ACE2 Rat

What is the tissue distribution pattern of ACE2 in rat models?

ACE2 mRNA is widely expressed across rat tissues, with particularly high expression in organs involved in blood pressure homeostasis, including the lung, heart, and kidney. Using multiplex RT-PCR and in situ hybridization techniques, researchers have demonstrated that ACE2 is coregionalized with ACE in many tissues. Enzymatic assays confirm that both ACE2 and ACE are coactive in these critical tissues .

In the rat brain, ACE2 is ubiquitously present in brain vasculature, with highest expression density in specific regions including:

• Olfactory bulb

• Hypothalamic structures (paraventricular, supraoptic and mammillary nuclei)

• Midbrain areas (substantia nigra and ventral tegmental area)

• Hindbrain regions (pontine nucleus, pre-Bötzinger complex)

This distribution pattern suggests ACE2's involvement in multiple physiological processes beyond blood pressure regulation, including respiratory rhythm, arousal, reward, homeostasis, and cognitive functions.

How does ACE2 function within the rat renin-angiotensin system?

In rat physiology, ACE2 functions as a pivotal counter-regulatory enzyme to ACE by breaking down angiotensin II, which is the central component of the renin-angiotensin-aldosterone system (RAAS). ACE2 competes with ACE for angiotensin peptide hydrolysis, thereby potentially modulating blood pressure regulation. Like ACE, ACE2 is a zinc-dependent peptidase of the M2-metalloprotease family with enzymatic activity that has been detected in multiple tissues .

Studies in rat models with altered ACE2 expression have demonstrated its importance in cardiovascular homeostasis. When ACE2 activity is increased or decreased, there are measurable changes in blood pressure and angiotensin II levels. The enzymatic activities of ACE2 and ACE can be differentially regulated in specific tissues, suggesting complex tissue-specific control mechanisms that allow for fine-tuning of the RAAS in different physiological and pathological contexts .

Why are rat models valuable for studying ACE2 compared to other animal models?

Rat models offer several advantages for ACE2 research:

• Physiological similarity: Rats share significant cardiovascular and respiratory system similarities with humans, making findings more translatable than those from more evolutionarily distant models.

• Well-characterized ACE2 distribution: The extensive mapping of ACE2 expression across rat tissues allows for targeted investigations of specific physiological processes .

• Availability of specialized models: Humanized ACE2 (hACE2) knockin rats provide a sophisticated model system where the rat ACE2 gene promoter drives expression of the human ACE2 protein while terminating rat ACE2 expression, enabling more direct translational research .

• Established disease models: Rat models of hypertension, metabolic disorders, and other conditions associated with ACE2 function are well-developed and validated.

• Larger size compared to mice: This facilitates certain experimental procedures, particularly those requiring repeated sampling or surgical interventions.

These characteristics position rat models as valuable tools for studying both basic ACE2 biology and its roles in disease processes, including recent applications in COVID-19 research.

How does prenatal programming affect ACE2 expression and activity in rat offspring?

Research using intrauterine growth retardation (IUGR) rat models has revealed important insights into developmental programming of ACE2. In a study using 70% food-restricted dams throughout gestation (FR30 rats), the adult offspring (4-month-old) exhibited significant alterations in ACE2 biology. These rats developed mild hypertension, showed impaired renal morphology, and had elevated plasma angiotensin II and aldosterone levels, suggesting systemic RAAS dysregulation .

Remarkably, ACE2 and ACE activities were increased specifically in the lungs of FR30 rats, while their mRNA expression remained unaltered. This finding demonstrates that:

• ACE2 and ACE exhibit tissue-specific sensitivity to developmental programming

• Post-transcriptional or post-translational mechanisms likely regulate enzyme activity

• Lung-specific alterations in ACE2/ACE activity may contribute to programmed hypertension

These findings have important implications for understanding how early-life conditions can program cardiovascular health throughout the lifespan, potentially through tissue-specific alterations in ACE2 function.

What role does ACE2 play in the rat brain respiratory and arousal networks?

ACE2 expression in the rat brain shows a distinct pattern of localization within circuits critical for respiratory function and arousal. High expression of ACE2 has been documented in the neuropil and cells of the neurovascular unit clustered in nuclei that comprise the brainstem respiratory network .

