Effect of fiber source and particle size on chick performance and nutrient utilization.

The addition of fiber in chick feeds is known to dilute nutrients; as a result, this may reduce nutrient digestibility and performance. However, recent studies suggest that moderate inclusion of insoluble fibers (2 to 3%) may stimulate gizzard development, which could result in better nutrient utilization and chick growth. The previous fiber sources evaluated were subject to wide fluctuation in their nutritional and chemical composition due to variation in processing. Miscanthus giganteus is a C4 grass purposefully grown for its fiber content which has a consistent fiber composition compared to food process residues. The objectives of this study were to determine the effect of dietary fiber source and particle size on day-old chick performance and nutrient digestibility. Day-old chicks (8 chicks per cage, 5 cages per treatment) were fed diets containing 3% of either sepiolite (SEP), cellulose (CEL), coarse beet pulp (BP), fine BP, coarse Miscanthus grass (MG), and fine MG. At the end of days 7, 14, and 21, chicks and experimental diets were weighed to compute average daily gain and feed intake. In addition, excreta from the previous 48 h of each data capture point was collected to determine nutrient digestibility. In general, chicks fed diets containing fiber consumed more feed, gained more weight, and had better feed conversion rate than birds fed the SEP diet. Particle size of the fiber had no effect on chick performance; however, nutrient utilization was higher (P < 0.05) for chicks fed coarse fiber particles compared to these fed fine fiber particles. Birds fed diets containing MG performed similar to chicks fed CEL (P > 0.05), but digestibility coefficients of birds fed BP diets were generally higher than chicks fed MG diets. In conclusion, chicks performed better with fiber in their diet and MG was comparable to CEL.

The amino acid composition and protein quality of various egg, poultry meal by-products, and vegetable proteins used in the production of dog and cat diets.

New protein ingredients are used to support pet food market growth and the development of new products while maintaining animal dietary needs. However, novel protein sources (e.g., spray-dried chicken, and (or) rice, pea, and potato protein concentrates) have limited data available regarding their protein quality. The objective of this study was to evaluate protein ingredients used in the pet food industry by laboratory analysis and a chick growth assay as a model. Following analysis for proximate and amino acid composition, chicks (six birds per pen with four pens per treatment) were fed experimental diets for 10 d. Diets contained 10% crude protein from each of the experimental protein sources (spray-dried egg-SDEG; spray-dried egg white-SDEW, spray-dried inedible whole egg-SDIE, chicken by-product meal-CBPM, chicken meal-CKML, low-temperature fluid bed air-dried chicken-LTCK, low-temperature and pressure fluid bed dried chicken-LTPC, spray-dried chicken-SDCK, whey protein concentrate-WPCT, corn gluten meal-CGML, corn protein concentrate-CPCT, potato protein isolate-PPIS, rice protein concentrate-RPCT, pea protein isolate-PEPI, soy protein isolate-SPIS, and soybean meal-SBML) along with an N-free diet (negative control). Chicks fed SDEG, SDIE, and LTPC had the highest protein efficiency ratio (PER; P < 0.0001; 5.18, 5.37, and 5.33, respectively), LTCK and SDCK were intermediate (4.54 and 4.79), and the CBPM and CKML were the lowest among the poultry proteins for EAA:NEAA, PER, and Lys availability. Among the vegetable proteins, PPIS and SBML had the highest PER values (3.60 and 3.48, P < 0.0001). In general, the chick PER method ranked the quality of animal protein sources higher than vegetable proteins, and these results were consistent with the EAA:NEAA ratio and Lys availability.

Thrombotic microangiopathy without renal involvement: two novel mutations in complement-regulator genes.

UNLABELLED: ESSENTIALS: The differential diagnosis among thrombotic microangiopathies (TMAs) is challenging. We studied a case of TMA with neurologic symptoms, no renal impairment and normal ADAMTS-13 levels. Two novel mutations in complement factor I and thrombomodulin genes were identified. Complement-regulator genes can be involved in TMAs with normal ADAMTS-13 regardless of renal damage. BACKGROUND: Thrombotic microangiopathies (TMAs) often represent a challenge for clinicians, because clinical, laboratory, and even genetic features are not always sufficient to distinguish among different TMAs. OBJECTIVES: The aim of this study was to investigate the pathogenetic mechanisms underlying an acute case of TMA with features of both thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). PATIENTS/METHODS: We report the case of a 49-year-old woman who developed an acute TMA with neurologic involvement and no renal impairment. ADAMTS-13, von Willebrand factor, and complement-system biochemical characterization was performed on acute phase samples. Exome sequencing and direct Sanger sequencing of previously aHUS-associated genes were performed. The functional consequences of the thrombomodulin (THBD) mutation were investigated by in vitro expression studies. RESULTS: Despite a clinical diagnosis of TTP, the patient had normal ADAMTS-13 levels and increased VWF antigen levels with ultra-large von Willebrand factor multimers. C3, C4, and complement factors H and I (CFI) were normal. Molecular analysis confirmed two novel heterozygous mutations in CFI (c.805G>A, p.G269S) and THBD (c.1103C>T, p.P368L), and in vitro expression studies showed a reduction in the generation of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) caused by mutated THBD. This proinflammatory condition, associated with the p.G269S mutation in CFI, probably leads to a complement-mediated endothelial activation, with a relevant prothrombotic potential in case of transient environmental triggers. CONCLUSIONS: This study identified the first case of acute TMA without renal involvement but with neurological damage carrying two novel mutations in complement-regulator genes, highlighting the possible role of the complement system as a common pathogenetic mechanism in TMAs.

Protein traffic activates NF-kB gene signaling and promotes MCP-1-dependent interstitial inflammation.

Mononuclear cells accumulate in the renal interstitium and contribute to renal injury in proteinuric nephropathies. Angiotensin-converting enzyme (ACE) inhibitors reduce protein trafficking and also lessen renal structural and functional damage. Many proinflammatory genes, including monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes and T lymphocytes, are transcriptionally regulated by nuclear factor-kappa B (NF-kB). We aimed to study NF-kB activation and MCP-1 expression over time in two models of progressive proteinuric nephropathies (5/6 nephrectomy and passive Heymann nephritis [PHN]) and evaluate the effect of antiproteinuric therapy with an ACE inhibitor on these factors. In both models, increased urinary protein excretion over time was associated with a remarkable increase in NF-kB activity, which was almost completely suppressed by reducing proteinuria with lisinopril. NF-kB activation was paralleled by upregulation of MCP-1 messenger RNA and interstitial accumulation of ED-1-positive monocytes/macrophages and CD8-positive T cells. Lisinopril inhibited MCP-1 upregulation and limited interstitial inflammation. In a group of PHN rats with advanced disease and severe proteinuria, a dose of lisinopril high enough to inhibit renal ACE activity failed to reduce proteinuria and also did not limit NF-kB activation, which was sustained over time, along with MCP-1 gene overexpression and interstitial inflammation. These data suggest that NF-kB is activated in the presence of increased protein traffic, enhancing the nuclear transcription of the MCP-1 gene with potent chemotactic and inflammatory properties. This mechanism may help explain the long-term renal toxicity of filtered proteins.

Xenogeneic serum promotes leukocyte-endothelium interaction under flow through two temporally distinct pathways: role of complement and nuclear factor-kappaB.

