Application of Dolichos biflorus in immunoassay detection of kidney collecting duct biomarkers.

Currently there are no biomarkers for detecting collecting duct damage in man. Antibodies to several collecting duct-specific antigens exist but sandwich assays have been difficult to establish due to the need for two different antibodies to the same protein. We hypothesized that a collecting duct-specific lectin could be used in combination with a collecting duct-specific antibody to negate the need for two different antibodies. The collecting duct specificity of selected antibodies (NiCa II 13C2, Pap XI 3C7, HuPaP VII 2B11 and aquaporin 2), was verified by immunohistochemistry. Aquaporin 2 and Pap XI 3C7 were used successfully in setting up assays with the lectin Dolichos biflorus, using the Meso Scale Discovery (MSD) platform. Antigen expression was highest in the papillae of rat and human kidney (corresponding to the greatest density of collecting ducts) and was also present in normal urine. We propose that further qualification and validation would lead to an assay for detecting collecting duct damage in man.

Urinary antigens as markers of papillary toxicity. I. Identification and characterization of rat kidney papillary antigens with monoclonal antibodies.

Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the rat renal papilla. One IgG and five IgM monoclonal antibodies, reacting with antigens localized in the papilla were obtained. Three of the IgM class and the IgG class monoclonal antibodies were found to be specific for antigens localized in collecting ducts, two of them staining papillary collecting ducts more intensely than cortical collecting ducts. The IgG class antibody, termed Pap X 5C10, recognizes an antigen located at high density on the luminal side of papillary collecting duct epithelial cells and at lower density in cortical collecting duct cells. One of the IgM class monoclonal antibodies reacts with an antigen localized in epithelial cells as ascending and descending loops of Henle and of connecting tubules. Another of the IgM class monoclonal antibodies reacts with an antigen localized in the interstices of the inner medulla. All these monoclonal antibodies react with their antigens in native frozen as well as in Bouin-fixed and paraffin-embedded tissue slices. Molecular properties of the Pap X 5C10 antigen have been investigated by gel permeation chromatography, SDS-PAGE, Western blotting, and isoelectric focusing. The results indicate that the antigen in both its tissue-derived and urinary form is of large (150-200 kDa) molecular size and can be separated into two molecular species with isoelectric points of pH 7.2 and 7.3 respectively. In the urine the antigens recognized by the monoclonal antibodies form large complexes with Tamm-Horsfall protein. The antigen-containing complexes can be extracted from urine by adsorption to diatomaceous earth and elution with SDS-containing buffer. Using sandwich ELISA-type assays it is possible to determine the concentration of the antigens. In preliminary experiments we were able to show that at least three of the antigens are detected in the urine following toxic insults to the kidney. The monoclonal antibodies prepared and the tests developed thus may provide direct diagnostic access to the renal papilla and allow, for the first time, early detection of papillary damage.

Papillary necrosis and the antinuclear factor.

The cold-reacting antinuclear factor, specific to kidney tissues, was detected transiently in serum specimens from a diabetic patient during an episode of papillary necrosis. Determination of the cold-reacting antinuclear factor is suggested as being useful in evaluating the degree destruction in kidney tissues.

In vitro lysis of target cells by rat polymorphonuclear leukocytes isolated from acute pyelonephritic exudates.

Polymorphonuclear (PMN) leukocytes from acute pyelonephritic exudates of rats were examined for cytoxicity against various target cells. Pyelonephritic PMN leukocytes caused lysis in vitro of syngeneic renal epithelial cells in 24 to 48 hr. They also caused lysis of allogeneic and xenogeneic target cells. The susceptibility to PMN-induced cytolysis was different among various target cell lines. Cell lysis proceeded at low ratios of effector to target cells and was not dependent on the addition of antibody or lectins. PMN isolated from peritoneal exudates (starch or endotoxin induced) were also cytoxic for the target cells. Cell-free supernatants from pyelonephritic PMN-target cell mixtures, or pyelonephritic PMN leukocytes cultured alone contained cytolytic substances which were nondialyzable and heat stable Finally, the urine of pyelonephritic rats was cytotoxic. These results suggest that PMN leukocytes and their cytotoxic mediators are directly involved in the pathogenesis of renal injury in pyelonephritis.

Cellular immunity in pyelonephritis: identification of suppressor cell activity of spleen cells in response to concanavalin A and inhibition of lymphocyte-mediated L cell cytotoxicity.

A population of fast sedimenting spleen cells was identified as the cause of impaired in vitro response of spleen cells to concanavalin A (Con A) in acute pyelonephritis in rats. These fast-sedimenting cells responded to Con A by suppressing the DNA synthetic response of normal spleen cells to Con A. In addition, spleen cells from acute pyelonephritis rats were significantly less able to mediate in vitro cell destruction in xenogeneic aggressor lymphocyte: target L cell mixtures. On the basis of these findings, the hypothesis is proposed that a suppressor cell can protect the pyelonephritic kidney against immunologically mediated tissue damage.

Amount and avidity of antibody to Escherichia coli O antigen measured with the ammonium sulphate precipitation technique in children with urinary tract infections.

The antibody amounts and avidities were analyzed in 13 patients with acute primary pyelonephritis, 11 patients with acute primary cystitis, and one with ureterocele and recurrent infections, using the ammonium sulphate precipitation (ASP) technique. The ASP titrations did not discriminate as well between pyelonephritis and cystitis as do the determinations with the indirect hemagglutination technique. The increase of antibody titer and avidity detected by the ASP method in some of the cystitis patients suggested a deeper tissue involvement in some cases resulting in antibodies demonstrable with this technique. Since control patients also showed a somewhat heterogenous antibody response regarding titer and avidity it cannot be excluded that stimulation by Escherichia coli antigens in the gut is detected by the ASP method.