Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.

The release of microparticles from eukaryotic cells is a well-recognized phenomenon. We demonstrate here that the chemokine receptor CCR5, the principal co-receptor for macrophage-tropic human immunodeficiency virus (HIV)-1, can be released through microparticles from the surface of CCR5+ Chinese hamster ovary cells and peripheral blood mononuclear cells. Microparticles containing CCR5 can transfer the receptor to CCR5- cells and render them CCR5+. The CCR5 transfer to CCR5-deficient peripheral blood mononuclear cells homozygous for a 32-base-pair deletion in the CCR5 gene enabled infection of these cells with macrophage-tropic HIV-1. In monocytes, the transfer of CCR5 could be inhibited by cytochalasin D, and transferred CCR5 could be downmodulated by chemokines. A transfer of CCR5 from peripheral blood mononuclear cells to endothelial cells during transendothelial migration could be demonstrated. Thus, the transfer of CCR5 may lead to infection of tissues without endogenous CCR5 expression. Moreover, the intercellular transfer of membrane proteins by microparticles might have broader consequences for intercellular communication beyond the effects seen for HIV-1.

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

B cells and tertiary lymphoid organs in renal inflammation.

B lymphocytes are part of the inflammatory cells recruited to the human kidney in various disease settings. B cell infiltrates have been described in renal allografts, in acute and chronic interstitial nephritis, and the most common glomerular diseases like immunoglobulin A (IgA) and membranous nephropathy. These cells are almost exclusively recruited to the tubulointerstitium, but not the glomerular tuft. In addition to diffuse tubulointerstitial infiltrates, B cells together with T cells and dendritic cells form organized nodular aggregates surrounded by neo-lymphatic vessels. The functional significance of these tertiary lymphoid organs remains to be fully defined. Intrarenal B cells may be part of a local system to enhance the immunological response by functioning as antigen presenting cells, and as a source for cytokines promoting T-cell proliferation and lymphatic neoangiogenesis. In this way, they could enhance the local immune response to persisting autoantigens in the tubulointerstitium.

Compartment specific expression of dendritic cell markers in human glomerulonephritis.

Macrophages and dendritic cells are heterogenous and highly plastic bone marrow-derived cells that play major roles in renal diseases. We characterized these cells using immunohistochemistry in 55 renal biopsies from control patients or patients with glomerulonephritis as an initial step towards postulating specific roles for these cells in kidney disease. In proliferative glomerulonephritis numerous CD68 positive (pan monocyte, macrophage and dendritic marker) cells were found in both glomeruli and the tubulointerstitial space, however, a myeloid dendritic cell marker (DC-SIGN) was identified only in the tubulointerstitium. A significant number of plasmacytoid dendritic cells (identified as BDCA-2 positive cells) were seen at sites of interstitial inflammation, including follicular aggregates of inflammatory cells. Langerin positive cells (a marker of Langerhans' cells) were detectable but rare. The area of either CD68 or DC-SIGN positive interstitial cells correlated with serum creatinine. Low levels of DC-SIGN, DC-LAMP and MHC class II mRNA were present in the tubulointerstitial space in controls and increased only in that region in proliferative glomerulonephritis. We demonstrate that the CD68 positive cells infiltrating the glomerulus lack dendritic cell markers (reflecting macrophages), whereas in the tubulointerstitial space the majority of CD68 positive cells are also DC-SIGN positive (reflecting myeloid dendritic cells). Their number correlated with serum creatinine, which further emphasizes the significance of interstitial DCs in progressive glomerular diseases.

Activation and attenuation of transcription factor NF-kB in mouse glomerular mesangial cells in response to tumor necrosis factor-alpha, immunoglobulin G, and adenosine 3':5'-cyclic monophosphate. Evidence for involvement of reactive oxygen species.

