Identificazione di profili di rischio cardiovascolare nel trapianto di rene: polimorfismi di geni coinvolti nei processi di infiammazione e di apoptosi

Introduction. Cardiovascular disease (CVD) represents the main cause of morbidity and mortality in kidney recipients. This study was undertaken to assess the impact of functional polymorphisms located in cytokine and apoptosis genes on CVD after kidney transplantation. Cytokine polymorphisms, generally located in gene regulatory regions, are associated with high and low cytokine production and are likely to modulate the magnitude of inflammatory responses following transplantation, depending on the balance between the levels of pro-inflammatory and antiinflammatory cytokines. The role of apoptosis in atherosclerosis has not been completely elucidated, and here we explored the hypothesis that the heterogeneity in cardiovascular risk in kidney recipients may also be linked to functional polymorphisms involved in apoptosis induction. Purpose. In the search for relevant genetic markers of predisposition to CVD after renal transplant, the present investigation was undertaken to identify the clinical impact of polymorphisms of cytokines TNF-α, TGF-β, IL-10, IL-6, IFN-γ and IL-8 and of apoptosis genes Fas and Caspase 9 in a population of kidney transplant recipients. Materials and methods. The study involved 167 patients who received cadaveric kidney transplantation at our centre between 1997 and 2005 (minimum follow-up of 12 months); 35 of them had experienced cardiovascular events (CVD group) and 132 had no cardiovascular complications (non-CVD group). Genotyping was performed using RFLP (Restriction Fragment Length Polymorphism) for RFLP per IL-8/T-251A, Fas/G-670A e Casp9/R221Q polymorphism and SSP (Sequence Specific Primer) for TNF-α/G-308A, TGF-β/L10P, TGF-β/R25P, IL-10/G-1082A, IL- 10/C-819T, IL-10/C-592A, IL-6/G-174C, IFN-γ/T+874A polymorphisms.Results. We found a significant difference in TNF-α and IL-10 genotype frequencies between the patients who had suffered cardiovascular events and those with no CVD history. The high producer genotype for proflogistic cytokine TNF-α appeared to have a significantly superior prevalence in the CVD group compared to the non-CVD group (40.0% vs 21.2%) and it resulted in a 2.4-fold increased cardiovascular risk (OR=2.361; p=0.0289). On the other hand, the high producer genotype for the antiinflammatory cytokine IL-10 was found in 2.8% of the CVD group and in 16.7% of non-CVD group; logistic regression showed a 0.3-fold reduced risk of CVD associated with genetically determined high IL-10 production (OR=0.278; p<0.0001). The other polymorphisms did not prove to have any impact on CVD. Conclusions. TNF-α and IL-10 gene polymorphisms might represent cardiovascular risk markers in renal transplant recipients.

Effetti dell'acido urico sulle cellule glomerulari mesangiali: meccanismi intracellulari di trasduzione del segnale e possibili implicazioni nella progressione del danno renale e nella sindrome infiammatoria in corso di nefropatie croniche

Uric acid is a major inducer of inflammation in renal interstitium and may play a role in the progression of renal damage in hyperuricemic subjects with primary nephropathies, renal vascular disease, and essential hypertension. At the same time, UA also acts as a water-soluble scavenger of reactive oxygen species. We evaluated the cellular effects of UA on cultured HMC as a potential interstitial target for abnormally elevated levels in acute and chronic renal disease. Intracellular free Ca2+ ([Ca2+]i) was monitored by microfluorometry of fura 2-loaded cells, while oxidation of intracellularly trapped non-fluorescent 2’,7’-dichlorofluorescein diacetate (DCFHDA, 20 uM) was employed to assess the generation of reactive oxygen species during 12-hr incubations with various concentrations of UA or monosodium urate. Fluorescent metabolites of DCFH-DA in the culture media of HMC were detected at 485/530 nm excitation/emission wavelengths, respectively. UA dose-dependently lowered resting [Ca2+]i (from 102±9 nM to 95±3, 57±2, 48±6 nM at 1-100 uM UA, respectively, p <0.05), leaving responses to vasoconstrictors such as angiotensin II unaffected. The effect was not due to Ca2+/H+ exchange upon acidification of the bathing media, as acetate, glutamate, lactate and other organic acids rather increased [Ca2+]i (to max. levels of 497±42 nM with 0.1 mM acetate). The decrease of [Ca2+]i was abolished by raising extracellular Ca2+ and not due to effects on Ca2+ channels or activation of Ca2+-ATPases, since unaffected by thapsigargin. The process rather appeared sensitive to removal of extracellular Na+ in combination with blockers of Na+/Ca2+ exchange, such as 2’,4’-dichlorobenzamil, pointing to a countertransport mechanism. UA dose-dependently prompted the extracellular release of oxidised DCFH (control 37±2 relative fluorescence units (RFU)/ml, 0.1uM 47±2, 1 uM 48±2, 10 uM 51±4, 0.1 mM 53±4; positive control, 10 uM sodium nitroprusside 92±5 RFU/ml, p<0.01). In summary, UA interferes with Ca2+ transport in cultured HMC, triggering oxidative stress which may initiate a sequence of events leading to interstitial injury and possibly amplifying renal vascular damage and/or the progression of chronic disease.

Ruolo delle cellule staminali mesenchimali in modelli cellulari e animali di nefropatia

Background. Mesenchymal stem cells (MSC) may be of value in regeneration of renal tissue after damage, however lack of biological knowledge and variability of results in animal models limit their utilization. Methods. We studied the effects of MSC on podocytes ‘in vitro’ and ‘in vivo’ utilizing adriamycin (ADR) as a model of renal toxicity. The ‘in vivo’ experimental approach was carried out in male Sprague Dawley rats (overall 60 animals) treated with different ADR schemes to induce acute and chronic nephrosis. MSC were given a) concomitantly to ADR in tail vein or b) in aorta and c) in tail vein 60 days after ADR. Homing was assessed with PKH26-MSC. Results. MSC rescued podocytes from apoptosis induced by ADR ‘in vitro’. The maximal effect (80% rescue) was obtained with MSC/Podocytes co-culture ratio of 1:1 for 72 hours. All rats treated with ADR developed nephrosis. In no case MSC modified the clinical parameters (i.e. proteinuria, serum creatinine, lipids) but protected the kidney from severe glomerulosclerosis when given concomitantly to ADR. Rats given MSC 60 days after ADR developed the same severe renal damage. Only few MSC were found in renal tubule-interstitial areas after 1-24 hours from injection and no MSC was detected in glomeruli. Conclusions. MSC reduced apoptosis of podocytes treated with ADR ‘in vitro’. Early and repeated MSC infusion blunted glomerular damage in chronic ADR nephropathy. MSC did not modify proteinuria and progression to renal failure, that implies lack of regenerative potential in this model.

ATG based combination therapy - a novel clinically relevant approach to promote regulation and induce long-term allograft survival

Regulatory T cells (Treg) actively regulate alloimmune responses and promote transplantation tolerance. Polyclonal anti-thymocyte globulin (ATG), a widely used induction therapy in clinical organ transplantation, depletes peripheral T cells. However, resistance to tolerance induction is seen with certain T cell depleting strategies and is attributed to alterations in the balance of naïve, memory and regulatory T cells. Here we report a novel reagent, murine ATG (mATG), depletes T cells but preferentially spares CD25+ natural Tregs which limit skewing of T cell repertoire toward T-effector-memory (Tem) phenotype among the recovering T cells. T-cell depletion with mATG combined with CTLA4Ig and Sirolimus synergize to prolong graft survival by tipping the Treg/Tem balance further in favor of Tregs by preserving Tregs, facilitating generation of new Tregs by a conversion mechanism and limiting Tem expansion in response to alloantigen and homeostatic proliferation. These results provide the rationale for translating such novel combination therapies to promote tolerance in primate and human organ transplantation.

