My laboratory is interested in studying genetic, molecular and signaling mechanisms of angiotensin II-dependent hypertension and the key roles of the kidney proximal tubules in maintaining basal blood pressure homeostasis and the development of hypertension. Specifically, we are especially interested to determine the roles of endocrine (tissue-to-tissue), paracrine (cell-to-cell), and intracrine (endosomal, mitochondrial, and nuclear) angiotensin II (ANG II) and its G protein-coupled AT1 (AT1a) receptor (GPCR) signal mechanisms in the proximal tubules of the kidney in blood pressure control and hypertension. Supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), we are currently investigating: 1) the molecular and signaling mechanisms by which circulating and paracrine ANG II is taken up by the proximal tubule of the kidney to act as an intracellular peptide; 2) high resolution confocal and electron microscopic localization of the internalized ANG II and its receptors in intracellular organelles including endosomes, mitochondria and nucleus; 3) the effects and signaling mechanisms by which intracrine ANG II induces long-term genomic or transcriptional effects; 4) the role of the sodium and hydrogen exchanger 3 (NHE3) in the proximal tubule of the kidney in blood pressure control and hypertension; 5) the role and signaling mechanisms of proximal tubule AT1a receptors and sirtuin 3 in the pathogenesis of renal ischemia and reperfusion injury; and 6) the roles and signaling mechanisms of vasoactive peptide AT1a and bradykinin B2 receptors in renomedullary interstitial cells (RMICs) in blood pressure control and urine concentration etc. To test our hypotheses, we use complementary state of the art approaches including: 1) live cell confocal fluorescent imaging; 2) high resolution electron microscopic immunohistochemistry; 3) intravital multi-photon functional imaging; 4) proximal tubule cells derived from human kidney, wild-type, AT1a (AT1a-KO) and AT2 receptor-deficient mice (AT2-KO); 5) novel mouse models with proximal tubule-specific knockout of AT1a (PT-AT1a-KO), NHE3 (PT-NHE3-KO), sirtuin 3 (PT-SIRT3-KO), or RMIC-specific knockout of AT1a or B2 receptors; 6) proximal tubule-specific, mitochondria- or nucleus-targeting overexpression of an intracellular cyan fluorescent fusion of ANG II protein (mito- or NLS-ECFP/ANG II); and 7) digital droplet PCR, single cell RNA seq, epigenetic and proteomic, and Western blot analyses. The new knowledge generated from our studies may lead to new paradigm shifts on the roles of the proximal tubules of the kidney in renal mechanisms of hypertension, and aid the development of new classes proximal tubule-selective multifunctional drugs to treat ANG II-dependent hypertension by blocking not only endocrine and paracrine, but also intracellular or nuclear actions of ANG II in the proximal tubules.
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NIDDK · 1759942
Intratubular angiotensin II and AT1a receptors in the proximal tubules: roles in hypertension and kidney injury
Hypertension is the most important risk factor for cardiovascular diseases, stroke, and end-stage kidney failure. In the United States, nearly 46% of adults develop hypertension and will be treated with antihypertensive drugs in their lifetime. Only 50% of hypertensive patients are responsive to current antihypertensive drugs, whereas 1/3 of remaining hypertensive patients will develop cardiovascular, stroke and renal complications. The mechanisms responsible for the development of cardiovascular and kidney injury and the reasons for poor responses to current antihypertensive therapies remain incompletely understood. Thus, further studies are necessary in order to uncover new mechanisms, pathways, and therapeutic targets of uncontrolled hypertension and target organ injury. In preliminary studies, we used the state of the art SGLT2-Cre/LoxP approach to delete angiotensin II (ANG II) AT1a receptors, the Na+/H+ exchanger 3 (NHE3), or a key mitochondrial protein sirtuin 3 (SIRT3) selectively in the S1 and S2 segments of the proximal tubules in the kidney. We have evidence that proximal tubule-specific deletion of AT1a or NHE3 decreases basal blood pressure, augments the pressure natriuresis response, and attenuates ANG II-induced hypertension, and that proximal tubule-specific deletion of AT1a receptors significantly attenuated, whereas proximal tubule-specific deletion of SIRT3 significantly worsened renal ischemia and reperfusion (I/R) injury. These preliminary studies strongly suggest that intratubular ANG II and its AT1a receptors and SIRT3 in the proximal tubules play an important role in the development of hypertension and renal I/R injury. In this proposal, we will test the hypotheses that intratubular ANG II and AT1a receptors in the proximal tubules of the kidney are required for the development of ANG II-induced hypertension and renal I/R injury, and that deletion of AT1a receptors or angiotensinogen (AGT) selectively in the proximal tubules will attenuate ANG II-induced hypertension and renal I/R injury in two specific aims. Specific Aim 1 will test the hypothesis that intratubular ANG II and AT1a receptors in the proximal tubules play a key role in maintaining basal blood pressure homeostasis and the development of ANG II-induced hypertension, via the activation of the Na+/H+ antiporter (NHE3), Na+ and glucose cotransporter 2 (sglt2), and the regulation of the pressure natriuresis response. Specific Aim 2 will test the hypothesis that AT1a receptors in the proximal tubules play a key role in the pathogenesis of renal I/R injury, activated by intratubular and intracellular ANG II to upregulate Toll-Like receptor 4 (TLR-4), downregulate mitochondrial SIRT3 expression, and impair mitochondrial function in the proximal tubules. These hypotheses will be tested using highly innovative mouse models with global and proximal tubule-specific knockout of a) AT1a receptors; b) angiotensinogen; c) NHE3; d) SGLT2; e) TLR4; or f) SIRT3. ANG II-dependent hypertension and renal I/R injury will be induced by a) infusing a native ANG II; b) 2-kidney, 1-clip renal hypertension; and c) overexpressing a proximal tubule- specific, mitochondria-targeting intracellular ECFP/ANG II. Telemetry blood pressure, the pressure natriuresis response, noninvasive glomerular filtration rate, intravital two-photon imaging and XFe24 Extracellular Flux Analyzer to measure mitochondrial function, electron microscopic and immunohistochemical imaging, and Western blot analyses of proinflammatory, profibrotic and signaling proteins will be studied. The proposed studies are highly significant and clinically relevant, and the new knowledge will lead to a paradigm shift on understanding the pathogenesis of hypertension and renal I/R injury and help develop proximal tubule-targeting drugs to treat poorly controlled hypertension and renal I/R injury.
