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Sex-differences in renal expression of selected transporters and transcription factors in lean and obese Zucker spontaneously hypertensive fatty rats.

Babelova A, Burckhardt BC, Wegner W, Burckhardt G, Henjakovic M - J Diabetes Res (2015)

Bottom Line: Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1β and Hnf4α in both sexes.Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors.However, OAT2-dependent uptake of cGMP was inhibited by furosemide.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular Physiology (Physiology I), Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany ; Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 83391 Bratislava, Slovakia.

ABSTRACT
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.

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IC50 determination for the inhibition of OAT2-mediated cGMP uptake by furosemide and bumetanide. In HEK293 cells stably transfected with OAT2 or empty vector, intracellular cGMP accumulation was determined after coincubation with 10 μM cGMP (0.1 μM [3H]cGMP + 9.9 μM unlabeled cGMP) and 1–1000 μM furosemide (a) or 10–1000 μM bumetanide (b), respectively, for 5 min at 37°C. The furosemide and bumetanide concentrations causing half-maximal inhibitory effect (IC50) on cGMP accumulation in OAT2 expressing cells were calculated. Data are presented as mean ± SEM. nfurosemide = 2–4; nbumetanide = 2.
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fig4: IC50 determination for the inhibition of OAT2-mediated cGMP uptake by furosemide and bumetanide. In HEK293 cells stably transfected with OAT2 or empty vector, intracellular cGMP accumulation was determined after coincubation with 10 μM cGMP (0.1 μM [3H]cGMP + 9.9 μM unlabeled cGMP) and 1–1000 μM furosemide (a) or 10–1000 μM bumetanide (b), respectively, for 5 min at 37°C. The furosemide and bumetanide concentrations causing half-maximal inhibitory effect (IC50) on cGMP accumulation in OAT2 expressing cells were calculated. Data are presented as mean ± SEM. nfurosemide = 2–4; nbumetanide = 2.

Mentions: OAT2 transport function was abolished by indomethacin, a verified inhibitor of OAT2-mediated cGMP uptake [29] (Figure 3(b)). The antidiabetic drug sitagliptin inhibited OAT2-mediated cGMP accumulation by approx. 25% (Figure 3(b)). Miglitol showed no effect on OAT2-dependent cGMP uptake (Figure 3(b)). No or a small significant inhibition of OAT2-dependent cGMP accumulation was observed in presence of the ACE-inhibitors captopril and enalapril (Figure 3(b)). The uptake of cGMP in OAT2-expressing HEK293 cells was strongly reduced by the diuretics furosemide and bumetanide (Figure 3), showing IC50 values of 10.9 ± 0.6 μM (Figure 4(a)) and 130.4 ± 21.8 μM (Figure 4(b)), respectively.


Sex-differences in renal expression of selected transporters and transcription factors in lean and obese Zucker spontaneously hypertensive fatty rats.

Babelova A, Burckhardt BC, Wegner W, Burckhardt G, Henjakovic M - J Diabetes Res (2015)

IC50 determination for the inhibition of OAT2-mediated cGMP uptake by furosemide and bumetanide. In HEK293 cells stably transfected with OAT2 or empty vector, intracellular cGMP accumulation was determined after coincubation with 10 μM cGMP (0.1 μM [3H]cGMP + 9.9 μM unlabeled cGMP) and 1–1000 μM furosemide (a) or 10–1000 μM bumetanide (b), respectively, for 5 min at 37°C. The furosemide and bumetanide concentrations causing half-maximal inhibitory effect (IC50) on cGMP accumulation in OAT2 expressing cells were calculated. Data are presented as mean ± SEM. nfurosemide = 2–4; nbumetanide = 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325971&req=5

fig4: IC50 determination for the inhibition of OAT2-mediated cGMP uptake by furosemide and bumetanide. In HEK293 cells stably transfected with OAT2 or empty vector, intracellular cGMP accumulation was determined after coincubation with 10 μM cGMP (0.1 μM [3H]cGMP + 9.9 μM unlabeled cGMP) and 1–1000 μM furosemide (a) or 10–1000 μM bumetanide (b), respectively, for 5 min at 37°C. The furosemide and bumetanide concentrations causing half-maximal inhibitory effect (IC50) on cGMP accumulation in OAT2 expressing cells were calculated. Data are presented as mean ± SEM. nfurosemide = 2–4; nbumetanide = 2.
Mentions: OAT2 transport function was abolished by indomethacin, a verified inhibitor of OAT2-mediated cGMP uptake [29] (Figure 3(b)). The antidiabetic drug sitagliptin inhibited OAT2-mediated cGMP accumulation by approx. 25% (Figure 3(b)). Miglitol showed no effect on OAT2-dependent cGMP uptake (Figure 3(b)). No or a small significant inhibition of OAT2-dependent cGMP accumulation was observed in presence of the ACE-inhibitors captopril and enalapril (Figure 3(b)). The uptake of cGMP in OAT2-expressing HEK293 cells was strongly reduced by the diuretics furosemide and bumetanide (Figure 3), showing IC50 values of 10.9 ± 0.6 μM (Figure 4(a)) and 130.4 ± 21.8 μM (Figure 4(b)), respectively.

Bottom Line: Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1β and Hnf4α in both sexes.Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors.However, OAT2-dependent uptake of cGMP was inhibited by furosemide.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cardiovascular Physiology (Physiology I), Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt am Main, Germany ; Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 83391 Bratislava, Slovakia.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus