In vivo antibody-mediated modulation of aminopeptidase A in mouse proximal tubular epithelial cells.

Aminopeptidase A (APA) is one of the many renal hydrolases. In mouse kidney, APA is predominantly expressed on the brush borders and sparsely on the basolateral membranes of proximal tubular epithelial cells. However, when large amounts of monoclonal antibodies (MAbs) against APA were injected into mice, we observed strong binding of the MAbs to the basolateral membranes, whereas the MAbs bound only transiently to the brush borders of the proximal tubular epithelial cells. In parallel, APA itself disappeared from the brush borders by both endocytosis and shedding, whereas it was increasingly expressed on the basolateral sides. Using ultrastructural immunohistology, we found no evidence for transcellular transport of endocytosed APA to the basolateral side of the proximal tubular epithelial cells. The absence of transcellular transport was confirmed by experiments in which we used a low dose of the MAbs. Such a low dose did not result in binding of the MAbs to the brush borders and had no effect on the presence of APA in the brush borders of the proximal tubular epithelial cells. In these experiments we still could observe binding of the MAbs to the basolateral membranes in parallel with the local appearance of APA. In addition, treatment of mice with chlorpromazine, a calmodulin antagonist that interferes with cytoskeletal function, largely inhibited the MAb-induced modulation of APA. Our studies suggest that injection of MAbs to APA specifically interrupts the normal intracellular traffic of this enzyme in proximal tubular epithelial cells. This intracellular transport is dependent on the action of cytoskeletal proteins.

Induction of albuminuria in mice: synergistic effect of two monoclonal antibodies directed to different domains of aminopeptidase A.

BACKGROUND: Aminopeptidase A is an enzyme that is present on podocytes and is involved in the degradation of angiotensin II. In previous studies in mice, we administered single monoclonal antibodies directed against aminopeptidase A. We observed that only monoclonal antibodies that inhibited aminopeptidase A enzyme activity caused albuminuria. METHODS: In this study, the effects of the combined injections of two monoclonal anti-aminopeptidase A antibodies (mAbs) were studied, using a combination of anti-aminopeptidase A mAbs that were directed against two different domains involved in the aminopeptidase A enzyme activity (ASD-3 or ASD-37) and an anti-aminopeptidase A mAb not related to the enzyme active site (ASD-41). RESULTS: An injection of the combinations ASD-3/37 (total 4 mg, 1:1 ratio) and ASD-37/41 (total 4 mg, 1:1 ratio) in doses that do not cause albuminuria when given alone (4 mg) induced massive albuminuria at day 1 after injection. The combination ASD-3/41 had no effect. This albuminuria was not dependent on systemic immune mediators of inflammation and could not merely be related to a blockade of aminopeptidase A enzyme activity. However, a correlation was observed between the induction of albuminuria and the aggregation of the mAbs injected and aminopeptidase A on the podocytes. An injection of the combinations ASD-3/37 or ASD-37/41 did not cause an increase in systemic blood pressure. The treatment with a combination of enalapril and losartan lowered blood pressure (53 +/- 10 vs. 90 +/- 3 mm Hg in untreated mice) and reduced the acute albuminuria by 55% (11,145 +/- 864 vs. 24,517 +/- 2448 micrograms albumin/18 hr in untreated mice). However, similar effects were observed using triple therapy. Therefore, the reduction of albuminuria by the combined treatment of enalapril/losartan seems to be the consequence of the reduction in the systemic blood pressure. These findings argue against a specific role for angiotensin II in this model. CONCLUSIONS: The combined injection of two mAbs directed against different domains of aminopeptidase A induces a massive albuminuria in mice, which is not merely dependent on angiotensin II. We hypothesize that the direct binding of mAbs to at least two pathogenic domains on aminopeptidase A triggers the podocyte to release mediators that are involved in the observed albuminuria.

Mouse glomerular epithelial cells in culture with features of podocytes in vivo express aminopeptidase A and angiotensinogen but not other components of the renin-angiotensin system.

