Elena Ranieri

Disease-associated Bias in T Helper Type 1 (Th1)/Th2 CD4+ T Cell Responses Against MAGE-6 in HLA-DRB10401+ Patients With Renal Cell Carcinoma or Melanoma

T helper type 1 (Th1)-type CD4+ antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4+ T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4+ T cells from human histocompatibility leukocyte antigens (HLA)-DRβ1*0401+ patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-γ and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6–derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus– or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4+ T cell secretion of IL-10 and transforming growth factor (TGF)-β1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4+ subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4+ T cell responses to provide optimal clinical benefit.

MONOCYTE CHEMOTACTIC PEPTIDE-1 EXPRESSION AND MONOCYTE INFILTRATION IN ACUTE RENAL TRANSPLANT REJECTION

Mononuclear cell infiltration is a common histopathological feature of acute renal transplant rejection, in which it seems to play a key role in the pathogenesis of tubulointerstitial lesions. Monocyte chemotactic peptide-1 (MCP-1) is a specific chemotactic and activating factor for monocytes. Thus, the present study was aimed at evaluating MCP-1 gene and protein expression in renal biopsies of kidney transplant recipients with acute deterioration of graft function, and to correlate it with the extent of monocyte infiltration. We studied 20 kidney transplant recipients with acute graft dysfunction (13 with acute rejection, seven with acute tubular damage). MCP-1 gene and protein expression were analyzed by in situ hybridization and immunohistochemistry, respectively. CD68-positive cells were identified as monocytes. CD68-positive cell number and MCP-1 expression were quantified by a computerized image analysis system. MCP-1 gene expression, undetectable in normal human kidneys, was strikingly increased in patients with acute rejection. The chemokine localized mainly to the proximal tubular cells and to mononuclear-infiltrating cells. In patients with acute tubular damage, the MCP-1 expression, even if higher than in controls, was significantly lower than in acute rejection. The expression of the chemokine strictly correlated with the number of infiltrating monocytes (r=0.87, P<0.05). Moreover, we measured MCP-1 urinary excretion by ELISA, in eight normal subjects (36+/-16 pg/mg urine creatinine), in 13 clinically stable transplant recipients (33+/-9 pg/mg, ns vs. normal patients), in 12 transplant recipients with acute rejection (250+/-46 pg/mg, P<0.01 vs. normal patients), and in five transplant recipients with acute tubular damage (97+/-33 pg/mg, P<0.05 vs. controls and patients with acute rejection). Urinary MCP-1 excretion directly correlated with renal MCP-1 gene expression (r=0.65, P=0.05). Finally, we observed a significant reduction in MCP-1 urine levels in patients with acute rejection, who responded to the antirejection treatment. In conclusion, our data suggest that MCP-1 may play a critical role in modulating monocyte influx and consequent tubulointerstitial damage in acute rejection. Therefore, an increase in urinary MCP-1 excretion may represent an early signal of ongoing acute graft rejection.

Expression of platelet-derived growth factor receptors in normal and diseased human kidney. An immunohistochemistry and in situ hybridization study.

We studied the expression of PDGF-alpha and -beta receptors in 10 normal and 40 pathologic human kidneys (five minimal change disease, five membranous nephropathy, 25 IgA nephropathy, five lupus nephritis), by both immunohistochemistry and in situ hybridization techniques. In normal-appearing kidneys, both PDGF-alpha and -beta receptors were expressed at the glomerular and interstitial level, the latter receptor more intensely than the former. The distribution and degree of expression of both receptors in nonproliferative glomerulonephritides were comparable with those found in normal-appearing kidneys. PDGF-beta receptor gene and protein expression were upregulated in proliferative nephritides both at the glomerular and the interstitial level and strictly correlated with the grade of histologic lesions. Finally, PDGF beta receptor expression was observed at a low level in normal-appearing renal vessels, and strikingly increased in injured arteries. Diseased kidneys displayed only a slight increase of PDGF-alpha receptor expression, chiefly at the interstitial level. Noteworthy, a few cases of lupus nephritis showed a moderate increase of PDGF-alpha receptor also at the glomerular level. These data establish PDGF-beta receptor activation as a candidate for driving glomerular and interstitial proliferation and, probably, expansion of extracellular matrix in proliferative glomerulonephritis, while the role of PDGF-alpha receptor activation at the renal level remains to be elucidated.

