Down-regulation of ABCG2, a urate exporter, by parathyroid hormone enhances urate accumulation in secondary hyperparathyroidism.

Hyperuricemia occurs with increasing frequency among patients with hyperparathyroidism. However, the molecular mechanism by which the serum parathyroid hormone (PTH) affects serum urate levels remains unknown. This was studied in uremic rats with secondary hyperparathyroidism where serum urate levels were found to be increased and urate excretion in the intestine and kidney decreased, presumably due to down-regulation of the expression of the urate exporter ABCG2 in intestinal and renal epithelial membranes. These effects were prevented by administration of the calcimimetic cinacalcet, a PTH suppressor, suggesting that PTH may down-regulate ABCG2 expression. This was directly tested in intestinal Caco-2 cells where the expression of ABCG2 on the plasma membrane was down-regulated by PTH (1-34) while its mRNA level remained unchanged. Interestingly, an inactive PTH derivative (13-34) had no effect, suggesting that a posttranscriptional regulatory system acts through the PTH receptor to regulate ABCG2 plasma membrane expression. As found in an animal study, additional clinical investigations showed that treatment with cinacalcet resulted in significant reductions in serum urate levels together with decreases in PTH levels in patients with secondary hyperparathyroidism undergoing dialysis. Thus, PTH down-regulates ABCG2 expression on the plasma membrane to suppress intestinal and renal urate excretion, and the effects of PTH can be prevented by cinacalcet treatment.

Human organic anion transporters function as a high-capacity transporter for p-cresyl sulfate, a uremic toxin.

BACKGROUND: Recent clinical studies have shown that increased serum levels of p-cresyl sulfate (PCS), a uremic toxin, are associated with the progression of chronic kidney disease (CKD) and cardiovascular outcomes. Using rat renal cortical slices, we previously reported that the rat organic anion transporter (OAT) could play a key role in the renal tubular secretion of PCS. However, no information is currently available regarding the transport of PCS via human OAT (hOAT) isoforms, hOAT1 and hOAT3. METHODS: Uptake experiments of PCS were performed using HEK293 cells, which stably express hOAT1 or hOAT3. RESULTS: PCS was taken up by hOAT1/HEK293 and hOAT3/HEK293 cells in a time- and concentration-dependent manner. The apparent K m for the hOAT1-mediated transport of PCS was 128 µM, whereas in hOAT3/HEK293, saturation was not observed at the highest tested PCS concentration of 5 mM. Probenecid, an OAT inhibitor, inhibited PCS transport by hOAT1 and hOAT3. The uptake of p-aminohippurate by hOAT1 and estron-3-sulfate by hOAT3 was decreased with increasing PCS concentration. The apparent 50 % inhibitory concentrations for PCS were 690 and 485 µM for hOAT1 and hOAT3, respectively. CONCLUSION: PCS is a substrate for hOAT1 and hOAT3, and hOAT1 and hOAT3 appear to play a physiological role as a high-capacity PCS transporter. Since hOATs are expressed not only in the kidneys, but also in blood vessels and osteoblasts, etc., these findings are of great significance in terms of elucidating the renal clearance, tissue disposition of PCS and the mechanism of its toxicity in CKD.

FRET-based imaging of transbilayer movement of pepducin in living cells by novel intracellular bioreductively activatable fluorescent probes.

To elucidate the mechanisms of direct transmembrane penetration of pepducins, which are artificial lipopeptide G protein-coupled receptor (GPCR) modulators, we developed two types of FRET-based probes, Pep13-FL-SS-Dab (13) targeting the inner leaflet of the lipid bilayer and Pep13-Dab-SS-FL (14) targeting the cytosol, respectively. They are composed of a pepducin moiety and a fluorescent switch component consisting of 5(6)-carboxyfluorescein (FAM) as a fluorophore and dabcyl as a quencher connected through disulfide bond linkage. When they are internalized into the cytosol, intracellular glutathione can cleave the disulfide bond to release the quencher, which results in a turn-on fluorescence signal. Using these probes, we performed live cell imaging of transbilayer movements of pepducins on MCF-7 cells for the first time. The results suggested that the lipid moiety of the probes facilitated pepducin flipping across and tethering to the membrane. The present study raises the possibility of applying the probe architecture for direct intracellular drug delivery.

Parathyroid hormone contributes to the down-regulation of cytochrome P450 3A through the cAMP/PI3K/PKC/PKA/NF-κB signaling pathway in secondary hyperparathyroidism.

Chronic kidney disease (CKD), which affects, not only renal clearance, but also non-renal clearance, is accompanied by a decline in renal function. Although it has been suggested that humoral factors, such as uremic toxins that accumulate in the body under CKD conditions, could be involved in the changes associated with non-renal drug clearance, the overall process is not completely understood. In this study, we report on the role of parathyroid hormone (PTH), a middle molecule uremic toxin, on the expression of drug metabolizing or transporting proteins using rats with secondary hyperparathyroidism (SHPT) as models. In SHPT rats, hepatic and intestinal CYP3A expression was suppressed, but the changes were recovered by the administration of the calcimimetic cinacalcet, a PTH suppressor. Under the same experimental conditions, a pharmacokinetic study using orally administered midazolam, a substrate for CYP3A, showed that the AUC was increased by 5 times in SHPT rats, but that was partially recovered by a cinacalcet treatment. This was directly tested in rat primary hepatocytes and intestinal Caco-2 cells where the expression of the CYP3A protein was down-regulated by PTH (1-34). In Caco-2 cells, PTH (1-34) down-regulated the expression of CYP3A mRNA, but an inactive PTH derivative (13-34) had no effect. 8-Bromo-cyclic adenosine monophosphate, a membrane-permeable cAMP analog, reduced mRNA expression of CYP3A whereas the inhibitors of PI3K, NF-κB, PKC and PKA reversed the PTH-induced CYP3A down-regulation. These results suggest that PTH down-regulates CYP3A through multiple signaling pathways, including the PI3K/PKC/PKA/NF-κB pathway after the elevation of intracellular cAMP, and the effect of PTH can be prevented by cinacalcet treatment.

New rapid detection test with a combination of polymerase chain reaction and immunochromatographic assay for Mycobacterium tuberculosis complex.

Recently, we had developed a polymerase chain reaction (PCR)-immunochromatographic assay (ICA) method for Mycobacterium tuberculosis and examined its clinical utility among 138 sputa of patients under suspicion of pulmonary tuberculosis. According to the results of fluorochrome staining of acid-fast bacillus, which were confirmed by Ziehl-Neelsen staining, these were 83 specimens (-), 7 specimens (+/-), 30 specimens (1+), 8 specimens (2+), and 10 specimens (3+). These specimens included 4 groups: group 1, 41 specimens of smear (+/-) approximately (3+) with culture-positive M. tuberculosis; group 2, 11 specimens of smear (-) with culture-positive M. tuberculosis; group 3, 12 specimens of smear (+/-) approximately (1+) with culture-positive nontuberculosis mycobacterium (NTM); and group 4, 9 specimens of smear (-) with culture-positive NTM. The positive results of PCR-ICA test and Amplicor M. tuberculosis (Amplicor MTB) test for M. tuberculosis are as follows: group 1, 40 positive by PCR-ICA and 39 positive by Amplicor MTB from 41 specimens; group 2, 1 positive by PCR-ICA and 5 positive by Amplicor MTB from 11 specimens; group 3, 0 positive by both tests from 12 specimens; and group 4, 0 positive by both tests from 9 specimens. None of NTM-positive specimens from groups 3 and 4 reacted on the PCR-ICA test for M. tuberculosis.

New rapid polymerase chain reaction-immunochromatographic assay for Porphyromonas gingivalis.

BACKGROUND: A simple and rapid method for Porphyromonas gingivalis detection in clinical samples has been developed using polymerase chain reaction (PCR) and an immunochromatographic assay (ICA) with a lateral-flow device (strip) to detect species-specific 16S rRNA genes. METHODS: The PCR used a pair of primer sets labeled with fluorescein isothiocyanate (FITC) or biotin at each 5' terminus. The strip used a nitrocellulose membrane containing streptavidin conjugated to gold particles and anti-FITC line. RESULTS: PCR and ICA detected as few as 1 and 10 cells of P. gingivalis, respectively. ICA required 5 to 10 minutes more than the initial PCR. The amplifications were not observed in other oral black-pigmented bacteria at concentrations of 10(6) colony forming unit (CFU). The ICA strips showed bands at more than 10(4) CFU/ml equivalents in clinical samples from periodontitis. CONCLUSIONS: A diagnostic assay based on PCR-ICA was developed for the detection of P. gingivalis, and results were obtained visually in 3 hours. PCR-ICA will be a valuable tool for the rapid detection of target bacteria by chair side.