Mecanismos del daño endotelial en la microangiopatía trombótica / Mechanisms of endothelial damage in thrombotic microangiopathy

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Selective dietary restriction of protein and calorie intakes prevents spontaneous proteinuria in male MWF rats.

BACKGROUND/AIMS: Previous observations indicate that protein and calorie restrictions can affect the course of renal disease progression. We compared the effects of selective protein and calorie restriction on glomerular hemodynamics and proteinuria in a model of spontaneous glomerular injury in the rat. METHODS: Three groups of male MWF rats were assigned to three different diets: standard diet (ST, 19% protein, 3.4 kcal/g), low protein (LP) and low calorie (LC). Proteinuria and systolic blood pressure were periodically measured. Glomerular hemodynamics and tuft volume were determined after 2 months of dietary treatment. RESULTS: The effective mean protein intake was 3.4 +/- 0.4, 1.6 +/- 0.2, and 3.2 +/- 0.2 g/day/rat, respectively, for the ST, LP, and LC diets, while caloric intake averaged 60 +/- 7, 59 +/- 9, and 30 +/- 2 kcal/day/rat. Both LP and LC diets significantly prevented proteinuria (104 +/- 32, 36 +/- 9, and 18 +/- 8 mg/day, respectively, in the three groups). The systolic blood pressure was unaffected by the diets. The LC diet induced lower body and kidney weights than the ST diet. The glomerular filtration rate was slightly but significantly increased by the LP diet, but not by the LC diet (0.64 +/- 0.14, 0.81 +/- 0.08, and 0.67 +/- 0.12 ml/min, respectively, for ST, LP and LC diets). The glomerular hydraulic pressures were not affected by the diets. No differences were also observed in glomerular volume. The incidences of glomerulosclerosis and tubulointerstitial changes were comparable in ST and LP diets and completely absent in the LC diet group. CONCLUSION: These results indicate that restriction of both protein and calorie intakes prevents spontaneous proteinuria in male MWF rats by preventing deterioration of glomerular perm-selective functions.

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.

PAF mediates neutrophil adhesion to thrombin or TNF-stimulated endothelial cells under shear stress.

Platelet-activating factor (PAF) is known to modulate polymorphonuclear leukocyte (PMN) adhesion to endothelial cells cultured under static conditions and activated by thrombin. In contrast, there are no data on the role of PAF in PMN adhesion to cells exposed to flow conditions and activated by stimuli other than thrombin. Here we used the PAF receptor antagonist L-659,989 to evaluate PMN adhesion to human umbilical vein endothelial cells (HUVEC) in basal conditions or upon challenge with thrombin or tumor necrosis factor-alpha (TNF-alpha). Experiments were performed under dynamic flow using a parallel-plate flow chamber and a computer-based image analysis system. Rolling and adhesion of PMNs to endothelial cells significantly increased upon stimulation with thrombin. Thrombin-stimulated HUVEC also synthesized higher amounts of PAF than untreated cells. Pretreatment of PMNs with L-659,989 significantly reduced their rolling and adhesion to thrombin-activated HUVEC. Stimulation of HUVEC with TNF-alpha significantly increased the number of rolling and adherent PMNs as compared with untreated cells. Adhesion of PMNs to and migration across TNF-alpha-stimulated HUVEC were reduced by L-659,989, whereas cell rolling was unchanged. We conclude that PAF mediates leukocyte interaction under flow conditions with HUVEC activated by inflammatory stimuli.

Dissociation between antiproteinuric and antihypertensive effect of angiotensin converting enzyme inhibitors in rats.

To clarify whether angiotensin converting enzyme (ACE) inhibitors prevent progressive renal injury directly by their antihypertensive effect we administered the ACE inhibitor lisinopril to male MWF/Ztm rats as a single daily dose that lowered blood pressure for only 9 of 24 h. We investigated the effects of this treatment in short- and long-term studies and compared them with another antihypertensive drug, the calcium channel blocker nitrendipine, given to partially control blood pressure as done with the ACE inhibitor. In untreated animals systemic hypertension, proteinuria, and glomerulosclerosis developed spontaneously with age, and lisinopril reduced systemic hypertension and prevented proteinuria and glomerular lesions. Nitrendipine, despite similar blood pressure control, was ineffective in preventing both proteinuria and glomerulosclerosis. After 2 mo of treatment glomerular capillary pressure was significantly reduced by lisinopril and slightly but significantly increased by nitrendipine, compared with untreated controls. The ultrafiltration coefficient was significantly higher in lisinopril than in controls and not significantly changed by nitrendipine. With both drugs, however, glomerular hemodynamic effects were observed only at a few hours after administration and were abolished before the next administration. No significant changes in glomerular tuft volume were observed in treated and untreated animals after 2 and 6 mo of observation. Thus ACE inhibitor, despite only partial control of systemic blood pressure, effectively prevented proteinuria and glomerular injury. Comparable blood pressure control obtained with a calcium channel blocker was not associated with renal protection. These results suggest that ACE inhibitors could protect glomerular microcirculation by a mechanism that is not directly related to their antihypertensive action.

