Peripheral donor leukocytes prolong survival of rat renal allografts.

BACKGROUND: The development of strategies to enhance the survival of transplanted organs and to potentially lower or even discontinue immunosuppressive therapy would represent a significant advancement in post-transplant patient care. METHODS: We studied the effect of pretransplant infusion of donor leukocytes alone or in combination with a short course of cyclosporine on the long-term outcome of a rat model of kidney allograft. RESULTS: A single intravenous infusion of donor peripheral blood leukocytes (100x10(6) cells) from Brown-Norway (BN) rats into major histocompatibility complex (MHC) incompatible Lewis recipients largely failed to prolong kidney allograft viability from the same donor transplanted 60, 40, or 30 days after cell infusion. A short course of cyclosporine (per se, unable to prolong graft survival) was started at the same day of donor leukocyte infusion, but instead was able to prolong the survival of the BN kidney transplant-performed 40 days later-but not of a Wistar Furth (WF) third party, with some animals even developing tolerance. A mixed lymphocyte reaction of host cells from long-term surviving rats to BN stimulator cells was significantly reduced as compared with controls. Donor BN DNA was detected in the peripheral blood of Lewis rats until day 40 after BN leukocyte infusion. Microchimerism persisted (60 to 70 days post-transplant) in most long-term graft recipients. Reducing the time interval between donor leukocyte infusion and subsequent kidney transplant to 10 days still prolonged graft survival. Donor peripheral blood mononuclear cells, but not polymorphonuclear cells, in the leukocyte preparation contributed to prolong kidney allograft survival. CONCLUSIONS: Pretransplant donor leukocyte infusion under the appropriate conditions can tip the immune balance toward improved graft acceptance. This result could be relevant to the achievement of donor-specific tolerance of the graft with the maintenance of an intact response to third-party antigens.

Increased fragmentation of von Willebrand factor, due to abnormal cleavage of the subunit, parallels disease activity in recurrent hemolytic uremic syndrome and thrombotic thrombocytopenic purpura and discloses predisposition in families. The Italian Registry of Familial and Recurrent HUS/TTP.

We investigated here the changes in von Willebrand factor (vWF) multimers in recurrent, sporadic and familial forms of hemolytic uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) to see whether they are actually proteolyzed in vivo in these patients. Molecular determinants of fragments in vWF were also characterized to identify possible sites of cleavage of the subunit. Unusually large vWF multimers were found in blood of 8 of 10 patients with recurrent HUS/TTP, both in the acute phase and in remission, but never in familial and sporadic cases. Instead, all of the groups showed evidence of enhanced fragmentation of vWF multimers during the acute phase. Increased fragmentation was also shown by decrease in native 225-kD vWF subunit. In recurrent and sporadic HUS/TTP, enhanced fragmentation normalized at remission, but the abnormality persisted in familial HUS/TTP patients. The latter findings suggest that patients with familial HUS/TTP may have a congenital abnormality in vWF processing. Analysis with specific monoclonal antibodies showed the presence of the normal vWF fragments with apparent molecular mass of 189, 176, and 140 kD in all patients; however, in 6 recurrent and in 5 familial cases, novel fragments that differed in size from normal ones were found. The size of these abnormal fragments differed from one patient to another and none of them was ever found in normal plasma. These results documented, for the first time in HUS/TTP, an abnormal cleavage of the vWF subunit that might account for the increased fragmentation observed in these patients.

Hypocomplementemia discloses genetic predisposition to hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: role of factor H abnormalities. Italian Registry of Familial and Recurrent Hemolytic Uremic Syndrome/Thrombotic Thrombocytopenic Purpura.

