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Physiology

Welcome to the Nakagawa Lab

The Nakagawa lab combines tools from neuroscience and physiology to understand the fundamental mechanisms of neural and autonomic mechanisms of cardiovascular control and how dysregulation of these systems contributes to the development of cardiovascular diseases. 
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Our Mission

Our mission is to support the future generation of outstanding scientists by creating an optimal environment for personal, scientific, and professional growth.
Our laboratory focuses on understanding the neural mechanisms of blood pressure and autonomic control that can be involved in the development and progression of hypertension. The brain renin-angiotensin system (RAS) plays a key role in blood pressure regulation, water & salt intake, metabolic function & cognitive function. Thus, we aim to understand the mechanisms contributing to brain RAS dysfunction and to address the neural bases of hypertension.
To study this, we utilize 1) genetically engineered models to manipulate expression of key genes in the brain, 2) induce expression of fluorescent reporter genes or genetically encoded biosensors to label & trace distinct proteins and cells in the brain, 3) methods to study the activity of neuronal circuits, and 4) methods to activate and suppress these circuits such as optogenetics and chemogenetics. State-of-the-art radiotelemetric devices are implanted in these mice to monitor cardiovascular function 24/7 wirelessly.

Current Projects

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Hypertension: The Silent Killer

Despite significant progress, cardiovascular (CV) diseases are still the leading cause of death globally. One of the main risk factors for CV disease is elevated blood pressure (BP) or hypertension (HT). High BP is typically managed with a wide variety of drugs. However, a vast number of patients with HT do not achieve desirable BP with conventional therapeutic options. Notably, patients who exhibit resistant HT are associated with autonomic imbalance and therefore, novel strategies to ameliorate sympathoexcitation and restore the depressed vagal parasympathetic tone (renal nerve ablation, vagus nerve stimulation, carotid baroreflex therapy, acetylcholinesterase inhibitors, etc.) are now recognized as potential therapeutic options for diseases beyond HT.

Thus, our laboratory’s main focus is to understand the fundamental neural mechanisms that control BP and the autonomic nervous system. We believe that our findings might contribute to developing new alternative therapies to treat HT.

 

(Fig. 1 - right)

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The Brain RAS: A Fundamental Mechanism of Blood Pressure Control

The renin-angiotensin system (RAS) is one of the most important mechanisms of BP control and fluid and electrolyte homeostasis. As shown in Figure 2, the activation of the RAS requires a two-step enzymatic process to generate Angiotensin II, an octapeptide that exerts potent pressor effects through angiotensin type 1 receptors (AT1R)-mediated mechanism. Importantly, renin plays a critical role in the regulation of the RAS since it is the rate-limiting enzyme of this biosynthetic cascade. Besides the classical RAS found in the circulating system, the existence of an unconventional RAS that operates locally in specific tissues, such as the brain, has been proposed.

Although the critical role of the local activation of the RAS in the brain to control BP regulation, hydromineral balance, autonomic function, and metabolism is well accepted it remains unclear how and where angiotensin (ANG)-II, the main bioactive component of the RAS, is generated within the brain; Surprisingly, whether renin, the rate-limiting enzyme of ANG-II biosynthesis, is expressed and necessary for the cleavage of angiotensinogen (AGT) to generate ANG-I in the brain remains controversial.

(Fig. 2 - left)

Unraveling a Scientific Mystery That Has Persisted for 5 Decades: the Discovery of Renin-Expressing Neurons in the Nucleus Ambiguus

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For many years, conventional techniques that are normally used to detect the abundant amounts of renin in plasma and kidneys have been used in an attempt to detect renin in whole brain tissue homogenates. But these methods lack sensitivity, specificity, and especially the anatomical precision to detect neurons that produce extremely low amounts of renin. Thus, these questionable reports raised a misconception that renin is not expressed in the brain. Our team has mastered key technology from neuroscience and acquired expertise in neuroanatomy to address this important question with the appropriate tools and techniques. These advanced techniques and new knowledge helped us to obtain convincing evidence for the expression of renin in cholinergic neurons within the brainstem, specifically, the nucleus ambiguus (NuAm), which is a relatively understudied brainstem region in the field of hypertension (Fig. 3).

