Functional and structural insights into activation of TRPV2 by weak acids.

Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

Mechanisms and Consequences of Oxygen and Carbon Dioxide Sensing in Mammals.

Molecular oxygen (O2) and carbon dioxide (CO2) are the primary gaseous substrate and product of oxidative phosphorylation in respiring organisms, respectively. Variance in the levels of either of these gasses outside of the physiological range presents a serious threat to cell, tissue, and organism survival. Therefore, it is essential that endogenous levels are monitored and kept at appropriate concentrations to maintain a state of homeostasis. Higher organisms such as mammals have evolved mechanisms to sense O2 and CO2 both in the circulation and in individual cells and elicit appropriate corrective responses to promote adaptation to commonly encountered conditions such as hypoxia and hypercapnia. These can be acute and transient nontranscriptional responses, which typically occur at the level of whole animal physiology or more sustained transcriptional responses, which promote chronic adaptation. In this review, we discuss the mechanisms by which mammals sense changes in O2 and CO2 and elicit adaptive responses to maintain homeostasis. We also discuss crosstalk between these pathways and how they may represent targets for therapeutic intervention in a range of pathological states.

Phenotypic screen of sixty-eight colorectal cancer cell lines identifies CEACAM6 and CEACAM5 as markers of acid resistance.

Elevated cancer metabolism releases lactic acid and CO2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as "acid-sensitive"/"acid-resistant" versus "low metabolic acid production"/"high metabolic acid production." Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12. CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors.

Tumor acidosis-induced DNA damage response and tetraploidy enhance sensitivity to ATM and ATR inhibitors.

Tumor acidosis is associated with increased invasiveness and drug resistance. Here, we take an unbiased approach to identify vulnerabilities of acid-exposed cancer cells by combining pH-dependent flow cytometry cell sorting from 3D colorectal tumor spheroids and transcriptomic profiling. Besides metabolic rewiring, we identify an increase in tetraploid cell frequency and DNA damage response as consistent hallmarks of acid-exposed cancer cells, supported by the activation of ATM and ATR signaling pathways. We find that regardless of the cell replication error status, both ATM and ATR inhibitors exert preferential growth inhibitory effects on acid-exposed cancer cells. The efficacy of a combination of these drugs with 5-FU is further documented in 3D spheroids as well as in patient-derived colorectal tumor organoids. These data position tumor acidosis as a revelator of the therapeutic potential of DNA repair blockers and as an attractive clinical biomarker to predict the response to a combination with chemotherapy.

Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct.

The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.

The study of intercalated cells using ex vivo techniques: primary cell culture, cell lines, kidney slices, and organoids.

This review summarizes methods to study kidney intercalated cell (IC) function ex vivo. While important for acid-base homeostasis, IC dysfunction is often not recognized clinically until it becomes severe. The advantage of using ex vivo techniques is that they allow for the differential evaluation of IC function in controlled environments. Although in vitro kidney tubular perfusion is a classical ex vivo technique to study IC, here we concentrate on primary cell cultures, immortalized cell lines, and ex vivo kidney slices. Ex vivo techniques are useful in evaluating IC signaling pathways that allow rapid responses to extracellular changes in pH, CO2, and bicarbonate (HCO3-). However, these methods for IC work can also be challenging, as cell lines that recapitulate IC do not proliferate easily in culture. Moreover, a "pure" IC population in culture does not necessarily replicate its collecting duct (CD) environment, where ICs are surrounded by the more abundant principal cells (PCs). It is reassuring that many findings obtained in ex vivo IC systems signaling have been largely confirmed in vivo. Some of these newly identified signaling pathways reveal that ICs are important for regulating NaCl reabsorption, thus suggesting new frontiers to target antihypertensive treatments. Moreover, recent single-cell characterization studies of kidney epithelial cells revealed a dual developmental origin of IC, as well as the presence of novel CD cell types with certain IC characteristics. These exciting findings present new opportunities for the study of IC ex vivo and will likely rediscover the importance of available tools in this field.NEW & NOTEWORTHY The study of kidney intercalated cells has been limited by current cell culture and kidney tissue isolation techniques. This review is to be used as a reference to select ex vivo techniques to study intercalated cells. We focused on the use of cell lines and kidney slices as potential useful models to study membrane transport proteins. We also review how novel collecting duct organoids may help better elucidate the role of these intriguing cells.

