Salvatore Di Paolo

Addition of Sirolimus to Cyclosporine Delays the Recovery from Delayed Graft Function but Does not Affect 1-Year Graft Function

Delayed graft function (DGF) has long been identified as one of the main correlates of poor graft survival in cadaveric renal transplantation, but the factors that affect its onset and duration are not fully elucidated. The impact of two immunosuppressive protocols on the incidence and length of DGF among kidney transplant recipients of a suboptimal organ was evaluated. Patients were randomly treated with corticosteroids (CS); low-dose cyclosporine (CsA) and sirolimus (SRL; group 1; n = 42); or CS, full-dose CsA, and mycophenolate mofetil (group 2; n = 48). All recipients received immunoprophylaxis with basiliximab. After 3 mo, group 1 discontinued CsA and continued with SRL, whereas group 2 continued the same treatment. The incidence of DGF was similar in the two groups (group 1 = 52.4%; group 2 = 58.3%), whereas its duration was significantly higher in the group 1 (19.0 +/- 6.0 versus 10.3 +/- 3.2 d; P = 0.001). Both groups showed 100% actuarial graft and patient survival at 1-yr. Among DGF patients, serum creatinine (sCr) at discharge was significantly worse in group 1 (sCr, 3.0 +/- 1.0 versus 1.5 +/- 0.2 mg/dl; calculated creatinine clearance, 31.2 +/- 9.3 versus 61.1 +/- 10 ml/min; P = 0.001). During the first year, the former group displayed a significant improvement of graft function, such that at 1-yr, no difference could be measured between groups (sCr, 1.8 +/- 0.5 versus 1.7 +/- 0.4 mg/dl; calculated creatinine clearance, 51.5 +/- 10.2 versus 53.3 +/- 9.4 ml/min). In conclusion, in de novo renal transplanted patients, the administration of SRL, in combination with low-dose CsA, is associated with a delayed recovery from DGF but does not worsen 1-yr graft function.

Early withdrawal of cyclosporine A improves 1-year kidney graft structure and function in sirolimus-treated patients.

Background. Chronic allograft nephropathy (CAN) represents the most common cause of late graft loss. Nephrotoxicity from chronic use of calcineurin inhibitors (CNI) has the potential to contribute to CAN. The present investigation aimed to evaluate the impact of early CNI withdrawal on kidney graft function and structure at 1 year in sirolimus (SRL)-treated patients. Methods. Forty consecutive kidney transplant recipients were initially treated with corticosteroids, cyclosporine A (CsA), and SRL (2 mg/day). After 3 months, patients were randomly assigned to either continue the same treatment (group I) or to withdraw CsA and continue SRL (group II). All patients underwent kidney graft biopsy immediately after graft reperfusion (0-hr biopsy) and 12 months after engraftment. Results. Baseline graft biopsy showed a higher degree of renal damage in group II patients (total score, 4±1.6 vs. 2±0.9;P <0.05). Twelve months after engraftment, CAN was diagnosed in 55% of all patients, of whom 64% were in group I and 36% in group II. CAN lesions were scored as moderate to severe in 90% of group I patients but only 32% of group II patients (P <0.05). A vascular score greater than or equal to 2 occurred in 90% of group I patients and in 38% of group II patients (P <0.05). At 1 year, group I patients showed a significantly worse kidney graft function (serum creatinine, 2.0±0.3 vs. 1.3±0.3 mg/dL; creatinine clearance, 54±14 vs. 66±17 mL/min; both P <0.002). Conclusions. These results suggest that early withdrawal of CsA is a safe option, which allows a significant reduction of chronic histologic damage, particularly vascular injury, of cadaveric kidney allografts.

Ischemia-Reperfusion Induces Glomerular and Tubular Activation of Proinflammatory and Antiapoptotic Pathways: Differential Modulation by Rapamycin

Ischemia-reperfusion (I-R) injury in transplanted kidney, a key pathogenic event of delayed graft function (DGF), is characterized by tubular cell apoptosis and interstitial inflammation. Akt-mammalian target of rapamycin-S6k and NF-kappaB-inducing kinase (NIK)-NF-kappaB axis are the two main signaling pathways regulating cell survival and inflammation. Rapamycin, an immunosuppressive drug inhibiting the Akt axis, is associated with a prolonged DGF. The aim of this study was to evaluate Akt and NF-kappaB axis activation in patients who had DGF and received or not rapamycin and in a pig model of I-R and the role of coagulation priming in this setting. In graft biopsies from patients who were not receiving rapamycin, phosphorylated Akt increased in proximal tubular, interstitial, and mesangial cells with a clear nuclear translocation. The same pattern of activation was observed for S6k and NIK. However, in rapamycin-treated patients, a significant reduction of S6k but not Akt and NIK activation was observed. A time-dependent activation of phosphatidylinositol 3-kinase, Akt, S6k, and NIK was observed in the experimental model with the same pattern reported for transplant recipients who did not receive rapamycin. Extensive interstitial and glomerular fibrin deposition was observed both in pig kidneys upon reperfusion and in DGF human biopsies. It is interesting that the activation of both Akt and NIK-NF-kappaB pathways was induced by thrombin in cultured proximal tubular cells. In conclusion, the data suggest that (1) coagulation may play a pathogenic role in I-R injury; (2) the Akt axis is activated after I-R, and its inhibition may explain the prolonged DGF observed in rapamycin-treated patients; and (3) NIK activation in I-R and DGF represents a proinflammatory, rapamycin-insensitive signal, potentially leading to progressive graft injury.

Interference of angiotensin-converting enzyme inhibitors on erythropoiesis in kidney transplant recipients: role of growth factors and cytokines.

BACKGROUND Recent data indicate that factors other than erythropoietin (EPO), such as insulin-like growth factor 1 (IGF-1), can promote erythropoiesis in vitro and correct the anemia of chronic renal failure in vivo. IGF-1 is produced by the liver under growth hormone control, as well as by other sources, including the kidney. The erythropoietic role of growth factors and cytokines and their possible modulation by angiotensin-converting enzyme inhibitors (ACEI) has never been explored. METHODS This study evaluated the serum levels of EPO, IGF-1, interleukin (IL)-2, IL-3, and granulocyte macrophage-colony-stimulating factor in 40 kidney transplanted patients with or without posttransplant erythrocytosis (PTE) and in 10 living kidney donors. Then, the effect of ACEI therapy on the above pattern was examined in patients with PTE. RESULTS EPO and IGF-1 serum levels were significantly higher in patients with PTE than in patients without PTE and in living kidney donor subjects. ACEI therapy significantly reduced hematocrit (Hct) as well as circulating IGF-1 and EPO levels. Of note, the decrease in IGF-1 was prominent mainly in those patients whose EPO levels were not significantly modified by ACEI therapy. In all of the patients Hct levels displayed a direct relationship with circulating IGF-1 levels, but not with EPO concentration. Growth hormone did not significantly differ among the groups examined, whereas it steeply increased under ACEI. Finally, no significant difference in IL-2, IL-3, and granulocyte macrophage-colony-stimulating factor serum levels was detected. CONCLUSIONS IGF-1 seems to play a role in the ACEI-related decrease of Hct in patients with PTE, chiefly in patients without any modification of EPO serum levels.

Urine Proteome Analysis May Allow Noninvasive Differential Diagnosis of Diabetic Nephropathy

OBJECTIVE Chronic renal insufficiency and/or proteinuria in type 2 diabetes may stem from chronic renal diseases (CKD) other than classic diabetic nephropathy in more than one-third of patients. We interrogated urine proteomic profiles generated by surface-enhanced laser desorption/ionization-time of flight/mass spectrometry with the aim of isolating a set of biomarkers able to reliably identify biopsy-proven diabetic nephropathy and to establish a stringent correlation with the different patterns of renal injury. RESEARCH DESIGN AND METHODS Ten micrograms of urine proteins from 190 subjects (20 healthy subjects, 20 normoalbuminuric, and 18 microalbuminuric diabetic patients and 132 patients with biopsy-proven nephropathy: 65 diabetic nephropathy, 10 diabetic with nondiabetic CKD [nd-CKD], and 57 nondiabetic with CKD) were run using a CM10 ProteinChip array and analyzed by supervised learning methods (Classification and Regression Tree analysis). RESULTS The classification model correctly identified 75% of patients with normoalbuminuria, 87.5% of those with microalbuminuria, and 87.5% of those with diabetic nephropathy when applied to a blinded testing set. Most importantly, it was able to reliably differentiate diabetic nephropathy from nd-CKD in both diabetic and nondiabetic patients. Among the best predictors of the classification model, we identified and validated two proteins, ubiquitin and β2-microglobulin. CONCLUSIONS Our data suggest the presence of a specific urine proteomic signature able to reliably identify type 2 diabetic patients with diabetic glomerulosclerosis.

Protease-activated receptor 1 and plasminogen activator inhibitor 1 expression in chronic allograft nephropathy : The role of coagulation and fibrinolysis in renal graft fibrosis

BACKGROUND Chronic allograft nephropathy (CAN), the major cause of renal graft failure, frequently displays extensive interstitial fibrin deposition. Little is known in regard to the cause of the altered coagulation/fibrinolysis balance and its relevance in the pathogenesis of CAN. Thrombin, present within the fibrin clots, can interact with a specific receptor, protease-activated receptor 1 (PAR-1), and modulate a variety of cell functions. On the other hand, the derangement of the fibrinolytic system may directly affect extracellular matrix (ECM) degradation. METHODS In the present study, we investigated, by in situ hybridization, PAR-1 gene expression and the mRNA levels for tissue factor and plasminogen activator inhibitor 1 (PAI-1), two key regulatory molecules of coagulation and fibrinolysis, in 16 CAN biopsies and in 10 normal human kidney grafts. The thrombin-induced transforming growth factor beta (TGF-beta) gene and protein expression in proximal tubular cells (PTC) was investigated by Northern blotting and ELISA, respectively. RESULTS Fibrin deposits, absent in normal grafts, were observed in the interstitial space and arterial wall of CAN. Tissue factor gene expression was not increased either at the vascular or at the interstitial level in CAN. On the contrary, PAI-1 gene expression, barely detectable in control tissue, was strikingly increased in CAN, with a distribution resembling the pattern of fibrin deposition. Note that PAI-1 gene expression was directly correlated with the degree of interstitial fibrosis. In addition, fibrin deposits were strictly associated with a marked increase of PAR-1 gene expression in endothelial cells and PTC. The tubular expression of PAR-1 was significantly higher in Banff grade II-III than in grade I. In vitro, incubation of PTC with thrombin caused a significant up-regulation of TGF-beta gene expression, followed by an increased TGF-beta release into the supernatant. Interestingly, urine from CAN patients contained significantly higher levels of TGF-beta. CONCLUSIONS Fibrin deposits in CAN may result from the increased expression of PAI-1 and the subsequent inhibition of fibrinolysis. The reduced fibrinolysis may cause, in turn, a decreased ECM turnover. Finally, thrombin, preserved in the active form within the fibrin clots, may interact with PAR-1 highly expressed on PTC and induce an up-regulation of ECM deposition in a TGF-beta-dependent manner.

HYPERTENSION IS AN INDEPENDENT PREDICTOR OF DELAYED GRAFT FUNCTION AND WORSE RENAL FUNCTION ONLY IN KIDNEYS WITH CHRONIC PATHOLOGICAL LESIONS

Background. Delayed graft function (DGF) has been identified as one of the principal correlates of poor graft survival in cadaveric renal transplantation. However, its risk factors and clinical predictors have been poorly elucidated. Methods. We analyzed the risk factors of DGF with a specific emphasis on the role of histological damage of donor kidney. Then, we also studied the impact of DGF, and donor factors affecting DGF, on kidney graft function over the first year after engraftment in 100 consecutive cadaveric renal transplant (Tx) recipients. Results. The organs displaying DGF (n=48) had a significantly higher degree of glomerular sclerosis and tubular atrophy (P <0.01), as well as of interstitial fibrosis and vascular damage (P <0.02) in time-zero biopsies. In patients who received an “ideal” organ for Tx (total histological score ≤4), DGF showed a strong relationship with &Dgr;age D-R (70% increase of risk for donors 10 years older than recipients), and with the histological score (odds ratio 1.34). In contrast, donor hypertension was the most relevant variable independently associated with DGF (odds ratio 19.4) in patients receiving a suboptimal organ (histological score >4). Moreover, DGF and donor hypertension adversely affected graft function at 1 year, but only in Tx patients with a histological score >4 in time-zero biopsy. Of note, both patients with and those without DGF showed a very low incidence of biopsy-proven acute rejection (8.5 and 6.8%, respectively) and a rather short cold ischemia time (<16 hr). Conclusion. Our findings suggest that the quality of the transplanted organ and the occurrence of DGF are strictly related to each other and can influence graft function through apparently nonimmune mechanisms. In addition, long-standing donor hypertension is a strong independent variable affecting both DGF and graft function of suboptimal cadaveric kidneys, at least up to 1 year.

Incidence of erythropoietin antibody-mediated pure red cell aplasia: the Prospective Immunogenicity Surveillance Registry (PRIMS)

Background Subcutaneous administration of Eprex® (epoetin alfa) in patients with chronic kidney disease (CKD) was contraindicated in the European Union between 2002 and 2006 after increased reports of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA). The Prospective Immunogenicity Surveillance Registry (PRIMS) was conducted to estimate the incidence of antibody-mediated PRCA with subcutaneous administration of a new coated-stopper syringe presentation of Eprex® and to compare this with the PRCA incidence with subcutaneous NeoRecormon® (epoetin beta) and Aranesp® (darbepoetin alfa). Methods PRIMS was a multicentre, multinational, non-interventional, parallel-group, immunogenicity surveillance registry. Adults with CKD receiving or about to initiate subcutaneous Eprex®, NeoRecormon® or Aranesp® for anaemia were enrolled and followed for up to 3 years. Unexplained loss or lack of effect (LOE), including suspected PRCA, was reported, with antibody testing for confirmation of PRCA. Results Of the 15 333 patients enrolled, 5948 received Eprex® (8377 patient-years) and 9356 received NeoRecormon®/Aranesp® (14 286 patient-years). No treatment data were available for 29 patients. Among 23 patients with LOE, five cases of PRCA were confirmed (Eprex®, n = 3; NeoRecormon®, n = 1; Aranesp®, n = 1). Based on exposed time, PRCA incidence was 35.8/100 000 patient-years (95% CI 7.4–104.7) for Eprex® versus 14.0/100 000 patient-years (95% CI 1.7–50.6) for NeoRecormon®/Aranesp®. The incidence of PRCA with Eprex® was not significantly different versus comparator ESAs (rate ratio: 2.56; 95% CI 0.43–15.31). An analysis based on observed time produced similar findings. Conclusion This large, prospective registry demonstrates that PRCA is rare with subcutaneous administration of either the new coated-stopper syringe presentation of Eprex®, or NeoRecormon® or Aranesp®.

Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS): from sample collection to data analysis.

Abstract Background: Saliva is one of the most promising and easy-to-collect source of potential biomarkers of oral and systemic disease. We standardized a protocol suitable for pre-analytical treatment and for the analysis of whole normal saliva by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS). Methods: We evaluated the impact of storage time, freeze/thaw cycles, denaturing agents, glycoproteins depletion, centrifugation, type of matrix and ProteinChip® used on the quality of the SELDI protein profile. Moreover, we explored the inter-individual and between-sex differences and the changes in the sample composition over the day. Results: Saliva was qualitatively stable, in the absence of protease inhibitors, for up to 3 h from the collection at room temperature, although the intensity of a number of peaks slightly decreased between 0 and 3 h and the addition of protease inhibitors did not completely revert this trend. The saliva proteome changed during the day and showed relevant between-sex differences. The protein profile remained stable for up to five freeze/thaw cycles. The addition of denaturing solutions and the depletion of glycoproteins improved the quality of the spectra without affecting their reproducibility. Conclusions: We defined a protocol that improved the quality and the reproducibility of SELDI-TOF/MS analysis, thus potentially supporting the search for putative biomarkers of disease. Clin Chem Lab Med 2008;46:89–99.

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells.

Glucocorticoids have long been used as first‐line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein‐1 (MCP‐1), and its relationship with interleukin‐12 and interleukin‐10 synthesis, in human monocyte‐derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN‐γ+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real‐time PCR and ELISA, respectively. Our results showed that dexamethasone‐primed mature dendritic cells expressed low levels of interleukin‐12, and, at the opposite, high levels of interleukin‐10 and MCP‐1. Transfection experiments confirmed the ability of dexamethasone to activate MCP‐1 gene promoter. Dexamethasone increased also MCP‐2, but not MCP‐3 synthesis, and the gene expression of CC chemokine receptor‐2 by mature dendritic cells. The addition of anti‐MCP‐1 blocking antibody depressed MCP‐1 release, and increased interleukin‐12 production in dexamethasone‐treated dendritic cells, thus demonstrating that interleukin‐12 downregulation is largely dependent on MCP‐1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T‐helper‐biased immune response and, possibly, to favor type 2 T‐helper‐skewed response.