Increased prostaglandin production by glomeruli isolated from rats with streptozotocin-induced diabetes mellitus.

Abnormalities in glomerular function have been observed frequently in the early stages of both clinical and experimental diabetes mellitus. Because prostaglandins (PGs) are present in the glomerulus and have profound effects on glomerular hemodynamics, and because abnormalities of PG metabolism have been noted in other tissues from diabetics, we studied PG biosynthesis in glomeruli obtained from rats in the early stages of experimental diabetes mellitus. Streptozotocin, 60 mg/kg, was administered intravenously to male Sprague-Dawley rats. Control rats received an equal volume of the vehicle. Glomeruli were isolated 9-23 d later. Production of eicosanoids was determined by two methods: by direct radioimmunoassay after incubation of glomeruli under basal conditions and in the presence of arachidonic acid (C20:4), 30 microM, and by radiometric high-performance liquid chromatography (HPLC) after incubation of glomeruli with [14C]C20:4. When assessed by radioimmunoassay, mean basal production of both prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was twofold greater in the diabetic animals whereas production of thromboxane B2 (TXB2) was not significantly greater than control. In response to C20:4, both PGE2 and PGF2 alpha were also greater in the diabetic animals, but these differences were not statistically significant. The increased rate of basal PG production did not appear to be related directly to the severity of the diabetic state as reflected by the degree of hyperglycemia at the time of sacrifice. In fact, the rates of glomerular PG production in the individual diabetic animals correlated inversely with the plasma glucose concentration. The increased rate of PG synthesis did not appear to be due to a nonspecific effect of streptozotocin inasmuch as glomerular PG production was not increased significantly in streptozotocin-treated rats which were made euglycemic by insulin therapy. Furthermore, addition of streptozotocin, 1-10 mM, to the incubation media had no effect on PGE2 production by normal glomeruli. PGE2 production by normal glomeruli was also not influenced by varying the glucose concentration in the incubation media over a range of 1-40 mM. When metabolism of [14C]C20:4 was evaluated by high-performance liquid chromatography conversion to labeled PGE2, PGF2 alpha, TXB2, and hydroxyheptadecatrienoic acid by diabetic glomeruli was two- to threefold greater compared with that in control glomeruli, whereas no significant difference in conversion to 12- and 15-hydroxyeicosatetraenoic acid occurred. These findings indicate that glomerular cyclooxygenase but not lipoxygenase activity was increased in the diabetic animals. A concomitant increase in glomerular phospholipase activity may also have been present to account for the more pronounced differences in PG production noted in the absence of exogenous unlabeled C20:4. These abnormalities in PG biosynthesis by diabetic glomeruli may contribute to the altered glomerular hemodynamics in this pathophysiologic setting.

Leukotriene C4 binds to human glomerular epithelial cells and promotes their proliferation in vitro.

In human and experimental glomerulonephritis, glomerular hypercellularity results both from accumulation of macrophages and proliferation of resident glomerular cells. The recent identification of macrophage-derived factors that stimulate mesangial and epithelial cell proliferation suggests that these factors might contribute to the hypercellularity. To determine the identity of such macrophage-derived growth factors, we studied the effect of leukotrienes (LTs), products that are released from macrophages and leukocytes, on proliferation of human glomerular epithelial cells in culture. Dose-dependent (1-100 nM) stimulation of [3H]thymidine incorporation, an index of cell proliferation, was observed in cells incubated with the sulfidopeptide LTs, LTC4 and LTD4, but not with LTB4. The response was 248 and 172% of control values at 100 nM LTC4 and LTD4, respectively. This effect of LTC4 was abolished by FPL 55712. Subsequent binding studies demonstrated that glomerular epithelial cells possess specific receptors for LTC4. [3H]LTC4 bound rapidly at 8 degrees C to the cells. There was a plateau after 40 min incubation. Maximum specific binding was 70-90% of total binding. Specific binding was totally reversible with addition of an excess of unlabeled LTC4. Analysis of time-course association slopes at two concentrations of [3H]LTC4 and of the competition between a single concentration of [3H]LTC4 and increasing concentrations of unlabelled LTC4 allowed calculation of dissociation constants (Kd) of 220 and 217 nM, respectively. Both LTD4 and LTE4 exhibited ED50 values that were at least one order of magnitude higher than for LTC4. Thus, our findings suggest that LTC4 binds to specific receptors of glomerular epithelial cells, promotes proliferation of these cells, and could contribute to epithelial hypercellularity found in glomerulonephritis.

High affinity binding of 125I-angiotensin II to rat glomerular basement membranes.

