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

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.

Figure 1

PAS staining in renal cortical slices. Renal sections of lean (le) and obese (ob) ZSF1 rats were stained with PAS and structural changes were analyzed. Examples for histological changes in renal tissue are marked as follows: #, tubular atrophy and dilatation; ##, hyaline protein casts in tubular lumen; §, pressure-induced deformation of glomerulus; §§, dilatation of Bowman's capsule and protein cast in Bowman's space; §§§, glomerulosclerosis. The arrows mark the thickening of tubular and glomerular basement membranes. Magnification: 200x. Representative images from three different rats under each condition are shown.

Figure 2

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.

Figure 3

Influence of dicarboxylates, metabolites, and drugs on OAT2-mediated cGMP uptake in HEK293 cells. The uptake of cGMP was determined after 5 min incubation with 10 μM cGMP (0.1 μM [³H]cGMP + 9.9 μM unlabeled cGMP) alone (controls) or in the presence of 500 μM potential inhibitors at 37°C. (a) Dicarboxylates and metabolites; (b) drugs. Data are presented as mean ± SEM. n = 3. ^* P < 0.05; ^*** P < 0.001, compared to control.

Figure 4

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. n _furosemide = 2–4; n _bumetanide = 2.