This exciting discovery is critical since the existence of a select population of neurons expressing renin incorporates new evidence that ANG might operate as a neurotransmitter in the brain. There are two main theories about this topic: a) the volume transmission hypothesis where ANG peptides are generated in the extracellular space acting as neurohormones, and b) the wiring transmission hypothesis where ANG generated by neurons acts as a neurotransmitter itself. Although several studies support the second possibility, the lack of evidence that renin is expressed in neurons has been the major limitation in defining ANG as a neurotransmitter. Thus, the identification of the renin-neurons in the NuAm could be an important finding to solve this critical question. Cellular, molecular, and neurobiological characterization of renin-expressing neurons will provide key information to understand how the RAS operates in the brain and ensure definitive evidence of the functional role of renin in the brain.

 

(Fig. 3)

New Advances: Characterizing the Novel Renin-Expressing Neurons Using State-of-the-Art Technology

Current studies are designed to understand the physiological role of the novel population of neurons that expresses renin in the NuAm. Since the laboratory was established at MCW in 2022, we have progressively advanced in our technical repertory to target genes with superior selectivity and neuroanatomical precision. We also incorporated new techniques to detect transcriptomic data in brain sections with high fidelity. Currently, we are conducting stereotactic microinjections and using conditional knockout mice to genetically ablate renin from the NuAm or cholinergic neurons (Fig. 4). These new techniques will allow us to define the function of renin in these cells. New mouse models carrying renin-reported genes are now available to map the entire brain for renin-expressing cells. These mice, which express a red fluorescent protein TdTomato in renin cells, were crossed with AT1R reporter mice, where green fluorescent protein expression is controlled by AT1R promoter. These triple transgenic mice will allow us to identify cells producing ANG II (renin) and cells responding to ANG II (AT1R) simultaneously and assess the anatomical proximity between them (Fig. 5). Retrograde and anterograde tracing studies revealed that neurons expressing renin in the NuAm extend projections to the cardiac ganglion suggesting that renin-expressing neurons could be cardiomodulatory. Considerable effort is currently invested in developing optogenetic and chemogenetic tools to modulate neural activity of renin-expressing neurons using either DREADDs (chemogenetics) or light (optogenetics). These tools will provide critical information about the physiological role of these novel neuronal populations. To study neuronal activity in awake animals, we recently acquired an in vivo calcium imaging system (miniscope) and mice carrying genetically encoded calcium sensors (GCaMP). These will allow us to identify what kind of external stimuli activate renin-expressing neurons. Finally, we aim to understand the cellular identity and transcriptomic profile of these neurons. In addition to bulk and single-cell RNA sequencing, we recently acquired spatial transcriptomics (10x Xenium), which will allow us to profile renin-expressing neurons in the NuAm and other brain regions.
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(Fig. 4)
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(Fig. 5)

Meet Our Team

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Pablo Nakagawa, PhD

Assistant Professor

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Eva Fekete, PhD

Research Scientist

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Mina Ghobrial

G2 Student

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Ana Hantke Guixa, BS

Research Technologist I

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Katie Kaminski

Graduate Student

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Haruka Okabe, MS

Research Technologist I

Publications

Publications of Pablo Nakagawa PhD from the Faculty Collaboration Database

Nakagawa Lab Image Gallery

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Stephanie Watts Career Development Award Finalist

Stephanie Watts Career Development Award Finalist

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Mina Ghobrial at The 2023 Cardiovascular Center Research Retreat

Mina Ghobrial at The 2023 Cardiovascular Center Research Retreat

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Nakagawa Lab_2024 AHA Hypertension Scientific Sessions Team Photo

2024 AHA Hypertension Scientific Sessions Team Photo

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Nakagawa Lab_2024 Team Photo

2024 Team Photo

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Nakagawa LabAzeez Ganiyu And Cotes Royson 2024 Aspire Program Students

Azeez Ganiyu and Cotes Royson - 2024 ASPIRE Program Students

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