Pflügers Archiv - European Journal of Physiology

Published by Springer Nature
Online ISSN: 1432-2013
Print ISSN: 0031-6768
Learn more about this page
Recent publications
  • David H. VandorpeDavid H. Vandorpe
  • John F. HeneghanJohn F. Heneghan
  • Joshua S. WaitzmanJoshua S. Waitzman
  • [...]
  • Seth L. AlperSeth L. Alper
Two heterozygous missense variants (G1 and G2) of Apolipoprotein L1 (APOL1) found in individuals of recent African ancestry can attenuate the severity of infection by some forms of Trypanosoma brucei. However, these two variants within a broader African haplotype also increase the risk of kidney disease in Americans of African descent. Although overexpression of either variant G1 or G2 causes multiple pathogenic changes in cultured cells and transgenic mouse models, the mechanism(s) promoting kidney disease remain unclear. Human serum APOL1 kills trypanosomes through its cation channel activity, and cation channel activity of recombinant APOL1 has been reconstituted in lipid bilayers and proteoliposomes. Although APOL1 overexpression increases whole cell cation currents in HEK-293 cells, the ion channel activity of APOL1 has not been assessed in glomerular podocytes, the major site of APOL1-associated kidney diseases. We characterize APOL1-associated whole cell and on-cell cation currents in HEK-293 T-Rex cells and demonstrate partial inhibition of currents by anti-APOL antibodies. We detect in primary human podocytes a similar cation current inducible by interferon-γ (IFNγ) and sensitive to inhibition by anti-APOL antibody as well as by a fragment of T. brucei Serum Resistance-Associated protein (SRA). CRISPR knockout of APOL1 in human primary podocytes abrogates the IFNγ-induced, antibody-sensitive current. Our novel characterization in HEK-293 cells of heterologous APOL1-associated cation conductance inhibited by anti-APOL antibody and our documentation in primary human glomerular podocytes of endogenous IFNγ-stimulated, APOL1-mediated, SRA and anti-APOL-sensitive ion channel activity together support APOL1-mediated channel activity as a therapeutic target for treatment of APOL1-associated kidney diseases.
The fibroblast growth factor FGF-23 is a member of the FGF-15/19 subfamily with hormonal functions. Besides its well-known role for bone mineralization, FGF-23 is discussed as a marker for cardiovascular disease. We investigated whether FGF-23 has any effects on the endocrine pancreas of mice by determining insulin secretion, electrical activity, intracellular Ca²⁺, and apoptosis. Acute application of FGF-23 (10 to 500 ng/ml, i.e., 0.4 to 20 nM) does not affect insulin release of murine islets, while prolonged exposure leads to a 21% decrease in glucose-stimulated secretion. The present study shows for the first time that FGF-23 (100 or 500 ng/ml) partially protects against impairment of insulin secretion and apoptotic cell death induced by glucolipotoxicity. The reduction of apoptosis by FGF-23 is approximately twofold higher compared to FGF-21 or FGF-15/19. In contrast to FGF-23 and FGF-21, FGF-15/19 is clearly pro-apoptotic under control conditions. The beneficial effect of FGF-23 against glucolipotoxicity involves interactions with the stimulus-secretion cascade of beta-cells. Electrical activity and the rise in the cytosolic Ca²⁺ concentration of islets in response to acute glucose stimulation increase after glucolipotoxic culture (48 h). Co-culture with FGF-23 further elevates the glucose-mediated effects on both parameters. Protection against apoptosis and glucolipotoxic impairment of insulin release by FGF-23 is prevented, when calcineurin is inhibited by tacrolimus or when c-Jun N-terminal kinase (JNK) is blocked by SP600125. In conclusion, our data suggest that FGF-23 can activate compensatory mechanisms to maintain beta-cell function and integrity of islets of Langerhans during excessive glucose and lipid supply.
Potential mechanisms for the coupling of paracellular water and ion transport. i Single file soft knock-on mechanism: alternation of water and ion. ii Permeation of fully hydrated ions. iii Solvent drag: dragging of ions by the bulk movement of water. iv Pseudo solvent drag due to unstirred layer effects that cause local gradients. In i and ii ions (orange circles) and water molecules are depicted within a claudin-based paracellular channel. In iii and iv blue shading depicts overall osmolarity (sum of all osmolytes, gradient, e.g., generated by transcellular transport) outside and within a paracellular channel. Arrows indicate the direction of water movement, orange and green circles illustrate two different ion species, one being able (orange) and one not being able (green) to cross the barrier. *, #, unstirred layers with osmolyte enrichment on the cis face (*) and depletion on the trans face (#) of the barrier, respectively
Ion and water transport in the proximal tubule. Upper: Immunofluorescence staining of claudin-2 (red) and claudin-10a (green) in a mouse proximal tubule showing the alternating arrangement of the two claudins in the tight junction (detail from [8]). Lower: Schematic drawing of trans- and paracellular transport in the proximal tubule. Transcellular solute transport leads to the build-up of local gradients through unstirred layer effects. These gradients drive the coupled H2O and cation reabsorption through the claudin-2-based paracellular channels by a solvent drag-like mechanism. The resulting local interstitial Cl⁻ depletion causes paracellular Cl⁻ reabsorption through the H2O impermeable claudin-10a-based paracellular Cl⁻ channels (pseudo solvent drag mechanism)
The concept of solvent drag, i.e., water and solutes sharing the same pore and their transport being frictionally coupled, was first proposed in the early 1950s. During the following decades, it was applied to transport processes across cell membranes as well as transport along the paracellular pathway. Water-driven solute transport was proposed as the major mechanism for electrolyte and nutrient absorption in the small intestine and for Cl- and HCO3- reabsorption in the renal proximal tubule. With the discovery of aquaporins as transcellular route for water transport and the claudin protein family as the major determinant of paracellular transport properties, new mechanistic insights in transepithelial water and solute transport are emerging and call for a reassessment of the solvent drag concept. Current knowledge does not provide a molecular basis for relevant solvent drag-driven, paracellular nutrient, and inorganic anion (re-)absorption. For inorganic cation transport, in contrast, solvent drag along claudin-2-formed paracellular channels appears feasible.
