Effect of chronic renal failure medium on the ubiquitin­proteasome pathway of arterial muscle cells.

The ubiquitin­proteasome pathway (UPP) is involved in the occurrence and development of atherosclerosis through inhibitor of κB (IκB) degradation which activates nuclear factor-κB (NF­κB). However, the correlation between UPP and vascular complications of uramia remains unknown. The aim of the present study was to determine whether the UPP is activated in aortic smooth muscle cells (ASMCs) when cultured with uremic serum and to examine the role of the UPP on the dysfunction of ASMCs in uremia. ASMCs were cultured with pooled normal sera or chronic renal failure sera. The mRNA expression levels for ubiquitin (Ub) and Ub-activating enzyme (E1) were analyzed using reverse transcription PCR and levels of the ubiquitinated proteins E1 and IκBα were measured using western blot analysis. The enzymatic activities of three 20S proteasomes were examined using specific fluorogenic peptide substrates. Compared with normal serum, chronic renal serum increased E1 mRNA and protein expression of rabbit ASMCs (both P<0.01). In addition, the mRNA expression of Ub also increased and the expression of IκBα was observed to decrease significantly (both P<0.01). Ubiquitinated proteins in the normal and chronic renal failure groups were not found to be significantly different, but the activity of proteasomes increased significantly (P<0.01). Chronic renal failure medium induced the activation of the UPP in ASMCs.

Uraemic serum induces dysfunction of vascular endothelial cells: role of ubiquitin-proteasome pathway.

The ubiquitin-proteasome pathway (UPP) has been indicated to contribute to dysfunction of endothelial cells (ECs). Nevertheless, the relationship between UPP and vascular complications of uraemia remains unknown. We aimed to determine whether the UPP is activated in vascular ECs when cultured with uraemic serum, and to examine the role of the UPP on dysfunction of ECs in uraemia. Rabbit aortic endothelial cells (RAECs) were cultured with normal serum or different concentrations of uraemic serum. The expression of the ubiquitin-activating enzyme (E1), an indicator of the UPP, was detected by real-time RT-PCR and Western blot; proteasome activity was determined by fluorescence spectrophotometry; and nuclear factor-κB (NF-κB) activity and expression, as well as tumour necrosis factor-α (TNF-α) expression, were also detected. We found that the expression of E1 and the activities of three kinds of proteasomes were increased significantly in RAECs after incubation with uraemic serum. Proliferation of RAECs was increased significantly by incubation with 3-15% uraemic serum but decreased markedly when incubated with uraemic serum above 15% (increased apoptosis). Incubation of RAECs with uraemic serum induced increased NF-B DNA-binding activity and nuclear translocation of NF-κB, decreased nitric oxide production and increased expression of TNF-α, which is the final effector of inflammatory activation of cells. All of these responses in RAECs were suppressed by the specific proteasome inhibitor, MG132. The inhibition of inflammatory responses by MG132 was further supported by a parallel experiment with pyrrolidine dithiocarbamate, a specific inhibitor of κNF-B. These findings suggest that the UPP was activated in RAECs by administration of uraemic serum, and played a pivotal role in the dysfunction of vascular ECs, such as inflammatory activation.

[Potential effect of uremic medium on cell proliferation and apoptosis and the activation of nuclear factor-kappa B pathway of aortic endothelial cells in rabbit culture].

OBJECTIVE: To investigate the potential effect of uremic medium on cell proliferation and apoptosis of aortic endothelial cell (AEC), two key processes in the development of atherosclerosis, in rabbit culture. And to understand the effects of uremic medium on the activation of nuclear factor-kappa B (NF-κB) pathway and cytokines expression of AEC. METHODS: Rabbit AEC were cultured with growth media supplemented with pooled sera from normal rabbits or those with chronic renal failure. The 80% confluent AEC were incubated for 24 h with media supplemented with pools of control or uremic sera. Cell proliferation was assessed by a MTT assay and cell cycle detected by flow cytometry. Hoechst33342 assay and flow cytometry were used to investigate the apoptotic effect of uremic medium in AEC. The expression of mRNA and protein levels for NF-κB, IκBα were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. NF-κB P65 nuclear translocation was analyzed by immunofluorescence. The activity of NF-κB was measured by electrophoretic mobility shift assay (EMSA). Concentrations of TNF-α and IL-6 in culture supernatants were evaluated by ELISA, and the expression of protein for TNF-α in cell lysates by Western blot. RESULTS: Uremic medium induced proliferation in the lower concentration range of 3%-10% while promoted apoptosis in the higher concentrations (> 10%). Uremic serum increased NF-κB mRNA (0.35 ± 0.05 vs 0.26 ± 0.02, P < 0.01) and protein (1.67 ± 0.15 vs 0.41 ± 0.05, P < 0.01) expression, decreased IκBα mRNA (0.13 ± 0.03 vs 0.24 ± 0.04, P < 0.01) and protein (0.29 ± 0.06 vs 0.65 ± 0.08, P < 0.01) expression. Uremic serum enabled NF-κB p65 nuclear translocation and increased NF-κB DNA binding activity. An increased secretion of cytokines IL-6 and TNF-α. in AEC was observed after a treatment of 10% uremic sera in a time dependent manner. The expression of TNF-α in AEC exposed to 10% uremic sera also increased significantly (0.37 ± 0.04 vs 0.14 ± 0.03, P < 0.01). CONCLUSION: Uremic medium induces the activation of AEC. A lower level of uremic medium accelerates the proliferation of AEC while a higher level induces the apoptosis of AEC. The increased proliferation may be related to a higher NF-κB activity and the expression of inflammation cytokines. Although the enhanced atherosclerosis can not be explained on the basis of an apoptotic process, the proliferative status can contribute to intimal proliferation, an earlier step in the development of atherosclerosis.

[Protective effects of metanephric mesenchymal cells on acute renal tubular damage: experiment with rats].

OBJECTIVE: To study the protective effects of metanephric mesenchymal cells (MMCs) on acute renal tubular damage and explore its possible mechanism. METHODS: MMCs were isolated and cultured from 13-day-old embryonic rats and labeled with 5-bromodeoxyuridine. Seventy-two male SD rats were randomly divided into 3 equal groups: MMC group, receiving MMC injection instantaneously when ischemia/reperfusion (I/R) renal injury was induced, I/R group, undergoing I/R to establish acute renal tubular damage models, and sham operation group. Six rats from each group were killed at different time points: 24 h, 48 h, 72 h, and 96 h later. Blood sample was collected from the vena cava inferior, to examine the serum creatinine (SCr) and blood urea nitrogen (BUN). Specimens of kidney underwent microscopy. Apoptosis was conformed by TUNEL assay. Immunohistochemistry was used to detect the protein expression of Bcl-2 and Bax. The distribution of MMCs labeled with 5-bromodeoxyuridine in kidney was observed by immunofluorescence technique. RESULTS: The SCr and BUN levels in different time points of the MMC group were both significantly lower than those of the I/R group (both P <0.05), HE staining showed that pathological damage of the MMC group was less than that of the I/R group (P <0.05). TUNEL results investigated that the number of apoptosis renal tubular epithelial cells of the MMC group was (13.4 +/- 3.2/HPF), significantly less than that of the I/R group [(25.4 +/- 5.2/HPF)]. In comparison with the I/R group, there were more Bcl-2 positive cells and fewer Bax positive cells in the MMC group. BrdU-labeled MMCs began to occur in the renal tissue (60 +/- 6/HP) In the 72 h subgroup of MMC group, and number of BrdU-labeled MMCs, the 96 h subgroup was (143 +/- 8/HP), significantly higher than that of the 72 h subgroup (P<0.05). CONCLUSION: MMCs have the ability to protect renal function in acute renal tubular damage in rats, migrate and repopulate in the I/R injured renal tubules, and inhibits renal tubular epithelial cell apoptosis. The mechanism may be involved in regulating the expression of Bcl-2 and Bax.