Regulation Of Insulin On Dopamine D₅ Receptor Expression And Natriuresis Function In Renal Proximal Tubule Cells From WKY And SHR Rats

BackgroundThe data about inhabitant nutrition and health investigation by national Ministry of Public Health shows that with the change of food and other life styles, the incidence of many diseases is increasing, including hypertension, diabetes, obesity and metabolism syndrome. Evidence indicates that insulin resistance and hyperinsulinemia play an important role in the development of these diseases and in the lesion of target organs. Kidney is the main target organ of insulin resistance and hyperinsulinemia. Insulin direcly results in the decrease of renal Na+ and water excretion, and renal natruresis is impaired.Stable Na+ and water metabolic balance is one of the basic nutritional status and normal physiological condition. As the key organ of natruresis, kidney can regulate Na+ and water reabsorb, stable Water-Electrolyte balance and affect the blood pressure. Natriuresis is regulated by many factors such as dopamine , insulin and their recptors.The dopaminergic system exerts a regulatory role on renal sodium transport and activation of dopamine receptors inhibits sodium reabsorption. When sodium is repleted, dopamine takes care of more than 50% sodium excretion and play an important role in the regulation of natruresis and blood pressure level. The function is obviously disordered in the condition of hypertension. Dopamine receptors based on their structure and pharmacology are classified into D1-like (D1, D5) and D2-like (D2, D3 and D4) subtypes. In the renal proximal tubule (RPT), D1-like (D1, D5) receptors is more important in renal natriuresis and decreasing blood pressure. Evidences showed dopamine D5 receptor is important to natriuresis and Water-Electrolyte balance. Dopamine D5 receptor, also expressed in RPT, has a higher affinity for dopamine than the Dl receptor and its natruresis function is supported by the outcome of D5 receptor gene deficient (D5-/-) mice. Dopamine D5 receptor can also interact with other systems about natruresis and blood pressure regulation. Dopamine D5 receptor impacts oxidative stress in the kidney. D5 receptor has a linkage with G protein subunit Gα12 or Gα13 and affect the activity of Na+/H+ exchange 3(NHE 3). And in the kidney, the degradation of angiotensin II receptor 1 (AT1R) induced by D1-like agonist is also mediated by dopamine D5 receptor.More and more evidence indicate the interaction between insulin and dopamine receptors. Now, most of the knowledge on the interaction between insulin and dopamine receptors been focused on the D1 dopamine receptor. There is no report about the interaction between insulin and dopamine D5 receptor. The D5 receptor belong to D1-like dopamine subtype and has some similarity with Dl receptor. Insulin receptor and D5 receptor, belong to membrane receptors, are both expressed in the kidney. Dopamine D5 receptor mediates the renal natruresis and insulin leads to water-sodium retention. So we can presume that insulin also has some effects on D5 receptor expression and its natruresis function in renal sodium excretion, and it may impact renal natruresis, Water-electrolyte balance and blood pressure. The regulations may has a difference between normotensive and hypertension.To test the above hypothesis, we evaluated insulin and D5 receptor interaction in immortalized RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs), and its physiological significance in D5 receptor-transfected HEK293 (HEK293-D5) cells and contrast the physiological effect between WKY and SHR RPT cells with transfected dopamine D1 receptor Antisense oligodeoxyribonucleotide (D1 receptor AS-ODN). We also observed the co-conjunction between dopamine D5 receptor and insulin receptor in RPT cell from WKY and SHR rats by the method of confocal microscopy and immunoprecipitation and approached the molecule mechanism of the regulation of insulin on dopamine D5 receptor. These have some significance to explain the interaction between insulin and dopamine D5 receptor on the regulation of renal natruresis, Water-electrolyte balance and blood pressure. And it is helpful to know the pathogenesy of hypertension and the renal damage or protection, the target organ of insulin resistance, hyperinsulinemia or hypertension. Investigation content and methods1. Immortalized renal proximal tubule (RPT) cells of WKY and SHR were used in this experiment.Effects of insulin on dopamine D5 receptor protein expression was observed by immunoblotting and immunohistochemistry and D5 receptor mRNA expression was checked by reverse transcription-polymerase chain reaction (RT-PCR). The difference between WKY and SHR cells was evaluated.2. Effects of dopamine D1-like agonist fenoldopam and insulin respectively on Na+-K+-ATPaes activity were checked in HEK293 cells and dopamine D5 receptor-transfected hek293 cells (HEK-D5 cells ). And in the condition of pretreatment with insulin , the changed effect of D5 receptor on Na+-K+-ATPase activity in D5 receptor-transfected HEK293 cells was also observed.3. Dopamine D1 receptor Antisense oligodeoxyribonucleotides were transfected into the RPT cells. In the cells transfeted with D1 receptor AS-ODN, the