Biogenesis of exosomes: (1) ESCRT-dependent pathway: ESCRT 0-III work closely together to facilitate the second budding step and sort ubiquitin-tagged proteins into the ILVs. They are assisted by the syndecan-syntenin-ALIX adapter complex, which stabilizes ESCRT III at the neck of the vesicle. Finally, ESCRT III mediates the sequestration of the vesicles into the lumina of the endosomes. (2) ESCRT independent pathway: Budding is mainly mediated by a modified lipid composition of the vesicle membrane, with ceramides, cardiolipids, or cholesterol. Flotillins and Rab31 help to internalize tyrosine kinase receptors besides acting as scaffold proteins and preventing lysosomal degradation, respectively. (3) A third major contribution is provided by tetraspanins: they mediate the sorting of various proteins into the ILVs by forming of microdomains. The image was created with CorelDRAW Graphics Suite (Corel, Ottawa, ON, Canada)
Exosomes are extracellular vesicles that are formed by two invaginations of the plasma membrane and can be released by all eukaryotic cells. Because of their bioactive contents, including nucleic acids and proteins, exosomes can activate a variety of functions in their recipient cells. Due to the plethora of physiological and pathophysiological functions, exosomes have received a lot of attention from researchers over the past few years. However, there is still no consensus regarding isolation and characterization protocols of exosomes and their subtypes. This heterogeneity poses a lot of methodical challenges but also offers new clinical opportunities simultaneously. So far, exosome-based research is still mostly limited to preclinical experiments and early-stage clinical trials since the translation of experimental findings remains difficult. Exosomes could potentially play an important role as future diagnostic and prognostic agents and might also be part of the development of new treatment strategies. Therefore, they have previously been investigated in a variety of nephrological and urological conditions such as acute kidney injury or prostate cancer.
The middle cerebral artery dilates after the decrease in intra- and extravascular sodium ion concentration from 145 to 121 mmol L⁻¹ without correction for the decreased osmolality (black bar). The correction of osmolality with N-methyl D-glucamine (NMDG) or Tris–HCl does not affect the response of the vessel to lowering of intra- and extraluminal sodium ion concentrations. Values are mean ± S.E. The number of vessels per group is between 6 and 8
Removal of the endothelium (-Endo), inhibition of the synthesis of nitric oxide (L-NAME, 10 µmol L⁻¹), or inhibition of guanylyl cyclase (ODQ, 5 µmol L⁻¹) abolishes the dilation of the middle cerebral artery in the low-sodium buffer. Vessels pretreated with L-NAME or ODQ constricted during the lowering of Na⁺ to 121 mmol L⁻¹ by 10 ± 5% (p < 0.05) and 12 ± 2% (p < 0.05), respectively. The horizontal line represents the reference diameter of the vessel during normonatremia, set at 100%. Values are mean ± S.E. The number of vessels per group is between 6 and 8
Administration of 1 µmol L⁻¹ KR-R7943 did not affect the response of the MCA to low sodium buffer, whereas inhibition of the reverse mode of Na⁺/Ca²⁺ exchanger with 10 µmol L⁻¹ KR-R7943 abolished the dilation of the MCA observed under low sodium conditions. Buffering intracellular Ca²⁺ in the endothelial cells with 10 µmol L⁻¹ BAPTA-AM did not affect the response of the MCA to low sodium buffer. The horizontal line represents the reference diameter of the vessel during normonatremia, set at 100%. Values are mean ± S.E. The number of vessels per group is between 6 and 8
List of the experimental series
Stability of the MCA diameter in series 0 and I
A decrease in serum sodium ion concentration below 135 mmol L ⁻¹ is usually accompanied by a decrease in plasma osmolality (hypoosmotic hyponatremia) and leads to the disorder of intracranial homeostasis mainly due to cellular swelling. Recently, using an in vitro model of hypoosmotic hyponatremia, we have found that a decrease in sodium ion concentration in the perfusate to 121 mmol L ⁻¹ relaxes the isolated rat middle cerebral artery (MCA). The aim of the present study was to explore the mechanism responsible for this relaxation. Isolated, pressurized, and perfused MCAs placed in a vessel chamber were subjected to a decrease in sodium ion concentration to 121 mmol L ⁻¹ . Changes in the diameter of the vessels were monitored with a video camera. The removal of the endothelium and inhibition of nitric oxide-dependent signaling or the reverse mode sodium-calcium exchanger (NCX) were used to study the mechanism of the dilation of the vessel during hyponatremia. The dilation of the MCA (19 ± 5%, p < 0.005) in a low-sodium buffer was absent after removal of the endothelium or administration of the inhibitor of the reverse mode of sodium-calcium exchange and was reversed to constriction after the inhibition of nitric oxide (NO)/cGMP signaling. The dilation of the middle cerebral artery of the rat in a 121 mmol L ⁻ ¹ Na ⁺ buffer depends on NO signaling and reverse mode of sodium-calcium exchange. These results suggest that constriction of large cerebral arteries with impaired NO-dependent signaling may be observed in response to hypoosmotic hyponatremia.
A de novo mutation in Cav1.3 channels causes neurological and metabolic syndroms.  A Location of the F747S mutation (orange dot) in the cryo-EM structure of the human Cav1.3 channel in complex with Cavb3 and Cava2d1 ancillary subunits (PDB: 7UHG and [9]). The F747 is located at the end of the second S6 segment which is part of the activation gate of the channel. B Amino acid alignment of part of the second S6 region showing the conservation of the phenylalanine (F) residue across all high-voltage-activated calcium channels. C Window current of Cav1.3 WT (black) and F747S (orange). Activation and inactivation curves were reproduced according to experimental parameters provided in [6] adjusted by -18 mV to take into account the depolarizing shift caused by recordings in 15 mM calcium compared to physiological 2 mM calcium concentration. D Computer modeling of the effect of the F747S variant on the pacemaking activity of dopaminergic (DA) neurons. The modeling was performed using the NEURON simulation environment where the relative alterations of the voltage dependence of activation properties (half activation potential (V1/2) and slope factor (k) values) caused by the F747S variant were introduced in a substantia nigra pars compacta (SNc) DA neuron model as previously described [1]. E Homologous phenylalanine (F) variants in Cav1.3 (brown), Cav1.4 (blue), Cav2.3 (green), and Cav2.1 (purple) and their associated clinical syndromes. DEE, development and epileptic encephalopathy; CSNB, congenital stationary night blindness; FHM-1, familial hemiplegic migraine type 1.
