Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes

Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive

Human Cytomegalovirus Induces TGF-β1 Activation in Renal Tubular Epithelial Cells after Epithelial-to-Mesenchymal Transition

Human cytomegalovirus (HCMV) infection is associated epidemiologically with poor outcome of renal allografts due to mechanisms which remain largely undefined. Transforming growth factor-β1 (TGF-β1), a potent fibrogenic cytokine, is more abundant in rejecting renal allografts that are infected with either HCMV or rat CMV as compared to uninfected, rejecting grafts. TGF-β1 induces renal fibrosis via epithelial-to-mesenchymal transition (EMT) of renal epithelial cells, a process by which epithelial cells acquire mesenchymal characteristics and a migratory phenotype, and secrete molecules associated with extracellular matrix deposition and remodeling. We report that human renal tubular epithelial cells infected in vitro with HCMV and exposed to TGF-β1 underwent morphologic and transcriptional changes of EMT, similar to uninfected cells. HCMV infected cells after EMT also activated extracellular latent TGF-β1 via induction of MMP-2. Renal epithelial cells transiently transfected with only the HCMV IE1 or IE2 open reading frames and stimulated to undergo EMT also induced TGF-β1 activation associated with MMP-2 production, suggesting a role for these viral gene products in MMP-2 production. Consistent with the function of these immediate early gene products, the antiviral agents ganciclovir and foscarnet did not inhibit TGF-β1 production after EMT by HCMV infected cells. These results indicate that HCMV infected renal tubular epithelial cells can undergo EMT after exposure to TGF-β1, similar to uninfected renal epithelial cells, but that HCMV infection by inducing active TGF-β1 may potentiate renal fibrosis. Our findings provide in vitro evidence for a pathogenic mechanism that could explain the clinical association between HCMV infection, TGF-β1, and adverse renal allograft outcome

Characterization of the macrophage transcriptome in glomerulonephritis-susceptible and -resistant rat strains

Crescentic glomerulonephritis (CRGN) is a major cause of rapidly progressive renal failure for which the underlying genetic basis is unknown. WKY rats show marked susceptibility to CRGN, while Lewis rats are resistant. Glomerular injury and crescent formation are macrophage-dependent and mainly explained by seven quantitative trait loci (Crgn1-7). Here, we used microarray analysis in basal and lipopolysaccharide (LPS)-stimulated macrophages to identify genes that reside on pathways predisposing WKY rats to CRGN. We detected 97 novel positional candidates for the uncharacterised Crgn3-7. We identified 10 additional secondary effector genes with profound differences in expression between the two strains (>5-fold change, <1% False Discovery Rate) for basal and LPS-stimulated macrophages. Moreover, we identified 8 genes with differentially expressed alternatively spliced isoforms, by using an in depth analysis at probe-level that allowed us to discard false positives due to polymorphisms between the two rat strains. Pathway analysis identified several common linked pathways, enriched for differentially expressed genes, which affect macrophage activation. In summary, our results identify distinct macrophage transcriptome profiles between two rat strains that differ in susceptibility to glomerulonephritis, provide novel positional candidates for Crgn3-7, and define groups of genes that play a significant role in differential regulation of macrophage activity

lmmunomorphologica characteristics of renal cell carcinoma

Immunomorphological characteristics of 27 renal cell carcinoma (RCC): 18 clear cell, 6 granular (chromophilic), 2 chromophobe, 1 spindle cell (sarcomatoid) as well as of 1 oncocytoma, were analyzed. The investigation was performed on cryostat sections by immunoperoxidase technique applying a panel of monoclonal antibodies which defined: proximal (TNE3, TN5, 5D9) and distal (TN8, TN9, 7C2) tubular antigens; intercellular adhesion molecule 1 (ICAMl); HLA class I1 (-DQ, -DR and -DP) antigens, intermediary filaments (cytokeratin and vimentin); and antigens on tumour infiltrating mononuclear leucocytes (TT1, TT2 and LeuM3 for CD4, CD8 and CD14 antigens, respectively). All RCC with exception of chromophobe CO-expressed cytokeratin and vimentin. In addition, they were usually positive for all proximal and two distal tubular markers (TN8, TN9) indicating primitive cells which could differentiate into the epithelium of both parts of tubule system as the most probable originators of in RCC. Almost all RCC but the chromophobe aberrantly expressed HLA class I1 antigens which great variability from case to caie. The presence of HLA-DR antigens was more intensive and widespread than of HLA-DQ and-DP antigens. Expression of ICAMl mostly correlated with presence of HLA class I1 antigens, particularly with -DR on tumour cells of RCC HLA-DR antigen expression was always more prominent than mononuclear cell infiltrate (among which macrophages prevailed over T cells) which could suggest that increased histocompatibility antigen expression precedes mononuclear cell influx. In contrast to all other RCC, chromophobe tumours had quite distinct features revealing the most intense reaction with 7C2 (MAb that produced the weakest reaction with other tumour types), absence of vimentin and very weak reaction with antibodies for HLA class Il Ag and ICAM 1. Since oncocytoma has similar immunohistological properties it could be supposed that both tumours have common histogenesis