The transcription factor NF-kB may play an important role in the response to tissue injury and activation of cytokines. We therefore examined the regulation of NF-kB in mesangial cells. Treatment of mesangial cells with TNF-alpha increased nuclear proteins that bound to an NF-kB-specific DNA oligonucleotide. IgG aggregates also increased nuclear NF-kB demonstrating Fc-tau receptor-mediated activation of NF-kB. Treatment of a cytosolic preparation with the detergent deoxycholate also activated NF-kB. The binding characteristics were typical for NF-kB transcription factors as determined by competition experiments with NF-kB-binding wild type kB DNA oligonucleotides or mutated oligonucleotides. Furthermore, a monoclonal antibody against the p65 subunit of NF-kB prevented the binding of NF-kB to the kB oligonucleotide. To evaluate the potential role of reactive oxygen intermediates in the activation of NF-kB, we used PDTC as a scavenger and HMAP as an inhibitor of NADPH-dependent oxidase. Both PDTC and HMAP attenuated the increase in nuclear NF-kB in response to either TNF-alpha or IgG complexes. Finally, generation of superoxide anion by xanthine oxidase activated NF-kB, an effect also mitigated by PDTC. In contrast, exogenous H2O2 did not activate NF-kB. Preincubation of cells with 8 br-cAMP, forskolin, or PGE2 attenuated the increase in nuclear NF-kB in response to TNF-alpha, aggregated IgG, or superoxide anion. Our results provide support for a role of reactive oxygen intermediates as mediators for activation of NF-kB in MC after stimulation with TNF-alpha or IgG aggregates. As an unexpected novel finding we report that cAMP can inhibit activation of NF-kB in MC. These observations may help to explain effects of TNF-alpha, IgG aggregates and cAMP on generation of cytokines by mesangial cells and the resulting glomerular pathophysiology.

Inhibition of vasopressin-stimulated water flow in toad bladder by phorbol myristate acetate, dioctanoylglycerol, and RHC-80267. Evidence for modulation of action of vasopressin by protein kinase C.

The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium-phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C.

Interaction of vasopressin and prostaglandins in the toad urinary bladder.

Prostaglandins are important modulators of the action of vasopressin. Others researchers have proposed that vasopressin stimulates prostaglandin synthesis, completing a negative feedback loop and thereby limiting vasopressin's antidiuretic effect. We have re-examined this question, using specific radioimmunoassay and thin-layer radiochromatography to determine prostaglandin synthesis by the toad bladder. Under control conditions, the bladder synthesizes prostaglandin (PG)E2 and thromboxane (TX)B2. There was no evidence for synthesis of PGE1 or PGF2 alpha by radioimmunoassay, or of other prostaglandins by radiochromatography. Furthermore, there was no evidence for metabolism of PGE2 by the bladder. Using a variety of protocols, in isolated epithelial cells as well as intact bladders, we were unable to detect any significant increase in PGE2 or TXB2 synthesis after stimulation with arginine vasopressin (AVP) or deamino-8-D-arginine vasopressin (DDAVP). Arachidonic acid, the specific precursor of prostaglandin synthesis, increased PGE2 synthesis twofold, and significantly inhibited AVP- and DDAVP-stimulated water flow by 60 and 75%, respectively. Naproxen and acetaminophen inhibited prostaglandin synthesis and enhanced water flow in response to AVP and DDAVP (44-54%). Our findings indicate that the toad bladder produces tow prostaglandins, PGE2 and TXB2, and that vasopressin does not alter their rate of synthesis. Because agents such as acetaminophen and naproxen inhibit prostaglandin synthesis and enhance vasopressin- and DDAVP-stimulated water flow, we suggest that it is the inhibitory effect of these agents on the hormone-independent rate of prostaglandin synthesis that is responsible for their enhancement of water flow. Furthermore, because AVP appears to increase prostaglandin synthesis by the intact kidney, we suggest that cells other than those of the collecting tubule are responsible for the increased prostaglandin production.

Renal vasoconstriction caused by short-term cholesterol feeding is corrected by thromboxane antagonist or probucol.

Recent studies indicate that short-term cholesterol feeding causes vascular hyperreactivity and/or increased tone in certain vascular beds. The present study in rats examined the effect of 3 wk of cholesterol-supplemented diet (CSD) on renal hemodynamics. We tested the hypothesis that LDL oxidized in vivo is causally related to increased renal vascular tone by adding the antioxidant drug probucol to the CSD (CSD + P). Micropuncture of surface nephrons in the CSD rats demonstrated that single nephron glomerular filtration rate (SNGFR) and single nephron afferent plasma flow (QA) were markedly lower than in normal rats, whereas glomerular capillary pressure (PGC), afferent arteriolar resistance (RA), and single nephron filtration fraction (SNFF) were higher. In the CSD + P animals, almost all of these hemodynamic abnormalities were absent. TXB2 and PGE2 were increased in proximal tubule fluid and urine in the CSD rats, but normal in the CSD + P group. Infusion of a TXA2 receptor antagonist into the suprarenal aorta of CSD rats caused a rapid return to normal of RBF (renal blood flow), GFR (glomerular filtration rate), SNGFR, QA, RA, PGC, and Kf (ultrafiltration coefficient). Our observations demonstrate that cholesterol feeding leads to renal vasoconstriction, which appears to be mediated largely by increased TXA2 production. The fact that probucol prevented the hemodynamic abnormalities as well as the increased TX production is consistent with the hypothesis that LDL oxidized in vivo initiates events leading to TX mediated vasoconstriction.

Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE/MCP-1, and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor-alpha and immunoglobulin G. Evidence for involvement of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase.

The potential involvement of reactive oxygen species in the expression of genes involved in immune response was examined in mesangial cells. Tumor necrosis factor (TNF-alpha) and aggregated (aggr.) IgG increased mRNA levels for the monocyte chemoattractant protein, JE/MCP-1, and the colony-stimulating factor, CSF-1. Scavengers for free radicals such as di- and tetra-methylthiourea (DMTU and TMTU) attenuated the increase in mRNA levels in response to TNF-alpha and aggr. IgG. Generation of superoxide anion by xanthine oxidase and hypoxanthine increased mRNA levels of these genes, but exogenous H2O2 did not. Addition of NADPH to activate a membrane-bound NADPH-oxidase generated superoxide and caused a dose-dependent increase in mRNA levels and further enhanced the stimulation by TNF-alpha or aggr. IgG. An inhibitor of NADPH-dependent oxidase 4'-hydroxy-3'-methoxy-acetophenone attenuated the rise in mRNA levels in response to TNF-alpha and aggr. IgG. By nuclear run-on experiments TNF-alpha, aggr. IgG and NADPH increased the transcription rates for JE/MCP-1 and CSF-1, effects inhibited by TMTU. We conclude that generation of reactive oxygen species, possibly by NADPH-dependent oxidase, are involved in the induction of the JE/MCP-1 and CSF-1 genes by TNF-alpha and IgG complexes. The concerted expression of leukocyte-directed cytokines represents a general response to tissue injury.

Effect of platelet-activating factor and serum-treated zymosan on prostaglandin E2 synthesis, arachidonic acid release, and contraction of cultured rat mesangial cells.

The interaction of inflammatory cells and glomerular prostaglandins (PG) may be important during glomerulonephritis. We therefore examined the influence of platelet-activating factor (PAF), (a mediator of inflammation released from leukocytes) and of phagocytosis of zymosan on arachidonic acid metabolism and on cell contractility in rat glomerular mesangial cells in culture. PAF increased PGE2 synthesis (determined by radioimmunoassay) within minutes (threshold: 10(-10)M; maximal effect: 10(-7)M). Serum-treated zymosan also stimulated PGE2, but with a slower onset. In cells prelabeled with [14C]arachidonic acid both PAF and serum-treated zymosan released 14C from phospholipids and increased free [14C]arachidonate. The ratio of 14C-release to PGE2 was, however, different with PAF and serum-treated zymosan, indicating different phospholipid pools. Under phase-contrast microscopy, PAF caused contraction of mesangial cells with a dose-response and time-course parallel to that for PGE2 synthesis. Serum-treated zymosan caused no contraction. The PAF-induced contraction was enhanced by PG synthesis inhibition and was attenuated by addition of PGE2, indicating a feedback mechanism. The mesangial contraction by PAF may be important in favoring deposition of immune complexes, while the PGE2 synthesis stimulated by PAF and by phagocytosis of zymosan may counteract the deleterious effects of PAF during induction of glomerulonephritis.

Functional profile of the isolated uremic nephron. Impaired water permeability and adenylate cyclase responsiveness of the cortical collecting tubule to vasopressin.

Resistance of the chronically diseased kidney to vasopressin has been proposed as a possible explanation for the urinary concentrating defect of uremia. The present studies examined the water permeability and adenylate cyclase responsiveness of isolated cortical collecting tubules (CCT) from remnant kidneys of uremic rabbits to vasopressin. In the absence of vasopressin the CCTs of both normal and uremic rabbits were impermeable to water. At the same osmotic gradient, addition of a supramaximal concentration of vasopressin to the peritubular bathing medium led to a significantly lower net water flux per unit length (and per unit luminal surface area) in uremic CCTs than in normal CCTs. Transepithelial osmotic water permeability coefficient, P(f), was 0.0232 +/-0.0043 cm/s in normal CCTs and 0.0059+/-0.001 cm/s in uremic CCTs (P < 0.001). The impaired vasopressin responsiveness of the uremic CCTs was observed whether normal or uremic serum was present in the bath. Basal adenylate cyclase activity per microgram protein was comparable in normal and uremic CCTs. Stimulation by NaF led to equivalent levels of activity in both, whereas vasopressin-stimulated activity was 50% lower in the uremic than in the normal CCTs (P < 0.025). The cyclic AMP analogue, 8-bromo cyclic AMP, produced an increase in the P(f) of normal CCTs closely comparable to that observed with vasopressin. In contrast, the P(f) of uremic CCTs was only minimally increased by this analogue and was not further stimulated by theophylline. These studies demonstrate an impaired responsiveness of the uremic CCT to vasopressin. This functional defect appears to be a result, at least in part, of a blunted responsiveness of adenylate cyclase to vasopressin. The data further suggest that an additional defect in the cellular response to vasopressin may exist, involving a step (or steps) subsequent to the formation of cyclic AMP.A unifying concept of the urinary concentrating defect of uremia is proposed which incorporates a number of hitherto unexplained observations on the concentrating and diluting functions of the diseased kidney.

Effects of trifluoperazine on function and structure of toad urinary bladder. Role of calmodulin vasopressin-stimulation of water permeability.

Calcium ion plays a major regulatory role in many hormone-stimulated systems. To determine the site of calcium's action in the toad urinary bladder, we examined the effect of trifluoperazine, a compound that binds specifically to the calcium binding protein, calmodulin, and thereby prevents activation of enzymes by the calcium- calmodulin complex. 10 microM trifluoperazine inhibited vasopressin stimulation of water flow, but did not alter vasopressin's effects on urea permeability or short-circuit current. Trifluoperazine also blocked stimulation of water flow by cyclic AMP and methylisobutylxanthine, implying a "postcyclic AMP" site of action. Consistent with these results, trifluoperazine did not decrease epithelial cyclic AMP content or the cyclic AMP-dependent protein kinase activity ratio. Assay of bladder epithelial supernate demonstrated calmodulin-like activity of 1.5 U/microgram protein. Morphologic studies of vasopressin-treated bladders revealed that trifluoperazine did not alter the volume density of cytoplasmic microtubules or significantly decrease the number of fusions between cytoplasmic, aggregate-containing, elongated vesicles and the luminal membrane. Nonetheless, the frequency of luminal membrane aggregates, structures that correlate well with luminal membrane water permeability, was decreased by greater than 50%. Thus, trifluoperazine appears to inhibit the movement of intramembranous particle aggregates from the fused intracellular membranes to the luminal membrane, perhaps by blocking an effect of calcium on microfilament function.

Evidence for immunoglobulin Fc receptor-mediated prostaglandin2 and platelet-activating factor formation by cultured rat mesangial cells.

The possibility of Fc-dependent uptake of IgG immune complexes was examined in subcultured rat mesangial cells free of monocytes. 195Au-labeled colloidal gold particles were coated either with BSA only or with BSA followed by rabbit anti-BSA-IgG or the F(ab')2 fragment of the IgG. Mesangial cells preferentially took up 195Au particles covered with BSA-anti-BSA-IgG over those covered with BSA or the F(ab')2 fragment. This uptake was a time-dependent and saturable process inhibitable by sodium azide or cytochalasin B. Using phase-contrast microscopy in the light reflectance mode, it was established that essentially all mesangial cells took up IgG-coated gold particles. By electron microscopy the process was shown to consist of vesicular uptake with delivery to endosomes. Mesangial binding-uptake of the IgG-covered particles was associated with stimulation of PGE2 synthesis and production of platelet-activating factor, a lipid mediator of inflammation. To characterize the potential Fc receptor for IgG we used the rosetting technique with sheep red blood cells coated with IgG subclass-specific mouse monoclonal antibodies. 50% of mesangial cells exhibited rosetting with red cells coated with mouse IgG2a, whereas negligible rosetting was observed with IgG2b or IgG1. Competition experiments confirmed the specificity of IgG2a binding. We conclude that cultured rat mesangial cells exhibit specific receptors for IgG and that occupancy of Fc receptors results in endocytosis and is associated with generation of PGE2 and platelet-activating factor. These observations may be of significance for immune-mediated glomerular diseases.

