The Effect Of Extracellular Potassium On The Channels In The Distal Tubula In Mice

Objective:Different levels of blood potassium can modulate the blood sodium via sodium chloride co-transporter?NCC?in the distal tubule in mice.Recent studies have shown that NCC not only plays the critical role in the maintenance of sodium homeostasis,but also can act as the sensor of blood potassium.The major K⁺secretion in the distal tubule is attributed to the small-conductance K⁺channel ROMK,which is modulated by aldosterone;however,enhanced urinary potassium excretion was detectable as early as spot urines could be collected followed by a significant rise of plasma aldosterone in mice?the abundance of ROMK will not increase until 6hr after the high K⁺intake?.Except ROMK,the large conductance Ca²⁺-Activated K⁺Channel?BK?is the only channel for facilitating K⁺secretion,which is not modulated by aldosterone;however,BK just has been considered to act as the supplemental role for ROMK.Thus,this study aims to discuss the mechanism of rapid K+secretion in mice via channels in the distal tubule.Methods:Kidneys of wild-type C57/Bl6 between 8 and 10 weeks old were removed under deep isoflurane anaesthesia and 300?m thick slices were cut with a vibrating microslicer performed in ice-cold Ringer-type solution bubbled with 95%O2 and 5%CO2.These slices were incubated in a similar Ringer-type solution for 30min.Then,slices were randomly transferred to the incubation chambers containing the high K⁺or low K⁺solutions.Slices collected from 5,15 and 30min were processed for immunoblotting to analyze the abundance and phosphorylation of NCC.The effect of BaCl2 and RbCl on the NCC abundance and phosphorylation was also detected.Slices collected from 2hr were processed for immunoblotting to analyze the abundance of different subunits of BK.Statistics were performed with Image J and GraphPad Prism software.The data are shown as mean values±s.e.m.Student's t-test was used to compare between two groups.One-way ANOVA test was used to compare more groups in the same experimental series,and p<0.05 was considered significant.Results:1)We studied the abundance of NCC and phosphorylated NCC in kidney slices incubated with high K⁺solution for 5,15 and 30min?p<0.01&p<0.001&p<0.01?.The abundance of NCC remained stable between 5 and 30min of incubation,whereas pNCC decreased continuously with time?p<0.001?.2)After incubation with BaCl2 or RbCl solution,pNCC was obviously decreased?p<0.001&p<0.01?.3)The total abundance of BK?-and?4-subunits were not changed during the course of 2h with high K⁺solution.Moreover,BK channels should be on the membrane for potassium secretion.Thus,the membrane proteins of BK?-subunit was detected;however,the result did not show significant difference.Conclusions:1)Extracellular potassium rapidly dephosphorylates and inactivates NCC,and this effect decreases continuously with time.2)Incubation with BaCl2 or RbCl solution affects potassium sensing by Kir4.1 channel in renal distal tubules,which finally causes the decrease of phosphorylated NCC.3)Considering no significant changes in the total abundance of BK?-and?4-subunits and the membrane abundance of BK?-subunit,patch-clamp study via distal tubule may be useful to verify the distribution and open probability under high K+condition.