Sandra Rodrigo Blomqvist

A randomized double-blind study was made in 67 modestly hypercholesterolemic subjects by replacing 50 g of daily dietary fat by the same amount of a rapeseed oil preparation without and with fat-soluble sitostanol esters. The diet became relatively rich in dietary fat (37%) especially in subjects with a low basal calorie intake. The esters were prepared by transesterification of sitostanol with rapeseed oil fatty acids. The effects of sitostanol esters were studied on serum cholesterol and cholesterol synthesis (measuring cholesterol precursors in serum) and absorption (measuring serum plant sterols). The results were related to different apoE phenotypes. A 6-week regimen of about 3.4 g/day of sitostanol lowered total and low density lipoprotein (LDL) cholesterol levels by 7.5% and 10%, respectively, over that due to rapeseed oil alone. High density lipoprotein (HDL) cholesterol and triglyceride concentrations were unchanged. Thus, the HDL/LDL cholesterol ratio was significantly increased. The decrease in LDL cholesterol level was more consistent in subjects with the epsilon 4 allele than in those with homozygous epsilon 3 alleles. Sitostanol markedly decreased serum campesterol (-46%) and sitosterol (-30%), especially in subjects with the epsilon 4 alleles known to have high cholesterol absorption . The decreases of LDL cholesterol and plant sterols were interrelated, suggesting that reduced cholesterol absorption contributed to the lowering of LDL cholesterol. Serum sitostanol was unchanged, while the serum cholesterol precursors, delta 8-cholestenol, desmosterol, and lathosterol, were compensatorily increased by 10% (P < 0.05), most consistently in the subjects with epsilon 4 alleles, indicating an increase in cholesterol synthesis. The study demonstrates that sitostanol esters dissolved in dietary fat can be recommended for treatment of modest primary hypercholesterolemia and are apparently practical and suitable for cholesterol lowering in a general population.

While macro- and microscopic kidney development appear to proceed normally in mice that lack Foxi1, electron microscopy reveals an altered ultrastructure of cells lining the distal nephron. Northern blot analyses, cRNA in situ hybridizations, and immunohistochemistry demonstrate a complete loss of expression of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells of the collecting ducts, many of which have been implicated in hereditary forms of distal renal tubular acidosis (dRTA). In Foxi1-null mutants the normal epithelium with its two major cell types - principal and intercalated cells - has been replaced by a single cell type positive for both principal and intercalated cell markers. To test the functional consequences of these alterations, Foxi1(-/-) mice were compared with WT littermates in their response to an acidic load. This revealed an inability to acidify the urine as well as a lowered systemic buffer capacity and overt acidosis in null mutants. Thus, Foxi1(-/-) mice seem to develop dRTA due to altered cellular composition of the distal nephron epithelium, thereby denying this epithelium the proper gene expression pattern needed for maintaining adequate acid-base homeostasis.

Mice that lack the winged helix/forkhead gene Foxi1 (also known as Fkh10) are deaf and display shaker/waltzer behavior, an indication of disturbed balance. While Foxi1 is expressed in the entire otic vesicle at E9.5, it becomes gradually restricted to the endolymphatic duct/sac epithelium and at E16.5 Foxi1 expression in the inner ear is confined to this epithelium. Histological sections, paintfill experiments and whole-mount hybridizations reveal no abnormality in inner ear development of Foxi1(-/-) mice before E13.5. Between E13.5 and E16.5 the membranous labyrinth of inner ears from null mutants starts to expand as can be seen in histological sections, paint-fill experiments and three-dimensional reconstruction. Postnatally, inner ears of Foxi1(-/-) mice are extremely expanded, and large irregular cavities, compressing the cerebellum and the otherwise normal middle ear, have replaced the delicate compartments of the wild-type inner ear. This phenotype resembles that of the human sensorineural deafness syndrome Pendred syndrome, caused by mutations in the PDS gene. In situ hybridization of Foxi1(-/-) endolymphatic duct/sac epithelium shows a complete lack of the transcript encoding the chloride/iodide transporter pendrin. Based on this, we would like to suggest that Foxi1 is an upstream regulator of pendrin and that the phenotype seen in Foxi1 null mice is, at least in part, due to defective pendrin-mediated chloride ion resorption in the endolymphatic duct/sac epithelium. We show that this regulation could be mediated by absence of a specific endolymphatic cell type--FORE (forkhead related) cells--expressing Foxi1, Pds, Coch and Jag1. Thus, mutations in FOXI1 could prove to cause a Pendred syndrome-like human deafness.

The vacuolar H(+)-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.