Calcium channelopathies are a group of human diseases resulting from the dysfunction of calcium channels or their regulatory subunits and are caused by either genetic or acquired factors. Hence, many pathogenic mutations in genes encoding voltage-gated calcium channels (VGCCs) are linked to a large spectrum of neurological, cardiac, and metabolic disorders. Of the 10 mammalian VGCCs, the Cav1.3 channel is abundantly expressed in neuroendocrine cells including pancreatic b and adrenal chromaffin cells, but is also found in brain, retina, ovaries, cochlear hair cells of the ear, and in cardiac atrial myocytes. It is involved in diverse physiological functions such as neurotransmitter and hormone release, synaptic plasticity, and control of cardiac rhythm and atrioventricular node conductance. Over the recent years, thank to next-generation sequencing approaches, numerous CACNA1D variants have been identified and are associated with an increase in allelic burden in neurological and neuroendocrine disorders. Importantly, a number of rare de novo disease-causing variants were identified. These variants are localized in the cytoplasmic ends of the first and second S6 segments of Cav1.3 which form the activation and inactivation gate and cause alterations in the gating properties that are in general consistent with a gain-of-function (GoF) of the channel.
One side effect of cisplatin, a cytotoxic platinum anticancer drug, is peripheral neuropathy; however, its central nervous system effects remain unclear. We monitored respiratory nerve activity from the C4 ventral root in brainstem and spinal cord preparations from neonatal rats (P0–3) to investigate its central effects. Bath application of 10–100 μM cisplatin for 15–20 min dose-dependently decreased the respiratory rate and increased the amplitude of C4 inspiratory activity. These effects were not reversed after washout. In separate perfusion experiments, cisplatin application to the medulla decreased the respiratory rate, and application to the spinal cord increased the C4 burst amplitude without changing the burst rate. Application of other platinum drugs, carboplatin or oxaliplatin, induced no change of respiratory activity. A membrane potential analysis of respiratory-related neurons in the rostral medulla showed that firing frequencies of action potentials in the burst phase tended to decrease during cisplatin application. In contrast, in inspiratory spinal motor neurons, cisplatin application increased the peak firing frequency of action potentials during the inspiratory burst phase. The increased burst amplitude and decreased respiratory frequency were partially antagonized by riluzole and picrotoxin, respectively. Taken together, cisplatin inhibited respiratory rhythm via medullary inhibitory system activation and enhanced inspiratory motor nerve activity by changing the firing property of motor neurons.
Experiment procedure. The rat hearts were cannulated and mounted onto the Langendorff perfusion system and were perfused with KH solution for 5 min to remove the blood. The hearts were then perfused with 30 ml HTK or HTK + pl-MT (1 μg/μl, 150 μl) solution, respectively. After a thorough washout of KH solution, the hearts were preserved at cold HTK and HTK + pl-MT solution for 9 h at 4 °C. After 9 h of preservation, KH solution was perfused for 5 min. The heartbeat, the volume of coronary circulation, and myocyte viability were observed for the functional analysis of the heart
Characterization of pl-MT. A pl-MT are identified inside platelets by MitoTracker (green), a membrane potential-dependent dye. B MitoTracker staining of pl-MT was shown to remain even after purification. Scarle bar = 10 μm. C ATP synthase activity of pl-MT. Results show when ADP was added, mitochondria maintained the ability for ATP synthesis, but the ATP synthesis ability was lost after being heated. D Citrate synthase activity of pl-MT was maintained when acetyl-CoA was added, but citrate synthase activity was lost after being heated. Results are presented as mean ± SEM and analyzed by one-way analysis of variance. ***P < 0.001 compared with controls
pl-MT increased the viability of cardiomyocytes. A pl-MT was transferred into cardiomyocytes after 9 h of preservation. Mitochondria labeled with MitoTracker (red). Nuclei (DAPI, blue), endogenous mitochondria (green), and the merged image. Scale bar = 10 μm. B Cardiomyocyte viability was measured continuously for 720 min (by WST-1 assay). Absorbance was recorded per 20 min. C Intracellular ATP was measured by ATP synthesis assay. The HTK + pl-MT group showed higher ATP synthesis activity compared to those with the HTK group. Each group was replicated four times. Results are presented as mean ± SEM and analyzed by one-way analysis of variance. * < 0.05 compared with HTK, ***P < 0.001 compared with HTK
Mitochondrial function was improved with pl-MT in rat hearts. The function of mitochondria isolated from pl-MT preserved hearts was measured as described in the “Methods and materials” section. A Mitochondrial membrane potential was analyzed by TMRM. B ROS generation was analyzed by Amplex as an H2O2 probe. A, B Oligo. (oligomycin) 25 μM, CCCP 5 μM, and A.A (Antimycin A) 2 μM were applied in sequence, and the signals were detected for 10 min. C The activity of ATP synthesis was measured by the increased ATP ratio after ADP treatment for 20 min. D The activity of citrate synthesis was measured by the decreased DTNB ratio after acetyl-CoA with DTNB treatment for 20 min. HTK + pl-MT have higher mitochondria activity compared with the HTK control group. Each group was replicated five times. Results are presented as mean ± SEM and analyzed by one-way analysis of variance. ** < 0.01 compared with HTK, ***P < 0.001 compared with HTK
Cardiac function was improved in pl-MT incubated rat hearts. A Pl-MT were observed in the heart of the HTK + pl-MT group but not in the heart of the HTK group after 9 h of preservation (pl-MT was labeled with MitoTracker, red, and nuclei with DAPI, blue). Scale bar = 10 μm. B Perfusion volume of coronary circulation was measured at 0-, 9-, 12-, 29-h time points. C Myocytes were isolated from HTK and HTK + pl-MT groups and imaged at a bright field. Scale bar = 100 μm. Results are presented as mean ± SEM and analyzed by one-way analysis of variance. * < 0.05 compared with HTK
Mitochondria transplantation emerges as an effective therapeutic strategy for ischemic-related diseases but the roles in the donor hearts for transplant remain unidentified. Here, we investigated whether the preservation of the donor heart with human platelet-derived mitochondria (pl-MT) could improve mitochondrial and cardiac function. Incubation with pl-MT resulted in the internalization of pl-MT and the enhancement of ATP production in primary cardiomyocytes. In addition, incubation of rat hearts with pl-MT ex vivo for 9 h clearly demonstrated pl-MT transfusion into the myocardium. Mitochondria isolated from the hearts incubated with pl-MT showed increased mitochondrial membrane potential and greater ATP synthase activity and citrate synthase activity. Importantly, the production of reactive oxygen species from cardiac mitochondria was not different with and without pl-MT incubation. Functionally, the heartbeat and the volume of coronary circulation perfusate were significantly increased in the Langendorff perfusion system and the viability of cardiomyocytes was increased from pl-MT hearts. Taken together, these results suggest that incubation with Pl-MT improves mitochondrial activity and maintains the cardiac function of rat hearts with prolonged preservation time. The study provides the proof of principle for pl-MT application as an enhancer of the donor heart.
An aberrant late sodium current ( I Na,Late ) caused by a mutation in the cardiac sodium channel (Na v 1.5) has emerged as a contributor to electrical remodeling that causes susceptibility to atrial fibrillation (AF). Although downregulation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with AF, the molecular mechanisms underlying the negative regulation of I Na,Late in AF remain unclear, and potential therapeutic approaches are needed. In this work, we constructed a tachypacing-induced cellular model of AF by exposing HL-1 myocytes to rapid electrical stimulation (1.5 V/cm, 4 ms, 10 Hz) for 6 h. Then, we gathered data using confocal Ca ²⁺ imaging, immunofluorescence, patch-clamp recordings, and immunoblots. The tachypacing cells displayed irregular Ca ²⁺ release, delayed afterdepolarization, prolonged action potential duration, and reduced PI3K/Akt signaling compared with controls. Those detrimental effects were related to increased I Na,Late and were significantly mediated by treatment with the I Na,Late blocker ranolazine. Furthermore, decreased PI3K/Akt signaling via PI3K inhibition increased I Na,Late and subsequent aberrant myocyte excitability, which were abolished by I Na,Late inhibition, suggesting that PI3K/Akt signaling is responsible for regulating pathogenic I Na,Late . These results indicate that PI3K/Akt signaling is critical for regulating I Na,Late and electrical remodeling, supporting the use of PI3K/Akt-mediated I Na,Late as a therapeutic target for AF.
The concentration of inorganic phosphate (Pi) in plasma is under hormonal control, with deviations from normal values promptly corrected to avoid hyper- or hypophosphatemia. Major regulators include parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23), and active vitamin D3 (calcitriol). This control is achieved by mechanisms largely dependent on regulating intestinal absorption and renal excretion, whose combined actions stabilise plasma Pi levels at around 1–2 mM. Instead, Pi concentrations up to 13 and 40 mM have been measured in saliva from humans and ruminants, respectively, suggesting that salivary glands have the capacity to concentrate Pi. Here we analysed the transcriptome of parotid glands, ileum, and kidneys of mice, to investigate their potential differences regarding the expression of genes responsible for epithelial transport of Pi as well as their known regulators. Given that Pi and Ca²⁺ homeostasis are tightly connected, the expression of genes involved in Ca²⁺ homeostasis was also included. In addition, we studied the effect of vitamin D3 treatment on the expression of Pi and Ca²⁺ regulating genes in the three major salivary glands. We found that parotid glands are equipped preferentially with Slc20 rather than with Slc34 Na⁺/Pi cotransporters, are suited to transport Ca²⁺ through the transcellular and paracellular route and are potential targets for PTH and vitamin D3 regulation.
We recorded spontaneous extracellular action potentials (eAPs) from rat chromaffin cells (CCs) at 37 °C using microelectrode arrays (MEAs) and compared them with intracellularly recorded APs (iAPs) through conventional patch clamp recordings at 22 °C. We show the existence of two distinct firing modes on MEAs: a ~ 4 Hz irregular continuous firing and a frequent intermittent firing mode where periods of high-intraburst frequency (~ 8 Hz) of ~ 7 s duration are interrupted by silent periods of ~ 12 s. eAPs occurred either as negative- or positive-going signals depending on the contact between cell and microelectrode: either predominantly controlled by junction-membrane ion channels (negative-going) or capacitive/ohmic coupling (positive-going). Negative-going eAPs were found to represent the trajectory of the Na⁺, Ca²⁺, and K⁺ currents passing through the cell area in tight contact with the microelectrode during an AP (point-contact junction). The inward Nav component of eAPs was blocked by TTX in a dose-dependent manner (IC50 ~ 10 nM) while the outward component was strongly attenuated by the BK channel blocker paxilline (200 nM) or TEA (5 mM). The SK channel blocker apamin (200 nM) had no effect on eAPs. Inward Nav and Cav currents were well-resolved after block of Kv and BK channels or in cells showing no evident outward K⁺ currents. Unexpectedly, on the same type of cells, we could also resolve inward L-type currents after adding nifedipine (3 μM). In conclusion, MEAs provide a direct way to record different firing modes of rat CCs and to estimate the Na⁺, Ca²⁺, and K⁺ currents that sustain cell firing and spontaneous catecholamines secretion.