CD2AP mutations are associated with sporadic nephrotic syndrome and focal segmental glomerulosclerosis (FSGS)

BACKGROUND CD2-associated protein (CD2AP) is a crucial protein for the slit-diaphragm assembly and function. In spite of the fact that CD2AP knockout causes nephrotic syndrome in mice and the heterozygous +/- mouse is prone to proteinuria, little is known about the relevance of this molecule in human renal pathology. METHODS A total of 80 Italian patients with idiopathic nephrotic syndrome were enrolled and screened for changes in the CD2AP gene. A normal control group of 200 healthy donors was also studied. The coding region of the CD2AP gene was analysed by polymerase chain reaction, denaturing high-performance liquid chromatography and sequencing. Peripheral blood mononuclear cells from patients with CD2AP mutations and from healthy donors were isolated by the Ficoll-Hypaque gradient, and the CD2/CD2AP interaction was studied on T-lymphocytes by confocal laser scanning microscopy analysis. The expression levels of CD2AP, nephrin and podocin proteins were evaluated by indirect immunofluorescence on renal biopsies from a patient with p.delGlu525 mutation and from control subjects. Moreover, the effect of the p.K301M mutation on cell viability was evaluated by flow cytometry and annexin V/propidium iodide staining. RESULTS Three heterozygous mutations (c.904A>T; c.1120A>G; c.1573delAGA) producing respectively aminoacidic changes (p.K301M, p.T374A) or a deletion in functional domains (p.delGlu525) were found in three unrelated patients. One (p.K301M) produced a lysine to methionine change in the third interactive SH3 domain (position 301) and resulted in the defective CD2-CD2AP interaction and clustering; the other (c.1573delAGA) caused the deletion of the glutamic acid in position 525 in the COOH-terminal region of binding with nephrin and was associated with down-modulation of CD2AP, podocin and nephrin glomerular expression. CONCLUSIONS Our findings suggest that CD2AP mutations modify the interaction with CD2 in lymphocytes and alter the composition of the renal slit diaphragm.

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.

Protease-Activated Receptor-2 Expression in IgA Nephropathy: A Potential Role in the Pathogenesis of Interstitial Fibrosis

An increasing body of evidence suggests that proteases may play a key role in the pathogenesis of tissue fibrosis. Protease-activated receptor-2 (PAR-2) is cleaved and activated by trypsin-like proteolytic enzymes, including tryptase and activated coagulation factor X (FXa). Both these soluble mediators have been demonstrated, directly or indirectly, at the interstitial level in progressive renal diseases, including IgA nephropathy (IgAN). PAR-2 mRNA and protein levels were investigated by RT-PCR and immunohistochemistry, respectively, in 17 biopsies from IgAN patients and 10 normal kidneys. PAR-2 expression was also evaluated, by RT-PCR and western blotting, in cultured human mesangial and proximal tubular cells. Finally, gene expression of plasminogen activator inhibitor-1 (PAI-1) and TGF-beta, two powerful fibrogenic factors, was evaluated in FXa-, trypsin-, and PAR-2 activating peptide-stimulated human proximal tubular cells by Northern blot. In normal kidneys, PAR-2 gene expression was barely detectable, whereas in IgAN biopsies the mRNA levels for this protease receptor were strikingly increased and directly correlated with the extent of interstitial fibrosis. Immunohistochemical staining demonstrated that PAR-2 protein expression in IgAN biopsies was mainly localized in the proximal tubuli and within the interstitial infiltrate. Proximal tubular cells in culture expressed PAR-2. Activation of this receptor by FXa in tubular cells induced a striking increase in intracellular calcium concentration. In addition, incubation of both cell lines with trypsin, FXa, or PAR-2 activating peptide caused a marked upregulation of PAI-1 gene expression that was not counterbalanced by an increased expression of plasminogen activators. Finally, PAR-2 activation induced a significant upregulation of TGF-beta gene and protein expression in both mesangial and tubular cells. On the basis of our data, we can suggest that PAR-2 expressed by renal resident cells and activated by either mast cell tryptase or FXa may induce extracellular matrix deposition modifying the PAI-1/PA balance and inducing TGF-beta expression. These molecular mechanisms may underlie interstitial fibrosis in IgAN.

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.

