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P Verroust

Publications and source records attributed to P Verroust.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization.

By use of immunodepletion studies, we characterized four monoclonal antibodies reactive with rabbit brush-border (BB) as specific for aminopeptidase N (AP), dipeptidylpeptidase IV (DPPIV), neutral endopeptidase (EP), and angiotensin-converting enzyme (ACE), and we used these antibodies for immunohistochemical detection of these four hydrolases. Expression within the kidney was studied by light and electron microscopy. All four hydrolases are expressed on the various segments of the proximal tubule. In addition, EP and DPPIV are detectable on visceral epithelial cells of the glomerulus and AP on the cells of Bowman's capsule. Outside the kidney, the four hydrolases are expressed within the digestive and genital tracts, where AP, EP, and DPPIV predominate on epithelial structures, whereas ACE is essentially located in vascular structures. The latter localization is also characteristic of ACE in the other organs studied, where clear-cut systematic distribution of the other hydrolases was often difficult to demonstrate. In addition, AP, DPPIV, and EP were detected on lymphoid cells. As compared to reports of data obtained essentially by enzymatic or immunoradiometric assays, these observations suggest considerable interspecies variations of extrarenal expression of the major BB hydrolases. This should be taken into account in attempting to define a general physiological role for a given enzyme.

Aminopeptidases↗
PubMed1988

Distribution of enkephalinase (membrane metalloendopeptidase, E.C. 3.4.24.11) in rat organs. Detection using a monoclonal antibody.

A monoclonal antibody to enkephalinase (membrane metalloendopeptidase E.C. 3.4.24.11) raised by immunizing mice with rabbit renal cortical cells (mAb 85A2) was used to assess the tissue distribution of the enzyme in adult rats. When incubated with enkephalinase purified from renal brush border or with solubilized brush border proteins in the presence of antimouse IgG, the mAb specifically precipitates active enkephalinase as assessed by thiorphan inhibitable hydrolysis of [3H]-(D.Ala2, Leu5)]enkephalin. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies of radiolabeled brush border proteins indicate that mAb 85A2 binds specifically an antigen with an apparent molecular weight of 90 kilodaltons, corresponding to published values for enkephalinase. This antibody was used to localize enkephalinase by autoradiographic and immunofluorescence techniques. Immunohistochemical data show intense expression of this ubiquitous enzyme in brain, kidney, thyroid, parts of intestine, lung, seminal vesicle, and prostate. Specific areas of the brain contain more enkephalinase than others. Similarly, specific intense localization demonstrated in pulmonary alveolar cells and along the brush borders of the gut and the pars recta of the renal proximal tubule indicates that only select cell populations and/or cell compartments express this enzyme.

Animals↗
PubMed1987

Enkephalinase (EC 3.4.24.11) is highly localized to a striatonigral pathway in rat brain.

Autoradiographic visualization of enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) in sagittal sections of rat brain using a 125I-labelled monoclonal antibody showed the presence of a dense immunoreactivity in a tract joining the striatum to the substantia nigra. Unilateral kainate injections into the striatum elicited a strong ipsilateral decrease in enkephalinase activity and immunoreactivity in both the injected area and substantia nigra, particularly its pars compacta. This demonstrates the presence of enkephalinase all along fibers of a striatonigral pathway.

Animals↗
PubMed1987

Characterisation of two probes for the localisation of enkephalinase in rat brain: [3H]thiorphan and a 125I-labeled monoclonal antibody.

We studied the binding of two radioactive probes, i.e. [3H]thiorphan and a 125I-labeled monoclonal antibody raised against the rabbit kidney enzyme, to enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) from rat cerebral membranes. [3H]Thiorphan binding at equilibrium to striatal membranes was monophasic with a KD (0.7 nM) and a pharmacology consistent with a selective labeling of the enzyme. The ratio of Vmax/Bmax was in the same range as the Kcat of the enzyme purified from peripheral tissues. The monoclonal antibody immunoprecipitated to a similar extent the solubilised enkephalinase activity and [3H]thiorphan binding sites from striatum. The regional distributions of binding sites for the two probes established either on isolated membranes or autoradiographic sections were highly heterogeneous and similar to that of enkephalinase activity. Hence the two probes appear to label membrane-bound enkephalinase in rat brain but, from a technical point of a view, the 125I-monoclonal antibody is a more sensitive and flexible tool.

Amino Acids, Sulfur↗
PubMed1987

Indomethacin reduces proteinuria in passive Heymann nephritis in rats.

