Complement Alternative Pathway Deficiency in Recipients Protects Kidney Allograft From Ischemia/Reperfusion Injury and Alloreactive T Cell Response.

Despite the introduction of novel and more targeted immunosuppressive drugs, the long-term survival of kidney transplants has not improved satisfactorily. Early antigen-independent intragraft inflammation plays a critical role in the initiation of the alloimmune response and impacts long-term graft function. Complement activation is a key player both in ischemia/reperfusion injury (IRI) as well as in adaptive antigraft immune response after kidney transplantation. Since the alternative pathway (AP) amplifies complement activation regardless of the initiation pathways and renal IR injured cells undergo uncontrolled complement activation, we speculated whether selective blockade of AP could be a strategy for prolonging kidney graft survival. Here we showed that Balb/c kidneys transplanted in factor b deficient C57 mice underwent reduced IRI and diminished T cell-mediated rejection. In in vitro studies, we found that fb deficiency in T cells and dendritic cells conferred intrinsic impaired alloreactive/allostimulatory functions, respectively, both in direct and indirect pathways of alloantigen presentation. By administering anti-fB antibody to C57 wt recipients in the early post Balb/c kidney transplant phases, we documented that inhibition of AP during both ischemia/reperfusion and early adaptive immune response is necessary for prolonging graft survival. These findings may have implication for the use of AP inhibitors in clinical kidney transplantation.

Active Subjects With Autoimmune Type 1 Diabetes Have Better Metabolic Profiles Than Sedentary Controls.

Previous studies in humans with type 1 diabetes mellitus (T1D) and in nonobese diabetic mice have investigated the beneficial immunomodulatory potential of aerobic physical activity. Performing high volume of aerobic exercise may favorably regulate autoimmunity in diabetes. We tested whether increased physical activity is a self-sufficient positive factor in T1D subjects. During a 3-month observational period, active (six males; 40.5 ± 6.1 years; BMI: 24.5 ± 2.1) and sedentary (four males, three females; 35.9 ± 8.9 years; BMI: 25.7 ± 3.8) T1D individuals on insulin pump therapy were studied for metabolic, inflammatory, and autoimmune parameters. At baseline and at the end of a 3-month period, glycosylated hemoglobin (HbA1c), autoantibodies (anti-GAD, anti-ZnT8, anti-IA2, and ICA) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. During the third month of the period, physically active T1D patients showed a significant reduction in the average glucose levels (-9%, p = 0.025, by CGM) compared to the first month values, and even their hyperglycemic episodes (>180 mg/dl) diminished significantly (-24.2%, p = 0.032 vs. first month). Moreover, active T1D subjects exhibited an improved body composition with respect to sedentary controls. No significant changes were detected as to the autoimmune and inflammatory profiles. This study confirms the beneficial role of physical exercise associated with insulin pump therapy in order to improve metabolic control in individuals with T1D. These preliminary positive observations need to be challenged in a prolonged interventional follow-up.

Impact of obesity severity and duration on pancreatic ß- and α-cell dynamics in normoglycemic non-human primates.

OBJECTIVE: Obesity is associated with high insulin and glucagon plasma levels. Enhanced ß-cell function and ß-cell expansion are responsible for insulin hypersecretion. It is unknown whether hyperglucagonemia is due to α-cell hypersecretion or to an increase in α-cell mass. In this study, we investigated the dynamics of the ß-cell and α-cell function and mass in pancreas of obese normoglycemic baboons. METHODS: Pancreatic ß- and α-cell volumes were measured in 51 normoglycemic baboons divided into six groups according to overweight severity or duration. Islets morphometric parameters were correlated to overweight and to diverse metabolic and laboratory parameters. RESULTS: Relative α-cell volume (RαV) and relative islet α-cell volume (RIαV) increased significantly with both overweight duration and severity. Conversely, in spite of the induction of insulin resistance, overweight produced only modest effects on relative ß-cell volume (RßV) and relative islet ß-cell volume (RIßV). Of note, RIßV did not increase neither with overweight duration nor with overweight severity, supposedly because of the concomitant, greater increase in RIαV. Baboons' body weights correlated with serum levels of interleukin-6 and tumor necrosis factor-α soluble receptors, demonstrating that overweight induces abnormal activation of the signaling of two cytokines known to impact differently ß- and α-cell viability and replication. CONCLUSION: In conclusion, overweight and insulin resistance induce in baboons a significant increase in α-cell volumes (RαV, RIαV), whereas have minimal effects on the ß cells. This study suggests that an increase in the α-cell mass may precede the loss of ß cells and the transition to overt hyperglycemia and diabetes.

Localization of mesenchymal stromal cells dictates their immune or proinflammatory effects in kidney transplantation.

Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell-based immunotherapy in solid-organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg-dependent mechanism. MSC-infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation.

Role of thymic- and graft-dependent mechanisms in tolerance induction to rat kidney transplant by donor PBMC infusion.

We previously demonstrated the presence of regulatory T cells (Tregs) in lymph nodes (LNs) from rats made tolerant to a kidney allograft by donor peripheral blood mononuclear cell (PBMC) infusion. Here, we investigated the origin of Treg and characterized their phenotype and mechanisms underlying their suppressive effect. At different points after PBMC infusion, thymus, LN, and graft-infiltrating -lymphocyte's (GIL) alloreactivity was evaluated in mixed lymphocyte reaction (MLR), coculture, and transwell experiments. GIL phenotype (by fluorescence-activated cell sorting and immunohistochemistry) and cytokines mRNA expression were analyzed. Before transplantation, CD4(+) thymocytes and LN cells from donor PBMC-infused rats showed a reduced anti-donor but a normal anti-third-party proliferation. Anti-donor hyporesponsiveness was reverted by interleukin (IL)-2. CD4(+) thymocytes had no regulatory activity on a naïve MLR. Treg appeared in LN at 60 days post-transplant. CD4(+)-GIL isolated early (5 days) and late post-transplant (days 60-80) were hyporesponsive and suppressed a naïve MLR. IL-10 mRNA was upregulated in GIL and an anti-IL-10 monoclonal antibody reverted their inhibitory effect. Cell-to-cell contact potentiated the suppressive activity of CD4(+)-GIL. We suppose that allograft tolerance in this model is mediated by pretransplant generation of anergic cells in the thymus, which may have a permissive role to prevent early graft disruption. The healed graft is a source of donor antigens, which led to early selection of Treg. In the late phase, tolerance is maintained by appearance of Treg in LN.

Mycophenolate mofetil combined with a cyclooxygenase-2 inhibitor ameliorates murine lupus nephritis.

BACKGROUND: Approaches to the treatment of lupus nephritis include immunosuppressants associated with anti-inflammatory drugs, mainly steroids, which, however, cause major side effects. Mycophenolate mofetil (MMF) has been described as being less toxic than conventional immunosuppressants, and it was effective in preventing progressive nephritis in lupus mice. Our study evaluated the therapeutic effect of MMF in NZB/W F1 hybrid mice with established disease. We also examined the combination of MMF with a selective cyclooxygenase-2 (COX-2) inhibitor, DFU, based on previous findings of excessive renal production of COX-2-derived thromboxane A2 (TXA2) in lupus nephritis. METHODS: Four groups of NZB/W mice (N = 30 each group), starting at five months of age, were given daily by gavage the following: vehicle, MMF 60 mg/kg, DFU 3 mg/kg, or MMF + DFU. Fifteen mice for each group were used for the survival studies, and the remaining mice were sacrificed at nine months. RESULTS: MMF or DFU alone partially delayed the onset of proteinuria compared with vehicle. Combined therapy was significantly (P < 0.05) more effective than single drugs. Animal survival was partially ameliorated by MMF and significantly improved by the drug combination in comparison with the vehicle (P = 0.005) and DFU alone (P < 0.03). At nine months, serum blood urea nitrogen (BUN) levels were lower in all of the treated groups than in the vehicle group. Renal damage was also limited, but to a greater extent in mice given the combined therapy. In untreated mice, renal COX-2 mRNA expression was up-regulated, and generation of TXB(2), the stable breakdown product of TXA(2), increased. DFU prevented the abnormal renal TXB(2) production, confirming the COX-2 origin of this eicosanoid, whereas renal 6-keto-PGF(1 alpha) and prostaglandin E(2) (PGE(2)) were not affected substantially. CONCLUSIONS: These results offer a strong case for exploring the possibility that in humans MMF combined with COX-2 inhibitors has a role in the treatment options for lupus nephritis. This combined drug therapy may be at least as effective as steroids but without the obvious nephrotoxicity of the latter.

Thymic dendritic cells express inducible nitric oxide synthase and generate nitric oxide in response to self- and alloantigens.

Thymocytes maturing in the thymus undergo clonal deletion/apoptosis when they encounter self- or allo-Ags presented by dendritic cells (DCs). How this occurs is a matter of debate, but NO may play a role given its ability of inducing apoptosis of these cells. APC (a mixed population of macrophages (Mphi) and DCs) from rat thymus expressed high levels of inducible NO synthase (iNOS) and produced large amounts of NO in basal conditions whereas iNOS expression and NO production were very low in thymocytes. Analysis by FACS and by double labeling of cytocentrifuged preparations showed that DCs and MPhi both express iNOS within APC. Analysis of a purified preparation of DCs confirmed that these cells express high levels of iNOS and produce large amounts of NO in basal conditions. The capacity of DCs to generate NO was enhanced by exposure to rat albumin, a self-protein, and required a fully expressed process of Ag internalization, processing, and presentation. Peptides derived from portions of class II MHC molecules up-regulate iNOS expression and NO production by DCs as well, both in self and allogeneic combinations, suggesting a role of NO in both self and acquired tolerance. We also found that NO induced apoptosis of rat double-positive thymocytes, the effect being more evident in anti-CD3-stimulated cells. Altogether, the present findings might suggest that DC-derived NO is at least one of the soluble factors regulating events, in the thymus, that follow recognition of self- and allo-Ags.

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.

Glomerular size-selective dysfunction in NIDDM is not ameliorated by ACE inhibition or by calcium channel blockade.

BACKGROUND: In patients with insulin-dependent diabetes mellitus (IDDM) and overt nephropathy glomerular barrier size-selectivity progressively deteriorates with time and is effectively improved by angiotensin converting enzyme (ACE) inhibition. Whether similar glomerular functional changes develop in proteinuric patients with non-insulin-dependent diabetes mellitus (NIDDM), and whether antihypertensive agents can favorably affect glomerular filtration of macromolecules in these patients, has not been documented yet. METHODS: We investigated renal hemodynamics and fractional clearance of neutral dextrans of graded sizes, in nine proteinuric patients with NIDDM and renal biopsy findings of typical diabetic glomerulopathy. Six healthy volunteers served as controls. We also investigated the effects of an ACE inhibitor and of a calcium channel blocker, both given in doses targeted to achieve a comparable level of systemic blood pressure control, on glomerular hemodynamics and sieving function. Theoretical analysis of glomerular macromolecule transport was adopted to evaluate intrinsic glomerular membrane permeability properties. RESULTS: Fractional clearance of large macromolecules (42 to 66 A in radius) was significantly higher in diabetic patients than in controls, and the distribution of membrane pore radii was calculated to be shifted towards larger pore sizes in diabetics (mean radius increased from 55 to 60 A). Despite effective blood pressure control, neither antihypertensive affected glomerular hemodynamics to any significant extent. Fractional clearance of dextrans, as well as of albumin and IgG, and total urinary proteins were not modified by either treatments. CONCLUSIONS: These data indicate that patients with NIDDM and overt nephropathy develop abnormalities in size-selective function of the glomerular barrier and, at variance to IDDM, such changes were not ameliorated either by ACE inhibition or calcium channel blockade.

Effect of acetate, bicarbonate dialysis, and acetate-free biofiltration on nitric oxide synthesis: implications for dialysis hypotension.

The effect of acetate dialysis (AD), bicarbonate dialysis (BD), and acetate-free biofiltration (AFB) on nitric oxide (NO) synthesis and the implications for dialysis hypotension was studied. The finding that uremic plasma is a potent inducer of NO synthesis by endothelial cells in vitro suggested that the cardiovascular instability of dialysis patients might result from excessive NO formation. Cardiovascular instability is more frequent in patients undergoing AD than BD. To see whether these differences were attributable to NO, we studied the NO synthetic pathway ex vivo in patients undergoing different dialysis procedures. Five patients were treated, in a random order, with AD, BD, and AFB, a technique using a buffer-free dialysate and postdilution of a sterile bicarbonate solution. Each type of dialysis was used for 1 week, comprising three dialysis sessions. A polyacrylonitrile dialyzer was used for all three methods. Before and after the third dialysis, plasma was collected, added to [3H]L-arginine, and incubated with human umbilical vein endothelial cells (HUVECs) for 24 hours. NO synthesis was evaluated as [3H]L-citrulline formation. Plasma concentrations of interleukin-1beta (IL-1beta), a potent inducer of inducible NO synthase (iNOS) in endothelial cells, were also measured. Plasma collected from patients after AD stimulated endothelial NO synthesis more than plasma from the same patients before the dialysis session (pre-AD, 0.173+/-0.028 nmol/10(5) cells v post-AD, 0.280+/-0.093 nmol/10(5) cells; P < 0.05). A slight, although not significant, increase was also observed when HUVECs were incubated with plasma drawn after BD (pre-BD, 0.151+/-0.014 nmol/10(5) cells; post-BD, 0.230+/-0.055 nmol/10(5) cells). AFB did not aggravate the stimulatory effect of uremic plasma on endothelial NO synthesis (pre-AFB, 0.184+/-0.038 nmol/10(5) cells; post-AFB, 0.189+/-0.040 nmol/10(5) cells). Plasma IL-1beta was greater (P < 0.01) after AD than after BD and AFB (post-AD, 0.234+/-0.028 pg/mL; post-BD, 0.124+/-0.019 pg/mL; post-AFB, 0.120+/-0.013 pg/mL). With AD, there was a greater intradialytic decrease in systolic blood pressure than with BD or AFB. Weight and blood volume loss and sodium balance were similar in AD, BD, and AFB. These data were consistent with the possibility that NO and cytokines, released in excessive amounts during AD, may contribute to hemodynamic instability.

Upregulation of renal and systemic cyclooxygenase-2 in patients with active lupus nephritis.

In lupus nephritis (LN), renal thromboxane A2 (TXA2) production is increased, and inhibition of TXA2 activity improves renal function. In patients with LN, renal function depends very much on vasodilatory prostaglandins, and indeed inhibiting the prostaglandin-forming enzyme cyclooxygenase (COX) with aspirin or related compounds was detrimental on renal hemodynamics in these patients. There are no data so far on whether the excessive TXA2 production in LN derives from upregulation of type I or type II isoforms of COX. It was found that TXB2 synthesis and COX-2 gene expression were higher in peripheral blood mononuclear cells from patients with active LN compared to patients in the inactive form of the disease and to healthy subjects. Unlike COX-2, levels of COX-1 mRNA were comparable in lupus patients and control subjects and were not influenced by the disease activity. Immunoperoxidase studies on kidney biopsies showed COX-1 staining in glomerular arterioles and other renal vessels, with no evident difference between lupus biopsies and control specimens taken from either individuals who were free of renal disease or patients with non-lupus nephropathies. In contrast, COX-2 staining was definitely stronger in specimens from patients with active LN than control specimens. In active LN, COX-2-specific staining was localized mainly in the glomeruli, with a weaker signal on tubuli and in the interstitium. Double-staining studies with an antibody against the macrophage marker CD68 and an anti-COX-2 antibody definitely showed that COX-2 and CD68 often colocalized on the same cell, with only occasional glomerular COX-2-stained mesangial areas. Patients with non-lupus nephropathies had no increase in renal COX-2 expression. These results indicate that COX-2 upregulation is a specific finding of active LN and that monocytes infiltrating the glomeruli contribute to the exaggerated local synthesis of TXA2. If this is correct, COX-2 may soon become a target for therapeutic intervention in this disease.

Individualized anticoagulation with dermatan sulphate for haemodialysis in chronic renal failure.

BACKGROUND: Dermatan sulphate (DS) is a selective thrombin inhibitor with antithrombotic properties and low bleeding potential. In preliminary studies it was reported to be effective for preventing clot formation in the haemodialysis circuit. METHODS: Ten patients on maintenance haemodialysis for chronic renal failure underwent three consecutive investigation phases. In phase 1 (individual dose titration), repeated dialyses were performed with increasing doses of DS until successful dialysis was obtained in two sessions at the same dose. In phase 2, individualized DS doses were validated by a randomized crossover comparison with the individual heparin dose of each patient. In phase 3, each patient underwent 24 consecutive dialyses with DS over 8 weeks. Successful dialysis was defined as completion of the procedure without visible clot formation in the bubble traps and lines or a greater than 20% decrease in dialyser capacity. Dialysis efficiency (decrease in serum urea and creatinine, Kt/V), APTT prolongation, bleeding time, and DS plasma concentrations were also assessed. RESULTS: Phase 1: successful dialysis was achieved in nine patients with 4 mg/kg DS as a predialysis intravenous bolus followed by continuous infusion of 0.65 mg/kg/h. One patient required 5 mg/kg plus 1.3 mg/kg/h. Phase 2: no statistically significant differences were found between DS and heparin in any of the investigated variables. Residual dialyser capacity and dialysis efficiency indexes indicated equivalent efficacy. Phase 3: residual dialyser capacity and dialysis efficiency did not change with time. There was no accumulation of DS in plasma. No bleeding or thrombocytopenia were observed. CONCLUSIONS: The dose of DS can be individually titrated to suppress clot formation during haemodialysis as efficiently as with individualized heparin. Such an individualized DS regimen maintains its anticoagulant efficacy and is safe in prolonged use.

Sequential monitoring of urine-soluble interleukin 2 receptor and interleukin 6 predicts acute rejection of human renal allografts before clinical or laboratory signs of renal dysfunction.

BACKGROUND: The significance of noninvasive techniques to the early diagnosis of acute rejection in kidney transplants remains elusive. In this study, we examined whether an early posttransplant increase in serum- and urine-soluble interleukin (IL) 2 receptor (sIL-2R) and IL-6 levels predicted acute rejection. METHODS: Sequential determinations of serum and urine sIL-2R and IL-6 were performed in the first 30 postoperative days in 40 renal transplant patients. Changes during the posttransplant period observed in 26 patients who had one or more episodes of acute rejection (group A) were compared with those recorded in 14 patients who did not experience acute rejection of their graft (group B). RESULTS: Serum sIL-2R was higher than normal in patients of groups A and B without statistical differences between the two groups. In the first 3 days after transplantation, urinary sIL-2R was higher than normal in group A but not in group B. Urinary sIL-2R at days 2 and 3 was significantly higher (P<0.05) in group A than in group B. In the first 5 days after transplantation, urinary IL-6 was persistently higher than normal in group A, whereas it progressively decreased to normal value on day 4 in group B. Sudden increases (doubling within 24 hr) in urine IL-6 preceded clinical diagnosis of acute rejection by a mean period of 2 days, with an 87% sensitivity and a 64% specificity, and also predicted recurrent rejection episodes. CONCLUSIONS: Sequential monitoring of urinary sIL-2R and IL-6 levels does allow very early diagnosis of rejection without invasive procedures. Specifically, high urinary sIL-2R in the first 5 posttransplant days identifies the subgroup of patients at risk. In the subsequent days, a sudden increase in urinary IL-6 occurs in those of the above patients who will indeed reject their graft.