Familial hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) carry a very poor outcome and have been reported in association with decreased serum levels of the third complement component (C3). Uncontrolled consumption in the microcirculation, possibly related to genetically determined deficiency in factor H--a modulator of the alternative pathway of complement activation--may account for decreased C3 serum levels even during disease remission and may predispose to intravascular thrombosis. In a case-control study by multivariate analysis, we correlated putative predisposing conditions, including low C3 serum levels, with history of disease in 15 cases reporting one or more episodes of familial HUS and TTP, in 25 age- and gender-matched healthy controls and in 63 case-relatives and 56 control-relatives, respectively. The relationship between history of disease, low C3, and factor H abnormalities was investigated in all affected families and in 17 controls. Seventy-three percent of cases compared with 16% of controls (P < 0.001), and 24% of case-relatives compared with 5% of control-relatives (P = 0.005) had decreased C3 serum levels. At multivariate analysis, C3 serum level was the only parameter associated with the disease within affected families (P = 0.02) and in the overall study population (P = 0.01). Thus, subjects with decreased C3 serum levels had a relative risk of HUS or TTP of 16.56 (95% confidence interval [CI], 1.66 to 162.39) within families and of 27.77 (95% CI, 2.44 to 314.19) in the overall population, compared to subjects with normal serum levels. Factor H abnormalities were found in four of the cases, compared with three of the healthy family members (P = 0.02) and none of the controls (P = 0.04) and, within families, factor H abnormalities were correlated with C3 reduction (P < 0.05). Reduced C3 clusters in familial HUS and TTP is likely related to a genetically determined deficiency in factor H and may predispose to the disease. Its demonstration may help identify subjects at risk in affected families.

Gene polymorphism of the renin-angiotensin system and progression of diabetic nephropathy.

Good evidence exists that genetic predisposition is a major determinant in the development of renal and cardiovascular complications of diabetes. In particular, the role of familial predisposition is well established in diabetic nephropathy which may cluster within families, both in type I (IDDM) and in type II (NIDDM) diabetes. The genes responsible for predisposition to renal and cardiovascular complications are not known, but those of the renin-angiotensin system (RAS) are plausible candidates. Beside the large number of studies aimed at evaluating the role of polymorphisms in these genes, particularly in angiotensin-converting enzyme (ACE) gene, in development of renal disease, no clear-cut evidence has been provided until now. Furthermore, a number of trials have shown that ACE-inhibitors (ACEi) may reduce the rate of progression of renal failure. If the RAS genotype were able to foresee the response to ACEi it would provide new strategies for a specific treatment of subjects at higher risk.

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.

Blocking both type A and B endothelin receptors in the kidney attenuates renal injury and prolongs survival in rats with remnant kidney.

Renal disease progression in the rat is associated with a time-dependent upregulation of renal endothelin-1 (ET-1) gene expression and synthesis. We have previously demonstrated that endothelin A receptor subtype (ETA) blockade in rats with remnant kidney reduced signs of disease activity, suggesting that ET-1 exerts part of its deleterious effects on the kidney through ETA. No data are available so far on the role of ETB receptor in progressive renal injury. We first studied renal ETA and ETB receptor gene expression in rats with remnant kidney on days 7, 30, and 120 after the surgical procedure. While renal expression of ETA was unaffected, ETB receptor gene was significantly upregulated with time in rats with remnant kidney, being 3.5-fold and sixfold higher than shamoperated rats at days 30 and 120. We also evaluated whether bosentan, a nonpeptidic ETA and ETB receptor antagonist, offered better protection against renal disease progression than reported for ETA-selective blockers and whether it improved survival in animals with renal ablation. Two groups of rats with renal mass reduction (n = 11 each) were given bosentan 100 mg/kg/d orally or its vehicle (carboxymethyl cellulose) beginning day 7 after the surgical procedure and were followed until the death of the vehicle-treated animals. Sham-operated animals comprised the control group. Bosentan partially prevented increases in blood pressure and proteinuria, but had a remarkable protective effect on renal function and significantly prolonged animal survival. These data suggest that blocking both renal ETA and ETB receptors might have major implications in the treatment of human progressive nephropathies.

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 role of endothelin in the progression of the renal disease in an experimental model of chronic kidney failure]. / Ruolo dell'endotelina nella progressione della malattia renale in un modello sperimentale di insufficienza renale cronica.

Recent in vitro and in vivo data suggest that endothelin, a potent vasoconstrictor peptide originally isolated from cultured porcine aortic endothelial cells, may be one of the factors accounting for progressive glomerulosclerosis in experimental and human glomerulopathies. Endothelin is expressed and produced by glomerular endothelial and mesangial cells in culture, has a mitogenic effect on mesangial cells and stimulates mesangial synthesis of extracellular matrix components such as type I, III and IV collagen and laminin. In rats with renal mass ablation, a model of chronic renal disease characterized by systemic hypertension, proteinuria and progressive glomerulosclerosis, a significant increase in urinary excretion of endothelin was documented 45 days after surgery as compared with basal values. Experiments involving the infusion of labeled endothelin into renal arteries of rats with renal mass ablation have suggested that the enhanced urinary excretion rate of endothelin could reflect an increased renal production of the peptide. Recent experiments done with Northern blot analysis have evidenced a 2.5 fold increase in pre-proendothelin transcript in kidney homogenates from rats with remnant kidney at 30 days after surgery. The increase averaged 4-5 fold at 60 and 120 days. In the same animals a significant correlation was found between urinary endothelin excretion and the percent of glomeruli affected by glomerulosclerosis. We conclude that the progression of renal disease after surgical ablation of renal mass is associated with an increased renal endothelin gene expression together with excessive urinary excretion of the corresponding protein. It is speculated that endothelin may mediate glomerular structural abnormalities associated with the progression of renal disease.

A specific endothelin subtype A receptor antagonist protects against injury in renal disease progression.

We have recently reported that renal preproendothelin-1 gene is up-regulated in rats with renal mass reduction (RMR) and that time-dependent increase in urinary excretion of the corresponding peptide correlates with renal disease progression. Here we evaluated whether a specific endothelin subtype A (ETA) receptor antagonist, FR139317, reduced signs of disease activity in this model. Two groups of rats were given FR139317 or its vehicle (saline) from day 7 to day 60 after the surgical procedure. Sham-operated animals were the control group. Blood pressure, urinary protein excretion and serum creatinine were evaluated at days 0, 7 (before FR139317 or saline administration), 30, 45 and 60. At sacrifice, histological evaluation of renal tissue was performed. The results showed that ETA receptor blocker reduced the abnormal permeability to proteins, limited glomerular injury and prevented renal function deterioration thus confirming the working hypothesis. These findings suggest that this class of compounds may eventually prove useful in the treatment of human progressive nephropathies.

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.

Renal cyclophilin-like protein gene expression parallels changes in sodium excretion in experimental nephrosis and is positively modulated by atrial natriuretic peptide.

Experimental evidence is available to indicate that intrarenal mechanisms play a role in the impaired salt excretion of nephrotic syndrome by multiple and still incompletely defined mediators. It is documented herein that the gene encoding for cyclophilin-like protein (Cy-LP) is up-regulated in renal medulla from adriamycin (ADR)-treated rats as compared with control animals. In the cortex of rats with ADR nephrosis, no change in Cy-LP as compared with that in controls was found for the entire observation period. By contrast, in the medulla of nephrotic rats, Cy-LP gene expression was significantly higher than in controls. Values of urinary Na excretion were inversely correlated to Cy-LP mRNA expression levels. Because in ADR nephrosis a blunted natriuretic response to ANP has been previously reported, it was investigated whether ANP infusion modulated Cy-LP mRNA in the renal medulla. ADR-treated rats, but not control rats, infused for 1 h with ANP (1 microgram/kg.min) had a significant (P < 0.05) increase in medullary Cy-LP mRNA as compared with nephrotic animals receiving the vehicle alone. These findings might be taken to suggest that renal Cy-LP gene expression is positively modulated in nephrotic syndrome and parallels changes in sodium excretion.