This systematic review and meta-analysis aimed at evaluating acute and chronic effects of physical exercise on IgA and IgG levels, as well as its relationship with the susceptibility to develop upper respiratory tract infections (URTI). This systematic review and meta-analysis was conducted and reported in accordance with PRISMA statement. A systematic search of PubMed, Web of Science, and EMBASE was performed in July 2020. This systematic review and meta-analysis included studies in which participants performed acute exercise or chronic physical training and were subjected to analyses of URTI incidence and concentrations of IgA and IgG. The selected studies for systematic review were divided into the following three groups: (I) trials that evaluated the effects of acute exercise in sedentary subjects, (II) trials that evaluated the effects of acute exercise in athletes/trained individuals, and (III) trials that evaluated the effects of chronic physical training on the incidence of URTI, as well as on the levels of IgA and IgG. Acute exercise increases the IgA levels in trained subjects but does not affect its levels in untrained subjects. Such increase in IgA levels induced by acute exercise is greater in trained individual that performed ultramarathon. On the other hand, chronic physical training reduces IgA levels in both trained and untrained subjects, does not change IgA levels in non-military subjects, besides from not affecting IgG levels. The present systematic review and meta-analysis indicates that acute exercise positively influences IgA levels in trained individuals, being this effect pronounced when a strenuous exercise such as ultramarathon is executed. Chronic physical training, in turn, does not affect IgG levels.
Ultrastructure of blood vessels in the neocortex of cuprizone-treated mice. a, b Normal-appearing blood vessels in the neocortex of control mice. Note the distinct structure of the basal lamina (black arrows). a Low-power image; bar represents 1000 nm. b High-power image; bar represents 500 nm. c, d After 5 weeks of feeding cuprizone pellets, edemas start to appear adjacent to the basal lamina (black arrows). These edemas represent swollen astrocytic endfeet (black asterisks), as they contain organelles, e.g., mitochondria (white asterisk in d). c Low-power image; bar represents 2500 nm. d High-power image; bar represents 500 nm. e, f After 5 weeks of cuprizone feeding with an additional week of feeding with normal food, large edemas are visible and even increased as compared to (c) and (d) (black asterisks). Black arrows indicate the basal lamina. e Low-power image; bar represents 1000 nm. f High-power image; bar represents 500 nm. g, h After 5 weeks of cuprizone feeding with additional 5 weeks of feeding with normal food, large edemas are visible (asterisks). Black arrows indicate the basal lamina. g Low-power image; bar represents 1000 nm. h High-power image; bar represents 500 nm. L, vessel lumen; e, endothelial cell
Area with edema in all experimental groups. Quantification (number of occurrence) and size distribution (plotted on y-axis) of swellings occurring directly adjacent to the basement membrane and thus localized in astrocytic endfeet in all experimental groups. Ultrastructural images from n = 3 mice per group were analyzed and the size of the vacuoles was measured (µm²). Each symbol represents one vacuole. The number of symbols represents the frequency, with which vacuoles and edemas were detected on the images taken from each group. The number is lower in the control group, as there was hardly any vacuole detectable next to the basement membranes. Shapiro–Wilk test revealed non-normal distribution of the data. Kruskal–Wallis test was applied with subsequent Dunn’s multiple comparison test. P values (as compared to control) are indicated in the graph
Freeze-fracture analysis of orthogonal arrays of particles. a Orthogonal arrays of particles (OAP, circles and arrows) along blood vessels in control mice. b After 5 weeks of cuprizone feeding plus additional 5 weeks on normal food, clusters of particles are still present (circles and arrows), but more dispersed and not arranged in arrays anymore, as compared to the control. Scale bars represent 100 nm
The cuprizone model is a widely used model to study the pathogenesis of multiple sclerosis (MS). Due to the selective loss of mature oligodendrocytes and myelin, it is mainly being used to study demyelination and the mechanisms of remyelination, as well as the efficiency of compounds or therapeutics aiming at remyelination. Although early investigations using high dosages of cuprizone reported the occurrence of hydrocephalus, it has long been assumed that cuprizone feeding at lower dosages does not induce changes at the blood–brain barrier (BBB). Here, by analyzing BBB ultrastructure with high-resolution electron microscopy, we report changes at astrocytic endfeet surrounding vessels in the brain parenchyma. Particularly, edema formation around blood vessels and swollen astrocytic endfeet already occurred after feeding low dosages of cuprizone. These findings indicate changes in BBB function that will have an impact on the milieu of the central nervous system (CNS) in the cuprizone model and need to be considered when studying the mechanisms of de- and remyelination.
The transverse-axial tubular system (tubular system) of cardiomyocytes plays a key role in excitation–contraction coupling. To determine the area of the tubular membrane in relation to the area of the surface membrane, indirect measurements through the determination of membrane capacitances are currently used in addition to microscopic methods. Unlike existing electrophysiological methods based on an irreversible procedure (osmotic shock), the proposed new approach uses a reversible short-term intermittent increase in the electrical resistance of the extracellular medium. The resulting increase in the lumen resistance of the tubular system makes it possible to determine separate capacitances of the tubular and surface membranes. Based on the analysis of the time course of the capacitive current, computational relations were derived to quantify the elements of the electrical equivalent circuit of the measured cardiomyocyte including both capacitances. The exposition to isotonic low-conductivity sucrose solution is reversible which is the main advantage of the proposed approach allowing repetitive measurements on the same cell under control and sucrose solutions. Experiments on rat ventricular cardiomyocytes (n = 20) resulted in the surface and tubular capacitance values implying the fraction of tubular capacitance/area of 0.327 ± 0.018. We conclude that the newly proposed method provides results comparable to the data obtained by the currently used detubulation method and, in addition, by being reversible, allows repeated evaluation of surface and tubular membrane parameters on the same cell.
