Study of childhood renal tumours using a monoclonal antibody to Tamm-Horsfall protein.

A monoclonal antibody to Tamm-Horsfall glycoprotein was used for the immuno-localization of Tamm-Horsfall protein in formalin fixed, paraffin embedded tissue sections of childhood renal tumours, normal children's kidneys, and human fetal kidneys. The procedure was a dinitrophenyl hapten sandwich staining method. The antibody, diluted 1/100,000, gave a very strong and specific staining of the loop of Henle and distal tubules of normal and fetal kidneys. No staining was seen in Wilms' tumour, mesoblastic nephroma, and bone metastasizing renal tumour of childhood. In contrast, two of seven renal carcinomas and three of four rhabdoid renal tumours were positive for Tamm-Horsfall protein.     

Benign epithelial cells and Tamm-Horsfall protein in lymph nodes from nephrectomy specimens with nephroblastoma: a diagnostic pitfall

When a lymph node metastasis is discovered during the pathology examination of surgical specimens with nephroblastoma, the tumour is classified as stage III, according to the classification of the International Society of Paediatric Oncology (SIOP 2001), and post-operative intensive treatment of the patient includes irradiation and chemotherapy. Benign epithelial cells in perinephric lymph nodes in cases of renal tumours in childhood may be confused with metastases. They are often associated with an accumulation of Tamm-Horsfall protein in lymph node sinuses. This case illustrates latero-aortic lymph node complexes of benign epithelial cells and Tamm-Horsfall protein in a 16-month-old girl with surgical resection of a nephroblastoma after pre-operative chemotherapy. The nephroblastoma was predominantly epithelial and was classified as SIOP stage I. There were accumulations of Tamm-Horsfall protein in lymph node sinuses, in lymphatic vessels, in the kidney outside the tumour, and in the renal sinus. This association of epithelial cells with deposits of Tamm-Horsfall protein and their resemblance to cells of the distal convoluted tubules favour a diagnosis of benignity. A definitive diagnosis is supported by the small size of the cells, their bland nuclei, and morphological features differing from those of tumour cells, but deposits of Tamm-Horsfall protein may be associated with true lymph node metastases.     

The Sda Antigen in the Human Kidney and Colon

The range of concentration of the Sd^a blood group antigen has been determined in the human adult kidney and colon. No Sd^a antigen was found in 2% of kidneys. Immunofluorescent studies of the kidney showed that Sd^a is present in the distal convoluted tubules and collecting ducts and occurs in the same location as Tamm-Horsfall protein. No Sd^a antigen was detected in about 2% of colons. In an additional 6%, no antigen was detected in saline extracts but was present in an insoluble form. In the colon, Sd^a is sited on the brush borders of the epithelial cells and in the goblet cells. No Tamm-Horsfall protein was identified in the colon.     

Tamm-Horsfall protein

Human Tamm-Horsfall glycoprotein, the major urinary protein, is a glycosyl-phosphatidyl-inositol (GPI) - anchored membrane protein which mainly resides at the luminal face of cells of the thick ascending limb of Henle's loop (TAL) and early distal convoluted tubules of nephron. Tamm-Horsfall protein contains exclusively N-linked glycans, mainly of polyantennary type largely sialylated and fucosylated, but also high-mannose glycans. Only a portion of the Tamm-Horsfall protein is released as soluble protein by the action of proteases and in a minor amount by a cell-associated GPI-specific phospholipase. The physiological function of Tamm-Horsfall glycoprotein has not been clarified to date. Since the urinary Tamm-Horsfall protein has a high gel-forming tendency, it has been postulated that it takes part in the water impermeability of TAL. It is also proposed that the Tamm-Horsfall protein plays a protective role towards pyelonephritogenic pathogens such as Escherichia coli. The Tamm-Horsfall protein may inhibit the colonization of these pathogens in the renal mucosa in that the soluble form competes with that exposed at the plasma membrane. Recently, urinary Tamm-Horsfall protein has been implicated in tubulointerstitial nephritis.     

