Renal microcirculation and hypertension induced renal injury

描述（由申请人提供）：糖尿病和高血压是慢性肾脏疾病（CKD）的主要原因，其发病率正以惊人的速度增长。两者都与肾血流（RBF）的自动调节障碍和肾小球毛细血管压力升高有关，从而促进肾损伤的发展。然而，只有一半的糖尿病或高血压患者会发生肾脏疾病，对其易感基因知之甚少。多年来，我们的研究小组一直在研究小鹿兜帽高血压（FHH）大鼠，这是一种高血压诱导的CKD的遗传模型，可发展为进行性蛋白尿和肾小球硬化。我们已经报道了FHH大鼠在肾和脑循环中表现出肌源性反应和血流的自我调节受损。然而，所涉及的基因和途径是未知的。该建议建立在我们令人兴奋的初步结果的基础上，该结果表明，由于大电导、钙活化钾（BK）通道活性的升高，FHH大鼠的传入小动脉的肌源性反应和RBF的自动调节受到损害，并且棕色挪威大鼠的1号染色体仅含有15个基因的区域替换到FHH遗传背景上，使BK通道活性正常化。恢复RBF的肌源性反应和自我调节，并减轻肾脏疾病的发展。该区域基因的测序和表达分析发现了内收蛋白3 （Add3）的序列变异，预计会损害蛋白质功能。本项目的目的是利用分子和转基因方法探讨Add3在FHH大鼠肌生成反应受损中的作用，研究其细胞和离子机制，以及RBF自调节受损对高血压或糖尿病后肾损害发展的功能后果。之所以选择Add3作为研究对象，是因为米兰正常血压大鼠与FHH大鼠在Add3中具有相同的K572Q突变，而且它们也极易发生肾脏疾病。此外，在人类关联研究中，Add基因家族的突变多次与高血压和血管功能障碍的发生有关。Add3的作用将通过补充方法进行评估，包括：在肾小动脉中siRNA敲除Add3，新型的Add3转基因救援FHH大鼠和我们创建的大鼠锌指核酸酶敲除（KO）菌株。我们将在分离的传入小动脉中表征肌源性反应，并测量从FHH大鼠、Add3 KO和转基因菌株分离的血管平滑肌细胞中的BK通道活性。这些研究将揭示Add3及其相关的下游通路如何调节BK通道活性和肌生成反应，并将为开发预防糖尿病和高血压患者CKD的新疗法提供重要信息，这些患者的肾脏自身调节经常受损。

DESCRIPTION (provided by applicant): Diabetes and hypertension are the leading causes of chronic kidney disease (CKD) and the incidence is increasing at an alarming rate. Both are associated with impairments in the autoregulation of renal blood flow (RBF) and elevations in glomerular capillary pressure that promote the development of renal injury. However, only half of patients with diabetes or hypertension develop renal disease and very little is known about the susceptibility genes. For many years our group has been studying the Fawn-Hooded Hypertensive (FHH) rat which is a genetic model of hypertension-induced CKD that develops progressive proteinuria and glomerulosclerosis. We have reported that FHH rats exhibit an impaired myogenic response and autoregulation of blood flow in both the renal and cerebral circulations. However, the genes and pathways involved are unknown. This proposal builds upon our exciting preliminary results indicating that the myogenic response of the afferent arteriole and autoregulation of RBF is impaired in FHH rats due to an elevation in the activity of the large conductance, calcium-activated potassium (BK) channel and that substitution of a region of chromosome 1 containing just 15 genes from the Brown Norway rat onto the FHH genetic background normalizes BK channel activity, restores the myogenic response and autoregulation of RBF and attenuates the development of renal disease. Sequencing and expression analysis of the genes in this region identified a sequence variant in adducin 3 (Add3) that is predicted to damage protein function. The goal of this project is to use molecular and transgenic approaches to explore the role of Add3 in mediating the impaired myogenic response in FHH rats, to study the cellular and ionic mechanisms involved and the functional consequences of impaired autoregulation of RBF to the development of renal damage following the development of hypertension or diabetes. Add3 was chosen for study since Milan Normotensive rats share the same K572Q mutation in Add3 as FHH rats and they are also highly susceptible to the development of renal disease. In addition, mutations in the Add gene family have been repeatedly linked to the development of hypertension and vascular dysfunction in human association studies. The role of Add3 will be evaluated using complementary approaches including: siRNA knockdown of Add3 in renal arterioles, novel Add3 transgenic rescue FHH rats and Zn-finger nuclease Add3 knockout (KO) strains of rats that we created. We will characterize the myogenic response in isolated afferent arterioles and measure BK channel activity in vascular smooth muscle cells isolated from FHH rats and the Add3 KO and transgenic strains. The proposed studies will reveal how Add3 and its associated downstream pathways regulate BK channel activity and the myogenic response, and will provide information critical to the development of new treatments for the prevention of CKD in diabetic and hypertensive patients in which renal autoregulation is often impaired.