Urinary Tamm-Horsfall Glycoprotein Research Articles

The relationship between urinary Tamm-Horsfall glycoprotein excretion and urinary activity of glycosidases in normal pregnancy and pre-eclampsia

Electrophoretic analyses of the urinary proteins of pre-eclamptic patients revealed a decrease in the staining intensity of the protein band representing the Tamm-Horsfall glycoprotein (THP). In the present study the quantitative analysis of the THP excretion rate and the urinary activity of THP oligosaccharide metabolizing glycosidases were investigated. The median THP excretion rate of non-pregnant women ( n = 24) was 20 mg/g creatinine (crea.). An increase in the THP excretion rate was seen in pregnancy to a level between 43 mg/g crea. (II. trimester) and 32 mg/g crea. (III. trimester) ( n = 29). Hypertension in pregnancy was associated with a decrease in the THP excretion rate to 9 mg/g crea. ( n = 85). Post partum, a transient elevated THP excretion rate up to 109 mg/g crea was recorded in the group of hypertensive patients. The urinary activities of the lysosomal β-mannosidase, α-fucosidase, α-mannosidase (pH 4.5) and β-galactosidase increased in normal pregnancy. This effect was most pronounced in the β-galactosidase activity which increased from 50 U/mg crea. before pregnancy to 280 U/mg crea. at term. Hypertension in pregnancy was associated with a further increase in the activities of the lysosomal glycosidases. In the case of the β-galactosidase a significant rise from 68 to 310 U/mg crea. was found. The urinary activity of the α-mannosidase (pH 5.5) originating from the Golgi apparatus was only elevated in patients with severe pre-eclampsia. Casuistic post partum recordings demonstrated that an elevation of the lysosomal glycosidases activities was followed by a transient increase in the THP excretion rate.

Antibody-dependent cell-mediated cytotoxicity against Tamm-Horsfall protein in acute pyelonephritis.

The cytotoxic activity of leukocytes from humans and rats with pyelonephritis were examined in an antibody-dependent cell-mediated cytotoxicity assay (ADCC) with CrCl3-treated erythrocytes coated with Tamm-Horsfall (TH) as target cells. The specificity of the ADCC was confirmed by absorption with TH urinary glycoprotein and inhibition of the ADCC activity seen with polyclonal rabbit anti-TH antisera by monoclonal mouse antibodies. The ADCC activity detected in children with acute pyelonephritis was low in the initial phase of the disease, but increased significantly 9 days after the start of antibacterial treatment. In rats with experimental pyelonephritis, ADCC activity decreased significantly with increased duration of infection. Depletion of cells adhering to carbonyl iron led to higher ADCC activity. During the course of the infection the difference in ADCC activity between effector cell preparations depleted using carbonyl iron and those not depleted decreased. The decreased ADCC activity demonstrated during acute pyelonephritis may point to mechanisms operating to diminish the risk of tissue damage.

Uromodulin (Tamm-Horsfall glycoprotein): a renal ligand for lymphokines.

The protein portion of the immunosuppressive glycoprotein uromodulin is identical to the Tamm-Horsfall urinary glycoprotein and is synthesized in the kidney. Evidence that the glycoproteins are the same is based on amino acid sequence identity, immunologic cross-reactivity, and tissue localization to the thick ascending limb of Henle's loop. Nucleic acid sequencing of clones for uromodulin isolated from a complementary DNA bank from human kidney predicts a protein 639 amino acids in length, including a 24--amino acid leader sequence and a cysteine-rich mature protein with eight potential glycosylation sites. Uromodulin and preparations of Tamm-Horsfall glycoprotein bind to recombinant murine interleukin-1 (rIL-1) and human rIL-1 alpha, rIL-1 beta, and recombinant tumor necrosis factor (rTNF). Uromodulin isolated from urine of pregnant women by lectin adherence is more immunosuppressive than material isolated by the original salt-precipitation protocol of Tamm and Horsfall. Immunohistologic studies demonstrate that rIL-1 and rTNF bind to the same area of the human kidney that binds to antiserum specific for uromodulin. Thus, uromodulin (Tamm-Horsfall glycoprotein) may function as a unique renal regulatory glycoprotein that specifically binds to and regulates the circulating activity of a number of potent cytokines, including IL-1 and TNF.

Loop-acting diuretics do not bind to Tamm-Horsfall urinary glycoprotein.

