Effects of amino acids and glucose on mesangial cell aminopeptidase a and angiotensin receptors.
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BACKGROUND: High protein diets and diabetes increase renal renin angiotensin system (RAS) activity, which is associated with glomerular injury. Aminopeptidase A (APA) is a cell surface metalloprotease that degrades angiotensin II (AII) in the mesangium. Mesangial cells (MC) also possess receptors for AII; the type 1 (AT1 receptor) promotes proliferation and fibrosis, while the type 2 (AT2 receptor) opposes these effects. We evaluated whether amino acids and glucose alter expression of APA, AT1 receptor and AT2 receptor in a manner that further augments RAS activity. METHODS: Confluent rat MC were grown in serum-free media for 48 hours prior to exposing to experimental conditions: control (C), high amino acids (HA, mixed amino acid solution added to raise concentrations 5- to 6-fold over C), high glucose (HG 30, mM glucose). Semi-quantitative RT-PCR was used to assess mRNA for APA, AT1 receptor, AT2 receptor, and beta-actin. Values are expressed relative to beta actin. RESULTS: Both HA and HG reduced APA mRNA (HG 1.13 plus minus 0.19, HA 1.12 plus minus 0.16 versus C 1.27 plus minus 0.16 P < 0.05, N = 8). HA increased AT1 receptor mRNA (HA 2.11 plus minus 0.43 versus C 1.14 plus minus 0.28 P < 0.05, N = 8). HG increased AT2 receptor mRNA (HG 1.31 plus minus 0.43 versus C 0.82 plus minus 0.33 P < 0.05, N = 6). CONCLUSIONS: A reduction of APA, in response to high levels of amino acids or glucose, could contribute to increased AII as a result of decreased degradation in MC. The effect of amino acids to increase AT1 receptor expression may further enhance adverse hemodynamic and pro-fibrotic actions of AII. Conversely, glucose increased AT2 receptor expression, which could modulate responses mediated by the AT1 receptor. (+info)
Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G.
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BACKGROUND: In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved. METHODS: Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated. RESULTS: The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial beta-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid beta-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-independent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis. CONCLUSIONS: Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA. (+info)
Antibodies against mesangial cells in a rat model of chronic renal allograft rejection.
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BACKGROUND: Chronic renal allograft rejection (CR) is the leading cause of late renal transplant failure. The histological lesions of CR may comprise glomerular basement membrane (GBM) duplications and mesangiolysis. Its pathogenesis is not yet completely understood, although lately humoral responses have been suggested to be important. Recently, we identified antibody responses directed against GBM antigens in the Fischer (F344) to Lewis (LEW) renal transplantation model. Immunofluorescent studies in this model also suggested deposition of antibodies on mesangial cells. Therefore, we hypothesized that antibodies were not only directed at GBM antigens but also to mesangial cell antigens. METHODS: F344 to LEW renal transplantations were performed and sera were collected. Pre- and post-transplantation sera were tested for antibody binding to donor rat mesangial cells (RMCs) cultured from F344 kidneys. Anti-mesangial cell antibodies were compared with anti-GBM antibodies measured in the same sera. RESULTS: Post-transplant sera of F344 to LEW renal transplantations, but not LEW to F344, bound to F344 RMC in a dose-dependent manner. Whereas antibodies reactive with RMCs were not present before transplantation, all rats with CR developed antibodies. The antibodies were predominantly of the IgG1 isotype. Antibody binding to RMCs correlated with binding to F344 GBM. Pre-incubation with RMCs partially inhibited GBM binding, and RMC binding was inhibited by GBM. Antibody binding to RMCs did not result in complement activation or cell lysis. CONCLUSION: LEW recipients of F344 grafts produce antibodies reactive with F344 RMCs. The antigens involved are similar to or at least share antigenic epitopes with antigens recognized in the GBM. (+info)
Tubular expression of angiotensin II receptors and their regulation in IgA nephropathy.
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Enhanced renal expression for the renin-angiotensin system (RAS) is detected in IgA nephropathy (IgAN). Previous data showed an altered glomerular expression of angiotensin II type 1 receptor (AT1R), suggesting a regulatory response to high intrarenal angiotensin II (Ang II) concentration in IgAN. In this study, the expression and regulation of Ang II receptors were examined in human proximal tubular epithelial cells (PTEC) in IgAN. Tubular expression of AT1R and Ang II type 2 receptor (AT2R) was increased in IgAN. In vitro culture experiment showed that the upregulation of Ang II receptors was not due to the direct effect of IgA but the indirect effect after IgA deposition on human mesangial cell. When PTEC were cultured with conditioned culture medium from human mesangial cells activated with IgA, Ang II production was upregulated, leading to inflammation and apoptosis via the AT1R and AT2R, respectively. Sequential expression of Ang II receptors determined the injury of PTEC induced by mediators in the conditioned medium. The initial interaction between Ang II and AT1R activated both protein kinase C and mitogen-activated protein kinase pathways, leading to inflammatory responses. This early AT1R-dependent event was followed by upregulation of AT2R expression and continued Ang II release. The interaction between Ang II and AT2R subsequently led to expression of cleaved poly[ADP-ribose] polymerase through downregulation of the mitogen-activated protein kinase pathway. The data suggest that appropriate control of Ang II receptor activities in PTEC may ameliorate tubulointerstitial injury in IgAN. (+info)
Similar effects on rat renal mesangial cells by expressing different fragments of adrenomedullin gene in vitro.
