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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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Diabetes- and sex-dependent renal gene expression in lean (le) and obese (ob) ZSF1 rats. Gene expressions were analyzed using TaqMan real-time PCR and presented as mean ± SEM. n = 6–8. n.s., not significant; **P < 0.01; and ***P < 0.001, for the comparison of ΔCt values between lean and obese ZSF1 rats. #P < 0.05; ##P < 0.01; and ###P < 0.001, for comparison of ΔCt values between females and males.
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fig2: Diabetes- and sex-dependent renal gene expression in lean (le) and obese (ob) ZSF1 rats. Gene expressions were analyzed using TaqMan real-time PCR and presented as mean ± SEM. n = 6–8. n.s., not significant; **P < 0.01; and ***P < 0.001, for the comparison of ΔCt values between lean and obese ZSF1 rats. #P < 0.05; ##P < 0.01; and ###P < 0.001, for comparison of ΔCt values between females and males.

Mentions: Superscript II Reverse Transcriptase (Life Technologies, Darmstadt, Germany) and Oligo dT-Primers (Eurofin MWG Operon, Ebersberg, Germany) were used for reverse transcription of RNA. Genes of interest were analyzed using TaqMan Master Mix and TaqMan Gene Expression Assays (Life Technologies): Sodium-dependent glucose cotransporter 1 (Sglt1), Rn00564718_m1; Sglt2, Rn00574917_m1; Oat1, Rn00568143_m1; Oat2, Rn00585513_m1; Oat3, Rn00580082_m1; Oct1, Rn00562250_m1; Oct2, Rn00580893_m1; Mrp2, Rn00563231_m1; Mrp4, Rn01465702_m1; P-glycoprotein (Mdr1b), Rn00561753_m1; Hnf1α, Rn00562020_m1; Hnf1β, Rn00447453_m1; Hnf4α, Rn00573309_m1. The mRNA levels of hypoxanthine phosphoribosyltransferase 1 (Hprt1, Rn01527840_m1), β-actin (Rn00667869_m1), and cyclophilin A (Rn00690933_m1) were tested as housekeeping control genes for sample normalization. For all tested genes, PCR conditions were as follows: 2 min at 50°C followed by 10 min at 95°C and 40 amplification cycles (95°C for 15 s and 60°C for 60 s), using Mx3000P real-time PCR cycler (Agilent Technologies). Signals detected between 35–40 amplification cycles were defined as low gene expression. The amplification efficiencies of used assays were 100% (+/− 10%), in accordance with manufacturer's information. The real-time PCR data were analyzed as ΔCt = housekeeping gene (Hprt1)-gene of interest (Figure 2), using the 2−ΔΔCt method (see Supplementary Figures in Supplementary Material available online at http://dx.doi.org/10.1155/2015/483238) [25].


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)

Diabetes- and sex-dependent renal gene expression in lean (le) and obese (ob) ZSF1 rats. Gene expressions were analyzed using TaqMan real-time PCR and presented as mean ± SEM. n = 6–8. n.s., not significant; **P < 0.01; and ***P < 0.001, for the comparison of ΔCt values between lean and obese ZSF1 rats. #P < 0.05; ##P < 0.01; and ###P < 0.001, for comparison of ΔCt values between females and males.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Diabetes- and sex-dependent renal gene expression in lean (le) and obese (ob) ZSF1 rats. Gene expressions were analyzed using TaqMan real-time PCR and presented as mean ± SEM. n = 6–8. n.s., not significant; **P < 0.01; and ***P < 0.001, for the comparison of ΔCt values between lean and obese ZSF1 rats. #P < 0.05; ##P < 0.01; and ###P < 0.001, for comparison of ΔCt values between females and males.
Mentions: Superscript II Reverse Transcriptase (Life Technologies, Darmstadt, Germany) and Oligo dT-Primers (Eurofin MWG Operon, Ebersberg, Germany) were used for reverse transcription of RNA. Genes of interest were analyzed using TaqMan Master Mix and TaqMan Gene Expression Assays (Life Technologies): Sodium-dependent glucose cotransporter 1 (Sglt1), Rn00564718_m1; Sglt2, Rn00574917_m1; Oat1, Rn00568143_m1; Oat2, Rn00585513_m1; Oat3, Rn00580082_m1; Oct1, Rn00562250_m1; Oct2, Rn00580893_m1; Mrp2, Rn00563231_m1; Mrp4, Rn01465702_m1; P-glycoprotein (Mdr1b), Rn00561753_m1; Hnf1α, Rn00562020_m1; Hnf1β, Rn00447453_m1; Hnf4α, Rn00573309_m1. The mRNA levels of hypoxanthine phosphoribosyltransferase 1 (Hprt1, Rn01527840_m1), β-actin (Rn00667869_m1), and cyclophilin A (Rn00690933_m1) were tested as housekeeping control genes for sample normalization. For all tested genes, PCR conditions were as follows: 2 min at 50°C followed by 10 min at 95°C and 40 amplification cycles (95°C for 15 s and 60°C for 60 s), using Mx3000P real-time PCR cycler (Agilent Technologies). Signals detected between 35–40 amplification cycles were defined as low gene expression. The amplification efficiencies of used assays were 100% (+/− 10%), in accordance with manufacturer's information. The real-time PCR data were analyzed as ΔCt = housekeeping gene (Hprt1)-gene of interest (Figure 2), using the 2−ΔΔCt method (see Supplementary Figures in Supplementary Material available online at http://dx.doi.org/10.1155/2015/483238) [25].

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