Tamm-Horsfall protein is a critical renal defense factor protecting against calcium oxalate crystal formation

BACKGROUND: The tubular fluid of the mammalian kidney is often supersaturated with mineral salts, but crystallization rarely occurs under normal conditions. The unique ability of the kidney to avoid harmful crystal formation has long been attributed to the inhibitory activity of the urinary macromolecules, although few in vivo studies have been carried out to examine this hypothesis. Here we examined the role of Tamm-Horsfall protein (THP), the principal urinary protein, in urinary defense against renal calcium crystal formation, using a THP knockout model that we recently developed. METHODS: Wild-type and THP knockout mice were examined for the spontaneous formation of renal calcium crystals using von Kossa staining. The susceptibility of these mice to experimentally induced renal crystal formation was evaluated by administering mice with ethylene glycol, a precursor of oxalate, and vitamin D(3), which increases calcium absorption. Renal calcium crystals were visualized by von Kossa stain, dark field microscopy with polarized light and scanning electron microscopy. RESULTS: Inactivating the THP gene in mouse embryonic stem cells results in spontaneous formation of calcium crystals in adult kidneys. Excessive intake of calcium and oxalate, precursors of the most common type of human renal stones, dramatically increases both the frequency and the severity of renal calcium crystal formation in THP-deficient, but not in wild-type mice. Under high calcium/oxalate conditions, the absence of THP triggers a marked, adaptive induction in renal epithelial cells of osteopontin (OPN), a potent inhibitor of bone mineralization and vascular calcification. Thus, OPN may serve as an inducible inhibitor of calcium crystallization, whereas THP can serve as a constitutive and apparently more effective inhibitor. CONCLUSION: These results provide the first in vivo evidence that THP is a critical urinary defense factor and suggest that its deficiency could be an important contributing factor in human nephrolithiasis, a condition afflicting tens of millions of people in the world annually.     

Urothelial cytoprotective activity of Tamm–Horsfall protein isolated from the urine of healthy subjects and patients with interstitial cystitis

PURPOSE: Tamm-Horsfall protein (THP) is a ubiquitous urinary protein with essentially no known function. We propose that THP is a cytoprotective agent that protects the urothelium from cationic species. To test this hypothesis we isolated THP from normal and interstitial cystitis urine to see if it could protect cultured cells from damage induced by the polyamine, protamine sulfate (PS). METHODS: Tamm-Horsfall protein was extracted from the urine of interstitial cystitis (IC) patients (N=28) and normal volunteers (N=5). Urothelial target cells (T24) were radiolabeled with 51Cr and then exposed to PS (0-1.0 mg/mL) for either 1.5 or 20 h. The resulting cytotoxicity data (dose-response curves) were then compared with the data obtained when PS was preincubated with 0-0.5 mg/mL of THP (IC vs normal), the semisynthetic polysaccharide, pentosan polysulfate (Elmiron), or human serum albumin. RESULTS: Toxicity of PS was significantly reduced by incubation with THP (or Elmiron) prior to evaluation by the chromium release assay, but not reduced by incubating with another protein, albumin. Tamm-Horsfall protein from IC patients' urine was less protective than an equal quantity of THP from normal urine. CONCLUSIONS: These experiments suggest that THP has an important role in bladder mucosal defense mechanisms, protecting the bladder surface from injury. Inability of THP to prevent cytotoxic damage by urinary polyamine or other urinary toxins (cationic species) may be relevant in the etiology of interstitial cystitis, as putative urinary toxic components have been described in the urine of some patients.     

Isolation and purification of a new glycoprotein from human urine inhibiting calcium oxalate crystallization

Summary A calcium oxalate crystal growth inhibitor was isolated from human urine using DEAE-Sephacel gel followed by Sephacryl S-300 chromatography and FPLC column. The isolated inhibitor was a uronic-acid-rich protein (UAP). It was found to be a glycoprotein with a molecular weight of 35 000 Da as determined by SDS-polyacrylamide gel electrophoresis. Inhibitory activity was demonstrated using a calcium oxalate crystallization system. In addition UAP, nephrocalcin (NC) or nephrocalcin-like (NC-like) activity was an effective inhibitor in this system. However, the inhibitory activity of UAP appeared to be higher than that of NC or NC-like activity. This finding suggests that NC or NC-like activity is not only urinary protein with strong inhibitory activity. UAP and probably other proteins also play a role in the control of urinary crystal growth.     

Immunocytochemical localization of Tamm-Horsfall protein in the kidneys of normal and nephrolithic rats

Studies using in vitro systems have indicated that Tamm-Horsfall protein (THP) can interact with calcium oxalate (CaOx) crystals during kidney stone formation. However, information regarding the nature of its participation in this process remains controversial and unclear. In order to better understand the putative interaction of THP and crystals in vivo, we compared the localization of THP in normal rats and in chronic and semi-acute rat models of nephrolithiasis. In these rats, CaOx crystal deposits were induced in the kidneys by administering ethylene glycol (EG) in drinking water. The formation of CaOx mono- and dihydrate aggregates in the urine was confirmed by scanning electron microscopy. Immunohistochemical localization, as well as protein A-gold labeling at the ultrastructural level, demonstrated that in addition to its normal distribution, THP specifically associated with the renal crystal deposits. The THP-containing, organic matrix-like material consisted of a fine, fibrillar meshwork surrounding individual crystals and their aggregates. In addition, THP also appeared in the papilla, where it is normally absent, concurrent with the appearance of crystal deposits in the kidneys. These observations indicate that in nephrolithic rats the normal localization of THP is altered. Such an alteration may indicate an important physiological event related to crystal aggregation and kidney stone formation.     

Characterization of Tamm-Horsfall protein in a rat nephrolithiasis model

PURPOSE: The role of Tamm-Horsfall protein in calcium oxalate stone formation is controversial. It is unclear whether Tamm-Horsfall protein has a role in crystallization. If it does, does it act as an inhibitor or promoter of crystallization? To elucidate the nature of its involvement we characterized Tamm-Horsfall protein in a rat model of calcium oxalate nephrolithiasis by in vivo and in vitro techniques. MATERIALS AND METHODS: Calcium oxalate nephrolithiasis was induced in male Sprague-Dawley rats. The amino acid and carbohydrate composition of Tamm-Horsfall protein from normal rats and those with nephrolithiasis was determined. The Tamm-Horsfall protein gene and protein expression in the kidneys were examined by in situ hybridization and immunohistochemistry. Furthermore, the interaction of Tamm-Horsfall protein and calcium oxalate crystals was assessed by an in vitro crystal aggregation assay. RESULTS: Tamm-Horsfall protein from rats with nephrolithiasis was biochemically similar to that from normal rats. Although Tamm-Horsfall protein was associated with crystal deposits in the renal papillae of rats with nephrolithiasis, Tamm-Horsfall protein messenger RNA expression in the kidneys remained unchanged. In each group Tamm-Horsfall protein inhibited calcium oxalate crystal aggregation by 47%, indicating no change in functional capabilities. CONCLUSIONS: The results of this study indicate that urinary excretion, and the biochemical nature and functional capabilities of Tamm-Horsfall protein remain unchanged during experimental calcium oxalate nephrolithiasis. Although staining for Tamm-Horsfall protein was evident in the papillae of rats with nephrolithiasis, the site of Tamm-Horsfall protein synthesis remained cells of the thick ascending limbs of the loop of Henle.     

Changes in urine macromolecular composition during processing

PURPOSE: To determine the urinary crystallization inhibitory activity, urine is generally centrifuged and/or filtered. These preparative procedures may result in a total or partial removal of many macromolecular constituents implicated in crystallization. The main purpose of this study was to investigate the changes in urinary macromolecular composition following centrifugation and filtration. MATERIALS AND METHODS: Twenty-four hour urine samples were collected from human volunteers. Each was divided into 4 aliquots; one was filtered, the other was centrifuged, another was centrifuged and filtered. The control sample was neither filtered nor centrifuged. Total protein and lipid contents of each sample were determined. Proteins were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using antibodies against osteopontin (OPN), prothrombin (PT) related proteins, inter-alpha-inhibitor (IalphaI) related proteins, Tamm-Horsfall protein (THP), and albumin (ALB). The effect of processing on incorporation of urinary proteins in crystal matrices was also examined. Calcium oxalate crystals were produced in processed and unprocessed urine samples by the addition of sodium oxalate. Crystals were harvested, de-mineralized and their proteins analyzed by SDS-PAGE and Western blotting. RESULTS: Processing reduced the amounts of both proteins and lipids in the urine. Previously we identified phospholipids in the matrix of calcium oxalate crystals as well as the filtrate and retentate removed during filtration and centrifugation. Phospholipids have a high affinity for calcium-containing crystals. In the case of proteins, those with high molecular weights appeared to be clearly affected by filtration and centrifugation. Processing also appeared to influence the incorporation of proteins in the crystals. The matrix of crystals produced in processed urine contained less THP than those made in unprocessed urine, apparently a result of the loss of this higher molecular weight protein during processing. Incorporation of PT-related proteins, particularly fragment 1, was increased. CONCLUSIONS: We propose that selective inclusion of macromolecules is a result of an increase in available binding sites on crystal surfaces because of the removal of certain calcium binding substances such as phospholipids and proteins. Removal of larger macromolecules from the milieu may also provide a better access to the crystal surfaces.     

Tamm-Horsfall protein excretion and its relation to citrate in urine of stone-forming patients

OBJECTIVES: To evaluate the relation of Tamm-Horsfall protein (THP) and citrate, both potent actors in the urinary stone forming process. METHODS: Quantitative determination of THP in calcium oxalate (CaOx) stone-forming patients and healthy subjects was carried out according to the enzyme-linked immunosorbent assay method. RESULTS: THP excretion in 24-hour urine samples of CaOx stone-forming patients was significantly reduced compared with healthy subjects. A significant correlation exists between the concentration of THP and citrate in the stone-forming group, as well as in the group of healthy subjects, and for the 24-hour excretion, this correlation persists in the group of CaOx stone-forming patients. CONCLUSIONS: Decreased THP and citrate excretions were found in CaOx stone-forming patients. They indicate a tubular dysfunction of the distal section.     

Antisera to a rabbit urinary tract antigen also react with human bladder and kidney tissue

The mucin layer covering the bladder transitional cell mucosa appears to function as a primary defense mechanism against bacterial infection. We have previously prepared a glycoprotein fraction (GP1) from the urinary bladder mucosa of NZW rabbits and raised murine antisera against it. These antisera react with bladder, ureter and kidney tissue from rabbits, rats, guinea pigs, and hamsters. We now show that a similar substance occurs in human kidneys and bladder. In order to remove antibodies reactive with the Tamm-Horsfall protein (THP), the antisera were initially absorbed with an immunoadsorbent composed of purified human THP covalently bound to Sepharose CL-4B gel. Using an enzyme linked immunosorbent assay (ELISA) it could be shown that the absorbed antisera did not react with THP but retained a high titer in binding to GP1. Immunohistochemical procedures involving avidin-biotin-immunoperoxidase staining demonstrated that the absorbed anti-GP1 reacted well with six human urinary bladder biopsy specimens and two kidney autopsy specimens while normal murine sera showed little or no binding. Although this reactivity was not as strong as that found with homologous tissue (rabbit) these studies suggest that GP1, an antigen common to several animal species, is also related to a human urinary tract component.     

Tamm-Horsfall protein in patients with kidney damage and diabetes

Tamm-Horsfall protein (THP) is a glycoprotein present abundantly in human urine. It is localized in the thick ascending limb of the loop of Henle (TAL) and the early distal convoluted tubule (DCT). The rate of urinary excretion of THP has been studied in various diabetic groups. It has been postulated that urinary THP may be a useful marker for renal damage. The aim of this study was to compare directly the immunogold localization of THP in diabetic and control kidney tissue specimens with or without kidney damage. Immunogold labeling was performed on archival tissue samples of 34 diabetic and 18 control human kidneys at the light microscope level. Slides were ranked as having a high, moderate or low degree of reaction. The majority of diabetic samples had a slightly lower degree of THP, while patients with known renal dysfunction had lowest THP. Previous studies have found a decreased excretion of urinary THP in diabetics. Our results show that decreased gold labeling is associated with known renal damage and may indicate damage to the thick ascending limb of the loop of Henle and the early distal convoluted tubule, irrespective of presence or absence of diabetes.     

Decreased renal expression of the putative calcium oxalate inhibitor Tamm-Horsfall protein in the ethylene glycol rat model of calcium oxalate urolithiasis

PURPOSE: Tamm-Horsfall protein is believed to inhibit calcium oxalate crystallization, aggregation or adhesion to the renal epithelium. We determined whether ethylene glycol induced urolithiasis changes the expression of renal and urinary Tamm-Horsfall protein. For comparison the expression of another calcium oxalate inhibitor, osteopontin, was also analyzed. MATERIALS AND METHODS: Male rats were treated with 0.75% ethylene glycol plus an AIN-76 diet (Dyets, Bethlehem Pennsylvania) (ethylene glycol group) or standard rat chow and water (control group) for up to 8 weeks (6 per group for 8 weeks and 3 per group for 3 days to 6 weeks). Kidneys and urine (8 weeks only) were harvested and analyzed by Northern and Western blot analysis, and immunohistochemistry. RESULTS: Tamm-Horsfall protein message and protein (membrane bound form) were decreased, while those of osteopontin were increased in the kidneys of rats treated with ethylene glycol for 8 weeks. As judged by immunochemistry Tamm-Horsfall protein and osteopontin were consistently present in a few tubules in rats in the ethylene glycol and control groups, respectively. In urine expression of the free form of Tamm-Horsfall protein (approximately 75 kDa.) was decreased but detectable in ethylene glycol treated rats. Although readily detected in tissue, osteopontin was not detected in the urine of control or ethylene glycol treated rats. In the time course experiment Tamm-Horsfall protein did not decrease until 4 weeks, when calcium oxalate crystals were detectable in the kidneys of treated rats. In contrast, osteopontin was increased, although inconsistently, beginning at 3 days. CONCLUSIONS: Unlike other calcium oxalate inhibitors, such as osteopontin, renal message and protein for Tamm-Horsfall protein was decreased in ethylene glycol treated rats. Tamm-Horsfall protein expression did not decrease until aggregates of crystals had been deposited in the kidneys, while osteopontin expression began to increase almost immediately. Comparisons of the data on kidneys and urine obtained by RNA or protein blot analysis and immunochemistry underscore the need to examine tissue and urine by multiple techniques to obtain the most accurate assessment of how protein expression is changed by a given treatment.     

Tamm-Horsfall glycoprotein-inhibitor or promoter of calcium oxalate monohydrate crystallization processes?

Summary The processes of calcium oxalate monohydrate (COM) crystal nucleation, growth and aggregation (agglomeration) generally have been studied using a wide variety of assay systems/conditions. This paper reviews the apparently conflicting data on the effects of Tamm-Horsfall glycoprotein (THP) on COM crystallization processes in vitro, with the main emphasis on crystal aggregation. According to its well-known physico-chemical properties. THP has a dual role in modifying crystal aggregation: at high pH and low ionic strength (IS), THP is a powerful crystal aggregation inhibitor. Upon lowering pH and rasing IS. THP viscosity increases, leading to reduced crystal aggregation inhibition. In the presence of additional calcium ions, some THPs even become strong promoters of crystal aggregation. This phenomenon seems to be more pronounced in THPs isolated from recurrent calcium stone formers whose proteins exhibit an abnormally high tendency of polymerization. Recent studies suggest an inherited molecular abnormality of THP among some severe recurrent calcium stone formers.     

Investigations on macromolecular precipitation inhibitors of calcium oxalate

Summary Certain important aspects of the urine oxalate tolerance test (OTT) have been revised. The stirring system has been changed and the test has been adapted to the kinetics of calcium oxalate precipitation. True equilibrium conditions are now ensured during the measurements. Furthermore, the endogenous oxalate concentration is determined and taken into consideration. As a result of these changes, the significance of the test results has greatly improved. The effects of the addition of small amounts of zinc on the precipitation of calcium oxalate have been used in a new variation of the OTT. This new test makes it possible to discriminate much faster and more simply between recurrent stone-formers and other subjects. Tamm-Horsfall protein (THP) has been tested for its effect on the precipitation of calcium oxalate by means of OTT. THP inhibits the precipitation of calcium oxalate, but THP of stone-formers has a diminished inhibitory activity. The inhibitory activity of this protein strongly depends on the method by which it is isolated.     