effect of dopamine D1-like agonist fenoldopam on Na+-K+-ATPaes activity was checked and in the condition of pretreatment with insulin, the changed effect of fenoldopam on Na+-K+-ATPase activity was also observed. The difference between WKY and SHR cells was evaluated.4. To investigate the signal pathway of insulin on dopamine D5 receptor in the WKY RPT cells, the signal channel of protein tyrosine kinase, phosphatidyl inositol 3 kinase(PI3K) and protein kinase C(PKC) were blocked , and the changed effect of insulin on D5 receptor were evaluated5. Immortalized RPT cells of WKY and SHR were used in this experiment. The respective localization of insulin receptor and dopamine D5 receptor and the colocalization of them were checked by laser confocal microscopy.6. Immunoprecipitation was used to detect the physiological linkage between the dopamine D5 receptor and insulin receptor. The effect of insulin on the physiological linkage was observed and the difference between WKY and SHR cells was evaluated.7. In the RPT cells of WKY and SHR rats, immunoblotting was used to check the regulation of insulin on the insulin receptors and the difference between WKY and SHR cells was evaluated. Results1. In RPT cells of WKY rats, insulin increased the expression of the D5 receptor in a concentration and time dependent manner and on the contrary, insulin decreased the D5 receptor expression in RPT cells of SHR rats. The basal protein expression of D5 receptor in WKT RPT cells was higher than SHRs and in both WKY and SHR cells, the effect of insulin on the D5 receptor accompanied with alterations of messenger RNA level.2. In the HEK-D5 cells, insulin obviously elevated Na+-K+-ATPase activity. But after the cells were treated with insulin for 24 hours and then changed into serum-free culture medium for 3 hours, Na+-K+-ATPase activity was recovered and has no difference with contrast.3. In the HEK-D5 cells, dopamine D1-like agonist fenoldopam obviously decreased Na+-K+-ATPase activity by the interaction with the D5 receptor. But on the contrary, fenoldopam had no effect on the Na+-K+-ATPase activity in HEK293 cells.4. Compared with the depression effect by only fenoldopam stimulation, pretreatment with insulin for 24 hours increased the inhibitory effect of the D5 receptor on Na+-K+ ATPase activity in the D5 receptor-transfected cells.5. Dopamine D1 receptor Antisense oligodeoxyribonucleotides effectively blocked the expression of the D1 receptor. In the WKY RPT cells transfeted with D1 receptor AS-ODN, pretreatment with insulin increased the inhibitory effect of dopamine D1-like agonist fenoldopam on Na+-K+ ATPase activity. And in the SHR RPT cells transfeted with D1 receptor AS-ODN, the inhibitory effect of the D5 receptor on Na+-K+ ATPase activity was impaired after pretreatment with insulin.6. The regulation of insulin on dopamine D5 receptor in RPT cells of WKY rats is related with the the signal channel of protein tyrosine kinase, phosphatidyl inositol 3 kinase(PI3K) and protein kinase C(PKC). In the HEK-D5 cells or PRT cells of WKY and SHR rats , the effect of insulin may has related with the decrease of its own insulin receptor.7. Dopamine D5 receptor and insulin receptor were expressed respectively in the PRT cells of WKY and SHR rats, and the receptors have the colocalization in the RPT cells. There is a physiological linkage between dopamine D5 receptor and insulin receptor , and the stimulation of insulin decreased the linkages in both RPT cells of WKY and SHR rats. The decreasing extent in WKY cells was higher than SHR cells.On the whole,insulin increased D5 receptor expressions in WKY RPT cells, enhanced the inhibitory effect of the D5 receptor on Na+-K+-ATPase activity and it was also confirmed in the D5 receptor-transfected cells. Because insulin increases sodium reabsorption, the increased D5 receptor expression following insulin treatment could counter-regulate the stimulatory effect of insulin on Na+-K+- ATPase activity in RPTs. Hence, a balance between the expression and activity of both insulin receptor and D5 receptor would be an important mechanism to keep sodium reabsorption in the proximal tubule within the normal extent. The increase of D5 receptor expression and function may be the compensatory mechanism in the state of insulin resistance or hyperinsulinemia. However, the compensatory increase in D5 receptor expression following insulin treatment is lost in SHR cells. Insulin impaired D5 receptor expression and natruresis and it leads to more sodium reabsorption and higher blood pressure. The regulation of insulin on dopamine D5 receptor is related with protein tyrosine kinase, PI3K and PKC signal channel. And the regulation of insulin receptor expression and the physiological linkage between D5 receptor and insulin receptor may also take part in the regulation of insulin on D5 receptor. Our study revealed the interaction between insulin and dopamine D5 receptor on the regulation of renal natruresis, Water-electrolyte balance and blood pressure. And this aberrant regulation of insulin on D5 receptor in the SHR might provide a mechanism for the pathogenesis of hypertension and damage or protection of the kidney, the target organ of insulin resistance, hyperinsulinemia or hypertension.