125I-angiotensin II (AII) specifically bound to rat glomerular basement membrane (GBM). The kinetics of binding were similar to those obtained with the total glomeruli. The apparent dissociation constant was close to 50 pM with both preparations. The number of sites related to the amount of protein was two times greater with GBM than with total glomeruli. Since the amount of GBM protein extracted from a given amount of glomerular protein was about 10%, it was possible to estimate the share of the GBM binding sites for AII as representing 20% of the total number present in the entire glomerulus. Binding studies at equilibrium as a function of 125I-AII concentration and competitive binding experiments suggested either multiplicity of the binding sites or cooperativity in the binding reaction. Degradation of 125I-AII in the presence of GBM was slight and did not increase with time. The difference in the degrees of degradation of 125I-AII was too small to account for the observed difference in binding when the results obtained with GBM and isolated glomeruli preparations were compared. 125I-AII binding to GBM was increased after treatment of these membranes with collagenase, slightly diminished with neuraminidase, and almost completely abolished with trypsin suggesting the proteic nature of the receptor. 125I-AII binding to GBM was diminished after incubation of GBM with anti-GBM antibodies as a result of a decrease in the number of binding sites. 125I-AII binding was even more diminished in preparations of glomeruli isolated from rats passively immunized with anti-GBM antibodies when compared with glomeruli from control animals. This resulted from both smaller affinity for AII and decrease in the number of the binding sites. The present data provides evidence for specific binding sites for AII localized on GBM. This is noteworthy since receptors for polypeptide hormones are currently observed on the surface of cell membranes. These findings also suggest a new physiological role for AII which might involve modification of GBM permeability.

Quantification of collagen synthesis by cultured human glomerular cells.

This study examines the amount of total collagen and its different fractions synthesized by cultured human glomerular epithelial and mesangial cells. Two quantitative techniques were used, namely estimation of proline (Pro) plus hydroxyproline (Hyp) present in the collagenase-sensitive proteins and ELISA or RIA of the different types of collagen. In addition, the pattern of collagen synthesis for both cell types was further examined using immunofluorescence methods and polyacrylamide gel electrophoresis. Glomerular epithelial cells synthesized mainly type IV collagen and it was, for the better part, cell-associated. Mesangial cells synthesized approx. 4-times more collagen than epithelial cells. Type I collagen was predominant, but there were also type IV and III collagens. Secreted and cell-associated collagens were present in roughly equivalent amounts. In both cell lines 10-14% of the newly synthesized collagen had been degraded within the cells. These results provide quantitative data on collagen synthesis by human glomerular cells in vitro and represent the first necessary stage before studying which factors mediate the development of glomerular sclerosis.

Response of adenylate cyclase to parathyroid hormone and prostaglandins by human isolated glomeruli.

A role for hormonal substances and other biochemical messengers in the regulation of the glomerular filtration rate has been inferred from the results of micropuncture studies in the rat and from the demonstration of hormone-responsive adenylate cyclase activity in glomeruli isolated from the renal cortex of rats and rabbits. To investigate whether such hormonal factors may contribute to the regulation of glomerular function in humans, we studied the response of adenylate cyclase activity to the administration of human PTH-(1-34) and prostaglandins (PG) by glomeruli isolated from the renal cortex of four human kidneys. PTH and PGs (PGE2, PGI2, and, to a lesser extent, PGF2 alpha) stimulated human glomerular adenylate cyclase activity. Basal adenylate cyclase activity ranged from 0.2-1.2 nmol 20 min-1 mg-1. For each agonist, the percent increase above basal values (at the maximum concentration tested) and the concentration of the agonist that elicited 50% of the maximum stimulation (ED50) were as follows: for PTH 300-460% (10,000 mIU/ml); ED50, 100-550 mIU/ml; for PGE2, 160-380% (10 microM); ED50, 0.4-1.6 microM; and for PGI2, 180-650% (10 microM); ED50, 0.09-0.46 microM. The synthetic guanylnucleotide 5'-guanylylimidodiphosphate [Gpp(NH)p] potentiated the effect of PTH and PGI2, since the combined effects were greater than the sums of the effects of the individual agonists, and there was a significant interaction between Gpp(NH)p and PTH or PGI2, as indicated by three-factor analysis of variance. Additivity, but not potentiation, was observed for PGE2. The effects of PTH plus PGE2 or PGI2 were also additive, a finding that suggests that PTH and PGs are not linked to the same pool of adenylate cyclase. In contrast, the combination of PGE2 and PGI2 resulted in a significantly lower effect than the sum of their individual effects, a finding indicating that these PGs share in part a common pool of adenylate cyclase. Demonstration of PTH- and PG-dependent adenylate cyclase activity in human isolated glomeruli suggests a role for these agonists in the regulation of the glomerular filtration rate in man.