Defective glomerular [3H]lysoPAF metabolism in the autologous phase of rabbit nephrotoxic nephritis.

Glomerular infiltration of blood-derived mononuclear cells contributes to the glomerular injury in the autologous phase of nephrotoxic nephritis (NTN). LysoPAF has recently been shown to be chemotactic for human monocytes, thus its accumulation might account for monocyte recruitment. We investigated [3H]lysoPAF metabolism in isolated glomeruli from normal and NTN rabbits studied both in the heterologous and in the autologous phases of the disease. [3H]lysoPAF was converted to [3H]1-O-alkyl-glycerol and [3H]1-O-alkyl-2-acyl-GPC by phospholipase C and acyltransferase, respectively, both in normal and NTN glomeruli. Glomerular metabolism of [3H]lysoPAF was normal during the heterologous phase of NTN. By contrast, in isolated glomeruli from NTN rabbits studied in the autologous phase of the disease, a significantly lower [3H]lysoPAF degradation occurred with respect to normal ones. This defective degradation resulted in a significantly reduced formation of [3H]1-O-alkyl-glycerol. The apparent Km for enzymatic conversion of [3H]lysoPAF to [3H]1-O-alkyl-glycerol, determined at 15 minutes as a function of [3H]lysoPAF concentration, was doubled in glomeruli from rabbits studied in the autologous phase of NTN as compared to normal ones, while Vmax values were similar in the two groups. These results show a defective glomerular lysoPAF degradation in the autologous phase of NTN, likely due to a decreased affinity of phospholipase C to lysoPAF. Altered lysoPAF metabolism results in glomerular accumulation of lysoPAF in the autologous phase of NTN, as shown by significantly higher levels of lysoPAF measured in nephritic glomeruli as compared to normal ones.

Methylprednisolone normalizes superoxide anion production by polymorphs from patients with ANCA-positive vasculitides.

It has been convincingly documented that reactive oxygen species released from activated neutrophils mediate glomerular damage in experimental glomerulonephritis. Recent findings that antineutrophil cytoplasmic autoantibodies (ANCA) induce neutrophils to degranulate and produce oxygen radicals in vitro led us to explore whether neutrophils from patients with ANCA-positive vasculitides and necrotizing glomerulonephritis generated an increased amount of superoxide anion (O2-). Since glucocorticoids inhibit oxygen radicals generation in vitro we also evaluated the effect of intravenous pulses of methylprednisolone. Polymorphs were isolated from peripheral blood collected before (basal), 6 and 24 hours after the first infusion of methylprednisolone and 24 hours after the third one. O2- release by cells was assessed after 30 minute incubation without specific stimuli. Basal O2- release was significantly higher in patients than in controls (P < 0.01). Intravenous infusion of high doses of methylprednisolone markedly reduced O2- production with respect to the basal value, and the difference was statistically significant at various time interval considered after the steroid infusion. Besides reducing the excessive O2- formation, methylprednisolone induced an increase in polymorph expression of the gene encoding for manganese superoxide dismutase (Mn-SOD) enzyme. We conclude that polymorphs taken from patients with ANCA-positive vasculitides and necrotizing glomerulonephritis generate higher amounts of O2- than those from normal subjects. Methylprednisolone normalizes the abnormal generation of O2-, likely through its ability to up-regulate the gene for Mn-SOD, a potent antioxidant enzyme.

Pathophysiologic implications of proteinuria in a rat model of progressive glomerular injury.