Specifically, ACE2 is prominently expressed in:

• Parabrachial nucleus (PBN)

• Nucleus of tractus solitarius (NTS)

• Pre-Bötzinger complex (pre-BötC)

• Retrotrapezoid nucleus (RTN)

• Bötzinger complex

Within the pre-BötC, some ACE2-positive neurons have been characterized as glycinergic, expressing the glycine transporter type 2 (GlyT2) . Additionally, ACE2-positive cells are abundant in arousal-related structures of the reticular formation, particularly in the pontine reticular nucleus (PRN) and gigantocellular reticular nucleus (GRN) .

This expression pattern suggests ACE2 may modulate respiratory rhythm generation and arousal state regulation, potentially explaining why respiratory and sleep disturbances are common in conditions with altered ACE2 function, including COVID-19.

How can researchers effectively manipulate ACE2 activity in rat models to study its physiological roles?

Researchers have several methodological approaches to manipulate ACE2 activity in rat models:

• Genetic Approaches:

  • Humanized ACE2 (hACE2) knockin rats: These models have the rat ACE2 gene promoter driving human ACE2 expression while terminating rat ACE2 expression .

  • CRISPR/Cas9 gene editing: Can be used to create tissue-specific knockouts or modifications of ACE2.

• Pharmacological Interventions:

  • ACE2 activators (e.g., diminazene aceturate)

  • ACE2 inhibitors (e.g., MLN-4760, DX600)

  • RAS modulators that indirectly affect ACE2 activity

• Viral Vector Administration:

  • Adeno-associated virus (AAV) vectors carrying ACE2 constructs for overexpression

  • siRNA or shRNA approaches for targeted knockdown

• Environmental Modulation:

  • Dietary interventions (e.g., high-fat diet, sodium restriction)

  • Prenatal programming models (e.g., maternal food restriction)

When designing these manipulations, researchers should consider:

• Tissue specificity of the intervention

• Timing relative to developmental stages

• Potential compensatory mechanisms

• Method-specific limitations

• Appropriate physiological readouts to assess ACE2 function

Combining multiple approaches often provides the most robust evidence regarding ACE2's physiological roles.

What techniques are most effective for quantifying ACE2 expression and activity in rat tissues?

Multiple complementary techniques provide comprehensive assessment of ACE2 expression and activity in rat tissues:

For ACE2 Expression:

• Multiplex RT-PCR: Enables simultaneous quantification of ACE2 and ACE mRNA expression, allowing for direct comparison of their relative abundance .

• In situ hybridization: Provides spatial information about ACE2 mRNA distribution within tissue architecture, revealing cell-specific expression patterns .

• Immunohistochemistry: Visualizes ACE2 protein distribution in tissues, especially valuable for identifying specific cell types expressing ACE2 (e.g., glycinergic neurons in the pre-Bötzinger complex) .

• Western blotting: Quantifies ACE2 protein levels and can identify post-translational modifications.

For ACE2 Activity:

• Enzymatic assays: Measure ACE2 catalytic activity using specific fluorogenic or chromogenic substrates, providing functional data beyond mere expression levels .

• Mass spectrometry: Quantifies ACE2 products (e.g., Ang 1-7) to assess functional activity in complex biological samples.

For comprehensive analysis, researchers should combine multiple approaches. For instance, in studies of hypertension programming, both mRNA expression analysis and enzymatic activity measurements revealed that ACE2 activity can be altered independently of mRNA levels in specific tissues like the lung .

How should researchers design experiments to distinguish between the roles of ACE and ACE2 in rat models?

Designing experiments to differentiate ACE and ACE2 functions requires careful methodological considerations:

• Selective inhibition studies:

  • Use ACE-specific inhibitors (e.g., captopril, lisinopril)

  • Use ACE2-specific inhibitors (e.g., MLN-4760, DX600)

  • Compare physiological responses to selective vs. combined inhibition

• Genetic approaches:

  • Compare ACE knockout/knockdown with ACE2 knockout/knockdown

  • Use humanized ACE2 rat models that express human ACE2 while terminating rat ACE2 expression

• Substrate specificity analysis:

  • ACE primarily converts Angiotensin I to Angiotensin II

  • ACE2 primarily converts Angiotensin II to Angiotensin 1-7

  • Measure these specific conversion products to differentiate activity

• Tissue-specific analysis:

  • Leverage the distinct but overlapping tissue distribution patterns of ACE and ACE2

  • Focus on tissues where differential expression occurs

• Dynamic response studies:

  • Examine temporal changes in ACE vs. ACE2 expression/activity following physiological perturbations

  • Study compensatory changes in one enzyme when the other is inhibited

When interpreting results, consider that ACE and ACE2 form part of a balanced system, so alterations in one enzyme often trigger compensatory changes in the other.

What considerations are important when working with humanized ACE2 (hACE2) knockin rat models?

When utilizing humanized ACE2 (hACE2) knockin rat models, researchers should consider several important factors:

• Expression pattern verification:

  • Confirm that the human ACE2 expression pattern matches the expected rat ACE2 distribution

  • Verify that expression is driven by the rat ACE2 promoter as designed

• Functional validation:

  • Test whether human ACE2 is enzymatically active in the rat background

  • Compare activity levels to wild-type rat ACE2 in multiple tissues

• Baseline phenotyping:

  • Characterize cardiovascular parameters (blood pressure, heart rate)

  • Assess renal function (important for RAAS studies)

  • Evaluate behavioral and neurological function given ACE2's expression in the brain

• Age and sex considerations:

  • ACE2 expression is influenced by age and sex in humans and likely in rats

  • Design studies to include both sexes and relevant age groups

• Environmental factors:

  • Control for dietary factors that may influence RAAS

  • Consider housing conditions that might affect stress levels and RAAS activation

• Experimental design for infectious disease studies:

  • When using these models for SARS-CoV-2 research, consider differences in viral binding affinity to human vs. rat ACE2

  • Account for potential differences in downstream signaling pathways

The hACE2 knockin rat model is commercially available from Inotiv (formerly Envigo) with different pricing for academic/non-profit ($260.00 USD for 4-12 weeks) and commercial use ($545.00 USD for 4-12 weeks) .

How does ACE2 expression in the rat brain compare to other tissues, and what are the functional implications?

ACE2 expression in the rat brain exhibits distinct patterns compared to peripheral tissues, with important functional implications:

Brain-specific expression patterns:

• In the brain, ACE2 is primarily expressed in the vasculature, with highest density in specific nuclei rather than uniform distribution

• Highest expression is found in regions critical for autonomic functions: olfactory bulb, hypothalamic nuclei (paraventricular, supraoptic, mammillary), midbrain structures (substantia nigra, ventral tegmental area), and hindbrain respiratory centers (pontine nucleus, pre-Bötzinger complex)

• Within the respiratory control network, ACE2 is expressed in glycinergic neurons in the pre-Bötzinger complex, suggesting direct involvement in respiratory rhythm generation

Comparison with peripheral tissues:

• Unlike the heart, lungs, and kidneys where ACE2 is expressed in parenchymal cells and vasculature, brain ACE2 appears more restricted to the neurovascular unit

• The lung shows particularly high ACE2 activity that can be modulated by developmental programming, suggesting tissue-specific regulatory mechanisms

Functional implications:

• Neurorespiratory control: The high expression in brainstem respiratory centers suggests ACE2 modulates central respiratory drive

• Blood-brain barrier function: Vascular expression implies a role in maintaining BBB integrity

• Autonomic regulation: Expression in hypothalamic and brainstem nuclei suggests involvement in blood pressure and stress responses

• Reward circuitry: Presence in the ventral tegmental area points to potential roles in motivation and addiction-related processes

• Neuroinflammation: May mediate inflammatory responses in the CNS during conditions like COVID-19

This brain-specific expression pattern helps explain why neurological symptoms occur in conditions affecting ACE2 function and provides rationale for targeted neurological investigations in ACE2-related research.

What differences exist in ACE2 enzymatic activity across rat tissues despite similar mRNA expression levels?

Research has revealed important discrepancies between ACE2 mRNA expression and enzymatic activity across rat tissues:

Tissue-specific activity vs. expression patterns:

• Despite similar mRNA expression levels, ACE2 enzymatic activity can vary significantly between tissues, suggesting post-transcriptional or post-translational regulation

• In FR30 rat models (offspring from food-restricted dams), lung tissue showed increased ACE2 activity without corresponding increases in mRNA expression, demonstrating tissue-specific regulation mechanisms

• Heart, kidney, and lungs all express high levels of both ACE2 mRNA and protein, but the relative enzymatic activity doesn't always correlate directly with expression levels

Factors influencing activity-expression discrepancies:

• Post-translational modifications: Tissue-specific glycosylation, phosphorylation, or other modifications may alter enzyme activity

• Presence of endogenous inhibitors or activators: Tissue-specific cofactors may modulate ACE2 activity

• Subcellular localization: Differences in membrane vs. soluble ACE2 abundance

• Developmental programming: Early-life influences may alter ACE2 activity independently of expression

• Pathological states: Disease conditions can selectively affect either expression or activity

Methodological considerations for researchers:

These findings highlight the importance of comprehensive assessment of both expression and activity when studying ACE2's role in physiological and pathological processes.

How has ACE2 expression mapping in rats contributed to understanding SARS-CoV-2 infection pathophysiology?

Detailed mapping of ACE2 expression in rat tissues has provided valuable insights into SARS-CoV-2 pathophysiology:

Respiratory system insights:

• High ACE2 expression in rat lung tissues correlates with primary respiratory involvement in COVID-19

• Developmental programming effects on lung ACE2 activity may help explain age-related differences in COVID-19 severity

Neurological manifestations:

• Mapping of ACE2 in rat brain revealed high expression in respiratory control centers (pre-Bötzinger complex, nucleus tractus solitarius), explaining respiratory control abnormalities in COVID-19

• Presence in arousal-regulating regions of the reticular formation provides mechanisms for altered consciousness states in severe COVID-19

• Olfactory bulb expression correlates with anosmia (loss of smell) in COVID-19 patients

Multi-organ involvement:

• Widespread ACE2 distribution in rats mirrors the multi-system effects of COVID-19 in humans

• The discovery that ACE2-expressing organs don't equally participate in COVID-19 pathophysiology suggests additional factors beyond ACE2 expression determine tissue susceptibility

Pathophysiological mechanisms:

• Studies in ACE2-knockout models suggest that viral-induced ACE2 downregulation through internalization contributes to disease severity by disrupting the protective actions of ACE2

• This downregulation mechanism helps explain the paradoxical worsening of symptoms despite ACE2 being the entry point for the virus

Research applications:

• Humanized ACE2 rat models provide platforms for testing therapeutic interventions

• The detailed ACE2 mapping guides targeted investigations of organ-specific COVID-19 complications

These findings demonstrate how basic research on ACE2 distribution in rat models has directly informed our understanding of human COVID-19 pathophysiology and points to potential therapeutic targets.

How do findings from rat ACE2 studies translate to human neurodevelopmental disorders?

Rat ACE2 studies have revealed important connections to human neurodevelopmental disorders:

Established associations:

• The rat Ace2 gene has been linked through comparative genetics to several human neurodevelopmental conditions, including autism spectrum disorder, neurodevelopmental disorders, and syndromic X-linked intellectual disability Lubs type

• These associations are primarily established through isogenic (ISO) evidence comparing rat ACE2 with human ACE2 functional similarities

Mechanistic insights:

• Brain development regulation: ACE2's expression in key brain regions suggests roles in neurodevelopmental processes

• Neurovascular interface: ACE2 may influence blood-brain barrier formation and function during development

• Neuroimmune interactions: ACE2's role in inflammatory modulation may affect neurodevelopmental trajectories

• RAAS influence on neurogenesis: The ACE2/Ang-(1-7)/Mas axis may regulate neural progenitor proliferation and differentiation

Translational significance:

• Rat models allow for detailed investigation of developmental timing and region-specific effects that cannot be easily studied in humans

• The high conservation of ACE2 function between species supports translational relevance of findings

• Multiple neurodevelopmental disorders showing ACE2 associations suggest a common pathway that may be therapeutically targetable

Research applications:

• Humanized ACE2 rat models provide opportunities to study neurodevelopmental disorder mechanisms in a more translationally relevant system

• Developmental studies in rats can help establish critical periods when ACE2 function is most important for proper neurodevelopment

These connections highlight how basic ACE2 research in rat models contributes to understanding complex human neurodevelopmental disorders and may guide development of novel therapeutic approaches.

What lessons from intrauterine growth restriction rat models with altered ACE2 function apply to human developmental programming?

Studies of intrauterine growth restriction (IUGR) in rat models have revealed significant insights about ACE2's role in developmental programming with important human implications:

Key findings from rat models:

• Offspring from 70% food-restricted dams throughout gestation (FR30 rats) develop mild hypertension, impaired renal morphology, and elevated plasma angiotensin II and aldosterone levels as adults

• These FR30 rats show increased ACE2 and ACE activities specifically in the lung, while mRNA expression remains unchanged, demonstrating tissue-specific post-transcriptional regulation

• The programmed alterations in ACE2 persist into adulthood, suggesting permanent remodeling of the RAAS

Translational implications for humans:

• Fetal origins of adult disease: Supports the DOHaD (Developmental Origins of Health and Disease) hypothesis in humans, linking low birth weight to adult hypertension

• Tissue-specific vulnerability: Suggests certain organs may be more susceptible to developmental programming of ACE2 function

• Intervention timing: Identifies pregnancy as a critical window for interventions to prevent long-term cardiovascular consequences

• Molecular mechanisms: Reveals post-transcriptional regulation as a key mechanism in programming that may be targeted therapeutically

• Screening opportunities: Suggests monitoring ACE2 activity might identify individuals at risk for programmed hypertension

Clinical relevance:

• May explain increased cardiovascular disease risk in individuals born with low birth weight

• Suggests maternal nutritional status during pregnancy could influence offspring's ACE2 function and subsequent disease susceptibility

• Provides rationale for early-life interventions targeting the RAAS to prevent programmed hypertension

These findings illustrate how rat models of developmental programming provide mechanistic insights into human developmental pathophysiology that would be impossible to obtain directly from human studies.

How do rat ACE2 models contribute to understanding COVID-19 pathophysiology and potential treatments?

Rat ACE2 models have provided crucial insights into COVID-19 pathophysiology and treatment approaches:

Pathophysiological insights:

• Tissue tropism mechanisms: Detailed mapping of ACE2 expression across rat tissues helps explain the multi-organ effects of SARS-CoV-2 infection

• Neurological manifestations: ACE2 expression in rat brain respiratory centers and olfactory regions provides mechanisms for COVID-19 neurological symptoms

• Disease severity factors: Studies showing tissue-specific ACE2 regulation help explain variable disease presentation

• ACE2 downregulation consequences: Research suggests viral binding causes ACE2 internalization, removing its protective effects and exacerbating pathology

Therapeutic implications:

• RAAS-targeting approaches: Understanding of ACE2's counter-regulatory role to ACE provides rationale for RAAS inhibitor investigations

• Soluble ACE2 strategies: Rat studies support the concept of using soluble ACE2 as a decoy receptor

• Tissue-specific interventions: Knowledge of differential ACE2 expression guides development of organ-targeted therapies

• Developmental considerations: Findings about age-related ACE2 differences inform age-stratified treatment approaches

Research applications:

• Humanized ACE2 models: Rats expressing human ACE2 provide valuable platforms for testing COVID-19 therapies and vaccines

• Drug screening: Rat models allow for rapid assessment of compounds that modulate ACE2 expression or activity

• Long COVID investigations: ACE2 distribution in rats helps identify potential mechanisms of persistent symptoms

Prevention strategies:

• Vaccine development: Understanding ACE2-viral interactions informs vaccine design

• Risk stratification: Knowledge of factors affecting ACE2 expression helps identify high-risk populations

• Targeted protective measures: Understanding of tissue-specific ACE2 expression guides protective interventions

These contributions demonstrate the critical role of rat ACE2 research in advancing our understanding of COVID-19 and developing evidence-based prevention and treatment strategies.