Cellular origin and differentiation of renal carcinomas. A fluorescence microscopic study with kidney-specific antibodies, antiintermediate filament antibodies, and lectins

Frozen sections of human renal carcinomas were studied in indirect immunofluorescence using antibodies against intermediate filaments of cytokeratin, desmin and vimentin type, and against proximal tubular brush border and distal tubular Tamm-Horsfall glycoprotein antigens, as well as with fluorochrome-labeled lectins in an attempt to study the origin and stage of differentiation of renal carcinomas. Eighty per cent of the renal carcinomas expressed the brush border antigens, whereas the Tamm-Horsfall glycoprotein could not be found. Antibodies against epidermal cytokeratins reacted only with collecting ducts in normal kidney, whereas antibodies against cytokeratins of Madin-Darby canine kidney epithelial cell line also reacted with glomerular and tubular epithelium. In 93% of the carcinomas tumor cells showed reactivity with both types of antikeratin antibodies. Vimentin, the cytoskeletal protein of mesenchymal cells, was present in the carcinoma cells of 53% of the tumors, although it was not present in normal tubular epithelium. Moreover, vimentin was expressed together with cytokeratin in the carcinoma cells in 57% of the keratin-positive samples as judged by double immunostaining, whereas the muscle type of intermediate filament protein, desmin, was not seen in the malignant cells. Binding sites for Lotus tetragonolobus agglutinin and soybean agglutinin, normally present in the cells of proximal tubules, were lacking or only faintly detectable in the neoplastic cells. Dolichos biflorus agglutinin, normally present in collecting ducts, was not detected in the tumors. The results show that most renal carcinomas express cytokeratin antigens as a sign of their epithelial origin and also show characteristics of proximal tubular cells. On the other hand, the results indicate that lectin-binding sites typical for normal differentiated tubular cells are profoundly modified in renal carcinomas. Ulex europaeus agglutinin did not bind to the malignant cells but decorated the endothelial cells of the tumors.     

Immunoanatomic distribution of cytostructural and tissue-associated antigens in the human urinary tract

The main objective of the present study is to define the expression and/or modulation of antigenic phenotypes in cells of the normal human kidney and urothelium according to cell type. Fourteen antibodies detecting differentiation and structural antigens expressed in the human urinary tract have been used to define the immunoanatomic distribution of these antigenic systems. They include urinary tract antigens (Tamm-Horsfall glycoprotein and prostate-specific antigen), tissue-associated antigens (epithelial membrane antigen, Factor VIII antigen, and Protein S-100), and cytoskeletal antigens of the intermediate filament classes (cytokeratins, vimentin, desmin, glial fibrillary acidic protein, and neurofilaments. Immunofluorescence and immunoperoxidase analyses performed on normal human fetal and adult tissue sections have demonstrated that these antigens are expressed by different cell types and domains of the nephron. Studies correlating normal fetal and adult tissues reveal that some of the antigens appear at distinct stages of maturation, representing early and late antigenic expression events. These antibodies offer a wide range of potential applications that include studies of embryogenesis of the human urinary tract and immunopathologic analyses of neoplastic and nonneoplastic diseases of the human kidney and urothelium.     

Distinctive patterns of renal neoplasms containing Tamm-Horsfall protein

Sections of 114 renal neoplasms from adults, and 2 renal rhabdoid tumours from children, were examined by an indirect immunoperoxidase method using two antibodies to Tamm-Horsfall protein. Forty-five of the adult neoplasms were also examined with an antibody to proximal tubular brush border. Tamm-Horsfall protein is normally only found in the cells of the thick limb of the loop of Henle, and there are widely divergent reports on its occurrence in renal neoplasms. In the present series, Tamm-Horsfall protein was detected in parts of 31 neoplasms. Four distinctive patterns of cell contained the protein: cells with a paranuclear inclusion typical of rhabdoid tumours; plasma rich cells, which were large cells with cytoplasm that was centrally dense and peripherally clear; eosinophilic cells forming one type of papillary structure; and giant cells. The areas containing Tamm-Horsfall protein did not express markers of proximal tubular brush border, and appeared white to the naked eye, rather than the yellow of typical clear cell carcinomas. Tamm-Horsfall protein can therefore be found in renal neoplasms. The four distinctive patterns of positive cells appear to represent neoplastic phenotypes of thick limb cells. This has implications for the classification of renal neoplasms and for theories of their origin.     

Tamm-Horsfall protein outside the kidney

Tamm-Horsfall protein is the main protein in normal urine and readily precipitates. Previously there has been little study of the protein outside the kidney. An antiserum to the protein was used in an immunohistological investigation of human tissues. No Tamm-Horsfall protein was detected in normal organs other than the kidney. The protein was detected outside the kidney in abnormal organs in three general sites: (i) in ulcers of epithelial surfaces exposed to urine, (ii) deep in the wall of a bladder that had had previous surgery and (iii) in lymph nodes at the hilum of kidneys that had been obstructed or contained extra-tubular deposits of the protein. There was little evidence of a cellular response to the deposits of Tamm-Horsfall protein. The antiserum to Tamm-Horsfall protein is a good immunohistological marker of extravasated urine and is useful in the study of such conditions as fistulas of the urinary tract, operations on the urinary tract and reflux of urine into lymphatics and lymph nodes.     

Influence of altered glomerular permeability on renal tubular immune complex formation and clearance

Proteinuria was induced in rats to determine whether intravenously injected antibodies to a distal tubular antigen would bind to the luminal surfaces of distal tubular cells in vivo. Rats with proteinuria induced by an intravenous injection of sheep antisera to Fx1A (passive Heymann model) were injected 10 days later with rabbit antisera to Tamm-Horsfall protein. Linear rabbit IgG deposits along the luminal cell surfaces in the initial portion of the thick ascending limb of the loop of Henle (ALH) were demonstrated by immunofluorescence microscopy at 4 hours and were maximal 1 to 3 days after injection of antibodies to Tamm-Horsfall protein. The distance that these immune deposits extended along the ALH was directly proportional to the magnitude of proteinuria. Light microscopy showed periodic acid-Schiff-positive luminal deposits and an increased number of mitoses confined to the early ALH. Ultrastructural studies revealed continuous very electron-dense deposits initially covering the luminal surfaces of ALH cells. During the clearance phase, these deposits were surrounded and separated from ALH cell surfaces by a less electron-dense fibrillar material with the ultrastructural characteristics of Tamm-Horsfall protein. The mechanism of immune complex formation in the present study appears to involve the in situ combination of rabbit antibodies to Tamm-Horsfall protein in the glomerular filtrate with a tubular surface membrane antigen, Tamm-Horsfall protein.     

Histochemical evidence for tubule segmentation in a case of Wilms' tumor

A lectin histochemical and immunohistochemical approach was used to compare the different histologic elements of an unusual case of Wilms' tumor with normal kidneys. This case was stained with ten lectin-horseradish peroxidase conjugates; immuno-stained for Tamm-Horsfall glycoprotein, epidermal keratin, and vimentin; and compared with 19 control kidneys. The morphologic and cytochemical properties of various tumor elements in this specimen served to identify them as tumor analogs of all segments of the normal kidney tubules except distal tubules. Evidence for Wilms' tumor differentiation is provided by this case, whose epithelium histochemically resembles normal human epithelial cells.     

Tamm-Horsfall protein in the glomerular capsular space.

Tamm-Horsfall protein was detected within the capsular space by immunofluorescence in 7 of 72 consecutive patients on whom renal immunopathological studies were performed. Three patients showed prominent aggregates or crescentic collections affecting 30-50% glomeruli; the remaining four patients showed smaller aggregates between lobules. All patients showed pathological evidence of tubulointerstitial disease. It is suggested that Tamm-Horsfall protein in the capsular space is a sign of intratubular urinary backflow and that Tamm-Horsfall antiserum is a useful addition to the reagents used in the immunofluorescence study of renal biopsies.     

Pathways of urinary backflow in obstructive uropathy: Demonstration by pigmented gelatin injection and Tamm-Horsfall uromucoprotein markers

The exact pathways of urinary reflux into the renal veins were studied in four cases of clinical obstructive uropathy and in 50 normal human cadaver kidneys. In the four clinical cases Tamm-Horsfall uromucoprotein was used as a marker for location of urine. Routine light microscopy and indirect immunofluorescence for Tamm-Horsfall uromucoprotein using rabbit antiserum showed tubular backflow up to the glomerulus. Dilated tubules filled with Tamm-Horsfall uromucoprotein ruptured into thin walled veins, forming tubulovenous anastomoses with extrusion of their contents into veins. The uromucoprotein was present in interlobar and arcuate veins with superimposed thrombosis and thrombophlebitis. Injection studies using pigmented gelatin in 45 normal cadaver kidneys and pigmented vinylite with corrosion casts in five additional kidneys complemented the clinical studies. Two types of urovascular communication were produced: the less frequent direct pyelovenous communication between a rupturing fornix and an adjacent small vein, and the more common indirect pyelovenous communication in which a ruptured fornix produced a sinus extravasate, which extended along the perivenous spaces of interlobar and arcuate veins. This extravasate gained access into the veins at points of rupture where venous tributaries joined the major veins in the renal medulla. The clinical implications of these tubulovenous and pyelovenous pathways of urinary reflux include backflow of whole urine and continued nephronic function in obstructive uropathy, "reverse backflow" of blood and hematuria, and a direct access for infectious agents into the circulation. These channels provide anatomic correlates for radiologic findings of extravasates and some backflow patterns of contrast material in pyelograms of clinical obstructive uropathy. The possible immunologic consequences of refluxing Tamm-Horsfall uromucoprotein gaining access to tissues and circulation are speculative.     

Rapid isolation of Tamm-Horsfall glycoprotein (uromodulin) from human urine

An isolation method for Tamm-Horsfall protein is described which is based on the observation that a diatomaceous earth filter is able to retain most of the glycoprotein present in urine and that the glycoprotein is easily desorbed from the filter by deionized water. This behaviour depends on the tendency of Tamm-Horsfall glycoprotein at normal urinary concentrations to form a gel in a solution containing mono- and divalent ions. By means of two-step filtration, the glycoprotein was purified to homogeneity. The yield was of about 20 mg/l of urine, and the time required for the isolation was approximately 5–6 h. This procedure should be particularly useful for preparing large amounts of Tamm-Horsfall glycoprotein oligosaccharides in order to investigate their potential use as immunosuppressive agents both in vitro and in vivo.     

Study of antibody production to Tamm-Horsfall protein in renal transplant donors and recipients

The content of Tamm-Horsfall protein was measured in the urine of humans without renal diseases, pregnant women, and donors and recipients of renal transplant using a new test system for measuring Tamm-Horsfall protein including antigenic diagnostic agent and immune serum. Production of antibodies to Tamm-Horsfall protein was characterized using antigen diagnostic system. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 559–562, May, 2006     

Heterogeneity of Proteins in Commercial Preparations of Human Chorionic Gonadotropin (hCG) Demonstrated by Western Blotting

ABSTRACT: Immunoblotting has been utilized to detect the presence of human chorionic gonadotropin (hCG), its α and β subunits, the Tamm-Horsfall protein (uromodulin), immunoglobulins G (IgG) and M (IgM), kappa (K) and lambda (L) chains, and serum albumin in commercially available preparations of hCG intended for human use. Concentrated pregnancy, postpartum, and normal urines were studied as a comparison. Those hCG batches prepared from pooled first-trimester pregnancy urine contained all of the hCG and non-hCG proteins listed above or their fragments, with the single exception of the IgM μ chain. The conflicting literature regarding the immunomodulatory properties of hCG requires reevaluation, since many previous reports of immunologic activity utilized these preparations, containing intact and degraded IgG, K, and L chains, and the Tamm-Horsfall protein, all of which may contribute to an altered immune response. Since hCG can be prepared in high yields, free of these contaminants, these data suggest that patients are being unnecessarily exposed to contaminating substances when receiving parenteral injections of hCG.     

Alkaline phosphatase activity of normal and cystic fibrosis fibroblasts.

Alkaline phosphatase (ALP) activities were compared in fibroblasts from three cystic fibrosis patients and two normal controls after culturing the cells in normal growth medium and in medium containing Tamm-Horsfall glycoprotein, isoproterenol, and theophylline. No consistent alterations in ALP activities were noted, either between the same cell lines grown under different conditions, or between normal and cystic cell lines. It is concluded that it is not possible to use changes in ALP activity in cultured cells for the prenatal diagnosis of cystic fibrosis.     

Benign salivary gland tissue inclusion in a pulmonary hilar lymph node from a patient with invasive well-differentiated adenocarcinoma of the lung: a potential misinterpretation for the staging of carcinoma

Benign epithelial and nonepithelial inclusions have been found in lymph nodes in multiple body sites. These inclusions have been seen in cervical, axillary, mediastinal, abdominal, and pelvic lymph nodes. They appear as benign epithelial, parathyroid, decidual, mesothelial, angiolipomatous, nevus cells, or Tamm-Horsfall protein. Although heterotopic salivary gland tissue is not infrequent in paraparotid lymph nodes, it has only been described in lymph nodes of the pulmonary hilum once. A 68-year-old woman with gastric lymphoma now in remission presented for routine follow-up and was found to have a lung mass. After a fine needle aspiration biopsy diagnosis of adenocarcinoma, lobectomy and lymph node dissection were performed. Histological sections of lung demonstrated a well-differentiated adenocarcinoma and one lymph node, which displayed a subcapsular nest of well-formed salivary glands occupying approximately one third of the nodal tissue. The inclusion was composed of acinar cells of both serous and mucinous types, but ductal type of cells were not seen. Identification of heterotopic tissue in lymph nodes is of great importance for patient management. Misdiagnosing benign glandular inclusions for metastasis could potentially lead to incorrect tumor staging. Benign salivary gland tissue inclusions should be considered in the differential diagnosis when evaluating for metastatic adenocarcinoma. The salivary gland inclusion in pulmonary hilar lymph node may be histogenetically related to the minor salivary glands, which are located within the bronchial submucosa.     

Tamm-Horsfall protein in lymph nodes

We found masses of amorphous waxy pale staining material in the peripheral sinuses of lymph nodes removed with renal tumors. The material was histochemically and immunohistochemically identical to Tamm-Horsfall protein. It was found also in tubular casts, renal tubules, and intrarenal lymphatics in the resected kidneys. Tamm-Horsfall protein was restricted to the compressed residual kidney and was not seen in the tumors.Under certain circumstances Tamm-Horsfall protein escapes from the tubules into the renal interstitium and may produce a local inflammatory reaction. It also gains access to intrarenal lymphatics and thence to regional lymph nodes. This may be relevant to the evolution of an antibody response.     

Tamm-Horsfall protein. Abnormal localization in renal disease.

Interstitial deposits of periodic acid-Schiff positive fibrillar material have been detected in a variety of diseases associated with tubulointerstitial pathology. This material has been shown to be Tamm-Horsfall protein by immunofluorescent, immunochemical, and electron microscopic studies. Prospective evaluation of 133 kidneys revealed deposits in 11 per cent. These deposits included medullary cystic disease (50 per cent), obstructive uropathy and vesicoureteral reflux (21 per cent), and tubulointerstitial disease (29 per cent). Tamm-Horsfall protein was also detected in Bowman's space in four cases of obstructive uropathy. It is proposed that these deposits result from severe tubular damage with release of Tamm-Horsfall protein from its normal intracellular and intralumenal locations into the renal interstitium. We speculate that the intersitial deposition of this sequestered protein may result in a continued inflammatory response and progressive renal damage.     

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.