Biomedical subjects

C Zoja

Publications and source records attributed to C Zoja.

At least 19 recordsLinked to original sources

PubMed1998

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.

Cell Adhesion↗

PubMed1998

Renoprotective effect of contemporary blocking of angiotensin II and endothelin-1 in rats with membranous nephropathy.

BACKGROUND: We previously showed that chronic administration of an angiotensin converting enzyme (ACE) inhibitor to rats with passive Heymann nephritis (PHN), a model of membranous nephropathy with proteinuria and increased renal synthesis of endothelin-1 (ET-1), reduces urinary proteins and partially limits the exaggerated ET-1 renal synthesis. Here we compared the effect of an ETA receptor antagonist and an ACE-inhibitor given as single therapies with a combination of the two drugs in uninephrectomized PHN rats. METHODS: PHN was induced with a single i.v. injection of rabbit anti-Fx1A antibody in 40 male Sprague Dawley rats. To accelerate the onset of renal damage rats underwent uninephrectomy seven days later and were subsequently treated until eight months with the ETA receptor antagonist LU-135252 (50 mg/kg b.i.d. p.o.) or the ACE-inhibitor trandolapril (1 mg/kg in the drinking water) or the combination of the two drugs. RESULTS: Either LU-135252 or trandolapril given alone prevented the increase in systolic blood pressure (SBP). Combined therapy was even more effective than single drugs. While LU-135252 and trandolapril reduced proteinuria by 23 to 25%, the drug combination resulted in 45% lowering of urinary proteins. Serum creatinine was significantly decreased by the combination, but not by the single drugs. Glomerulosclerosis and tubulointerstitial damage were more reduced by combined therapy than by LU-135252 or trandolapril alone. CONCLUSIONS: These data suggest that contemporary blocking angiotensin II (Ang II) and ET-1 in an accelerated model of PHN had an additive renoprotective effect than single blocking Ang II or ET-1 and would represent a therapeutic advantage for renal disease patients who do not completely respond to ACE inhibitors.

Angiotensin II↗

PubMed1998

Experimental Goodpasture's syndrome in Wistar-Kyoto rats immunized with alpha3 chain of type IV collagen.

BACKGROUND: Glomerulonephritis and lung hemorrhage of autoimmune Goodpasture syndrome develop due to immune reactions against epitope(s) of the non-collagenous (NC1) domain of alpha3-chain of type IV collagen [alpha3(IV) NC1]. Whether thymic mechanisms have a role in the loss of tolerance to the Goodpasture epitope has not been established. We studied the renal and pulmonary effects of immunization with different forms (monomer, dimer, or hexamer) of alpha3(IV) NC1 collagen in Wistar-Kyoto (WKY) rats, and assessed whether the intrathymic inoculation of the antigen may protect against anti-GBM disease. METHODS: WKY rats were immunized with bovine alpha3(IV) monomer, dimer, or hexamer, or with alpha3(IV) NC1 synthetic peptide. Renal function, kidney and lung immunohistology, and circulating and tissue bound antibodies to type IV collagen chains were analyzed. Effects of intrathymic inoculation of antigen on subsequent disease induction were analyzed in WKY rats given alpha3(IV) NC1 dimer or GBM preparation intrathymically 48 hours before immunization. RESULTS: Proteinuria, linear IgG deposition in GBM, and crescentic glomerulonephritis developed in WKY rats immunized with alpha3(IV) NC1 dimer or hexamer. Lesions were dose-dependent upon injections of 10 to 100 microgram dimer. The alpha3(IV) NC1 monomer induced less severe proteinuria and no crescents. Pulmonary hemorrhage was detectable in 35% of rats immunized with 25 to 100 microgram alpha3(IV) NC1 dimer; alpha3(IV) synthetic peptide (36 carboxyl terminal) did not induce disease. Rats injected intrathymically with up to 100 microgram alpha3(IV) NC1 dimer or with GBM 48 hours before immunization were not protected against subsequent development of proteinuria and glomerulonephritis. CONCLUSIONS: These findings document that glomerulonephritis and lung hemorrhage can be elicited in WKY rats by immunization with alpha3(IV) NC1. Failure of the intrathymic inoculation of antigen to prevent disease suggests that immunological tolerance cannot be achieved by this intervention, in contrast to other autoimmune conditions, and may imply independent roles for cellular and humoral nephritogenic pathways in anti-GBM nephritis.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Angiotensin II↗

PubMed1998

Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B.

Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.

Animals↗

PubMed1998

In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation.

Progression to end-stage renal failure is the final common pathway of many forms of glomerular disease, independent of the type of initial insult. Progressive glomerulopathies have in common persistently high levels of urinary protein excretion and tubulointerstitial lesions at biopsy. Among the cellular mechanisms that may determine progression regardless of etiology, the traffic of excess proteins filtered from glomerulus in renal tubule may have functional importance by initiating interstitial inflammation in the early phase of parenchymal injury. This study analyzes the time course and sites of protein accumulation and interstitial cellular infiltration in two different models of proteinuric nephropathies. In remnant kidneys after 5/6 renal mass ablation, albumin and IgG accumulation by proximal tubular cells was visualized in the early stage, preceding interstitial infiltration of MHC-II-positive cells and macrophages. By double-staining, infiltrates developed at or near tubules containing intracellular IgG or luminal casts. This relationship persisted thereafter despite more irregular distribution of infiltrate. Similar patterns were found in an immune model (passive Heymann nephritis), indicating that the interstitial inflammatory reaction develops at the sites of protein overload, regardless of the type of glomerular injury. Osteopontin was detectable in cells of proximal tubules congested with protein in both models at sites of interstitial infiltration, and by virtue of its chemoattractive action this is likely mediator of a proximal tubule-dependent inflammatory pathway in response to protein load. Protein overload of tubules is a key candidate process translating glomerular protein leakage into cellular signals of interstitial inflammation. Mechanisms underlying the proinflammatory response of tubular cells to protein challenge in diseased kidney should be explored, as well as ways of limiting protein reabsorption/deposition to prevent consequent inflammation and progressive disease.

Albumins↗

PubMed1997

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.

Cells, Cultured↗

PubMed1997

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.

Angiotensin II↗

PubMed1997

Mycophenolate mofetil limits renal damage and prolongs life in murine lupus autoimmune disease.

Classical immunosuppressants like cyclophosphamide give excellent results in human lupus nephritis. However, they augment malignancies and viral infections. Here we investigated the effect of the new immunosuppressant agent, mycophenolate mofetil (MMF), in New Zealand Black x New Zealand White (NZBxW) F1 hybrid mice, a model of genetically determined immune complex disease that mimics systemic lupus in humans. MMF has a selective antiproliferative effect on T- and B-lymphocytes, inhibits antibody formation and blocks the glycosylation of lymphocyte glycoproteins involved in the adhesion of leukocytes to endothelial cells. Two groups of NZBxW mice were used: group 1 (N = 20) given daily MMF (60 mg/kg p.o.) and group 2 (N = 15) given daily vehicle alone. Treatment started at three months of age and lasted until the death of the animals. Results showed that percentage of proteinuric mice was significantly reduced by MMF treatment and serum BUN levels were also lower than vehicle. MMF had a suppressive effect on autoantibody production and protected animals from leukopenia and anemia. Life survival of MMF treated lupus mice was significantly improved in respect to untreated animals. Thus, MMF delayed renal function deterioration and prolonged life survival in murine lupus nephritis. MMF has been already recognized as reasonably well tolerated in renal transplant patients and despite its gastrointestinal toxicity its overall safety profile appears superior to azathioprine. Human studies are needed to establish whether MMF may function as a steroid-sparing drug in lupus nephritis.

Animals↗

PubMed1997

Renal and systemic nitric oxide synthesis in rats with renal mass reduction.

