Ultrastructural localization of Tamm-Horsfall protein in human kidney using immunogold electron microscopy.

The ultrastructural localization of Tamm-Horsfall protein (THP) was studied in paraformaldehyde-fixed human renal biopsies. Pre-embedding and post-embedding immunogold labelling techniques were developed utilizing a monoclonal antibody specific for human urinary THP. With the pre-embedding technique, membrane contrast was enhanced by osmification thus allowing precise localization of gold particles. Reasonable tissue penetration of antibodies was achieved without compromising ultrastructural detail. The hydrophilic resin LR White was used for post-embedding labelling to ensure maximum penetration of antibodies. However, sections had only mild osmification and consequently localization of label was less certain. Both labelling techniques gave similar results. THP was found to be associated with two renal cell types. Epithelial cells lining the thick ascending limb of Henle's loop had gold label closely associated with the whole cell plasmalemma, with some of these cells having an apparently random distribution of label throughout the cytoplasm. Only the luminal plasmalemma of epithelial cells lining distal convoluted tubules were found to be labelled. Basolateral membranes and the cytoplasm of these cells were negative. The use of a monoclonal antibody of defined specificity combined with the two immunolabelling procedures represents a precise reliable method for studying ultrastructural localization of THP in the human kidney.

Relationship of abnormal Tamm-Horsfall glycoprotein localization to renal morphology and function.

Tamm-Horsfall glycoprotein (TH) distribution was studied using a biotin-avidin immunoperoxidase technique in renal biopsies from 166 consecutive patients and 8 normal kidneys. Tubulointerstitial damage was independently assessed and graded. In 109 patients TH antibodies were measured by ELISA and in 30 of these urinary TH and beta 2-microglobulin excretions were measured by radioimmunoassay. In 124 biopsies only distal tubular epithelium and casts were stained. Glomerular space (8) or interstitial (34) deposits were seen in 42 biopsies; 16/68 with glomerulonephritis, 4/14 with systemic vasculitis, 12/33 with chronic interstitial nephritis, 1/8 with acute interstitial nephritis, 9/43 with other nephropathies. There was no correlation between TH distribution and the degree of tubulointerstitial damage (p greater than 0.5), urinary TH excretion (p greater than 0.05), urinary beta 2-microglobulin excretion (p greater than 0.05), glomerular filtration rate, urinary concentrating ability, or the incidence of pyuria. TH antibodies did not correlate with TH distribution (p greater than 0.5) or the degree of tubulointerstitial damage. Abnormal TH distribution showed no statistical relationship to the degree of tubulointerstitial damage, changes in renal function or levels of TH antibodies.

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/ml. Results from three control sera run on all plates used in the survey showed mean CV less than 7.6% while no binding was observed with sera from an anephric patient.

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.

Relationship of glomerular phagocytic cells to immune complexes and DNA autoantibodies in NZB/W mice.

Systematic study of groups of NZB/W mice aged from 1-8 mth showed progressive development of antibodies to ssDNA and dsDNA, accompanied at 5 mth by the appearance of serum immune complexes as detected by Clq liquid phase binding and immune deposits in glomeruli. Cellular outgrowths in cultures of isolated glomeruli showed increased numbers of phagocytic cells as well as epithelial and mesangial cells, reaching a maximum in mice aged 7 mth. These numbers exceeded those found in control CBA mice and, while consistent with the hypothesis that phagocytic cells are involved in the processing of immune complexes lodged in the glomeruli, also reflected the hypercellularity of the glomerular lesions.

Goodpasture's syndrome with normal renal function.

Two young men with anti-glomerular basement membrane (anti-GBM) antibody-induced Goodpasture's syndrome are described. Despite the characteristic renal morphological features and high titers of anti-GBM antibody, they had normal renal function. Both patients recovered spontaneously without treatment and remain well with normal renal function more than four years after presentation. The use of more sensitive diagnostic techniques has demonstrated that there is a broad spectrum of clinical severity in this disease and that mild cases can be expected to have a good outcome.

Mouse glomerular culture.

A method is described for the culture of isolated intact mouse glomeruli. Cultures showed adherent glomeruli after 2 days and from 2-4 days a monolayer of cells began to develop around the points of adherence. The predominant cell (Type I) was 80-200 micrometer in diameter with cytoplasmic extensions, later becoming polyhedral and often multinucleated. Type II spindle cells, 70-100 micrometer in length, were less prominent in the earlier stages and later formed sheaves between Type I cells. Small phagocytic Type III cells, 10-20 micrometer in diameter, were not prominent in cultures from normal kidneys and tended to form small clusters on top of the Type I cell monolayer.

Phagocytic cells in glomerular cultures from NZB/W mice.

Comparative studies of glomerular cultures from NZB/W and normal control mice aged 2, 4, 6, 8, 10 and 12 months revealed a significant increase in the number of phagocytic cells in cultures from NZB/W mice at all ages. These cells on electron microscopy had the appearance of macrophages and on immunofluorescence showed Fc receptors and reacted with rabbit anti-mouse macrophage serum. The cells appear to be of blood monocyte origin and it is likely that they play a role in the pathogenesis in NZB/W mice of the glomerulopathy associated with clearance of immune complexes.

Biochemical and mitogenic properties of Tamm-Horsfall urinary glycoprotein.

Thirty separate preparations of human Tamm-Horsfall urinary glycoprotein (THP) prepared from pooled urine of normal individuals were assessed for the capacity to stimulate in vitro blast transformation with normal human peripheral blood lymphocytes. Detailed studies were performed on some of these preparations by various biochemical, ultrastructural and in vitro techniques. Heterogeneity amongst the sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profiles of several electrophoresis (2D-IEP). Gas liquid chromatographic analysis (GLC) revealed significant amounts of glucose in two of these samples. Ultrastructural studies using direct staining or platinum carbon shadowed specimens confirmed the fibrillar polymeric nature of soluble THP in vitro studies with human peripheral blood lymphocyte populations showed THP to have mitogenic properties towards B cells associated with the production of intracytoplasmic immunoglobulin. THP also stimulated the migration of mouse peritoneal exudate macrophages in a micro macrophages migration assay.

A renal lesion in ankylosing spondylitis.

2 patients with ankylosing spondylitis (A Sp) were found to have renal lesions similar to those seen in IgA nephropathy. In 1 patient the changes were extremely severe and progressive and in the other they were mild. Vascular changes were also noted in 1 patient. The findings suggest an immune complex mediated glomerulonephritis and support an earlier report that there may be a specific renal lesion in patients with A Sp. The significance of IgA deposition in the mesangium, and of an increase in the serum levels of IgA in some patients with A Sp is unclear.

Isolation of human glomerular basement membrane antigens by affinity chromatography utilising Goodpasture's kidney antibody eluates.

The soluble material produced from a 96-hour digest of sonicated human glomerular basement membranes with purified collagenase was shown to contain at least 12 components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with molecular weights ranging from 20 x 10(3) to greater than 200 x 10(3) daltons. Five of these components produced antigenic peaks after two-dimensional immunoelectrophoresis into agar plates containing rabbit antiserum to the solubilised glomerular basement membrane. Two groups of antigenic components were demonstrated in these gels by two-dimensional immunoelectrophoresis autoradiographic techniques utilising 125I-labelled collagenase-digested human glomerular basement membranes reacted with antibodies eluted at acid pH from the kidney of the Goodpasture's patient. This acid eluate was shown to contain contaminants of alpha 1-antitrypsin and albumin co-eluting with the antibodies bound to the glomerular basement membrane. After removal of these contaminants, the antibodies were linked to cyanogen bromide-activated Sepharose and used to affinity purify four antigenic fractions from the collagenase-digested glomerular basement membrane. These fractions were eluted by 0.2 M glycine pH 2.8 and with 0.5 M ammonium thiocyanate and had molecular weights of 22-25, 43-45, 65-70 and 205 x 10(3) daltons. The smaller molecular weight components were shown to be common to both included and excluded fractions obtained by Ultragel AcA 44 chromatography of the digested material in 10 mM phosphate pH 8. This suggests that the larger molecular weight component would be an aggregate of a smaller component. Preliminary carbohydrate and amino acid analyses indicated that the different elution procedures elicited antigens with different chemical characteristics.

Macrophages in NZB/NZW mouse glomerular disease.

Glomerular cultures from mice have been established and the various cells associated with them observed microscopically by phase contrast time-lapse cinematography, after histological staining and ultrastructurally with the transmission and scanning electron microscope. The most common cell types were large 80-200 microns diameter irregular polyhedral cells appearing as flattened plate-like structures morphologically, possibly of epithelial origin. Smaller 70-100 microns diameter fusiform cells possibly derived from either endothelium or mesangium were also often seen. A third cell type, small 10-20 microns diameter circular, motile and actively phagocytic was especially noticeable in cultures of hybrid New Zealand Black/New Zealand White mice but was not present in appreciable numbers in control cultures from strains without renal disease. This third cell type appeared to have Fc receptors, stained positively with non-specific esterase and by indirect immunofluorescence with specific rabbit anti-mouse macrophage serum and had the morphological appearance of a macrophage by scanning and transmission electron microscopy. It is concluded that cells of macrophage origin are associated with the immune complex glomerulonephritis in these hybrid mice.

Mitogenic proteins of pokeweed. I. Purification, characterization and mitogenic activity of two proteins from pokeweed (Phytolacca octandra).

Saline extracts from the roots of the pokeweed species. Phytolacca octandra were separated by ion-exchange chromatography into three fractions, Po-1, Po-2 and Po-3. Po-1 contained two monomeric proteins with molecular weights of 36,000 and 29,000 and these were partially purified by gel filtration. Po-2 was purified as a single polymeric protein composed of approximately ten 14,000 mol. wt polypeptides and is a new pokeweed mitogen. Po-3 was purified as a single polymeric protein composed of approximately four 31,000 mol. wt subunits, and apart from its polymeric structure closely resembles commercial pokeweed mitogen (PWM). Po-2 and Po-3 were mitogenic for unseparated human peripheral blood lymphocytes but the degree of mitogenic activity in Po-2 preparations was dependent on storage following purification. Purified B cells were not stimulated by either mitogen. Po-3 was a potent mitogen for T cells but preparations of Po-2 required storage before they stimulated T cells. Higher responses were observed in co-cultures of B and T cells than in separated B and T cell cultures. It is suggested that human B and T lymphocytes show synergy in their responses to Po-2 and Po-3.

Mitogenic proteins of pokeweed. II. The differentiation of human peripheral blood B lymphocytes stimulated with purified pokeweed mitogens (Po-2 and Po-6) from pokeweed, Phytolacca octandra.

Purified pokeweed mitogens, Po-2 and Po-3, extracted from Phytolacca octandra, stimulated plasma cell formation in cultures of human peripheral blood lymphocytes. Plasma cell formation did not occur in cultures of purified B cells but was dependent on T-cell help. High T-cell numbers, however, suppressed plasma cell formation in mixed B- and T-cell cultures stimulated with Po-2. T-cell helper function was exerted across a major histocompatibility barrier and was not dependent on T-cell proliferation. Soluble helper factor(s) from activated T cells were not demonstrated. Po-3 was an effective B-cell stimulant only at concentrations between 0.1 and 1.0 micrograms/ml. In contrast, relatively high concentrations of Po-2 (50--100 micrograms/ml) were required to induce B-cell differentiation. More plasma cells were generated in Po-2-stimulated cultures than in Po-3-stimulated cultures and this was thought to reflect the more aggregated state of Po-2.

Stimulation of human peripheral blood lymphocytes by Tamm-Horsfall urinary glycoprotein.

Tamm-Horsfall urinary glycoprotein (THP), prepared by salt precipitation of pooled urine from normal individuals, stimulated purified human peripheral blood lymphocytes (PBL) to undergo blastoid transformation. the response was measured by tritiated thymidine uptake into DNA after 6 days in culture. Several batches of THP stimulated, in varying degrees, all samples of PBL tested and the response approached that seen with the mitogens phytohaemagglutinin (PHA), Concanavalin A (Con A) and pokeweed mitogen (PWM) after 4 days in culture. The response usually exceeded that seen after 6 days with tuberculin purified protein derivative (PPD) in Mantoux positive lymphocyte donors.