Effects Of Hydrogen Sulfide On Sodium And Sodium Excretion In Rat Kidney And Its Mechanism

Hydrogen sulfide (H2S) is well known as a toxic gas with the characteristic smell of rotten eggs. But it is becoming increasingly clear that mammalian cells also produce H2S. The H2S concentration in rat serum is about 46μmol/L, while it reaches 100μmol/L in their brain. Endogenous H2S is synthesized naturally in the body from L-cysteine mainly by the activity of two enzymes, cystathionine β-synthase (CBS) and cystathionine y-lyase (CSE). CBS seems to be main H2S-forming enzyme in the brain and nervous system, whereas CSE is the main H2S-synthesizing enzyme in the cardiovascular system. Endogenous H2S is involved in physiological regulation of nervous system, cardiovascular system, digestive system and respiratory system. Since H2S is a small molecule of gas, freely permeable to membrane, endogenously and enzymatically generated, has physiological functions, it is regarded as third gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO).H2S plays an important role in the maintenance and regulation of normal blood pressure. It is reported that the deficit of H2S/CSE system might be responsible for the development of hypertension. The administration of exogenous H2S can effectively anti-hypertension and slow down the process of hypertension. Kidney is an important organ involving in blood pressure regulation in vivo. It is of great significance that kidney can maintain and regulate normal blood pressure by regulating the excretion of water and sodium in body. When the reabsorption of water and sodium occur obstacles in kidney, the blood pressure of body will be increased significantly. At present, the close relation between salt and hypertension causes the attention of people increasingly. Excessive salt intake will affect the sodium homeostasis of normal physiology state resulting in the retention of water and sodium in kidney and lead to hypertension. This shows that the obstacle of the excretion of water and sodium in kidney and the retention of water and sodium are the important mechanism of hypertension. Over the years the diuretic drugs have been used in the treatment of hypertension, so there are important clinical values to research on the regulation of renal water and sodium excretion.There are rich in CBS and CSE which can synthesize endogenous H2S in kidney. They are mainly located in renal proximal tubular in cortex. However, the main part for the regulation of water and sodium excretion in kidney is just the renal proximal tubular in cortex. It is known that about 70% water and sodium was reabsorbed in the renal proximal tubular. So, we postulate that whether H2S can affect the renal water and sodium excretion or not? But it is not clear about the physiological function of H2S in kidney. There was only one literature on this domain in 2009. It is reported that H2S may be involved in the regulation of renal hemodynamics and excretion. Therefore, based on this dissertation that researches on the effect of H2S on the renal water and sodium excretion, we will more deeply recognize the new gasotransmitter.Firstly, the effect of H2S on the renal water and sodium excretion in normal SD rats is observed. Use the NaHS as the donor of H2S. Choose the method of continuous intravenous infusion and keep the stabilization of MAP and RBF so as to avoid affect the renal water and sodium excretion. Test UV, UNaV, UkV and UciV after continuous intravenous infusion of NaHS (20μmol/kg) to normal SD rats and the result is that H2S can increase the renal sodium and potassium excretion in normal SD rats without the change of renal blood flow.Secondly, the effect of H2S on the renal water and sodium excretion in salt loading rats is observed. It is established two animal models which are the acute and chronic salt loading rat models to research on whether H2S can increase renal water and sodium excretion and slow down the form of hypertension or not in the pathological state. Establish acute salt loading rat model by continuous intravenous infusion of 5% NaCl for one hour. Then observe the effect of H2S on the aspects of hemodynamic and excretion after continuous intravenous infusion of different concentrations of NaHS (5,10,20,30,60,90μmol/kg). The result is that H2S has no effect on MAP, RBF and GFR of hemodynamic while 10 and 20μmol/kg NaHS can promote renal natriuresis, kaliuresis