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Kidney fibrosis is marked by an epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs). Here we find that, during renal fibrosis, TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of the EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several solute and solvent transporters in TECs. We also found that transgenic expression of either Twist1 (encoding twist family bHLH transcription factor 1, known as Twist) or Snail (encoding snail family zinc finger 1, known as Snail) expression is sufficient to promote prolonged TGF-β1-induced G2 arrest of TECs, limiting the cells' potential for repair and regeneration. In mouse models of experimentally induced renal fibrosis, conditional deletion of Twist1 or Snail in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while also restoring cell proliferation, dedifferentiation-associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of the EMT program in TECs during chronic renal injury represents a potential anti-fibrosis therapy., Kidney fibrosis is associated with a reduction in the functional renal parenchyma, leading to compromised kidney functions and eventual organ death (1). There is no effective treatment for renal fibrosis [...]

In the present study, we determined the functional role of 15 positively charged amino acid residues at or within 1 of the predicted 11 transmembrane helixes of the flounder renal sodium-dicarboxylate cotransporter fNaDC-3. Using site-directed mutagenesis, histidine (H), lysine (K), and arginine (R) residues of fNaDC-3 were replaced by alanine (A), isoleucine (I), or leucine (L). Most mutants showed sodium-dependent, lithium-inhibitable [[sup.14]C]succinate uptake and, in two-electrode voltage-clamp (TEVC) experiments, [K.sub.m] and [DELTA]Imax values comparable to wild-type (WT) fNaDC-3. The replacement of R109 and R110 by alanine and isoleucine (RR109/110AI) prevented the expression of fNaDC-3 at the plasma membrane. When the lysines at positions 232 and 235 were replaced by isoleucine (KK232/235II), the transporter was expressed but showed small transport rates and succinate-induced currents. K1141, located within transmembrane helix 4, showed [[sup.14]C]succinate uptake similar to WT but relatively small inward currents. When K114 was replaced by arginine, glutamic acid (E), or glutamine (Q), all mutants were expressed at the cell surface. In [[sup.14]C]succinate uptake and TEVC experiments performed simultaneously on the same oocytes, uptake was similar to or higher than WT, whereas succinate-induced currents were either comparable (K114R) to, or considerably smaller (K114E, K1141, K114Q) than, those evoked by WT. These results suggest that a positively charged residue at position 114 is required for electrogenic sodium-dicarboxylate cotransport. sodium/dicarboxylate cotransport; electrogenicity; SLC13A3; mutational analysis; basic amino acids

Several important physiological functions are regulated by cortisol. Previously, we demonstrated the involvement of human organic anion transporter 3 (hOAT3) in cortisol release. In the present study, we investigated the influence of dehydroepiandrosterone sulfate (DHEA-S) and estrone sulfate on cortisol release in a human adrenocortical cell line (NCI-H295R) compared with forskolin stimulation. Additionally, we examined the impact of forskolin and DHEA-S on the expression of key enzymes in steroid biosynthesis and expression of hOAT3 and -4 in NCI-H295R cells. The cortisol release was increased 10-fold after 24-h incubation with DHEA-S, but incubation with estrone sulfate did not show any significant change in cortisol release. When cells were incubated with DHEA-S in the presence of forskolin, an additive influence of DHEA-S stimulation of cortisol was recorded over forskolin alone. The 24-h stimulation of NCI-H295R cells with forskolin increased the expression of steroidogenic acute regulatory protein (STAR), CYP17, CYP21A2, and CYP11A1, whereas only StAR mRNA expression was increased significantly by incubation with DHEA-S. Immunofluorescence analyses revealed strongly elevated expression of hOAT3 by forskolin as well as by DHEA-S stimulation. We conclude that the increased cortisol release of adrenocortical cells by DHEA-S and forskolin stimulation is probably due to high expression of the key enzymes of steroid biosynthesis and hOAT3. cortisol; NCI-H295R cells; organic anion transporter 3; organic anion transporter 4; adrenal gland; human adrenocortical cells; SLC22A8; SLC22A11; forskolin; dehydroepiandrosterone sulfate

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis. endothelial cells; estrone sulfate; dehydroepiandrosterone sulfate; hepatocytes; sinusoidal localization; sulfate/bicarbonate exchange

Tryptophan metabolites such as kynurenate (KYNA), xanthurenate (XA), and quinolinate are considered to have an important impact on many physiological processes, especially brain function. Many of these metabolites are secreted with the urine. Because organic anion transporters (OATs) facilitate the renal secretion of weak organic acids, we investigated whether the secretion of bioactive tryptophan metabolites is mediated by OAT1 and OAT3, two prominent members of the OAT family. Immunohistochemical analyses of the mouse kidneys revealed the expression of OAT1 to be restricted to the proximal convoluted tubule (representing S1 and S2 segments), whereas OAT3 was detected in almost all parts of the nephron, including macula densa cells. In the mouse brain, OAT1 was found to be expressed in neurons of the cortex cerebri and hippocampus as well as in the ependymal cell layer of the choroid plexus. Six tryptophan metabolites, including the bioactive substances KYNA, XA, and the serotonin metabollte 5-hydroxyindol acetate inhibited [[sup.3]H]p-aminohippurate (PAH) or 6-carboxyfluorescein (6-CF) uptake by 50-85%, demonswating that these compounds interact with OAT1 as well as with OAT3. Half-maximal inhibition of mOAT1 occurred at 34 [micro]M KYNA and 15 [micro]M XA, and it occurred at 8 [micro]M KYNA and 11.5 [micro]M XA for mOAT3. Quinolinate showed a slight but significant inhibition of [[sup.3]H]PAH uptake by mOAT1 and no alteration of 6-CF uptake by mOAT3. [[sup.14]C]-Glutarate (GA) uptake was examined for both transporters and demonstrated differences in the transport rate for this substrate by a factor of 4. Trans-stimulation experiments with GA revealed that KYNA and XA are substrates for mOAT1. Our results support the idea that OAT1 and OAT3 are involved in the secretion of bioactive tryptophan metabolites from the body. Consequently, they are crucial for the regulation of central nervous system tryptophan metabolite concentration. kidneys; brain; macula densa; transforming growth factor: N-methyl-D-aspartate receptor

In rats, the secretion of p-aminohippurate (PAH) by the kidney is higher in males (M) than in females (F). The role of the major renal PAH transporters, OAT1 and OAT3, in the generation of these gender differences, as well as the responsible hormones and mechanisms, has not been clarified. Here we used various immunocytochemical methods to study effects of gender, gonadectomy, and treatment with sex hormones on localization and abundance of OAT1 and OAT3 along the rat nephron. Both transporters were localized to the basolateral membrane: OAT1 was strong in proximal tubule S2 and weak in the S3 segments, whereas OAT3 was stained in proximal tubule S1 and S2 segments, thick ascending limb, distal tubule, and in principal cells along the collecting duct. Gender differences in the expression of both transporters in adult rats (M > F) were observed only in the cortical tubules. OAT1 in the cortex was strongly reduced by castration in adult M, whereas the treatment of castrated M with testosterone, estradiol, or progesterone resulted in its complete restitution, further depression, or partial restitution, respectively. In adult F, ovariectomy weakly increased, whereas estradiol treatment of ovariectomized F strongly decreased, the expression of OAT1. The expression of OAT3 in the M and F cortex largely followed a similar pattern, except that ovariectomy and progesterone treatment showed no effect, whereas in other tissue zones gender differences were not observed. In prepubertal rats, the expression of OAT1 and OAT3 in the kidney cortex was low and showed no gender differences. Our data indicate that gender differences in the rat renal cortical OAT1 and OAT3 (M > F) appear after puberty and are determined by both a stimulatory effect of androgens (and progesterone in the case of OAT1) and an inhibitory effect of estrogens. kidney; membrane transport; organic anion transporter; progesterone; sex differences; steroid hormones

The sodium dicarboxylate cotransporter located at the basolateral side supplies renal proximal tubule cells with Krebs cycle intermediates and maintains the driving force for the exchange of organic anions like PAH against [alpha]-ketoglutarate through the organic anion transporter-1. Recently, we cloned sodium dicarboxylate cotransporter-3 from winter flounder kidney (fNaDC-3). To understand the regulation of fNaDC-3, we preincubated fNaDC-3-expressing oocytes with PMA, a PKC activator. PMA dose and time dependently inhibited fNaDC-3-mediated succinate uptake. Simultaneous preincubation of fNaDC-3-expressing oocytes with 50 nM PMA and either staurosporine or RO 31-8220 for 30 min attenuated PKC-mediated inhibition of succinate uptake. Site-directed mutagenesis of the five putative PKC sites (S7, T167, SI74, T188, and S396) resulted in no change in PKC-mediated inhibition of the transporter. In electrophysiological studies performed at -60 mV, the [K.sub.0.5] for succinate was not significantly affected (56 [+ or -] 13 vs. 42 [+ or -] 19 [micro]M), but [DELTA][I.sub.max] was reduced from -139 [+ or -] 49 to -20 [+ or -] 8 nA by PMA (50 nM, 30 min). Immunofluorescence analysis of fNaDC-3-expressing oocytes revealed that PMA leads to an endocytosis of fNaDC-3 protein. In conclusion, fNaDC-3 expressed in oocytes is downregulated by PMA through endocytosis. PKC consensus sites appear not to be important for this process. endocytosis; phorbol 12-myristate 13-acetate

The present study was conducted for the period of six months, September, 2016 to February, 2017 so as to isolate and identify Escherichia (E.coli) from fresh fish samples collected from four different retailing shops in Mekelle city and determine the antibiotic resistance pattern of isolated bacteria. Isolation and identification of E.coli and antibiotic sensitivity test were performed following the standard microbiological techniques. The antibiotic resistance patterns of the E.coli isolates were tested against 6 different antibiotics, namely Ampicillin, Amoxicillin, Ciprofloxacin, Norfloxacin, Gentamicin and Streptomycin. Accordingly, out of the total 96 fish samples collected and analyzed, 9(9.4%) were positive for E.coli following the primary and secondary biochemical test results. Out of the four fish meat retailing shops, the highest and statistically significant prevalence (P=0.001) of E.coli was recorded in shop III as 5(20.8%) followed by shop II as 4(16.7%).Furthermore, results of the antimicrobial sensitivity test have shown that all of the isolates were completely significantly (P=0.001) resistant to two antibiotics (Ampicillin and Amoxicillin) and 100% significantly susceptible (P=0.001) to three of the antibiotics (Ciprofloxacin, Norfloxacin, and Gentamicin). Whereas, out of the 9 isolates, 2 isolates (22.2%) were intermediate and 7 isolates (77.8%) were susceptible to Streptomycin. Hence, it can be concluded that fishes from Mekelle shops do contain antimicrobial resistant pathogenic E.coli where the proportion of these isolates significantly (P<0.001) varies from shop to shop. In spite of the lesser practice of their utilization, Ciprofloxacin, Norfloxacin, and Gentamicin were found to be the best antimicrobials to treat E.coli associated infections in the study area. However, it is suggested that post-harvest hygienic management practices are to be practiced along the fish production-marketing chain. [ABSTRACT FROM AUTHOR]