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.

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.

High-energy microwave thermotherapy in the treatment of benign prostatic hyperplasia.

INTRODUCTION: Transurethral microwave thermotherapy is an anesthesia-free, outpatient method of treating lower urinary tract symptoms due to benign prostatic hyperplasia (BPH). Our results with the use of this technique in 25 patients are reported. MATERIALS AND METHODS: Twenty-five patients with BPH, 8 of whom with complete urinary retention, were treated with high-energy transurethral microwave thermotherapy (HE-TUMT) (Prostatron system). Preoperative investigations included digital rectal examination, urinary free flow rate, PSA, urinalysis, urine culture, transrectal ultrasonography, urodynamic evaluation, International Prostatic Symptom Score (IPSS) and quality of life (QoL). Main selection criteria included: large prostate, high surgical risk, reluctance to undergo surgery. All patients were obstructed according to the Abrams-Griffith's nomogram. For the statistical analysis a repeated-measures, one-way ANOVA was performed on previously non-catheterized patients. RESULTS: Six of the 8 patients with catheter before treatment were able to urinate spontaneously with no significant post-voiding residual urine. In the 17 remaining patients, IPSS decreased from a mean of 18.5 at baseline to 7.30 and QoL from a mean of 3.9 to 1.2. Mean maximum flow rates during uroflowmetry increased from 8.5 to 16.9 ml/s. P(det) Q(max) decreased from a mean of 83.0 cm H(2)O at baseline to 50.7 cm H(2)O and Q(max) increased from a mean of 6.8 ml/s at baseline to 15.1 ml/s during the pressure-flow study. After TUMT, 13 patients were unobstructed and 4 equivocal according to the Abrams-Griffith's nomogram. CONCLUSION: Our study performed in a selected population of patients with BPH documents the efficiency and safety of HE-TUMT. This technique appears to be a valid therapeutic option, particularly in patients with high surgical risk.

17beta-estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases.

Conjugated estrogens shorten the prolonged bleeding time in uremic patients and are similarly effective in a rat model of uremia. We have previously demonstrated that the shortening effect of a conjugated estrogen mixture or 17beta-estradiol on bleeding time was abolished by the nitric oxide (NO) precursor L-arginine, suggesting that the effect of these drugs on hemostasis in uremia might be mediated by changes in the NO synthetic pathway. The present study investigated the biochemical mechanism(s) by which conjugated estrogens limit the excessive formation of NO. 17beta-estradiol (0.6 mg/kg), given to rats made uremic by reduction of renal mass, significantly reduced bleeding time within 24 h and completely normalized plasma concentrations of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS) catalytic activity, determined by NADPH-diaphorase staining in the thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS) immunoperoxidase staining in the endothelium of uremic aortas of untreated rats was significantly more intense than in control rats, while in uremic rats receiving 17beta-estradiol staining was comparable to controls. Thus 17beta-estradiol corrected the prolonged bleeding time of uremic rats and fully normalized the formation of NO by reducing the expression of ecNOS and iNOS in vascular endothelium. These results provide a possible biochemical explanation of the well-known effect of estrogens on primary hemostasis in uremia, in experimental animals and humans.

Renoprotection by nitric oxide donor and lisinopril in the remnant kidney model.

Previous studies showed a renoprotective effect of l-arginine in experimental uremia. Whether this was caused by an increased nitric oxide (NO) release or depended on l-arginine per se is not clear. Here, we evaluated whether chronic administration of an NO donor, molsidomine, controlled systemic blood pressure and renal disease progression and prolonged survival in rats with renal mass reduction (RMR). Rats with RMR received the following daily in the drinking water: group 1 (n = 21), no specific therapy (vehicle); group 2 (n = 12), molsidomine, 120 mg/L; group 3 (n = 9), lisinopril, 25 mg/L; and group 4 (n = 12), reserpine, 5 mg/L, hydralazine, 80 mg/L, and hydrochlorothiazide, 25 mg/L, from day 21 after surgery, when rats had hypertension and proteinuria, until the death of the vehicle-treated rats. Molsidomine normalized systemic hypertension, only partially reduced proteinuria and serum creatinine levels, but significantly prolonged animal survival, particularly in the early stage of the disease. Lisinopril at a similar systemic blood pressure was even better than molsidomine in limiting proteinuria, preserving renal function, and prolonging survival, but triple therapy, despite being effective on blood pressure, offered no renoprotection or prolonged survival. Endothelin-1 (ET-1) levels, formed in excessive amounts by the kidneys of these animals, were reduced by molsidomine and lisinopril, but not by triple therapy. The prolongation of survival by NO donor could be attributed to its effect of reducing ET levels, which in turn may limit the smooth muscle cell proliferation and matrix accumulation responsible for organ and, especially, myocardial fibrosis in uremia.

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.

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

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

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.

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.

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.

Enhanced nitric oxide synthesis in uremia: implications for platelet dysfunction and dialysis hypotension.

Nitric oxide (NO), a potent vasodilator which also inhibits platelet adhesion and aggregation, is generated by endothelial cells and platelets from its precursor L-arginine. Since N-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis, normalizes the prolonged bleeding time of uremic rats, it has been suggested that bleeding associated with uremia was due to an excessive NO formation. With the present study we sought to evaluate whether in patients with chronic renal failure--like in uremic rats--defective platelet aggregation were associated with excessive formation of NO and whether uremic plasma promotes NO synthesis by cultured vascular endothelium. Data indicated that plasma L-arginine was higher in uremics than in controls, uremic platelets generated more NO than control platelets, and intraplatelet levels of cGMP (the NO second messenger) were also higher in uremic than in control platelets. Moreover, uremic plasma potently induced NO synthesis by cultured endothelial cells, a phenomenon which was further amplified by adding to uremic plasma endotoxin and interferon gamma. Increased NO biosynthesis may contribute to platelet dysfunction and possibly other manifestations of uremic syndrome, including hemodialysis hypotension.

Urinary excretion of platelet activating factor in patients with immune-mediated glomerulonephritis.

We have investigated whether human immune-mediated glomerulonephritis is associated with changes in platelet activating factor (PAF) biosynthesis. Urinary PAF, taken as a marker of its renal synthesis, was significantly higher in patients with membranous nephropathy (N = 9) than in healthy controls (N = 8). This was not due to a lower degradation of PAF since urinary acetylhydrolase activity was comparable in patients and controls. A significant positive correlation between urinary excretion of PAF and proteinuria was observed. PAF generation was comparable in polymorphonuclear cells isolated from patients with membranous nephropathy and controls. PAF levels in blood from patients with membranous nephropathy were significantly lower than in controls, suggesting that the excessive generation of PAF is confined to the kidney. The results document that signs of renal disease activity in human membranous nephropathy are associated with an excessive renal synthesis of PAF.