[show abstract][hide abstract] ABSTRACT: The 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).
CB2 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.
Podocytes 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.
The 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.
[show abstract][hide abstract] ABSTRACT: Monocyte 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.
Expression 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.
In 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.
These findings suggest that the MCP-1/CCR2 system may be relevant in the pathogenesis of proteinuria in diabetes.
[show abstract][hide abstract] ABSTRACT: In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases.
American Journal Of Pathology 01/2008; 171(6):1789-99. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The molecular events associated with acute and chronic exposure of mesangial cells (MC) to hyperglycemia were evaluated. We found that, unlike high glucose (HG) and Amadori adducts, advanced glycation end products (AGE) and transforming growth factor-beta (TGF-beta) induced p21waf expression and accumulation of MC in G0/G1. TGF-beta1 blockade inhibited AGE-mediated collagen production but only partially affected AGE-induced p21waf expression and cell-cycle events, indicating that AGE by binding to AGE receptor (RAGE) per se could control MC growth. Moreover, AGE and TGF-beta treatment led to the activation of the signal transduction and activators of transcription (STAT)5 and the formation of a STAT5/p21SIE2 complex. The role of STAT5 in AGE- and TGF-beta-mediated p21waf expression and growth arrest, but not collagen production, was confirmed by the expression of the dominant negative STAT5 (DeltaSTAT5) or the constitutively activated STAT5 (1*6-STAT5) constructs. Finally, in p21waf-/- fibroblasts both AGE and TGF-beta failed to inhibit cell-cycle progression. A potential in vivo role of these mechanisms was sustained by the increasing immunoreactivity for the activated STAT5 and p21(waf) in kidney biopsies from early to advanced stage of diabetic nephropathy. Our data indicate that AGE- and TGF-beta-mediated signals, by converging on STAT5 activation and p21waf expression, may regulate MC growth.
The FASEB Journal 09/2004; 18(11):1249-51. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The long pentraxin PTX3 has been recently involved in amplification of the inflammatory reactions and regulation of innate immunity. In the present study we evaluated the expression and role of PTX3 in glomerular inflammation. PTX3 expression was investigated in the IgA, type I membranoproliferative, and diffuse proliferative lupus glomerulonephritis, which are characterized by inflammatory and proliferative lesions mainly driven by resident mesangial cells, and in the membranous glomerulonephritis and the focal segmental glomerular sclerosis, where signs of glomerular inflammation are usually absent. We found an intense staining for PTX3 in the expanded mesangial areas of renal biopsies obtained from patients with IgA glomerulonephritis. The pattern of staining was on glomerular mesangial and endothelial cells. Scattered PTX3-positive cells were also detected in glomeruli of type I membranoproliferative glomerulonephritis. The concomitant expression of CD14 suggests an inflammatory origin of these cells. Normal renal tissue and biopsies from patients with the other glomerular nephropathies studied were mainly negative for PTX3 expression in glomeruli. However, PTX3-positive cells were detected in the interstitium of nephropathies showing inflammatory interstitial injury. In vitro, cultured human mesangial cells synthesized PTX3 when stimulated with TNF-alpha and IgA and exhibited specific binding for recombinant PTX3. Moreover, stimulation with exogenous PTX3 promoted mesangial cell contraction and synthesis of the proinflammatory lipid mediator platelet-activating factor. In conclusion, we provide the first evidence that mesangial cells may both produce and be a target for PTX3. The detection of this long pentraxin in the renal tissue of patients with glomerulonephritis suggests its potential role in the modulation of glomerular and tubular injury.
The Journal of Immunology 03/2003; 170(3):1466-72. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
American Journal Of Pathology 06/2001; 158(5):1723-31. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: A high incidence of alpha 1-antitrypsin (AAT) deficiency has been reported in patients with C-ANCA systemic vasculitis in association with antibodies against proteinase-3 (PR3). To clarify the role of AAT deficiency in the acute vasculitic process as well as in progression of the disease, we studied 84 patients with either C-ANCA or P-ANCA vasculitis with special reference to: (a) the AAT gene, (b) the phenotypic (Pi) variants and (c) the serum levels during both acute illness and remission. The PiZ gene was found in six patients (8% vs. 1.5% controls) irrespective of the type of autoantibodies (C-ANCA vs. P-ANCA). All PiZ patients displayed the ability to raise their AAT serum levels up to the normal range during acute illness. In contrast, 24 patients with the PiM phenotype presented low AAT serum levels during acute illness. In all these patients, the AAT levels returned to normal values during the remission. Low AAT levels were associated with low levels of C-reactive protein (PCR) (P < 0.001), with a less severe renal involvement or a minor risk of death, and, in one tested patient, with a novel point mutation (TCGA-->TCAA) at the enhancer-promoter region of the AAT gene. Low AAT serum levels did not correlate with either type/titre of autoantibody or distribution/severity of the vasculitis process. In the case-control study, high AAT levels emerged as a major determinant of progression towards end-stage renal failure [odds ratio 3 (95% CI 1.1-8.4)]. These results indicate: (a) a high incidence of the PiZ gene of AAT in systemic vasculitis irrespective of the type of autoantibodies; (b) a novel form of AAT deficiency associated with the normal PiM phenotype becoming manifest only during acute illness; (c) dysregulation of the acute-phase response affecting selectively AAT or both AAT and PCR; (d) correlation between low plasma levels of AAT and less severe renal involvement or risk of death.
