Lentiviral-mediated RNA interference against TGF-beta receptor type II in renal epithelial and fibroblast cell populations in vitro demonstrates regulated renal fibrogenesis that is more efficient than a nonlentiviral vector.

BACKGROUND: Lentiviral constructs reportedly can integrate into the genome of non-dividing, terminally differentiated cells and dividing cells, for long-term gene expression. This investigation tested whether a third generation lentiviral-mediated small interfering RNA (siRNA) delivered into renal epithelial and fibroblast cells against type II transforming growth factor-beta receptor (siRNA-TBRII) could better attenuate renal fibrogenesis in comparison with a non-lentiviral construct. METHODS: HIV-derived lentiviral and non-lentiviral constructs were used to transfect cells with siRNA-TBRII or siRNA-EGFP control. Human embryonic kidney (HEK-293T), renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were transfected and gene silencing quantified (fluorescence microscopy, Western blotting, fluorescence-activated cell sorting). Renal fibrogenesis was assessed using extracellular matrix protein synthesis (fibronectin and collagen-III; Western immunoblot), and α-smooth muscle actin (α-SMA) was analysed as a marker of fibroblast activation and epithelial-to-mesenchymal transdifferentiation (EMT). RESULTS: Lentiviral-mediated siRNA-TBRII significantly suppressed TBRII expression in all cell lines, and also significantly suppressed renal fibrogenesis. In comparison with the non-lentiviral construct, lentiviral-mediated siRNA-TBRII produced stronger and more persistent inhibition of collagen-III in NRK-49F cells, fibronectin in all renal cell lines, and α-SMA in renal epithelial cells. CONCLUSIONS: Lentiviral vector systems against TBRII can be delivered into renal cells to efficiently limit renal fibrogenesis by sequence-specific gene silencing.

Expression of apoptotic tumour necrosis factor receptor-associated factor, caspase recruitment domain and cell death-inducing DFF-45 effector genes in therapy-treated renal cell carcinoma.

AIM: Dysfunction in apoptosis plays a role in development of renal cell carcinoma (RCC). This investigation aimed to identify expression of apoptosis-related genes not previously characterized in human RCC. METHODS: The RCC ACHN cell line was treated with radiation plus interferon-alpha to induce significant apoptosis. Apoptosis RNA microarrays were used to compare control and treated RCC for apoptosis-regulatory genes with significantly altered expression (>or= twofold). Translational correlates were analysed using western blot. Immunohistochemistry of human RCC and non-cancerous kidney in tissue microarrays was also completed. RESULTS: Several gene families, not well characterized in RCC, were significantly upregulated in RNA microarray. These were the tumour necrosis factor receptor-associated factors (TRAF1, 3 and 4), caspase recruitment domain (NOL3 and PYCARD), and cell death-inducing DFF-45 effector domain (ICAD/CAD) genes. The protein expression patterns did not always increase similarly, perhaps indicating some post-transcriptional controls needing further investigation. TRAF1 had significantly increased expression for RNA and protein (P<0.01). NOL3 had significantly decreased whole-cell protein expression (P<0.05), but had strongly localized nuclear positivity in RCC in the immunohistochemistry. CONCLUSION: These newly identified RCC apoptosis genes have shown potential for improving outcome in other cancers and may prove to have the same potential in RCC with further study.

Phosphorylation of caveolin-1 is anti-apoptotic and promotes cell attachment during oxidative stress of kidney cells.

AIMS: Caveolin-1 (cav1) is reported to have both cell survival and pro-apoptotic characteristics. This may be explained by its localisation or phosphorylation in injured cells. This study investigated the role of cav1 in kidney cells of different nephron origin and developmental state after oxidative stress. METHODS: Renal MCDK distal tubular, HK2 proximal tubular epithelial cells and HEK293T renal embryonic cells were treated with 1 mM hydrogen peroxide. Apoptosis, loss of cell adhesion, and cell survival were compared with expression of cav1 in its non-phosphorylated and phosphorylated (p-cav1) forms. Cav1 was transfected into the HEK293T cells, or caveolae were disrupted with filipin or nystatin in HK2 cells, to investigate functions of cav1 and p-cav1. RESULTS: Oxidative stress induced more apoptosis in HK2s than MDCKs (p < 0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p < 0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p < 0.01). Cav1 was located mainly in the plasma membrane of untreated cells and translocated to the cytoplasm with oxidative stress in both cell lines, more so in MDCKs. Disruption of caveolae caused cytoplasmic translocation of cav1 in HK2s, but did not alter high levels of oxidative stress-induced apoptosis. When HEK293Ts lacking endogenous cav1 were transfected with cav1, oxidant-induced apoptosis and loss of cell adhesion was decreased (p < 0.01), and p-cav1 was induced by treatment. CONCLUSION: Cav1 expression and localisation in kidney cells is not anti-apoptotic, but increased expression of p-cav1 may promote cell survival after oxidative stress.

Increased constitutive activation of NF-κB p65 (RelA) in peripheral blood cells of patients with progressive multiple sclerosis.

The NF-κB signalling pathway plays an important role in controlling cellular immune responses, inflammation and apoptosis. In multiple sclerosis (MS), there is evidence of dysregulation of NF-κB signalling in patients with a relapsing-remitting disease course, but thus far there is little information on whether it is also dysregulated in patients with progressive disease. We hypothesised that patients with progressive MS would have more activation of NF-κB than relapsing-remitting MS patients. Using several different methods, we showed that there was more nuclear translocation of p65 in cells from progressive MS patients, particularly in T cells and monocytes. In addition, the amount of p65 translocated to the nucleus in cells of patients with progressive MS was not increased upon non-specific activation of the cells with the mitogen Con A. These results suggest that NF-κB dysregulation occurs in patients with progressive MS patients, as well as those with relapsing-remitting MS.

Comparative analysis of caspase activation and apoptosis in renal tubular epithelial cells and renal cell carcinomas.

BACKGROUND/AIMS: Treatment of renal cell carcinoma (RCC) is limited by its resistance to conventional chemotherapies. This may occur, in part, from resistance to apoptosis. The role of caspase activation in apoptosis resistance in treated RCCs was investigated. METHODS: Two human RCC cell lines (ACHN and SN12K1) and renal tubular epithelial cells (HK2) were treated with 5-fluorouracil (0.2-20 microg/ml) or cisplatin (1-100 microM). Activation of caspase-3 and -2 was analysed and compared with levels of apoptosis. Caspase function was analysed using pan-caspase inhibition (z-VAD-fmk) and caspase-2 inhibition (z-VDVAD-fmk). RESULTS: RCC apoptosis was significantly lower (p < 0.05) than in HK2s after treatment, confirming their chemoresistance. Pro-caspase-3 (32 kDa) was detected in all cell lines. Cleaved caspase-3 (19 kDa) was not detected by Western immunoblots in treated RCCs and only minimal activated caspase-3 was detected in treated RCCs using immunohistochemistry. All cells had pro-caspase-2 (48 kDa) and the activated form (33 kDa) appeared in all treated cells. Caspase inhibition caused a reduction in, but not negation of, therapy-induced apoptosis in HK2s and RCCs (p < 0.05 for HK2s and ACHN cells), indicating that a caspase activation pathway must occur in RCC apoptosis but this pathway does not act via caspase-3 cleavage. Inhibition of caspase-2 reduced apoptosis only in HK2s, indicating that the activated caspase-2, identified in treated RCCs, was not responsible for their apoptosis induction. CONCLUSION: Specific differences in caspase-3 and -2 activation were identified in renal tubular epithelium and RCCs after chemotherapy. Identification of RCC-specific caspase inactivation or redundancy may explain, in part, the resistance of RCCs to cancer therapies and may be useful in targeting apoptotic pathways to overcome RCC resistance to treatment.

Bcl-2 in endothelial cells is increased by vitamin E and alpha-lipoic acid supplementation but not exercise training.

Atherosclerotic plaque contains apoptotic endothelial cells with oxidative stress implicated in this process. Vitamin E and alpha-lipoic acid are a potent antioxidant combination with the potential to prevent endothelial apoptosis. Regular exercise is known to increase myocardial protection, however, little research has investigated the effects of exercise on the endothelium. The purpose of these studies was to investigate the effects of antioxidant supplementation and/or exercise training on proteins that regulate apoptosis in endothelial cells. Male rats received a control or antioxidant-supplemented diet (vitamin E and alpha-lipoic acid) and were assigned to sedentary or exercise-trained groups for 14 weeks. Left ventricular endothelial cells (LVECs) were isolated and levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were measured. Antioxidant supplementation caused a fourfold increase in Bcl-2 (P < 0.05) with no change in Bax (P > 0.05). Bcl-2:Bax was increased sixfold with antioxidant supplementation compared to non-supplemented animals (P < 0.05). Exercise training had no significant effect on Bcl-2, Bax or Bcl-2:Bax either alone or combined with antioxidant supplementation (P > 0.05) compared to non-supplemented animals. However, Bax was significantly lower (P < 0.05) in the supplemented trained group compared to non-supplemented trained animals. Cultured bovine endothelial cells incubated for 24 h with vitamin E and/or alpha-lipoic acid showed the combination of the two antioxidants increased Bcl-2 to a greater extent than cells incubated with the vehicle alone. In summary, vitamin E and alpha-lipoic acid increase endothelial cell Bcl-2, which may provide increased protection against apoptosis.

Fibrogenic stresses activate different mitogen-activated protein kinase pathways in renal epithelial, endothelial or fibroblast cell populations.

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in chronic fibrosis.