Indomethacin has been used to lower proteinuria in human glomerular diseases with controversial results. The mechanism of indomethacin beneficial effects has not been established. A possible explanation is that indomethacin reduces proteinuria by inhibiting the synthesis of renal prostaglandins (PGs); however, appropriate studies to address this issue have never been done. The objectives of the present study were: to investigate whether indomethacin influences protein excretion in an experimental model of immunologically-mediated glomerular disease; to establish if the possible favorable effect of indomethacin on proteinuria is related to a reduction in glomerular filtration rate (GFR); to establish the possible association between the antiproteinuric effect of indomethacin and its inhibitory effect on arachidonic acid (AA) metabolites of renal or extrarenal origin; and to further investigate the relationship between proteinuria and renal thromboxane (Tx) synthesis previously demonstrated in experimental models of nephrotoxic nephritis and adriamycin (ADR) nephrosis. To this purpose we used an experimental immune-complex disease, passive Heymann nephritis (PHN) which was induced in the rat by a single intravenous (i.v.) injection of heterologous serum directed against a brush border component (gp 330 antigen). Indomethacin at a dose of 6 mg/kg intraperitoneally (i.p.) administered for four consecutive days to PHN animals during the period of heavy proteinuria, effectively reduced urinary protein excretion. The reduction in proteinuria does not appear to be a consequence of a reduction in GFR as documented by inulin clearance. Glomerular synthesis and urinary excretion of vasodilatory prostacyclin (PGI2) and PGE2 were decreased or unchanged in PHN animals in respect to control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Down modulation of Heymann's nephritis by mercuric chloride.

The time course of Heymann's nephritis (HN), assessed on proteinuria and immunomorphology, has been compared in Lewis (LEW) rats immunized with BB alone (group A) or injected with HgCl2 and subsequently immunized in a similar manner (group B). Whereas all rats from group A developed typical HN characterized by heavy proteinuria and abundant glomerular immune deposits, rats from group B did not develop or developed a markedly attenuated form of HN; proteinuria was never detectable, immune deposits were absent or minimal. No abnormalities were found in rats injected with HgCl2 alone. In order to explain our findings, we have studied the glomerular and tubular expression of the 330 kD nephritogenic glycoprotein (gp330) as well as the corresponding antibody response. In rats receiving HgCl2, gp330 was normally expressed on BB and glomerular epithelial cells as indicated by in vitro and in vivo binding of anti-gp330 antibodies, but titers of anti-BB and anti-gp330 antibodies were considerably lower than in group A control rats. These findings therefore suggest that HgCl2 acts by its immunodepressive effect recently related to an increase in T suppressor cells. This effect is paradoxical since HgCl2 induces autoimmunity in Brown-Norway rats, and we suggest that it may be akin to observations reported in clinical practice where drugs may be immunostimulatory in some patients and immunodepressive in others. The mercury model may therefore represent a unique tool to evaluate the relationship between genetics and drug-induced immune dysregulation.

Animals↗
PubMed1987

Multiple myeloma and severe renal failure: a clinicopathologic study of outcome and prognosis in 34 patients.

Renal failure (RF) occurring in the course of multiple myeloma is often judged irreversible and generally considered an ominous complication. The aim of the present study was to re-evaluate the outcome, triggering conditions and prognostic factors of severe RF in a series of 34 patients, 33 to 90 years old. RF was totally reversible in 7 patients and partially reversible in 9 although 6 of them had to be temporarily dialyzed. However, the improvement in renal function was often very slow as indicated by an average recovery time of 115 days. The high rate of RF reversibility was associated with markedly lengthened survival. Review of triggering events confirmed the leading role of dehydration and hypercalcemia, but further suggested that intake of nonsteroidal anti-inflammatory drugs and renal infection might play a part in the development of RF. Systematic statistical analysis of potential prognostic factors showed that the outcome was significantly more severe in females, but age, myeloma characteristics including tumor mass, calcemia, and triggering events had no predictive value. The most reliable prognostic indicators were provided by analysis of kidney biopsy performed in 30 patients. Complete recovery from RF was observed only in the absence of global tubular atrophy and interstitial damage. In contrast, cast-induced tubular obstruction detected by the presence of Tamm-Horsfall protein in urinary space of glomeruli did not seem to influence the outcome of RF. Finally, we analyzed the prognostic value of immunochemical properties of light chains (LC). Lambda LC were unexpectedly detected in 2 of 3 patients, as compared to a ratio of 1 to 3 in the population of normal and monoclonal Ig, but LC type did not correlate with the course of RF. Isoelectric points of LC measured in 32 patients were dispersed from 5.2 to 8.9 and bore only weak prognostic significance. These results underline the value of kidney biopsy and justify aggressive treatment including dialysis and chemotherapy.

Acute Kidney Injury↗
PubMed1987

Immunological segmentation of the rabbit distal, connecting, and collecting tubules.

To obtain monoclonal antibodies (MAB) specific for the different cell types of distal and collecting tubules, BALB/c mice were immunized with cell suspensions highly enriched in cells from the distal segments of the rabbit nephron. Nine MAB were selected and cloned. Four groups could be identified on the basis of double-labeling immunofluorescence (IF) on frozen kidney sections and on microdissected tubules. In addition, binding specificity at the cellular level was studied by immunoelectronmicroscopy (IEM) for selected MAB. A single MAB (group 1) was specific for distal bright cells and a subpopulation of cortical ascending limb cells. Six MAB (group 2) reacted with connecting and collecting tubules. Five of these (group 2A) had similar binding patterns and reacted identically with the two tubular segments. The MAB studied by IEM was specific for connecting and principal cells. One antibody (group 2B) reacted with only a fraction of the cells associated with the connecting tubule (CNT), but with all cells of the cortical collecting tubule (CCT). By IEM, this antibody was found to be specific for intercalated cells in CNT and bound both principal and intercalated cells of the CCT. Two MAB (group 3) reacted with antigen(s) expressed by the various terminal segments of renal tubule. MAB of groups 1 and 2A, which define distal bright cells and connecting-principal cells from the CNT-CCT, respectively, were used for cell fractionation experiments. Heterogeneous rabbit cortical cells were first incubated with the selected MAB. MAB-bearing renal cells were separated on plastic dishes previously coated with an affinity-purified goat anti-mouse immunoglobulin. Using these procedures it was possible to obtain highly purified subpopulations of distal, bright, or connecting-principal cells.

Animals↗
PubMed1987

Lymphocyte expression of a 90 kD brush border antigen.

This study analyses the expression by rat lymphocytes of six renal brush border (BB) antigens defined by a set of monoclonal antibodies and demonstrates that a 90 kD protein (gp 90), synthesized by BB and glomeruli is found on the surface of T and B cells. The other antigens, including the 330 kD protein involved in Heymann's nephritis--a model of epimembranous glomerulonephritis--are not detectable on the surface of lymphocytes. Immunochemical studies indicate that gp 90 and the related protein immunoprecipitated from thymocyte membranes co-migrate in SDS-PAGE. Quantitative binding analysis shows that the number of antigenic sites is in the same order of magnitude on thymocytes, spleen and bone-marrow lymphocytes as well as on two lines of pre-B and pre-T cells, but considerably lower on a highly differentiated helper T cell line.

Animals↗
PubMed1986

Characterization of monoclonal antibodies to rabbit renal cortical cells.

The immunological heterogeneity of the rabbit nephron was investigated using monoclonal antibodies. Seventeen antibodies have been produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with unfractionated rabbit renal cortical cell preparations. Sixteen antibodies reacted with proximal tubular cells: 11 with the brush border and 5 with basolateral membrane or intracytoplasmic components. Only one of the latter was specific for constituents of the proximal tubule. One antibody reacted with the cortical collecting tubule. Eight of the anti-brush-border antibodies were further characterized by immunoprecipitation of detergent-solubilized radiolabeled brush-border membrane vesicles. Seven proteins with subunits ranging in molecular weight from 90,000 to greater than 340,000 were identified. Systematic survey showed that one of these proteins with a subunit molecular weight of 115,000 exhibited leucine aminopeptidase activity. Selected monoclonal antibodies bound to Sepharose 4B immunoadsorbents were used to deplete solubilized brush-border membrane vesicles of a given antigen and to identify leucine aminopeptidase. Furthermore, the obtention of specific antibodies directed against the proximal tubule allowed us to set up a simple method for renal cell separation: isolated renal cortical cells could be depleted by 80% in proximal cells by passage over columns of Sepharose 6MB covalently linked with three different monoclonal anti-brush-border antibodies, thus leading to cell suspensions considerably enriched in tubule cells originating from the more distal segments of the nephron.

Animals↗
PubMed1986

Indirect immunoselection of late distal cell populations from rabbit kidney cortex.

