anti-THP Antibodies Research Articles

Development of Lateral Flow Assay for the Detection of Tamm Horsfall Protein in Urine

Tamm Horsfall Protein (THP) is the most abundant urinary protein with concentrations of 20-200 mg/day and is known to be involved in a myriad of renal pathological processes such as renal stones and inflammation. THP is produced by renal epithelial cells and is the most abundant urinary protein, which tends to aggregate at higher concentrations. Therefore, our study was aimed to facilitate the detection of THP in urine by a rapid, efficient, and optimum detection using a lateral flow assay system. To develop the assay system, THP was isolated and purified from the urine of a healthy individual and used to generate antibodies in a white male New Zealand rabbit. These anti-THP antibodies were purified from the rabbit serum using the Protein-A column and further conjugated with gold nanoparticles. Gold nanoparticles (GNPs) of the size of 21 nm were synthesized by the chemical reduction method. Conjugation of gold nanoparticles and anti-THP antibodies was optimized by varying pH and concentrations. The developed lateral flow assay was tested with purified THP, and its sensitivity was calculated as 1 mg/ml of THP. This lateral flow immunoassay can detect THP in urine and can be beneficial for the detection of a various pathological or physiological phenomenon involving urinary Tamm Horsefall Protein secretions.

Targeting of Receptor for Advanced Glycation End Products Suppresses Cyst Growth in Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Although a myriad of research groups have attempted to identify a new therapeutic target for ADPKD, no drug has worked well in clinical trials. Our research group has focused on the receptor for advanced glycation end products (RAGE) gene as a novel target for ADPKD. This gene is involved in inflammation and cell proliferation. We have already confirmed that blocking RAGE function attenuates cyst growth in vitro. Based on this previous investigation, our group examined the effect of RAGE on cyst enlargement in vivo. PC2R mice, a severe ADPKD mouse model that we generated, were utilized. An adenovirus containing anti-RAGE shRNA was injected intravenously into this model. We observed that RAGE gene knockdown resulted in loss of kidney weight and volume. Additionally, the cystic area that originated from different nephron segments decreased in size because of down-regulation of the RAGE gene. Blood urea nitrogen and creatinine values tended to be lower after inhibiting RAGE. Based on these results, we confirmed that the RAGE gene could be an effective target for ADPKD treatment.

Tamm-Horsfall protein in recurrent calcium kidney stone formers with positive family history: abnormalities in urinary excretion, molecular structure and function

Tamm-Horsfall protein (THP) powerfully inhibits calcium oxalate crystal aggregation, but structurally abnormal THPs from recurrent calcium stone formers may promote crystal aggregation. Therefore, increased urinary excretion of abnormal THP might be of relevance in nephrolithiasis. We studied 44 recurrent idiopathic calcium stone formers with a positive family history of stone disease (RCSF(fam)) and 34 age- and sex-matched healthy controls (C). Twenty-four-hour urinary THP excretion was measured by enzyme linked immunosorbent assay. Structural properties of individually purified THPs were obtained from analysis of elution patterns from a Sepharose 4B column. Sialic acid (SA) contents of native whole 24-h urines, crude salt precipitates of native urines and individually purified THPs were measured. THP function was studied by measuring inhibition of CaOx crystal aggregation in vitro (pH 5.7, 200 mM sodium chloride). Twenty-four-hour urine excretion of THP was higher in RCSF(fam) (44.0 +/- 4.0 mg/day) than in C (30.9 +/- 2.2 mg/day, P = 0.015). Upon salt precipitation and lyophilization, elution from a Sepharose 4B column revealed one major peak (peak A, cross-reacting with polyclonal anti-THP antibody) and a second minor peak (peak B, not cross-reacting). THPs from RCSF(fam) eluted later than those from C (P = 0.021), and maximum width of THP peaks was higher in RCSF(fam )than in C (P = 0.024). SA content was higher in specimens from RCSF(fam) than from C, in native 24-h urines (207.5 +/- 20.4 mg vs. 135.2 +/- 16.1 mg, P = 0.013) as well as in crude salt precipitates of 24-h urines (10.4 +/- 0.5 mg vs. 7.4 +/- 0.9 mg, P = 0.002) and in purified THPs (75.3 +/- 9.3 microg/mg vs. 48.8 +/- 9.8 microg/mg THP, P = 0.043). Finally, inhibition of calcium oxalate monohydrate crystal aggregation by 40 mg/L of THP was lower in RCSF(fam) (6.1 +/- 5.5%, range -62.0 to +84.2%) than in C (24.9 +/- 6.0%, range -39.8 to +82.7%), P = 0.022, and only 25 out of 44 (57%) THPs from RCSF(fam )were inhibitory (positive inhibition value) vs. 25 out of 34 (74%) THPs from C, P < 0.05. In conclusion, severely recurrent calcium stone formers with a positive family history excrete more THP than healthy controls, and their THP molecules elute later from an analytical column and contain more SA. Such increasingly aggregated THP molecules predispose to exaggerated calcium oxalate crystal aggregation, an important prerequisite for urinary stone formation.

Immunomorphometric Studies of Proteinuria in Individual Deep and Superficial Nephrons of Rats

Heterogeneity of structure and function among nephrons is a well-recognized feature of chronic renal diseases. However, only a small number of superficial nephrons per kidney are accessible for micropuncture analysis and relationships of proteinuria to structural change in individual nephrons of experimental models are not clearly established. To directly evaluate proteinuria in many individual nephrons, we developed an immunomorphometric method of analysis. This method is based on the uniformly abundant renal synthesis of Tamm-Horsfall protein (THP) in the thick ascending limb of Henle's loop (TAL). Luminal rabbit immunoglobulin G (IgG) deposits are formed in TALs of proteinuric nephrons in rats injected with heterologous IgG anti-THP antibodies. This immunomorphometric luminal deposit method of assessing proteinuria was previously validated through analysis of heterologous immune complex nephropathy. Glomerular dysfunction in several models—spontaneously hypertensive rats (SHR), aging Sprague-Dawley (SD) rats, rats with adriamycin nephropathy (ADR), and rats subjected to subtotal nephrectomy (NX)—was characterized by immunomorphometric analysis after injection of anti-THP antibodies. Luminal IgG deposits were used to identify nephrons with increased proteinuria. Nephrons were identified histologically as either long looped (LL) or short looped (SL), and frequency of luminal deposits in these nephrons was determined. Glomerular size and sclerosis in deep and superficial zones of renal cortex were determined. Luminal deposits in LL nephrons were more frequent than luminal deposits in SL nephrons in SHRs (p < .001) and aging rats (p < .001) and SL nephrons in ADR rats (p < .02). Whole kidney levels of albuminuria correlated closely with the frequency of luminal deposits in both LL and SL nephrons of SHRs and ADR rats and in LL nephrons of aging rats (p < .005). In contrast, LL and SL deposits were equal in NX rats and did not correlate with albuminuria. A majority of luminal deposits extended beyond the first medullary TAL zone of NX rats, but was confined to this zone in the other 3 models. Deep cortical glomeruli were larger with more glomerulosclerosis than superficial cortical glomeruli. Albuminuria correlated with sclerosis of both deep (p < .002) and superficial (p < .01) glomeruli in NX rats, but not in the other three models. These studies provide a detailed characterization of a new method that allows comparison of proteinuria derived from deep and superficial nephrons. They also provide evidence that pathogenesis of the glomerulosclerosis in NX rats differs from that of the other three models. Glomerulosclerosis was closely linked to the overall level of albuminuria in NX rats, but not to luminal deposits. In the other three models, albuminuria and luminal deposits were closely linked but did not correlate with glomerulosclerosis. Furthermore, LL and SL nephron proteinuria of NX rats was comparable while LL proteinuria was markedly greater than SL proteinuria in the other three models. The luminal deposit method provides a new way to analyze heterogeneity of proteinuria among nephrons and the mechanisms underlying structural change in experimental glomerular diseases.