Angiotensin II receptors in human isolated renal glomeruli.

125I-Labeled angiotensin II ([125I]A II) binds specifically to glomeruli isolated from human kidneys that were obtained at nephrectomy or early autopsy. Equilibrium was reached after 30 min, and specific binding represented more than 90% of the total binding. Dissociation after dilution with the addition of an excess of unlabeled hormone was more rapid than after dilution alone. The effect increased as a function of the A II concentration. The Scatchard plot derived from saturation experiments was curvilinear, with an upward concavity. Two groups of receptor sites could be defined by the Kd values (0.1 and 2 nM, respectively) and the number of receptor sites (40 and 300 fmol mg glomerular protein-1, respectively). Alternatively, binding could be considered to follow a negative cooperative type of hormone-receptor interaction. [Asn1, Val5]A II, [Asp1,Ile5]A II, [des, Asp1,Ile5]A II, [Sar1, Ala8]A II, and [Sar1, Ile8]A II were all equally effective as competitive inhibitors of [125I]A II binding. Both calcium and magnesium (0.5-5 mM) produced an increase in [125I]A II specific binding, whereas guanylylimidodiphosphate, an analog of GTP, inhibited it. Degradation of the [125I]A II present in the incubation medium was estimated by three different techniques. It increased linearly with time and reached 20% at 30 min. Specific binding of A II to human glomeruli at plasma concentrations observed in man under physiological conditions and during the iv administration of A II demonstrates that human renal glomeruli include target cells for A II and thus suggests a role for A II in regulation of the glomerular filtration rate in man.

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide.

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.

Stimulation of guanylate cyclase by atrial natriuretic factor in isolated human glomeruli.

A 23 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) stimulated guanylate cyclase activity in isolated human glomeruli in a concentration- and time-dependent manner. ANF activated particulate guanylate cyclase whereas it had no effect on soluble guanylate cyclase. These results demonstrate that the glomerulus is a target structure for ANF in humans. They also suggest that ANF-induced increase in glomerular filtration rate is due to a direct effect of this peptide on the glomerular cells mediated by activation of glomerular guanylate cyclase.

A method for the quantitative assessment of malaria parasite development in organs of the mammalian host.

A non-radioactive PCR method was developed to quantify the development of malaria parasites in the infected host. This was achieved by using Plasmodium genus-specific primers corresponding to the parasite's small subunit ribosomal RNA genes. The quantification of the PCR product was performed by high performance liquid chromatography, and calibration curves were obtained by amplification from defined quantities of purified Plasmodium genomic DNA. Using this method, it was possible to quantify development of P. berghei and P. yoelii blood-stage parasites from blood and brain samples of infected mice, and of hepatic stage parasites, from liver samples of mice infected with different numbers of sporozoites.

Histamine H2 receptors in rat renal glomeruli.

The aim of this study was to demonstrate histamine-H2 receptors in glomeruli isolated from rat renal cortex and to correlate binding to stimulation by histamine of glomerular cyclic AMP concentration. Binding studies were performed at 10-12 degrees C using [3H]cimetidine as a tracer. Specificity of binding relies on the following: inhibition of [3H]cimetidine binding by the unlabelled drug, other H2-antagonists and agonists in contrast with the very weak inhibitory effects of H1 agonists and antagonists; reversibility of steady-state binding after addition of unlabelled drug; half inhibition of the glomerular cyclic AMP response to histamine at concentrations of cimetidine close to the KD value derived from the binding studies (3 microM); calculated KD value in agreement with the therapeutical concentration of cimetidine and the physiological concentration of histamine. [3H]Cimetidine binding concentration of cimetidine and the physiological concentration of histamine. [3H]Cimetidine binding strikingly increased in the presence of copper chloride (20-300 microM) due to an increase both in number of sites and affinity. However this greater binding did not influence either the inhibitory effect of cimetidine on histamine-induced glomerular cyclic AMP concentration or the stimulatory effect of histamine itself. [3H]Cimetidine binding was temperature-dependent since it progressively diminished from 0 to 37 degrees. This was not due to [3H]cimetidine degradation as shown by thin layer chromatography but rather to a change in drug-receptor interaction at higher temperatures. Glumerular concentration of cyclic AMP increased progressively in the presence of histamine (0.1-1000 microM). This stimulatory effect was markedly inhibited by H2 antagonists. These data demonstrate the presence in rat glomeruli of H2 receptors linked to adenylate cyclase.

Quantitative assessment of murine retrovirus LP-BM5 infection in MAIDS by PCR and anion exchange HPLC.

In vitro amplification of DNA by PCR is a powerful tool to detect small amounts of DNA. It is now widely used for detection of pathogenic agents from extracellular fluids and organs. The use of anion exchange HPLC to quantify the PCR product resulting from the specific amplification of the DNA from the replicative-defective viral DNA responsible for MAIDS is described. This technique allows precise quantification of MAIDS virus DNA in different organs and circumvents the use of radioactivity and gel electrophoresis.

Influence of calcium infusion on calcitonin and parathyroid hormone concentrations in normal and hemodialyzed subjects.

Plasma calcitonin and parathyroid hormone concentrations were compared before and after calcium infusion in both hemodialyzed patients and control subjects. Levels of both hormones were significantly higher in patients with chronic renal failure. In the uremic group, calcium infusion inhibited parathyroid hormone secretion but did not affect calcitonin. In the control group, calcium infusion stimulated calcitonin secretion but had no effect on parathyroid hormone.

Prostaglandin synthesis by human glomerular cells in culture.

PG synthesis by cultured human glomerular mesangial and epithelial cells incubated with [1- 14C] arachidonic acid was determined by radioimmunoassay (RIA) after high performance liquid chromatography purification. Both dissociated cells and cell monolayers were studied under basal conditions. PG synthesis by epithelial cells was undetectable. Mesangial cells produced low amounts of PGE2, PGF2 alpha and 6 keto-PGF1 alpha and no TXB2. We also examined the effects of several agents on PG synthesis in these two types of cells scraped away from their flasks using direct RIA. Arachidonic acid produced a slight stimulation only with mesangial cells whereas angiotensin II, cyclic AMP and calcium ionophore were inactive with both cell lines. Homogenization of the cells did not enhance the stimulatory effect of arachidonic acid. Alkalinization of the incubation medium produced an increase of PG production by mesangial cells. These results suggest that two types of human glomerular cells, particularly epithelial cells, possess low cyclooxygenase activity. The low capacity of human mesangial and epithelial cells to produce PG may have consequences for the endocrine control of the glomerular microcirculation in man.

Hepatic binding sites for angiotensen II in the rat.

1. 125I-labelled (Asn1,Val5)-angiotensin II (125I-labelled AII) incubated with purified rat liver membranes was degraded with time, as estimated by three techniques: binding to an excess of specific antibody, polyacrylamide-gel electrophoresis and rebinding to fresh membranes. Degradation was inhibited in the presence of an excess of beta 1-24-corticotrophin but still very marked. 2. 125 I-labelled AII became bound to purified rat liver membranes. Association and dissociation rates were slow. Binding was competitively inhibited by (Asn1,Val5)-AII, (Asp1,Ile5)-AII and (Des,Asp1, Ile5)-AII. Apparent KD was approximately 0.1 nmol/l. 3. Bound hormone was also partly degraded independently of time. 4. Angiotensinases inhibitors had different effects on 125I-labelled AII binding. A clear increase was observed in the presence of beta 1-24-corticotrophin and phenylmethylsulphonylfluoride whereas binding was decreased in the presence of EDTA or 8-hydroxyquinoline. 5. These results demonstrate the presence of high-affinity binding sites for AII and of angiotensinases in hepatic membranes.

Stimulation by oxygen radicals of prostaglandin production by rat renal glomeruli.

Polymorphonuclear leukocytes secreting oxygen radicals are found in the glomerular capillaries at an early stage of experimental acute glomerulonephritis. The aim of this work was to study the effects of these radicals on prostaglandin (PG) production by the glomeruli. Glomeruli were isolated from rat renal cortex and incubated in the presence of a biochemical system capable of generating oxygen radicals (addition to 100 microM xanthine of increasing concentrations of xanthine oxidase). Synthesis of PGE2, PGF2alpha, 6 keto PGF1alpha, and TXB2 estimated using specific radioimmunoassays was twofold greater in the presence of oxygen radicals. This effect was inhibited by catalase, slightly stimulated by superoxide dismutase, unaffected by hydroxyl radical scavengers, thus suggesting that hydrogen peroxide was the by-product responsible. This was confirmed by the stimulatory effect of hydrogen peroxide itself (1 to 100 microM) on PG synthesis. The effect of mepacrine, an inhibitor of phospholipase activity, on PG production was more marked in the presence of hydrogen peroxide and the stimulation of PG synthesis by hydrogen peroxide or oxygen radicals was progressively inhibited in the presence of arachidonic acid. Moreover, oxygen radicals stimulated the release of 14C-arachidonic acid previously incorporated in isolated glomeruli. This demonstrates that the increase in PG synthesis in response to oxygen radicals is due to activation of glomerular phospholipase by these radicals. This effect that is likely to occur at an early stage of experimental glomerulonephritis could play a role in the mechanism of the inflammatory process.