Renal endothelin gene expression is increased in remnant kidney and correlates with disease progression.

We previously demonstrated that urinary endothelin excretion is increased in rats with extensive renal mass reduction, a model of progressive renal disease. Here we explored whether the increased urinary endothelin in this model were due to induction of renal pre-pro-endothelin-1 gene and whether changes in endothelin synthetic pathway correlated with the development of glomerulosclerosis. Four groups of rats with renal mass reduction and four groups of sham-operated control rats were studied 7, 30, 60 and 120 days after the surgical procedure. Urinary protein excretion in renal mass ablation animals did not differ from controls at seven days, but was already significantly elevated (P < 0.01) 30 days after surgery. Then proteinuria progressively increased in rats with remnant kidney at values above 400 mg/day at day 120. Serum creatinine concentration also progressively increased with time in renal mass ablation rats, unlike sham-operated animals, and values were significantly different (P < 0.01) at each of the points considered. Rats with renal mass reduction, unlike sham-operated animals, developed focal glomerulosclerosis that affected 8% of glomeruli at day 30, and 24% of glomeruli at day 120. Seven days after renal mass reduction renal pre-pro-endothelin-1 (pre-pro ET) mRNA was comparable to that of sham-operated rats, while a 2.5-, 5- and fourfold increase in 2.3 Kb pre-proET-1 transcript was observed at 30, 60 and 120 days, respectively. Urinary excretion of endothelin was significantly elevated (P < 0.01) in rats with renal mass reduction with respect to sham-operated rats, starting from 30 days after surgery and increased further thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence against a pathogenetic role for endothelin in pre-eclampsia.

OBJECTIVE: To assess whether increased placental or systemic endothelin synthesis has a pathogenic role in pre-eclampsia (gestational proteinuric hypertension). DESIGN: Prospective observations study. SUBJECTS: 19 women with pre-eclampsia and 10 healthy pregnant women were studies. All were in the last trimester. MAIN OUTCOME MEASURES: Preproendothelin-1 gene expression by Northern blot analysis and generation of endothelin-1 precursor, big-endothelin-1, and endothelin isoforms, namely endothelin-1, 2 and 3, were assessed by specific radio-immunoassays, in placental tissue. Plasma endothelin-1 levels and urinary excretion of big-endothelin-1 and endothelin-1 were measured. RESULTS: Placental preproendothelin-1 gene expression and immunoreactive big-endothelin-1 and endothelin-1, 2 and 3, were comparable in placental tissue from pre-eclamptic and normal pregnant women. Plasma levels of endothelin-1 did not differ between pre-eclamptic and normal pregnancies. In contrast, urinary excretion of endothelin-1, which is likely to reflect the renal synthesis of the peptide, was significantly decreased in pre-eclamptic, as compared with normal pregnant women. This was not due to a decreased renal generation of endothelin-1 precursor, since urinary excretion of big-endothelin-1 did not differ between pre-eclamptic and normal pregnancies. These data suggest an increased renal endothelin-1 breakdown in pre-eclampsia. CONCLUSIONS: Endothelin is unlikely to play a role in the pathogenesis of pre-eclampsia. Instead, an increased renal breakdown may have a role in limiting the negative effects of other vasoactive factors on the renal circulation.

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.

Turnour necrosis factor stimulates endothelin-1 gene expression in cultured bovine endothelial cells.

We have studied the effect of human recombinant tumour necrosis factor-alpha (TNF-alpha) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-alpha (10 and 100 ng ml(-1)) increased in a time dependent manner the preproendothelin-1 mRNA levels in respect to unstimulated endothelial cells. TNF-alpha induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.

Human placenta expresses endothelin gene and corresponding protein is excreted in urine in increasing amounts during normal pregnancy.

Systemic and renal hemodyanmic changes in normal pregnancy have been attributed in part to altered vascular synthesis of vasodilatory prostaglandins. Besides vasodilatory substances, endothelium also generates vasoconstrictors, including endothelin. We evaluated the capacity of placental tissue from normal pregnant women to express endothelin gene and to generate endothelin. Placental tissue expressed a single 2.3 kb preproendothelin messenger ribonucleic acid and produced comparable amounts of endothelin 3, Big endothelin 1, and endothelin 1 and a minor quantity of endothelin 2. To investigate the possible influence of placental endothelin production on plasma levels of the peptide, plasma endothelin concentrations were measured in normal pregnant women at delivery and were found to be numerically higher than those measured in nonpregnant subjects. Urinary excretion of endothelin, taken as a marker of the renal synthesis of the peptide, tended to increase, although not significantly, in the first 14 weeks of pregnancy. This trend continued throughout pregnancy, resulting in a significant increase from the second trimester to delivery.

Constitutive expression of endothelin gene in cultured human mesangial cells and its modulation by transforming growth factor-beta, thrombin, and a thromboxane A2 analogue.

The present study was designed to assess whether human glomerular mesangial cells in culture express preproendothelin gene and whether endothelin gene expression in the mesangium is regulated by factors potentially released by inflammatory cells and platelets infiltrating the glomerular tuft during the course of various types of glomerulonephritis. For this purpose mesangial cells were incubated for 6 hours in the presence of absence of interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TBF-beta), the thromboxane A2 analogue U-46619, and thrombin. Resting mesangial cells expressed a 2.3-kilobase mRNA on blot hybridization analysis with a human cDNA preproendothelin probe, indicating that this type of cells, in addition to glomerular endothelial cells, constitutively expresses endothelin gene. IL-1 beta did not change endothelin mRNA levels in respect to unstimulated mesangial cells. At variance, TGF-beta, U-46619, and thrombin had a marked effect on endothelin mRNA, stimulating a 3- to 8-fold increase over basal levels. Quantification of actin mRNA and analysis of the autoradiographic signals provided validation of the difference in the endothelin mRNA levels. Expression of preproendothelin mRNA in either resting or stimulated mesangial cells was associated with synthesis and release of the corresponding peptide in the cell supernatant as determined by a specific radioimmunoassay for endothelin. Endothelin production from IL-1 beta stimulated mesangial cells was not different from that of unstimulated cells, whereas a significant (p less than 0.01) increase in endothelin production was observed after cell stimulation with TGF-beta, U-46619, and thrombin. The demonstration that mesangial cells constitutively express mRNA for preproendothelin and release endothelin into culture medium, together with the finding that endothelin gene expression and production in mesangial cells are regulated by molecules potentially released at glomerular level during an inflammatory reaction may suggest that endothelin participates in the complex process of glomerular disease progression.

Susceptibility to type I diabetes in women is associated with the CD3 epsilon locus on chromosome 11.

Type I diabetes is associated with the DQ loci of the MHC and to a lesser extent with the T cell antigen receptor (TcR) beta chain genes. The non-obese diabetic (NOD) mouse is an animal model of human diabetes, in which up to 90% of female mice develop overt insulin-dependent diabetes. Genetic studies in the NOD mouse suggest that there are at least three diabetogenic genes; one that maps to the MHC, another that may map to the mouse Thy-I locus, and a third that has still to be identified. We have investigated loci in the vicinity of the human Thy-I locus on chromosome 11q23 and report here the results of restriction fragment length polymorphism (RFLP) analysis of the CD3 epsilon locus of 168 Caucasoid patients with type I diabetes. While no association was found between this locus and type I diabetes, a significant difference in the frequency of the CD3 epsilon 8-kb allele was found between male and female patients (0.268 versus 0.430; P less than 0.0025, Pc = 0.02) and between female patients and healthy female controls (0.430 versus 0.267; P less than 0.015). These results suggest that a gene residing on chromosome 11q23 may confer susceptibility to type I diabetes in women.

Effect of low-dose aspirin on fetal and maternal generation of thromboxane by platelets in women at risk for pregnancy-induced hypertension.

There is evidence that aspirin in low doses favorably influences the course of pregnancy-induced hypertension, but the mechanism, although assumed to involve suppression of the production of thromboxane by platelets, has not been established. We performed a randomized study of the effect of the long-term daily administration of 60 mg of aspirin (n = 17) or placebo (n = 16) on platelet thromboxane A2 and vascular prostacyclin in women at risk for pregnancy-induced hypertension. Low doses of aspirin were associated with a longer pregnancy and increased weight of newborns. Serum levels of thromboxane B2, a stable product of thromboxane A2, were almost completely (greater than 90 percent) inhibited by low doses of aspirin. The urinary excretion of immunoreactive thromboxane B2 was significantly reduced without changes in the level of 6-keto-prostaglandin F1 alpha, a product of prostacyclin. Mass spectrometric analysis showed that aspirin reduced the excretion of the 2,3-dinor-thromboxane B2 metabolite--mainly of platelet origin--by 81 percent and of thromboxane B2, probably chiefly of renal origin, by 59 percent. The urinary excretion of 6-keto-prostaglandin F1 alpha and of its metabolite 2,3-dinor-6-keto-prostaglandin F1 alpha was not affected. Low doses of aspirin only partially (63 percent) reduced neonatal serum thromboxane B2. No hemorrhagic complications were observed in the newborns. Thus, in women at risk for pregnancy-induced hypertension, low doses of aspirin selectively suppressed maternal platelet thromboxane B2 while sparing vascular prostacyclin, but only partially suppressed neonatal platelet thromboxane B2, allowing hemostatic competence in the fetus and newborn.

Selective inhibition of platelet thromboxane generation with low-dose aspirin does not protect rats with reduced renal mass from the development of progressive disease.

Rats with extensive renal mass reduction develop hypertension, proteinuria and progressive glomerulosclerosis. Previous studies have demonstrated that these changes are associated with an increased urinary excretion of thromboxane compared with normal rats and that the administration of a thromboxane synthetase inhibitor prevents glomerulosclerosis and progressive renal function deterioration. On this basis it has been speculated that the thromboxane synthetase inhibitor, by inhibiting platelet thromboxane, reduces platelet aggregation and prevents the generation of substances that can influence glomerular functional properties. Because the thromboxane synthetase inhibitor also inhibits thromboxane synthesis by resident glomerular cells and lowers blood pressure in these animals, the question of whether platelet thromboxane is indeed the factor implicated in the development of renal disease after renal ablation remains unanswered. To address this issue the authors administered at different time intervals from the surgical procedure a low-dose of oral aspirin (ASA) to rats with remnant kidney. This approach resulted in selective inhibition of platelet cyclooxygenase leading to an almost complete prevention of platelet thromboxane generation. Low-dose ASA spared renal cyclooxygenase as documented by a lack of significant inhibition of glomerular and urinary 6-keto-PGF1 alpha and did not lower blood pressure. Renal function studies showed that low-dose ASA, despite inhibiting platelet aggregation, had no effect on proteinuria and progressive renal insufficiency irrespectively if administered late (ie, 80 days after surgery) and given daily for all the observation period (ie, 20 days) or earlier in the course of the disease (ie, 40 and 10 days after surgery). Histologic data showed that the degree of glomerulosclerosis and tubulo-interstitial damage was not significantly different in rats with reduction of renal mass alone compared with rats with remnant kidney given low-dose ASA. In conclusion, the present findings indicate that inhibition of platelet aggregation and thromboxane formation does not prevent the progressive glomerulosclerosis that develops in rats with surgical reduction of renal mass. It is suggested that the beneficial results obtained previously in the same model by the use of a thromboxane synthesis inhibitor must be attributed either to an effect on resident glomerular cell thromboxane synthesis or to lowering systemic blood pressure.