Antibodies to Tamm-Horsfall protein in endemic nephropathy.

The aim of the study was to investigate the possible role of antibodies to Tamm-Horsfall protein (anti-THP) in the early diagnosis of endemic nephropathy (EN). Anti-THP (IgA, IgM, IgG classes) antibodies were determined by direct ELISA in a random sample of 159 subjects from the endemic village of Kaniza who were divided into four groups according to the WHO criteria, i.e., 'diseased', 'suspect', 'at risk', and 'others'. These groups were compared to subjects from the non-endemic village of Klakar and healthy subjects from Zagreb. No differences for all the classes of antibody were observed among the groups of subjects from the endemic village of Kaniza (P>0.05) or between these subjects and those from the non-endemic village of Klakar (P>0.05). The values of IgM anti-THP antibodies exceeded those of the IgA and IgG classes in all groups of subjects (P<0.05). The values for all three classes of antibodies were higher in the rural than the urban population (P<0. 05). Determination of anti-THP antibodies was not found to be useful in the early diagnosis of endemic nephropathy. The results suggest that most of the anti-THP antibodies are 'natural' and/or cross reactive. The highest values observed in the rural population could probably be explained by exposure to some ubiquitous antigen or more likely they are consequences of fever.

Decreased Tamm-Horsfall Protein in Lithiasic Patients

Abstract Objectives: Tamm-Horsfall glycoprotein (THP) is the most abundant substance of renal origin appearing in urine. It seems to be one of the major inhibitors of calcium oxalate crystal nucleation and/or aggregation, but its role in the pathogenesis of stone formation has not yet been clarified. The present work was undertaken to quantify THP excretion in lithiasic (L) patients, so as to deterline if these levels are different from those of control subjects (C). Design and Methods: THP was isolated from human urine by reprecipitation steps, rabbit antiTHP antibody was obtained and its specificity determined, an ELISA was developed and technical conditions standardized, and quantitative measurements of urinary THP were performed on samples from L patients, who had suffered more than one lithiasic episode, and from C subjects. Microtiter plates coated with THP or diluted urine samples were subjected to successive incubation with antiTHP and alkaline phosphatase antirabbit IgP. Results: A good correlation between measured absorbance and THP concentration in standard and urine samples was observed. Data, expressed as Median and Interquartile Range, are 388/209–626 μg THP/mmol creatinine for C ( n = 85) and 124/82–171 μg THP/mmol creatinine for L ( n = 23). Conclusions: We have obtained an antiserum antiTHP that can be used in the ELISA technique to determine reliable urinary THP values. The results show a significant decrease of THP excretion in recurrent stone formers compared to controls ( p

Decreased Tamm-Horsfall protein in lithiasic patients

Objectives: Tamm-Horsfall glycoprotein (THP) is the most abundant substance of renal origin appearing in urine. It seems to be one of the major inhibitors of calcium oxalate crystal nucleation and/or aggregation, but its role in the pathogenesis of stone formation has not yet been clarified. The present work was undertaken to quantify THP excretion in lithiasic (L) patients, so as to deterline if these levels are different from those of control subjects (C). Design and Methods: THP was isolated from human urine by reprecipitation steps, rabbit antiTHP antibody was obtained and its specificity determined, an ELISA was developed and technical conditions standardized, and quantitative measurements of urinary THP were performed on samples from L patients, who had suffered more than one lithiasic episode, and from C subjects. Microtiter plates coated with THP or diluted urine samples were subjected to successive incubation with antiTHP and alkaline phosphatase antirabbit IgP. Results: A good correlation between measured absorbance and THP concentration in standard and urine samples was observed. Data, expressed as Median and Interquartile Range, are 388/209–626 μg THP/mmol creatinine for C ( n = 85) and 124/82–171 μg THP/mmol creatinine for L ( n = 23). Conclusions: We have obtained an antiserum antiTHP that can be used in the ELISA technique to determine reliable urinary THP values. The results show a significant decrease of THP excretion in recurrent stone formers compared to controls ( p < 0.001) and may have interesting implications in the pathogenesis of urolithiasis.

