Empirical Study On Change Rule And Regulating Mechanism Of Potassium During Hypokalemia

Objective: K⁺-depleted rabbit models were prepared with a low-K cotent diet in this study, plasma potassium ion (K⁺) concentration, K⁺ content, Na⁺-K⁺-ATPase activity and Na⁺-K⁺-ATPaseα₁,α₂ isoform protein expression in erythrocyte(RBC), myocardium and skeletal muscle, Na⁺-K⁺-ATPaseα₁,α₂ isoform mRNA expression in myocardium and skeletal muscle were measured when rabbits were fed different days, to investigate correlation between K⁺ content and Na⁺-K⁺-ATPase activity in RBC, myocardium and skeletal muscle, respectively, and to explore correlation between RBC K content and myocadial or skeletal muscle K⁺ content. Total K⁺ infusion dosage, renal K⁺ excretion and K⁺ uptake rate of myocardium and skeletal muscle were observed after clamping of plasma K⁺ by KCl infusion.Methods:1 Prepared animal models with a low-K content diet Forty-eight healthy male rabbits weighing 2.0².7kg were randomly divided into 6 groups (n=8/group) : normal control group (group T₀) ,K-depleted-3-day group (group T₁) , K-depleted-6-day group (group T₂) , K-depleted-9-day group (group T₃) , K-depleted-12-day group (group T₄) , K-depleted-15-day group (group T₅).All rabbits were fed a normal diet for a week before model preparation.Group T₀ were given a normal diet only(100g standard chow/day each rabbit,free access to normal water),groupT₁,T₂,T₃,T₄,T₅ were fed with a low-K content diet (100g low-K content chow/day each rabbit,free access to distilled water)for 3 days,6 days, 9 days, 12 days, 15 days,respectively. On the day that models were prepared, rabbits in group T₀,T₁,T₂,T₃,T₄,T₅ were anesthetized by 4% pentobarbital (1ml/kg) given through auricular vein, 1ml blood for measuring plama K⁺, sodium(Na⁺) and hemoglobin concentration was sampled through central auricular artery , their thoracic cavity were opend and hearts were excised, at the moment, soleus muscle and femoral vein were separated and exposed, then soleus muscle was amputated ,blood was taken from femoral vein for separating RBC,to measure K⁺ and Na⁺ content in RBC, myocardium and soleus muscle.Plasma K⁺ and Na⁺ concentrations were measured by ion-sensitive electrodes, hemoglobin concentration by spectrophotometric method. K⁺ contents and Na⁺ contents in RBC, myocardium and soleus muscle were measured by flame atomic absorption photometry.2 Protein and mRNA expression of Na⁺-K⁺-ATPaseα₁,α₂ isoform in RBC, myocardium and soleus muscle of K⁺-depleted rabbitsForty-eight healthy male rabbits weighing 2.0².7kg were randomly divided into 6 groups (n=8/group) : normal control group (group T₀) ,K⁺-depleted-3-day group (group T₁) , K⁺-depleted-6-day group (group T₂) , K⁺-depleted-9-day group (group T₃) , K⁺-depleted-12-day group (group T₄) , K⁺-depleted-15-day group (group T₅).All rabbits were fed a normal diet for a week before model preparation.Group T₀ were given a normal diet only(100g standard chow/day each rabbit,free access to normal water),groupT₁,T₂,T₃,T₄,T₅ were fed with a low-K content diet (100g low-K⁺ content chow/day each rabbit,free access to distilled water)for 3 days,6 days, 9 days, 12 days, 15 days,respectively. When animal models were prepared, hearts and soleus muscle of rabbits in group T₀,T₁,T₂,T₃,T₄,T₅ were cut off after blood sampling ,to measure plasma K⁺, Na, hemoglobin concentration, K⁺ and Na⁺ content in RBC, myocardium and soleus muscle.On the day that models were prepared, rabbits in group T₀,T₁,T₂,T₃,T₄,T₅ were anesthetized by 4% pentobarbital (1ml/kg) given through auricular vein, their thoracic cavity were opend and hearts were excised, at the moment, soleus muscle and femoral vein were separated and exposed, then soleus muscle was amputated ,blood was taken from femoral vein for separating RBC, to measure Na⁺-K⁺-ATPase activity and Na⁺-K⁺-ATPaseα₁,α₂ isoform protein and mRNA expression in RBC, myocardium, soleus muscle. Na⁺-K⁺-ATPase activity of RBC, myocardium and soleus muscle were measured with an ultramicro-ATPase test box after total protein quantity with a coomssie brilliant blue protein measurement test box. Na⁺-K⁺-ATPaseα₁ andα₂ isoform protein expression in RBC, myocardium and soleus muscle were measured by western blot. Na⁺-K⁺-ATPaseα₁ andα₂ isoform mRNA expression in myocardium and soleus muscle were measured by real-time PCR.3 Correlation between RBC K⁺ content and mycardial K content or soleus K⁺ content in K-depleted rabbitsForty-eight healthy male rabbits weighing 2.0².7 kg were randomly divided into 6 groups (n=8/group) : normal control group (group T₀) ,K⁺-depleted-3-day group (group T₁) , K⁺-depleted-6-day group (group T₂) , K⁺-depleted-9-day group (group T₃) , K⁺-depleted-12-day group (group T₄) , K⁺-depleted-15-day group (group T₅).All rabbits were fed a normal diet for a week before model preparation.Group T₀ were given a normal diet only(100g standard chow/day each rabbit,free access to normal water),groupT₁,T₂,T₃,T₄,T₅ were fed with a low-K content diet (100g low-K content chow/day each rabbit,free access to distilled water)for 3 days,6 days, 9 days, 12 days, 15 days,respectively. When animal models were prepared, hearts and soleus muscle of rabbits in group T₀,T₁,T₂,T₃,T₄,T₅ were cut off after blood sampling ,to measure plasma K⁺, Na, hemoglobin concentration, K⁺ and Na⁺ content in RBC, myocardium and soleus muscle.On the day that models were prepared, rabbits in group T₀,T₁,T₂,T₃,T₄,T₅ were anesthetized by 4% pentobarbital (1ml/kg) given through auricular vein, their thoracic cavity were opend and hearts were excised, at the moment, soleus muscle and femoral vein were separated and exposed, then soleus muscle was amputated ,blood was taken from femoral vein for separating RBC, to measure K⁺ content in RBC, myocardium and soleus muscle.K⁺ contents in RBC, myocardium and soleus muscle were measured by flame atomic absorption photometry.Analyze relationship between RBC K⁺ content and mycardial K⁺ content or soleus K⁺ content4 Change of K⁺ content in myocardium and soleus muscle in K⁺-depleted rabbits after K⁺ repletionSixteen healthy male rabbits weighing 2.0².7kg were randomly divided into 2 groups (n=8/group) : repletion control group (group RC) and repletion experimental group (group RE).All rabbits were fed a normal diet for a week before model preparation.Group RC were given a normal diet only(100 g standard chow/day each rabbit,free access to normal water),group RE were fed with a low-K⁺ content diet (100 g low-K⁺ content chow/day each rabbit,free access to distilled water)for 15 days. Rabbits in group RC and RE were used for intravenous potassium repletion experiment. After rabbits were anesthetized, a urinary catheter was incerted for collecting urine to measure renal K⁺ excretion in 1 hour, then KCl infusions began and continued until the plasma K⁺ reached the set-point 5.5mmol/L and were stable for 1 h.KCl infusions were then immediately stopped,the hearts and soleus muscle of animals were excised. Urine during KCl infusion was collected to measure K⁺ content in urine.Total K uptake of grop RC and RE was expressed as total K infusion dosage after K⁺ clamping was stopped, renal K⁺ excretion was expressed as total K⁺ content in urine .Myocardial(Soleus) K⁺ uptake of group RC during KCl infusions was calculated as the difference between myocardial (soleus) K⁺ content in each heart (soleus muscle) after KCl infusion and mean myocardial (soleus) K⁺ content in group T₀; myocardial(soleus) K⁺ uptake of group RE during KCl infusions was calculated as the difference between myocardial (soleus) K⁺ content in each heart (soleus muscle) after KCl infusion and mean myocardial (soleus) K⁺ content in group T₅;mean myocardial (soleus) K⁺ uptake rate of each rabbit was calculated by respective myocardial (soleus) K⁺ uptake divided by duration of K⁺ infusion .Result:1 Prepared animal models with a low-K ⁺content diet1.1 Animals were clinically unaffected and the low-K⁺ content dietary regimens were well tolerated.1.2 There is no difference of the rabbits'weight and ages in each groups.1.3 The change of plasma K⁺, Na⁺ and hemoglobin concentrationCompared with group T₀, plasma K⁺ concentration in group T₁,T₂,T₃,T₄,T₅ gradually decreased,the statistical differcence was significant(P<0.05); no significant difference in plasma Na⁺ and hemoglobin concentration was observed in group T₀,T₁,T₂,T₃,T₄,T₅(P>0.05).1.4 The change