Altered renal sodium transporter expression in an animal model of type 2 diabetes mellitus

Abstract

Hemodynamic factors play an important role in the development and/or progression of diabetic nephropathy. We hypothesized that renal sodium transporter dysregulation might contribute to the hemodynamic alterations in diabetic nephropathy. Otsuka Long Evans Tokushima Fatty (OLETF) rats were used as an animal model for type 2 diabetes. Long Evans Tokushima (LETO) rats were used as controls. Renal sodium transporter regulation was investigated by semiquantitative immunoblotting and immunohistochemistry of the kidneys of 40-week-old animals. The mean serum glucose level in OLETF rats was increased to 235+/-25 mg/dL at 25 weeks, and the hyperglycemia continued up to the end of 40 weeks. Urine protein/ creatinine ratios were 10 times higher in OLETF rats than in LETO rats. At 40th week, the abundance of the epithelial sodium channel (ENaC) beta-subunit was increased in OLETF rats, but the abundance of the ENaC gamma-subunit was decreased. No significant differences were observed in the ENaC alpha-subunit or other major sodium transporters. Immunohistochemistry for the ENaC beta-subunit showed increased immunoreactivity in OLETF rats, whereas the ENaC gamma-subunit showed reduced immunoreactivity in these rats. In OLETF rats, ENaC beta-subunit upregulation and ENaC gamma-subunit downregulation after the development of diabetic nephropathy may reflect an abnormal sodium balance.

Fig. 1

Serial changes in plasma glucose (A), body weight (B), systolic blood pressure measured using a tail cuff (C), 24-hr urine volume per kg body weight (D), fractional excretion of sodium (E), and daily urine sodium excretion (F) in LETO and OLETF rats at 12, 25, 30, and 40 weeks of age. Symbols are ▪, OLETF; ○, LETO. ^*p<0.05.

Fig. 2

Immunoblots (A-C) and immunoblot data summaries (D) for NHE3, NKCC2, and NCC. Representative blots are from 40 week-old LETO and OLETF rats. Each lane was loaded with whole kidney homogenate from a different rat. Equal amounts of protein were loaded in all lanes. Immunoblots were probed with (A) anti-NHE3 (L546), (B) anti-NKCC2 (L320), and (C) anti-NCC (573) antibodies. The relative abundance of NHE3, NKCC2, and NCC was not different between LETO and OLETF rats.

Fig. 3

Immunoblots (A-C) and immunoblot data summaries (D) for ENaC subunits. Representative blots are from 40 week-old LETO and OLETF rats. Each lane was loaded with whole kidney homogenate from a different rat. Equal amounts of protein were loaded in all lanes. Immunoblots were probed with (A) anti-ENaC α-subunit (3560-2-4), (B) anti-ENaC β-subunit (3755-2), and (C) anti-ENaC γ-subunit (A881) antibodies. Band densities of the ENaC β-subunit were significantly increased, and band densities for the ENaC γ-subunit were significantly decreased in OLETF rats vs. LETO rats. The relative abundances of ENaC α-subunit were not affected by rat type. ^*p<0.05

Fig. 4

Immunohistochemistry of ENaC β and γ-subunits in the cortex of LETO and OLETF rats. The ENaC β-subunit showed enhanced immunostaining (A, B) and the ENaC γ-subunit diminished immunostaining (C, D) in 40 week-old OLETF rats vs. LETO rats (×100).