The binding of antibodies to podocytic antigens such as the Heymann antigen or aminopeptidase A may lead to the induction of a membranous glomerulonephritis in several species. To study the possible future interactions of antibodies with antigens on these podocytes, epithelial cells from isolated mouse glomeruli were cultured. By indirect immunofluorescence, the cells were positive for cytokeratin, vimentin, desmin, and the ZO-1 protein, a component of the tight junction complex. When rat monoclonal antibodies were used, the cells were also positive for the hydrolases aminopeptidase A and dipeptidyl peptidase IV, and they stained with ASD-33, a monoclonal antibody that recognized an epitope only present on the cell membranes of mouse podocytes. They were negative for the von Willebrand factor and did not stain with a monoclonal antibody (ASD-13) that binds to endothelial cells of glomeruli and peritubular capillaries. By electron microscopy, the cells showed tight junctions but lacked Weibel Palade bodies (endothelium), desmosomes, and cilia (parietal epithelium). The mRNA expression of several components of the renin-angiotensin system was also examined, and some factors indirectly coupled to the renin-angiotensin system component angiotensin II in this podocytic culture by RT-PCR analysis. mRNA Expression for the angiotensin II degrading hydrolase aminopeptidase A and angiotensinogen was found, but this was not found for any other component of this system, such as renin, angiotensin-converting enzyme, or the angiotensin II receptors AT1a, AT1b, and AT2. Low mRNA expression for dipeptidyl peptidase IV was observed. In addition, expression of the growth factors transforming growth factor-beta and interleukin-7, and the extracellular matrix components fibronectin, laminin B2, perlecan, and collagen IV alpha 1, was observed. Given these characteristics, a glomerular epithelial cell culture with features of podocytes in vivo that will allow future studies on the interaction of anti-aminopeptidase A monoclonal antibodies and angiotensin II with aminopeptidase A was established. This is of interest in light of the observation that injection of mice with anti-aminopeptidase A antibodies causes an acute albuminuria.

Inhibition of aminopeptidase A activity causes an acute albuminuria in mice: an angiotensin II-mediated effect?

The hydrolase aminopeptidase A is an important regulator of the renin-angiotensin system, since it inactivates its most vasoactive component angiotensin II (Ang II). A single i.v. injection of a monoclonal antibody against mouse aminopeptidase A (ASD-4) induces a membranous-like glomerulonephritis in mice, characterized by an acute albuminuria, that is not dependent on complement, the coagulation system, or inflammatory cells. We hypothesized that this albuminuria is the consequence of a reduction in aminopeptidase A enzyme activity, that might subsequently lead to an increase in Ang II levels. Aminopeptidase A enzyme activity was analysed in vitro by a fluorimetric enzyme assay and in vivo by enzyme histochemistry. The role of Ang II in the induction of albuminuria in this model was studied by measuring the renal aminopeptidase A mRNA expression in our model by a competitive PCR assay as an indirect measure of Ang II levels. In addition, the role of Ang II in this model was studied by preventing the formation of Ang II with the angiotensin-converting enzyme inhibitor enalapril or by blocking of the Ang II receptor with the AT1 receptor antagonist losartan. Only antibodies that were able to inhibit the aminopeptidase A enzyme activity in vitro and in vivo induced an acute albuminuria in mice. Renal aminopeptidase A mRNA expression was increased by injection of the anti-aminopeptidase A antibody. Both enalapril and losartan treatment reduced the acute albuminuria, measured 1 day after injection of a monoclonal antibody against aminopeptidase A, by 91% and 83%, respectively. It is concluded that the induction of acute albuminuria is correlated to the enzyme-inhibiting capacity of the anti-aminopeptidase A antibodies. This impaired enzymatic activity most likely leads to an increase in the levels of Ang II, the best known substrate of aminopeptidase A. The results of our additional experiments are in keeping with our hypothesis that Ang II mediates this acute albuminuria. Whether this occurs by an increase of blood pressure or by a growth factor-like effect remains to be defined by further studies in this model.

Organ distribution of aminopeptidase A and dipeptidyl peptidase IV in normal mice.

The hydrolases aminopeptidase A and dipeptidyl peptidase IV, both present in the kidney on the brush borders of the proximal tubule epithelial cells and podocytes, are involved in the induction of experimental membranous glomerulonephritis in the mouse. However, little is known about their (co)distribution in other tissues and their function in health and disease. A detailed insight into the localization of these two enzymes is a prerequisite to elucidation of their function. Therefore, we investigated the presence and co-distribution of aminopeptidase A and dipeptidyl peptidase IV by immunohistology with two different rat monoclonal antibodies, the specificity of which was determined by an immunodepletion technique. In addition, the molecular weight of the hydrolases; was analyzed by SDS-PAGE after isolation by solid-phase immunoprecipitation from glomeruli, renal brush borders, and thymus. Both hydrolases showed different molecular weights in renal corpuscle, renal brush borders, and thymic cells. A widespread organ distribution of the two hydrolases was observed, with co-localization in kidney, liver, small intestine, thymus, brain, spleen, and lymph nodes, either on the same cells or on different cells in the same organ. This distribution and partial co-localization suggests that the two hydrolases, acting either alone or in concert, have a role in many diverse biological processes.