Monocyte chemoattractant protein-1 is excreted in excessive amounts in the urine of patients with lupus nephritis.

BACKGROUND: In patients with lupus nephritis, mononuclear inflammatory cells infiltrate the renal interstitium and glomeruli, and the degree of leukocyte infiltration correlates with the severity of the renal dysfunction. The precise mediator signaling inflammatory cells to migrate into the kidney is not known. Recent findings that monocyte chemoattractant protein-1 (MCP-1), a chemotactic cytokine with a high degree of specificity for lymphocytes and monocytes, is overexpressed in glomeruli from rats with immunecomplex glomerulonephritis prompted us to explore the possibility that MCP-1 could be implicated in the renal inflammatory response of lupus erythematosus. EXPERIMENTAL DESIGN: Serum and urine levels of MCP-1 were evaluated in 10 patients with active lupus nephritis. Patients were studied at admission, before therapy, and at various time points after the first administration of high dose methylprednisolone. There was an additional observation of the four patients who underwent remission of clinical signs of the disease after chronic steroid treatment. Three additional groups, one (n = 9) of patients with inactive lupus nephritis, one (n = 9) of patients with nonlupus glomerulonephritis and high degree proteinuria, and one (n = 10) of healthy subjects, were also studied. RESULTS: In patients with active lupus nephritis, urinary MCP-1 was significantly higher than in lupus patients studied in the inactive phase of the disease or in healthy volunteers. High doses of i.v. methylprednisolone significantly lowered urinary MCP-1 in patients with active lupus nephritis. In patients undergoing remission of lupus nephritis after chronic steroid treatment, urinary MCP-1 did not correlate with either serum MCP-1 levels or proteinuria. Unlike in lupus, in patients with nonlupus forms of glomerulonephritis, urinary MCP-1 was comparable to controls. CONCLUSIONS: Altogether, the present data suggest a role for MCP-1 in mononuclear cell migration into the kidney in lupus nephritis.

Renal prostacyclin biosynthesis is reduced in children with hemolytic-uremic syndrome in the context of systemic platelet activation.

Previous studies have reported various abnormalities in prostacyclin (PGI2) synthesis and metabolism in hemolytic-uremic syndrome (HUS). However, the conclusions of most of these studies are based on in vitro or ex vivo experiments that only give an indirect estimate of the actual biosynthesis in vivo. We studied the urinary excretion of PGI2 metabolites, taken as a marker of the actual biosynthesis, in six children with HUS during the acute phase of the disease and again when remission was achieved. Eight age- and sex-matched healthy children were studied as controls. Since HUS is also associated with platelet activation and consumption, we also studied the urinary excretion of thromboxane A2 (TxA2) metabolites. Urinary PGI2 and TxA2 metabolites were assessed by radioimmunoassay after high-performance liquid chromatography (HPLC) purification. Urinary excretion of the PGI2 hydrolysis product, 6-keto-PGF1 alpha, was significantly reduced in children with acute HUS as compared with controls, indicating a defective renal synthesis of PGI2. A significant inverse correlation was found between urinary 6-keto-PGF1 alpha and blood urea nitrogen (BUN), as well as plasma creatinine. At remission, urinary 6-keto-PGF1 alpha levels increased to values higher than those of controls. By contrast, the urinary excretion of the major PGI2 beta-oxidation product, 2,3-dinor-6-keto-PGF1 alpha, was comparable to controls, indicating normal systemic PGI2 biosynthesis. The urinary excretion of both TxA2 hydrolysis product, TxB2, and the major beta-oxidation metabolite, 2,3-dinor-TxB2, were lower than normal in the acute phase of HUS if expressed as absolute values.(ABSTRACT TRUNCATED AT 250 WORDS)