CD4+ T cells participate in the nephropathy of canine visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent. The renal lesions of humans and dogs are similar but their pathogenesis has not been clearly elucidated. There is growing evidence that the cellular immune response is involved in the pathogenesis of immunologically mediated glomerulonephritis. Since T cells could participate in the pathogenesis of nephropathy, in the present study we investigated the possible involvement of CD4+ and CD8+ T cells in the nephropathy of canine VL. Six dogs naturally infected with Leishmania (Leishmania) chagasi from the endemic area in the Northeast of Brazil, the town of Teresina in the State of Piauí, were studied. An expressive inflammatory infiltrate of CD4+ T cells both in glomeruli and in interstitium was present in 4 animals and absent in 2. CD8+ T cells were detected only in one animal. CD4+ T cells alone were observed in 3 animals; when CD8+ T cells were present CD4+ T cells were also present. CD4+ T cells were observed in cases of focal segmental glomerulosclerosis, diffuse membranoproliferative glomerulonephritis, diffuse mesangial proliferative glomerulonephritis and crescentic glomerulonephritis. CD8+ T cells were present only in a case of crescentic glomerulonephritis. Leishmania antigen was detected in glomeruli and in interstitial inflammatory infiltrate in 4 animals and immunoglobulins were observed in 4 dogs. In this study we observed that T cells, in addition to immunoglobulins, are present in the renal lesion of canine VL. Further studies are in progress addressing the immunopathogenic mechanisms involving the participation of immunoglobulins and T cells in canine VL nephropathy

Patterns of interstitial inflammation during the evolution of renal injury in experimental aristolochic acid nephropathy.

BACKGROUND: Interstitial inflammation is a prominent feature associated with the severity of renal injury and progressive kidney failure. We utilized an animal model of aristolochic acid (AA)-induced nephropathy (AAN) to assess patterns of infiltration and inflammation during the evolution of tubulointerstitial damage and to relate them to the development of fibrosis. METHODS: Male Wistar rats receiving sc daily AA or vehicle were sacrificed between Days 1 and 35. Infiltrating mononuclear cells were characterized by immunohistochemistry. The kidney infiltrating T lymphocytes were phenotyped by flow cytometry. Urinary levels of Th-1/ Th-2 cytokines, of monocyte chemoattractant protein-1 and of active transforming growth factor-beta (TGF-beta) were measured. Tissue expression of phosphorylated smad 2/3 protein was used to examine the TGF-beta signalling pathway. RESULTS: In AA rats, monocytes/macrophages and T lymphocytes predominantly infiltrated areas of necrotic proximal tubular cells. The coexpressions of ED1 and/or Ki-67/MHCII by infiltrating cells reflected monocyte/macrophage proliferation and their activation, respectively. The accumulation of cytotoxic T lymphocytes was attested by severe signs of CD8+ cell tubulitis. The CD8/E-cadherin costaining confirmed intrarenal homing of CD8+CD103+ cells. Urinary levels of proinflammatory cytokines and of active TGF-beta significantly increased at Days 10 and 35. An early and persistent nuclear overexpression of phosphorylated smad 2/3 protein was detected in tubular and interstitial compartments. CONCLUSION: An early and massive interstitial inflammation characterized by activated monocytes/macrophages and cytotoxic CD8+CD103+ T lymphocytes is demonstrated for the first time during the progression of experimental AAN. The involvement in an interstitial fibrosis onset of active TGF-beta is highly suggested, at least via the psmad 2/3 intracellular signalling pathway.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe