Skip Navigation


NDT Advance Access originally published online on July 21, 2006
Nephrology Dialysis Transplantation 2006 21(10):2938-2942; doi:10.1093/ndt/gfl330
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
21/10/2938    most recent
gfl330v1
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 ISI Web of Science
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 arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gersch, M. S.
Right arrow Articles by Johnson, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gersch, M. S.
Right arrow Articles by Johnson, R. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Does Tamm–Horsfall protein–uric acid binding play a significant role in urate homeostasis?

Michael S. Gersch1,, Yuri Y. Sautin1, Christine M. Gersch1, George Henderson1, Lise Bankir2 and Richard J. Johnson1

1University of Florida, Department of Medicine, Gainesville, Florida, USA and 2Institut National de la Sante et de la Recherche Médicale, Unit 652, Paris, France

Correspondence and offprint requests to: Michael S. Gersch, MD, University of Florida, Room CG98, 1600 SW Archer Rd, PO Box 100224, Gainesville, FL 32610-0224, USA. Email: gerscms{at}medicine.ufl.edu



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Mutations in Tamm–Horsfall protein (THP), also known as uromodulin, lead to a group of diseases known as the uromodulin storage disorders. Clinically, these diseases present with tubulo-interstitial damage, progressive renal dysfunction, hyperuricaemia, and gout. However, it remains unclear how a mutation in THP, a protein produced in the thick ascending limb, can cause hyperuricaemia when most of the uric acid transport is believed to occur in the proximal tubule. However, one study in humans suggests that uric acid could also be secreted in the distal tubule. Thus, an attractive hypothesis could be that THP would bind to uric acid in the distal tubule, and decrease its subsequent reabsorption in the distal nephron.

Methods. We screened for uric acid binding to THP using four independent binding assays.

Results. There was no evidence that uric acid could bind to THP.

Conclusion. THP–uric acid binding does not seem to play a significant role in the regulation of urate homeostasis.

Keywords: binding; familial juvenile hyperuricaemic nephropathy (FJHN); medullary cystic kidney disease (MCKD); thick ascending limb; uromodulin



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Tamm–Horsfall protein (THP), which is also known as uromodulin, is the most abundant protein found in normal urine. The primary amino acid sequence of human THP was determined in 1987 [1,2]. The mature form of THP is 616 aminoacids long (MW = 85 kDa) and contains six glycosylation sites [3]. It contains a large number of cysteine residues, which form the disulfide bonds responsible for its complex 3-D structure. In the urine, THP forms polymeric chains and easily forms a gel in solution. It is synthesized exclusively in the thick ascending limb (TAL) of the loop of Henle and secreted into the tubular lumen [3].

This glycoprotein was first described more than 50 years ago, but its physiological role is still not fully understood. It was first assumed to play a role in protecting the urinary tract from calcium nephrolithiasis by preventing the aggregation of calcium oxalate crystals [4,5]. A role in TAL ion transport in relation with the diluting function of the TAL was hypothesized but not confirmed [6]. More recently, several studies have shown that THP plays an important role in defending the urinary tract against bacterial infections [7,8].

In recent years, attention has been drawn to a possible role of THP in uric acid handling by the kidney. Several genetic diseases (called uromodulin storage diseases) including medullary cystic kidney disease (MCKD), familial juvenile hyperuricaemic nephropathy (FJHN) and glomerulocystic kidney disease (GCK), presenting with hyperuricaemia, reduced fractional excretion of uric acid and gout, have been shown to result from mutations of the THP/uromodulin gene [9–11]. More that 30 uromodulin mutations have been described, most of which disrupt highly conserved cysteine residues in the uromodulin protein [12]. In affected patients, a mutant form of THP is produced that cannot efficiently exit the cell resulting in intracellular THP accumulation and a decreased amount of THP secreted in the urine [13].

Despite these advances in the genetics of the uromodulin storage diseases, several physiological aspects of these diseases, including the possible link between THP protein and uric acid handling, remain a mystery. Because THP is produced exclusively in the TAL and secreted in the urine, a possible interaction with uric acid would have to take place in the TAL or distal to this site. However, the generally accepted theory of uric acid transport states that all uric acid exchange is completed by the time it reaches the end of the proximal tubule [14]. Thus, it remains unclear how a mutation of a protein produced exclusively in the thick ascending limb (TAL) could alter urate homeostasis as it does in uromodulin storage diseases.

However, some older evidence that supports a role for distal uric acid transport may have received too little attention [15,16]. In this study, inulin, radiolabelled uric acid, para-aminohipuric acid (PAH) and potassium were injected simultaneously into hypertensive subjects, and serial urine samples were collected. They found that uric acid appeared in the urine before the PAH but after potassium, indicative of secretion of uric acid in the nephron lumen at a site located after the proximal tubule and before the collecting duct [15,16]. Reviewing this literature, along with the recent observations regarding the uromodulin storage diseases, gave rise to the hypothesis that THP may bind to uric acid and act in a way that could favour its efficient excretion. The complex 3–D structure of the THP, imparted by its disulfide bonds and its capacity to form gels in solution, could favour THP–uric acid binding. This interaction could take place in the cells of the TAL, allowing THP to participate in uric acid secretion into the nephron lumen. This binding could also take place in the tubular lumen, preventing subsequent reabsorption of uric acid along the distal nephron as the urine is progressively concentrated in the collecting duct.

THP has been previously shown to bind to the Bence–Jones protein (BJP), where it plays a role in the formation of casts in multiple myeloma [17]. THP also binds to the fimbrae from uropathogenic strains of E. Coli [18] where it plays a physiological role in defending the urinary tract from infection [7,18–23]. Thus, THP binding interactions play important physiological roles in at least two areas. We undertook the present series of experiments to determine if THP is also able to bind to uric acid, a property that could provide part of the mechanism for the hyperuricaemia seen in uromodulin storage diseases.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
THP was isolated from the urine of healthy males by three cycles of precipitation with 0.58 molar sodium chloride (Sigma, St Louis) and dialysed to remove the sodium chloride as previously described [24]. Protocols involving human subjects were approved by the University of Florida institutional review board. The protein was run on a 7.5% polyacrilamide gel (Sigma, St Louis, MO, USA) and stained with Commassie Blue (Bio-Rad, Hercules, CA, USA).

Binding method 1—immunoblotting with radiolabel detection
About 2 µg of THP, albumin and BJP were placed on a nitrocellulose membrane (Bio-Rad, Hercules CA, USA) and allowed to dry. The membrane was washed three times in phosphate buffered saline (PBS) (Sigma, St Louis). The membrane was blocked with 2% non-fat dried milk in tris-buffered saline (Bio-Rad, Hercules CA, USA) with 0.1% Tween-20 (Sigma, St Louis) (TBST) for 2 h at room temperature. The membrane was then incubated with 5000 dpm 14C-uric acid (American Radiochemicals, St Louis, MO) at 4°C overnight. The membranes were then rinsed three times with TBST and counted in a scintillation counter (Beckman-Coulter, Fullerton, CA, USA). For the control experiment, the membrane was incubated in BJP (Bethyl laboratories, Montgomery, TX, USA) at 1 µg/ml TBST with 2% milk for 1 h. The membrane was rinsed three times with TBST. Immunoblotting of the membrane was performed with (1) primary antibody goat anti-human BJP (A80-116a, Bethyl laboratories, Montgomery, TX) overnight at 4°C at 1/1000 dilution in TBST with 0.1%, (2) bovine serum albumin rabbit anti-goat horseradish peroxides linked secondary antibody (Dako) for 1 h at room temperature at 1/2000 dilution in TBST with 0.1% BSA and (3) West Pico luminol reagents (Pierce, Rockford, IL, USA).