Current insights in renal cell cancer pathology

In recent years molecular biologists and pathologists have described new entities of renal cell cancer (RCC) with a totally different morphology and biology among the histotypes of renal carcinoma, but always referring to the same renal cancer disease. The evidence of a distinct biological behavior and long-term prognosis among these makes the correct pathological diagnosis of renal cancer critically important for the clinician. Advances in understanding of the pathogenesis, behavior, and importance of prognostic factors for RCC have paved the way for a revision of its classification and staging. We reviewed the role of histological classification, microscopic tumor necrosis, microscopic venous invasion, lymph node involvement and, particularly, pathological stage. In our series of patients who underwent renal surgery for neoplasm, a retrospective study established the predictive role of tumor size on recurrence rate, compared with other known prognostic factors, and we conclude that histological grade, pathological stage and tumor size remain relevant prognosticators in early stage RCC patients. In order to optimize the management of patients with RCC it is necessary to develop an interdisciplinary approach (surgeon, radiologist, pathologist, oncologist) and find new prognostic parameters at molecular and cellular levels. Many efforts are ongoing to integrate molecular data (from tissue microarrays) and clinical data (traditional prognosticators) into a molecular integrated staging system. In the postgenomic era, new tumor-associated antigens and molecules can be identified at the protein level using proteomics, providing a major opportunity for screening and finding novel targets that are the basis of new emerging therapies for RCC.

TRPC6 Mutations in Children with Steroid-Resistant Nephrotic Syndrome and Atypical Phenotype

BACKGROUND AND OBJECTIVES Mutations in the TRPC6 gene have been recently identified as the cause of late-onset autosomal-dominant focal segmental glomerulosclerosis (FSGS). To extend the screening, we analyzed TRPC6 in 33 Italian children with sporadic early-onset SRNS and three Italian families with adult-onset FSGS. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS TRPC6 mutation analysis was performed through PCR and sequencing. The effects of the detected amino acid substitutions were analyzed by bioinformatics tools and functional in vitro studies. The expression levels of TRPC6 and nephrin proteins were evaluated by confocal microscopy. RESULTS Three heterozygous missense mutations (c.374A>G_p.N125S, c.653A>T_p.H218L, c.2684G>T_p.R895L) were identified. The first new mutation, p.H218L, was found in a 18-year-old boy who presented a severe form of FSGS at the age of 8 years. The second, p.R895L, a new de novo mutation, was identified in a girl with collapsing glomerulosclerosis at the age of 2 years. The former mutation, p.N125S, was found in two siblings with early-onset steroid-resistant nephrotic syndrome (SRNS) at the ages of 4 and 14 years. Renal immunofluorescence revealed upregulated expression of TRPC6 and loss of nephrin in glomeruli. The intracellular calcium concentrations were significantly higher in the cells expressing all mutant TRPC6 channels compared with cells expressing wild-type TRPC6. CONCLUSIONS Our findings suggest that TRPC6 variants can also be detected in children with early-onset and sporadic SRNS (4 of 33 patients). Moreover, in one patient a new de novo TRPC6 mutation was associated with a rare severe form of childhood collapsing glomerulosclerosis with rapid progression to uremia.

RAPAMYCIN INHIBITS PAI-1 EXPRESSION AND REDUCES INTERSTITIAL FIBROSIS AND GLOMERULOSCLEROSIS IN CHRONIC ALLOGRAFT NEPHROPATHY

Background. Chronic allograft nephropathy (CAN) is characterized by deposition of extracellular matrix (ECM) in all renal compartments. PAI-1 seems to play a pivotal role in ECM turnover in CAN. Rapamycin has been shown to improve long-term graft survival in patients with CAN. The aim of the study was to evaluate the molecular mechanisms underlying the beneficial effects of rapamycin on CAN progression at glomerular and tubulointerstitial level. Methods. After a biopsy-proven CAN diagnosis (T0), 18 patients on calcineurin inhibitors (CNI) were randomly assigned in a 2:1 ratio to continue CNI (6 patients) or to receive rapamycin (RAPA; 12 patients). After 2 years of treatment (T24), all patients underwent a second renal biopsy. Morphometric analysis was conducted at T0 and at T24. PAI-1 expression was evaluated at T0 and T24 by immunohistochemistry. We evaluated the effect of rapamycin on PAI-1 gene expression in cultured proximal tubular cells incubated with CD40L or thrombin, two potential CAN pathogenic mediators. Results. The RAPA group showed a significant regression of glomerulosclerotic lesions and only a 26% increase in interstitial fibrosis after 2 years compared to baseline, whereas the CNI group showed progression of glomerulosclerosis and 112% increase in fibrosis. Glomerular and tubulointerstitial PAI-1 expression was reduced compared to the baseline in the RAPA group, while they were unchanged in the CNI group. In vitro data showed that rapamycin significantly reduced PAI-1 gene expression induced by both CD40L and thrombin in proximal tubular epithelial cells. Conclusions. These data suggest that rapamycin may modulate ECM deposition in CAN reducing PAI-1 expression.