Foto del docente

Gaetano La Manna

Associate Professor

Department of Experimental, Diagnostic and Specialty Medicine - DIMES

Academic discipline: MED/14 Nephrology

Head of Scuola di Specializzazione Nefrologia (DI 68/2015)

Research

  1. Genetic cardiovascular risk factors after renal transplantation: role of cytokine polymorphisms
  2. Prevention of ischemia/reperfusion injury in renal transplantation: small/medium size animal models. Organ preservation and use of antioxidizing agents
  3. Reduction of oxidative damage and kidney transplantation outcome
  4. VDR expression of on endothelial progenitor cells in dialysis patients
  5. Effects of heparin and on osteoprotegerin and RANKL plasma levels in haemodialysis patients
  6. Restless-leg sindrome in uremia
  7. Stem cells in renal regeneration: in vitro and in vivo models for nephrogenic differentiation
  8. Urinary neutrophil gelatinase-associated lipocalin at birth predicts early renal function in very low birth weight infants
  9. Risk for contrast nephropathy in patients undergoing coronarography
  10. Hyperhomocysteinemia in dialysis: therapeutic strategies


1) Genetic cardiovascular risk factors after renal transplantation: role of cytokine polymorphisms

Cardiovascular disease (CVD) represents the main cause of morbidity and mortality after renal transplantation. A pathogenetic link between inflammation, atherosclerosis and CVD have been demonstrated. For this reason, genetic factors regulating cytokine production and the balance between pro- and anti- inflammatory molecules could have a deep influence on CVD risk. In the view of the modern paradigm of atherosclerosis as an inflammatory disease, the present study was undertaken to investigate the impact of inflammatory cytokine polymorphisms on post-renal transplant CVD. To evaluate the association between cytokine polymorphisms and CVD, a case-control study was carried out on 798 renal allograft recipients transplanted between 1997 and 2008, 196 of them with CVD (CVD group) and 602 controls free of cardiovascular complications (no-CVD group). TNF-α/G-308A, TGF-β1/L10P, TGF-β1/R25P, IL-10/G-1082A, IL-10/C-819T, IL-10/C-592A, IL-6/G-174C, IFN-γ/T+874A, IL-8/T-251A and VEGF/C+936T polymorphisms were genotyped using PCR-SSP and RFLP. The patients in CVD and no-CVD group differed significantly in IL-10 and TNF-α genotype frequencies. Using multivariate analyses to test the association with CVD, the high producer genotype of the pro-inflammatory cytokine TNF-α was significantly associated with a 4.41-fold increased cardiovascular risk. Conversely, the carriage of high producer genotype of the anti-inflammatory cytokine IL-10 resulted protective against post-transplant CVD, with an adjusted OR of 0.2 (0.02-0.29). This work seems to indicate that gene polymorphisms involved in inflammatory response might represent cardiovascular risk markers in renal allograft recipients and suggests a prognostic value of TNF-α and IL-10 genotypes. Further studies are necessary to evaluate if patients with a CVD "predisposing" genotype should be treated more aggressively.

 

2) Prevention of ischemia/reperfusion injury in renal transplantation: small/medium size animal models. Organ preservation and use of antioxidizing agents

There is increasing evidence that organ preservation by machine perfusion is able to limit ischemia/reperfusion injury in kidney transplantation. This study was designed to compare the efficiency in hypothermic organ preservation by machine perfusion or cold storage in an animal model of kidney autotransplantation. Twelve pigs underwent left nephrectomy after warm ischemic time; the organs were preserved in machine perfusion (n=6) or cold storage (n=6) and then autotransplanted with immediate contralateral nephrectomy. The following parameters were compared between the two groups of animals: hematological and urine indexes of renal function, histological features, tissue adenosine-5'-triphosphate (ATP) content, perforin gene expression in kidney biopsies, and organ weight changes before and after preservation. The amount of cellular ATP was significantly higher in organs preserved by machine perfusion; moreover, the study of apoptosis induction revealed an enhanced perforin expression in thsoe kidneys which underwent simple hypothermic preservation compared to the machine-preserved ones. Organ weight was significantly decreased after cold storage, but it remained quite stable for machine-perfused kidneys. The present model seems to suggest that organ preservation by hypothermic machine perfusion is able to better control cellular impairment in comparison with cold storage A similar study is being performed in small size animals, also evaluating the role of anti-oxidizing molecules.

 

3) Reduction of oxidative damage and kidney transplantation outcome

DNA fragmentation is one of the typical features of apoptosis, frequently induced by oxidative stress. Increased oxidative stress is known to be related to several pathological processes. In this study, oxidative damage in the early follow-up period after kidney transplantation was assessed measuring DNA oxidation and fragmentation of mononuclear cells and the circulating levels of inflammatory cytokines. Blood samples from 30 kidney transplant recipients were collected before transplantation and after 2 days, 1 month and 6 months. Oxidative DNA fragmentation was measured by Comet Assay, whereas DNA oxidation was evaluated measuring 8-OHdG leukocyte levels. Serum IL-1beta, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF-alpha were assayed using a multiplex ELISA analysis. At 6 months after transplantation, a significant reduction in DNA fragmentation and IL-6 plasma levels was observed; DNA oxidation was higher in patients with a worse outcome, with delayed graft function and low nutritional status. We also found a correlation of IL-6 and IL-10 levels with DNA fragmentation and of IL-10 levels with DNA oxidation.

These results seem to suggest that lower levels of oxidation and apoptosis at 6 months after transplantation correlate with a better recovery of renal function in kidney allografts.

 

4) VDR expression of on endothelial progenitor cells in dialysis patients

Vitamin D deficiency in uremic patients has been associated with endothelial dysfunction. The basis of this link is probably an alteration of endothelial progenitor cells (EPCs), in terms of both function and number. The purpose of this study was to evaluate in a population of 89 hemodialysis patients the factors associated with the number of circulating EPCs, either in less differentiated EPCs (CD45 ±/CD34/CD133/KDR) or in more differentiated EPCs (CD45-/CD34/CD133-/ KDR), the presence of vitamin D receptor (VDR) in these cells and factors that influence the expression of the VDR in EPCs, in particular treatment with calcitriol.

EPC counts, the percentage of cells positive for VDR and the expression of the VDR in EPCs were determined by flow cytometry. Cells isolated from a subgroup of patients were cultured to the analysis of colony-forming units, immunofluorescence for specific markers (CD34, CD133, KDR), flow cytometry for CD34, CD133, KDR, VDR, CD14, CD31, and test the ability to form tubular-like structures on Matrigel.

The study demonstrates for the first time the presence of the VDR on EPCs. In dialysis patients, the expression of VDR either in less differentiated or in more differentiated EPCs seems to be influenced by uremia-related factors such as anemia, inflammation, diabetes, levels of 25(OH) vitamin D and therapy with calcitriol.

 

5) Effects of heparin and on osteoprotegerin and RANKL plasma levels in haemodialysis patients

A crossover study was carried out to analyze the effects of unfractioned heparin (UFH) and low-molecular-weight heparin (LMWH) on intra- and post-dialytic blood levels of osteoprotegerin, RANKL and inflammatory cytokines. Forty patients on hemodialysis for at least 12 months were studied.

The results have shown that heparin infusion, regardless of the type, led to an increase of osteoprotegerin levels within 8 hours following administration, with a return to baseline levels after 24 hours. No effect on the levels of RANKL and inflammatory cytokines was observed. These cyclic increases of osteoprotegerin might play a role in the vascular pathology of hemodialysis patients.

 

6) Restless-leg sindrome in uremia

Restless legs syndrome (RLS) is a sleep-related movement disorder involving an urge to move the legs frequently in association with unpleasant sensory symptoms. RLS can occur in 2 forms: idiopathic or secondary to other conditions including end-stage renal disease. RLS is highly prevalent in patients on maintenance dialysis and ESRD patients with sleep disorders which are independently linked to a higher mortality rate. We evaluated the impact of chronic inflammation on RLS in dialysis patients. One hundred patients in dialysis for at least 3 months were included in this study. A specific questionnaire based on the criteria of the International Restless Legs Syndrome Study Group (IRLSSG) was used to asses the prevalence of RLS. Gender, age on starting dialysis, time on dialysis, BMI, blood pressure, smoking, comorbidities and drugs were recorded. The basal values of hemoglobin, hematocrit, CRP, PTH, calcium, phosphorus, albumin, alkaline phosphatase, total-, HDL- and LDL-cholesterol, triglycerides, uric acid, fibrinogen, erythrocyte sedimentation rate (ESR), iron, ferritin, transferrin, electrolytes were assayed in each patient. RLS was observed in 31/100 (31%). Univariate analysis revealed positive correlations between RLS and female gender (p=0.048), polyneuropathy (p=0.031), fibrinogen (p=0.004), ESR (p=0.004), CRP (p=0.025), white blood cells (WBC) (p=0.023), albumin (p=0.018). After adjustment for age, dialysis vintage, gender and Charlson score, multivariate analysis showed that CRP (p=0.041), fibrinogen (p=0.002), ESR (p=0.005), albumin (p=0.026) and WBC (p=0.022) were significant independent predictors for RLS. This study highlighted a higher prevalence of RLS in dialysis patients, thus suggesting a role in the pathogenesis of RSL of chronic inflammation, besides the well-known associations with atherosclerosis, malnutrition, longer hospitalizations and cardiovascular mortality.

 

7) Stem cells in renal regeneration: in vitro and in vivo models for nephrogenic differentiation

The destruction of a tissue is the cause of many pathologies which affect a significant part of the population. At present, the best therapeutic option is organ transplantation, but with two important limits: the lack of available organs and the need for chronic immunosuppression to prevent rejection. Hence, stem cells represent an interesting opportunity for the regeneration of damaged organs, including kidneys. A current area of interest is the study of novel differentiating agents to induce transdifferentiation into renal progenitors of mesenchymal stem cells isolated from fetal membranes of human term placenta. The rationale for the choice of synthetic molecules as hyaluronan monoesters with butyric acid (HB) and mixed esters with retinoic and butyric acid (HBR) is based on their internalization through the hyaluronan CD44 receptor, and the consequent increase of transcription factor accessibility to target cis-acting regulatory sites, associated with the histone deacetylase inhibitory action of the butyrate moiety. The in vitro phase includes the treatment of cells with HB or HBR and the successive analysis of eventual variations in gene expression profile involving the markers of metanephric differentiation. The preliminary data shows that the differentiation of mesenchymal stem cells induced by HBR is effective in producing a high throughput of cardiogenesis, but not metanephric differentiation. In contrast, cell treatment with HB at the concentration of 1 mg/ml resulted in a significant gene expression increase of all the metanephric differentiation markers. Subsequently, the cells pretreated with HB were investigated for their ability to regenerate the renal tissue after induced damage, using in vivo rat models. The results of the research have been published on Cell Transplantation with an impact factor of 5.126.

 

8) Urinary neutrophil gelatinase-associated lipocalin at birth predicts early renal function in very low birth weight infants

Preterm infants are exposed to a number of conditions that can impair renal function. We evaluated the ability of serum and urinary neutrophil gelatinase-associated lipocalin (sNGAL and uNGAL) to predict renal function in the first weeks of life. In infants weighing ≤1500 g at birth with no major congenital anomalies or sepsis, we measured sNGAL and uNGAL levels at birth. To evaluate renal function, we determined changes in serum creatinine (sCreat) and estimated GFR (eGFR) from birth to day 21. Forty neonates (gestational age: 27 ± 2 weeks) completed the study. Renal function improved in 32 of 40 (80%) infants (normal renal function, NRF group) (sCreat range: from 0.97 ± 0.2 to 0.53 ± 0.13 mg/dl; eGFR range: from 15.3 ± 4.1 ml/min to 28.6 ± 7.9 ml/min), whereas renal function worsened in 8 of 40 (20%) infants (impaired renal function, IRF group) (sCreat range: from 0.71 ± 0.27 mg/dl to 0.98 ± 0.43 mg/dl; eGFR from 23 ± 14.7 ml/min to 16.4 ± 9.1 ml/min). The uNGAL/urinary creatinine (uCreat) ratio at birth was higher in the IRF group (31.05 ng/mg) than the NRF group (6.0 ng/mg), and uNGAL was significantly higher in IRF group, detecting IRF with a cutoff of 100 ng/ml. This study suggest that uNGAL levels at birth may have a predictive role on early renal function in very low birth weight infants.

 

9) Risk for contrast nephropathy in patients undergoing coronarography

Another research has analyzed the incidence of contrast-induced nephropathy in the Cardiology Intensive Care Unit. Contrast-induced nephropathy is the third in prevalence among the causes of acute renal failure. The study recruited patients submitted to coronarography at the Bentivoglio hospital from April 2007 to April 2008 who required at least 3 days of hospitalization due to the presence of potential risk factors; 136 out of 784 patients were included. Among the selected patients, 21 (15.44%) developed a renal impairment compatible with contrast-induced nephropathy. The risk factors that seemed to display the best correlation with risk of contrast nephropathy were advanced age and ventricular failure (ejection fraction <40%); however, the critical condition did not appear to be due to a single risk factor, but it resulted from the association of more contextual risk factors. Particularly, the concomitant presence of ventricular failure, anemia, diabetes, previous myocardial infarction and advanced age (>70 years) determined a 3-fold increased risk of contrast nephropathy. These data suggest that the development of contrast nephropathy following coronarography is associated with worse renal function during hospitalization and at discharge.

 

10) Hyperhomocysteinemia in dialysis: therapeutic strategies

One study in the field of dialysis analysed the risk factors of cardiovascular morbidity and mortality in 341 patients hemodialysis patients. Given the role of hyperhomocysteinemia as cardiovascular risk factor, the study also investigated whether high doses of folates or 5-methyltetrahydrofolate (5-MTHF) could improve survival in hemodialysis patients. The patients were divided into two groups: group A was treated with 50 mg i.v. 5-MTHF, and group B was treated with 5 mg/day oral folic acid. All the patients received i.v. vitamin B(6) and B(12). By dividing patients into C-reactive protein (CRP) quartiles, group A had the highest survival for CRP<12 mg/l, whereas no survival difference was found for group B. CRP was the only predictive risk factor for death. Dialysis age, hyperhomocysteinemia, methylenetetrahydrofolate reductase polymorphism, albumin, lipoprotein(a) and folate did not influence mortality risk. Survival in group A was significantly higher than that in group B. These data suggest that CRP, but not hyperhomocysteinemia, is the main risk factor for mortality in hemodialysis patients receiving vitamin supplements. Intravenous 5-MTHF seems to improve survival in hemodialysis patients independent from homocysteine lowering.

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