BACKGROUND: Males of a substrain of Munich-Wistar rats (MWF/Ztm), selected for their large number of superficial glomeruli, develop spontaneous proteinuria with age, whereas females of this strain have a normal urinary protein excretion rate. EXPERIMENTAL DESIGN: We investigated the relationship between functional and structural glomerular alterations in four groups of male and female MWF/Ztm rats, respectively at 20 and 35 weeks of age. Systolic blood pressure, urinary protein excretion and composition of urinary proteins were periodically measured during the study. At the end of the observation period, renal function was evaluated with solute clearance technique and kidney tissue processed for morphologic and morphometrical analysis using light and electron microscopy. RESULTS: Systolic blood pressure in males was significantly higher than in females, and progressed toward systemic hypertension with age. Urinary protein excretion became spontaneously abnormal in males. About 50% of urinary proteins consisted of albumin, whereas 35% was a sex-dependent low molecular weight protein. Albumin excretion increased with age in these animals, whereas excretion of the sex-dependent protein decreased. Urinary protein excretion in females was significantly lower than in males of the same age, remaining near normal levels. No decline in renal function with age was observed in all animal groups. Glomerular structural alterations developed progressively with age in males, leading to important glomerular and tubular alterations. Female rats maintained almost intact glomerular morphology until the end of the study. Morphometric analysis showed an important increase in glomerular volume with age in males but not in females. This glomerular tuft enlargement was the result of an increase in the number of glomerular cells and a concomitant increase in cell volume. CONCLUSIONS: Male MWF/Ztm rats develop spontaneously systemic hypertension, proteinuria of glomerular origin, and glomerulosclerosis. Female rats develop less severe hypertension and are protected from proteinuria and glomerulosclerosis.

Defective platelet aggregation in response to platelet-activating factor in uremia associated with low platelet thromboxane A2 generation.

The bleeding tendency associated with uremia is likely due to a qualitative platelet dysfunction. So far the data available on platelet aggregation are conflicting. Since platelet-activating factor (PAF) plays a role in primary hemostasis, we studied platelet aggregation in response to PAF in 40 patients with chronic uremia on regular hemodialysis and 12 control subjects. Our results showed that in 28 of 40 uremics, platelet aggregation response to PAF was normal, whereas in the remaining 12 it was defective in that no second wave of aggregation was elicited even if the PAF concentrations were increased by a factor of 10,000. This abnormal response was peculiar to PAF and only partially related to factor(s) of plasma origin. The number of platelet PAF receptors and their affinity for the agonist were comparable in controls and "PAF-unresponsive" patients. The defective platelet aggregation in response to PAF was associated with a statistically significant reduction (P less than 0.01) in thromboxane A2 (TxA2) generation in platelet-rich plasma (PRP) challenged with PAF (10 and 100 nmol/L). When PRPs from PAF-unresponsive patients were preincubated with a stable analogue of prostaglandin endoperoxides/TxA2 U-46619, an irreversible platelet aggregation in response to PAF was obtained. Thus in a subpopulation of uremics, platelet aggregation in response to PAF is selectively abnormal as a consequence of a reduced TxA2 generation.

Platelet-activating factor receptor blocking reduces proteinuria and improves survival in lupus autoimmune mice.

Platelet-activating factor (PAF) has been suggested recently to play an important role in immune glomerulonephritis, favoring the formation of immune deposits in glomeruli and contributing to the local inflammatory reaction. Here we sought to investigate whether urinary PAF excretion was modified in New Zealand Black x New Zealand White mice a model of genetically determined immune complex disease which mimics systemic lupus in humans and whether changes in PAF urinary excretion values correlated with the extent of proteinuria. To clarify the possible "in vivo" relevance of these findings we evaluated whether PAF receptor antagonist has any influence on the evolution of renal disease and survival of these mice. Our results showed that: 1) in lupus mice urinary PAF excretion increased progressively with age in New Zealand Black x White; 2) the increase in PAF excretion correlated with the severity of proteinuria; and 3) the chronic administration of a PAF receptor antagonist [L-659,989 [(+/- )-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5- (3,4,5-trimethoxyphenyl)tetrahydrofuran]] starting from 26 weeks of age significantly delayed the onset of proteinuria and prolonged survival.

Increased urinary excretion of platelet activating factor in mice with lupus nephritis.

Platelet activating factor (PAF) is present in urine from humans and experimental animals in normal conditions. Very little is known about changes in PAF urinary excretion under pathologic conditions and no data are available about the origin of PAF in the urine. In the present study we explored the possibility that immunologic renal disease is associated with an increase in PAF urinary excretion using gas chromatography-mass spectrometry technique. To clarify the renal or extrarenal origin of urinary PAF we evaluated whether exogenously administered PAF (1-[1', 2'-3H]alkyl) is filtered through the glomerulus and excreted in the urine. The results show that: 1) urine from mice with lupus nephritis in the early phase of the disease contained amounts of PAF comparable to those excreted in normal mouse urine, 2) PAF levels increased when animals started to develop high grade proteinuria, 3) after intravenous injection of [3H] PAF in nephritic mice, a negligible amount of [3H] ether lipid, corresponding to [3H]1-alkyl -2-acyl-3-phosphocholine (alkyl-2-acyl-GPC), was recovered from the 24 h urine extract.

