Functional And Structural Remodeling Of Intrarenal Artery And Its Potential Mechanisms In Simulated Microgravity Rats

The weightless environment during space flight could cause a series of adaptations in cardiovascular system, including hypovolemia, redistribution of blood flows, cardiac atrophy and decreased cardiac function as well as region-specific changes in arterial system, etc. And these adaptations may further induce postflight cardiovascular dysfunction upon returning back to the earth or encountering with other weightless environment. Evidence from real spaceflight and ground-based animal studies indicates that, real/simulated microgravity leads to region-specific structural and functional remodeling of the arterial system. In particular, the arteries in the fore body would undergo hypertrophy and enhanced vasoconstriction, while arteries in the hind body would undergo hypotrophy and reduced vasoconstriction. Meanwhile, the plasma volume is also decreased when exposed to the real/simulated microgravity. It has been demonstrated that even short-term space flight could decrease plasma volume by 10-17%. Kidney, which is well recognized in balancing water-electrolyte metabolism, plays a crucial role in maintenance of plasma volume and long-term regulation of blood pressure. A decrease in plasma volume would result in enhanced vasoconstriction in renal vessels, especially in pre-glomerulus intrarenal arteries via a series of compensatory reactions of the kidney, which in turn ameliorates the plasma volume lost. Additionally, intrarenal arteries, which are also considered as resistant vessels, play an important part in maintaining total peripheral resistance. Thus, the functional remodeling of intrarenal arteries might be involved in the postflight cardiovascular dysfunction. However, what happens to the vasoreactivity of intrarenal arteries under real/simulated microgravity has not been reported yet. Therefore,to clarify these changes and the underlining mechanism may be of great importance for revealing mechanisms of postflight cardiovascular dysfunction.The contraction of vascular smooth muscle cells（VMSCs） is determined by intracellular free Ca²⁺ concentration([Ca²⁺]i) and sensitivity of VSMCs to Ca²⁺. The former way in inducing VMSCs contraction is called Ca²⁺-dependent pathway and the latter one is considered to be Ca²⁺-independent pathway and named as Ca²⁺-sensitization pathway, which is regulated by Rho-associated protein kinase（ROCK）. Evidence from previous vascular biological research has demonstrated that both the two pathways are involved in pathologically functional and structural remodeling of the artery system in hypertension, pulmonary arterial hypertension and other vascular dysfunctional diseases. Moreover, our previous studies have also indicated that altered Ca²⁺ signaling also plays an important role in the vascular dysfunction associated with real/simulated microgravity. Therefore, we speculate that both these two pathways are involved in vasoconstrictive changes of intrarenal artery induced by simulated microgravity.NO is considered to be the most important endothelium-derived relaxing factor（EDRF）. Under physiological conditions, NO is produced by endothelial NO synthase（e NOS） catalyzing L-arginine in endothelial cells and comes into effects in VMSCs. Previous studies from our lab have found that the reduced production or the accelerated inactivation of endogenous NO may account for impaired vasodilatation of abdominal aorta and common carotid arteries induced by simulated microgravity. Thus, we speculate that the endothelium-dependent vasodilatation may also be impaired in intrarenal artery by simulated microgravity and NO, the most important EDRF, may also be involved in it.To confirm the above hypothesis, 4-week hindlimb unweighting rats were used to simulate the cardiovascular dysfunctional effects evoked by simulated microgravity. The second branches of intrarenal artery were applied as the targeting vessel. Through experiments including isometric force measurement, Ca²⁺ image, Western blot, ELISA and Immunohistochemistry, etc., the functional and structural changes of the intrarenal arteries induced by simulated microgravity were detected and the underlining mechanisms were also discussed.The major findings are presented as follows:1. The intrarenal artery underwent enhanced vasoconstriction, impaired endothelium-dependent vasodilatation and no significant changes of the vessel wall after exposure to simulated microgravity.Compared with CON rats,phenylephrine（PE）- and KCl-evoked vasoconstriction were both significantly consolidated in HU rats; ACh-evoked endothelium-dependent vasodilatation was significantly impaired in HU rats; no significant changes in sodium nitroprusside（SNP）-evoked vasodilatation or in intraluminal diameter（D）, media thickness（T）, media cross-sectional area（CSA） of the vessel wall were observed between the two groups.2. Increased IP₃R-mediated sarcoplasmic reticulum（SR） Ca²⁺ release may be involved in enhanced vasoconstriction of intrarenal artery induced by simulated microgravity.Freshly isolated VSMCs of intrarenal arteries from two groups were loaded with fluo-3/AM and used for Ca²⁺ imaging under laser scanning confocal microscope with the combination of different Ca²⁺ channel agonists and antagonists. Compared with CON rats, PE-induced Ca²⁺ release in VSMCs was significantly augmented by HU in PSS buffer solution; when ryanodine receptor（Ry R） was suppressed, ATP-Na2-induced Ca²⁺ release was also significantly increased by HU in Ca²⁺-free PSS buffer solution. The result of Western blot indicated that the expression of IP₃ receptor type 1（IP₃R1） was significantly upregulated.3. ROCK-mediated Ca²⁺ sensitization pathway may be involved in enhanced vasoconstriction of intrarenal artery induced by simulated microgravity.Y-27632, a ROCK antagonist, can significantly diminish the vasoconstrictive discrepancy between CON and HU in both PE- and KCl-evoked contraction. Compared with CON rats, vasoconstriction evoked by U-46619, a non-specific ROCK agonist, was significantly enhanced in HU rats. The result of Western blot and IHC both indicated that the expression of ROCK was downregulated in HU rats. The result of ROCK activity tests demonstrated that, as compared with CON rats, the phosphorylation of myosin light chain phosphatase target-1（MYPT-1） and MLC was both significantly increased.4. NO-e NOS system may account for the impairment of intrarenal artery vasodilatation induced by simulated microgravity.Compared with CON rats, both the expression of e NOS and the relative nitrate and nitrite content, representing the amount of NO production were significantly decreased.Above all, the present study has found that intrarenal artery underwent enhanced vasoconstriction, impaired endothelium-derived vasodilatation after exposure to simulated microgravity. Both increased IP₃R-mediated SR Ca²⁺ release and activated ROCK mediated Ca²⁺ sensitization pathway may be involved in enhanced vasoconstriction of intrarenal artery induced by simulated microgravity. NO-e NOS system may account for decreased endothelium-dependent vasodilatation by simulated microgravity. We estimate that the altered vasoreactivity of intrarenal artery by simulated microgravity may be an important compensatory response to the decreased circulatory blood volume. The specificity of kidney which plays key roles in the regulation of water-electrolyte metabolism may offset the bad effects of redistribution of transmural pressure under simulated microgravity. Meanwhile, as resistant arteries, enhanced vasoconstriction of intrarenal arteries may neutralize the decrement of total peripheral resistance under simulated microgravity.