Time-dependent inhibition of SOCE by CFTRinh-172 in Calu-3 cells. A–D Representative Ca²⁺ fluorimetry traces tracking changes in [Ca²⁺]i following a Tg-activated, repeated Ca²⁺ addback protocol. Cells were exposed to the vehicle DMSO (A), or 20 µM CFTRinh-172 (CFinh172, B-D), in a nominally Ca²⁺ free solution for 3 (B), 10 (C), or 30 (D) min before the second SOCE was induced by adding back 1 mM Ca.²⁺ (black bars). The agents were removed from the perfusing solution for 15 (B, C) or 30 (D) min before the third SOCE was activated. (E) Box and whiskers summary of percentage change in SOCE peak amplitude and rate, from SOCE #1 to SOCE #2, following DMSO (control) or CFTRinh-172 treatment of different durations. One-way ANOVA with Holm-Sidak multiple comparisons tests was performed across the four groups. Boxes represent median ± 25th/75th percentiles, while whiskers represent minimum/maximum. * = p < 0.05 vs. control; # = p < 0.05 vs. 3 min. F Frequency of > 10% inhibition of SOCE amplitude and rate by DMSO or CFTRinh-172 treatment of different durations. Percentage inhibition was calculated as percentage of SOCE #2 amplitude/rate over that of SOCE #1, and the frequency of experiments with over and under 10% inhibition was tallied. A chi-square test was performed across the four groups. χ = p < 0.05 for chi-square test. n = 4–14
Distinct CFTR inhibitors reduce SOCE in HEK293T cells. A, B HEK293T cells do not express CFTR. A Raw CT values for amplification of CFTR, and the housekeeping gene GAPDH, in Calu-3 and HEK293T cell samples. Each sample was run in duplicate. B Western blot image of Calu-3 and HEK293T samples probed for CFTR expression (Band C ~ 180 kDa, fully glycosylated CFTR). C–E Representative Ca²⁺ fluorimetry traces tracking changes in [Ca²⁺]i following a Tg-activated, repeated Ca.²⁺ addback protocol, with pre-treatment of DMSO (C), 20 µM CFTRinh-172 for 30 min (D) or 10 µM GlyH-101 for 10 min (E) before the second SOCE was activated. F Box and whiskers summary of percentage change in SOCE peak amplitude and rate, from SOCE #1 to SOCE #2, following treatment of DMSO (control) or the CFTR inhibitors. One-way ANOVA with Holm-Sidak multiple comparisons tests was performed across the three groups. Boxes represent median ± 25th/75th percentiles, while whiskers represent minimum/maximum. * = p < 0.05 vs. control. n = 5–6
CFTRinh-172 and GlyH-101 inhibit Orai1/Stim1-mediated whole cell currents in HEK293T cells. A, B, D, E Current–voltage relationships for whole cell currents measured in cells pre-exposed to CPA (10 µM) to activate Orai1 channels, followed by CFTR inhibitors. The inhibitors were present for 3 min at each concentration tested. Cells were mock-transfected (black symbols) or overexpressed Orai1/Stim1 (red symbols). Increasing concentrations of CFTRinh-172 (CFinh172, 0.2, 2, 20 µM) or GlyH-101 (GlyH101, 0.5, 5, 50 µM) were applied in the continuous presence of CPA. Application of the highest concentration of CFTRinh-172 (20 µM) or GlyH-101 (50 µM) significantly inhibited the inward currents, but had no effect in mock-transfected cells. C, F Summary of the concentration-dependent inhibition of inward currents by CFTRinh-172 or GlyH-101. * = p < 0.05 vs. control (mock). Un-paired t-test
CFTRinh-172 and GlyH-101 affect ENaC currents in Xenopus oocytes. A Left panel: representative current trace of a human αβγ-ENaC expressing oocyte. The application of amiloride (100 µM; ‘a’) is represented by the black bars and was used to determine amiloride-sensitive fractions of IM (ΔIami; right panel), before and after application of CFTRinh172 (20 µM; CFinh172). n = 9. B Similar experiments as shown in panel A, with human δβγ-ENaC expressing oocytes (n = 9). C/D Similar experiments as shown in panels A/B, where ΔIami were determined before and after application of GlyH-101 (GlyH101, 10 µM) n = 9 for both conditions. Student’s paired t-test was employed for all statistical analyses, with ** indicating p < 0.01 and *** indicating p < 0.001. E Left panel: Representative current trace of a water-injected control oocyte. The application of amiloride (100 µM; ‘a’) is represented by the black bars, the application of CFTR inhibitors by grey bars. Neither of the CFTR-inhibitors had any significant effect on transmembrane currents (IM). n = 6. Wilcoxon matched-pairs signed rank test was employed for statistical analyses
Putative CFTR-inhibitor binding sites in human ENaC. Molecular docking experiments identified two potential binding pockets for CFTRinh-172 (yellow) and GlyH-101 (orange) in the human α-ENaC structure. α-ENaC is shown in blue, γ-ENaC in brown and β-ENaC in green. The structure of each ENaC subunit represents a clenched hand holding a ball of β-sheets [32]. One binding pocket is located at the ‘finger’/’ thumb’ domain interface, the second binding pocket is located between the ‘β-ball’ and ‘palm’ domains
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and the epithelial Na + channel (ENaC) play essential roles in transepithelial ion and fluid transport in numerous epithelial tissues. Inhibitors of both channels have been important tools for defining their physiological role in vitro. However, two commonly used CFTR inhibitors, CFTR inh-172 and GlyH-101, also inhibit non-CFTR anion channels, indicating they are not CFTR specific. However, the potential off-target effects of these inhibitors on epithelial cation channels has to date not been addressed. Here, we show that both CFTR blockers, at concentrations routinely employed by many researchers, caused a significant inhibition of store-operated calcium entry (SOCE) that was time-dependent, poorly reversible and independent of CFTR. Patch clamp experiments showed that both CFTR inh-172 and GlyH-101 caused a significant block of Orai1-mediated whole cell currents, establishing that they likely reduce SOCE via modulation of this Ca 2+ release-activated Ca 2+ (CRAC) channel. In addition to off-target effects on calcium channels, both inhibitors significantly reduced human αβγ-ENaC-mediated currents after heterologous expression in Xenopus oocytes, but had differential effects on δβγ-ENaC function. Molecular docking identified two putative binding sites in the extracellular domain of ENaC for both CFTR blockers. Together, our results indicate that caution is needed when using these two CFTR inhibitors to dissect the role of CFTR, and potentially ENaC, in physiological processes.