1. Binding between the radiolabelled loop-acting diuretics ([14C]frusemide, [14C]ethacrynic acid and [3H]bumetanide) and human Tamm-Horsfall glycoprotein or human serum albumin in vitro was evaluated by equilibrium dialysis. 2. The diuretic action and binding to urinary Tamm-Horsfall glycoprotein of the radiolabelled diuretics in vivo, after intravenous administration, were examined in rabbits. 3. In vitro, all three radiolabelled diuretics bound strongly to human serum albumin, but not to Tamm-Horsfall glycoprotein. 4. Radiolabelled frusemide and bumetanide, but not ethacrynic acid, caused a diuresis in rabbits, but no binding between the drugs and Tamm-Horsfall glycoprotein was seen in vivo. 5. Binding to Tamm-Horsfall glycoprotein does not appear to be an important mechanism in the action of loop diuretics.

Excretion of urinary Tamm-Horsfall glycoprotein in girls with recurrent urinary tract infections.

Tamm-Horsfall glycoprotein (THG) might constitute a bacteria-fixing mucus which contributes to the non-immunological anti-infectious mechanisms of the lower urinary tract. To evaluate the role of THG in girls with idiopathic recurrent lower urinary tract infections, the THG excretion and concentration in 24-h urine were measured by a radial immunodiffusion method in 16 patients with a median age of 9 years and in 14 healthy age-matched girls. The results showed no significant differences in the THG excretion or concentration between the patients and the controls. Transiently decreased THG excretion rates as well as functional changes in the ability and/or capacity to trap bacteria, however, may leave girls periodically prone to colonization of the bladder. Thus, further studies are warranted to evaluate the importance of THG in the bladder defence mechanism.

Urinary Tamm-Horsfall glycoprotein in normal subjects.

Urinary Tamm-Horsfall glycoprotein in normal subjects.

A sensitive and specific ELISA using a monoclonal capture antibody for detection of Tamm-Horsfall urinary glycoprotein in serum

An enzyme-linked immunosorbent assay (ELISA) has been established using Nunc polystyrene immunoplates coated with a monoclonal antibody to human Tamm-Horsfall urinary glycoprotein (THP) to detect and measure THP in human serum. Optimal reaction conditions for both the monoclonal capture antibody and the affinity-purified rabbit anti-human THP second antibody were established to produce standard curves which showed linearity between 20–90 ng/ml with a sensitivity of 2–3 ng/ml. The plate-to-plate standard curve mean coefficient of variation (CV) was 5.9 ± 2.9% on assays performed on the same day while day to day mean CV was 13.3 ± 2.4%. The specificity of the ELISA was demonstrated by inhibition of binding after preincubation of both urinary THP standards and serum with monoclonal anti-THP antibody. Sera from 195 blood donors tested by the ELISA had a mean concentration of THP antigenic determinants of 260 ± 105 ng/ l. Results from three control sera run on all plates used in the survey showed mean CV < 7.6% while no binding was observed with sera from an anephric patient.

Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study

Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted.

Effect of Tamm-Horsfall urinary glycoprotein on phagocytosis and killing of type I-fimbriated Escherichia coli.

Human polymorphonuclear leukocytes (PMN) ingest type I (mannose sensitive) fimbriated Escherichia coli even in the absence of antibody, complement, or other serum opsonins. Our studies suggest that the Tamm-Horsfall urinary glycoprotein (THP) interferes with serum-independent ingestion. Electron micrographs showed that dissolved THP adhered to type I fimbriae and formed a pseudocapsule around bacteria bearing type I fimbriae. Phase-variant bacteria grown on blood agar neither expressed fimbriae nor bound THP. Affinity column chromatography demonstrated mannose-sensitive binding between purified type I fimbriae and purified THP. The ability of human PMN to bind and ingest type I-fimbriated E. coli was diminished if the bacteria had been coated by exposure to THP at physiologic concentrations. At 1 h, PMN were associated with an average of 2.62 uncoated bacteria, but with only 0.18 coated bacteria (P less than 0.001). alpha-Methyl mannoside blocked the observed effect of THP on binding and phagocytosis in a dose-dependent fashion: increased mannoside led to increased blocking. PMN preincubated with THP were able to bind and phagocytose normally. There did not appear to be any significant clumping of bacteria in suspension to account for these effects. Bactericidal assays with leukocytes in suspension demonstrated protection of THP-coated bacteria. At 1 h, PMN killed 42% of noncoated E. coli (a decrease of 0.24 log), but the number of THP-coated bacteria increased by 75% (an increase of 0.24 log). These observations may partially explain the virulence of E. coli in the bladder and kidney, where serum activity is low and THP is abundant.

Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

Human Tamm-Horsfall glycoprotein: urinary and plasma levels in normal subjects and patients with renal disease determined by a fully validated radioimmunoassay.

A recently developed, simple and sensitive radioimmunoassay has been used to examine 24 h excretion and plasma levels of Tamm-Horsfall glycoprotein (THG) in normal subjects, stone formers and patients with stable chronic renal disease. In normal subjects THG excretion ranged from 22 to 66 mg/24 h, with no sex difference and no correlation with creatinine clearance or body surface area. There was no correlation between 24 h THG excretion and urine volume, pH or osmolality, excretion of Na+, K+ or Ca2+ or free-water clearance. There was a small significant correlation between plasma THG concentration and urinary THG excretion. A good correlation was obtained between the THG/creatinine ratio in 24 h and random samples. This made possible the use of random samples to establish a reference range for THG excretion of 0.15-0.50 micrograms/ml of creatinine clearance which did not depend on sex or age. The excretion rate of THG in stone formers was generally within the reference range. It was not significantly different in those who were hypercalciuric or in those taking thiazides. In patients with chronic renal disease there was a good correlation between 24 h THG excretion, plasma THG concentration and creatinine clearance. The range of excretion of THG per ml of creatinine clearance was greater than in normal subjects, independent of the type of renal disease and unrelated to proteinuria. In patients with glomerulonephritis the excretion of THG per ml of creatinine clearance was significantly higher in those with well-preserved tubules compared with those with tubular atrophy.

Radioimmunoassay for serum and urinary Tamm-Horsfall glycoprotein in the rat.

Rat Tamm-Horsfall glycoprotein (THG) was isolated from urine and disaggregated by gel filtration. Purity was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Antibodies were raised and used in the development of a specific radioimmunoassay for rat THG. Parallelism could be demonstrated between the standard preparation and urine (diluted to 1:320 with distilled water) and serum (at all dilutions) samples. Recovery of standard added to urine or serum was on average 93% and inter- and intra-assay variation were both below 7%. There was a positive correlation between the mean urinary excretory rate of THG (microgram/24h) and body weight in female rats (p less than 0.001) but not in male rats. Serum THG levels were similar in both sexes and showed a positive correlation with body weight (p less than 0.001; range 43-240 ng/ml). It is concluded that the rat urinary THG excretion rate is similar to that in man and that rat serum also contains a THG-like protein.

Tamm-Horsfall glycoprotein excretion and aggregation during intravenous urography. Relevance to acute renal failure.

The effect of intravenous urography (IVU) on the excretion rate and precipitation of Tamm-Horsfall glycoprotein (THG) has been evaluated in 19 patients. Urine samples were collected immediately before the administration of contrast medium (U1) and then during the period of the excretion urogram (U2). The urinary THG: creatinine clearance ratio decreased in 15 patients by a mean of 28.2% (range 2.9-70.8) and increased in four by a mean of 22.8% (range 13.5-36.4). In 18 of 19 patients the amount of THG precipitated following the IVU decreased from an average of 75.6% of the total in U1 (range 2.2-100) to 19.5% (range 2.0-59) in U2. In the one exception the fraction precipitated was only 2.2% in U1 and 2.5% in U2. It is concluded that during routine IVU there is no marked increase in the excretion rate of THG. More importantly, the percentage of THG precipitated actually decreases.

Glycosyl transferases of baby hamster kidney cells and ricin-resistant mutants. O-glycan biosynthesis.

Extracts of baby hamster kidney (BHK) cells catalyzed the incorporation of N-acetylgalactosamine from UDP-N-acetyl[14C]galactosamine into myelin basic protein and an acylated tetrapeptide, N-acetylthreonyl-triproline, based on the threonine residue 98, glycosylated in myelin basic protein. The incorporated N-acetylgalactosamine residues were shown to be in alpha linkage to the peptide moieties. Several ricin-resistant BHK cell lines contained enhanced (approximately twofold) levels of the transferase activity. Apomucins obtained from bovine submaxillary gland mucin by chemical or enzymic degradation were relatively poor acceptors. Using asialomucin as acceptor, galactosyl, transferase activities and a weak sialyl transferase activity were detected in BHK cell extracts. Galactose transfer occurred at two sites: to peptide-linked N-acetylgalactosamine residues to form the linkage, galactosyl-(beta 1 leads to 3)-N-acetylgalactosamine and to terminally linked N-acetylglucosamine residues that exist as a minor constituent in bovine submaxillary mucin O-glycans, to form a galactosyl N-acetylglucosamine linkage. This reaction was not inhibited by ovalbumin, an efficient acceptor of the beta 1 leads to 4 galactosyl transferase involved in N-glycan assembly. Incorporation of galactose and N-acetylgalactosamine into endogenous proteins of BHK cell extracts was also detected. Sialic acid, fucose and N-acetylglucosamine residues were not incorporated. The incorporated N-acetylgalactosamine residues were shown to be in alpha linkage to polypeptide, and galactose incorporation represented synthesis of the galactosyl-(beta 1 leads to 3)-N-acetylgalactosamine sequence linked to polypeptide. The major endogenous protein labelled by either sugar had a molecular weight of approximately 80 000. A BHK-cell-associated glycoprotein, analogous to the urinary Tamm-Horsfall glycoprotein of molecular weight similar to the major endogenous acceptor of glycosylation, was not glycosylated in the experiments in vitro.

