Progenitori endoteliali nei pazienti con Chronic Kidney Disease - Mineral and Bone Disorder (CKD-MBD) in fase uremica: effetti del trattamento con vitamina D.

L'insufficienza renale cronica (CKD) è associata ad un rischio cardiovascolare più elevato rispetto alla popolazione generale: fattori come uremia, stress ossidativo, età dialitica, infiammazione, alterazioni del metabolismo minerale e presenza di calcificazioni vascolari incidono fortemente sulla morbosità e mortalità per cause cardiovascolari nel paziente uremico. Diversi studi hanno verificato il coinvolgimento dei progenitori endoteliali (EPC) nella malattia aterosclerotica ed è stato dimostrato che esprimono osteocalcina, marcatore di calcificazione. Inoltre, nella CKD è presente una disfunzione in numero e funzionalità delle EPC. Attualmente, il ruolo delle EPC nella formazione delle calcificazioni vascolari nei pazienti in dialisi non è stato ancora chiarito. Lo scopo della tesi è quello di studiare le EPC prelevate da pazienti con CKD, al fine di determinarne numero e fenotipo. È stato anche valutato l'effetto del trattamento in vitro e in vivo con calcitriolo e paracalcitolo sulle EPC, dato il deficit di vitamina D dei pazienti con CKD: il trattamento con vitamina D sembra avere effetti positivi sul sistema cardiovascolare. Sono stati valutati: numero di EPC circolanti e la relativa espressione di osteocalcina e del recettore della vitamina D; morfologia e fenotipo EPC in vitro; effetti di calcitriolo e paracalcitolo sull’espressione di osteocalcina e sui depositi di calcio. I risultati dello studio suggeriscono che il trattamento con vitamina D abbia un effetto positivo sulle EPC, aumentando il numero di EPC circolanti e normalizzandone la morfologia. Sia calcitriolo che paracalcitolo sono in grado di ridurre notevolmente l’espressione di OC, mentre solo il paracalcitolo ha un effetto significativo sulla riduzione dei depositi di calcio in coltura. In conclusione, il trattamento con vitamina D sembra ridurre il potenziale calcifico delle EPC nell’uremia, aprendo nuove strade per la gestione del rischio cardiovascolare nei pazienti affetti da CKD.

Recupero della funzione renale in pazienti con acute kidney injury (AKI) sottoposti a terapia sostitutiva renale

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.

Circulating Fibrocytes, their role in renal fibrosis and molecular pathways involved. Possible biomarkers of fibrogenesis in chronic kidney disease

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.

The medical management after surgery for advanced heart failure

The aim of the Research of this Ph D Project is to improve the medical management after surgery for advanced heart failure, both after left ventricular assist devices (LVAD) implantation, and after heart transplantation in the long-term. Regarding heart transplantation (HTx), the Research Project is focused on diagnostics, classification, prevention and treatment of cardiac allograft vasculopathy (CAV), and on treatment of post-HTx cancers; the results are presented in the first part of this Thesis. In particular, the main aspect investigated are the prognostic role of information derived from coronary angiography, coronary tomography and intravascular ultrasound, and the different sensitivity of these techniques in predicting outcomes and in diagnosing CAV. Moreover, the role of mTOR inhibitors on CAV prevention or treatment is investigated, both alone and in combination with different anti-CMV prevention strategies, as well as the impact of mTOR inhibitors on clinical outcomes in the long term. Regarding LVAD, the main focus is on the role of transthoracic echocardiography in the management of patients with a continuous-flow, centrifugal, intrapericardial pump (HVAD, Heartware); this section is reported in the second part of this Thesis. The main aspects investigated are the use of echocardiography in patients with HVAD device and its interaction with the information derived from pump curves' analysis in predicting aortic valve opening status, a surrogate of the condition of support provided by the LVAD.

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.

New polymeric materials for vascular surgery

The dramatic impact that vascular diseases have on human life quality and expectancy nowadays is the reason why both medical and scientific communities put great effort in discovering new and effective ways to fight vascular pathologies. Among the many different treatments, endovascular surgery is a minimally-invasive technique that makes use of X-ray fluoroscopy to obtain real-time images of the patient during interventions. In this context radiopaque biomaterials, i.e. materials able to absorb X-ray radiation, play a fundamental role as they are employed both to enhance visibility of devices during interventions and to protect medical staff and patients from X-ray radiations. Organic-inorganic hybrids are materials that combine characteristics of organic polymers with those of inorganic metal oxides. These materials can be synthesized via the sol-gel process and can be easily applied as thin coatings on different kinds of substrates. Good radiopacity of organic-inorganic hybrids has been recently reported suggesting that these materials might find applications in medical fields where X-ray absorption and visibility is required. The present PhD thesis aimed at developing and characterizing new radiopaque organic-inorganic hybrid materials that can find application in the vascular surgery field as coatings for the improvement of medical devices traceability as well as for the production of X-ray shielding objects and garments. Novel organic-inorganic hybrids based on different polyesters (poly-lactic acid and poly-ε-caprolactone) and polycarbonate (poly-trimethylene carbonate) as the polymeric phase and on titanium oxide as the inorganic phase were synthesized. Study of the phase interactions in these materials allowed to demonstrate that Class II hybrids (where covalent bonds exists between the two phases) can be obtained starting from any kind of polyester or polycarbonate, without the need of polymer pre-functionalization, thanks to the occurrence of transesterification reactions operated by inorganic molecules on ester and carbonate moieties. Polyester based hybrids were successfully coated via dip coating on different kinds of textiles. Coated textiles showed improved radiopacity with respect to the plain fabric while remaining soft to the touch. The hybrid was able to coat single fibers of the yarn rather than coating the yarn as a whole. Openings between yarns were maintained and therefore fabric breathability was preserved. Such coatings are promising for the production of light-weight garments for X-ray protection of medical staff during interventional fluoroscopy, which will help preventing pathologies that stem from chronic X-ray exposure. A means to increase the protection capacity of hybrid-coated fabrics was also investigated and implemented in this thesis. By synthesizing the hybrid in the presence of a suspension of radiopaque tantalum nanoparticles, PDMS-titania hybrid materials with tunable radiopacity were developed and were successfully applied as coatings. A solution for enhancing medical device radiopacity was also successfully investigated. High metal radiopacity was associated with good mechanical and protective properties of organic-inorganic hybrids in the form of a double-layer coating. Tantalum was employed as the constituent of the first layer deposited on sample substrates by means of a sputtering technique. The second layer was composed of a hybrid whose constituents are well-known biocompatible organic and inorganic components, such as the two polymers PCL and PDMS, and titanium oxide, respectively. The metallic layer conferred to the substrate good X-ray visibility. A correlation between radiopacity and coating thickness derived during this study allows to tailor radiopacity simply by controlling the metal layer sputtering deposition time. The applied metal deposition technique also permits easy shaping of the radiopaque layer, allowing production of radiopaque markers for medical devices that can be unambiguously identified by surgeons during implantation and in subsequent radiological investigations. Synthesized PCL-titania and PDMS-titania hybrids strongly adhered to substrates and show good biocompatibility as highlighted by cytotoxicity tests. The PDMS-titania hybrid coating was also characterized by high flexibility that allows it to stand large substrate deformations without detaching nor cracking, thus being suitable for application on flexible medical devices.

Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures

Impatto della glomerulopatia cronica da trapianto sulla disfunzione tardiva del rene trapiantato

INTRODUCTION. Late chronic allograft disfunction (CAD) is one of the more concerning issues in the management of patients (pts) with renal transplant (tx). Humoral immune response seems to play an important role in CAD pathogenesis. AIM OF THE STUDY. To identify the causes of late chronic allograft disfunction. METHODS. This study (march 2004-august 2011) enrolled pts who underwent renal biopsy (BR) because of CAD (increase of creatininemia (s-Cr) >30% and/or proteinuria >1g/day at least one year after tx). BR were classified according to 1997/2005 Banff classification. Histological evaluation of C4d (positive if >25%), glomerulitis, tubulitis, intimal arteritis, atrophy/fibrosis and arteriolar-hyalinosis were performed. Ab anti-HLA research at BR was an inclusion criteria. Pts were divided into two groups: with or without transplant glomerulopathy (CTG). RESULTS. Evaluated BR: 93/109. BR indication: impaired s-Cr (52/93), proteinuria (23/93), both (18/93). Time Tx-BR: 7.4±6.3 yrs; s-Cr at BR: 2.7±1.4 mg/dl. CTG group(n=49) not-CTG group(n=44) p Time tx-BR (yrs) 9.3±6.7 5.3±5.2 0.002 Follow-up post-BR (yrs) 2.7±1.8 4.1±1.4 0.0001 s-Cr at BR (mg/dl) 2.9±1.3 2.4±1.5 NS Rate (%) of pts: Proteinuria at BR 61% 25% 0.0004 C4d+ 84% 25% <0.0001 Ab anti-HLA+ 71% 30% 0.0001 C4d+ and/or Ab antiHLA 92% 43% 0.0001 Glomerulitis 76% 16% <0.0001 Tubulitis 6% 32% 0.0014 Intimal arteritis 18% 0% 0.002 Arteriolar hyalinosis 65% 50% NS Atrophy/fibrosis 80% 77% NS Graft survival 45% 86% 0.00005 Histological Diagnosis: CTG group (n=49:Chronic rejection 94%;IgA recurrence + humoral activity 4%;IIA acute rejection + humoral activity 2%. Not-CTG group (n=44: GN recurrence 27%;IF/TA 23%; acute rejection 23%;BKV nephritis 9%; mild not specific alterations 18%. CONCLUSIONS: CTG is the morphological lesion mainly related to CAD. In the 92% of the cases it is associated with markers of immunological activity. It causes graft failure within five years after diagnosis in 55% of pts.

Molecular genetics of inherited cystic kidney diseases: new diagnostic approaches

Background. Hhereditary cystic kidney diseases are a heterogeneous spectrum of disorders leading to renal failure. Clinical features and family history can help to distinguish the recessive from dominant diseases but the differential diagnosis is difficult due the phenotypic overlap. The molecular diagnosis is often the only way to characterize the different forms. A conventional molecular screening is suitable for small genes but is expensive and time-consuming for large size genes. Next Generation Sequencing (NGS) technologies enables massively parallel sequencing of nucleic acid fragments. Purpose. The first purpose was to validate a diagnostic algorithm useful to drive the genetic screening. The second aim was to validate a NGS protocol of PKHD1 gene. Methods. DNAs from 50 patients were submitted to conventional screening of NPHP1, NPHP5, UMOD, REN and HNF1B genes. 5 patients with known mutations in PKHD1 were submitted to NGS to validate the new method and a not genotyped proband with his parents were analyzed for a diagnostic application. Results. The conventional molecular screening detected 8 mutations: 1) the novel p.E48K of REN in a patient with cystic nephropathy, hyperuricemia, hyperkalemia and anemia; 2) p.R489X of NPHP5 in a patient with Senior Loken Syndrome; 3) pR295C of HNF1B in a patient with renal failure and diabetes.; 4) the NPHP1 deletion in 3 patients with medullar cysts; 5) the HNF1B deletion in a patient with medullar cysts and renal hypoplasia and in a diabetic patient with liver disease. The NGS of PKHD1 detected all known mutations and two additional variants during the validation. The diagnostic NGS analysis identified the patient’s compound heterozygosity with a maternal frameshift mutation and a paternal missense mutation besides a not transmitted paternal missense mutation. Conclusions. The results confirm the validity of our diagnostic algorithm and suggest the possibility to introduce this NGS protocol to clinical practice.