Multiomics Signatures of Coagulopathy in a Polytrauma Swine Model Contrasted with Severe Multisystem Injured Patients.

Trauma-induced coagulopathy (TIC) is a leading contributor to preventable mortality in severely injured patients. Understanding the molecular drivers of TIC is an essential step in identifying novel therapeutics to reduce morbidity and mortality. This study investigated multiomics and viscoelastic responses to polytrauma using our novel swine model and compared these findings with severely injured patients. Molecular signatures of TIC were significantly associated with perturbed coagulation and inflammation systems as well as extensive hemolysis. These results were consistent with patterns observed in trauma patients who had multisystem injuries. Here, intervention using resuscitative endovascular balloon occlusion of the aorta following polytrauma in our swine model revealed distinct multiomics alterations as a function of placement location. Aortic balloon placement in zone-1 worsened ischemic damage and mitochondrial dysfunction, patterns that continued throughout the monitored time course. While placement in zone-III showed a beneficial effect on TIC, it showed an improvement in effective coagulation. Taken together, this study highlights the translational relevance of our polytrauma swine model for investigating therapeutic interventions to correct TIC in patients.

Hyperglycaemic crises in adults with diabetes: a consensus report.

The American Diabetes Association (ADA), European Association for the Study of Diabetes (EASD), Joint British Diabetes Societies for Inpatient Care (JBDS), American Association of Clinical Endocrinology (AACE) and Diabetes Technology Society (DTS) convened a panel of internists and diabetologists to update the ADA consensus statement on hyperglycaemic crises in adults with diabetes, published in 2001 and last updated in 2009. The objective of this consensus report is to provide up-to-date knowledge about the epidemiology, pathophysiology, clinical presentation, and recommendations for the diagnosis, treatment and prevention of diabetic ketoacidosis (DKA) and hyperglycaemic hyperosmolar state (HHS) in adults. A systematic examination of publications since 2009 informed new recommendations. The target audience is the full spectrum of diabetes healthcare professionals and individuals with diabetes.

Lactate and hydrogen ions play a predominant role in evoking the exercise pressor reflex during ischaemic contractions but not during freely perfused contractions.

We investigated the role played by lactate and hydrogen in evoking the exercise pressor reflex (EPR) in decerebrated rats whose hindlimb muscles were either freely perfused or ischaemic. Production of lactate and hydrogen by the contracting hindlimb muscles was manipulated by knocking out the myophosphorylase gene (pygm). In knockout rats (pygm-/-; n = 13) or wild-type rats (pygm+/+; n = 13), the EPR was evoked by isometrically contracting the triceps surae muscles. Blood pressure, tension, blood flow, renal sympathetic nerve activity and blood lactate concentrations were measured. Intramuscular metabolites and pH changes induced by the contractions were quantified by 31P-magnetic resonance spectroscopy (n = 5). In a subset of pygm-/- rats (n = 5), contractions were evoked with prior infusion of lactate (pH 6.0) in an attempt to restore the effect of lactate and hydrogen ions. Contraction of freely perfused muscles increased blood lactate and decreased muscle pH in pygm+/+ rats only. Despite these differences, the reflex pressor and sympathetic responses to freely perfused contraction did not differ between groups (P = 0.992). During ischaemia, contraction increased muscle lactate and hydrogen ion production in pygm+/+ rats (P < 0.0134), whereas it had no effect in pygm-/- rats (P > 0.783). Likewise, ischaemia exaggerated the reflex pressor, and sympathetic responses to contraction in pygm+/+ but not in pygm-/- rats. This exaggeration was restored when a solution of lactate (pH 6.0) was infused prior to the contraction in pygm-/- rats. We conclude that lactate and hydrogen accumulation in contracting myocytes play a key role in evoking the metabolic component of the EPR during ischaemic but not during freely perfused contractions. KEY POINTS: Conflicting results exist about the role played by lactate and hydrogen ions in evoking the exercise pressor reflex. Using CRISP-Cas9, we rendered the myophosphorylase gene non-functional to block the production of lactate and hydrogen ions. The exercise pressor reflex was evoked in decerebrated rats by statically contracting the triceps surae muscles with or without muscle ischaemia. Static contraction elevated the concentration of lactate and hydrogen ions in pygm+/+ but not in pygm-/- rats. Despite these differences, the exercise pressor reflex was not different between groups. Acute muscle ischaemia exaggerated the concentration of lactate and hydrogen ions in pygm+/+ but not in pygm-/- rats. Likewise, acute muscle ischaemia exaggerated the exercise pressor reflex in pygm+/+ but not in pygm-/- rats. We conclude that lactate and hydrogen play a key role in evoking the exercise pressor reflex during ischaemic but not during freely perfused contractions.

hERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs.