Tamm-Horsfall protein in bladder tissue. Morphologic spectrum and clinical significance.

Tamm-Horsfall protein (THP) is a high-molecular-weight glycoprotein synthesized exclusively by the ascending loop of Henle and the distal tubule of normal kidney. In pathologic conditions, THP may accumulate in renal parenchyma, perirenal soft tissue, or renal hilar lymph nodes. Our recent finding of a cystectomy specimen showing large mural deposits of THP prompted a pertinent literature search, which uncovered only a single article in which THP deposition in bladder is briefly mentioned. In the current study, the clinical and morphologic features of THP were studied in 247 consecutive bladder biopsies and 15 cystectomies obtained in a 1-year period. A total of 18 cases were found (an incidence of 6.9%), with cystectomy specimens being much more frequently affected than biopsy specimens (60 versus 3.6%). Most patients were elderly men (45-78 years, with a mean 61 years; a male/female ratio of 16:2). In seven cases, THP appeared as large, "waxy," pale or weakly eosinophilic masses, so characteristic that the diagnosis could be readily made without any special studies. THP in these cases was strongly positive by periodic acid-Schiff, pale blue on Masson's trichrome stain, and ultrastructurally composed of nonbranching 4-nm-wide fibrils arranged in a parallel fashion. In 11 cases, THP appeared as inconspicuous flecks or interconnecting strands of eosinophilic material obscured by a large amount of adjacent fibrinous exudate or necrotic tissue. In these cases, the PAS and trichrome stains were not always helpful in the diagnosis. Immunostaining using an anti-THP antibody clearly identified even small amounts of THP in all 18 cases. This immunostaining was not only sensitive but also specific, giving a negative result for 64 control cases containing such materials as amyloid, fibrin, dense fibrosis, tissue necrosis, and edema fluid, all of which can potentially simulate THP. Although diagnoses for the specimens with THP were variable (nine transitional cell carcinomas, one squamous cell carcinoma, two nephrogenic adenomas, and five cases of cystitis), the areas where THP was deposited in each of these cases invariably showed necrosis, inflammation, fibrinous exudate, ulcer, or crystalline material. The mechanism for THP precipitation is not clear but is probably related to the mucosal changes, including inflammation and necrosis, which is always seen in areas of THP deposition. Follow-up study did not show any bladder abnormalities pertinent to THP deposition. In summary, THP is frequently seen in bladder tissue and most probably represents an incidental finding of morphologic interest but of no clinical significance.

Determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation

A method for enzyme-antibody conjugation using a new maleimide derivative as coupling reagent has been developed. Since a monomeric conjugate of horseradish peroxidase and Fab' antibody could be readily prepared with high efficiency and reproducibility, the enzyme activity and antigen-binding activity were well preserved and nonspecific staining was greatly reduced. The conjugate is suitable for use in both ELISA procedures and immunohistochemistry. Using both methods we examined the pathophysiological significance of Tamm-Horsfall protein (THP) and the present study describes the ELISA method to quantify urinary THP using the new method with rabbit anti-THP antibody. A low concentration (0.04 M) of urea added to the urine samples increased the linearity of the standard curve and the sensitivity of the assay, permitting the detection of as little as 20 ng/ml THP. Freezing and thawing the urine resulted in variable or lower values of THP concentration. THP concentrations in urine as determined by ELISA were stable for at least one month after −70°C storage, but not after −30°C storage. There was no correlation between THP concentrations in 24 h urine samples and the morning urine of the same patient. These results suggest that it is essential to use fresh or −70°C stored 24 h urine samples with added urea (0.04 M) for the determination of THP concentrations in urine by the present enzyme-antibody conjugation method. The THP concentration in normal 24 h urine of young children was found to be less than 51.8 mg/g Cr.