Pathophysiology of the trigeminal neuralgia-associated TRPM7 A931T mutation.  A Location of the A931T mutation in the cryo-EM structure of TRPM7 (PDB: 5ZX5). Alanine 931 is located in the S3 transmembrane domain of the channel at the interface with the S4 voltage sensor. B While TRPM7 is essentially permeable to Ca2+ and Mg2+ through the canonical pore, the A931T produces an additional non-canonical omega pore causing a passive influx of Na2+ under physiological conditions. C An A931T-mediated Na+ leak current causes the depolarization of the resting membrane potential of trigeminal ganglion (TG) neurons and an increased evoked excitability possibly contributing to the sensitization of the trigeminal nerve.
Trigeminal neuralgia (TN), traditionally referred as tic douloureux, is a rare (incidence of about 4 per 100,000 cases per year) form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While the etiology of TN is not fully understood, most cases occur in a sporadic manner and are associated with intracranial vascular compression of the trigeminal nerve root that is often assumed to be the pain-initiating mechanism. However, although compression of the trigeminal root appears to be common in the aging population, only few individuals develop TN. Conversely, many TN patients do no show compression of the trigeminal nerve. TN can also occur in a familial manner suggesting the existence of predisposing genetic factors]. Whole genome sequencing studies have indeed revealed numerous genetic variants especially in genes encoding ion channels. However, the pathological relevance of ion channel variants in the etiology of TN remains largely unknown. In a recent study published in the Proceedings of the National Academy of Sciences, Gualdani and colleagues report on a new heterozygous missense mutation c.2791G > A in TRPM7 identified in a 73-year-old man presenting with familial TN.
Respiration modulates local gamma oscillations. A Respiration (Resp) recording and ECoG signals from the olfactory bulb (OB) and frontal cortex (FC) in quiet awake animals. B Respiration-entrained low-frequency oscillations in OB and FC of representative animals. Top: Power spectrum for OB (purple) and FC (black). Bottom: Coherence spectrum between Resp and OB (purple) and between Resp and FC (black). C Mean phase-amplitude comodulograms for each species (top OB; bottom: FC). White traces show the average respiratory frequency for each species. N = 6 mice, 8 rats and 3 cats. D Normalized modulation index values for the ECoG respiratory rhythm phase and the amplitude-modulated frequencies. Mean ± SEM. Top: OB. Bottom: FC. For each animal, the normalization consisted of dividing the modulation index values by the maximum value
Method for measuring respiratory modulation of long-range gamma synchronization. Example recording taken from an awake mouse and analyzed for modulation of fast gamma. A Respiration (Resp) recording and fast-gamma signals (100–120 Hz) from the olfactory bulb (OB) and frontal cortex (FC). B Top: Resp phase (black); the band-pass filtered Resp signal is also shown in arbitrary scale (gray). Middle: OB and FC fast-gamma phases. Bottom: fast-gamma phase difference Δϕγ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left({\Delta\upphi }_{\upgamma }\right)$$\end{document} between OB and FC. The gray box highlights a period where the OB-FC phase difference remains roughly constant (near 0°) close to the peak of the respiratory signal. C Polar histograms of OB-FC Δϕγ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\Delta\upphi }_{\upgamma }$$\end{document} distributions. Each plot was obtained using a different phase interval of the respiratory cycle. Gray (left), all respiration phases; red (middle), close to the respiration peak; blue (right), respiration trough. Notice a greater concentration of Δϕγ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\Delta\upphi }_{\upgamma }$$\end{document} close to the respiration peak, and that the phase-locking value (PLV, informed in the title of the panels) computed from all respiration phases (0.32) lies between the maximal PLV near the respiration peak (0.46) and the minimal value near the trough (0.24). D Fast-gamma PLV as a function of the respiration phase. The colored bars correspond to the respective polar histograms in (C). Notice a variation of PLVs within the respiration cycle, which is captured by applying the modulation index, the final step of the method
Respiration modulates long-range gamma synchrony in mice, rats, and cats during quiet wakefulness. A Top traces: examples of raw (black) and filtered (blue) respiration (Resp) and gamma-filtered signals from the olfactory bulb (OB) and frontal cortex (FC) in quiet awake animals. Middle: polar histograms depicting OB-FC gamma phase difference across all Resp phases (gray), and when only employing Resp phases around the peak (red) or the trough (blue). Bottom: bar plots showing gamma OB-FC PLVs obtained for the different phases of the respiratory cycle (schematically represented by the white lines; 0° corresponds to inspiration). The respective modulation indexes (MI) are shown on top of each subplot. B Group data of normalized PLV distributions as a function of the respiration phase (mean ± SEM). The analyzed gamma frequency varied across species: fast gamma in mice (N = 6), middle gamma in rats (N = 8), and slow gamma in cats (N = 3). C Mean OB-FC PLV comodulograms. White traces show the average respiratory frequency. In mice, the respiration signal was estimated from plethysmograph recordings; in rats, respiration was estimated from OB recordings; in cats (head-fixed), respiration was estimated from chest movements
Respiration provides a fixed-latency time window for long-distance synchronization of gamma oscillations. A Example respiration recordings in a mouse during the states of quiet (QW, blue) and active (AW, orange) wakefulness. The superimposed black diagonal traces show the respiratory phase, from 0° to 360°. B Respiration-triggered OB-FC fast-gamma PLV during both awake states in mice (mean ± SEM). For each cycle, the timestamp of the maximal slope of the respiratory signal was used as a trigger. The top dashed lines depict the respiratory cycle length in each state. The normalization consisted of dividing each PLV value by the sum of all PLVs across time. C Normalized PLV as a function of the respiratory phase (mean ± SEM). The normalization consisted of dividing each PLV value by the sum of all PLVs across all phases