In rats undergoing renal mass reduction (RMR) oral supplementation with the nitric oxide (NO) precursor L-arginine increases glomerular filtration rate and ameliorates signs of glomerular injury, suggesting that chronic renal failure in the rats is a condition of low NO formation in the kidney. On the contrary, data are available that in the systemic circulation of uremics, both rats and human beings, NO is formed in excessive amounts and may contribute to platelet dysfunction and bleeding tendency, well-known complications of uremia. The present study was designed to clarify the pathophysiology of renal and systemic NO synthesis in uremia. We showed that renal ex vivo NO generation, measured as the conversion of [3H] L-arginine to [3H] L-citrulline, was lower than normal in RMR rats, seven days after surgery, and progressively worsened with time in close correlation with signs of renal injury. Consistent with these results, urinary excretion of the stable NO metabolites, NO2-/NO3-, significantly decreased in rats with RMR. To go deeper into the cellular origin and biochemical nature of this abnormality we used two histochemical approaches that could locate either NO synthase (NOS) catalytic activity (NADPH-diaphorase) or NOS isoenzyme expression (immunoperoxidase). NADPH-diaphorase documented a progressive loss of renal NOS activity in RMR rats that co-localized with a strong progressive decrease of inducible NOS isoenzyme (iNOS) immunostaining. At variance with iNOS, endothelial cell NOS (ecNOS) staining was rather comparable in RMR and control kidneys. At variance to the kidney, in the systemic circulation of RMR rats the synthesis of NO increased as reflected by higher than normal plasma NO2-/NO3- concentrations. High systemic NO likely derives from vessels as documented by the increased NOS activity and higher expression of both iNOS and ecNOS in the aorta of RMR rats. Up-regulation of systemic NO synthesis might be an early defense mechanism against hypertension of uremia. On the other hand, more NO available to circulating cells may sustain the bleeding tendency, a well-known complication of uremia.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Time course and localization of endothelin-1 gene expression in a model of renal disease progression.

Experimental and human proteinuric glomerulopathies are associated with tubulo-interstitial injury that correlates with the decline of renal function even better than glomerular lesions do. Mechanism(s) leading to tubulo-interstitial damage are unknown. It has been proposed that excessive reabsorption of filtered proteins activates renal cells to produce vasoactive and inflammatory molecules including endothelin-1. The aim of the present study was twofold: we first evaluated the cellular origin of excessive renal endothelin-1 production in the renal mass reduction model and then related endothelin-1 distribution to the development of kidney lesions. Four groups of renal mass reduction (n = 15) and four groups of control rats (n = 5) were studied at 7, 14, 21, and 28 days after surgery. Urinary proteins in renal mass reduction rats were comparable with controls at day 7 but became significantly higher thereafter. Renal mass reduction rats first developed tubulo-interstitial changes, which were already evident at day 14 in the majority of them. At 28 days, renal mass reduction rats also developed glomerulosclerosis. A parallel increase of renal endothelin-1 gene expression and synthesis of the corresponding peptide in renal mass reduction rats versus controls was observed from day 14. Nonradioactive in situ hybridization confirmed a pattern of endothelin-1 mRNA consistent with the distribution of lesions. At day 14, endothelin-1 staining was stronger in renal mass reduction than in control kidneys and mainly localized to the cytoplasm of tubular cells, whereas glomeruli were negative. At day 28, endothelin-1 expression further increased in renal mass reduction rats as compared with controls, and the staining was apparent also in glomeruli. Thus, in renal mass reduction, a progressive up-regulation of endothelin-1 occurs during the development of renal injury, that first involves the tubules and, only in a subsequent phase, the glomeruli.

Animals↗

PubMed1996

Cyclosporine enhances leukocyte adhesion to vascular endothelium under physiologic flow conditions.

We investigated the effect of cyclosporine (CyA) on leukocyte adhesion to endothelium under flow conditions. Confluent human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with CyA (1, 5, and 10 micromol/L) and then exposed to a total human leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2. Human umbilical vein endothelial cells stimulated with interleukin-1beta (20 U/mL) were used as a positive control. Adherent cells were measured by digital image analysis. Results showed that CyA dose-dependently increased the number of leukocytes adhering to HUVECs compared with control cells. Leukocyte adhesion markedly increased on HUVECs incubated with interleukin-lbeta, one of the most potent inducers of endothelial cell adhesiveness. Exposure of endothelial cells to CyA did not affect the number of rolling leukocytes, which was similar to control values. To examine the role of adhesion molecules in CyA-induced leukocyte adhesion, HUVECs were incubated with monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin before adhesion assay. Functional blocking of ICAM-1, VCAM-1, and E-selectin on endothelial cells significantly inhibited CyA (10 micromol/L)-induced leukocyte adhesion. Confocal fluorescence microscopy studies showed that CyA induced an increase in the endothelial surface expression of ICAM-1, VCAM-1, and E-selectin. Pretreatment of leukocytes with the platelet activating factor receptor antagonist L659,989 significantly reduced the number of leukocytes adhering to CyA-treated HUVECs. We suggest that CyA enhances leukocyte adhesion to endothelium by upregulating adhesive proteins on endothelial surface membrane. Blocking leukocyte receptor for platelet-activating factor partially prevents adhesion, suggesting a role for endothelial cell-associated platelet-activating factor in the interaction between leukocytes and CyA-treated endothelium.

Cell Adhesion↗

PubMed1996

Increased nitric oxide formation in recurrent thrombotic microangiopathies: a possible mediator of microvascular injury.

The term thrombotic microangiopathy (TMA) has been used extensively to encompass hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, two syndromes of hemolytic anemia, and thrombocytopenia associated with renal or brain involvement or both. There is evidence that endothelial damage is a crucial feature in the sequence of events that precedes the development of microvascular lesions. More recent studies would suggest that endothelial dysfunction could be a consequence of neutrophil activation. Activated neutrophils generate superoxide anions (O2-) that, combining with endothelial-derived nitric oxide (NO), form the highly cytotoxic hydroxyl radical. Seven patients with recurrent forms of TMA and seven healthy volunteers were studied. Plasma concentrations of the NO metabolites, nitrites/nitrates, were elevated in the acute phase of TMA, indicating an increased NO synthesis in vivo. In addition, elevated serum concentrations of tumor necrosis factor, a potent inducer of endothelial NO synthase, were found in acute TMA. Serum from patients with acute TMA induced NO synthesis in cultured endothelial cells more than normal serum. Enhanced stimulatory activity was no longer found in the recovery phase. Release of O2- by neutrophils ex vivo was higher than normal in patients with acute TMA, but decreased in the recovery phase. Exactly the same trend was observed for plasma malondialdehyde and conjugated dienes, indicating that excessive oxygen radical formation in acute TMA is associated with increased lipid peroxidation. Thus, in recurrent forms of TMA, NO formation was increased as compared with controls. This was associated with signs of lipid peroxidation, likely the consequence of the interaction of NO with neutrophil-derived oxygen products.

3,4-Methylenedioxyamphetamine↗

PubMed1996

A study of low-nutrient diets used for aging studies in the rat.

The effects of diets of low caloric value on rats used in aging studies were investigated. Groups of 85 Sprague-Dawley rats were fed ad libitum from 3 months of age on three different diets containing 8 or 10 Megajoule (MJ) of metabolizable energy and 80 or 100 g of crude protein/kg. Body weights, food consumption, and morphological and biochemical parameters were monitored throughout life. Kidneys were examined histologically. Rats given the diet with highest energy and protein ate less food, attained greater weights, and had larger abdominal fat deposits than those on the lower energy diets. They had a raised proteinuria, and nearly half developed glomerulosclerosis and tubulo-interstitial damage by 26 months. There was no significant difference in mortality between the groups, and no other serious abnormalities were observed. It is concluded that rats can be maintained into old age with no signs of nutritional inadequacy on diets with lower energy and protein contents than those in general use.

Abdomen↗

PubMed1996

Colchicine interferes with L-selectin and leukocyte function-associated antigen-1 expression on human T lymphocytes and inhibits T cell activation.

Colchicine, which inhibits cell microtubule assembly by preventing polymerization of tubulin monomers, inhibits cell-mediated immune responses and promotes long-term survival of major histocompatibility complex-incompatible renal allografts in rats. Here we evaluated the effect of blocking cell microtubule assembly by colchicine on T cell and endothelial cell adhesion receptors involved in transducing signals for T cell activation. By using immunofluorescence flow cytometry analysis, evidence is presented that colchicine, in a dose-dependent fashion, downregulated L-selectin and leukocyte function-associated antigen-1, but not CD2 and CD44 on the surface of naive human peripheral blood lymphocytes. This effect was confirmed in two subsets of T lymphocytes, namely, CD45RA- and CD45RO-positive cells. However, colchicine did not influence the rapid shedding of L-selectin from T lymphocytes exposed to activating stimuli. Colchicine inhibited expression of interleukin-2 receptor on activated T lymphocytes. This effect was observed when T lymphocytes were stimulated with both anti-CD3 and anti L-selectin monoclonal antibodies. Colchicine also inhibited lymphocyte function in vitro as documented by inhibition of the human mixed lymphocyte response in a dose-dependent fashion. Moreover, colchicine downregulated surface expression of intercellular adhesion molecule-1 and E-selectin on activated human umbilical vein endothelial cells. These results indicate that blocking cell microfubule assembly inhibits surface expression of adhesion molecules on T cells and endothelial cells, and provides insights into the complex mechanisms of the action of colchicine in vivo.

Antibodies, Monoclonal↗