Fabrizio Fop

Endothelial progenitor cell-derived microvesicles improve neovascularization in a murine model of hindlimb ischemia.

Paracrine mediators released from endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis following ischemia. Recently, we demonstrated that microvesicles (MVs) derived from EPCs are able to activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of RNA. In this study we aim to investigate whether EPC-derived MVs are able to induce neoangiogenesis and to enhance recovery in a murine model of hindlimb ischemia. Hindlimb ischemia was induced in severe combined immunodeficient (SCID) mice by ligation and resection of the left femoral artery and mice were treated with EPC-derived MVs (MVs), RNase-inactivated MVs (RnaseMVs), fibroblast-derived MVs or vehicle alone as control (CTL). Since MVs contained the angiogenic miR-126 and miR-296, we evaluated whether microRNAs may account for the angiogenic activities by treating mice with MVs obtained from DICER-knock-down EPC (DICER-MVs). The limb perfusion evaluated by laserdoppler analysis demonstrated that MVs significantly enhanced perfusion in respect to CTL (0.50±0.08 vs 0.39±0.03, p < 0.05). After 7 days, immunohistochemical analyses on the gastrocnemius muscle of the ischemic hindlimb showed that MVs but not fibroblast-MVs significantly increased the capillary density in respect to CTL (MVs vs CTL: 24.7±10.3 vs 13.5±6, p < 0.0001) and (fibroblast-MVs vs CTL: 10.2±3.4 vs 13.5±6, ns); RNaseMVs and DICER-MVs significantly reduced the effect of MVs (RNaseMVs vs CTL: 15.7±4.1 vs 13.5±6, ns) (MVs vs DICER-MVs 24.7±10.3 vs 18.1±5.8, p < 0.05), suggesting a role of RNAs shuttled by MVs. Morphometric analysis confirmed that MVs enhanced limb perfusion and reduced injury. The results of the present study indicate that treatment with EPC-derived MVs improves neovascularization and favors regeneration in severe hindlimb ischemia induced in SCID mice. This suggests a possible use of EPCs-derived MVs for treatment of peripheral arterial disease.

Bacterial- and fungal-positive cultures in organ donors: Clinical impact in liver transplantation

Infection transmission from donor to recipient is a dreadful complication in transplantation. Although bacteremia was previously detected in 5% of donors without negative impact on recipient outcome, the current expansion of graft pool requires consideration of the infectious risk associated with suboptimal donors. This study aims to evaluate the incidence and risk factors of infection in unselected cadaveric liver donors, the occurrence of microorganism transmission to recipient and its influence on patient survival. Results of microbiologic cultures obtained before harvesting in intensive care unit (ICU) and routinely at harvesting from 610 consecutive liver donors were retrospectively analyzed. Evidence for bacterial and fungal transmission to the recipient was searched for in each culture‐positive donor. One or more cultures were positive in 293 donors (48%), while bacteremia was present in 128 (21%). Culture‐positive and bacteremic donors were of significantly older age and had longer ICU stays. At multivariate analysis, an ICU stay of 3 or more days was the only significant predictor of donor infection. Although 1‐year patient/graft survival rates were not influenced by donor culture positivity, pathogen transmission occurred in 11 cases with high recipient 1‐year mortality (45%). In those 11 cases, median donor age was 74 years, significantly much older than that of the other culture‐positive donors. In conclusion, donors with a prolonged ICU stay are at increased risk of infection, while older donor age is associated with pathogen transmission to the recipient. Adequate donor maintenance and careful microbiologic surveillance and treatment, especially of elderly donors, may limit transmission of donor infection. Liver Transpl 12:1253‐1259, 2006.

Correlation between Cytomegalovirus Infection and Raynaud’s Phenomenon in Lupus nephritis

Relationships between viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) are still elusive. Recent reports demonstrated the association of some viral infections with peculiar clinical events in the general population, such as cytomegalovirus (CMV) with arterial damage and Parvovirus B19 (PV-B19) with hematologic abnormalities. We planned to look for this kind of viral imprinting in SLE, hypothesizing that traces of specific features of some viral infections might be found in some subsets of seropositive SLE patients. In 60 SLE patients recruited at our nephrologic center, serology for CMV, PV-B19, Epstein-Barr virus viral capsid antigen (EBV-VCA), Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr virus early antigen (EBV-EA) was performed. χ2 and ANOVA were employed to compare the frequency and titers of antiviral antibodies in SLE patients with groups of transplant, hemodialysis and blood donor subjects. χ2, Fisher’s test, Bonferroni and Scheffe’s test were employed to compare the different biochemical/clinical features between seropositive and seronegative SLE patients. Univariate and multivariate analysis (logistic regression models) were employed to evaluate the odds ratio (OR) of different risk factors for vascular events (including Raynaud’s phenomenon, deep venous thrombosis) and hematologic abnormalities (including severe anemia, leukopenia and thrombocytopenia). Anti-CMV (82%), anti-PV-B19 (60%), anti-EBV-VCA (92%) and EBV-EA (45%) IgG antibodies were frequent in SLE, with higher prevalence in comparison with the blood donor group and higher titers in comparison with transplant and hemodialysis groups. CMV seropositivity was a highly significant risk factor for Raynaud’s phenomenon (OR +α in univariate and multivariate analysis = 13.51 using a correction of 0.5 in case of a zero event), but not for venous vascular events (OR = 1.31). An increased though not significant risk factor was found for antiphospholipid antibodies (OR = 2.71, p = 0.19), while the presence of nephrotic syndrome during the follow-up was a significant protective factor (OR = 0.15, p = 0.035). There was no significantly increased OR for PV-B19 seropositivity in cases with severe anemia (OR = 2.09, p = 0.29). No significant associations were found with the status of EBV reactivation. In conclusion, our results support the hypothesis that viral infection may imprint the course of SLE leading to specific clinical subsets (i.e. CMV and ‘vascular’ SLE, with more frequent Raynaud’s phenomenon and a less frequent typical histological renal picture responsible for nephrotic syndrome). Further prospective studies are justified to validate these correlations, mainly dealing with associations between acute viral infections and vascular events, thus eventually leading to a better understanding of mutual relationships between viruses and SLE.

Angiotensin I-converting enzyme genotype significantly affects progression of IgA glomerulonephritis in an italian population.

To evaluate the role of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the progression of immunoglobulin A glomerulonephritis (IgA-GN), genotype distribution in 81 biopsy-proven cases of IgA-GN was studied. A logistic regression model showed that the risk for homozygous DD was not significantly elevated in patients with IgA-GN compared with healthy subjects (odds ratio = 1.16; confidence interval [CI], 0.4 to 3.3). However, the 5-year (78% v 90%) and 10-year (52% v 82%) renal survival rates for 47 patients with serum creatine (Cr) levels of 1.5 mg/dL or less at biopsy was significantly less in DD patients (n = 18; chi2 = 5.41; P = 0.02). The hazard ratio (HR) for DD (multivariate analysis from Cox proportional model after adjustment for known factors of progression, such as hypertension [HPT] and proteinuria [PTO]) was 3.07 (CI, 1.1 to 9.4). The HR for heavy PTO was 6.1 (CI, 1.9 to 19). The association of DD genotype with progression was even more striking when patients with other risk factors (heavy proteinuria) were excluded, as shown by DD-related risk in the absence (HR = 3.6; CI, 1.1 to 12) and presence (HR = 2; CI, 0.4 to 10) of PTO. The risk ratio was further increased by the coexistence of DD + PTO (HR = 9.16; CI, 1.8 to 15.7). Furthermore, in a cross-sectional study among patients with IgA-GN, a logistic regression model showed that the risk for homozygous DD was greater, although not at a statistically significant level in the end-stage renal failure subgroup compared with the normal renal function subgroup (odds ratio = 3.16; CI, 0.7 to 13.7) after adjustment by sex, age at biopsy, HPT, PTO, and therapy. Last, DD was significantly more frequent in those patients who started hemodialysis at an earlier age (chi2 for trend = 6.81; P = 0.009). Our study further supports that ACE genotype is a risk factor not for the development, but for the worsening of IgA-GN clinical course. However, on the basis of current knowledge, we cannot exclude that I/D polymorphism may simply serve as a prognostic marker, eventually linked with other discrete loci involved in the progression of renal damage.

Loss of nephrin expression in glomeruli of kidney-transplanted patients under m-TOR inhibitor therapy.

The development of proteinuria has been observed in kidney‐transplanted patients on m‐TOR inhibitor (m‐TORi) treatment. Recent studies suggest that m‐TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney‐transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit‐diaphragm. In a group of patients on ‘de novo’ m‐TORi‐treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m‐TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m‐TORi‐treatment, a protocol biopsy performed before introduction of m‐TORi was also available. The expression of nephrin in the pre‐m‐TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m‐TORi(s). Proteinuria increased after the m‐TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m‐TORi(s) may affect nephrin expression in kidney‐transplanted patients, consistently with the observation in vitro on cultured podocytes.

Urinary CD133+ extracellular vesicles are decreased in kidney transplanted patients with slow graft function and vascular damage.

Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133+ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133+ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133+ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133+ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133+ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.

Relationship among C1q-fixing de novo donor specific antibodies, C4d deposition and renal outcome in transplant glomerulopathy.

BACKGROUND The C1q-binding properties of donor specific antibodies (DSA) may be related to antibody-mediated rejection and poor outcome. METHODS We retrospectively studied 35 kidney transplant recipients with transplant glomerulopathy (TG) and de novo DSA (dnDSA). C1q dnDSA were measured in the serum stored at renal biopsy and the association among C1q-fixing dnDSA, C4d deposition and graft loss was examined. RESULTS Of the 35 patients with dnDSA and TG, 15 (42.9%) had C1q-positive dnDSA and 20 (57.1%) had C1q-negative dnDSA. Ten out of 15 patients with C1q-positive dnDSA (66.6%) and 5 with C1q-negative dnDSA (25%) had C4d positive staining renal biopsies (P=0.02), being the C1q-negative dnDSA/C4d-negative TG 42.9% of the total. The C1q-positive dnDSA group has significantly higher IgG DSA Class II MFI than the C1q-negative dnDSA group (P=0.004). Patients with C4d deposits have significantly higher IgG DSA MFI for both Class I and Class II than those without C4d deposits (P=0.02). We found a trend toward higher graft loss in the C1q-positive dnDSA group (60%) versus the C1q-negative dnDSA group (40%) without a statistical significance (P=0.31). CONCLUSION Our study provides further characterization of TG associated with dnDSA. The major part of dnDSA-associated TG was C1q-negative and the presence of C1q-fixing dnDSA did not significantly correlate with graft outcome.

Comparison between 24‐h proteinuria, urinary protein/creatinine ratio and dipstick test in patients with nephropathy: Patterns of proteinuria in dipstick‐negative patients

Objective. Three main tests are commonly employed for the measurement of proteinuria: the dipstick test, the urinary protein/creatinine ratio (P/C) and the 24‐h urine collection. The aim of this study was to evaluate the correlation between these methods, comparing linear regression and ROC curve data. Material and methods. A total of 297 consecutive outpatients with different renal diseases were included in the study. Twenty‐four‐hour proteinuria was considered the reference test. Results. A high degree of correlation was observed between all the tests (p<0.0001), the highest regression coefficient being between 24‐h proteinuria and P/C (R = 0.82), and the lowest between P/C and the dipstick test (R = 0.72). The dipstick test failed to detect pathological proteinuria in 94 patients (31.6 %). Therefore, in these subjects, the patterns of proteinuria were assessed by immunofixation and sodium dodecyl sulphate (SDS) electrophoresis. Conclusions. Our data strongly support the use of urinary P/C for the detection of proteinuria, at least in nephrology units, where the prevalence of proteinuria is likely to be high.

Evaluation of CMV-specific cellular immune response by EliSPOT assay in kidney transplant patients.

BACKGROUND Immunological monitoring for CMV can be useful in transplant patients; however, few centers perform it on a routine basis. OBJECTIVES In this study, CMV-specific cellular response was evaluated in a population of kidney transplant recipients and related to viral infection/reactivation and other demographic and clinical features. STUDY DESIGN Three hundred and twenty-eight patients were studied by EliSPOT assay: 201 prospectively monitored in the first year posttransplantation, 127 with a single determination at >1 year. Clinical features, including occurrence of CMV-DNAemia, CMV serostatus, anti-viral strategies and immunosuppressive protocols, were evaluated. RESULTS Overall, 66.5% of patients were CMV-responders at EliSPOT assay. No episode of infection occurred at follow-up (mean 24.5 months) in 73.4% responders versus 55.5% non-responders (p<0.005); CMV-free period was significantly longer in responders (p<0.001). Although no significant difference of peak viral load was found, prevalence of CMV-DNAemia values >10(5)copies/mL was significantly higher in non-responders versus responders (8.2% and 2.3%, p<0.05). Non-responder status was significantly associated to CMV-seronegativity (p<0.0001), anti-viral prophylaxis use (p<0.0001), and immunosuppression induction with basiliximab (p<0.005). No significant association was found for other clinical features and immunosuppressive protocols. CONCLUSIONS Immunological data for CMV could be used in the clinical evaluation and decision-making process, in combination with virological monitoring, in kidney transplant recipients.

Assessment of cardiovascular risk in waiting-listed renal transplant patients: a single center experience in 558 cases

Abstract:  Cardiac screening is recommended to prevent cardiovascular death after renal transplantation. This retrospective observational study illustrates the results of application of a cardiac assessment algorithm in a series of 558 renal transplant candidates at a single center in Turin, Italy. A dipyridamole‐stress sestamibi myocardial scintiscan (DMS) performed in 302/558 (54.1%) cases was positive in 52 (17.2%), negative in 200 (66.2%), borderline in 16 (5.3%), and with signs of previous necrosis in 34 (11.4%). Coronary lesions detected by angiography in 48.1% of the 52 positives were treated medically (13.5%) or by percutaneous/surgical procedure (34.6%). Coronary lesions were detected in 14.1% of asymptomatic population subgroup. The minor and major cardiovascular event rates and the cardiovascular death rate were 1.9%, 0%, and 0%, respectively, in positive DMS group (high‐cardiological risk) vs. 10%, 4.5%, and 3.5% in the negatives (p > 0.5; n.s.). It is suggested that not increased cardiovascular event or deaths rates in the high‐risk group reflect early coronary lesion detection and correction. Since 55.9% of cardiovascular events or deaths occurred in the negative group more than 24 months after the DMS, its mandatory repetition every two yr after a negative finding is recommended.