CKD中硫酸吲哚酚通过抑制SIRT1活性上调时钟基因DBP促进高血压的机制研究

The regulatory role and the underlying mechanism of gut microbiota on hypertension with disturbed blood pressure circadian rhythm in CKD patients remain unclear. Our study suggests that indoxyl sulfate (IS), the metabolite of gut microbiota, may inhibit the deacetylation of histone 3 (Lys9/14) in clock gene DBP promoter by SIRT1, thus causing the arrhythmic high expression of adrenal DBP and related adosterone synthase gene Hsd3b6. As a result, the abnormal aldosterone synthesis may aggravate hypertension and impairs blood pressure circadian rhythm in CKD patients. To verify this mechanism, firstly, low IS models are performed by colonizing B.thetaiotaomicron with tryptophan knockout in the gut of CKD mice, while high IS models are conducted by intraperitoneal injection of IS. The effects of IS on blood pressure level and rhythm, DBP and aldosterone level are observed in these models. Then, IS is used to interfere with primary adrenal cortical cells to verify the role of IS inhibiting SIRT1 and up-regulating DBP. DBP knockout/overexpression cells are used to confirm that IS induced Hsd3b6 and aldosterone synthesis by up-regulating DBP. Finally, SIRT1 agonist is used to treat CKD mice to clarify the reversal effect of SIRT1 activity improving on hypertension with disturbed blood pressure circadian rhythm in CKD. The expected outcomes of this research can elucidate the new regulation mechanism of CKD gut microbiota on hypertension with disturbed blood pressure circadian rhythm, and also provide new evidence for the regulation of host biological clock by gut microbiota.

肠道菌群在CKD患者高血压伴血压昼夜节律异常中的作用及机制尚不明确。我们研究提示，CKD中肠道菌群产物硫酸吲哚酚（IS）可能通过抑制SIRT1对时钟基因DBP启动子区组蛋白的去乙酰化，致肾上腺DBP及其调控的醛固酮合成关键酶基因Hsd3b6失节律性高表达，引起醛固酮合成异常，促进血压水平及节律异常。本研究首先用色氨酸酶敲除菌定植于CKD小鼠构建低IS-CKD模型，并用IS腹腔注射构建高IS-CKD模型，观察IS对血压水平和节律、DBP及醛固酮的影响；其次用IS干预肾上腺皮质细胞，验证IS抑制SIRT1从而上调DBP的机制，并在DBP敲除/过表达细胞中，证实IS通过上调DBP诱导Hsd3b6及醛固酮合成；最后用SIRT1激动剂干预CKD小鼠，明确SIRT1活性改善对CKD高血压伴血压节律异常的逆转作用。预期成果可阐明CKD肠道菌群紊乱促进高血压的新机制，也为肠道菌群调控宿主生物钟增添新证据。

慢性肾脏病（CKD）患者中普遍存在高血压，且多数伴血压昼夜节律受损。尽管已有许多研究试图阐明CKD中促进高血压的因素及机制，但现有研究对血压升高伴随昼夜节律受损的机制认识不足，更缺乏针对这一特点的血压调定方法。我们在前期中研究发现，CKD状态下调控醛固酮合成的时钟基因D位点结合蛋白（DBP）呈失节律性高表达，而其这一变化可能与肠道菌群代谢产物硫酸吲哚酚（IS）有关。在本研究中，我们利用粪菌移植干预5/6肾切除术构建的CKD大鼠模型，发现粪菌移植可有效降低CKD大鼠粪便及血清中IS水平，同时使CKD大鼠血压水平明显下降、昼夜节律异常改善，时钟基因DBP及其靶基因醛固酮合成关键酶HSD3B1表达水平下降、节律震荡部分恢复。进一步，在体外肾上腺皮质细胞（NCI-H295R）中，我们证实IS可剂量、时间依赖性地诱导ALD合成分泌，而这一过程伴随SIRT1去乙酰化酶（HDAC）活性降低、DBP及HSD3B1表达升高；在NCI-H295R细胞中敲低DBP或给予SIRT1 HDAC活性激动剂白藜芦醇（RV），HSD3B1表达显著减少、ALD合成分泌减少，表明IS可通过抑制SIRT1 HDAC活性，进而通过DBP-HSD3B1通路促进ALD合成分泌。最后，以SIRT1 HDAC激动剂RV定时喂养CKD大鼠，发现CKD大鼠肾上腺DBP节律性表达部分恢复、血压水平和节律明显改善，反向验证了IS引起的SIRT1 HDAC活性抑制在CKD大鼠高血压及血压节律异常中的重要作用。综合上述结果，我们认为CKD状态下肠道菌群代谢产物的IS异常升高，可通过抑制SIRT1 HDAC活性而诱导时钟基因DBP失节律性高表达，进而以DBP-HSD3B1通路促进ALD合成分泌，引起CKD患者血压升高及血压节律异常。本项目为基于肠道菌群调节或靶向关键代谢物的新型降压方法奠定了理论基础，研究成果为CKD患者高血压及血压昼夜节律受损提供了新的治疗思路及潜在靶点。