Effect of vasopressin on cyclic AMP-dependent protein kinase in toad urinary bladder.

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.

Prostaglandin synthesis linked to phosphatidylinositol turnover in isolated rat glomeruli.

Prostaglandins produced by the glomerulus are important factors in controlling glomerular function. The controlling step, i.e., the release of arachidonic acid from the phospholipids by either phospholipase A2 and/or C, remains poorly defined. The present studies were designed to determine which factors control arachidonic acid turnover and prostaglandin synthesis in glomeruli. As tools we used the calcium ionophore A23187, mepacrine, a phospholipase inhibitor, trifluoperazine, a calmodulin antagonist, and angiotensin II. A23187 (2 microM) caused a significant stimulation of both prostaglandin E2 and prostaglandin F2 alpha synthesis (measured by radioimmunoassay), which was associated with increased phosphatidylinositol turnover (measured by [14C]arachidonic acid and [32P]orthophosphate incorporation). Surprisingly, trifluoperazine (10-100 microM) also progressively increased synthesis of both prostaglandins, which was accompanied by increased phosphatidic acid/phosphatidylinositol turnover and decreased phosphatidylinositol content. In contrast, phosphatidylcholine and phosphatidylethanolamine turnover were significantly inhibited by trifluoperazine and their total content remained unaffected. Mepacrine (1 mM) decreased prostaglandin synthesis and both phosphatidylcholine and phosphatidylethanolamine turnover, and had no consistent effect on phosphatidylinositol turnover in control glomeruli. Mepacrine did, however, inhibit both A23187 or trifluoperazine-induced increase in phosphatidylinositol turnover. Angiotensin II increased turnover of phosphatidylinositol and also phosphatidylcholine, as determined by incorporation of [14C]arachidonic acid. Thus, all agents that increased prostaglandin synthesis also enhanced phosphatidylinositol turnover. The exact pathway of arachidonic acid release remains to be determined.

Comparison of glomerular and mesangial prostaglandin synthesis and glomerular contraction in two rat models of diabetes mellitus.

Enhanced prostaglandin production is postulated to contribute to altered vascular reactivity and glomerular hyperfiltration in early insulin-deficient diabetes mellitus. Rats with streptozocin-induced diabetes (STZ-D) show glomerular hyperfiltration and develop renal disease. BB rats with genetic diabetes (BB-D) also hyperfilter but have only minor renal lesions. We therefore compared glomerular and mesangial prostaglandin E2 (PGE2) production and glomerular contractility in response to pressors as a reflection of in vitro vascular reactivity in these models. Glomeruli isolated from rats with 3 wk of STZ-D produced significantly more PGE2 under basal and ionophore A23187-stimulated conditions than those from control rats. Glomeruli from BB-D rats under basal and stimulated conditions, however, generated amounts of PGE2 that were comparable to either those of nondiabetic littermates or of normal Wistar rats. Mesangial cells cultured from glomeruli of STZ-D, BB-D, and control rats all had identical prostaglandin profiles judged by conversion of [14 C]arachidonic acid. They also produced comparable amounts of PGE2 under basal conditions and after stimulation with angiotensin II or A23187, as determined by radioimmunoassay. Planar surface area of glomeruli isolated from control rats showed a dose-dependent decrease in response to angiotensin II (10(-11)-10(-9) M). This response to angiotensin II was at least as great in glomeruli from STZ-D rats. Contraction of glomeruli from control and STZ-D rats was also comparable after vasopressin or norepinephrine. Similarly, glomeruli from BB-D and BB control rats contracted in a comparable fashion to angiotensin II and norepinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of glucose on proteoglycans produced by cultured mesangial cells.

Altered proteoglycan metabolism may play a role in the development of diabetic glomerulopathy. This study was conducted to examine the effects of glucose on the production and physical characteristics of proteoglycans generated by rat mesangial cells in culture. Rat mesangial cells were exposed to elevated glucose media (500 mg/dl) or standard glucose media (200 mg/dl) for 8-10 days, and proteoglycan synthesis was determined using 35S-labeling in conjunction with anion exchange and sizing chromatography. Rat mesangial cells generated predominantly chondroitin/dermatan sulfate proteoglycans, with small amounts of heparan sulfate proteoglycans. High glucose did not alter the number of rat mesangial cells after 24 h or after 8-10 days, compared with cells grown under standard glucose conditions. The total amount of glycosaminoglycan generated and the sizes of the major proteoglycans were not different between cultures grown in standard and elevated glucose medium. Levels of mRNA for the proteoglycan, biglycan (as assessed by Northern blot analysis), also were comparable between the standard and elevated glucose conditions. Exposure to media high in glucose did not change the rate of secretion of proteoglycans from the cell layer to the medium, but did result in a greater quantity of radiolabeled proteoglycan deposited in the extracellular matrix. The cell, extracellular matrix and medium proteoglycans isolated from the elevated glucose cultures, consistently eluted from the anion exchange column at a lower [NaCl] compared with those generated under standard glucose conditions, indicating a loss of anionic charges.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of nonenzymatic glycosylation of mesangial matrix on proliferation of mesangial cells.

Cross-linking of cell matrix components by nonenzymatic glycosylation may contribute to diabetic glomerulopathy. We examined the effects of modification of matrix by nonenzymatic glycosylation on mesangial cell function. Matrix was generated by growing mesangial cells in tissue culture for 2 wk and removing the cells with a detergent cell-lysis solution. By indirect immunofluorescence and Northern-blot analysis, the remaining matrix contained laminin, fibronectin, and collagens type I and IV. The matrix was modified by incubation for 24 h with 50 mM glycolaldehyde, a highly reactive cross-linking nonenzymatic glycosylation product, or for 2 wk with 200 mM glucose-6-phosphate (G6P). Modification was carried out with or without equimolar aminoguanidine, an inhibitor of cross-link formation. Nonenzymatic glycosylation of the matrix by glycolaldehyde or G6P was confirmed by fluorometry and [14C]G6P incorporation and was prevented by aminoguanidine. [3H]thymidine incorporation for 24 h by mesangial cells plated onto unmodified or modified matrix was then performed. Modification of matrix had no effect on attachment of mesangial cells, determined 4 h after plating. Nonenzymatic glycosylation of matrix by glycolaldehyde or G6P significantly inhibited thymidine incorporation by mesangial cells. This effect was partially reversible by aminoguanidine. Aminoguanidine-modified matrix had no effect on thymidine incorporation. Thymidine-incorporation results were confirmed by direct cell counting. We conclude that modification of matrix by nonenzymatic glycosylation influences growth of mesangial cells, which could contribute to the mesangial abnormalities of diabetic glomerulopathy.

Effect of testosterone on compensatory renal hypertrophy in the rat.

The effect of testosterone on compensatory renal hypertrophy (CRH) remains controversial. We therefore examined the effect of exogenous testosterone on CRH in adult male and female rats after unilateral nephrectomy. The influence of endogenous testosterone was studied by comparing the degree of CRH in normal male, castrated male, and in testosterone receptor deficient male pseudohermaphrodite rats. Furthermore, serial determinations of serum testosterone levels were performed after unilateral nephrectomy in male rats. Compensatory renal hypertrophy was comparable between male rats--with or without exogenous testosterone administration--and between normal male, castrated male, and psuedohermaphrodite male rats. In contrast, exogenous testosterone administration in female rats enhanced CRH. Serum testosterone levels fell markedly after unilateral nephrectomy or sham surgery, but increased to 183% and 234% of control values at 1 and 2 days after surgery in the unilaterally nephrectomized rats. At no time, however, did they exceed the range of normal values. The results indicate a different effect of testosterone on CRH in male and female rats.

Modulation of HIV-1 enhancer activity and virus production by cAMP.

The effect of cAMP on the transcriptional activity of the HIV-1 long terminal repeat/enhancer was investigated and compared to the effect of cAMP on virus replication. In culture cAMP repressed virus replication in vivo using different cell types. Transient transfection studies with HIV-1 enhancer-derived luciferase reporter gene constructs identified the minimal DNA sequence mediating the negative regulatory effect of cAMP on HIV-1 transcription. A single nuclear factor kappaB element from the HIV-1 enhancer mediates the repressive effect on transcription. AP-2 is not involved in cAMP repression. Stable transfection of Jurkat T cells with the co-activators CREB binding protein (CBP) and p300 completely abolished the cAMP repressive effect, supporting the hypothesis that elevation of intracellular cAMP increases phosphorylation of CREB, which then competes with phosphorylated p65 and Ets-1 for limiting amounts of CBP/p300 thereby mediating the observed repressive effect on transcription. These findings suggest an important role of cAMP on HIV-1 transcription.