The Regulation Of Kidney Tropomodulin1 On Water Balance And Its Mechanism

The reabsorption of water by the renal tubules and collecting tube of the kidney determines the water balance of the body.The osmotic pressure formed by solutes in the tubular fluid of the renal tubule is the force against the reabsorption of water.Therefore,the reabsorption and excretion of water and electrolytes by the kidney are mutually influencing processes.Their balance determines the normal function of the kidney.Since the water reabsorption in proximal tubules is constant fraction reabsorption,those in distal convoluted tubule and collecting duct are adjustable.Therefore,reabsorption and excretion of water and electrolytes in these segments determines the water balance of body.It is important to study its regulating mechanisms.Actin cytoskeleton can regulate cell processes,such as cell shape and cell motility,etc.Literatures have shown that actin and its related proteins play important roles in the regulation of water and salt metabolism in the kidney.Proteomics analysis of the apical membrane of the cortex collecting tube of mice treated with vasopressin showed that ~100 proteins had obvious changes,20% of which were related to the cytoskeleton structure of actin,which highlighted the important role of actin cytoskeleton in regulating renal function.Our previous study found that Tropomodulin 1(Tmod1),a capping protein of actin filaments(F-actin),was specifically expressed in the distal tubules and collecting ducts of the kidney,but its role remains to be studied.Objective:By studying the effect of tubule-specific Tmod1 knockout on the water reabsorption function of mouse kidney,analyzing the protein regulatory networks associated with Tmod1 in kidney,and examining the effect of Tmod1 on the expressions of water channels and ion channels,the present study was aimed to explore the regulation of Tmod1 on body water balance and its mechanism.Methods:1.Immunohistochemistry was performed to determine the expression and localization of Tmod1 in mouse kidney.2.Tmod1flox/flox mice was created and bred with Ksp-Cre mice to generate tubule-specific Tmod1 knockout mice,Tmod1flox/flox/Ksp-Cre+(designated as TFK).Tmod1 flox/ flox/ Ksp-Cre-mice(designated as TF)was used as control.PCR and Western blot were performed to validate the mice.3.Metablism cages were used to collect the urine of TF and TFK mice in basal,water deprivation and water loading conditions.The urine volume and osmotic pressure of urine were measured.Non-invasive blood pressure measuring device was used to measure blood pressures of the mice.The concentrations of elestrolytes were measured by biochemical analyses and their excretions were calculated.4.ELISA kits were used to measure the concentration of arginine vesopressin(AVP)in blood of TF and TFK mice.The mice were injected by AVP and their urine volume and osmotic pressure were measured after 2 hours.5.Kidneys of TF and TFK mice were isolated and proteins were extracted for mass spectrometric proteomics analysis.Bioinformatics analysis was performed on the differentially expressed proteins.The expressions of some selected proteins were verified by Western blot.6.By using real-time quantitative PCR(q PCR)and Western blot,the expressions of water and ion channels were determined in kidneys of TF and TFK mice.Results:1.Immunohistochemistry results showed that Tmod1 was specifically expressed in the distal convoluted tubules and collecting tubules of the kidney.2.PCR and Western blot results showed that Tmod1 was completely knocked out from renal tubules and collecting tubes of TFK mice.3.The results of metabolic cage experiments showed that,in the basic state,the 24-hour urine volume of TFK mice was 0.81± 0.14 m L,which was much less than that of TF mice(1.59± 0.12 m L,P < 0.01).The urine osmotic pressure of TFK mice was 5494± 259 m Osm/kg,which was significantly higher than that of TF mice(3843± 106 m Osm/kg,P < 0.01).In water deprivation condition,the urine volume of TFK mice was 0.40± 0.10 m L,significantly lower than that of TF mice(0.93± 0.18 m L,P < 0.05).The urine osmotic pressure was 6512± 977 m Osm/kg,greatly higher than that of TF mice(3709±319 m Osm/kg,P < 0.05).In water loading condition,24-hour urine volume was 8.50±0.85 m L,still significantly lower than that of TF mice(10.92±0.45 m L,P<0.05).The urine osmotic pressure of TFK mice was 878±145 m Osm/kg,much higher than that of TF mice(492±16 m Osm/kg,P<0.05).Blood pressure measurements showed that TFK mice had higher systolic pressure than TF mice(97.9± 0.8 mm Hg v.s 105.0± 1.6 mm Hg,P<0.05).4.In basal condition,the 24-hour excretions of major electrolytes(including Na+,K+,Cl-,Ca2+,P)and urea were significantly decreased in TFK mice as compared to TF mice(P<0.05).5.ELISA data showed that the plasma AVP levels were comparable in TF and TFK mice.After injection of AVP,urine volume decreased and urine osmotic pressure increased in both TF and TFK mice.6.Mass spectrometry analyses showed that 83 proteins were differentially expressed(change multiple>2 or <0.5,P <0.05)in the kidneys of TFK mice,among which 13 were up-regulated and 70 were down-regulated.Gene Ontology analysis showed that differentially expressed genes were related to protein phosphorylation,metabolic process,phosphorylation process,intracellular signal transduction and other biological processes,as well as cell components such as exosomes,mitochondria and lysosomes.Western blot data showed that the expressions of three selected proteins,including TGFβR2,Sl C25A11 and MTFP1,were significantly decreased in TFK mice as compared with TF mice.7.q PCR and Western blot results showed that the expressions of AQP2,AQP4 and chloride ion channel CFTR were up-regulated in TFK mice kidney as compared with TFK mice.Conclusion:The knockout of Tmod1 caused changes in the urine concentration ability of mice,making the mice show the characteristics of oliguria,hyperosmolality urine,and high blood pressure.Tmod1 in the kidney could affect the expression of a variety of proteins and participate in the regulation of a variety of biological processes in the kidney.It could also regulate the expression of water channels and ion channels,and thereby affect the water homeostasis of the body.