Antibodies to Tamm-Horsfall urinary glycoprotein in patients with urinary tract infection, reflux nephropathy, urinary obstruction and paraplegia.

Antibodies to Tamm-Horsfall urinary glycoprotein in patients with urinary tract infection, reflux nephropathy, urinary obstruction and paraplegia.

Development of a radioimmunoassay for ‘Tamm-Horsfall-like’ glycoprotein in serum and cerebrospinal fluid

Affinity chromatography purification was combined with a radioimmunoassay for ‘Tamm-Horsfall-like’ glycoprotein. This enabled serum concentrations to be established and to demonstrate its presence in cerebrospinal fluid for the first time. This assay method used in different circumstances suggests a multifocal synthesis. Nevertheless, urinary Tamm-Horsfall glycoprotein so far must be distinguished from the serum or cerebrospinal fluid Tamm-Horsfall-like glycoprotein.

A blood group Sd a-active pentasaccharide isolated from Tamm-Horsfall urinary glycoprotein

Human Tamm-Horsfall urinary glycoprotein from an individual of the blood group Sd(a+) phenotype was tritium-labelled by treatment with galactose oxidase and sodium boro[3H]hydride and was then digested with endo-beta-galactosidase. A series of dialysable, labelled fragments was released from which a pentasaccharide was isolated that strongly inhibited the agglutination of Sd(a+) red cells by human anti-Sda serum and hence contained the Sda determinant structure. Reduction, methylation analysis and sequential exo-glycosidase digestion established the structure of the pentasaccharide as: GalNAc beta(1 leads to 4)[NeuAc(2 leads to 3)]Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3)Gal

Tamm-Horsfall glycoprotein in human amniotic fluid

Radioimmunoassay of material cross-reacting with urinary Tamm-Horsfall glycoprotein in human amniotic fluid was carried out. The cross-reacting material was shown to be identical with urinary Tamm-Horsfall glycoprotein by double immunodiffusion and by crossed immunoelectrophoresis. The concentrations of the substance from 16–25 weeks pregnancy were found to increase from average levels of 0.9 mg · 1 −1 to 4.9 mg · 1 −1 over this period. The concentrations were considerably higher (up to 33 mg · 1 −1) close to term. Measurements of the glycoprotein in amniotic fluid may be a useful index for development of renal function in the foetus.

Forensic Identification of Human Urine by Radioimmunoassay for Tamm-Horsfall Urinary Glycoprotein

A solid phase radioimmunoassay for the identification of human urine has been developed, utilizing affinity-purified rabbit anti-human Tamm-Horsfall protein. This antiserum exhibited no cross-reactivity with human body fluids and showed some specificity for human urine over urine from several animal species. Conditions for the reliable identification of human urine in stains on cotton cloth have been determined. The effects of age and storage conditions on this and other materials are also described.

An improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein. Investigation and resolution of factors affecting its quantification

A rapid, specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. A dilution of 1 in 100 was chosen for routine assay. Whole urine was centrifuged and the dissolved precipitate and supernatant assayed to quantify the proportion of TH glycoprotein of TH glycoprotein initially present in highly aggregated form. This correlated positively and significantly with increasing osmolality, decreasing pH and increasing TH glycoprotein concentration. When the urine was diluted 1 in 100 in water, no TH glycoprotein was precipitated by centrifugation and the measured concentrations were unaffected by alterations of urine pH or calcium concentration or by addition of sodium dodecyl sulphate. Parallelism was demonstrated between the diluted samples and the disaggregated standard preparation. Recovery of added standard to diluted urine varied between 96 and 114%. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4 degrees C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22--56 mg/24 h, based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance.