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Sex Differences in Maxi‐K channel and Klotho Expressions in ROMK Bartter’s Mouse Kidney

Renal outer medullary potassium channel (ROMK) function is critical to maintain normal electron homeostasis and blood pressure. Mutations of ROMK cause Bartter’s syndrome with volume depletion, electrolyte imbalance and is postnatally life‐threatening in humans. ROMK knockout (KO) mice produced the same phenotypes to Bartter’s syndrome with postnataldeath. Previously we reported that female ROMK KO mice manifest more severe Bartter II phenotype, and males had better survival rates than female ROMK KO mice. We have also reported that thecalcium activated potassium channel BK, also known as Maxi‐K channels, contributes to urinary potassium excretion in the ROMK KO mouse. It is well documented that Klotho is an aging‐suppression protein, which is also expressed in the kidney and functions to increase ROMK1 activity by reducing ROMK1 endocytosis. However, whether BK or Klotho is altered in ROMK KO mice and whether there is any sex or gender difference in Klotho or BK expression in the kidney has never been studied. We examined the α‐subunit of BK channel (BKα) and Klotho expressions at both mRNA and the protein levels in the kidneys by QPCR, Western Blotting and immunocytochemistry methods in male and female ROMK WT and KO mice. Western Blotting results show in WT mice that BKα abundance was 6‐fold higher in males than females (2.0 vs. 0.27 BKa/β‐actin). In ROMK KO mice, BKα abundance was significantly increased in both males (from 2.0 to 3.1 BKα/β‐actin, P<0.05) and females (from 0.27 to 0.39 BKα/β‐actin, P<0.05) compared to the WT, despite the lower expression level in female WT. The Klotho abundance was also higher in male over female WT and was also upregulated in both males (3.3 vs. 2.3 BKa/β‐actin, P<0.05) and females (1.8 vs. 1.5 BKα/β‐actin, P<0.05) KO mice compared to the WT. QPCR results show that BKα mRNA expression was higher in males over females in both WT and KO mice. It was significantly elevated in both male (3.6‐fold) and female (2.4‐fold) ROMK KO compared to the WT mice. The Klotho expression at the mRNA level was slightly elevated in both male and female ROMK KO compared to the WT. Immunocytochemistry results show higher BKα and Klotho staining intensity in male over female WT mice kidney, and the intensity increased in both male and female ROMK KO mice compared to the WT. Such an increase of BKa and Klotho was observed as early as the age of 2 weeks, and more increases were observed at 4 weeks in the KO compared to the WT. There was no additional increase in either BKa or Klotho at the age of 12 weeks compared to the 4 weeks KO mice. We conclude that i) higher expressions of BKα and Klotho at both mRNA and protein levels in male over female mice kidney; ii) BKα and Klotho expression was upregulated in both male and female ROMK KO mice; iii) The significant sex difference in BKα and Klotho expression may be responsible for better survival rate of male ROMK Bartter’s mice.