We studied the distribution of nephrin in renal biopsies from 17 patients with diabetes and nephrotic syndrome (7 type 1 and 10 type 2 diabetes), 6 patients with diabetes and microalbuminuria (1 type 1 and 5 type 2 diabetes), and 10 normal subjects. Nephrin expression was semiquantitatively evaluated by measuring immunofluorescence intensity by digital image analysis. We found an extensive reduction of nephrin staining in both type 1 (67 ؎ 9%; P < 0.001) and type 2 (65 ؎ 10%; P < 0.001) diabetic patients with diabetes and nephrotic syndrome when compared with control subjects. The pattern of staining shifted from punctate/linear distribution to granular. In patients with microalbuminuria, the staining pattern of nephrin also showed granular distribution and reduction intensity of 69% in the patient with type 1 diabetes and of 62 ؎ 4% (P < 0.001) in the patients with type 2 diabetes. In vitro studies on human cultured podocytes demonstrated that glycated albumin and angiotensin II reduced nephrin expression. Glycated albumin inhibited nephrin synthesis through the engagement of receptor for advanced glycation end products, whereas angiotensin II acted on cytoskeleton redistribution, inducing the shedding of nephrin. This study indicates that the alteration in nephrin expression is an early event in proteinuric patients with diabetes and suggests that glycated albumin and angiotensin II contribute to nephrin downregulation.

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

OBJECTIVEThe cannabinoid receptor type 2 (CB2) has protective effects in chronic degenerative diseases. Our aim was to assess the potential relevance of the CB2 receptor in both human and experimental diabetic nephropathy (DN).RESEARCH DESIGN AND METHODSCB2 expression was studied in kidney biopsies from patients with advanced DN, in early experimental diabetes, and in cultured podocytes. Levels of endocannabinoids and related enzymes were measured in the renal cortex from diabetic mice. To assess the functional role of CB2, streptozotocin-induced diabetic mice were treated for 14 weeks with AM1241, a selective CB2 agonist. In these animals, we studied albuminuria, renal function, expression of podocyte proteins (nephrin and zonula occludens-1), and markers of both fibrosis (fibronectin and transforming growth factor-β1) and inflammation (monocyte chemoattractant protein-1 [MCP-1], CC chemokine receptor 2 [CCR2], and monocyte markers). CB2 signaling was assessed in cultured podocytes.RESULTSPodocytes express the CB2 receptor both in vitro and in vivo. CB2 was downregulated in kidney biopsies from patients with advanced DN, and renal levels of the CB2 ligand 2-arachidonoylglycerol were reduced in diabetic mice, suggesting impaired CB2 regulation. In experimental diabetes, AM1241 ameliorated albuminuria, podocyte protein downregulation, and glomerular monocyte infiltration, without affecting early markers of fibrosis. In addition, AM1241 reduced CCR2 expression in both renal cortex and cultured podocytes, suggesting that CB2 activation may interfere with the deleterious effects of MCP-1 signaling.CONCLUSIONSThe CB2 receptor is expressed by podocytes, and in experimental diabetes, CB2 activation ameliorates both albuminuria and podocyte protein loss, suggesting a protective effect of signaling through CB2 in DN.

Platelet-activating factor (PAF)' is a mediator of inflammation with a wide range of biological activities (see reference 1 for review) . PAF was initially recognized as a product of IgE-sensitized rabbit basophils (2) and was identified with 1-0-alkyl-2-sn-glyceryl-3-phosphorylcholine (3-5). It was subsequently shown that PAF is synthesized after appropriate stimulation by monocytes/macrophages (6-9), polymorphonuclear neutrophils (7, 10, 11), platelets (12) and endothelial cells (13-15) . PAF induces aggregation and degranulation of platelets (2, 16), stimulates contraction of smooth muscle (17), promotes chemotaxis and granule secretion of neutrophils (18-19) and monocytes (20), increases vascular permeability, and alters the vascular tone (21).It was recently suggested that PAF is a mediator of endotoxic shock (22-24) on the basis of the following observations : (a) PAF is produced during endotoxic shock and experimental sepsis by Gram-negative bacteria (23-25) ; (b) infusion of experimental animals with PAF results in hypotension, decrease in cardiac output, and hypovolemic shock (26-28) ; (c) three PAF receptor antagonists (CV3988, kadsurenone, and SRI 63072) inhibit or reverse endotoxin-induced hypotension and, in this way, prolong the survival of rats (22,23,29) .Tumor necrosis factor/cachectin (TNF) is a mediator of endotoxic shock (30). Because TNF administration to experimental animals reproduces several aspects of PAF infusion (30), it seems possible that PAF is synthesized in response to

OBJECTIVEMonocyte chemoattractant protein-1 (MCP-1), a chemokine binding to the CC chemokine receptor 2 (CCR2) and promoting monocyte infiltration, has been implicated in the pathogenesis of diabetic nephropathy. To assess the potential relevance of the MCP-1/CCR2 system in the pathogenesis of diabetic proteinuria, we studied in vitro if MCP-1 binding to the CCR2 receptor modulates nephrin expression in cultured podocytes. Moreover, we investigated in vivo if glomerular CCR2 expression is altered in kidney biopsies from patients with diabetic nephropathy and whether lack of MCP-1 affects proteinuria and expression of nephrin in experimental diabetes.RESEARCH DESIGN AND METHODSExpression of nephrin was assessed in human podocytes exposed to rh-MCP-1 by immunofluorescence and real-time PCR. Glomerular CCR2 expression was studied in 10 kidney sections from patients with overt nephropathy and eight control subjects by immunohistochemistry. Both wild-type and MCP-1 knockout mice were made diabetic with streptozotocin. Ten weeks after the onset of diabetes, albuminuria and expression of nephrin, synaptopodin, and zonula occludens-1 were examined by immunofluorescence and immunoblotting.RESULTSIn human podocytes, MCP-1 binding to the CCR2 receptor induced a significant reduction in nephrin both mRNA and protein expression via a Rho-dependent mechanism. The MCP-1 receptor, CCR2, was overexpressed in the glomerular podocytes of patients with overt nephropathy. In experimental diabetes, MCP-1 was overexpressed within the glomeruli and the absence of MCP-1 reduced both albuminuria and downregulation of nephrin and synaptopodin.CONCLUSIONSThese findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.