Tubulointerstitial nephritis induced by Tamm-Horsfall protein sensitization in guinea pigs

Experimental tubulointerstitial nephritis (TIN) was induced in guinea pigs by immunization with homologous Tamm-Horsfall protein (THP). This disease was characterized by focal interstitial mononuclear cell infiltration around the distal nephron segments with degeneration of renal tubular cells. Although concomitant granular immunoglobulin deposition on the tubular basement membrane and a rise of serum anti-THP antibodies were recognized, they were related to the severity of the lesion. Lymphocytes from lymph nodes of animals with TIN showed blast transformation in the presence of THP in vitro. Following the transfer of lymphocytes and spleen cells from guinea pigs with THP-induced TIN to nonimmunized animals, the recipient animals developed TIN 7 days later. These observations suggest that TIN induced in guinea pigs by challenge with homologous THP may, at least in part, be related to a cell-mediated immune response.

A new ELISA method for the rapid quantification of Tamm-Horsfall protein in urine.

The authors present a simplified enzyme-linked immunosorbent assay (ELISA) technique for the quantitative measurement of urinary Tamm-Horsfall protein (THP). Microtiter plates are coated with THP and urine samples at various dilutions without the need for a capture antibody. The bound glycoprotein is then incubated with a monoclonal anti-THP antibody and an alkaline phosphatase conjugated anti-IgG antibody. The assay was validated and gave reproducible results over a wide range of absorbance values. The sensitivity of the assay for THP was 2-5 micrograms/L, the coefficient of variation between assays 7.5%, and the day-to-day variability 11.1% for THP concentrations between 6.25 and 200 micrograms/L. THP excretion was assayed in five volunteers over five days comparing THP concentration in spot urines and in 24-hour urine collections.

Studies to assess the biological relevance of anti-Tamm-Horsfall protein antibodies detected by direct-binding enzyme-linked immunosorbent assay.

1. A role has been suggested for anti-Tamm-Horsfall protein (THP) antibodies in renal disease based on the results of immunoassays of pathological sera. The putative autoantibodies have not been isolated from such sera nor have definitive inhibition studies of their binding been carried out. We have carried out such studies using rabbit anti-THP antibodies as control reagents. 2. Urinary THP prepared by salt precipitation was used to prepare four immunoabsorbent columns by covalent coupling to CNBr-activated Sepharose 4B. After washing with a variety of dissociating agents to remove any non-covalently bound subunit THP, each column was incubated with normal and immune rabbit serum. Fractions washed and eluted from columns were tested for anti-THP antibodies by enzyme-linked immunosorbent assay (ELISA) and THP antigen by radioimmunoassay, and showed NH4SCN (3 mol/l) and guanidine hydrochloride (GuHCl) (6 mol/l) equivalent and sodium dodecyl sulphate (20 g/l) to be inferior in their capacity to produce immunoabsorbent THP capable of isolating specific antibodies from immune rabbit serum. 3. The column treated with GuHCl (6 mol/l) was used further in attempts to isolate putative anti-THP antibodies from five patients, who had a history of urinary tract infections and whose sera showed strong binding by ELISA. 4. Results from direct and inhibition ELISA experiments on fractions collected after washing and elution with all sera suggested that the putative human anti-THP antibodies were of very low affinity and/or directed against non-subunit THP. 5. The pathological relevance of human anti-THP antibodies measured by ELISA remains to be established.

