Role Of AT1a Receptor In Regulation Of Renal Sodium And Chloride Cotransporter Function In Male And Female Mice

Sodium and chloride co-transporter（NCC）,coded by SLC12A3 gene,primary expresses in kidney and mediates the majority of sodium absorption in distal tubules.Alteration of NCC directly affects electron balance and blood pressure.NCC activity had gender difference and can be regulated by multiple hormones,both female hormone and angiotensin Ⅱ stimulated NCC activity.Angiotensin Ⅱ regulated renal salt and water balance by its type 1 receptors,which had gender difference on expression in kidney,however,the role of angiotensin Ⅱ in gender difference of NCC function is unclear.Angiotensin Ⅱ regulated NHE3 expression in proximal tubule,but if it correlated with NCC function is still unknown.We studied gender differences in NCC activity and expression in mae and female wild type(AT1a^+/+)and AT1 a knockout(AT1a^-/-)mice,to investigate the role of AT1 a receptor in NCC function regulation and sex-related difference,we further studied NHE3 expression in male and female AT1a^+/+ and AT1a^-/-mice,to investigate the correlated regulation mechanism.ObjectiveTo study the role of AT1 a receptor in regulation of gender difference in NCC function and NHE3 expression.Materials and MethodsRenal clearance experiments were performed on both age-paired male and female AT1a^+/+ and AT1a^-/-mice,6-8 mice for each group.Urine volume（UV）,glomerular filtration rate（GFR）,absolute Na and K（ENa;EK）and fractional Na and K（FENa;FEK）excretion were measured and compared at time and peak changes after bolus iv injection of hydrochlorothiazide（HCTZ;30mg/kg）.Real-time RT-PCR（q-PCR）was used for NCC and NHE3 mRNA expression measurement,western-blotting was used for NCC and NHE3 membrane protein expression measurement.Student’s t-test and one-way ANOVA was for different groups comparison.The differences between the mean values were considered significant if P < 0.05.ResultsⅠn AT1a^+/+ mice,HCTZ produced greater diuretic and natriuretic effects in females than males.The fractional increases of UV,ENa,FENa and EK by HCTZ were 3.4-vs.7.8-folds;5.6-vs.11.7-folds;4.9-vs.7.9-folds and 1.4-vs.3.1-folds in males and females,respectively（P<0.05）.Ⅰn contrast,there was no gender difference in AT1a^-/-mice,since HCTZ produced stronger effects on male AT1a^-/-than their AT1a^+/+,and similar effect on female AT1a^+/+ and AT1a^-/-mice.q-PCR result showed that both in AT1a^+/+ and AT1a^-/-mice,there was gender difference in NCC mRNA expression,females have higher NCC mRNA expression than males;NHE3 mRNA expression was not difference between male and female AT1a^+/+ and AT1a^-/-mice.Western-blotting result showed,in AT1a^+/+ mice,the total NCC（tNCC）and phosphorylated NCC（pNCC）were 1.7-folds and 4.6-folds higher in female compared to males（P<0.05）,consistent with the larger response to HCTZ.Ⅰn AT1a^-/-mice,tNCC and pNCC increased 2.2-folds and 6.2-folds in males but no significant increase in females,resulted in no gender difference of tNCC and pNCC expression in AT1a^-/-mice.There was no gender difference of Na/H-Exchanger（NHE3）expression in AT1a^+/+ mice and with 61%decreased in male AT1a^-/-mice and 46% decreased in female AT1a^-/-mice,but the different ratio of decrease in male and female AT1a^-/-mice was not significant.Conclusion1.Higher NCC mRNA and protein expression in female correlated with activity in AT1a^+/+mice;2.The gender-specific differences in NCC protein and activity expression were absent in AT1a^-/-mice,AT1 a receptor regulated NCC protein expression and activity.3.Male AT1a^-/-mice significantly increased NCC activity and expression may due to stronger AT1a-mediated NHE3 expression decreased in kidney proximal tubules.