We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish α globin gene clusters and compared them with the corresponding region in man. This has defined a small segment (∼135–155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate α globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized α globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the α globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

Background. Tubulointerstitial fibrosis is a relatively common and sinister complication of cyclosporine A (CsA) therapy that limits its clinical use. CsA may have direct effects on renal tubular epithelial cells by promoting epithelial–mesenchymal transition (EMT). EMT plays an important role in embryonic development and tumourigenesis and has been described in organ remodelling during fibrogenesis. In this study, we investigated the effects of CsA on a human renal cell line as a model system to test the hypothesis that CsA can induce renal EMT. Methods. Human renal proximal tubular cells were treated with CsA (0.42–42 µm) for periods up to 72 h. Viability was assessed by the Alamar Blue assay. Morphological changes were assessed by phase contrast microscopy. The effects on epithelial adherens molecule, β-catenin and stress fibre protein, F-actin were analysed by indirect immunofluorescence. Reverse transcription––polymerse chain reaction was performed to measure the mRNA levels of extracellular matrix components. Expression of transforming growth factor-β was measured by western blotting. Expression and activity of matrix metalloproteinases were measured by gelatin zymography. Results. CsA induced striking morphological changes in epithelial cells, including changes in cellular morphology, F-actin stress fibre formation, delocalization of the adherens junction protein β-catenin and increased levels of collagen IV and fibronectin. In addition, CsA-induced EMT was associated with increased TGF-β1 protein levels and EMT was markedly attenuated in the presence of anti-TGF-β1 antibody. CsA-induced EMT was also associated with increased expression of connective tissue growth factor (CTGF) suggesting that this molecule may serve as downstream mediator of TGF-β1 pro-fibrotic activity in this setting. Conclusions. In aggregate, these data suggest that CsA is a direct stimulus for EMT in renal tubule epithelial cells and implicate TGF-β1 and CTGF as mediators of this response. The further delineation of the molecular components of this pro-fibrogenic response may suggest novel strategies through which to prevent CsA-induced tubulo-interstitial fibrosis in vivo.

The incidence of Type 2 diabetes is increasing rapidly worldwide, and understanding the mechanisms of its complications including diabetic nephropathy (DN) is important in the discovery of early biomarkers, understanding the causative mechanisms of its complications and identifying therapeutic targets. DN is characterized by glomerulosclerosis, tubulointerstitial fibrosis and tubular atrophy. The tubular component of the disease is important in progression of disease. In vitro models are a valuable alternative to animal studies and an effective way to explore mechanisms of human disease. Several proximal tubular cell lines have been used in studying mechanisms of DN. Key extracellular conditions that contribute to damage to the proximal tubule in DN include hyperglycaemia, proteinuria, and hypoxia and inflammation. According to current knowledge, these exert their effects through changes in transforming growth factor beta signalling, the renin–angiotensin system, dysregulation of pathways such as the polyol pathway, hexosamine pathway and protein kinase C pathway and through formation of advanced glycation end products. Studies in cell culture models have been instrumental in the delineation of these processes. However, all of the existing cell culture models have limitations including dedifferentiation. To bring research forward along with technological advances, such as major advances in ‘omics’ methodologies, a more suitable model is necessary. The RPTEC/TERT1 cell line is a promising alternative to previous proximal tubular epithelial cell lines due to features that resemble the cell type in vivo, such as its epithelial characteristics, maintenance of functional capabilities, glucose handling, expression of the primary cilium and transport activity including albumin. This cell line will facilitate identification of mechanisms of DN with potential to identify new therapeutic targets.

Primary cilia are microtubule‑based organelles that are expressed on almost all mammalian cells. It has become apparent that these structures are important signaling hubs that serve crucial roles in Wnt, hedgehog, extracellular signal‑regulated kinase (ERK)/mitogen‑activated protein kinase (MAPK) and Notch signaling pathways. A number of diseases have been found to involve dysfunctional primary cilia; collectively these diseases are called ciliopathies. In recent years, there has been more focus on the association between primary cilia and cancer, including renal, pancreatic and breast cancer. Numerous studies have demonstrated that various types of cancer cells fail to express cilia. Notably, it has also been indicated that a number of renal carcinogens induce a significant loss of cilia in renal epithelial cells. The present review focuses on the existing literature regarding primary cilia and their involvement with cancer signaling pathways, providing a brief overview of the structural features and functions of primary cilia, then discussing the evidence associating primary cilia with cancer, and presenting the available information on the ERK/MAPK, hedgehog and Wnt signaling pathways, and their involvement in primary cilia in association with cancer.