Characterization of atrial natriuretic factor receptors in human glomerular epithelial and mesangial cells.

To evaluate the distribution and functions of receptors of atrial natriuretic factor (ANF) in human glomeruli, we studied the binding sites of ANF-(1-28) in homogeneous populations of human glomerular epithelial cells or mesangial cells. 125I-labeled ANF bound specifically to both cell types. Equilibrium saturation binding curves suggested one group of receptor sites in mesangial cells (Kd = 99 +/- 32 pmol/l, Bmax = 15.3 +/- 3.5 fmol/mg) but multiple groups in glomerular epithelial cells. Binding was greater at 37 than at 4 degrees C in mesangial cells. The reverse was observed in glomerular epithelial cells due to marked degradation of the tracer at 37 degrees C. The fractions of undisplaceable tracer in a hypertonic acid medium after 60 min incubation were 45 and 16% at 37 degrees C for glomerular epithelial and mesangial cells, respectively. ANF-(1-28) and C-ANF-(4-23), a specific ligand of clearance receptors, similarly inhibited 125I-ANF binding to mesangial cells, whereas [Ala7-Ala23]-ANF, a linear analogue, was slightly less potent. In epithelial cells, C-ANF-(4-23) competitively inhibited 125I-ANF binding but with a lower potency than ANF, whereas linear ANF at low concentrations (10-100 pmol/l) stimulated 125I-ANF binding. In addition, linear ANF markedly inhibited the degradation of 125I-ANF in the incubation medium of epithelial and mesangial cells, whereas thiorphan, an inhibitor of enkephalinase, was inactive. ANF-(1-28) stimulated cGMP production in glomerular epithelial cells but not in mesangial cells. Both analogues were inactive in both cell types and did not modify ANF-(1-28)-dependent cGMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

High and low molecular weight tau proteins are differentially expressed from a single gene.

Both high and low molecular weight (HMW and LMW) tau proteins are expressed in the immature and adult mouse spinal cord. Northern blot analysis, performed with probes complementary to domains common and uncommon to the LMW and HMW entities, suggested that HMW tau proteins found in the immature mouse spinal cord are not translated from the single transcript of 6 kb expressed at these stages, but are transported within this nervous structure by axons arising in the periphery. In contrast, another minor transcript of 8 kb was detected in the adult mouse spinal cord by a HMW tau specific probe, suggesting that a small fraction of the HMW tau forms present in adulthood are translated within mouse spinal cord neurons. LMW spinal cord tau forms are encoded by mRNAs of 6 kb that contain three and four homologous repeats at immature and mature stages, respectively, whereas adult HMW entities contain four repeats. PCR analysis performed with mouse genomic DNA also showed that the nonhomologous region specific for HMW tau is a single exon. Southern blot and gene mapping showed that the same gene, located on the murine chromosome 11, encodes all the LMW and HMW tau variants. All these tau forms, therefore, are produced by an alternative splicing mechanism that is neuron-specific and developmentally regulated.

Altered PGE2 production by glomeruli and papilla of rats with hereditary diabetes insipidus.

PGE2 synthesis rate was studied in vitro in isolated glomeruli and in papillary homogenates prepared from kidneys of Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) and of Brattleboro heterozygous control rats. Incubations were carried out in isotonic buffer at 37 degrees C in the presence or absence of arachidonic acid for 15, 30, 60 and 90 min. PGE2 production was measured in the supernatant by specific radioimmunoassay. Results were compared by analysis of variance. PGE2 production was significantly decreased in the papilla (p < 0.01) and increased in the glomeruli (p < 0.01) of DI rats compared to controls. Stimulation by arachidonic acid was similar in both groups. Chronic ADH deficiency thus modifies the ability of the kidney to produce PGE2 in vitro. The opposite effects observed in glomeruli and papilla suggest a different hormonal control of PGE2 synthesis in both tissues.

Increased plasma calcitonin in early acute renal failure.

1. High plasma immunoreactive calcitonin concentrations were observed in ten of eleven patients with acute renal failure, particularly in the oliguric phase. 2. Immunoreactive calcitonin decreased progressively with time, independently of recovery of renal function. 3. Radioimmunoassay curves obtained with serial dilutions of plasma from these patients were parallel to or superimposed upon those obtained with calcitonin standards.