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.

Reverse transcriptase inhibitors induce cell differentiation and enhance the immunogenic phenotype in human renal clear-cell carcinoma.

Reverse transcriptase (RT) inhibitors are emerging as a novel class of anticancer differentiating agents, active in several human tumor cell models, such as melanoma and prostate, thyroid and colon carcinoma. Indeed, much evidence suggests that they may act by inhibiting endogenous RT, a gene highly expressed in undifferentiated and transformed cells. We therefore evaluated whether endogenous RT may represent a new molecular target in the treatment of human renal clear-cell carcinoma, a neoplasm with very low sensitivity to standard pharmacological therapies. Efavirenz and nevirapine, 2 non-nucleosidic RT inhibitors commonly used in HIV patients, either induced a reversible downregulation of cell proliferation or enhanced cell differentiation in primary cultures of human renal carcinoma cells characterized by high levels of endogenous RT activity. Both agents upregulated the expression of the vitamin D receptor and calbindin 28k genes, which are constitutively expressed in renal tubular cells, and induced vitamin D signaling by enhancing the ability of tumor cells to upregulate the vitamin D-dependent gene, CYP24. Furthermore, efavirenz- and nevirapine-differentiated tumor cells exhibited an immunogenic phenotype with an increased expression of HLA-I and CD40 antigens and an enhanced ability to elicit a specific T-cell response in mixed lymphocyte/tumor-cell cultures. Indeed, renal carcinoma cells exposed to efavirenz induced a CD8(+)CCR7-CD45RA(-) effector memory T-cell phenotype, whereas untreated RCC cells induced a CD8(+)CCR7(+)CD45RA(-) central memory T-cell phenotype. These data suggest that RT inhibitors may be a novel tool in the treatment of human renal clear-cell carcinoma, potentially able to enhance the immunogenic potential of tumor cell.

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-gamma+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.