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (IKr) and is critical for human cardiac repolarization. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased IKr. Silencing hERG1b's impact on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of IKr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines IKr proton sensitivity in hiPSC-CMs.

Dietary acid load in health and disease.

Maintaining an appropriate acid-base equilibrium is crucial for human health. A primary influencer of this equilibrium is diet, as foods are metabolized into non-volatile acids or bases. Dietary acid load (DAL) is a measure of the acid load derived from diet, taking into account both the potential renal acid load (PRAL) from food components like protein, potassium, phosphorus, calcium, and magnesium, and the organic acids from foods, which are metabolized to bicarbonate and thus have an alkalinizing effect. Current Western diets are characterized by a high DAL, due to large amounts of animal protein and processed foods. A chronic low-grade metabolic acidosis can occur following a Western diet and is associated with increased morbidity and mortality. Nutritional advice focusing on DAL, rather than macronutrients, is gaining rapid attention as it provides a more holistic approach to managing health. However, current evidence for the role of DAL is mainly associative, and underlying mechanisms are poorly understood. This review focusses on the role of DAL in multiple conditions such as obesity, cardiovascular health, impaired kidney function, and cancer.

Strengthening the basics: acids and bases influence vascular structure and function, tissue perfusion, blood pressure, and human cardiovascular disease.

Acids and their conjugate bases accumulate in or dissipate from the interstitial space when tissue perfusion does not match the metabolic demand. Extracellular acidosis dilates most arterial beds, but associated acid-base disturbances-e.g., intracellular acidification and decreases in HCO3- concentration-can also elicit pro-contractile influences that diminish vasodilation and even dominate in some vascular beds to cause vasoconstriction. The ensemble activities of the acid-base-sensitive reactions in vascular smooth muscle and endothelial cells optimize vascular resistance for blood pressure control and direct the perfusion towards active tissue. In this review, we describe the mechanisms of intracellular pH regulation in the vascular wall and discuss how vascular smooth muscle and endothelial cells sense acid-base disturbances. We further deliberate on the functional effects of local acid-base disturbances and their integrated cardiovascular consequences under physiological and pathophysiological conditions. Finally, we address how mutations and polymorphisms in the molecular machinery that regulates pH locally and senses acid-base disturbances in the vascular wall can result in cardiovascular disease. Based on the emerging molecular insight, we propose that targeting local pH-dependent effectors-rather than systemic acid-base disturbances-has therapeutic potential to interfere with the progression and reduce the severity of cardiovascular disease.

What can we learn about acid-base transporters in cancer from studying somatic mutations in their genes?

Acidosis is a chemical signature of the tumour microenvironment that challenges intracellular pH homeostasis. The orchestrated activity of acid-base transporters of the solute-linked carrier (SLC) family is critical for removing the end-products of fermentative metabolism (lactate/H+) and maintaining a favourably alkaline cytoplasm. Given the critical role of pH homeostasis in enabling cellular activities, mutations in relevant SLC genes may impact the oncogenic process, emerging as negatively or positively selected, or as driver or passenger mutations. To address this, we performed a pan-cancer analysis of The Cancer Genome Atlas simple nucleotide variation data for acid/base-transporting SLCs (ABT-SLCs). Somatic mutation patterns of monocarboxylate transporters (MCTs) were consistent with their proposed essentiality in facilitating lactate/H+ efflux. Among all cancers, tumours of uterine corpus endometrial cancer carried more ABT-SLC somatic mutations than expected from median tumour mutation burden. Among these, somatic mutations in SLC4A3 had features consistent with meaningful consequences on cellular fitness. Definitive evidence for ABT-SLCs as 'cancer essential' or 'driver genes' will have to consider microenvironmental context in genomic sequencing because bulk approaches are insensitive to pH heterogeneity within tumours. Moreover, genomic analyses must be validated with phenotypic outcomes (i.e. SLC-carried flux) to appreciate the opportunities for targeting acid-base transport in cancers.

Acute sodium bicarbonate administration improves ventilatory efficiency in experimental respiratory acidosis: clinical implications.

Administering sodium bicarbonate (NaHCO3) to patients with respiratory acidosis breathing spontaneously is contraindicated because it increases carbon dioxide load and depresses pulmonary ventilation. Nonetheless, several studies have reported salutary effects of NaHCO3 in patients with respiratory acidosis but the underlying mechanism remains uncertain. Considering that such reports have been ignored, we examined the ventilatory response of unanesthetized dogs with respiratory acidosis to hypertonic NaHCO3 infusion (1 N, 5 mmol/kg) and compared it with that of animals with normal acid-base status or one of the remaining acid-base disorders. Ventilatory response to NaHCO3 infusion was evaluated by examining the ensuing change in PaCO2 and the linear regression of the PaCO2 vs. pH relationship. Strikingly, PaCO2 failed to increase and the ΔPaCO2 vs. ΔpH slope was negative in respiratory acidosis, whereas PaCO2 increased consistently and the ΔPaCO2 vs. ΔpH slope was positive in the remaining study groups. These results cannot be explained by differences in buffering-induced decomposition of infused bicarbonate or baseline levels of blood pH, PaCO2, and pulmonary ventilation. We propose that NaHCO3 infusion improved the ventilatory efficiency of animals with respiratory acidosis, i.e., it decreased their ratio of total pulmonary ventilation to carbon dioxide excretion (VE/VCO2). Such exclusive effect of NaHCO3 infusion in animals with respiratory acidosis might emanate from baseline increased VD/VT (dead space/tidal volume) caused by bronchoconstriction and likely reduced pulmonary blood flow, defects that are reversed by alkali infusion. Our observations might explain the beneficial effects of NaHCO3 reported in patients with acute respiratory acidosis.

Novel functions of the anion exchanger AE4 (SLC4A9).

The kidney plays a crucial role in acid-base homeostasis. In the distal nephron, α-intercalated cells contribute to urinary acid (H+) secretion and ß-intercalated cells accomplish urinary base (HCO3-) secretion. ß-intercalated cells regulate the acid base status through modulation of the apical Cl-/HCO3- exchanger pendrin (SLC26A4) activity. In this review, we summarize and discuss our current knowledge of the physiological role of the renal transporter AE4 (SLC4A9). The AE4, as cation-dependent Cl-/HCO3- exchanger, is exclusively expressed in the basolateral membrane of ß-intercalated cells and is essential for the sensing of metabolic acid-base disturbances in mice, but not for renal sodium reabsorption and plasma volume control. Potential intracellular signaling pathways are discussed that might link basolateral acid-base sensing through the AE4 to apical pendrin activity.

Trimethoprim inhibits renal H+-K+-ATPase in states of K+ depletion.

There is growing consensus that under physiological conditions, collecting duct H+ secretion is independent of epithelial Na+ channel (ENaC) activity. We have recently shown that the direct ENaC inhibitor benzamil acutely impairs H+ excretion by blocking renal H+-K+-ATPase. However, the question remains whether inhibition of ENaC per se causes alterations in renal H+ excretion. To revisit this question, we studied the effect of the antibiotic trimethoprim (TMP), which is well known to cause K+ retention by direct ENaC inhibition. The acute effect of TMP (5 µg/g body wt) was assessed in bladder-catheterized mice, allowing real-time measurement of urinary pH, electrolyte, and acid excretion. Dietary K+ depletion was used to increase renal H+-K+-ATPase activity. In addition, the effect of TMP was investigated in vitro using pig gastric H+-K+-ATPase-enriched membrane vesicles. TMP acutely increased natriuresis and decreased kaliuresis, confirming its ENaC-inhibiting property. Under control diet conditions, TMP had no effect on urinary pH or acid excretion. Interestingly, K+ depletion unmasked an acute urine alkalizing effect of TMP. This finding was corroborated by in vitro experiments showing that TMP inhibits H+-K+-ATPase activity, albeit at much higher concentrations than benzamil. In conclusion, under control diet conditions, TMP inhibited ENaC function without changing urinary H+ excretion. This finding further supports the hypothesis that the inhibition of ENaC per se does not impair H+ excretion in the collecting duct. Moreover, TMP-induced urinary alkalization in animals fed a low-K+ diet highlights the importance of renal H+-K+-ATPase-mediated H+ secretion in states of K+ depletion.NEW & NOTEWORTHY The antibiotic trimethoprim (TMP) often mediates K+ retention and metabolic acidosis. We suggest a revision of the underlying mechanism that causes metabolic acidosis. Our results indicate that TMP-induced metabolic acidosis is secondary to epithelial Na+ channel-dependent K+ retention. Under control dietary conditions, TMP does not per se inhibit collecting duct H+ secretion. These findings add further argument against a physiologically relevant voltage-dependent mechanism of collecting duct H+ excretion.

Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

Altered ventilatory responses to hypercapnia-hypoxia challenges in a preclinical SUDEP model involve orexin neurons.

Failure to recover from repeated hypercapnia and hypoxemia (HH) challenges caused by severe GCS and postictal apneas may contribute to sudden unexpected death in epilepsy (SUDEP). Our previous studies found orexinergic dysfunction contributes to respiratory abnormalities in a preclinical model of SUDEP, Kcna1-/- mice. Here, we developed two gas challenges consisting of repeated HH exposures and used whole body plethysmography to determine whether Kcna1-/- mice have detrimental ventilatory responses. Kcna1-/- mice exhibited an elevated ventilatory response to a mild repeated hypercapnia-hypoxia (HH) challenge compared to WT. Moreover, 71% of Kcna1-/- mice failed to survive a severe repeated HH challenge, whereas all WT mice recovered. We next determined whether orexin was involved in these differences. Pretreating Kcna1-/- mice with a dual orexin receptor antagonist rescued the ventilatory response during the mild challenge and all subjects survived the severe challenge. In ex vivo extracellular recordings in the lateral hypothalamus of coronal brain slices, we found reducing pH either inhibits or stimulates putative orexin neurons similar to other chemosensitive neurons; however, a significantly greater percentage of putative orexin neurons from Kcna1-/-mice were stimulated and the magnitude of stimulation was increased resulting in augmentation of the calculated chemosensitivity index relative to WT. Collectively, our data suggest that increased chemosensitive activity of orexin neurons may be pathologic in the Kcna1-/- mouse model of SUDEP, and contribute to elevated ventilatory responses. Our preclinical data suggest that those at high risk for SUDEP may be more sensitive to HH challenges, whether induced by seizures or other means; and the depth and length of the HH exposure could dictate the probability of survival.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

How Reciprocal Interactions Between the Tumor Microenvironment and Ion Transport Proteins Drive Cancer Progression.

Solid tumors comprise two major components: the cancer cells and the tumor stroma. The stroma is a mixture of cellular and acellular components including fibroblasts, mesenchymal and cancer stem cells, endothelial cells, immune cells, extracellular matrix, and tumor interstitial fluid. The insufficient tumor perfusion and the highly proliferative state and dysregulated metabolism of the cancer cells collectively create a physicochemical microenvironment characterized by altered nutrient concentrations and varying degrees of hypoxia and acidosis. Furthermore, both cancer and stromal cells secrete numerous growth factors, cytokines, and extracellular matrix proteins which further shape the tumor microenvironment (TME), favoring cancer progression.Transport proteins expressed by cancer and stromal cells localize at the interface between the cells and the TME and are in a reciprocal relationship with it, as both sensors and modulators of TME properties. It has been amply demonstrated how acid-base and nutrient transporters of cancer cells enable their growth, presumably by contributing both to the extracellular acidosis and the exchange of metabolic substrates and waste products between cells and TME. However, the TME also impacts other transport proteins important for cancer progression, such as multidrug resistance proteins. In this review, we summarize current knowledge of the cellular and acellular components of solid tumors and their interrelationship with key ion transport proteins. We focus in particular on acid-base transport proteins with known or proposed roles in cancer development, and we discuss their relevance for novel therapeutic strategies.