Luca Giovannini

C-Reactive Protein and Interleukin-6 Levels Are Related to Renal Function in Predialytic Chronic Renal Failure

Background: Several studies have provided convincing evidence that in apparently healthy subjects elevated serum levels of plasma C-reactive protein (CRP) are associated with an increased risk of experiencing myocardial infarction and sudden cardiac death. It has been claimed that, in dialytic patients, the hepatic synthesis of this ‘acute phase response’ plasma protein is primarily induced by the macrophage-derived interleukin 6 (IL-6). Little information is available, however, regarding CRP and IL-6 plasma levels in pre-dialytic renal failure. Methods: Plasma CRP by a modification of the laser nephelometry technique, IL-6 and serum albumin were determined in 103 chronic pre-dialytic patients (mean age 50 ± 6.3 years; creatinine clearance (Cr.cl.) 36.3 ± 23.1 ml/min). Results: CRP was >5 mg/l (normal upper range) in 42% of the global population. CRP and IL-6 were significantly related (r = 0.35, p < 0.0004). CRP and IL-6 were related to renal function (CRP vs. Cr.cl., r = –0.56, p < 0.0001; IL-6 vs. Cr.cl., r = –0.55, p < 0.0001, Spearman correlation coefficient). When patients were divided in tertiles according to renal function, CRP median value resulted 7.9 mg/l (interquartile interval: 5–12) in the first tertile (Cr.cl. <18.5 ml/min), 4.0 mg/l (3–6) in the second tertile (Cr.cl. 18.5–45 ml/min) and 3.2 mg/l (2.7–4.0) in the last tertile (Cr.cl. >45 ml/min) (p < 0.0001). A negative correlation between CRP and S-albumin was also found (r = –0.52, p < 0.0001, Spearman correlation coefficient). Conclusions: IL-6 and CRP were increased and were inversely related to creatinine clearance in our population of 103 chronic predialytic patients. The possibility of a decreased renal clearance of CRP and/or cytokines as a cause of an activated acute-phase response is discussed. A negative correlation between CRP and S-albumin was found confirming the link between chronic inflammation and malnutrition in chronic renal patients.

C REACTIVE PROTEIN IN PATIENTS WITH CHRONIC RENAL DISEASES

Base-line serum levels of plasma C-reactive protein (CRP) are predictive of future myocardial infarction and sudden cardiac death in apparently healthy subjects, suggesting the hypothesis that chronic inflammation might be important in the pathogenesis of atherothrombosis. CRP production is mediated by several inflammatory mediators: interleukin 6 (IL-6) is currently felt to be the major cytokine influencing the acute phase response. CRP and other acute phase proteins are elevated in dialysis patients and cardiovascular diseases represent the single largest cause of mortality in chronic renal failure patients. Little information is available, however regarding CRP and IL-6 plasma levels in pre-dialysis renal failure. Plasma CRP was determined by a modification of the laser nephelometry technique; IL-6 by immunoassay (RD System); and fibrinogen, serum albumin, cholesterol, triglycerides, hematocrit, white blood cell count, erythrocytic sedimentation rate (ESR) and urinary protein levels by standard laboratory techniques. Results were obtained in 102 chronic pre-dialysis patients whose mean age was 53 ± 5.8 years with a mean creatinine clearance (CCr) of 52 ± 37 mL/min). CRP was greater than 5 mg/L in 25% of the global population. CRP and IL-6 were 4.0 ± 4.6 mg/L and 5.8 ± 5.6 pg/mL, respectively and were not significantly correlated (r = 0.11, p = n.s.). CRP and IL-6 were however related with renal function (CRP versus CCr r = −0.40 p < 0.001; IL-6 versus CCr r = −0.45; p < 0.001). When patients were divided in two groups according to renal function, CRP resulted 7.4 ± 6.3 mg/L in the group of patients with a CCr lower than 20 mL/min (n = 32) and 2.76 ± 4.35 in the group of patients with a CCr higher than 20mL/min (n = 70) (p < 0.0001). CRP and IL-6 were positively related with ESR (r = 0.32 and 0.46 respectively). Serum albumin levels were not significantly different in the two groups of patients (3.2 ± 0.4 versus 3.0 ± 0.5 g/dL). CRP and serum albumin were not significantly related (r = 0.17). CRP and IL-6 correlated positively with ESR (r = 0.32 and 0.46 respectively). In pre-dialysis patients we have demonstrated an increase in both CRP and IL-6 that occurs as renal function decreases. These data provided evidence of the activation – even in the predialysis phase of renal failure – of mechanisms known to contribute to the enhanced cardiovascular morbidity and mortality of the uremic syndrome.

