The immunosuppressive therapy still remains the only therapeutic strategy to control excessive immune activation following renal transplantation, but remain the problems related to excessive immunosuppression and in particular the toxicity due to high doses of immunosuppressive drugs such as calcineurin inhibitors. The present study has the aim of documenting, in a porcine animal model, the histological damage from calcineurin inhibitors using incremental doses of Tacrolimus, achieved in a limited amount of time, until it reaches toxic blood concentrations. We perform the study under different condition, like oral administration, intravenous infusion and with or without kidney transplant. It is noted that the damage is early, predominantly vascular and that affects different organs in addition to the kidneys. We also observe that the functional damage underestimates the structural damage. The search for non-invasive methods for the identification of biomarkers of nephrotoxicity and rejection, and to better characterize the inflammation status, led us to conduct analysis of the exosomal content, allowing us to observe the presence of serum cytokines which, although in low amounts, suggest a possible role of these in the inflammatory process mediated by exosomal vesicles. Finally, following the genetic SLA typing for the determination of the donors and the recipients, in an accessory project, we got a controlled and stable colony of pigs with SLA defined in homozygosis through the coupling of specific pathogen free pigs (SPF) for DQB-1 and SLA-1 genes.

L’insufficienza renale acuta(AKI) grave che richiede terapia sostitutiva, è una complicanza frequente nelle unità di terapia intensiva(UTI) e rappresenta un fattore di rischio indipendente di mortalità. Scopo dello studio é stato valutare prospetticamente, in pazienti “critici” sottoposti a terapie sostitutive renali continue(CRRT) per IRA post cardiochirurgia, la prevalenza ed il significato prognostico del recupero della funzione renale(RFR). Pazienti e Metodi:Pazienti(pz) con AKI dopo intervento di cardiochirurgia elettivo o in emergenza con disfunzione di due o più organi trattati con CRRT. Risultati:Dal 1996 al 2011, 266 pz (M 195,F 71, età 65.5±11.3aa) sono stati trattati con CRRT. Tipo di intervento: CABG(27.6%), dissecazione aortica(33%), sostituzione valvolare(21.1%), CABG+sostituzione valvolare(12.6%), altro(5.7%). Parametri all’inizio del trattamento: BUN 86.1±39.4, creatininemia(Cr) 3.96±1.86mg/dL, PAM 72.4±13.6mmHg, APACHE II score 30.7±6.1, SOFAscore 13.7±3. RIFLE: Risk (11%), Injury (31.4%), Failure (57.6%). AKI oligurica (72.2%), ventilazione meccanica (93.2%), inotropi (84.5%). La sopravvivenza a 30 gg ed alla dimissione è stata del 54.2% e del 37.1%. La sopravvivenza per stratificazione APACHE II: <24=85.1 e 66%, 25-29=63.5 e 48.1%, 30-34=51.8 e 