| Sodium retention is one of the core areas in the pathogenesis of hypertension, and the renal reabsorption function of sodium ions is closely related with blood pressure and volume of extracellular fluid. As a key component to the adjustment of sodium ions level in vivo, the sodium transport channel abnormalities of the tubules may directly result in sodium retention. In the past 50 years, thiazide diuretics are commonly selected as first-line drugs for hypertension in clinic. However, pathogenesis of hypertension has not been clearly explained when developing these drugs. The poor efficacy in some patients with hypertension may be partely related to the inadequate understanding of the sodium excretion mechanisms. Recent studies have found that thiazide diuretics are in fact the specific inhibitor of thiazide-sensitive Na-Cl cotransporter (NCC; SLC12A3). Thus the exploration of NCC mechanisms and regulatory pathways is quite important to the clinical therapy and the development of new antihypertensive drugs.Recently people pay more and more attention to the fuction that With No Lysine Kinase (WNK kinase) plays in the pathogenesis of hypertension. WNK is named after its lack of a highly conserved catalytic lysine in subdomain No. Hand is consists of four family members including WNK1～4. WNK1 may phosphorylate ERK5 through MEKK2/3, and WNK3 kinases can also phosphorylate SPAK, a STE20/SPS1-related kinase. WNK4 can directly inhibit the activity of NCC; WNK1 may prevent this function of WNK4; and the kidney Special WNK1 (KS-WNK1) may inhibit the full-length WNK1. All the above form a mutual restraint homeostasis in vivo and finish the coordinate regulation of ion channels, such as NCC. The mutation of WNK1 and WNK4 may result in a hereditary hypertension named Pseudohypoaldosteronism Ⅱ (PHA Ⅱ).Newly research show that WNK3 is also one of the important members in adjusting the NCC and this function of WNK3 is closely related to the work of WNK4 and WNK1. WNK3 may antagonize the activity of WNK4 and vice versa. KS-WNK1 may inhibit the activity of WNK3. Rinehart et al found by Tracking method using radioactively label (22Na flow) in Xenopus oocytes, Wild-type WNK3 (WNK3-WT) can enhance the expression of the NCC while WNK3 without kinase activity may inhibit the NCC. The effect of the induced mutants PHAⅡWNK3 on the NCC is similar to that of WNK3-WT, indicates that WNK3 kinase may imply a a new pathway in the regulation of blood pressure and maintenance of electrolyte balance. In this present study, we constructed transfected tubular cells with several WNK3 and NCC plasmids with various markers, to observe the distribution of WNK3 and NCC over the cells and explore the possible regulatory mechanisms. We believe that this study has a significantly realistic meaning in elucidating the pathogenesis of hypertension and developing of new thiazide diuretics. It is also helpful to design new treatment and provide more drugs with fewer side effects for patients with hypertension and thereby improving their health.WNK3 involves in the formation of the cytoskeleton, cell apoptosis, and invasion and metastasis of tumor cells in vivo. After transfected by plasmids with Enzyme defect area WNK3 (WNK3-KD), HEK293 cells were found to crenate obviously under confocal microscopy which indicates WNK3-KD can not maintain normal morphology of the cells. At the same time, the function of WNK1, WNK3 of maintaining cell survival become more obviously when there is metabolic disorders such as lactic acidosis and abnormal changes in cell volume. The survival time of the cervical cancer cell HeLa transfected by plasmids with WT-WNK3 is significantly longer than that of the cell without WT-WNK3 when facing the actinomycin-D -induced apoptosis, showing WNK3 may prolong cell survival time by caspase-3 -dependent pathway and therefore delay apoptosis. In addition, Lingo-1 receptors may promote neuronal apoptosis by inhibiting the activity of WNK3 suggesting WNK3 express the anti -apoptotic effect by maintaining procaspase-3 activity in neuronal cells. As a vital organs to maintain water and electrolyte balance, the kidney is quite vulnerable to a variety of factors such as hypoxic-ischemic injury and drugs, thereby resulting in impaired function of different kidney cells, even apoptosis and necrosis. Different to the other three family members, WNK3 has been shown a protective effect on apoptotic cells such as cervical cancer cells, nerve cells et al., although the mechanism is not very clear. But it