Oxidative Stress and Inflammatory Reaction Modulation by White Wine

Abstract: Wine and olive oil, essential components of the Mediterranean diet, are considered important factors for a healthy life style. Tyrosol (T) and caffeic acid (CA) are found in both extra virgin olive oil and in white wine. Three white wines from the northeast Italy and four white wines from Germany were analyzed for their content of T and CA. These compounds were tested for their antioxidant activity and their capacity to modulate three different cytokines: IL‐1β, IL‐6, and TNF‐α, which are currently considered to be the major cytokines influencing the acute phase of the inflammatory response. Furthermore, the antioxidant activity of T and CA was analyzed by monitoring the oxidation of a redox‐sensitive probe by using laser scanning confocal microscopy. T and CA, applied at nanomolar range, were found to significantly reduce the generation of oxidants induced by azobis‐amidinopropanedihydrochloride. Peripheral blood mononuclear cells (PBMC) from healthy volunteers were incubated at 37°C for 12 hours with 100 ng LPS (E. coli and P. maltofilia). Increasing doses of T and CA (150 nM to 300 μM) were added and cell‐associated IL‐1β and TNF‐α were determined by immunoreactive tests after three freeze‐thaw cycles. IL‐6 release was also determined in cell surnatants. LPS‐stimulated PBMC showed a significant increase in cytokine release, while T and CA, used at nanomolar concentrations, were able to modulate their expression. Taken together, these results suggest a remarkable effect of white wine non‐alcoholic compounds on oxidative stress and inflammatory reaction.

C-Reactive Protein as a Marker of Chronic Inflammation in Uremic Patients

Cardiovascular complications caused by an accelerated atherosclerotic disease represent the largest single cause of mortality in chronic renal failure patients. The rapidly developing atherosclerosis of the uremic syndrome appears to be caused by a synergism of different mechanisms, such as malnutrition, oxidative stress and genetic factors. Recent studies provide evidence that chronic inflammation plays an important role in the pathogenesis of cardiovascular diseases. Elevated serum levels of plasma C-reactive protein (CRP) are associated with an increased risk of experiencing myocardial infarction and sudden cardiac death in apparently healthy subjects. Several recently published papers have confirmed this strong association between CRP and the extent and severity of the atherosclerotic processes. In patients affected by predialytic renal failure, increased levels of CRP and interleukin (IL)-6 were recorded in 25% of our population; CRP and IL-6 were inversely related with renal function. These data suggest the activation – even in the predialytic phase of renal failure – of mechanisms known to contribute to the enhanced cardiovascular morbidity and mortality of the uremic syndrome. In recent years we have investigated the hypothesis that the chronic inflammatory state of the uremic patient could at least in part be due to the dialytic technique. We provide evidence suggesting that the increase of CRP in stable dialytic patients may be due to the stimulation of monocyte/macrophage by backfiltration of dialysate contaminants.

Rescue of retinal function by BDNF in a mouse model of glaucoma.

Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs) and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP) elevation that mimics primary open-angle glaucoma (POAG). IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG) and visual cortical evoked potentials (VEP). RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl); the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks) and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks) were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension.

Melatonin prevents cyclosporine-induced nephrotoxicity in isolated and perfused rat kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 m +N O 3 m were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.

Endothelial progenitor cell-derived extracellular vesicles protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis

BACKGROUND Endothelial progenitor cells (EPCs) are known to induce tissue repair by paracrine mechanisms including the release of growth factors and extracellular vesicles (EVs), nanoparticles able to carry proteins and genetic information to target cells. The aim of this study was to evaluate whether EVs derived from EPCs may protect from complement-mediated mesangial injury in experimental anti-Thy1.1 glomerulonephritis. METHODS EVs were isolated by serial ultracentrifugation from supernatants of cultured human EPCs and characterized for their protein and RNA content. In vivo, EVs were injected i.v. in the experimental rat model of mesangiolytic anti-Thy1.1 glomerulonephritis evaluating renal function, proteinuria, complement activity and histological lesions. In vitro, the biological effects of EPC-derived EVs were studied in cultured rat mesangial cells incubated with anti-Thy1.1 antibody and rat or human serum as complement source. RESULTS After i.v. injection in Thy1.1-treated rats, EVs localized within injured glomeruli and inhibited mesangial cell activation, leucocyte infiltration and apoptosis, decreased proteinuria, increased serum complement haemolytic activity (CH50) and ameliorated renal function. EV treatment decreased intraglomerular deposition of the membrane attack complex (MAC or C5b-9) and expression of smooth muscle cell actin and preserved the endothelial antigen RECA-1 and the podocyte marker synaptopodin. The protective effect of EVs was significantly reduced by pre-treatment with a high dose of RNase (1 U/mL), suggesting a key role for EV-carried RNAs in these mechanisms. Indeed, EPC-derived EVs contained different mRNAs coding for several anti-apoptotic molecules and for the complement inhibitors Factor H, CD55 and CD59 and the related proteins. The in vitro experiments aimed to investigate the mechanisms of EV protection indicated that EVs transferred to mesangial cell mRNAs coding for Factor H, CD55 and CD59 and inhibited anti-Thy1.1 antibody/complement-induced apoptosis and C5b-9/C3 mesangial cell deposition. CONCLUSIONS EVs derived from EPCs exert a protective effect in Thy1.1 glomerulonephritis by inhibition of antibody- and complement-mediated injury of mesangial cells.

Caffeic acid, a phenol found in white wine, modulates endothelial nitric oxide production and protects from oxidative stress-associated endothelial cell injury.

Introduction Several studies demonstrated that endothelium dependent vasodilatation is impaired in cardiovascular and chronic kidney diseases because of oxidant stress-induced nitric oxide availability reduction. The Mediterranean diet, which is characterized by food containing phenols, was correlated with a reduced incidence of cardiovascular diseases and delayed progression toward end stage chronic renal failure. Previous studies demonstrated that both red and white wine exert cardioprotective effects. In particular, wine contains Caffeic acid (CAF), an active component with known antioxidant activities. Aim of the study The aim of the present study was to investigate the protective effect of low doses of CAF on oxidative stress-induced endothelial injury. Results CAF increased basal as well as acetylcholine—induced NO release by a mechanism independent from eNOS expression and phosphorylation. In addition, low doses of CAF (100 nM and 1 μM) increased proliferation and angiogenesis and inhibited leukocyte adhesion and endothelial cell apoptosis induced by hypoxia or by the uremic toxins ADMA, p-cresyl sulfate and indoxyl sulfate. The biological effects exerted by CAF on endothelial cells may be at least in part ascribed to modulation of NO release and by decreased ROS production. In an experimental model of kidney ischemia-reperfusion injury in mice, CAF significantly decreased tubular cell apoptosis, intraluminal cast deposition and leukocyte infiltration. Conclusion The results of the present study suggest that CAF, at very low dosages similar to those observed after moderate white wine consumption, may exert a protective effect on endothelial cell function by modulating NO release independently from eNOS expression and phosphorylation. CAF-induced NO modulation may limit cardiovascular and kidney disease progression associated with oxidative stress-mediated endothelial injury.

Nitric Oxide–Dependent Renal Vasodilatation Is Not Altered in Rat with rHuEpo–Induced Hypertension

Background: Recombinant human erythropoietin (rHuEpo) is the treatment of choice in anemia associated with end–stage renal disease. Its major side effect is hypertension, which occurs in 8–30% of uremic patients. The exact mechanism of rHuEpo–induced hypertension has not been fully elucidated, and several possibilities have been proposed, such as a direct vascular effect of the drug with a shift in the balance of constrictor and relaxing endothelial factors (endothelins and nitric oxide (NO)). Recent papers suggested an enhanced rather than reduced activity of endogenous NO system in rats with normal renal function and rHuEpo–induced hypertension. Our study was designed to verify whether, in spite of enhanced activity of the renal NO system, rHuEpo may affect endothelium–dependent (acetylcholine–induced) and/or endothelium–independent (sodium nitroprusside–induced) vasorelaxation and to evaluate basal NO release by the infusion of NG–nitro–L–arginine methyl ester (L–NAME) in an isolated and perfused rat kidney model. Methods: To investigate this hypothesis, we have determined systemic and renal NO activity in Wistar rats treated with a hypertensive dose of rHuEpo (150 IU/kg b.w. every other day for 2 weeks) by measuring stable NO metabolites (NO2+NO3) in the urine and have also evaluated variations in renal vascular resistance after the injection of Ach, SNP and the infusion of L–NAME. Results: Hematocrit, hemoglobin concentration and arterial blood pressure were significantly increased in the treated group as compared with the controls. Urinary excretion of NO2+NO3 was significantly higher in treated than in the controls (438±66 vs. 294±36 nM/ml/min, p<0.01, respectively). There were no significant differences in the dose–response curves to Ach and SNP between the two groups. The renal vasoconstriction following the infusion of L–NAME was also similar in the two groups. Conclusions: The analysis of our results seems to indicate that the endogenous NO system activity was enhanced in rHuEpo–induced hypertension in rats with normal renal function and a resistance to NO was not developed in renal circulation. Further studies seem to be necessary to better clarify the exact mechanisms underlying the development of rHuEpo–induced hypertension.

Immunomodulatory activity of shikimic acid and quercitin in comparison with oseltamivir (Tamiflu) in an in vitro model.

The risk of an avian influenza pandemic has put oseltamivir (Tamiflu®) in the spotlight and has given rise to rumors that shikimic acid (SK), which is used for the synthesis of Tamiflu®, possesses therapeutic activity. This study was undertaken to determine whether SK, either alone or in combination with quercitin (QT) is able to modulate the release of IL‐6 and IL‐8 from peripheral blood mononuclear cells (PBMCs). The experiments were conducted comparing the properties of SK, both alone and in combination, with those of Tamiflu®. The incubation of PBMCs with 100 nM Tamiflu® or SK at two concentrations (10 nM; 100 nM) did not produce any change in IL‐6 and IL‐8 baseline levels (data expressed as incremental change vs. baseline). On the contrary, incubation with SK and QT at both concentrations (10 and 100 nM) produced a significant increase in the release of IL‐8 as compared to other groups (4.19 ± 0.82, SK‐QT 10 nM; 3.83 ± 1.17 SK‐QT 100 nM, P < 0.05 vs. baseline 1.00 ± 0.10, Tamiflu® 100 nM 1.35 ± 0.16, SK 10 nM 1.68 ± 0.15 and SK 100 nM 1.80 ± 0.48). The SK‐QT combination also proved to be effective in the upregulation of IL‐6 (3.08 ± 0.46, SK‐QT 10 nM; 3.60 ± 0.74 SK‐QT 100 nM, P < 0.05 vs. baseline 1.00 ± 0.26). According to these findings SK alone is not able to modulate innate immunity in antiviral terms. However, the data show that the SK + QT combination, even at low doses, may be effective for the modulation of innate immunity. J. Med. Virol. 80:741–745, 2008.