Charakterisierung allergeninduzierter immunologischer Reaktionen im Asthmamodell und ex vivo in Precision cut lung slices (PCLS)

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In female rats, ethylene glycol treatment elevates protein expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) without inducing hyperoxaluria

Aim To investigate whether the sex-dependent expression of hepatic and renal oxalate transporter sat-1 (Slc26a1) changes in a rat model of ethylene glycol (EG)-induced hyperoxaluria. Methods Rats were given tap water (12 males and 12 females; controls) or EG (12 males and 12 females; 0.75% v/v in tap water) for one month. Oxaluric state was confirmed by biochemical parameters in blood plasma, urine, and tissues. Expression of sat-1 and rate-limiting enzymes of oxalate synthesis, alcohol dehydrogenase 1 (Adh1) and hydroxy-acid oxidase 1 (Hao1), was determined by immunocytochemistry (protein) and/or real time reverse transcription polymerase chain reaction (mRNA). Results EG-treated males had significantly higher (in μmol/L; mean ± standard deviation) plasma (59.7 ± 27.2 vs 12.9 ± 4.1, P < 0.001) and urine (3716 ± 1726 vs 241 ± 204, P < 0.001) oxalate levels, and more abundant oxalate crystaluria than controls, while the liver and kidney sat-1 protein and mRNA expression did not differ significantly between these groups. EG-treated females, in comparison with controls had significantly higher (in μmol/L) serum oxalate levels (18.8 ± 2.9 vs 11.6 ± 4.9, P < 0.001), unchanged urine oxalate levels, low oxalate crystaluria, and significantly higher expression (in relative fluorescence units) of the liver (1.59 ± 0.61 vs 0.56 ± 0.39, P = 0.006) and kidney (1.77 ± 0.42 vs 0.69 ± 0.27, P < 0.001) sat-1 protein, but not mRNA. The mRNA expression of Adh1 was femaledominant and that of Hao1 male-dominant, but both were unaffected by EG treatment. Conclusions An increased expression of hepatic and renal oxalate transporting protein sat-1 in EG-treated female rats could protect from hyperoxaluria and oxalate urolithiasis

Male-Dominant Activation of Rat Renal Organic Anion Transporter 1 (Oat1) and 3 (Oat3) Expression by Transcription Factor BCL6

Background: Organic anion transporters 1 (Oat1) and 3 (Oat3) mediate the transport of organic anions, including frequently prescribed drugs, across cell membranes in kidney proximal tubule cells. In rats, these transporters are known to be maledominant and testosterone-dependently expressed. The molecular mechanisms that are involved in the sex-dependent expression are unknown. Our aim was to identify genes that show a sex-dependent expression and could be involved in male-dominant regulation of Oat1 and Oat3. Methodology/Principal Findings: Promoter activities of Oat1 and Oat3 were analyzed using luciferase assays. Expression profiling was done using a SurePrint G3 rat GE 8660K microarray. RNA was isolated from renal cortical slices of four adult rats per sex. To filter the achieved microarray data for genes expressed in proximal tubule cells, transcription database alignment was carried out. We demonstrate that predicted androgen response elements in the promoters of Oat1 and Oat3 are not functional when the promoters were expressed in OK cells. Using microarray analyses we analyzed 17,406 different genes. Out of these genes, 56 exhibit a sex-dependent expression in rat proximal tubule cells. As genes potentially involved in the regulation of Oat1 and Oat3 expression, we identified, amongst others, the male-dominant hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1), B-cell CLL/lymphoma 6 (BCL6), and polymerase (RNA) III (DNA directed) polypeptide G (Polr3g). Moreover, our results revealed that the transcription factor BCL6 activates promoter constructs of Oat1 and Oat3

Counter-flow suggests transport of dantrolene and 5-OH dantrolene by the organic anion transporters 2 (OAT2) and 3 (OAT3)

Dantrolene is the only available drug for the treatment of malignant hyperthermia, a life-threatening inborn sensitivity of the ryanodine receptor (RyR1) in skeletal muscles to volatile anesthetics. Dantrolene is metabolized in the liver to 5-OH dantrolene. Both compounds are zwitterions or net negatively charged. Here, we investigated interactions of dantrolene and 5-OH dantrolene with solute carrier (SLC) family members occurring in skeletal muscle cells, hepatocytes, and renal proximal tubule cells. SLC22A8 (organic anion transporter 3, OAT3) was very sensitive to both compounds exhibiting IC50 values of 0.35 +/- 0.03 and 1.84 +/- 0.34 mu M, respectively. These IC50 concentrations are well below the plasma concentration in patients treated with dantrolene (3-28 mu M). SLC22A7 (OAT2) was less sensitive to dantrolene and 5-OH dantrolene with IC50 values of 15.6 +/- 2.1 and 15.8 +/- 3.2 mu M, respectively. SLCO1B1 (OATP1B1), SLCO1B3 (OATP1B3), and SLCO2B1 (OATP2B1) mainly interacted with 5-OH dantrolene albeit with higher IC50 values than those observed for OAT2 and OAT3. Dantrolene and 5-OH dantrolene failed to inhibit uptake of 1-methyl-4-phenylpyrimidinium (MPP) by OCT1 and of carnitine by OCTN2. In counter-flow experiments on OAT3, dantrolene and 5-OH dantrolene decreased pre-equilibrated cellular [H-3]estrone-3-sulfate (ES) content as did the transported substrates glutarate, furosemide, and bumetanide. With OAT2, dantrolene and 5-OH dantrolene slightly decreased the pre-equilibrated [H-3] cGMP content. If no other transporter markedly contributes to uptake or release of ES or cGMP, respectively, these data suggest that OAT3 and OAT2 may be involved in absorption, metabolism, and excretion of dantrolene and its metabolite 5-OH dantrolene

Sex-Differences in Renal Expression of Selected Transporters and Transcription Factors in Lean and Obese Zucker Spontaneously Hypertensive Fatty Rats

The aim of this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman’s space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated from kidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obese males. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1β and Hnf4α in both sexes. The highest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2-dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide

Sex-Differences in Renal Expression of Selected Transporters and Transcription Factors in Lean and Obese Zucker Spontaneously Hypertensive Fatty Rats

The aim of this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman&apos;s space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated from kidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obese males. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1 and Hnf4 in both sexes. The highest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2-dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide

Estrogen receptor alpha (ER alpha) indirectly induces transcription of human renal organic anion transporter 1 (OAT1)

Organic anion transporter 1 (OAT1) is a polyspecific transport protein located in the basolateral membrane of renal proximal tubule cells. OAT1 plays a pivotal role in drug clearance. Adverse drug reactions (ADR) are observed more frequently in women than in men, especially ADR are higher in women for drugs which are known interactors of OAT1. Sex-dependent expression of Oat1 has been observed in rodents with a tendency to male-dominant expression. This study aims at elucidating the transcriptional regulation of human OAT1 and tests the effect of estrogen receptor alpha (ER alpha). Promoter activation of OAT1 was assessed by luciferase assays carried out by Opossum kidney (OK) cells, transiently transfected with promoter constructs of human OAT1 and expression vectors for ER alpha and exposed to 100 nmol/L 17 beta-estradiol. Furthermore, a transcription factor array and proteomic analysis was performed to identify estrogen-induced transcription factors. Human OAT1 was significantly activated by ligand activated ER alpha. However, activation occurred without a direct interaction of ER alpha with the OAT1 promoter. Our data rather show an activation of the transcription factors CCAAT-box-binding transcription factor (CBF) and heterogeneous nuclear ribonucleoprotein K (HNRNPK) by ER alpha, which in turn bind and initiate OAT1 promoter activity. Herewith, we provide novel evidence of estrogen-dependent, transcriptional regulation of polyspecific drug transporters including the estrogen-induced transcription factors CBF and HNRNPK