Endothelial cell activation and mononuclear cell infiltration are consistent features of discordant xenograft rejection. This study evaluated whether xenogeneic serum--as a source of xenoreactive natural antibodies and complement--induced endothelial activation with consequent leukocyte adhesion and transmigration under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 30 min, 1 h 30 min, or 5 h with 10% human serum or 10% porcine serum and then perfused with human leukocytes in a parallel plate flow chamber under flow (1.5 dynes/cm2). Adherent and transmigrated cells were counted by digital image analysis. Results showed that human serum significantly (P < 0.01) increased over time the number of adherent leukocytes compared with porcine serum. Stimulation of PAEC with human serum also promoted a progressive increase in leukocyte transmigration that reached statistical significance (P < 0.01) at 1 h 30 min and at 5 h compared with porcine serum. Studying the role of complement in leukocyte-endothelium interaction in xenogeneic conditions, a marked complement C3 deposition on PAEC exposed to human serum was shown by immunofluorescence, whereas cells incubated with porcine serum were negative. Next, it was documented that human serum decomplemented by heating and C3-deficient human serum failed to promote both leukocyte adhesion and transmigration, results that were comparable to porcine serum. To elucidate the intracellular mediators involved in endothelial cell activation by xenogeneic serum, this study focused on transcriptional factor nuclear factor-kappaB (NF-kappaB), a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. Positive nuclear staining of NF-kappaB (p65 subunit) found by confocal fluorescence microscopy of PAEC exposed to human serum was taken to reflect NF-kappaB activation. NF-kappaB was instead strictly localized in the cell cytoplasm in PAEC incubated with the homologous serum. Heat-inactivated human serum failed to activate NF-kappaB. Electrophoretic mobility shift assay of nuclear extracts from PAEC exposed to human serum revealed an intense NF-kappaB activation that was inhibited by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. The NF-kappaB inhibitors pyrrolidinedithiocarbamate and tosyl-phe-chloromethylketone did not affect the number of adherent and transmigrated leukocytes in PAEC exposed to human serum for 30 min and 1 h 30 min. Both inhibitors instead significantly reduced leukocyte adhesion and transmigration induced by human serum at 5 h. Confocal fluorescence microscopy studies showed that human serum induced an increase in the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. Functional blocking of these adhesive molecules with the corresponding antibodies significantly inhibited xenogeneic serum-induced leukocyte adhesion. These data suggest that leukocyte adhesion and transmigration are directly dependent on complement deposited on PAEC in the early phase of cell activation (30 min and 1 h 30 min) induced by xenogeneic serum, whereas leukocyte adhesive events observed after 5 h of incubation of endothelial cells with xenogeneic serum are possibly regulated by transcription of NF-kappaB-dependent genes. The finding that xenogeneic serum promotes leukocyte-endothelial interaction depending on NF-kappaB activation might be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Protein overload stimulates RANTES production by proximal tubular cells depending on NF-kappa B activation.

Abnormal traffic of proteins through the glomerular capillary has an intrinsic renal toxicity possibly linked to the subsequent process of proximal tubular reabsorption. Here we investigated in vitro the effect of protein overload on proximal tubular cell production of RANTES, a nuclear factor-kappa B (NF-kappa B)-dependent chemokine with potent chemotactic activity for monocytes/macrophages and T lymphocytes. Confluent pig LLC-PK1 cells were incubated for 24 and 48 hours with Eagle's MEM plus 0.5% FCS containing bovine serum albumin (BSA, 1 to 30 mg/ml). Tumor necrosis factor-alpha (TNF-alpha; 100 U/ml) was used as a positive control. RANTES was measured in cell supernatants by ELISA. Bovine serum albumin (BSA) induced a time- and dose-dependent increase in proximal tubular cell RANTES production. Selected experiments using transwells showed that the RANTES release was predominantly basolateral. The stimulatory effect on tubular RANTES was not specific to albumin but was shared by immunoglobulin (Ig) G. We then explored the role of NF-kappa B on BSA-induced RANTES. The NF-kappa B inhibitors pyrrolidine dithiocarbamate (PDTC; 25 microM) and sodium salicylate (10 mM) significantly reduced BSA-induced RANTES production. Electrophoretic mobility shift assay of nuclear extracts of LLC-PK1 exposed to BSA revealed an intense NF-kappa B activation as early as 30 minutes in a dose-dependent fashion, which was inhibited by PDTC. Supershift analysis revealed that the protein subunits of activated NF-kappa B were p65/p65 homodimer, p65/cRel, p50/p65 heterodimers. Given its chemotactic activity, RANTES released into the interstitium might promote inflammatory cell recruitment and contribute to interstitial inflammation and renal disease progression.

Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion.

We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 microg/ml), or hyperglycemic (174-316 mg/dl) sera from patients with diabetes and abnormal hemoglobin A1c (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dyn/cm2). Rolling and adherent cells were evaluated by digital image processing. Results showed that 30 mM glucose significantly (P < 0. 01) increased the number of adherent leukocytes to endothelial cells in respect to control (5 mM glucose; 151+/-19 versus 33+/-8 cells/mm2). A similar response was induced by endothelial stimulation with IL-1beta, here used as positive control (195+/-20 cells/mm2). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1beta-treated endothelial cells, was significantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 microm). Functional blocking of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion (67+/-16, 83+/-12, 62+/-8 versus 144+/-21 cells/ mm2). Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation. Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate (100 microM) and tosyl-phe-chloromethylketone (25 microM) significantly reduced (P < 0.05) leukocyte adhesion in respect to HUVEC treated with glucose alone. A significant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine (5 nM). When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte adhesion as compared with controls, despite normal levels of IL-1beta and TNFalpha in these sera. These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins, possibly depending on NF-kB activation.