Binding assay 2—immunoprecipitation with radiolabel detection
THP solution (106 µg/ml) in TBST was pretreated with sepharose protein A beads (Rockland, Gilbertsville PA) for 1 h at 4°C, then these beads were removed by centrifugation for 5 min at 5000 g. About 10 µl mouse anti-human THP antibody (CL1032a lot 2964, Cedarlane, Ontario, Canada) was then added to the THP solution and incubated for 2 h at 4°C and 400 µl freshly washed sepharose beads were added to the solution for 1 h. The beads were then washed three times with PBS. The beads were then incubated with 10 000–100 000 dpm of 14C-labelled uric acid for 1 h at room temperature or 24 h at 4°C with rotation. In other experiments, the effect of the buffer on binding was examined. In these experiments PBS, TBS, TBST and Hanks buffered salt solution (HBSS) without phenol red, calcium or magnesium (GIBCO, Grand Island, NY, USA) were tested. After the incubation period, the beads were washed three times with PBS, and were then placed in scintillation fluid (Sigma, St Louis) and counted in the scintillation counter.

Binding assay 3—immunoprecipitation with LC–MS/MS detection
The THP bound beads were prepared as above. They were then incubated in PBS containing 10 µM uric acid (Sigma, St Louis, MO, USA). The beads were then rinsed three times in PBS. They were then suspended in 300 µl distilled water and heated to 100°C for 10 min to detach the protein from the beads. The samples were then filtered through a 10 kDa filter (Millipore, Bedford, MA, USA) to remove the proteins and the supernatant was analysed on a LC–MS/MS system.

Binding assay 4—molar equivalent binding, filter separation and LC–MS/MS analysis
THP solution was mixed with a molar equivalent uric acid in PBS pH 7.4. After 1 h of binding, the solution was filtered through a 10 kDa filter to remove proteins. The filtrate was analysed on an LC–MS/MS system for the determination of uric acid concentration.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
THP was purified from normal urine. Electropheresis under non-reducing conditions revealed that the THP was in polymeric form (data not shown). THP was also run on a gel under reducing conditions to confirm purity (Figure 1). Four distinct techniques were used for the determination of binding of THP to uric acid. Using an immunoblotting method, we found there was no evidence of binding of THP to uric acid. As a control however, BJP was able to bind to the THP but not to albumin (Figure 2). Using an immunoprecipitation method with radiolabel detection, we found there was also no evidence of THP binding to uric acid. As a control, the presence of the THP could be demonstrated on the beads after the immunoprecipitation (Figure 3). Alteration of the composition of the buffer from PBS to TBS and HBSS, with or without calcium and magnesium, had no effect on the binding data (not shown). Similarly, alteration of the pH of the binding solution from pH 6 to pH 8 did not lead to any binding (data not shown). Using an immunoprecipitation method in conjunction with LC–MS/MS detection, no THP-bound uric acid could be detected, even though uric acid could be detected with this method down to a concentration of 5 nM (Figure 4). Finally, a filter separation technique and LC–MS/MS detection was used to assess the binding of uric acid to THP. After incubation of THP with an equal molar amount of uric acid, THP was separated from uric acid solution by filtration through a 10 kDa filter. THP binding to uric acid would be expected to alter the concentration of uric acid recovered in the filtrate; however, no significant change in concentration could be detected.


Figure 1
View larger version (112K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. THP run on 7.5% polyacrilamide gel and stained with Commassie blue.

 

Figure 2
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Purified THP and albumin placed on nitrocellulose membrane. Incubated with Bence–Jones protein for 1 h at room temperature, then immunoblotted with anti-Bence–Jones protein antibody. Bence–Jones protein bound to THP and not to albumin.

 

Figure 3
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Western blot with anti-THP antibody showing presence of THP on the beads after immunoprecipitation (IP), lack of THP on beads before IP and depletion of THP from supernatant of the THP solution by IP.

 

Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. LC–MS/MS analysis of uric acid (RT = 4.5 in) in negative electrospray mode [the reactions monitored were: m/z 167–96 (25V) and m/z 167–124 (25V)].

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The hypothesis that THP could bind to uric acid and that this may prevent distal urate transport is intriguing. Generally, it is believed that uric acid transport is complete by the end of the proximal tubule. However, there is some older evidence supporting the concept of distal uric acid transport [15,16]. As mutations in THP produce a phenotype that frequently has a defect in uric acid excretion, it is tempting to hypothesize that there may be an interaction between uric acid and THP.

Nevertheless, in the present study, four independent methods were used to test the hypothesis that THP might bind to uric acid under physiological conditions. While each of the methods employed has some limitations, the combined results of these four experimental methods provide good evidence against the hypothesis that THP can bind to uric acid under physiological conditions. However, it is still possible that THP modulates uric acid transport in the distal tubule via a mechanism that does not involve binding. In vivo or in vitro studies would need to be designed to answer this question.

Another hypothesis that has been advanced to explain the hyperuricaemia seen in some uromodulin storage diseases is compensation for renal salt wasting [11,20,25]. The uromodulin storage diseases are frequently associated with renal salt wasting [11,26]. Compensatory mechanisms for this renal salt wasting could lead to increased proximal tubular resorption of uric acid, similar to hyperuricaemia often observed in Bartter's syndrome and Gittleman's syndrome [27]. There is some evidence from THP knockout mice to support this theory [25], but more experiments are required to definitively answer this question. In conclusion, we found no evidence that uric acid can bind to THP under physiological conditions.

Conflict of interest statement. Dr Johnson is a consultant for SCIOS and TAP pharmaceuticals.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Hession C, Decker JM, Sherblom AP, et al. (1987) Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines. Science 237:1479–1484.[Abstract/Free Full Text]
  2. Pennica D, Kohr WJ, Kuang WJ, et al. (1987) Identification of human uromodulin as the Tamm–Horsfall urinary glycoprotein. Science 236:83–88.[Abstract/Free Full Text]
  3. Serafini-Cessi F, Malagolini N, Cavallone D. (2003) Tamm-Horsfall glycoprotein: biology and clinical relevance. Am J Kidney Dis 42:658–676.[CrossRef][Web of Science][Medline]
  4. Hess B. (1991) The role of Tamm–Horsfall glycoprotein and Nephrocalcin in calcium oxalate monohydrate crystallization processes. Scanning Microsc 5:689–695 discussion 696.[Web of Science][Medline]
  5. Hess B. (1994) Tamm–Horsfall glycoprotein and calcium nephrolithiasis. Miner Electrolyte Metab 20:393–398.[Web of Science][Medline]
  6. Bachmann S, Dawnay AB, Bouby N, et al. (1991) Tamm–Horsfall protein excretion during chronic alterations in urinary concentration and protein intake in the rat. Ren Physiol Biochem 14:236–245.[Web of Science][Medline]
  7. Saemann MD, Weichhart T, Horl WH, et al. (2005) Tamm–Horsfall protein: a multilayered defence molecule against urinary tract infection. Eur J Clin Invest 35:227–235.[CrossRef][Web of Science][Medline]
  8. Bates JM, Raffi HM, Prasadan K, et al. (2004) Tamm–Horsfall protein knockout mice are more prone to urinary tract infection: rapid communication. Kidney Int 65:791–797.[CrossRef][Web of Science][Medline]
  9. Lens XM, Banet JF, Outeda P, et al. (2005) A novel pattern of mutation in uromodulin disorders: autosomal dominant medullary cystic kidney disease type 2, familial juvenile hyperuricaemic nephropathy, and autosomal dominant glomerulocystic kidney disease. Am J Kidney Dis 46:52–57.[CrossRef][Web of Science][Medline]
  10. Bleyer AJ, Hart TC, Willingham MC, et al. (2005) Clinico-pathologic findings in medullary cystic kidney disease type 2. Pediatr Nephrol 20:824–827.[CrossRef][Web of Science][Medline]
  11. Scolari F, Caridi G, Rampoldi L, et al. (2004) Uromodulin storage diseases: clinical aspects and mechanisms. Am J Kidney Dis 44:987–999.[CrossRef][Web of Science][Medline]
  12. Bleyer AJ, Trachtman H, Sandhu J, et al. (2003) Renal manifestations of a mutation in the uromodulin (Tamm Horsfall protein) gene. Am J Kidney Dis 42:E20–E26.[CrossRef][Medline]