Effects of chondroitin sulphate,human serum albumin and Tamm-Horsfall mucoprotein on calcium oxalate crystallization in undiluted human urine

Summary The effects of physiological concentrations of chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein on the crystallization of calcium oxalate in undiluted, ultrafiltered human urine were investigated using particle size analysis and scanning electron microscopy. Neither the amount of oxalate required to induce detectable calcium oxalate crystal nucleation nor crystal morphology was affected by the presence of any of these macromolecules. Chondroitin sulphate had no effect on the amount of crystalline material deposited or on the size of the particles precipitated in response to a standard oxalate load. Human serum albumin slightly reduced the size of the crystal aggregates and caused a small increase in the amount of crystal matter precipitated. By contrast, Tamm-Horsfall mucoprotein significantly inhibited crystal aggregation and markedly increased the volume of matter deposited, although this could not be attributed to a promotion of solute precipitation. It was concluded that chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein cannot account for the inhibitory effects of macromolecules with a relative mass greater than 10 kDa in spun and filtered urine. Nonetheless, Tamm-Horsfall mucoprotein is likely to inhibit crystal aggregation in whole urine in vivo and may therefore be instrumental in preventing calcium oxalate stone formation.     

Uric acid-binding proteins in calcium oxalate stone formers and their effect on calcium oxalate crystallization.

OBJECTIVES: To study the effect of urinary uric acid-binding proteins of controls and stone formers on calcium oxalate crystal nucleation and aggregation. MATERIALS AND METHODS: Urine samples were collected over 24 h from 20 stone formers and from 20 age-matched normal controls. Uric acid crystallization was induced by adding equal volumes of 2.5 mmol/L uric acid. The bound proteins were separated on a cellulose column, and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The effect of the separated fractions on calcium oxalate crystal nucleation and aggregation was assessed. RESULTS: The protein bound to unit mass of uric acid crystals was higher in hyperoxaluric urine than in control urine. On cellulose-column separation, the uric acid-crystal binding proteins produced three major protein peaks, i.e. fraction I (buffer), fraction II (0.05 mol/L sodium chloride in Tris-HCl buffer) and fraction III (0.3 mol/L sodium chloride in buffer), with a minor peak obtained on elution with increasing concentrations of sodium chloride in Tris-HCl buffer (pH 7.0). Fraction I derived from either stone formers or controls promoted calcium oxalate crystallization. Fraction II from the control samples was a strong inhibitor, whereas hyperoxaluric fraction II was less inhibitory. CONCLUSION: Uric acid-binding proteins isolated either from the urine of stone formers or controls modulated calcium oxalate crystal growth. Proteins isolated from stone formers were less inhibitory of crystal nucleation and aggregation. These proteins may act as a bridge, leading to the epitaxial deposition of calcium oxalate over a urate core.     

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998     

Possible role of Tamm-Horsfall glycoprotein in calcium oxalate crystallisation

The role of Tamm-Horsfall glycoprotein (THGP) in the crystallisation of calcium oxalate was investigated. The results showed THGP to have a weak inhibitory effect on crystal growth. In contrast, urinary macromolecules showed a strong inhibitory effect. THGP should, therefore, not be considered the main component of urinary macromolecules which have a strong inhibitory effect on crystal growth. However, THGP did enhance crystal production in the early phase of crystallisation. It appears that THGP should be viewed as a promoter rather than an inhibitor of calcium oxalate crystallisation at physiological concentrations.     

Urinary macromolecular inhibition of crystal adhesion to renal epithelial cells is impaired in male stone formers

BACKGROUND: Retention of microcrystals that form in tubular fluid could be a critical event in kidney stone formation. This study was performed to determine if urinary macromolecules from stone-forming (SF) individuals have reduced ability to inhibit crystal adhesion to renal cells. METHODS: A first morning whole urine (WU) sample was obtained from 24 SF subjects (17 males and 7 females) and 24 age-, race-, and sex-matched controls (C). An aliquot of urine was centrifuged and an ultrafiltrate (UF) free of macromolecules >10 kD and 10x concentrate (U(conc)) were prepared. RESULTS: Supplementing UF with increasing amounts of U(conc) to return the macromolecule concentration to 0.25x, 0.5x, or 1x of baseline progressively decreased crystal binding to cells. This effect was blunted in the male SF group compared to controls (P < 0.05, SF vs. C, for UF plus 0.25x macromolecules). No difference was apparent in the female groups. In order to identify responsible macromolecule(s), calcium oxalate monohydrate (COM) crystals were coated with U(conc) and adherent proteins then released and probed by Western blot. Coated COM crystals from male controls contained 3.5-fold more Tamm-Horsfall protein (THP) than SF subjects (P < 0.01). COM crystal coating with other proteins did not consistently differ between the groups. COM crystal coating by urinary prothrombin fragment 1 (UPTF1, P < 0.05) and crystal adhesion inhibitor (CAI) (P= 0.09) correlated with decreased crystal binding to cells, whereas coating with osteopontin (OPN) correlated with increased adhesion tendency (P < 0.05). CONCLUSION: Urinary macromolecules >10 kD coat COM crystals and block their adhesion to renal cells. This capacity appears to be blunted in male but not female SF individuals. Multiple urinary proteins may play a role in renal cell-urinary crystal interactions, and THP appears to be one of the more important ones.     

Modifiers of calcium oxalate crystallization found in urine. I. Studies with a continuous crystallizer using an artificial urine

Various modifiers of the rate of crystallization of calcium oxalate (CaOx) known to be present in urine were studied in a continuous crystallizer system under conditions approximating those in whole urine. Of the small ions examined, magnesium and citrate were only weak inhibitors of the crystal growth rate and degree of agglomeration of CaOx within the urinary range of concentration of these ions. At and beyond the upper limit of the normal, however, both ions became much more active mainly through their ability to complex oxalate and calcium ions respectively. Pyrophosphate showed similar characteristics although its ability to act as an inhibitor at high concentrations was not due to complexation of calcium ions but to adsorption onto the crystal surface of the freshly generated CaOx crystals. The polyanionic inhibitors, chondroitin-4-sulphate, heparin and ribonucleic acid (RNA), also acted as adsorption inhibitors of growth and agglomeration. Of all the inhibitors tested RNA was the most active within the concentration range found in normal urine. Under the conditions of ionic strength employed, it was not possible to measure the effect of Tamm-Horsfall mucoprotein satisfactorily since it polymerized to form particles sufficiently large to interfere with the true crystal counts of CaOx in the test system.     

Effects of Tamm-Horsfall protein and albumin on the inhibition of free radicals.

INTRODUCTION: Oxalate in urine can cause tubular cellular damage by the production of free radicals. Then, cell death and cellular debris may promote the retention of calcium oxalate crystals and finally the formation of stones. The two most abundant urinary proteins, Tamm-Horsfall protein (THP) and albumin, were tested for the effects of antioxidants. MATERIALS AND METHODS: By using xanthine-xanthine oxidase reaction, purified THP and albumin were tested for the inhibitory effect. OD(295) was used as a spectrophotometric method to measure the production of uric acid during the reaction. RESULTS AND CONCLUSIONS: Both proteins can inhibit the reaction of xanthine oxidase on xanthine, although the effect was decreased after enzymatic deglycosylation of sialic acid. Albumin has an IC(50) of 10.7 nM in native condition and 11.9 nM after deglycosylation, whereas THP has 69.6 nM in native condition and 102.0 nM in deglycosylated condition. The data indicates that THP and albumin have an antioxidant effect. Sialic acid in THP has partly an inhibitory effect and is associated with calcium oxalate formation. Studies have indicated that further investigation of the role of free radicals in the formation of urolithiasis and of sialic acid in protein function is needed.