Interaction of Tamm–Horsfall protein with bacterial extracts

Crude extracts of uropathic Escherichia coli have been reported to inhibit the binding of human Tamm-Horsfall protein (THP) to homologous and heterologous anti-THP antibody in immunoassays. This phenomenon was believed to be due to immunologic cross reactivity between THP and the bacterial antigens for the same antibody. Our attempts to further purify and characterize these "cross reactive" antigens with ion exchange and molecular sieve chromatography were unsuccessful. When purified anti-THP antibody was conjugated to sepharose beads forming an immunoadsorption column capable of isolating THP and cross reactive antigens from solution, the bacterial extracts did not react with the affinity column. However, binding between THP and the bacterial extracts and between THP and whole bacteria were demonstrated. These findings suggest that the cross reactivity seen in the immunoassays is caused by the interaction between the bacterial extracts and THP, and is not representative of true immunologic cross reaction for a common antibody.

Association between fever and the antibody response to Tamm-Horsfall protein in urinary tract infection.

The usefulness of measuring serum antibodies to Tamm-Horsfall protein (THP) for diagnosing the level of acute urinary tract infections in adult women was evaluated. There was a significant (p less than 0.001) elevation of IgG and IgA anti-THP in pregnant and non-pregnant women with acute pyelonephritis compared to women with acute cystitis. The changes in anti-THP antibody titre among patients with acute pyelonephritis varied considerably, limiting the value of determining such antibodies for diagnostic purposes. Patients with febrile infections of non-renal origin demonstrated an antibody response comparable to the pyelonephritis patients. One explanation for these results could be that fever is associated with transient rental tubular damage, thus exposing THP to the immune system. Polyclonal antibody activation seems less likely since neither an IgM antibody response to THP, nor an increase in antibody titre to poliovirus could be demonstrated.

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.

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl- N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum—I : 7S and 19S anti-THP antibodies. Preparation, characterization and analytical application of immunoadsorption

Antibodies against the solvatochromic dye 4-(3-aminophenyl)-2,6-diphenyl- N-(4-hydroxy-phenyl)-pyridinium betain (ATHP ∗) we produced by immunization of pregnants cows with an THP-edestin conjugate. Large amounts of 7S and 19S anti-THP antibodies were isolated from the colostrum of one of the immunized animals. The specific isolation was performed with the aid of an immunoadsorbent prepared by coupling ATHP to bovine γ-globulin, previously cross-linked with glutaraldehyde. Various conditions for antibody binding to, and elution from, the adsorbent were studied and compared. Using the adsorbent a test was developed which enabled quantitative determination of anti-THP antibodies, including the non-precipitating ones, which were found to be present in the colostrum in large quantities. The isolated anti-THP antibodies were fractionated by chromatography on Sephadex G-200 into 7S and 19S antibodies. The 7S anti-THP antibodies proved to belong the type IgG s whereas the 19S anti-THP antibodies were composed of 19S IgG s and IgM.

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum—I 7S and 19S anti-THP antibodies. Preparation, characterization and analytical application of immunoadsorption

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum—I 7S and 19S anti-THP antibodies. Preparation, characterization and analytical application of immunoadsorption

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl- N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum—II : Characteristic differences in hapten binding kinetics of 7S and 19S anti-THP antibodies

The hapten binding properties of purified 7S and 19S 2,4,6-triphenyl- N-(4-hydroxyphenyl)-pyridinium (THP ∗-) specific antibodies were studied by temperature-jumped spectrometry. In both cases a nearly diffusion-controlled hapten-antibody recombination process in the μsec range was observed. In the case of the 19S immunoglobulins an additional relaxation process in the msec range was found to be independent of reactant concentrations. This slow first-order process, suggesting structural rearrangements of the antibodies, is discussed with respect to hapten-dependent conformational changes of the immunoglobulins.

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum—II Characteristic differences in hapten binding kinetics of 7S and 19S anti-THP antibodies

Isolation and dynamic hapten binding properties of 2,4,6-triphenyl-N-(4-hydroxyphenyl)-pyridinium (THP-) specific antibodies from bovine colostrum—II Characteristic differences in hapten binding kinetics of 7S and 19S anti-THP antibodies