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 α1 and β1 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 α1 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 α1 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 β1 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 α1 subunit and higher enzyme activity might be additional contributing factors to the improved postischaemic renal function of female rats.

Sex differences in heat shock protein 72 expression and localization in rats following renal ischemia-reperfusion injury

“…Previously, we have shown that the I/R-induced translocation of the NKA ␣ 1 -subunit is less pronounced in F rats than in M. In parallel with this, the enzyme was also more active in F rats, which could possibly contribute to the gender differences in postischemic survival (14) and renal recovery (4).…”

“…Tubular damage and leukocyte infiltration were semiquantitatively evaluated on a scale [0, none (no necrotic tubules, no infiltrating cells); 1, mild (Ͻ25%, necrotic tubules, Ͻ10 cells); 2, moderate (26 -50% necrotic tubules, 11-50 cells); 3, massive (51-75% necrotic tubules, 51-100 cells); and 4, severe (Ͼ76% necrotic tubules, Ͼ101 cells)]. Nucleus atypia, vacuolization and hyalinization in the tubular cells, and dissolving, sloughing off, and lack of tubular cells such as hyaline in the tubules were used for more detailed quantification as described previously (4).…”

“…All reagents, enzymes, and isolation kits were purchased from Qiagen (Hilden, Germany). Total RNA extraction and firststrand cDNA synthesis were performed as previously described (4). PCR was performed in PCR buffer, 2 mM MgCl 2, 2 mM dNTPs, 1.5 U Ampli Taq DNA polymerase, and 0.5 mol of the following HSP72 primers: F: 5Ј-cgc cgc tgt cgc tgg gtc tgg ag-3Ј; R: 5Ј-ggc ggc cct tgt gtc tgg tga tgg-3Ј(product length: 327 bp).…”

“…We also demonstrated that in female kidneys NKA stays in its physiological place, in the membrane fraction, and enzyme activity is preserved. This suggests that the enzyme is more stable and protected from the detrimental effects of postischemic injury than in males; however, the molecular mechanism still remains elusive (4).…”

Estrogen Protects against Oxidative Multiorgan Damage in Rats with Chronic Renal Failure

“…Several studies on acute renal injury have reported that females were relatively protected in comparison to males, have developed renal dysfunction in later periods of ischemic injury with lower BUN and creatinine levels, and had a lower mortality rate with a less severe tubular necrosis. [37][38][39] Despite the abundance of clinical and animal studies demonstrating that estrogens are renoprotective, the mechanisms by which estrogens exert their protective effects on the kidney remain poorly understood.…”