31.8%, >34=31.6 e 17.7%. RFR ha consentito l’interruzione della CRRT nel 87.8% (86/98) dei survivors (Cr 1.4±0.6mg/dL) e nel 14.5% (24/166) dei nonsurvivors (Cr 2.2±0.9mg/dL) con un recupero totale del 41.4%. RFR è stato osservato nel 59.5% (44/74) dei pz non oligurici e nel 34.4% dei pz oligurici (66/192). La distribuzione dei pz sulla base dei tempi di RFR è stata:<8=38.2%, 8-14=20.9%, 15-21=11.8%, 22-28=10.9%, >28=18.2%. All’analisi multivariata, l’oliguria, l’età e il CV-SOFA a 7gg dall’inizio della CRRT si sono dimostrati fattori prognostici sfavorevoli su RFR(>21gg). RFR si associa ad una sopravvivenza elevata(78.2%). Conclusioni:RFR significativamente piu frequente nei pz non oligurici si associa ad una sopravvivenza alla dimissione piu elevata. La distribuzione dei pz in rapporto ad APACHE II e SOFAscore dimostra che la sopravvivenza e RFR sono strettamente legati alla gravità della patologia. / Severe AKI requiring RRT frequently occurs in ICU and represents an independent risk factor for mortality. The aim was to prospectively evaluate, in critically ill undergoing CRRT for AKI following heart surgery, prevalence and prognostic significance of renal function recovery (RFR). Patients and Methods: Patients (pts) with AKI following elective or emergent cardiac surgery with dysfunction of 2 or more organs treated with CRRT. Results: From 1996 to 2011, 266 pts (M 195, F 71, age 65.5±11.3) underwent CRRT. Type of surgery: CABG (27.6%), aortic dissection (33%), valvular surgery (21.1%), CABG+valvular surgery (12.6%), others (5.7%). CRRT starting parameters: BUN 86.1±39.4, creatinine (Cr) 3.96±1.86 mg/dL, MAP 72.4±13.6 mmHg, APACHE II 30.7±6.1, SOFA 13.7±3. RIFLE staging: Risk (11%), Injury (31.4%), Failure (57.6%). Oliguric AKI (72.2%), ventilation (93.2%), inotropics (84.5%). At 30 days and at hospital discharge, total survival was 54.2% and 37.1% (APACHE II score clusters survival: <24= 85.1 and 66%, 25-29= 63.5 and 48.1%, 30-34= 51.8 and 31.8%, >34= 31.6 and 17.7%). RFR allowed to stop CRRT in 87.8% (86/98) of survivors (Cr 1.4±0.6 mg/dL) and in 14.5% (24/166) of nonsurvivors (Cr 2.2±0.9mg/dL) with an overall recovery of 41.4%. RFR has been observed in 59.5% (44/74) of non oliguric pts and in 34.4% of oliguric pts (66/192). Distribution of pts according to the timing of RFR (days from CRRT start): <8 (38.2%), 8-14 (20.9%), 15-21 (11.8%), 22-28 (10.9%), >28 (18.2%). Logistic regression selected occurrence of oliguria, age, CV-SOFA at 7 days from CRRT start as a prognostic factors for delayed RFR (> 21 days).RFR was associated with a high survival rate (78.2%). Conclusions: RFR, more frequently observed in pts with nonoliguric AKI, was mostly associated with a favourable outcome. Patient distribution according to APACHE II and SOFA score revealed that survival and RFR are strictly related to the severity of illness.

Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

Caratterizzazione di EPC in pazienti con malattia renale cronica. Background: Gli effetti del recettore della vitamina D (VDR)e dell'espressione dell' osteocalcina (OCN), così come VDR agonista terapia (VDRA) su circolanti cellule progenitrici endoteliali (EPCs) non è stato ancora chiarito. Metodi: sono state analizzat EPCs in 23 controlli sani e 53 pazienti sottoposti a dialisi. La percentuale di EPCs (CD34+KDR+CD133+/-CD45-) VDR+/- o OCN+/- è stata analizzata in citometria a flusso e correlata con molecole coinvolte nelle malattie. Risultati: EPCs aumentano nei pazienti con CKD trattati con VDRAs. EPCsOCN +/- in pazienti non trattati con VDRAs correlano positivamente con il calcio sierico e reticolociti, e negativamente con DKK1. La percentuale di EPCsVDR +/- correla negativamente con OPN.  EPCsOCN + nei pazienti trattati con VDRAs correla positivamente con la vitamina D. La percentuale di EPCsVDR +/- positivamente correla con IL-6. Conclusioni: la terapia VDRA influenza l'espressione VDR e di OCN su EPCs circolanti. Dal momento che l'espressione OCN può contribuire alla calcificazione vascolare, ipotizziamo un putativo effetto pro-calcificazione di VDRA. Effetti immunomodulatori di EPO. Background: Il cloruro di sodio spinge l'induzione di cellule TH17 patogeni e TH1. La correzione dell'anemia con eritropoietina (EPO) è associato ad un miglioramento della tolleranza del trapianto di rene. Prove emergenti indicano che queste osservazioni possano essere eritropoiesi-indipendente e che l'EPO presenta proprietà immunosoppressive. Metodi: esaminato gli effetti del trattamento con EPO e sale su cellule T umana, apoptosi e la produzione di INF-γ, l'effetto sulla iTh17 e iFoxP3. Le cellule sono state analizzate con l'analisi di citometria a flusso. Risultati: NaCl aumenta la proliferazione di CD4+ e CD8+, iTh17 e la produzione di INFγ. Questo effetto è prevenuto da EPO. EPO aumenta l'induzione di Foxp3. Non cambia l'apoptosi e la stabilità di FoxP3. EPO è in grado di contiene l'effetto pro infiammatorio del sale / First project: Characterization of EPCs in patients with CKD. Background: The effects of vitamin D receptor (VDR) and osteocalcin (OCN) expression as well as VDR agonist (VDRA) therapy on circulating endothelial progenitor cells (EPCs) has not been elucidated yet. Methods: we therefore analyzed EPCs in 23 healthy controls and 53 patients undergoing dialysis. The percentage of EPCs (CD34+CD133+/-KDR+CD45-) expressing VDR or OCN were analyzed using flow cytometry and correlated with molecules involved in diseases. Results: EPCs increase in CKD patients treated with VDRAs. EPCsOCN+/- in patients untreated with VDRAs correlated positively with serum calcium and reticulocytes, and negatively with DKK1. The percentage of EPCsVDR+/- correlated negatively with OPN. EPCsOCN+ in patients treated with VDRAs correlated positively with vitamin D. The percentage of EPCsVDR+/- correlated positively with IL-6. Conclusions: our data suggest that VDRA therapy influence VDR and OCN expression on circulating EPCs. Since OC expression may contribute to vascular calcification, we hypothesize a putative pro-calcifying effect of VDRA. Second project: Immunomodulatory effects of EPO. Background: Sodium chloride drives the induction of pathogenic TH17 cells and TH1. Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. Methods: examined the effects of treatment with EPO and salt on human T-cell alloimmunity, the apoptosis and the production to INF-γ, the effect on iTh17 and iFoxP3. The cells were analyzed with flow cytometry analysis. Results: NaCl increases the proliferation of CD4+ and CD8+ cells, iTh17 and the production of INFγ. This effect is prevented by EPO. EPO increases the induction of Foxp3. It does not change the apoptosis and stability of FoxP3. Epo contains the inflammatory effect of NaCl. EPO in the kidney, where NaCl is high, may have a tolerance effect.

Simkania negevensis is a bacterium belonging to the order Chlamydiales but with certain biological characteristics different from those of chlamydia, according to which it was classified in the family Simkaniaceae. It is widespread in the environment, due to its ability to survive in amoebae also in phase cystic, for which it was hypothesized a possible transmission after contact with water in which they are present amoebae. So far it is known its role in diseases of the lower respiratory tract, such as childhood bronchiolitis and pneumonia in adults of the community, following its transmission through infected aerosols. A recent American study showed, by PCR, a high prevalence of S. negevensis in patients with lung transplant than other transplant recipients, assuming an association between the presence of the bacterium in these patients, and transplant rejection, were more frequent in lung transplant recipients infected compared to uninfected. There are no data so far analyzed in Italy relative to the population of dialysis and kidney transplant recipients relative to simkania negevensis why this study was undertaken in order to start a specific location and evaluate the scientific implications. Because its ability to assume persistent forms of infection, which may lead to a prolonged inflammatory response, Simkania negevensis, similar to other persistent bacteria or viruses, may be ivolved in pathologic complication. Sn may be a factor in graft rejection in mmunesuppressed lung transplant recipients, and further studies are planned to explore the posible association of Sn infections with various in vivo pathologies.

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.

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.

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