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.

Effects of 4-phenylbutyric acid on the process and development of diabetic nephropathy induced in rats by streptozotocin: regulation of endoplasmic reticulum stress-oxidative activation.

Oxidative stress may contribute to the pathogenesis of diabetic nephropathy (DN), although the precise regulatory mechanism is still unclear. Recent reports have shown that chemical molecular chaperone 4-phenylbutyric acid (4-PBA) can suppress oxidative stress by attenuating endoplasmic reticulum (ER) stress. We therefore hypothesized that 4-PBA could provide renoprotection through the suppression of oxidative stress in DN rats. Male Sprague-Dawley (SD) rats were randomly divided into three groups: a normal control (NC) group, a streptozotocin (STZ)-induced DN model group, and a DN plus 4-PBA (1g/kg) treatment group. At the end of 4, 8, and 12 weeks, hydroxyproline content, NADPH oxidase activity and the expression of phosphorylation of inositol-requiring enzyme-1alpha (p-IRE1alpha), p47phox, nitrotyrosine (NT) and NF-E2-related factor 2 (Nrf2) in the kidneys of all rats were determined; malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity in serum and urine were also detected; renal nuclear factor kappaB (NF-kappaB) activity in all of the rats was examined at the end of 12 weeks. Compared with the NC group, the DN rats showed a significant increase in hydroxyproline content, NADPH oxidase activity, NF-kappaB activity, the expression of p-IRE1alpha, p47phox, NT and Nrf2 in renal tissue; markedly, MDA levels were higher and SOD activity was lower in serum and urine of DN rats than in NC rats for the indicated time. These alterations were inhibited by the administration of 4-PBA. These findings first demonstrated that treatment with 4-PBA significantly inhibits the process and development of diabetic nephropathy in rats through the regulation of ER stress-oxidative activation.

Prevention of diabetic nephropathy in rats through enhanced renal antioxidative capacity by inhibition of the proteasome.

AIMS: Oxidative stress may play an important role in the pathogenesis of diabetic nephropathy (DN). Recent studies have shown that the ubiquitin-proteasome pathway (UPP) and oxidative stress have interaction. We aimed to investigate whether inhibiting the proteasome has a preventive effect on DN through suppression of renal oxidative stress. MAIN METHODS: Male Sprague-Dawley rats were randomly divided into three groups: a normal control (NC) group, a streptozotocin-induced DN model group, and a DN plus MG132 (10 µg/kg) treatment group. KEY FINDINGS: Increased 24-h urinary protein excretion rate (UPER) and renal pathological changes were all improved after MG132 administration. Furthermore, enhanced renal 26S proteasome activity and concentration in DN rats were effectively reduced after MG132 administration. Increased p47phox and nitrotyrosine (NT) expressions in kidneys of DN rats were decreased after MG132 treatment. Renal mRNA and protein expressions of NF-E2 related factor 2 (Nrf2) were up-regulated by MG132 in comparison to DN alone. Decreased renal mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) in DN rats was heightened after MG132 intervention. Depressed activities of renal SOD, CAT and GPx in DN rats were also improved by MG132 treatment. Increased renal nuclear factor κB (NF-κB) activity was inhibited after MG132 administration in DN rats at the end of 12 weeks. SIGNIFICANCE: Our present data suggest that inhibition of the proteasome by low-dose MG132 has a preventive effect on DN development and progression in rats through the up-regulation of antioxidant genes.

Attenuation of diabetic nephropathy in diabetes rats induced by streptozotocin by regulating the endoplasmic reticulum stress inflammatory response.

The endoplasmic reticulum (ER) is capable of sensing metabolic and stress parameters and integrating intra- and extracellular signals to support a coordinated cell response. In the present study, we verified the hypothesis that 4-phenylbutyric acid (4-PBA), a chemical chaperone, prevented the progression of diabetic nephropathy (DN). Male Sprague-Dawley rats were randomly divided into 3 groups: a normal control group, a DN group, and a DN model plus 4-PBA treatment group (PBA). The DN model was induced by injection of streptozotocin with uninephrectomy. The dosage of 4-PBA treatment was gavaged at a dose of 1 g/kg body weight each day for 12 weeks. The expression of the ER stress indicators significantly increased in the kidney of DN rats within the indicated period. Moreover, the expression of phosphorylated c-JUN NH(2)-terminal kinase, the monocyte chemoattractant protein-1, and the final fibrotic effector all elevated markedly in the kidney of DN rats. Urinary protein excretion rate and the concentration of urinary monocyte chemoattractant protein-1 were higher than those in the normal control group. Treatment with 4-PBA can suppress the expression of the glucose-regulated protein 78 and the phosphorylation of the PKR-like ER kinase, both of which are ER stress indicators; renoinflammatory signal; and the expression of inflammatory cytokines and fibrosis factors. It also can inhibit the increase in urinary protein excretion rate and urinary monocyte chemoattractant protein-1. In conclusion, 4-PBA exerts a marked renoprotective effect possibly due to modulating ER stress and related inflammatory cascade.