Renal metabolism and urinary excretion of platelet-activating factor in the rat.

The origin of platelet-activating factor (PAF) in the urine remains ill defined. The present study documents that [3H]PAF (3.5 mu Ci) injected into the renal artery of isolated control rat kidney preparations perfused at constant pressure with a cell-free medium containing 1% bovine serum albumin (BSA) was excreted in negligible amounts (0.034%) in the urine, whereas 6% was retained by the kidney. When kidneys were perfused with a BSA-free medium, 0.029 and 71% of the total radioactivity added to the perfusate was recovered in the urine and in the renal tissue, respectively. [3H]PAF urine excretion in proteinuric kidneys from adriamycin-treated rats was still negligible (0.015%). Analysis of the renal tissue-retained radioactivity in control and proteinuric kidneys perfused with 1% BSA indicated metabolism into long chain acyl-sn-glycero-3-phosphorylcholine species, lyso-PAF, glycerols, and intact PAF. Thin layer chromatography analysis of [3H]glycerol fraction in these renal extracts showed two major components comigrating with 1-O-alkylglycerol and 1-O-alkyl-2-fatty acylglycerol. Isolated proximal tubules, but not glomeruli from nephrotic rats exposed to increasing concentrations of BSA (0-4%), had a higher PAF uptake than control tubules for BSA concentrations ranging from 0 to 0.1%. Our findings in the isolated perfused kidneys indicate that, in normal conditions, circulating PAF is excreted in the urine in negligible amounts and that the altered glomerular permeability to proteins does not affect this excretion rate. Moreover, analysis of renal tissue radioactivity documented that the renal metabolism of PAF is comparable in control and nephrotic kidneys.

Role of endothelium-derived nitric oxide in the bleeding tendency of uremia.

Endothelium-derived relaxing factor, now identified as nitric oxide (NO), is a labile humoral agent formed by vascular endothelial cells from L-arginine. NO mediates the action of substances that induce endothelium-dependent relaxation and plays a role in regulating blood pressure. In this study we investigated whether NO is involved in the pathogenesis of the bleeding tendency associated with renal failure. Rats with extensive surgical ablation of renal mass develop renal insufficiency due to progressive glomerulosclerosis. Like uremic humans, rats with renal mass reduction and uremia have a bleeding tendency that manifests itself by a prolonged bleeding time. We found that N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO formation from L-arginine, completely normalized bleeding time when given to uremic rats. L-NMMA injection also increased ex vivo platelet adhesion but did not affect ex vivo platelet aggregation induced by adenosine diphosphate, arachidonic acid, and calcium ionophore A23187. The shortening effect of L-NMMA on bleeding time was completely reversed by giving the animals the NO precursor L-arginine, but not D-arginine, which is not a precursor of NO. It thus appears that NO is a mediator of the bleeding tendency of uremia.

Tissue plasminogen activator therapy of rabbit nephrotoxic nephritis.

To study the efficacy of tissue plasminogen activator (PA) therapy to prevent deteriorating renal function in experimental proliferative glomerulonephritis we used a model of nephrotoxic nephritis induced in rabbits by injection of antiglomerular basement membrane antiserum. Saline or recombinant tissue plasminogen activator (rt-PA, 1.3 mg/kg body weight) was infused daily for 7 days starting from day 7 after the injection of antiglomerular basement membrane antiserum when the disease had been already triggered. Animals were killed on day 14. Rabbits given saline had abundant deposits of fibrin and crescents in about 67% of glomeruli. rt-PA significantly protected animals from glomerular fibrin deposition and crescent formation with respect to saline-treated animals. Renal function measured as creatinine clearance was dramatically impaired in rabbits given saline. Treatment with rt-PA ameliorated the renal function impairment of nephrotoxic nephritis. rt-PA did not produce a systemic fibrinolytic state as indicated by alpha 2-antiplasmin level measurement. These results suggest that tissue PA may have important implications in preventing renal function deterioration in humans with crescentic glomerulonephritis and fibrin depositions.

