The Involvement Of WNK4 In The Dietary Salt Regulation Of Sodium Transports In The Kidney Through Aldosterone

PartⅠThe involvement of WNK4 in the dietary salt regulation of sodium transports in the kidneyBackgroundHypertension and its complications are the major diseases that threaten human health. Volume metabolism in the pathogenesis of hypertension has an important meaning, Kidney is a vital organ in regulating water-salt metabolism. And the kidney regulates the capacity of body through filtration of glomerular as well as reabsorption of renal tubular. Na-Cl cotransporter (NCC) in distal convoluted tubule and epithelial sodium channel(ENaC) in the collecting duct of the kidney is the major site of reabsorption in this section. In physiological conditions the percentage of soudium in the distal nephron reabsorption of fluid filtration is only 5%,but this is a speed limit of site of renal sodium ion reabsorption. As theγ-subunit of ENaC is so important for ENaC membrane localization,γENaC can directly affect the serum sodium concentration.Distal renal tubular reabsorption of sodium ion dependent on the access of the sodium load. High load of sodium can cause distal tubule cell ultrastructure, such as DCT cell hypertrophy, basement membrane area increased, and enhanced the capacity of sodium chloride cotransporter in distal tubule.Aldosterone regulate water-salt metabolism,it can up-regulate NCC and ENaC expression in the kidney. In recent years, people found a new kinase family--With No lysine Kinase, called WNK kinase for short.The mutation of WNK1 and WNK4 in this family are causes of PHAⅡ(pseudohyperaldosteronism typeⅡ).Study had found that WNK4 can inhibit the expression of ENaC and NCC on cell membrane.Therefore, the purpose of the study is observing whether WNK4 involvement of the dietary salt regulation of sodium transports in the kidneyMethodsSD rats were divided into three groups:(1)high-salt group (H,4% NaCl) (2) normal salt group (N,0.4% NaCl) (3)low-salt group (L,0.07% NaCl),all rats were free access to water, fed for 2 weeks.Caudilis arterial blood pressure and 24-hour-urine were measured before and after the animals were made into a mold.The clinical and biochemical parameters were determined.Plasma aldosterone were detected by RIA. The variation of WNK4,NCC,AQP-2 andγENaC mRNA in kidey was performed by Real-time PCR and their protein expression was detected by both of immunohistochemistry and Western blot.ResultsThe blood pressure,serum creatinine,albumin, sodium ion, potassium ion of the rats in each group were in the normal range.Urine volume of High-salt rats was increase, while excretion of sodium ion has a marked increase.Excretion of sodium ion of Low-salt rats was reduced, while that of potassium in urine was increased. Westernblot and immunohistochemistry have shown that:AQP-2 in the high-salt group increased compared with that of in the normal salt group, while significantly reduced in the low-salt group.All these show that the model of salt diet is successful, Plasma aldosterone in high-salt reduced than normal salt group, while increased in low-salt group.Real-time PCR and Westernblot showed that WNK4 expression in kidney of low-salt group reduced compared with that of normal salt group, while had increased in the high-salt group. Real-time PCR and Western blot showed that the renal expression of NCC andγENaC increased compared with that of the normal salt group, while the high-salt group was reduced at the same time.ConclusionsDiet salt can regulate expression.of NCC andγENaC in kidney of rats. Aldosterone and WNK4 are involved in this physiological adjustment process. Part II The negative regulation of aldosterone on WNK4BackgroundStudy had found that WNK4 can inhibit the expression of ENaC and NCC on cell membrane. Aldosterone and WNK4 can all affect the expression of ENaC and NCC on cell membrane, the relationship of them is not clear. As mentioned in some literature, WNK4 can be phosphorylated by SGK1,and its role depends on the activity of SGK1. SGK1 is an aldosterone-induced major protein involved in the regulation ENaC in the cell membrane expression. The purpose of this study was to observe the function of aldosterone and WNK4 on ENaC and NCC in physiological conditions through the model of the salt diet, and to explore whether WNK4 was involved in aldosterone regulation of salt reabsorption.MethodsSD rats were divided into five groups:(1)high-salt group(H,4% NaCl) (2) normal salt group (N,0.4%NaCl) (3)low-salt group (L,0.07% NaCl) (4) high salt+ aldosterone group (H+A,4% NaCl+1 mg/kg/day aldosterone) (5)low-salt +aldosterone (L+ S,0.07% NaCl+0.1g/kg/day spironolactone),all rats were free access to water and fed for 2 weeks.Caudilis arterial blood pressure and 24-hour-urine were measured before and after the animals were made into a mold. At the end of the 2 week, the clinical and biochemical parameters were determined. Plasma aldosterone was detected by RIA.The expression of WNK4, NCC and yENaC mRNA in kidey was detected by Real-time PCR and their protein expression was detected by both of immunohistochemistry and Western blot. Distribution of NCC in the cell was observed by sucrose density gradient centrifugation.ResultsThe serum creatinine,albumin, sodium ion, potassium ion of the rats in each group were in normal range.The blood pressure of H+A increased. Plasma aldosterone in H+A group increased compared with that of high-salt group.Real-time PCR and Westernblot showed that WNK4 expression in L+S increased compared with the low-salt group, while H+A group decreased compared with the H group.The expression of NCC andγENaC of L+S decreased compared with that of the L group, while increased in H+A compared with the H group. Sucrose density gradient centrifugation showed that NCC had more tendency to express on membrane in the low-salt group compared with that of in the high salt. NCC of high-salt group had the same trend in distribution with Golgi body.ConclusionsDiet salt can regulate expression of NCC and yENaC,and affect the distribution of NCC in the cell.Aldosterone and WNK4 were involved in this physiological adjustment process.WNK4 was negative regulated by aldosterone. PartⅢThe regulation of WNK4 on the glycosylation of NCCBackgroundWNK4 can inhibit the NCC expression on the cell membrane.However, the impact mechanism is not clear. As only the complete-glycosylation NCC can properly express on the cell membrane and play a role. The purpose of this study was to observe the regulation of WNK4 on the glycosylation of NCC.MethodsThis study used a method of plasmid transfection in HEK293 cells.Samples were divided into three groups:(1)control group(NCC):HEK293 cells with transfected NCC plasmid (2)wild-type WNK4 Group(WT-WNK4-NCC):wild type WNK4 plasmid and NCC plasmid co-transfected into HEK293 cells,(3)mutant E562K-WNK4 group(E562K-WNK4-NCC):mutant E562K-WNK4 plasmid and NCC plasmid co-transfected into HEK293 cells. After transfected for 48 hours, cell protein were collected. Used immunofluorescence and Western Blot to see the expression of NCC on cell membrane. Then used sugar N peptidase F kit digested NCC to detect changes of NCC molecular.ResultsImmunofluorescence showed that compared with the control group, the expression of NCC in wild-type WNK4 group decreased on cell membrane, while the expression of NCC in the mutant E562K-WNK4 group did not change.Western Blot showed that two different molecular weight bands could be seen in the three groups, they were 130KD and 110KD, Compared with the control group,130KD of the NCC's expression was decreased 30%in wild-type WNK4 group, P<0.05.While there was no difference in mutant E562K-WNK4 group, P> 0.05.N peptidase F digestion results showed that three groups of NCC could be digested by N peptidase F and molecular weight was turned into 110KD.Conclusion:WNK4 could inhibit the expression of NCC on cell membrane.However, WNK4 did not affect the glycosylation of NCC in Golgi body.