Early Referral e prevenzione secondaria dell'insufficienza renale cronica benefici per il paziente e per la sostenibilità del sistema dialisi

Attività di ricerca nell'ambito dell'inidirizzo nefrologia clinica, valutando: la popolazione dei pazienti in trattamento sostitutivo in particolare incidenza e prevalenza dei pazienti in trattamento renale sostitutivo nell'Azienda USL di Cesena dal 2000 al 2009 confronto con i dati del registro italiano (analisi retrospettiva). Analisi di questa popolazione e in particolare referral nefrologico e applicazione di misure atte a ridurre la progressione dell'IRC (prevenzione secondaria, terziaria e riduzione dei costi). Confronto con i dati internazionali.

Studio prospettico sul significato clinico e prognostico delle infezioni da virus BK e virus JC nel trapianto di rene

Le infezioni post trapianto sono una complicanza frequente, tra queste l'infezione da Polyomavirus rappresenta una patologia molto frequente e causa di perdita del graft. Sono stati studiati 70 pazienti portatori di trapianto renale per valutare l'incidenza dell'infezione e la sua prognosi e la risposta alla terapia

Studio di sensibilità e specificità dei markers di acute kidney injury, per la diagnosi precoce delle complicanze nell'immediato decorso post trapianto renale

NGAL è considerato dalla comunità scientifica un marcatore di danno renale sia di tipo ischemico che tossico. In questo studio è stato effettuato un follow-up ad un mese di una popolazione sottoposta a trapianto di rene, sono state analizzate la fase post-operatoria e l’immediato periodo di stabilizzazione del trapianto. E' stato osservato l’andamento di NGAL in relazione alle principali variabili incidenti.

La Restless Legs Syndrome aumenta il rischio cardiovascolare e la mortalità a breve termine nei pazienti emodializzati

Abstract Restless legs syndrome (RLS) is a sensori-motor neurological disorder characterzed by paraesthesia, dysaesthesia and irresistibile urge to move the legs especially at night. Its prevalence is much higher among dialysis patients at 12 to 62% compared to 3 to 9% in the general population. In our study we investigated the association between RLS and cardiovascular events risk and laboratory parameters in End-stage kidney disease (ESKD) patients on dialysis. We studied 100 ESKD patients undergoing hemodialysis that were enrolled in an 18-months prospective observational study. The main outcomes were the association of RLS with new cardiovascular events and cardiovascular mortality. RLS affected 31% of the study population. It was associated with female gender, gradual reduction in residual dieresis, lower albumin (P=0.039) and inflammation, but not the dialysis parameters spKt/V and URR. During observation, 47% of patients experienced new cardiovascular events (64.5% with and 39.1% without RLS; P=0.019). Mortality was 20.0% in all patients, 32.3% in those with and 14.5% in patients without RLS (P=0.04). This study confirmed the high prevalence of RLS among dialysis patients and the associations between the severity of RLS and the risk of new cardiovascular events and higher short-term mortality. Abstract Restless legs syndrome (RLS) is a sensori-motor neurological disorder characterzed by paraesthesia, dysaesthesia and irresistibile urge to move the legs especially at night. Its prevalence is much higher among dialysis patients at 12 to 62% compared to 3 to 9% in the general population. In our study we investigated the association between RLS and cardiovascular events risk and laboratory parameters in End-stage kidney disease (ESKD) patients on dialysis. We studied 100 ESKD patients undergoing hemodialysis that were enrolled in an 18-months prospective observational study. The main outcomes were the association of RLS with new cardiovascular events and cardiovascular mortality. RLS affected 31% of the study population. It was associated with female gender, gradual reduction in residual dieresis, lower albumin (P=0.039) and inflammation, but not the dialysis parameters spKt/V and URR. During observation, 47% of patients experienced new cardiovascular events (64.5% with and 39.1% without RLS; P=0.019). Mortality was 20.0% in all patients, 32.3% in those with and 14.5% in patients without RLS (P=0.04). This study confirmed the high prevalence of RLS among dialysis patients and the associations between the severity of RLS and the risk of new cardiovascular events and higher short-term mortality.