has not been reported still whether WNK3 may resist the apoptosis of human kidney cells. So this paper also aims to discover the role WNK3 plays in interleukin -1β induced apoptosis of HEK293 cells and the data showed WNK3 may protect renal cell viability under adverse conditions. As a result, the excessive suppression of WNK3 should be avoid in the regulation so as not to affect renal cell viability.Part I Construction of plasmid with WNK3 kinase and NCCObjective:To Construct WNK3 kinase and NCC plasmids with marks such as HA、GFP、Myc.Methods:Extracted DNA from human whole blood with Human whole blood DNA extraction kit. Choosing 50ul reaction system, WNK3 fragments was extracted from human DNA template by a WNK3 primer as designed. Then PCR was performed and the products was retrieved and purified after analysis and identification by 1.5% agarose gel electrophoresis. EcoRI and Xho I were selected as restriction enzyme cutting sites for opening the blank plasmid ring and accessing the WNA3 fragment. The WNK3 kinase and pCMV-HA Vector, empty vector with HA-tagged,50ul respectively were put into the PCR instrument together for 4 h Enzyme digestion at 37℃ for complementary effects after digestion.20 ul liquids with T4 DNA ligase was put into the PCR instruments 16℃ overnight for connecting WNK3 fragments to pCMV-HA Vector. Competent E. coli was prepared and then the plasmid was transformed into competent E. coli. Next, the SOC broth was added in and shaking cultured under 37℃ 225～250 rpm until the broth became muddy before inoculated onto petri dish. The monoclonal colony was selected and the E.coli was amplified. The recombinant plasmid was extracted with QIAGEN Plasmid mini Kit for EcoRI and Xho I enzyme identification and enzyme digestion. The digestion products was retrieved and purified after analysis and identification by 1.5% agarose gel electrophoresis. The 4th, 5th,9th and 11th fragments were collected for sequencing by Agencourt Bioscience Corporation and the results were compared with WNK3 gene sequences in the human GenBank. The corresponding monoclonal plasmid of correct sequence was confirmed our target plasmid. The pCMV-HA-WNK3 plasmid, pCMV-Myc-WNK3 plasmid, PCMV-HA-NCC plasmid and pEGFP-N1-NCC plasmid were constructed in the similar way as described above. And then the embryonic kidney cells HEK293 and tubular Cos-7 cells were transfected and the fluorescence and protein expression were observed.Results:1) Gene sequencing of the constructed plasmids are detected to be correct; 2) Immunofluorescence observation and the follow-up experiments showed the plasmids express quite well in cells after transfected.Conclusion:The plasmids for the follow-up experiments were successfully constructed using genetic engineering. The results of restriction enzyme digestion identification and immunofluorescence observation of the transfected cells confirmed the the qualification.Part II Distribution and interaction of WNK3 and NCC in Cos-7 cellObjectives:To observe the distribution of WNK3 and NCC in the cytoplasm and on the cell membrane of Cos-7 cell; to explore dosage-depending effect of the NCC protein expression by WNK3 and whether there is direct interaction between WNK3 and NCC protein.Methods:1) Cos-7 cells were trasfected with pEGFP-NCC+ pCMV-HA-Vector plasmid for experimental group and with pCMV-HA-WNK3 plasmid as control. The distributions of WNK3 and NCC were observed under immunofluorescence microscopy after 36 h incubation.2) cos-7 cells were divided into 3 groups and incubated at 37℃ and plasmid DNA transfection was performed when the cell growth density reached about 40-50% within 24 h. A constant amount of NCC (0.5ug/group) was given to each group while the amounts of WNK3 were trasfected with increasing dosages (0.5μg、1μg、2μg/dish). Cells was transfected with single NCC as control group. The cells protein were collected for polyacrylamide gel electrophoresis after 36 h incubation. The expression of WNK3 and NCC was detected with a polyclonal rabbit anti- HA antibody. The regulation effept of WNK on NCC total protein levels was observed.3) The Cos-7 cells were passaged 24 h before transfection and incubated in 210 cm dishes at 37℃. Plasmid DNA transfection was performed when the cell growth density reached about 40-50% within 24 h. Cos-7 cells were trasfected with NCC+WNK3 plasmid for experimental group and with GFP-NCC plasmid as control. Using anti-HA and beads to collecte WNK3 and the serum as control, whether there is direct effect of WNK3 on NCC protein was observed by western blot.Results:1) The observation under immune confocal microscopy showed that WNK3 mainly distributed in the cytoplasm of Cos-7 while NCC may be observed in the cytoplasm and on the cell membrane. After transfected by WNK3, the expression of WNK3 in the cytoplasm and on the cell membrane was enhanced obviously.2) The results of Western blot showed with the increase of WNK dosage, the expression of NCC may be significantly increased after transfection.3) After transfected with GFP-NCC and HA-WNK3, anti-HA antibody can result in immunoprecipitation of NCC by WNK3. However, this reaction did not occur when serum worked as the primary antibody or without WNK3 transfected. The above results indicate that there is direct interaction between WNK3 and NCC.Conclusion:WNK3 mainly distributed in the cytoplasm of Cos-7 while NCC may occur in the cytoplasm and on the cell membrane. The strong expression of WNK3 may help increasing the expression of NCC in Cos-7 cells. The results of immunoprecipitation experiments suggest the possibility of a direct interaction between NCC and WNK3.PartⅢ The impact of WNK3 kinase on the NCC protein synthesis and degradation process.Objectives:To discover whether WNK3 kinase increases the expression of NCC by influencing the synthesis or degradation of NCC protein.Methods:1) Cos-7 cells were divided into 2 groups for tansfection of NCC and NCC+WNK3 respectively when reaching the cell intensity of about 30-40%. After 48 h incubation, the cells were removed into nutrient fluid with 100μg/ml Cycloheximide from microbial (CHX). Cells were collected after 0h,1 h,2 h,4 h,6 h,8 h incubation and proteins were extracted for detection. 2) Cos-7 cells were divided into 4 groups and a certain amount of NCC (0.5ug/group) was transfected into cells in each group. Cells in the 2nd and 4th group was then transfected with 1 μg of WNK3.16 h later, Baf A1 was added to the 3rd and 4th group while the same amount of DMSO was added to the 1st and 2nd group. The final concentration of Baf A1 was 0.5μmol/L and 1ml 10%DMEM was replaced in drug and DMSO administrating.12 h later, cells were lysed and proteins were extracted.Results:1) NCC protein expression was significantly reduced 2 h after CHX was added in Cos-7 cells transfected with NCC alone. But in Cos-7 cells transfected with WNK3 and NCC, the expression of NCC protein began to increase obviously 6 h after CHX was added in.2) Compared with Cos-7 transfected with NCC alone (the 1st group), the transfected WNK3 obviously increased the expression of NCC protein (the 2nd group) to 2 times (p<0.05, n=4). After pretreated with nutrient fluid with 0.5 μM Baf A1, the expression of NCC protein in Cos-7 cells transfected with NCC alone (the 3rd group) increased 50% (p<0.05, n=4) when comparing with those of cells without Baf A1 (the 1st group). However, the expression of NCC protein in Baf A1 pretreated with cells transfected with WNK3 and NCC (the 4th group) did not increase obviously when comparing with those without Baf A1 (the 2nd group), indicating that WNK3 may increase the expression of NCC by reducing lysosomal degradation of NCC.Conclusion:WNK3 kinase may enhance NCC protein expression by reducing lysosomal degradation of NCC rather than by increasing the protein synthesis.PartIV the role of E RK pathway in regulation of NCC by WNK3Objective:To explore whether WNK3 enhance the expression of total NCC protein through MAPK ERK1/2 signaling pathway.Methods:Cos-7 cells were divided into 3 groups and each group was transfected with 0.5μg pCMV-HA-NCC. Then the 2 groups were transfected with 0,1 or 3ug of pCMV-Myc-WNK3 respectively and cell proteins were collected after 36 h incubation. Anti-HA and anti Myc antibodies, t-ERK and pERK antibodies were used for detection of associated protein. Brands were formed with Microchemi chemiluminescence imaging system and the gray values of target brand and internal control were detected by AlphaEaseFC Software.Results:1) The expression level of p-ERK1/2 decreased with the increase of WNK3 dosage indicating WNK3 inhibited the expression of p-ERK1/2 in a dose-dependent manner. t-ERK1/2, working as the internal reference protein sample for p-ERK1/2, remained unchanged, showing that the total ERK1/2 protein did not change.2) overexpressing WNK3 alone significantly increased NCC protein expression in mDCT cells compared to that in mDCT cells without WNK3. Knock-down of ERK 1/2 expression itself also increased NCC protein expression. However, knock-down of ERK 