  13. Rampoldi L, Caridi G, Santon D, et al. (2003) Allelism of MCKD, FJHN and GCKD caused by impairment of uromodulin export dynamics. Hum Mol Genet 12:3369–3384.[Abstract/Free Full Text]
  14. Rafey MA, Lipkowitz MS, Leal-Pinto E, et al. (2003) Uric acid transport. Curr Opin Nephrol Hypertens 12:511–516.[Web of Science][Medline]
  15. Podevin R, Ardaillou R, Paillard F, et al. (1968) Study in man of the kinetics of the appearance of uric acid 2-14C in the urine. Nephron 5:134–140.[Web of Science][Medline]
  16. Podevin R, Paillard F, Hornych A, et al. (1968) Kinetics of the appearance in the urine of para-aminohippuric acid (PAH) and of 2-14C uric acid in man. Effects of benziodarone, acetylsalicylic acid and pyrazinamide. Rev Fr Etud Clin Biol 13:513–522.[Web of Science][Medline]
  17. Huang ZQ, Kirk KA, Connelly KG, et al. (1993) Bence Jones proteins bind to a common peptide segment of Tamm–Horsfall glycoprotein to promote heterotypic aggregation. J Clin Invest 92:2975–2983.[Web of Science][Medline]
  18. Leeker A, Kreft B, Sandmann J, et al. (1997) Tamm–Horsfall protein inhibits binding of S- and P-fimbriated Escherichia coli to human renal tubular epithelial cells. Exp Nephrol 5:38–46.[Web of Science][Medline]
  19. Bachmann S, Mutig K, Bates J, et al. (2005) Renal effects of Tamm–Horsfall protein (uromodulin) deficiency in mice. Am J Physiol Renal Physiol 288:F559–F567.[Abstract/Free Full Text]
  20. Devuyst O, Dahan K, Pirson Y. (2005) Tamm-Horsfall protein or uromodulin: new ideas about an old molecule. Nephrol Dial Transplant 20:1290–1294.[Free Full Text]
  21. Dou W, Thompson-Jaeger S, Laulederkind SJ, et al. (2005) Defective expression of Tamm–Horsfall protein/uromodulin in COX-2-deficient mice increases their susceptibility to urinary tract infections. Am J Physiol Renal Physiol 289:F49–F60.[Abstract/Free Full Text]
  22. Saemann MD and Weichhart T. (2005) Causes of recurrent urinary tract infections: new insights. Dtsch Med Wochenschr 130:2031–2034.[CrossRef][Medline]
  23. Saemann MD, Weichhart T, Zeyda M, et al. (2005) Tamm–Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4-dependent mechanism. J Clin Invest 115:468–475.[CrossRef][Web of Science][Medline]
  24. Serafini-Cessi F, Dall'Olio F, Malagolini N. (1984) High-mannose oligosaccharides from human Tamm–Horsfall glycoprotein. Biosci Rep 4:269–274.[CrossRef][Web of Science][Medline]
  25. Nephrol Dial Transplant Gersch M, Mutig K, Bachmann S, et al. Is salt-wasting the long awaited answer to the hyperuricaemia seen in uromodulin storage diseases? Epub Jan 18, 2006.
  26. Wolf MT, Mucha BE, Attanasio M, et al. (2003) Mutations of the Uromodulin gene in MCKD type 2 patients cluster in exon 4, which encodes three EGF-like domains. Kidney Int 64:1580–1587.[CrossRef][Web of Science][Medline]
  27. Peters N, Bettinelli A, Spicher I, et al. (1995) Renal tubular function in children and adolescents with Gitelman's syndrome, the hypocalciuric variant of Bartter's syndrome. Nephrol Dial Transplant 10:1313–1319.[Abstract/Free Full Text]
Received for publication: 8. 3.06
Accepted in revised form: 9. 5.06


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



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
21/10/2938    most recent
gfl330v1
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 ISI Web of Science
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 arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gersch, M. S.
Right arrow Articles by Johnson, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gersch, M. S.
Right arrow Articles by Johnson, R. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?