Renal efflux transporter expression in pregnant mice with Type I diabetes

Abstract

Prior research suggests that sex hormones and metabolic changes, such as obesity and hyperglycemia, can alter renal transporter expression in rodents. The purpose of this study was to characterize the expression of kidney efflux transporters and regulatory transcription factors in response to Type I diabetes and pregnancy. Female C57BL/6 mice were treated with multiple low doses of streptozotocin (STZ) to induce hyperglycemia and then mated with normoglycemic male mice. Transporter mRNA and protein expression were quantified in kidneys from vehicle- and STZ-treated non-pregnant and pregnant mice on gestation day 14. Pregnancy decreased the expression of Mdr1b, Mrp4, and 5 proteins and increased the mRNA and protein expression of Mrp3 by 50-60%. STZ treatment elevated Mrp1, 2, 4, and 5 and reduced Mrp3, 6, and Mdr1b mRNA and/or protein in non-pregnant mice. Pregnancy had little effect on STZ-mediated changes in renal efflux transporter expression. Transcriptional profiles of Hnf1α, PXR, AhR, and Nrf2 were altered in patterns similar to some efflux transporters suggesting potential involvement in their regulation. Taken together, these results suggest that renal drug efflux transporters and regulatory signaling pathways are altered by endocrine and metabolic changes that occur during pregnancy and Type I diabetes.

Figure 1. Serum glucose levels in vehicle- and STZ-treated mice during pregnancy

Serum glucose levels of vehicle- and STZ-treated fed mice were quantified colorimetrically and presented as mean ± SE (n= 3-8 animals). Asterisks (*) represent a statistical difference compared to vehicle non-pregnant mice (p ≤ 0.05); Daggers (†) represent a statistical difference between vehicle and STZ-treated pregnant mice (p ≤ 0.05);

Figure 2. Renal mRNA expression of efflux transporters in vehicle- and STZ-treated mice during pregnancy

Total RNA was isolated from kidneys and mRNA levels were quantified using real-time quantitative PCR. The mRNA data for (A) apical transporters and (B) basolateral transporters are presented as means normalized to Rpl13a ± SE (n= 3-8 animals). Asterisks (*) represent a statistical difference compared to vehicle non-pregnant mice (p ≤ 0.05); Daggers (†) represent a statistical difference between vehicle and STZ-treated pregnant mice (p ≤ 0.05); Double daggers (‡) represent a statistical difference between STZ-treated non-pregnant and pregnant mice (p ≤ 0.05).

Figure 3. Renal protein expression of efflux transporters in vehicle- and STZ-treated mice during pregnancy

Immunoblots are presented for (A) apical transporters and (B) basolateral transporters in the upper portion (protein loading amount 50 μg/lane). Protein band intensity was quantified by densitometry using an Alpha Innotech Fluorochem and presented as mean relative protein expression (normalized to the vehicle-treated non-pregnant experimental group) ± SE (n = 4 animals). β-actin was used as a loading control. Asterisks (*) represent a statistical difference compared to vehicle non-pregnant mice (p ≤ 0.05); Daggers (†) represent a statistical difference between vehicle and STZ-treated pregnant mice (p ≤ 0.05); Double daggers (‡) represent a statistical difference between STZ-treated non-pregnant and pregnant mice (p ≤ 0.05).

Figure 4. Immunofluorescent detection of renal Mrp1, Mrp3, Mrp4, and Mrp6 efflux transporters in vehicle- and STZ-treated mice during pregnancy

Indirect immunofluorescence staining using MRPr1, M₃II-2, M₄I-10, and M₆II-68 antibodies was conducted on kidney cryosections (6 μm) obtained from gestation day 14 vehicle- and STZ-treated mice during pregnancy. Images are shown at 10X magnification. Images were cropped, enlarged, and provided as insets.

Figure 5. Renal mRNA expression of transcription factors in vehicle- and STZ-treated mice during pregnancy

Total RNA was isolated from kidneys and mRNA levels were quantified using real-time quantitative PCR. The data are presented as means normalized to Rpl13a ± SE (n= 3-8 animals). Asterisks (*) represent a statistical difference compared to vehicle non-pregnant mice (p ≤ 0.05); Daggers (†) represent a statistical difference between vehicle and STZ-treated pregnant mice (p ≤ 0.05); Double daggers (‡) represent a statistical difference between STZ-treated non-pregnant and pregnant mice (p ≤ 0.05).

Figure 6. Renal mRNA expression of antioxidant enzymes Nqo1 and Ho-1 and the Nrf2 transcription factor in vehicle- and STZ-treated mice during pregnancy

Total RNA was isolated from kidneys and mRNA levels were quantified using real-time quantitative PCR. The data are presented as means normalized to Rpl13a ± SE (n= 3-8 animals). Asterisks (*) represent a statistical difference compared to vehicle non-pregnant mice (p ≤ 0.05); Daggers (†) represent a statistical difference between vehicle and STZ-treated pregnant mice (p ≤ 0.05); Double daggers (‡) represent a statistical difference between STZ-treated non-pregnant and pregnant mice (p ≤ 0.05).

Figure 7. Differential expression of renal efflux transporters during diabetes and pregnancy

Relative changes in mRNA and/or protein expression of efflux transporters in STZ-treated diabetic or pregnant mice are summarized.