1/2 expression prevented the WNK3-mediated enhancement of NCC expression.Conclusion:WNK3 may inhibit expression of p-ERK1/2 protein in a dose- dependent manner. WNK3 regulates the expression level of the NCC through MAPK ERK1/2 signaling pathway.PartV The protective effect of WNK3 kinase on interleukin -1β induced apoptosis in HEK293 cellObjective:To observe whether high expression of WNK3 can prevent interleukin -1β induced apoptosis in HEK293 cells.Methods:1) The HERK293cells were divided into 3 groups and the tansfection was performed when the passaging cells reaching the cell intensity of about 40%. Liposome 2000 was added to the 1st group as the blank control and 1μg PCMV-HA-Vector was added to the 2nd group as the negative control. 3μg WNK3 was added to the 3rd group to observe the regulation effect of WNK3 on total NCC protein levels.4 h after transfecttion, all the cells were moved into the normal culture medium for 20 h incubation. Next, the cells were seeded in 96 -well plates with the density of 1×104 cells/per well. The culture medium for the administration group was DMEM+10% FBS+10ng/ ml IL-1β,100μl per well; and the culture medium for the control groups was DMEM+10% FBS of the same amount. CCK8 analysis was performed for the detection of cell activity after incubation for 0 h,12 h,24 h and 36 h at 37℃ respectively.2) HERK293 cells were grouped and transfected as described above.4 h after transfecttion, all the cells were moved into the normal culture medium for 20 h incubation. 10ng/ml IL-1β was added to the culture medium of the 2nd and the 3rd group while the same amount of normal saline was added to the 1st group. Cell proteins were collected after 0 h,18 h and 36 h incubation respectively for detection of Caspase-3、Cleaved Caspase-3, Caspase-9、Cleaved Caspase-9,Bcl-2、Bax、LC3A/B、Beclin-1 protein expression levels.3) HERK293 cells were grouped and transfected as described in step 2. Cell protein were collected 0 min,30 min and 60 min after the treatment of IL-1β(Saline for the control group) for detect JNK and ERK.Results:1) No significant difference of cell activity was observed in the Vector+NS group within 48 h. In the Vector+IL-1β group, cell activity began to decline soon after administration.24 h later significant difference could be found (93.21±3.83 VS 77.25±4.60, P<0.05) and the cell activity reached the lowest at 48 h (93.21±3.83 VS 57.26±6.71, P<0.01).Cell activity of WNK3+IL-1β group declined in a moderate manner and the significant difference appeared at 36 h and 48 h when comparing with that at 0 h in the same group (both P<0.05). However, in comparison with the Vector+IL-1β group at the same time point, cell activity of WNK3+IL-1β group was still a little higher and the significant difference could be found at 48 h (57.26±6.71 VS 80.35±5.34, P<0.01).2) In the Vector+IL-1β group, Bcl-2 expression began to decline 18 h after the administration, and the significant difference appeared at 36 h (P<0.05); The expression of Caspase-9 and Caspase-3 also began to decline 18 h after the administration with an increasing peak of Cleaved Caspase-9 which decreased slightly at 36 h. An obvious Cleaved Caspase-3 could also be observed 36 h after the administration. In the WNK3+IL-1β group, the decline of Caspase-3 was more moderate when comparing with that in the Vector+IL-1β group. There was no apparent changed of Caspase-9 and the expression of Cleaved Caspase-9 showed no significant although weak expression could be observed. A small quantity of Cleaved Caspase-3 could be detected and was showed an statistical significance in comparison with that of the same group at 0 h (P<0.05), indicating IL-1βactivated the Caspase depending apoptosis pathway. Bcl-2 expression decreased while Bax remained unchanged in the WNK3+IL-1β group. After the action of IL-1β, the expression of Beclin-1 and LC3 both increased and the high expression of WNK3 could partly reverse expression of the above 2 autophagy proteins.3) Phosphorylation of JNK and ERK on HEK293 cell transfected WNK3 is lower than which without WNK3.Conclusion:1) IL-1β may decrease activity of HEK cells while transfection of WNK3 may partly reverse this affection of IL-1β.2) WNK3 prevent the excessive autophagy through Beclin, reduce apoptosis by decrease theactivation of Caspase pathway, and finally protect the kidney cells under adverse conditions.3) The over expression of WNK3 may inhibit the activation of JNK and ERK pathways induced by IL-1β. |
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