Identification of a novel gene--SSK1--in human endothelial cells exposed to shear stress.

To identify transcriptionally regulated genes potentially involved in the effect of shear stress on endothelial gene expression, we performed a differential display analysis of mRNAs from human umbilical vein endothelial cell (HUVEC) exposed to laminar shear stress (12 dynes/cm2) in comparison to HUVEC maintained in static condition. We identified a cDNA fragment overexpressed by laminar shear stress. The full-length, SSK1, was 3653 long and encoded for a novel protein of 1050 amino acids. Northern blot demonstrates that SSK1 mRNA is expressed at high levels also in placenta, a weak transcript was present in heart, skeletal muscle, kidney and pancreas. Homology searches of the protein databases showed that SSK1 is related to numerous serine-threonine kinases. The highest homology was found with a very recently described gene, BUBR1, an analogue of BUB1, which is a kinase involved in the regulation of cell cycle. The most conserved residues in catalytic domains II, III, VIb, VII, VIII and IX of serine-threonine protein kinases were found in the C terminal region of SSK1 which further supports the kinase nature of the new protein. The putative serine-threonine kinase SSK1 may represent a tool by which mechanical forces regulates phosphorylation events within endothelial cells.

Bindarit retards renal disease and prolongs survival in murine lupus autoimmune disease.

As an alternative to classical immunosuppressants in experimental lupus nephritis, we looked at bindarit, 2-methyl-2-[[1-phenylmethyl)-1H-indazol-3-y1]methoxy]propanoic acid, a novel molecule devoid of immunosuppressive effects, which selectively reduces chronic inflammation in rat adjuvant arthritis. Two groups of NZB/W mice (N = 55 for each group) were given bindarit, (50 mg/kg/day p.o.) or vehicle starting at 2 months of age. Mice were sacrificed at 2, 6, 8 and 10 months or used for survival studies. Bindarit delayed the onset of proteinuria (% proteinuric mice, bindarit vs. vehicle, 6 months: 0 vs. 33% and 8 months: 7% vs. 60%, P < 0.005; 10 months: 53% vs. 80%) and significantly (P < 0.05) protected from renal function impairment (serum BUN, bindarit vs. vehicle: 8 months, 30 +/- 3 vs. 127 +/- 42; 10 months, 53 +/-5 vs. 140 +/- 37 mg/dl). Appearance of anti-DNA antibodies was retarded and survival significantly (P < 0.0001) prolonged by bindarit (% survival, bindarit vs. vehicle: 8 months, 100% vs. 80%; 10 months, 87% vs. 40%; 12 months, 27% vs. 20%). Bindarit significantly limited glomerular hypercellularity, interstitial inflammation and tubular damage. Renal expression of monocyte chemoattractant protein (MCP-1) mRNA (Northern blot) markedly increased (7 - 12-fold in 8- 10-month-old mice vs. 2-month-old) during the progression of nephritis in association with mononuclear cell infiltration. Bindarit completely prevented MCP-1 up-regulation. In another series of experiments, bindarit (0.25% and 0.5% medicated diet, N = 16 for each group) when started at 4.5 months of age in NZB/W mice improved survival in respect to untreated mice (N = 17) in a dose-dependent manner (% survival: 8 months, 94% and 100%, respectively, vs. 47%; 10 months, 75% and 100% vs. 35%; 12 months, 31% and 75% vs. 12%). Survival was even more prolonged when bindarit (0.5% medicated diet) was combined with a low dose of methylprednisolone (1.5 mg/kg i.p.), which that only partially modifies proteinuria and survival of lupus mice, in an additional group of animals (N = 16). Thus, at 14.5 months when all mice given bindarit alone died, 50% of mice on combined therapy were still alive (P < 0.023). Studies are needed to establish whether bindarit may function as a steroid sparing drug in human lupus.

Pharmacologic control of angiotensin II ameliorates renal disease while reducing renal TGF-beta in experimental mesangioproliferative glomerulonephritis.

We evaluated the effect of blocking angiotensin II (AngII) on the development of proteinuria and glomerular injury in antithymocyte serum (ATS) glomerulonephritis. Disease was induced in Sprague-Dawley rats by a single intravenous injection of rabbit ATS. Three groups of rats were considered: group 1 (n = 13), ATS rats with no therapy; group 2 (n = 13), ATS rats treated with angiotensin-converting enzyme inhibitor (40 mg/L lisinopril in the drinking water); and group 3 (n = 13), ATS rats treated with AngII receptor antagonist (50 mg/L L-158,809 in the drinking water). Treatment started 3 hours after ATS injection and lasted 4 days. An additional group of control rats (group 4, n = 13) received preimmune serum. At day 4, ATS rats developed proteinuria (46+/-5 mg/d v control 12+/-1 mg/d; P < 0.01), which was prevented by both lisinopril and L-158,809 (14+/-0.2 mg/d and 15+/-1.6 mg/d, respectively, P < 0.01 v ATS). Systolic blood pressure was comparable in ATS rats and in controls (119+/-4 mm Hg v 120+/-2 mm Hg). Systolic blood pressure values were significantly decreased after either lisinopril or L-158,809 (104+/-3 mm Hg and 101+/-5 mm Hg, respectively; P < 0.01 v ATS). Serum creatinine levels were similar in all groups. Quantitation of proliferating cells and macrophages by analysis of proliferating cell nuclear antigen-positive and ED1-positive cells/glomerular cross-section showed a marked increase in proliferating cell nuclear antigen-positive cells in glomeruli of ATS rats over controls (12.6+/-0.5 cells/glomerular cross-section v 1.9+/-0.2 cells/glomerular cross-section; P < 0.01), which was significantly (P < 0.01) prevented by both treatments (lisinopril, 5.7+/-1.0 cells/glomerular cross-section; L-158,809, 4.8+/-1.5 cells/glomerular cross-section). The increase in ED1-positive cells (10+/-0.7 cells/glomerular cross-section v controls, 1.8+/-0.2 cells/glomerular cross-section; P < 0.01) was also significantly (P < 0.01) reduced by lisinopril and L-158,809 (4.1+/-0.7 cells/glomerular cross-sections and 2.6+/-0.6 cells/glomerular cross-section, respectively). Blocking of AngII activity prevented almost completely the formation of microaneurysms in ATS rats (percent of glomeruli with microaneurysms: ATS, 11.5%+/-3.5%; ATS + lisinopril, 0.4%+/-0.2%; ATS + L-158,809, 0.8%+/-0.8%; controls, 0%). Because AngII is a potent inducer of renal transforming growth factor-beta (TGF-beta), a cytokine involved in the regulation of cell proliferation, matrix deposition, and monocyte migration (which is overexpressed in the kidney of ATS rats), we then evaluated the effect of AngII inhibitors on renal gene expression of TGF-beta1 and on urinary TGF-beta1. TGF-beta1 mRNA levels in kidneys of ATS rats were 3.6-fold higher than those of controls and were reduced by 46% and 32% after treatment with lisinopril and L-158,809, respectively. Urinary TGF-beta1 excretion increased in ATS (37.3+/-6.0 ng/d v controls, 13.8+/-3.4 ng/d; P< 0.01) but was normalized by lisinopril and L-158,809 (7.6+/-1.9 ng/d and 6.4+/-0.4 ng/d, respectively; P < 0.01). Thus, in ATS, blocking AngII synthesis prevents proteinuria and reduces glomerular cell proliferation and inflammatory cell infiltration, possibly by reducing excessive renal TGF-beta synthesis. These findings may be relevant for future strategies in the treatment of human mesangioproliferative glomerulonephritis.

Fluid shear stress modulates von Willebrand factor release from human vascular endothelium.

Fluid shear stress generated by blood flow on arterial wall may play a role in the process of atherosclerosis, not only affecting the mass transport phenomena that take place in blood, but also by modulation of synthesis and secretion of humoral factors released by vascular endothelium that mediate platelet-vessel wall interactions. The present study was designed to investigate whether shear stress, induced by laminar flow, modulates von Willebrand factor (vWF) release from cultured human umbilical vein endothelial cells (HUVEC) and whether this physical stimulation can affect vWF synthesis. Monolayers of HUVEC were exposed to laminar flow of varying magnitude (from 2 to 12 dynes/cm2) using a cone-and-plate device. The release of vWF in cell supernatant and in extracellular matrix by cells exposed to flow or maintained in static conditions was evaluated by enzyme-linked immunosorbent assay. HUVEC exposed to laminar flow released higher amounts of vWF into the cell supernatant within few hours of exposure and vWF secretion was dependent on shear stress magnitude. vWF released in extracellular matrix was also higher in cell monolayers exposed to shear than in static controls. vWF mRNA expression in HUVEC was not affected by exposure of cells to laminar flow, indicating that shear-induced vWF release reflected enhanced secretion without de novo protein synthesis. Immunofluorescence studies showed that the release of vWF is due to exocytosis from Weibel-Palade bodies, the storage organelles of vWF. These data indicate a novel mechanism by which local hemodynamic shear forces modulate endothelial cell function and may play a role in development of arterial thrombotic events.

Renal expression of monocyte chemoattractant protein-1 in lupus autoimmune mice.

Mononuclear cell infiltration in glomeruli and renal interstitium is a prominent feature of some types of glomerulonephritis, including lupus nephritis. The mechanism(s) underlying monocyte influx into the kidney is not fully understood. Recently, monocyte chemoattractant protein-1 (MCP-1) has been identified as a chemotactic factor involved in the recruitment of monocytes/macrophages in the glomeruli of rats with mesangioproliferative as well as anti-glomerular basement membrane glomerulonephritis. In the study presented here, renal MCP-1 mRNA expression in New Zealand Black x New Zealand White (NZB/W) F1 mice, a model of genetically determined immune complex disease that mimics systemic lupus in humans, was investigated. Northern blot analysis revealed a single 0.7 kb MCP-1 transcript of very low intensity in kidneys from 2-month-old NZB/W mice that had not yet developed proteinuria nor renal damage. Message levels, which increased markedly with the progression of nephritis and in association with mononuclear cell infiltration, were 10- and 15- fold higher in 8-10-month-old mice than in 2-month-old mice. By in situ hybridization, increased expression of MCP-1 mRNA was demonstrated in glomeruli and, even more striking, in tubular epithelial cells. Western blot analysis demonstrated increased expression of MCP-1 protein in kidneys of 10-month-old NZB/W mice, consistent with MCP-1 mRNA data. When NZB/W mice were treated with cyclophosphamide up to 12 months of age, expression of MCP-1 in the renal tissue remained low, the influx of inflammatory cells did not appear, and glomerular and tubular structures remained well preserved. These data suggest that elevated MCP-1 might act as a signal for inflammatory cells to infiltrate the kidney in lupus nephritis.

The renoprotective properties of angiotensin-converting enzyme inhibitors in a chronic model of membranous nephropathy are solely due to the inhibition of angiotensin II: evidence based on comparative studies with a receptor antagonist.

In several models of renal disease progression, angiotensin-converting enzyme (ACE) inhibitors reduced proteinuria and limited glomerulosclerosis, which suggested that reduction of renal angiotensin II (Ang II) activity is crucial for the preservation of glomerular structure and function. However, it cannot be ruled out that other hormonal systems, including inhibition of the bradykinin breakdown, also play a role. We compared the effects of chronic treatment with the ACE inhibitor lisinopril with those of a specific Ang II receptor antagonist, L-158,809, on proteinuria and renal injury in passive Heymann nephritis (PHN), a model of immune renal disease that closely resembles human membranous nephropathy, with long-lasting proteinuria followed by tubulointerstitial damage and glomerulosclerosis. Passive Heymann nephritis was induced with 0.5 mL/100 g of rabbit anti-Fx1A antibody in 24 male Sprague-Dawley rats. The animals were divided into three groups of eight rats each, and were given the following in the drinking water on a daily basis: lisinopril (40 mg/L), L-158,809 (50 mg/L), or no therapy. Treatment started at day 7 (proteinuria was already present) and lasted 12 months. Eight normal rats were used as controls. Untreated PHN rats developed hypertension, while rats with PHN given lisinopril or L-158,809 all had systolic blood pressure values even lower than those of normal rats. Urinary protein excretion progressively increased with time in untreated PHN rats, who developed tubulointerstitial damage and glomerulosclerosis. Both lisinopril and L-158,809 exhibited a potent antiproteinuric effect and preserved glomerular and tubular structural integrity at a similar extent. Renal gene expression of transforming growth factor-beta and extracellular matrix proteins was also effectively reduced by the two treatments. These results indicate that ACE inhibitors and Ang II receptor antagonists are equally effective in preventing renal injury in PHN and suggest that the renoprotective effects of ACE inhibitors in this model are solely due to inhibition of Ang II.