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AIM: To construct pEGFP-N3 recombinant vectors carrying adrenomedullin (AM) or fragments of the AM gene, and to express AM or fragments of AM from the pEGFP-N3 recombinant vectors (pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3) and study their biological properties on cultured rat renal mesangial cells (RMC). METHODS: Total RNA of rat kidney was obtained using TriZol reagent. The cDNA was synthesized by reverse transcriptase using oligo-deoxythymidine as primer. The fragments of AM gene were then amplified by polymerase chain reaction (PCR) with specific upstream and downstream oligonucleotides. The PCR products were digested with EcoRI and BamHI and subcloned into the plasmid pEGFP-N3. Facilitated by cationic liposomes, RMC were transfected with pEGFP-N3-AM1-2 or pEGFP-N3-AM1-3. After 24 h, green fluorescent protein (GFP) fluorescent images were examined with a fluorescence microscope. After 48 h, the proliferation of RMC was detected using the MTT assay, and the mRNA expression of transforming growth factor-beta1 (TGF-beta1) was measured by semiquantitative PCR. RESULTS: DNA sequence reports verified that pEGFP-N3-AM1-2, which carried the full length AM gene translation fragment (preproadrenomedullin preproAM1-185), and pEGFP-N3-AM1-3, which carried the translation fragment of preproAM [without adrenotensin (ADT, preproAM150-185)], were constructed successfully. After 24 h, green fluorescence was observed in RMC into which either pEGFP-N3-AM1-2 or pEGFP-N3-AM1-3 was transfected, while in the control cells no fluorescence was observed. Either pEGFP-N3-AM1-2 or pEGFP-N3-AM1-3 delivery inhibited the proliferation of RMC (P<0.01) and decreased the mRNA transcription level of TGF-beta1 in RMC (P<0.05). However, no significant difference was observed between the effects of pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3. CONCLUSION: pEGFP-N3-AM1-2 and pEGFP-N3-AM1-3 were constructed successfully and were functionally expressed in RMC. Expressing the fragment of AM without ADT has similar inhibitory biological effects on RMS proliferation and TGF-beta1 transcription with full length preproAM. (+info)
Aldosterone stimulates proliferation of mesangial cells by activating mitogen-activated protein kinase 1/2, cyclin D1, and cyclin A.
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Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11beta-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease. (+info)
Expression of matrix metalloproteinase-9 associated with ets-1 proto-oncogene in rat tubulointerstitial cells.
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BACKGROUND: Ets-1 proto-oncogene exhibits multiple activities in the transcriptional regulation of numerous genes including metalloproteinase (MMP)-1, -3 and -9. MMPs play an important role in the remodelling of extracellular matrix in various renal diseases. However, the role of the Ets-1-MMP axis in advanced renal diseases is uncertain. In the present study, we investigated whether Ets-1 is involved in interleukin (IL)-1-mediated expression of MMPs in tubulointerstitial cells. METHODS: Rat renal fibroblasts (NRK-49F) and tubular epithelial cells (NRK-52E) were cultured and allocated to an IL-1beta-treated group (10 ng/ml), a platelet-derived growth factor (PDGF)-BB-treated group (25 ng/ml) and a control group. Protein and mRNA were extracted after 1, 6, 12 and 24 h of treatment. Parallel flasks were treated with 2 muM ets-1 antisense oligodeoxynucleotides (ODNs) before exposure to IL-1beta. The expression of Ets-1 protein was evaluated by western blotting. The activities of MMPs were evaluated by gelatin zymography. The expression of ets-1 and/or MMP-9 mRNA was evaluated semiquantitatively by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In NRK-49F cells, Ets-1 protein increased significantly by 6.8-fold at 6 h, and MMP-9 activity increased significantly by 9.9-fold at 12 h in the IL-1beta-treated group compared with controls. MMP-2 and -3 activities also increased significantly in the IL-1beta-treated group. In NRK-52E cells, Ets-1 protein was 3.1 times higher at 1 h, and the latent form of MMP-9 activity increased 3.4-fold at 6 h in the IL-1beta group compared with controls. However, MMP-2 or MMP-3 activities were not markedly altered by IL-1beta treatment compared with controls. When the cells were treated with ets-1 antisense ODNs before IL-1beta treatment, Ets-1 protein expression decreased at least 50%, and MMP-9 activity was clearly inhibited in both cells. We also confirmed that MMP-9 activity was upregulated on days 21 and 28 in renal cortex of rat crescentic glomerulonephritis. CONCLUSIONS: The Ets-1 transcriptional factor may participate in IL-1beta-mediated MMP-9 expression in tubulointerstitial cells. (+info)
IGF-1 induces rat glomerular mesangial cells to accumulate triglyceride.
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Rat glomerular mesangial cells (MC) become lipid-laden foam cells when they are exposed to IGF-1. IGF-1 increased accumulation of triglyceride (TG) 2.5-fold in MC after 7 days. TG accumulation resulted from enhanced macropinocytosis and decreased efflux secondary to a 40-50% reduction in peroxisome proliferator-activated receptor (PPAR)-delta (PPARdelta). There was no evidence of primary or secondary changes in cholesterol or TG synthesis, increased uptake by LDL or scavenger receptors, or reduced efflux via ATP-binding cassette A-1. Although the lipid moiety taken up can be influenced by the concentration of cholesterol or TG in the medium, in standard medium MC preferentially accumulate TG. TG-rich MC foam cells fail to contract in response to angiotensin II (Berfield AK, Andress DL, and Abrass CK. Kidney Int 62: 1229-1237, 2002); however, their migratory response to IGF binding protein-5 is unaffected. This differs from cholesterol loading, which impairs both phagocytosis and migration. These findings have important implications for understanding the mechanisms that contribute to lipid accumulation in MC and the functional consequences of different forms of foam cells. These observations are relevant to understanding vascular disease and progressive renal diseases that are accelerated by abnormalities in lipid metabolism. (+info)