Genetic mutation of Kcnj16 identifies Kir5.1-containing channels as key regulators of acute and chronic pH homeostasis.

Acute and chronic homeostatic pH regulation is critical for the maintenance of optimal cellular function. Renal mechanisms dominate global pH regulation over longer time frames, and rapid adjustments in ventilation compensate for acute pH and CO2 changes. Ventilatory CO2 and pH chemoreflexes are primarily determined by brain chemoreceptors with intrinsic pH sensitivity likely driven by K+ channels. Here, we studied acute and chronic pH regulation in Kcnj16 mutant Dahl salt-sensitive (SS Kcnj16-/-) rats; Kcnj16 encodes the pH-sensitive inwardly rectifying K+ 5.1 (Kir5.1) channel. SS Kcnj16-/- rats hyperventilated at rest, likely compensating for a chronic metabolic acidosis. Despite their resting hyperventilation, SS Kcnj16-/- rats showed up to 45% reduction in the ventilatory response to graded hypercapnic acidosis vs. controls. SS Kcnj16-/- rats chronically treated with bicarbonate or the carbonic anhydrase inhibitor hydrochlorothiazide had partial restoration of arterial pH, but there was a further reduction in the ventilatory response to hypercapnic acidosis. SS Kcnj16-/- rats also had a nearly absent hypoxic ventilatory response, suggesting major contributions of Kir5.1 to O2- and CO2-dependent chemoreflexes. Although previous studies demonstrated beneficial effects of a high-K+ diet (HKD) on cardiorenal phenotypes in SS Kcnj16-/- rats, HKD failed to restore the observed ventilatory phenotypes. We conclude that Kir5.1 is a key regulator of renal H+ handling and essential for acute and chronic regulation of arterial pH as determinants of the ventilatory CO2 chemoreflex.-Puissant, M. M., Muere, C., Levchenko, V., Manis, A. D., Martino, P., Forster, H. V., Palygin, O., Staruschenko, A., Hodges, M. R. Genetic mutation of Kcnj16 identifies Kir5.1-containing channels as key regulators of acute and chronic pH homeostasis.

RNASeq-derived transcriptome comparisons reveal neuromodulatory deficiency in the CO2 insensitive brown Norway rat.

Raphé-derived serotonin (5-HT) and thyrotropin-releasing hormone (TRH) play important roles in fundamental, homeostatic control systems such as breathing and specifically the ventilatory CO2 chemoreflex. Brown Norway (BN) rats exhibit an inherent and severe ventilatory insensitivity to hypercapnia but also exhibit relatively normal ventilation at rest and during other conditions, similar to multiple genetic models of 5-HT system dysfunction in mice. Herein, we tested the hypothesis that the ventilatory insensitivity to hypercapnia in BN rats is due to altered raphé gene expression and the consequent deficiencies in raphé-derived neuromodulators such as TRH. Medullary raphé transcriptome comparisons revealed lower expression of multiple 5-HT neuron-specific genes in BN compared to control Dahl salt-sensitive rats, predictive of reduced central nervous system monoamines by bioinformatics analyses and confirmed by high-performance liquid chromatography measurements. In particular, raphé Trh mRNA and peptide levels were significantly reduced in BN rats, and injections of the stable TRH analogue Taltirelin (TAL) stimulated breathing dose-dependently, with greater effects in BN versus control Sprague-Dawley rats. Importantly, TAL also effectively normalized the ventilatory CO2 chemoreflex in BN rats, but TAL did not affect CO2 sensitivity in control Sprague-Dawley rats. These data establish a molecular basis of the neuromodulatory deficiency in BN rats, and further suggest an important functional role for TRH signalling in the mammalian CO2 chemoreflex.

Racial differences in triage assessment at rural vs urban Maine emergency departments.

Data continue to accumulate demonstrating that those belonging to racialized groups face implicit bias in the emergency care delivery system across many indices, including triage assessment. The Emergency Severity Index (ESI) was developed and widely implemented across the US to improve the objectivity of triage assessment and prioritization of care delivery; however, research continues to support the presence of subjective bias in triage assessment. We sought to assess the relationship between perceived race and/or need for translator and assigned ESI score and whether this was impacted by hospital geography. We performed retrospective EMR-based review of patients presenting to urban and rural emergency departments of a health system in Maine with one of the top ten most common chief complaints (CC) across a 5-year period, excluding psychiatric CCs. We used multivariable regression to analyze the relationships between perceived race, need for translator, and gender with ESI score, wait time, and hallway bed assignments. We found that patients perceived as non-white were more likely to receive lower acuity ESI scores and have longer wait times as compared to patients perceived as white. Patients perceived as female were more likely to receive lower acuity scores and wait longer to be seen than patients perceived as male. The need for an interpreter was associated with increased wait times but not significantly associated with ESI score. After stratification by hospital geography, evidence of subjective bias was limited to urban emergency departments and was not evident in rural emergency departments. Further investigation of subjective bias in emergency departments in Maine, particularly in urban settings, is warranted.

Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels.

Ventilation is continuously adjusted by a neural network to maintain blood gases and pH. Acute CO2 and/or pH regulation requires neural feedback from brainstem cells that encode CO2/pH to modulate ventilation, including but not limited to brainstem serotonin (5-HT) neurons. Brainstem 5-HT neurons modulate ventilation and are stimulated by hypercapnic acidosis, the sensitivity of which increases with increasing postnatal age. The proper function of brainstem 5-HT neurons, particularly during post-natal development is critical given that multiple abnormalities in the 5-HT system have been identified in victims of Sudden Infant Death Syndrome. Here, we tested the hypothesis that there are age-dependent increases in expression of pH-sensitive ion channels in brainstem 5-HT neurons, which may underlie their cellular CO2/pH sensitivity. Midline raphe neurons were acutely dissociated from neonatal and mature transgenic SSePet-eGFP rats [which have enhanced green fluorescent protein (eGFP) expression in all 5-HT neurons] and sorted with fluorescence-activated cell sorting (FACS) into 5-HT-enriched and non-5-HT cell pools for subsequent RNA extraction, cDNA library preparation and RNA sequencing. Overlapping differential expression analyses pointed to age-dependent shifts in multiple ion channels, including but not limited to the pH-sensitive potassium ion (K+) channel genes kcnj10 (Kir4.1), kcnj16 (Kir5.1), kcnk1 (TWIK-1), kcnk3 (TASK-1) and kcnk9 (TASK-3). Intracellular contents isolated from single adult eGFP+ 5-HT neurons confirmed gene expression of Kir4.1, Kir5.1 and other K+ channels, but also showed heterogeneity in the expression of multiple genes. 5-HT neuron-enriched cell pools from selected post-natal ages showed increases in Kir4.1, Kir5.1, and TWIK-1, fitting with age-dependent increases in Kir4.1 and Kir5.1 protein expression in raphe tissue samples. Immunofluorescence imaging confirmed Kir5.1 protein was co-localized to brainstem neurons and glia including 5-HT neurons as expected. However, Kir4.1 protein expression was restricted to glia, suggesting that it may not contribute to 5-HT neuron pH sensitivity. Although there are caveats to this approach, the data suggest that pH-sensitive Kir5.1 channels may underlie cellular CO2/pH chemosensitivity in brainstem 5-HT neurons.

Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats.

Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 (Tph2) gene in dark agouti (DA(Tph2-/-)) rats. DA(Tph2-/-) rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DA(Tph2-/-) rats. Body temperature was also maintained in adult DA(Tph2-/-) rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DA(Tph2-/-) rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation (P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat.

Fluoxetine augments ventilatory CO2 sensitivity in Brown Norway but not Sprague Dawley rats.

The Brown Norway (BN; BN/NHsdMcwi) rat exhibits a deficit in ventilatory CO2 sensitivity and a modest serotonin (5-HT) deficiency. Here, we tested the hypothesis that the selective serotonin reuptake inhibitor fluoxetine would augment CO2 sensitivity in BN but not Sprague Dawley (SD) rats. Ventilation during room air or 7% CO2 exposure was measured before, during and after 3 weeks of daily injections of saline or fluoxetine (10mg/(kgday)) in adult male BN and SD rats. Fluoxetine had minimal effects on room air breathing in BN and SD rats (p>0.05), although tidal volume (VT) was reduced in BN rats (p<0.05). There were also minimal effects of fluoxetine on CO2 sensitivity in SD rats, but fluoxetine increased minute ventilation, breathing frequency and VT during hypercapnia in BN rats (p<0.05). The augmented CO2 response was reversible upon withdrawal of fluoxetine. Brain levels of biogenic amines were largely unaffected, but 5-HIAA and the ratio of 5-HIAA/5-HT were reduced (p<0.05) consistent with selective and effective 5-HT reuptake inhibition. Thus, fluoxetine increases ventilatory CO2 sensitivity in BN but not SD rats, further suggesting altered 5-HT system function may contribute to the inherently low CO2 sensitivity in the BN rat.