Renal microcirculation and hypertension induced renal injury

描述(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，其发病率正在以惊人的速度增加。两者都与肾血流量(RBF)自动调节功能受损和肾小球毛细血管压力升高有关，从而促进了肾损伤的发展。然而，只有一半的糖尿病或高血压患者会患上肾脏疾病，而且对易感基因知之甚少。多年来，我们小组一直在研究Fawn-hooded高血压(FHH)大鼠，这是一种高血压诱导的CKD的遗传模型，发展为进行性蛋白尿和肾小球硬化。我们已经报道，FHH大鼠表现出肌源性反应和肾和脑循环中血流的自动调节受损。然而，涉及的基因和途径尚不清楚。这一建议建立在我们激动人心的初步结果的基础上，这些结果表明，FHH大鼠的传入小动脉的生肌反应和RBF的自动调节受到损害，这是由于大电导钙激活钾(BK)通道的活性增加，并且将1号染色体上仅包含15个基因的区域替换到FHH遗传背景上可以使BK通道的活动正常化，恢复RBF的生肌反应和自动调节，并减缓肾脏疾病的发展。对该区域基因的测序和表达分析确定了内收蛋白3(ADD3)中的一个序列变异，该序列变异被预测为损害蛋白质功能。本项目的目标是利用分子和转基因方法，探讨ADD3在FHH大鼠肌源性反应受损中的作用，研究RBF自身调节受损对高血压或糖尿病大鼠肾脏损害的细胞和离子机制及其功能后果。之所以选择ADD3进行研究，是因为米兰正常血压的大鼠与FHH大鼠具有相同的ADD3基因K572Q突变，而且它们也非常容易发生肾脏疾病。此外，在人类关联性研究中，ADD基因家族的突变已多次被认为与高血压和血管功能障碍的发展有关。ADD3的作用将通过互补的方法进行评估，包括：肾小动脉中ADD3的siRNA敲除，新的ADD3转基因拯救FHH大鼠和我们创建的锌指核酸酶ADD3基因敲除(KO)品系。我们将研究FHH大鼠和ADD3KO转基因株分离的血管平滑肌细胞的肌源性反应，并测定其BK通道活性。这项拟议的研究将揭示ADD3及其相关的下游通路如何调节BK通道活性和生肌反应，并将为开发预防糖尿病和高血压患者CKD的新疗法提供关键信息，在糖尿病和高血压患者中，肾脏自动调节经常受损。

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.