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

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

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 1mM 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 (p0.05). HK2s had lower endogenous cav1 and p-cav1 than MDCKs (p0.05). Both cell lines had increased p-cav1, but not cav1, with oxidative stress. This increase was greatest in MDCKs (p0.01). 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 (p0.01), 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

A comparison of pathomolecular markers of fibrosis and morphology in kidney from autopsies of African Americans and whites

African Americans have an increased incidence of chronic kidney disease (CKD) due to hypertension and arteriosclerosis and increased death due to coronary artery disease, compared with whites. The pathogenesis of CKD involves the increased presence and activation of myofibroblasts and macrophages, promotion of tubulointerstitial fibrosis, and effects of tubulointerstitial cell mitosis and apoptosis. We hypothesized that increased risk of hypertensive vascular disease may be identified by renal pathomolecular markers that are associated with progressive CKD. Renal sections were available from 50 autopsies of 33 African Americans (55% males) and 17 whites (76% males) undergoing forensic autopsy for unexpected death. Sclerotic glomeruli, severity of cortical fibrosis, and renal arterioloselerosis, total glomerular number (N-glom), average glomerular volume (V-glom), birth weights, and blood pressure were known. Presence and locality of markers for myofibroblasts (alpha-SMA), macrophages (CD68), collagen, pro-fibrotic transforming growth factor-beta1 were scored in renal autopsies, and tubulointerstitial apoptosis was recorded. The results demonstrated a strong positive correlation between age, cortical fibrosis and alpha-SMA (p < 0.05), and between CD68 and hypertension and coronary artery disease (p < 0.05). The findings confirm the role of myofibroblasts and macrophages in pathogenesis of human CKD. However, the markers showed no significant relationships to V-glom, N-glom, birth weight, or race