European Journal of Clinical Investigation 08/1997; 27(8):696-702. · 3.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study focused on the utility of interferon gamma (IFN gamma) as an anti-fibrotic drug in renal experimental fibrosis; the nephropathy was induced by two doses of Adriamycin (ADR) in 20 Sprague Dawley rats, 10 of which were randomly assigned to receive IFN gamma (45,000 UI) on alternate day for 16 weeks. At the end of the follow up, ADR rats treated with IFN gamma developed massive proteinuria, slight renal insufficiency, and presented diffuse glomerulosclerosis, tubulo interstitial infiltration and fibrosis. No difference was found in the composition of tubulo-interstitial infiltrates, mainly consisting in CD4+T lymphocytes with a minor component of CD8+T cells, in comparison with rats treated with ADR alone. These observations demonstrate the inefficacy of a protracted high-dose treatment with IFN gamma in chronic experimental nephropathy with interstitial fibrosis.
Life Sciences 02/1994; 54(4):PL45-50. · 2.56 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, we examined the progression of chronic Adriamycin (ADR) nephropathy in mild leukopenic rats and tried to define the possible relationship between tubulointerstitial lesions and proteinuria in this model. Chronic ADR nephropathy was induced by 2 doses of ADR (2 mg/kg) in 32 Sprague-Dawley rats. Eight of these were randomly assigned to cyclophosphamide treatment (50 mg/kg), given intravenously every week, to keep the blood leukocyte count constantly lower than 5,000/mm3. Serial parameters were followed for 16 weeks including clearance studies with iothalamate and p-aminohippurate and the analysis of urinary protein composition by: (a) an enzymatic assay for beta-glucosidase; (b) specific ELISA using antibodies against rat albumin and RBP, and finally (c) two-dimensional electrophoresis. ADR-treated rats rapidly (within 2 weeks) developed massive proteinuria which was in constant increment throughout the disease evolution in each single component (i.e., high and low molecular weight proteinuria, enzymuria) and developed renal insufficiency. At week 8, in ADR rats, glomerulosclerosis was mild whereas tubulointerstitial infiltrates predominated, characterized mainly by CD4+ T lymphocytes while CD8+ T lymphocytes were inconspicuous, and macrophages were only occasionally present. All such alterations had worsened after 16 weeks when the tubulointerstitial infiltration was associated with marked interstitial fibrosis and tubular atrophy. Leukopenia induced by cyclophosphamide was in all cases associated with a net amelioration of renal histopathology reducing tubulointerstitial infiltrates (by 40%) and glomerulosclerosis (33 +/- 5 vs. 52.2 +/- 7.5% sclerotic glomeruli) and also ameliorated glomerular filtration indexes (Cl 780 +/- 40 vs. 447 +/- 66 microliters/min/kg-1). In spite of these differences, albuminuria and urinary-retinol-binding protein were comparable at weeks 4, 8 and 16 in this group, while urinary beta-glucosidase was decreased at week 16 (p < 0.001) in cyclophosphamide-treated rats. No other qualitative changes in urinary proteins were detectable by 2-dimensional electrophoresis during the disease development. We concluded that chronic leukopenia prevents interstitial cellular infiltration by lymphocytes, interstitial fibrosis and slows down the decline of renal function typical of chronic ADR nephropathy. Glomerulosclerosis is also reduced in leukopenic rats without any appreciable changes in the urinary excretion of high molecular weight proteins deriving from the glomerulus. Finally, the improvement in tubulointerstitial alteration is associated with the reduction in urinary lysosomal enzymes. Tubulointerstitial alterations are implicated with a prominent role in the progression towards renal failure in chronic ADR glomerulopathy.
[show abstract][hide abstract] ABSTRACT: Heymann nephritis in the rat is a proteinuric condition caused by glomerular lesions similar to those found in human membranous nephritis. In the present study the effect of two different receptor antagonists of platelet-activating factor (PAF) on the course of passive Heymann nephritis was assessed. It was found that rats treated with either antagonist showed the same degree of proteinuria and the same glomerular immunopathological features as untreated rats. Furthermore, at sacrifice, 7 days after the initiation of the disease, the concentration of circulating PAF in treated as well as untreated rats was normal. These results indicate that PAF is not crucial in the pathogenesis of Heymann nephritis.
[show abstract][hide abstract] ABSTRACT: Chlorpromazine blocks antibody-mediated redistribution of cell surface antigens in vitro and in vivo and inhibits the development of passive Heymann glomerulonephritis, a disease characterized by in situ formation of immune complexes (Camussi et al J Immunol 1986, 136:2127-2135). The aim of this study was to establish whether chlorpromazine exerts similar effects in other rat models characterized by in situ formation of immune complexes. In glomerulonephritis induced by antibodies reactive with an exogenous antigen "planted" in glomeruli pretreatment with chlorpromazine prevented formation of "humps" and exudative and proliferative lesions. Likewise, chlorpromazine prevented passive reverse Arthus reaction in the skin. In contrast, the drug was ineffective when these lesions were already established, and also failed to inhibit the fulminant course of nephrotoxic serum glomerulonephritis with an enhanced autologous phase. It is proposed that the antiinflammatory effect of chlorpromazine is due to its ability to block the recruitment of inflammatory cells and the release of inflammatory mediators.
American Journal Of Pathology 07/1988; 131(3):418-34. · 4.52 Impact Factor