Characterization of a 43 kD protein associated to aminopeptidase A from murine kidney.

SDS-PAGE of affinity-purified APA under reducing conditions showed in addition to the specific APA band of M(r) 130 kD, a second band of M(r) 43 kD. Internal amino acid sequencing of three tryptic peptides from this second band, that was cut out of the polyacrylamide gel, matched the actin sequence. The identity of the 43 kD band was also confirmed by Western blotting.

Antigen-specificity of antibodies bound to glomeruli of mice with systemic lupus erythematosus-like syndromes.

BACKGROUND: MRL/l mice with diffuse proliferative glomerulonephritis, and graft-versus-host (GVH) mice with membranous glomerulonephritis, are both regarded as models for human systemic lupus erythematosus. In these two models, the specificity of the nephritogenic antibodies was analyzed. EXPERIMENTAL DESIGN: The nephritogenic antibodies were eluted from isolated glomeruli with an acid buffer. The antibodies were purified from these eluates on protein-A sepharose under high salt conditions to exclude the presence of antigens potentially bound to the antibodies. The specificities of the antibodies were analyzed towards components of the glomerular basement membrane (GBM), nuclear antigens and brush border of proximal epithelial cells in the indirect immunofluorescence (IF), enzyme linked immunosorbent assay, and Crithidia luciliae assay. Furthermore, we studied the glomerular binding by IF and immunoelectron microscopy after intravenous injection of the eluted antibodies into control mice. RESULTS: Glomerular eluates of both MRL/l and GVH mice showed nuclear staining and linear to homogeneous binding to GBM and tubular basement membrane in IF on normal mouse kidney. In enzyme linked immunosorbent assay, both eluates reacted not only with DNA, but also with histones and laminin, an important component of the GBM, however not with brush border. Reactivity with dsDNA was also found in the Crithidia luciliae assay for both eluates. In vivo binding was determined by injection of eluates into control MRL/n and F1 hybrid mice. The GVH eluate showed linear to homogeneous binding to the GBM 1.5 hour after injection which changed into a granular, membraneous pattern after 5 days, as determined by IF and immunoelectron microscopy. The MRL/l eluate also demonstrated linear to homogeneous binding to the GBM 1.5 hour after intravenous injection, that had decreased after 5 days, but did not change into a granular pattern. CONCLUSIONS: In these two models of systemic lupus erythematosus nephritis not only antibodies to DNA and histones, but also antibodies to laminin are involved in the induction of murine systemic lupus erythematosus nephritis. The latter antibody specificity has not been identified before in renal or glomerular eluates obtained from diseased MRL/l mice.

A nephritogenic rat monoclonal antibody to mouse aminopeptidase A. Induction of massive albuminuria after a single intravenous injection.

Antibodies directed against antigens present on renal epithelial cells can cause membranous glomerulonephritis in experimental animals, which closely resembles the human form of this disease. However, most antibodies produced so far fail to cause the persistent and severe proteinuria that is seen in humans. In our search for new antibodies of this kind, we have now produced a monoclonal antibody (mAb) against mouse aminopeptidase A, a hydrolase that is present in the mouse kidney. The mAb (ASD-4) was prepared by fusion of mouse myeloma cells with splenocytes of Lou rats immunized with brush border (BB) membranes from mouse kidneys. ASD-4 is of the IgG1 subclass and reacts with a 140-kD protein as demonstrated by immunoprecipitation on radiolabeled BB membranes. In indirect immunofluorescence and immunoelectronmicroscopy of normal mouse kidneys, ASD-4 was diffusely present on the BB of the S1 and S2 segments of the proximal tubules, and on the cell membranes of the glomerular visceral epithelia. It also bound to cell membranes of nonglomerular endothelia, smooth muscle cells of arteries, and juxtaglomerular cells. After injection of ASD-4 into normal mice, an immediate homogeneous binding to the capillary wall was seen that gradually changed into a fine granular pattern after 1 d. This glomerular binding was followed by binding to the BB and basolateral membranes of the convoluted proximal tubules. Immediately after injection of ASD-4, a dose-dependent albuminuria occurred that lasted for at least 16 d. ASD-4 is thus a new rat mAb against a well-defined renal epithelial antigen that causes not only membranous glomerulonephritis after a single injection in the mouse, but also severe albuminuria.