Development of a mass spectrometric method to quantitate platelet activating factor in mouse urine.

Platelet activating factor (PAF) is a lipid mediator of inflammation released by a variety of stimulated inflammatory cells. It may be involved in immune glomerulonephritis. Thus, its measurement in urine could give information on the mechanism of this disease. We present here a method to measure PAF in mouse urine, using gas-liquid chromatography-mass spectrometry (GLC-MS) in the selected ion recording (SIR) mode. Before instrumental analysis, the extracted and purified samples were hydrolyzed and derivatized with pentafluorobenzoyl chloride. Different experimental conditions are presented and discussed to corroborate the analytical findings. PAF levels in mouse urine were 2.08 +/- 0.46 ng/24 h. This procedure might represent a new experimental tool to establish the possible role of PAF as mediator of tissue damage in renal disease.

Enhanced glomerular thromboxane A2 mediates some pathophysiologic effect of platelet-activating factor in rabbit nephrotoxic nephritis: evidence from biochemical measurements and inhibitor trials.

Previous studies have shown that platelet-activating factor (PAF) receptor blocking has a protective effect on rabbit nephrotoxic nephritis (NTN). We examined whether arachidonic acid (AA) metabolism is altered in NTN and whether a PAF receptor antagonist has any influence on such changes. Rabbits injected with anti-glomerular basement membrane antiserum in the heterologous phase had a markedly increased glomerular thromboxane B2 (TxB2) production level, whereas no changes have been detected in glomerular 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2). During the autologous phase of the disease, the glomerular TxB2 level was even higher than in the heterologous phase. The level of 6-keto-PGF1 alpha was significantly lower than normal, and the level of PGE2 was unchanged in respect to the basal values. The use of L-652,731 (a specific PAF receptor antagonist) reversed the abnormal generation of AA metabolites at glomerular level both in the heterologous and autologous phase of the disease. The effect of L-652,731 on AA metabolism is likely to be an indirect result of the PAF receptor blocking, because L-652,731 given to normal rabbits had no direct effect on glomerular AA metabolism. To assess whether the beneficial effect of L-652,731 in NTN is at least in part mediated by its capability of suppressing the excessive intrarenal synthesis of thromboxane A2 (TxA2), we compared the effect of L-652,731 with that of a selective TxA2-synthase inhibitor (FCE-22178). FCE-22178 ameliorated the morphologic expression of rabbit NTN and reduced function deterioration. The protective effect of L-652,731 on proteinuria in the autologous phase and on glomerular filtration rate in both phases was superior to that of FCE-22178. We conclude that an excessive intraglomerular synthesis of TxA2 occurs in rabbit NTN that can play a role in renal function deterioration. Both a specific PAF receptor antagonist and a TxA2-synthase inhibitor reduced the exaggerated TxA2 synthesis and favorably influenced the evolution of the disease.

Metabolism of arachidonic acid in isolated glomeruli from pig kidney.

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF1 alpha and PGF2 alpha, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B2 (TxB2) and PGE2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF1 alpha. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.

Platelet activating factor (PAF) as a mediator of injury in nephrotoxic nephritis.

Release of acetyl glyceryl ether phosphorylcholine, platelet-activating factor (PAF), has been demonstrated to be associated with glomerular inflammatory damage in acute serum sickness. Moreover, PAF can increase glomerular permeability to proteins and induce mesangial contraction. Thus PAF might be a good candidate as a mediator of glomerular damage. However the in vivo evidence that PAF might cause glomerular injury is lacking. To evaluate if PAF has a major role in promoting glomerular inflammatory reaction and fibrin deposition, we studied the effect of a molecule, L-652,731, which blocks the PAF receptor, on the evolution of an experimental model of anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN). GN was initiated by sheep-anti-rabbit nephrotoxic serum. A proliferative GN regularly occurred in which heavy proteinuria, intra and extracapillary proliferation of resident and inflammatory cells and fibrin deposition in Bowman's capsule were the prominent findings. Our results showed that the PAF receptor antagonist reduces the glomerular damage in anti-GBM GN, supporting the hypothesis that PAF is indeed a mediator of glomerular inflammatory reaction. PAF receptor blocking prevented renal function deterioration in the early phase of the disease, probably preserving glomerular hemodynamics. In the delayed phase of the disease the PAF receptor antagonist reduced proteinuria and prevented renal function deterioration and fibrin deposition. These effects appear to be mediated by an inhibitory action of PAF receptor blocking on macrophage accumulation and activation.