This study describes a method for the separation of distal cell populations based on the sequestration of proximal cells on immunoadsorbent columns (CNBr-activated Sepharose 6MB) bound with three brush-border monoclonal antibodies (S6-Mab). A high yield of isolated cell suspension from rabbit kidney cortex was prepared by mechanical dissociation after perfusion and incubation of the kidneys with 10(-3) M EDTA. The sequestration of the proximal cells was achieved in two sequential chromatographic steps. About 92% of the applied cells were first retained on an S6-Mab column after a 60-min stationary stage and the unbound cells were submitted by direct flow to a second S6-Mab column. In such conditions, 8 X 10(6) cells were recovered when starting with 331 X 10(6) cortical cells. The efficiency of the proximal cell depletion process was confirmed by an 80% decrease in brush-border enzymes, a very low phosphoenolpyruvate carboxykinase activity, and absence of cells bearing long microvilli, as ascertained by electron microscopy. This immunodepleted cell population presented the enzymatical characteristics of cells from the more distal segments. As compared with the initial cell suspension, these cells exhibited higher hexokinase (2.3 times), succinate dehydrogenase (1.5 times), and Na+-K+-ATPase (2.6 times) activities. In addition, adenylate cyclase activities remained sensitive to parathormone, arginine vasopressin, and isoproterenol. The functional capacity of these immunodepleted cells was assessed by an almost complete exclusion of eosin dye, a low Na+ and high K+ intracellular content, and a high respiratory rate of oxygen consumption. In conclusion, this immunoselective process makes it possible to obtain subpopulations of renal cortical cells possessing the main characteristics of the distal, connecting, and collecting cells for physiological and metabolic studies.

Adsorption↗
PubMed1986

An immunopathologic study of a 330-kD protein defined by monoclonal antibodies and reactive with anti-RTE alpha 5 antibodies and kidney eluates from active Heymann nephritis.

There is evidence indicating that the glomerular Ig deposits of Heymann's nephritis (HN)--a model of epimembranous glomerulonephritis--may be formed at least in part in situ by binding of free circulating antibody with brush border (BB) antigen expressed by glomerular epithelial cells. In this work, we provide evidence that a 330-kD protein defined by seven monoclonal antibodies is responsible for HN. 1) Ig eluted from glomeruli of rats with HN induced classically with crude BB preparation bind specifically the 330-kD antigen; 2) passive immunization with monoclonal antibodies induces epimembranous glomerular Ig deposits; 3) active immunization with the 330-kD antigen induces proteinuric glomerulonephritis; 4) the 330-kD antigen was present in the nephritogenic preparation purified by Edgington, Glassock, and Dixon, because it was identified by the corresponding heterologous antisera. These results, obtained by a completely different approach, confirm and extend those of Kerjaschki and Farquhar and provide a link with the classical studies on HN.

Animals↗
PubMed1986

Epitope diversity of angiotensin II analysed with monoclonal antibodies.

The antigenic heterogeneity of angiotensin II (AII) was studied with monoclonal antibodies. Twelve antibodies were produced and characterized. Association constants for AII varied from 1.2 X 10(8) to 1.1 X 10(10) M-1. The fine specificity of the Mab was studied by immunoenzymoassay using solid-phase AII. Using AII analogues in binding inhibition experiments, three groups of specificity could be characterized: (1) five antibodies reacted only with peptides in which phenylalanine is the carboxy terminal aminoacid; for two of these antibodies, tyrosine4 is closely associated with the binding site, since iodine labelling suppresses reactivity; (2) two antibodies also required phenylalanine in position 8, but, in addition, reacted with AI, a decapeptide in which phenylalanine is not terminal; (3) five antibodies reacted with analogues in which phenylalanine had been substituted for another amino acid. In addition, studies in which binding of a biotinylated Mab to solid-phase AII was analysed in the presence of various unlabelled Mab suggest further antigenic heterogeneity of AII.

Angiotensin I↗
PubMed1986

Effect of aspirin and sulindac in rabbit nephrotoxic nephritis.

Glomerular damage in nephrotoxic nephritis is mediated by circulating cells (neutrophils, platelets, and macrophages) infiltrating the glomerular tuft and responsible for the inflammatory reactions. It has been suggested that inflammatory cells may participate in glomerular inflammation through the synthesis and release of eicosanoids derived from the metabolism of arachidonic acid. On the other hand, old data and recent evidence indicate that eicosanoids derived from renal arachidonic acid metabolism play an important role in maintaining hemodynamics, especially in disease conditions. We wanted to evaluate the differential role of arachidonic acid metabolites derived from circulating or renal cells on the evolution of nephrotoxic nephritis. We used a nonselective cyclooxygenase inhibitor, aspirin, which blocks eicosanoid synthesis both in circulating cells and in renal tissue, as compared with a selective cyclooxygenase inhibitor, sulindac, which inhibits arachidonic acid metabolism in circulating cells, partially sparing renal cyclooxygenase. Aspirin, at a dosage that almost completely inhibits both circulating cell and renal arachidonate metabolites, worsens the morphologic expression of rabbit nephrotoxic nephritis and negatively influences the clinical course of the disease. Sulindac, at a dose that suppresses circulating platelet cyclooxygenase activity by 90%, but only partially affects renal prostaglandin synthesis, prevents extracapillary proliferation and reduces proteinuria without negative influence on glomerular hemodynamics.

Animals↗