Sex differences in the alterations of Na(+), K(+)-ATPase following ischaemia-reperfusion injury in the rat kidney

Abstract

Postischaemic acute renal failure (ARF) is influenced by sex. Na(+), K(+)-ATPase (NKA) plays a crucial role in the pathogenesis of postischaemic ARF. We tested the impact of sex on mRNA, protein expression, cellular distribution and enzyme activity of NKA following renal ischaemia-reperfusion (I-R) injury. The left renal pedicle of uninephrectomized female (F) and male (M) Wistar rats was clamped for 55 min followed by 2 h (T2) and 16 h (T16) of reperfusion. Uninephrectomized, sham-operated F and M rats served as controls (n= 6 per group). Blood urea nitrogen, serum creatinine and renal histology were evaluated to detect the severity of postischaemic ARF. mRNA expression of NKA alpha1 and beta1 subunits were detected by RT-PCR. The effect of I-R on cellular distribution was compared by Triton X-100 extraction. Cellular proteins were divided into Triton-insoluble and Triton-soluble fractions and assessed by Western blot. NKA enzyme activity was also determined. After the ischaemic insult blood urea nitrogen and serum creatinine were higher and renal histology showed more rapid progression in M versus F (P < 0.05). mRNA expression of the NKA alpha1 subunit decreased in I-R groups versus controls, but was higher in F versus M both in control and I-R groups (P < 0.05). However, protein levels of the NKA alpha1 subunit in total tissue homogenate did not differ in controls, but were higher in F versus M in I-R groups (P < 0.05). Triton X-100 extractability was lower in F versus M at T16 (P < 0.05). NKA enzyme activity was the same in controls, but was higher in F versus M in I-R groups (T2: 14.9 +/- 2.3 versus 9.15 +/- 2.21 U) (T16: 11.7 +/- 4.1 versus 5.65 +/- 2.3 U; P < 0.05). mRNA and protein expression of the NKA beta1 subunit did not differ between F and M in any of the protocol. We concluded that NKA is more protected from the detrimental effects of postischaemic injury in females. Higher mRNA and protein expression of the NKA alpha1 subunit and higher enzyme activity might be additional contributing factors to the improved postischaemic renal function of female rats.

Figure 1. Histopathological changes in postischaemic kidneys of F and M rats

Histopathological changes in postischaemic kidneys were determined and evaluated in PAS-stained kidneys of T2 female (A) and T2 male (B), and of T16 female (C) and T16 male (D) rats.

Figure 2. Effect of I-R injury on mRNA expression of Na⁺,K⁺-ATPase α1 and β1 subunits in F and M rat kidney

mRNA expression of Na⁺,K⁺-ATPase α1 and β1 subunits were determined in kidney samples from controls, and at T2 and T16 hours of reperfusion following 55 min of renal ischaemia in F and M rats (n= 6 per group). Top panel, examples of RT-PCR analysis of Na⁺,K⁺-ATPase α1 and β1 subunits and GAPDH in kidney. A and B, results are given as the ratio of intensity of Na⁺,K⁺-ATPase α1 or β1 subunit mRNA to GAPDH mRNA. □,F; ▪,M. Data are given as mean ±s.d. *P < 0.05 versus M; ⁺P < 0.05versus T2; ^§P < 0.05versus T16.

Figure 3. Effect of I-R injury on protein levels and cellular distribution of Na⁺,K⁺-ATPase α1 in F and M rat kidney

Protein levels of the Na⁺,K⁺-ATPase α1 subunit were determined in total kidney tissue homogenate (TOT), the Triton-soluble supernatant (SUP) and the Triton-insoluble pellet (PEL) fraction of F and M rats in controls, and at T2 and T16 hours of reperfusion following 55 min of renal ischaemia (n= 6 per group). Top panel, examples of Western blot analysis of Na⁺,K⁺-ATPase α1 in TOT, PEL and SUP fractions. A–C, results for Na⁺,K⁺-ATPase α1 protein levels in TOT, PEL and SUP were normalized to an internal standard. □,F; ▪,M. Data are given as mean ±s.d.*P < 0.05versus M; ^§P < 0.05versus T16.

Figure 4. Effect of I-R injury on protein levels and cellular distribution of Na⁺,K⁺-ATPase β1 in F and M rat kidney

Protein levels of the Na⁺,K⁺-ATPase β1 subunit were determined in total kidney tissue homogenate (TOT), Triton-soluble supernatant (SUP) and Triton-insoluble pellet (PEL) fractions of F and M rats in controls, and at T2 and T16 hours of reperfusion following 55 min of renal ischaemia (n= 6 per group). Top panel, examples of Western blot analysis of Na⁺,K⁺-ATPase β1 in TOT, PEL and SUP fractions. A–C, results for Na⁺,K⁺-ATPase β1 protein levels in TOT, PEL and SUP were normalized to an internal standard. □,F; ▪,M. Data are given as mean ±s.d.*P < 0.05versus M; ^†P < 0.05versus T2; ^§P < 0.05versus T16.

Figure 5. Effect of I-R injury on Na⁺,K⁺-ATPase enzyme activity in F and M rat kidney

Na⁺,K⁺-ATPase activity was determined in kidney samples from controls, and at T2 and at T16 hours of reperfusion following 55 min of renal ischaemia in F (□) and M (▪) rats (n= 6 per group). Data are given as mean ±s.d.*P < 0.05versus M; ⁺P < 0.05versus T2; ^§P < 0.05versus T16.