of RBC K⁺,Na ⁺contentCompared with group T₀, RBC K⁺ content in group T₁,T₂,T₃,T₄,T₅ gradually decreased,the statistical differcence was significant(P<0.05); compared with group T₀, RBC Na⁺ content in group T₁,T₂,T₃,T₄,T₅ gradually increased,the statistical differcence was significant(P<0.05).1.5 The change of myocardial K⁺,Na⁺ contentCompared with group T₀, there was no significant decrease in myocardial K⁺ content of group T₁,(P >0.05), myocardial K⁺ content in groupT₂, T₃,T₄,T₅ decreased,the statistical differcence was significant(P <0.05);there was no significant increse in myocardial Na⁺ content in T₁,T₂,T₃,T₄,T₅ compared with group T₀(P >0.05).1.6 The change of soleus K⁺,Na⁺ contentCompared with group T₀, soleus K⁺ content in group T₁,T₂,T₃,T₄,T₅ gradually decreased,the statistical differcence was significant(P<0.05); compared with group T₀, soleus Na⁺ content in group T₁,T₂,T₃,T₄,T₅ gradually increased,the statistical differcence was significant(P<0.05).2 Protein and mRNA expression of Na⁺-K⁺-ATPaseα₁,α₂ isoform in RBC, myocardium and soleus muscle of K⁺-depleted rabbits2.1 The change of RBC Na⁺-K⁺-ATPase activityCompared with group T₀, RBC Na⁺-K⁺-ATPase activity in group T₁ decreased, but the statistical differcence was not significant(P >0.05), RBC Na⁺-K⁺-ATPase activity in group T₂,T₃,T₄,T₅ decreased,the statistical differcence was significant(P <0.05)2.2 The change of myocardial Na⁺-K⁺-ATPase activity Compared with group T₀, there was no significant increase in myocardial Na⁺-K⁺-ATPase activity in group T₁,T₂ (P >0.05), but myocardial Na⁺-K⁺-ATPase activity in group T₃ , T₄,T₅ increased,the statistical differcence was significant(P <0.05).2.3 The change of soleus Na⁺-K⁺-ATPase activityCompared with group T₀, RBC Na⁺-K⁺-ATPase activity in group T₁,T₂,T₃,T₄,T₅ decreased, the statistical differcence was significant(P <0.05)2.4 Relationship between RBC K and Na⁺-K⁺-ATPase activityDuring K depletion,both RBC K content and Na⁺-K⁺-ATPase activity gradually decreased, linear regression analysis showed a positive linear relationship between RBC Na⁺-K⁺-ATPaseb activity and RBC K content, linear regression equation was Y=0.08X-0.31, R²=0.766, P < 0.05.2.5 Relationship between myocardial K and Na⁺-K⁺-ATPase activityDuring K depletion, myocardial K content gradually decreased ,however, myocardial Na⁺-K⁺-ATPase activity gradually increased, linear regression analysis showed a negative linear relationship between myocardial Na⁺-K⁺-ATPaseb activity and myocardial K content, linear regression equation was Y=18.41-0.09X,R²=0.434, P < 0.05.2.6 Relationship between soleus K and Na⁺-K⁺-ATPase activityDuring K depletion,both soleus K content and Na⁺-K⁺-ATPase activity gradually decreased, linear regression analysis showed a positive linear relationship between soleus Na⁺-K⁺-ATPaseb activity and soleus K content, linear regression equation was Y=0.25X-7.94,R²=0.870, P< 0.05.2.7 Protein expression of RBC Na⁺-K⁺-ATPaseα₁ andα₂ isoformCompared with group T₀, there was no significant decrease in RBC Na⁺-K⁺-ATPaseα₁ isoform protein expression in group T₃(P>0.05) ,RBC Na⁺-K⁺-ATPaseα₁ isoform protein expression in group T₅ decreased, the statistical differcence was significant(P<0.05); compared with group T₀, RBC Na⁺-K⁺-ATPaseα₂ isoform protein expression in group T₃,T₅ decreased, the statistical differcence was significant(P<0.05).2.8 Protein expression of myocardial Na⁺-K⁺-ATPaseα₁ andα₂ isoform Compared with group T₀, myocardial Na⁺-K⁺-ATPaseα₁ isoform protein expression in group T₃,T₅ increased, the statistical differcence was significant(P<0.05), myocardial Na⁺-K⁺-ATPaseα₂ isoform protein expression in group T₃,T₅ decreased, the statistical differcence was significant(P <0.05).2.9 Protein expression of soleus Na⁺-K⁺-ATPaseα₁ andα₂ isoformCompared with group T₀, there was no significant decrease in soleus Na⁺-K⁺-ATPaseα₁ isoform protein expression in group T₃(P>0.05) , soleus Na⁺-K⁺-ATPaseα₁ isoform protein expression in group T₅ decreased, the statistical differcence was significant(P<0.05); compared with group T₀, soleus Na⁺-K⁺-ATPaseα₂ isoform protein expression in group T₃,T₅ decreased, the statistical differcence was significant(P<0.05).2.10 mRNA expression of myocardial Na⁺-K⁺-ATPaseα₁ andα₂ isoformCompared with group T₀, myocardial Na⁺-K⁺-ATPaseα₁ isoform mRNA expression in group T₃,T₅ increased, the statistical differcence was significant(P<0.05), myocardial Na⁺-K⁺-ATPaseα₂ isoform mRNA expression in group T₃,T₅ decreased, the statistical differcence was significant(P <0.05).2.11 mRNA expression of soleus Na⁺-K⁺-ATPaseα₁ andα₂ isoformCompared with group T₀, there was no significant decrease in soleus Na⁺-K⁺-ATPaseα₁ isoform mRNA expression in group T₃(P>0.05) , soleus Na⁺-K⁺-ATPaseα₁ isoform mRNA expression in group T₅ decreased, the statistical differcence was significant(P<0.05); compared with group T₀, soleus Na⁺-K⁺-ATPaseα₂ isoform mRNA expression in group T₃,T₅ decreased, the statistical differcence was significant(P<0.05).3 Correlation between RBC K⁺ content and mycardial K⁺ content or soleus K⁺ content in K⁺-depleted rabbits3.1 Relationship between RBC K⁺ content and myocardial K⁺ content during K⁺ depletionLinear regression analysis showed a positive linear relationship between RBC K⁺ content and myocardial K⁺ content, linear regression equation was Y=0.37X⁺58.24,R²=0.666, P<0.05. 3.2 Relationship between RBC K⁺ content and soleus K⁺ content during K⁺ depletionLinear regression analysis showed a positive linear relationship between RBC K⁺ content and soleus K⁺ content, linear regression equation was Y=0.81X⁺15.01,R²=0.822, P<0.05.4 Change of K⁺ content in myocardium and soleus muscle in K⁺-depleted rabbits after K⁺ repletion4.1 When K⁺ repletion were stopped, total KCl infusion dosage was higher in group RE than in group RC, the statistical differcence was significant(P<0.05); total renal K excretion was higher in group RE than in group RC, the statistical differcence was significant(P<0.05).4.2 The comparison of myocardial K⁺ uptake and mean myocardial K⁺ uptake rate after potassium repletionCompared with group RC, myocardial K⁺ uptake in group RE was higher, the statistical differcence was significant(P <0.05), mean myocardial K⁺ uptake rate in group RE was higher, the statistical differcence was significant(P <0.05).4.3 The comparison of soleus K⁺ uptake and mean soleus K⁺ uptake rate after potassium repletionCompared with group RC, soleus K⁺ uptake in group RE was higher, the statistical differcence was significant(P <0.05), mean soleusl K⁺ uptake rate in group RE was higher, the statistical differcence was significant(P <0.05).4.4 During clamping plasma K⁺ concentration by KCl infusion, K⁺ uptake rate of myocardiaum was higher than that of soleus muscle, the statistical differcence was significant(P <0.05).4.5 Body total K⁺ uptake gradually decreased during clamping plasma K⁺ by KCl infusion.Conclusion:1 The present study prepared K⁺-depleted rabbit models successfully used a low-K⁺ content diet,which induced a plasma K⁺ decrease in experimental rabbits.2 Plasma K⁺ concentation,RBC K⁺ content,myocardial K⁺ content and soleus K⁺ content of experimental rabbits gradually decreased afer they were fed with the low-K⁺ content chaw.3 Potassium depletion induced a down regulation in RBC Na⁺-K⁺-ATPase activity and soleus Na⁺-K⁺-ATPase activity but an upregulation in myocardial Na⁺-K⁺-ATPase activity;linear regression analysis of data from K-depleted and contral rabbits showed a positive linear relationship between Na⁺-K⁺-ATPaseb activity and K⁺ content in RBC and soleus muscle respectively and a negative linear relationship between Na⁺-K⁺-ATPaseb activity and K content in myocardium.4 RBC K⁺ content was positively correlated with myocardial K⁺ content and soleus K⁺ content during K depletion.5 During K⁺ depletion, the responsivity of Na⁺-K⁺-ATPaseα₁ andα₂ isoform was different in RBC,myocardium and soleus muscle: mRNA and protein expression ofα₁ andα₂ isoform gradually decreased in RBC and soleus muscle, mRNA and protein expression ofα₁ isoform gradually increased but that ofα₂ isoform gradually decreased in myocardium.6 Total K⁺ uptake and renal K⁺ excretion incresed significantly in K⁺-deplted rabbits,and K⁺ uptake rate in myocardium was hiagher than that in soleus muscle, during K⁺ repletion. Body total K⁺ uptake gradually decreased during clamping plasma K⁺ by KCl infusion.