Tacrolimus induces fibroblast-to-myofibroblast transition via a TGF-ß-dependent mechanism to contribute to renal fibrosis.

Use of immunosuppressant calcineurin inhibitors (CNIs) is limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesized that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the transforming growth factor-ß (TGF-ß)/Smad signaling axis. To test this, kidney damage-resistant mice (C57BL/6) received tacrolimus (10 mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1 nM) or vehicle for 24 h. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-smooth muscle actin expression was significantly increased, suggesting the presence of fibroblast activation. TGF-ß receptor activation and downstream Smad2/3 signaling were also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-smooth muscle actin, actin stress fibers, cell motility, and collagen type IV expression were significantly increased. These findings were accompanied by concomitant induction of TGF-ß signaling. Pharmacological inhibition of the downstream TGF-ß effector Smad3 attenuated tacrolimus-induced phenotypic changes. Collectively, these findings suggest that 1) tacrolimus inhibits the calcineurin/nuclear factor of activated T cells axis while inducing TGF-ß1 ligand secretion and receptor activation in renal fibroblasts; 2) aberrant TGF-ß receptor activation stimulates Smad-mediated production of myofibroblast markers, notable features of FMT; and 3) FMT contributes to extracellular matrix expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts into the growing list of CNI-targeted cell types and identify renal FMT as a process mediated via a TGF-ß-dependent mechanism.NEW & NOTEWORTHY Renal fibrosis, a detrimental feature of irreversible kidney damage, remains a sinister consequence of long-term calcineurin inhibitor (CNI) immunosuppressive therapy. Our study not only incorporates renal fibroblasts into the growing list of cell types negatively impacted by CNIs but also identifies renal fibroblast-to-myofibroblast transition as a process mediated via a TGF-ß-dependent mechanism. This insight will direct future studies investigating the feasibility of inhibiting TGF-ß signaling to maintain CNI-mediated immunosuppression while ultimately preserving kidney health.

Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH.

The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.

Filtration of deformable emulsion droplets.

Oil-in-water (o/w) emulsions of different droplet size were filtered on membranes of various pore sizes to investigate the growth and behaviour of o/w filter cakes. The cake desorptivity S and the filter membrane resistance R were measured at various filtration pressures P. The variation of S with P shows that filter cake oil droplets of radius a are effectively rigid for P << gamma/a and fully deformable for P >> gamma/a, where gamma is the oil-water interfacial tension. For the largest P, when S became P-independent, the filter cake remained water-permeable as expected from theory.

The use of heavy metal soil analysis for archaeological surveying.

An evaluation of the geochemical analysis of soils as an archaeological investigative technique is illustrated with reference to a site with a known history of human use and settlement. The results show certain elements, including the heavy metals Pb, Zn, Cd, Cu etc., to be associated with locations of past human activity, and support such analyses as significant additions to the means by which archaeologists might investigate the archaeological record.

A study of the transpiration surfaces of Avena sterilis L. var. Algerian leaves using monosilicic acid as a tracer for water movement.

The sites and pathways of transpiration from leaves of Avena sterilis L. var. Algerian were studied using the accumulation of monosilicic acid as a tracer for water movement. Seedlings of Algerian oats were grown under silicon free conditions and fed monosilicic acid, in a normal nutrient solution, via the roots. The silicon component of monosilicic acid was located in freeze substituted tissue by means of x-ray microprobe analysis. Methods of tissue fixation preventing post treatment movement of tracer were developed and it was determined that monosilicic acid is a suitable tracer for water.Sites of water loss were marked by accumulation of silicon. Internal evaporating surfaces having a high intensity of water loss were demonstrated. Evaporation from epidermal surfaces was most intense over the guard and subsidiary cells with very little evaporation from the cuticular surfaces of normal epidermal cells. Moderately high evaporation occurred from epidermal fibre cells located above the veins. Evaporation from all exposed walls of guard cells including the wall adjacent to the pore was intense. Smaller amounts of tracer were located in the unexposed anticlinal walls of epidermal cells as well as within the unexposed walls of mesophyll cells. The results are interpreted as demonstrating the extent of internal transpiration surfaces and that cuticular epidermal transpiration is low. Strong support is given to the existence of peristomatal transpiration. Internal pathways of water movement are defined and the occurrence of these is discussed in relation to cuticular transpiration and lateral water movement in the epidermis.