Environmental influences on the composition and structure of the freshwater mussels in shallow lakes in the Cuiabá River floodplain.

The maintenance of the freshwater mussels' community in lakes is determined by abiotic factors at the local scale and at regional scale by interspecific relations between the larvae of bivalves and fish host. Whereas the distribution pattern at local scale, our goal was to understand the abundance and community composition of bivalves and relate the environmental agents structuring this community. We sampled 20 lakes in the floodplain of the Cuiabá River using a standardized method of sampling. To evaluate the effect of environment on the community we applied multivariate inferential analyses. We found 1.143 individuals alive belonging into six species distributed at the family Hyriidae, Mycetopodidae, Sphaeridae and Corbiculidae. The results showed that in the Pantanal the bivalve assemblage structure is influenced locally by organic matter and particle size, variables that reflect the intense interactions between water-sediment. However it is important to emphasize that these environmental characteristics are the result of the dynamics of this system which is dependent on the flood pulse, a regional factor.

Increase in renal and urinary low and high molecular weight kininogens during chromate-induced acute renal failure in the rat: evidence for renal kininogen production.

In the present study, we investigated the plasma, urinary and intrarenal concentrations of low and high molecular weight kininogens during sodium chromate (25 mg/kg body weight)-induced acute renal failure (ARF) in the rat. Urinary kininogen underwent a transient increase with a maximum on day 7 (78 +/- 22 versus 4.2 +/- 1.6 ng bradykinin/mg creatinine) whereas plasma kininogen did not and glomerular filtration rate decreased (92 +/- 8 versus 895 +/- 70 microliters/min). The tissue level of kininogen was enhanced both in the cortex (1,319 +/- 123 versus 86 +/- 8 pg bradykinin Eq/mg protein) and in the medulla (1,673 +/- 138 versus 44 +/- 9 pg bradykinin Eq/mg protein) but more in the medulla (36 +/- 4- versus 15 +/- 3-fold). As plasma kininogen level was unchanged and glomerular filtration rate decreased, the increase in both renal concentration and urinary excretion of kininogen probably reflects stimulated renal production of kininogen in this model of ARF. Whether the evoked renal production of kininogen results from a local inflammatory response only or may subserve another physiological purpose remains to be elucidated.

Cystatin C secretion by rat glomerular mesangial cells: autocrine loop for in vitro growth-promoting activity.

Cystatin C, the major inhibitor of the cysteine proteinases found in human and rat body fluids, is particularly abundant in seminal plasma and cerebrospinal fluid. In a precedent report, we have evidenced noteworthy levels of cystatin C in rat kidney cortex. In the present study, we show that rat mesangial glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeled cells and culture media showed that the synthesized cystatin C is a 15.5 +/- 0.5 kDa protein. The protein was released into the culture supernatant (1.6 +/- 0.26 micrograms/10(6) cells/24 h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequence of rat cystatin C) increased mesangial cell proliferation. Affinity chromatography on Ultrogel-avidin-biotin-PPPR of extracts of metabolically labeled cells indicate the existence of a PPPR binding protein of 46 kDa. The results described in this work suggest, for glomerular rat mesangial cells in vitro, an autocrine regulation of proliferation by cystatin C.

Cystatin C levels in sera of patients with human immunodeficiency virus infection. A new avidin-biotin ELISA assay for its measurement.

A solid-phase enzyme-linked immunosorbent assay (ELISA) for determining human serum cystatin C is described. In 50 normal samples, cystatin C concentration was 1247 +/- 224 micrograms/L (mean +/- SD) which is in agreement with previously reported levels. Serum levels of cystatin C and beta 2-microglobulin (beta 2-M) were investigated in a time-course study during the development of human immunodeficiency virus (HIV) infection. We found a persistent and uniform increase in the beta 2-M concentration (2762 +/- 1239 micrograms/L). In contrast to beta 2-M, on the basis of cystatin C levels, we found two distinct populations, one of which demonstrated an increased concentration (1620 +/- 618 micrograms/L). Interestingly a second group (21% of patients) exhibited an initial significant decrease in cystatin C concentration with a mean value of 377 (range 55-850) micrograms/L, followed by an increase. The biphasic pattern of cystatin C serum, a major cysteine proteinase inhibitor, during the course of HIV infection suggests a possible role for these proteinases (or proteinase inhibitors) in the development of this syndrome.

Early acute effects of mercuric chloride on synthesis and release of kallikrein and on distal tubular morphology of rat renal cortical slices.

The effect of mercuric chloride on kallikrein content and secretion of renal cortical slices was studied. Mercuric chloride showed dose-dependent inhibition of the secretion of immunoreactive and active kallikrein in the medium associated with a relative increase in the kallikrein tissue content of slices. However the net de novo biosynthesis was also reduced. Active and inactive kallikrein exhibited the same percentage of inhibition indicating that the activation mechanism of prokallikrein was not affected. These results suggest that mercuric chloride exerts an inhibition on tubular secretion but also on the tissular biosynthesis of kallikrein in these in vitro conditions. Morphological study of slices incubated in the presence of mercury also revealed significant tissular lesions which were located on the proximal tubule, but distal tubular changes were also observed. Distal nephrons were identified by the presence of immunoreactive kallikrein with the peroxidase-antiperoxidase method. These ultrastructural alterations included an increase in number and size of lysozomes, vacuoles and lipid droplets. These lesions were associated with an increased release of the lysozomal enzyme N-acetyl-beta-D-glucosaminidase. Since these distal tubular lesions are associated with inhibition of kallikrein secretion which is specifically located in the distal tubule, these results suggest that acute exposure of kidney cortical slices to mercuric chloride causes rapid and marked ultrastructural changes not only on the proximal tubule but also induced structural and biochemical effects at the distal tubule level. As incubation of mercuric chloride did not induce any direct alterations of immunoreactive and active kallikrein, it is likely that the observed inhibition of kallikrein synthesis and secretion are secondary to the morphological lesions.

Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteases.

Cystatin C, a cysteine proteinase inhibitor has recently been suggested to be a potent regulator of inflammatory processes and may act in defense against viral and bacterial infections. Two common forms of the protein were purified from the urine of a patient having received a renal transplant. The slow form of cystatin C possessed the N-terminal tetrapeptide Lys Pro Pro Arg, which was cleaved in the fast form. This peptide sequence, called postin, was synthesized. The three molecules, slow and fast forms of cystatin and the synthetic peptide, were tested for their effects on the migration activity of human polymorphonuclear neutrophils (PMNs). The slow form was found to display both chemotactic and chemokinetic activities, while the fast form and postin were only chemokinetic. Nevertheless, all the substances could induce a "motile" morphology. In addition, the two forms of cystatin C were powerful inhibitors of PMN chemotaxis induced by complement-derived chemotactic factors. This suggests that cystatin C in its two different cleaved forms and the N-terminal tetrapeptide can modulate PMN locomotion. Cysteine proteases may therefore play a role in neutrophil migration activity.

Modulation of phagocytosis-associated respiratory burst by human cystatin C: role of the N-terminal tetrapeptide Lys-Pro-Pro-Arg.

Cystatin C, a cysteine protease inhibitor, has recently been suggested to be a potent regulator in inflammatory processes. Human cystatin C was isolated from the urine of one patient suffering from tubular disorders and was tested for its effects on two functions of human polymorphonuclear neutrophils (PMN): O2- release and phagocytosis. Slow-form or (des 1-4) cystatin C and fast-form or (des 1-8) cystatin C differed by the presence in (des 1-4) cystatin C only of the N-terminal tetrapeptide Lys-Pro-Pro-Arg. Whereas (des 1-8) cystatin C did not seem to interfere with PMN functions at physiological concentrations, (des 1-4) cystatin C induced an inhibition of PMN phagocytosis-associated respiratory burst in response to opsonized zymosan particles. The inhibition may be attributed to the tetrapeptide Lys-Pro-Pro-Arg which has been synthesized and shown to have the same inhibitor effects, at concentrations similar to those required for (des 1-4) cystatin C. These results support a potential role for cystatin C as a modulator during inflammation.

Direct radioimmunoassay of rat cystatin C: increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy.

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. The two-steps purification procedure included a Carboxymethyl-papain affinity chromatography and anion exchange chromatography. The purified protein was identified as rat cystatin C by the following criteria: firstly retained on a Cm-papain affinity column, secondly an apparent molecular weight of 15 kDa and pI of 10.2. Antisera raised in rabbits against our purified rat cystatin C did not cross-react with other urinary proteins such as rat albumin and rat kallikrein, but partially cross-reacted with human cystatin C. A direct radioimmunoassay was developed and it enabled 8.32 fmol/ml of rat cystatin C to be detected. The detection range was between 0.125 and 62.5 ng/ml, with 10% intra-assay variation and 14% inter-assay variation. Physiological rat cystatin C excretion (40 +/- 18 micrograms/24 h) was found by the direct assay. In the chromate-intoxicated rat, urinary excretion increased twenty-fivefold (1017 +/- 391 micrograms/24 h) and returned to normal level one week after intoxication. This RIA will allow the study of rat cystatin C metabolism particularly during renal dysfunction.

Inverse relationship between renal and urinary kallikrein during chromate-induced acute renal failure in rat: urinary kallikrein excretion as a possible recovery index.

Acute renal failure (ARF) was induced in rat following a single injection of sodium chromate. A transient polyuria and a 10-fold decrease in glomerular filtration rate was immediately observed after sodium chromate administration. Urinary sodium and potassium excretion were reduced within 24 h and remained decreased for 8 to 10 days. Progressive recovery of normal renal functions, mainly electrolyte excretion and filtration rate was observed 12 days after sodium chromate administration. Urinary kallikrein excretion (UKE) was decreased only 48 h after sodium chromate administration. However the proportion of the active and inactive form excreted was unchanged. UKE remained also at a reduced level for 8 to 10 days and returned progressively to base-line level. The kallikrein content in the tissue was significantly increased immediately after sodium chromate administration and recovered normal values 12 days later. The increase of kallikrein in the tissue is more likely unspecific due to impaired protein transport than a specific stimulation of renal kallikrein biosynthesis. The decreased UKE may indicate a distal tubular reversible dysfunction in this ARF model. These reductions in electrolyte excretion, glomerular filtration and UKE were associated with selective morphological lesions. Whereas the glomeruli were intact, important damages affected proximal tubule cells which appeared necrotic and showed presence of vacuoles, liquefaction of cytoplasmic material and lost of microvilli. Less marked lesions were however observed in distal tubules, particularly large vacuoles were present at the apical poles of the tubule cells, the sites of kallikrein secretion. These distal damages may be involved in the increase of tissue concentration and in the decrease of UKE.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct radioimmunoassay of active and inactive human glandular kallikrein: some physiological and pathological variabilities.

We have developed a sensitive and specific radioimmunoassay which allows the detection of human glandular kallikrein in biologic fluids at a level of 40 pg/ml. The antisera did not recognize human plasma kallikrein and glandular kallikrein from other species including marmoset. Furthermore the antibody did not bind pro-kallikrein but was specific for the trypsin activated kallikrein. The antibody inhibited the kininogenase activity of standard kallikrein incubated with human kininogen. However active kallikrein inhibited by inhibitors bound at the active site is still detectable, indicating that the antibody is specific for the structure of the active form but not for the active site. In normotensive subjects, daily urinary kallikrein excretion increased with age until 30, then a decrease was observed. In renal transplanted recipients a progressive increase of the active form was found. A low concentration of immunoreactive active kallikrein was detected in lymphatic fluids of patients suffering from acute pancreatitis treated by lymphatic drainage; although this kallikrein is the active immunoreactive form, a very weak kininogenase activity was measured, suggesting a partial inhibition by anti-proteases. These data provide complementary evidence for the physiological and pathological role of glandular kallikrein.

A rapid two-step purification of rat cystatin C, one major inhibitor of cysteine proteinases.

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.

Isolation and characterization of rat kidney alanine aminopeptidase.

Rat alanine aminopeptidase was purified from kidney by isolation of the brush border membrane with CaCl2 followed by differential centrifugation and tryptic proteolysis. It is a glycoprotein with a molecular weight of approximately 210,000 daltons comprising two 110,000-dalton subunits and has an amino acid composition similar to that of the human enzyme. Two zinc atoms are covalently bound to each protein subunit.

Isolation and characterization of post gamma globulin in mouse.

A protein of about 13,000 daltons was isolated from mouse CBA urine after inducing a tubular renal dysfunction. This protein was demonstrated similar to human Post Gamma globulin by electrophoresis, aminoacid content and immunochemical criteria.

Biochemical characterization of an Fc receptor of rabbit lymphocytes.

Isolation of Fc-binding molecules was performed by complexing IgG antibodies and the corresponding antigens in lysates of radiolabeled rabbit lymphoid cells. A single-chain molecule of 110 000 apparent mol. wt. (unreduced) or 120 000 (reduced) was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis when the antibodies were in the IgG form but not as F(ab')2. This molecule was very susceptible to proteolysis, but the fragments thus produced remained associated by disulfide bridges, and the binding properties of the molecule were conserved. The molecule and its proteolytic fragments (mol. wts. 75 000, 45 000 and 20 000) were very similar to those obtained for the mouse Fc receptor under similar conditions, and therefore the molecule was designated as rabbit Fc receptor. Among several precipitating systems used, some Ig-anti-Ig complexes appeared to be the most efficient in coprecipitating the rabbit Fc receptor. Remarkably high titers of Fc receptor were found on lymphocyte membrane of infected rabbits, in agreement with a possible role of this molecule in immune regulation.

Lead poisoning in monkeys: functional and histopathological alterations of the kidneys.

Ten monkeys (Macacus Irus) were given 0--15 mg of lead acetate (in drinking water) 6 days a week for 9 months. Two of the monkeys were also put on a low calcium diet with 6 mg of lead acetate/day. The blood lead level usually increased from the third month according to the dose of lead ingested and more quickly in monkeys deprived of calcium. Some of the monkeys showed signs of alteration in protein glomerular filtration and/or proximal tubular reabsorption. Studies using optical and electron microscopy showed distinct pathological changes in the proximal tubular epithelium where heavy deposits of lead were seen in the nuclei.