Systemic and fetal-maternal nitric oxide synthesis in normal pregnancy and pre-eclampsia.

OBJECTIVE: To investigate systemic and fetal-placental nitric oxide synthesis by biochemical and molecular biology means in normal human pregnancy and pre-eclampsia. DESIGN AND PARTICIPANTS: Three groups of women were studied: healthy pregnant women (n = 8), pregnant women with pre-eclampsia (n = 8), and age-matched nonpregnant controls (n = 8). Pre-eclamptic patients were treated with nifedipine (30-60 mg/day) for severe hypertension. Systemic nitric oxide synthesis was assessed in normal pregnant women at weeks 18-21, 29-32 and 38-39 and in pre-eclamptic women on admission to the hospital (29-32 weeks, 30 on average), before the morning nifedipine administration. Nonpregnant women were studied twice at four-week intervals as controls. The pattern of nitric oxide biosynthesis in fetal-placental circulation was studied in normal and pre-eclamptic women at the delivery. SETTING: Mario Negri Institute for Pharmacological Research, Bergamo, and the Division of Obstetrics and Gynaecology of the University of Brescia. MAIN OUTCOME MEASURES: Plasma cGMP levels and platelet nitric oxide synthesis, assessed by measuring the conversion of [3H]L-arginine to [3H]L-citrulline as well as intracellular cGMP, were evaluated. Constitutive nitric oxide synthase (EC-NOS) gene expression by Northern blot analysis and nitric oxide release by the conversion of [3H]L-arginine to [3H]L-citrulline were assessed in umbilical vein endothelial cells (HUVEC) and in placenta. Inducible nitric oxide synthase activity was also evaluated in HUVEC exposed to tumour necrosis factor alpha (TNF alpha) and in placenta homogenates incubated in calcium free medium. RESULTS: Plasma cGMP was higher in both normal pregnant and pre-eclamptic women than in nonpregnant controls. In normal pregnancy cGMP rose as early as 18-21 weeks and remained elevated throughout pregnancy. [3H]L-citrulline production and intracellular cGMP were comparable in platelets from all women. EC-NOS gene expression and nitric oxide synthesis were identical in HUVEC and placenta from normal pregnant and pre-eclamptic women. CONCLUSIONS: Systemic levels of CGMP, the nitric oxide second messenger, are increased in normal pregnancy. Excessive nitric oxide production does not derive from platelets. Pre-eclampsia is not associated with changes in fetal-placental nitric oxide synthesis.

Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions.

In the present study, we examined the hypothesis that dynamic characteristics of flow modulate the production of vasoactive mediators, namely nitric oxide (NO) and endothelin-1 (ET-1), by human umbilical vein endothelial cells (HUVECs). Cells were exposed for 6 hours in a cone-and-plate apparatus to different types of flow: steady laminar, with shear stresses of 2, 8, and 12 dyne/cm2, pulsatile laminar, with shear stress from 8.2 to 16.6 dyne/cm2 and a frequency of 2 Hz; periodic laminar, with square wave cycles of 15 minutes and shear stress from 2 to 8 dyne/cm2, and turbulent, with shear stress of 8 dyne/cm2 on average. A second culture dish was kept in a normal incubator as a static control for each experiment. Laminar flow induced synthesis of NO by HUVECs that was dependent on shear-stress magnitude. Laminar shear stress at 8 dyne/cm2 also upregulated the level of NO synthase mRNA. As observed with steady laminar flow, pulsatile flow also induced an increase in NO release by endothelial cells. When HUVECs were subjected to step-change increases of laminar shear, a further increase of NO synthesis was observed, compared with steady laminar shear of the same magnitude. Turbulent flow did not upregulate NO synthase mRNA or increase NO release. Both laminar and turbulent shear stress reduced, although not significantly, ET-1 mRNA and ET-1 production compared with the static condition. These results indicate that local blood flow conditions modulate the production of vasoactive substances by endothelial cells. This may affect vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone.

The acute effect of FK506 and cyclosporine on endothelial cell function and renal vascular resistance.

We have previously documented that cyclosporine exerts a direct cytotoxic effect on endothelial cells and causes an increase in renal vascular resistance (RVR) in the rat. In the present study we investigated whether FK506, a novel immunosuppressive agent thought to be less nephrotoxic than CsA, impairs endothelial cell function in vitro and affects RVR in vivo. In vitro eicosanoid release and endothelin release were measured in bovine aortic endothelial cells in culture exposed for 1, 6, and 24 hr to increasing concentrations of FK506 (1 nM to 10 microM) or CsA (0.5, 10 microM). No significant changes in TxB2 and 6-keto-PGF1 alpha (the stable breakdown products of TxA2 and PGI2, respectively) and endothelin release were found after 1 and 6 hr of incubation with all the concentrations of FK506 and CsA considered. FK506 did not affect endothelin release even after 24 hr of incubation. In contrast, cell exposure to CsA was associated with a dose-dependent increase in TxB2, 6-keto-PGF1 alpha, and endothelin release that reached statistical significance after incubation with 10 microM CsA. FK506 did not induce cell detachment or lysis at any concentration and time considered, while 10 microM CsA induced a significant reduction in cell count accompanied by cell lysis. In vivo studies showed that a single i.v. injection of FK506 to rats within a broad range of doses (28 ng/kg to 2.8 micrograms/kg) did not modify RVR. This was true even for a dose as high as 20 mg/kg, while 20 mg/kg CsA caused a dramatic increase in RVR. We conclude that FK506, unlike CsA, does not induce endothelial cell injury in vitro. Whether this explains the differences in renovascular resistance observed in vivo after acute injection of FK506 and CsA is an attractive possibility that needs to be further explored.

Methylprednisolone dosage effects on peripheral lymphocyte subpopulations and eicosanoid synthesis.

Glucocorticoids have a major role in the treatment of glomerular diseases. Despite recent advances in understanding of their mechanism of action, very few studies have addressed the relative advantage of the wide range of different dose regimens employed in clinical practice. We studied the effects of methylprednisolone given intravenously for three consecutive days at the doses of 1 mg/kg (group 1, N = 7; group 2, N = 5), 5 mg/kg (group 3, N = 5) or 15 mg/kg (group 4, N = 6) on total blood peripheral leukocytes and on lymphocyte subsets in patients with glomerular diseases, and investigated whether such effects were a function of the drug concentration in the blood. Since glucocorticoids have an inhibitory effect on the formation of eicosanoids in different cells, we also investigated in the same patients the effect of 1 and 15 mg/kg methylprednisolone on systemic and renal eicosanoid synthesis. Results of pharmacokinetic study showed that the three different doses of methylprednisolone we used resulted in major differences in patient's exposure to the drug, and within the same dose there was a great individual variability. By contrast the three different doses of methylprednisolone induced a comparable drop in the absolute number of lymphocytes six hours after the first injection of methylprednisolone, while 24 hours later blood lymphocyte counts returned to the pre-injection values in all patients. Analysis of lymphocyte subsets showed a selective decrease in the number of circulating CD4+ and CD8+ cells six hours after methylprednisolone which was comparable in the four groups of patients studied. As for the effect of methylprednisolone on systemic and renal eicosanoid synthesis in patients with glomerular diseases, 1 and 15 mg/kg were equally unable to reduce thromboxane A2 (TxA2) and prostaglandin E2 (PGE2) release by circulating polimorphonuclear cells (PMNs). By contrast, methylprednisolone partially inhibited eicosanoid synthesis by PMNs in vitro. Consistent with the data on PMNs, urinary excretion of TxA2 and prostacyclin (PGI2) metabolites were unaltered by the different doses of methylprednisolone. By contrast urinary PGE2 was markedly and significantly reduced in patients given 15 but not 1 mg/kg. We conclude that 1 mg/kg methylprednisolone given to patients with glomerular diseases has the same effect on peripheral total blood leukocyte count and lymphocyte subsets than 5 and 15 mg/kg. The same is true for eicosanoid synthesis by PMNs. Renal synthesis of PGE2 is inhibited by 15 mg/kg but not by 1 mg/kg.(ABSTRACT TRUNCATED AT 400 WORDS)