, England R, Delpire E. Volume sensitivity of cation-Cl-cotransporters is modulated by the interaction of two kinases: Ste-20-related proline-alanine-rich kinase and WNK4. Am J Physiol Cell Physiol (2006) 290:C134–142.[Abstract/Free Full Text]
  25. Schambelan M, Sebastian A, Rector FC. Mineralocorticoid-resistant renal hyperkalaemia without salt wasting (type II pseudohypoaldosteronism): role of increased renal chloride reabsorption. Kidney Int (1981) 19:716–727.[Web of Science][Medline]
  26. Lee JW, Joo KW, Chang HR, et al. Urine immunoblotting of sodium chloride cotransporter in patients with Gitelman's syndrome: its implications in differential diagnosis. [Abstract]. J Am Soc Nephrol (2004) 15:312A.
  27. Hadchouel J, Delaloy C, Faure S, et al. Familial Hyperkalemic Hypertension. J Am Soc Nephrol (2006) 17:208–217.[Free Full Text]
  28. Achard JM, Warnock DG, Disse-Nicodeme S, et al. Familial hyperkalemic hypertension: phenotypic analysis in a large family with the WNK1 deletion mutation. Am J Med (2003) 114:495–498.[CrossRef][Web of Science][Medline]
Received for publication: 4. 7.07
Accepted in revised form: 22. 8.07


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
H. Mayan, S. Melnikov, I. Novikov, E. J. Holtzman, and Z. Farfel
Familial Hyperkalemia and Hypertension: Pathogenetic Insights Based on Lithium Clearance
J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 3010 - 3016.
[Abstract] [Full Text] [PDF]

Home page
 Nephrol Dial TransplantHome page
E. J. Hoorn, N. van der Lubbe, and R. Zietse
The renal WNK kinase pathway: a new link to hypertension
Nephrol. Dial. Transplant., April 1, 2009; 24(4): 1074 - 1077.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/2/492    most recent
gfm641v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Mayan, H.
Right arrow Articles by Farfel, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mayan, H.
Right arrow Articles by Farfel, Z.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?