and polychloruria of the acute salt loading rats without the change of UV. Then the concentration of ALD and ADH are tested and the result shows that H2S has no effect on their level.Then, establish DHR model as the chronic salt loading rat model. One week after nephrectomy, the rats were subcutaneously injected with DOCA (30mg·kg-1) once a week and fed with a chaw with 1%NaCl plus 0.2% KCl in drinking water. At the same time, the rats were subcutaneously injected with different concentrations of NaHS (10、30、60μmol/kg·d) for 8 weeks, the effects of H2S on MAP and excretion were observed. As a result, H2S can reduce MAP and slow the form of hypertension at the 8th week, while 10μmol/kg NaHS can promote dieresis, natriuresis, kaliuresis and polychloruria in kidney of chronic salt loading rats at the 4th week. Similarly, the levels of ALD and ADH in the chronic salt loading rats were measured at the 4th week, and the result showed that H2S had no effects on both of the hormones.The above results show that H2S can promote the renal water and sodium excretion in salt loading rats without the changes of renal blood flow, glomerular filtration rate, ALD and ADH. It suggests that the site where H2S can promote renal water and sodium excretion is likely to be the renal tubular. Therefore, the renal tubular epithelial cells were separated to measure their Na+-K+-ATPase activity and the result shows that their Na+-K+-ATPase activity were decreased. So, it is presumed that H2S can decrease the Na+-K+-ATPase activity of renal tubular epithelial cell and inhibit the sodium reabsorption of renal tubular, thereby promote the renal sodium excretion. Furthermore, the difference of H2S promoting renal natriuresis and kaliuresis between SD rats and acute and chronic salt loading rats are compared and found that in the pathological state, the effects of H2S are stronger.In order to further research the mechanism of H2S promoting the renal sodium excretion in rats, the renal tubular epithelial cells of rats are separated and primary cultured. Whether H2S can directly regulate the sodium transport in renal tubular will be studied on later. The experiment result shows that NaHS can directly inhibit the Na+-K+-ATPase activity of renal tubular epithelial cells. So, how does H2S inhibit the Na+-K+-ATPase of renal tubular epithelial cells? The activity of cAMP, cGMP, PKA and PKC and phosphorylation of PKA and PKC are tested. The result shows that H2S have no influence on them. Then Western blot result shows that H2S treatment induced a dose and time dependent increase in PI3K and Akt phosphorylation in renal tubular epithelial cells. Both the PI3K inhibitors LY294002 and wortmannin prevented H2S-induced Akt phosphorylation. Moreover, H2S-induced Na+-K+-ATPase inhibition in renal tubular epithelial cells was also blocked by either LY294002 or transfection with the dominant-negative Akt mutant. These data suggest that H2S inhibits Na+-K+-ATPase by activating PI3K/Akt pathway. Further research indicates that H2S can not only increase Na+-K+-ATPase α1 subunit tyrosine phosphorylation but also increase ERK phosphorylation. It suggests that Na+-K+-ATPase α1 subunit and ERK may be involved in the process of H2S regulating Na+-K+-ATPase.Finally, whether H2S directly combines with Na+-K+-ATPase to regulate its activity besides cellular signal transduction mechanism? Making use of the direct reaction between purified Na+-K+-ATPase and NaHS, the result shows that H2S induced a dose and time dependent increase in the activity of purified Na+-K+-ATPase. The reasons of the abnormal result should be the tissue difference, specie difference and conformational change of purified Na+-K+-ATPase. In order to further research on the interaction between H2S and Na+-K+-ATPase, molecular docking of H2S and Na+-K+-ATPase was processed using AutoDock software. The result shows that there are three possible binding sites between H2S and Na+-K+-ATPase. It is known that one of them is K+ binding site and suggest that H2S may affect the combination between K+ and Na+-K+-ATPase to regulate the Na pump function.In conlusion, on the base of experiments, We found that H2S can promote the renal sodium excretion in normal SD rats and salt loading rats; moreover, H2S promotes sodium excretion by inhibiting the Na+-K+-ATPase in renal tubular epithelial cells; while it is relevant to activate PI3K/Akt pathway.