Respiratory modulation of gamma synchrony decreases during sleep. A Mean modulation index (MI) values (± SEM) computed for the coupling of respiration phase and gamma amplitude in the olfactory bulb (OB, top) and frontal cortex (FC, bottom) of each species. B Mean MI (± SEM) for the coupling of respiration phase and gamma synchrony. Notice a decrease in gamma synchrony modulation during sleep. For these plots, the analyzed respiration and gamma frequency varied depending on the species to match the coupled frequency ranges observed in the comodulograms (c.f. Figure 3C). Also, note that a logarithmic y-scale was employed to better depict the data range. Wake, quiet wake state; REM, rapid eye movement sleep. NREM, non-REM sleep. #p < 0.10, *p < 0.05, **p < 0.01, paired t-tests against wake. N mice = 6, rats = 8, cats = 3
Nasal respiration influences brain dynamics by phase-entraining neural oscillations at the same frequency as the breathing rate and by phase-modulating the activity of faster gamma rhythms. Despite being widely reported, we still do not understand the functional roles of respiration-entrained oscillations. A common hypothesis is that these rhythms aid long-range communication and provide a privileged window for synchronization. Here we tested this hypothesis by analyzing electrocor-ticographic (ECoG) recordings in mice, rats, and cats during the different sleep-wake states. We found that the respiration phase modulates the amplitude of cortical gamma oscillations in the three species, although the modulated gamma frequency bands differed with faster oscillations (90-130 Hz) in mice, intermediate frequencies (60-100 Hz) in rats, and slower activity (30-60 Hz) in cats. In addition, our results also show that respiration modulates olfactory bulb-frontal cortex synchronization in the gamma range, in which each breathing cycle evokes (following a delay) a transient time window of increased gamma synchrony. Long-range gamma synchrony modulation occurs during quiet and active wake states but decreases during sleep. Thus, our results suggest that respiration-entrained brain rhythms orchestrate communication in awake mammals.
The protein-bound uremic toxin indoxyl sulfate has negative effects on a variety of physiological activities including vascular function. Uridine adenosine tetraphosphate (Up4A), a new dinucleotide molecule affects vascular function including induction of vasocontraction, and aberrant responsiveness to Up4A is evident in arteries from disorders such as hypertension and diabetes. The link between indoxyl sulfate and the Up4A-mediated response is, however, unknown. We used Wistar rat’s renal arteries to see if indoxyl sulfate will affect Up4A-mediated vascular contraction. In renal arteries of indoxyl sulfate, the contractile response generated by Up4A was dramatically reduced compared to the non-treated control group. Indoxyl sulfate increased endothelin-1-induced contraction but had no effect on phenylephrine, thromboxane analog, or isotonic K⁺-induced renal arterial contractions. UTP, ATP, UDP, and ADP-produced contractions were reduced by indoxyl sulfate. CH223191, an aryl hydrocarbon receptor (AhR) antagonist, did not reverse Up4A, and UTP contraction decreases caused by indoxyl sulfate. The ectonucleotidase inhibitor ARL67156 prevents indoxyl sulfate from reducing Up4A- and UTP-mediated contractions. In conclusion, we discovered for the first time that indoxyl sulfate inhibits Up4A-mediated contraction in the renal artery, possibly through activating ectonucleotidase but not AhR. Indoxyl sulfate is thought to play a function in the pathophysiology of purinergic signaling.
Solute carriers (SLC) are important membrane transport proteins in normal and pathophysiological cells. The aim was to identify amino acid SLC(s) responsible for uptake of sarcosine and glycine in prostate cancer cells and investigate the impact hereon of hyperosmotic stress. Uptake of ¹⁴C-sarcosine and ³H-glycine was measured in human prostate cancer (PC-3) cells cultured under isosmotic (300 mOsm/kg) and hyperosmotic (500 mOsm/kg) conditions for 24 h. Hyperosmotic culture medium was obtained by supplementing the medium with 200 mM of the trisaccharide raffinose. Amino acid SLC expression was studied using RT-PCR, real-time PCR, and western blotting. siRNA knockdown of SNAT2 was performed. Experiments were conducted in at least 3 independent cell passages. The uptake of Sar and Gly was increased approximately 8–ninefold in PC-3 cells after 24 h hyperosmotic culture. PAT1 mRNA and protein could not be detected, while SNAT2 was upregulated at the mRNA and protein level. Transfection with SNAT2-specific siRNA reduced Vmax of Sar uptake from 2653 ± 38 to 513 ± 38 nmol mg protein⁻¹ min⁻¹, without altering the Km value (3.19 ± 0.13 vs. 3.42 ± 0.71 mM), indicating that SNAT2 is responsible for at least 80% of Sar uptake in hyperosmotic cultured PC-3 cells. SNAT2 is upregulated in hyperosmotic stressed prostate cancer cells and SNAT2 is responsible for cellular sarcosine and glycine uptake in hyperosmotic cultured PC-3 cells. Sar is identified as a substrate for SNAT2, and this has physiological implications for understanding cellular solute transport in prostate cancer cells.
Mechanism of genome editing technologies (ZFN and TALEN)
Overview of CRISPR-Cas9 mechanism
Generation of gene-edited cell lines using CRISPR-Cas9
Patient-specific iPSCs for disease modeling. Regularly interspersed short palindrome (CRISPR) genome editing is used to correct mutations
Double-strand DNA breaks repair models. The cell uses non-homologous end-joining (NHEJ) or homology-directed repair (HDR) to repair double-strand DNA breaks. NHEJ is active throughout the cell cycle, whereas HDR is active in the S and G2 phases; as a result, NHEJ repair occurs more frequently than HDR. NHEJ is error-prone, resulting in insertions or deletions (indels) at the break site. HDR requires a homologous repair template; if the repair template contains a mutation, the mutation will be inserted into the genome
Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. However, the lack of human cardiomyocytes with proper genetic backgrounds limits the study of disease mechanisms. Human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) have significantly advanced the study of these conditions. Moreover, hPSC-CMs made it easy to study CVDs using genome-editing techniques. This article discusses the applications of these techniques in hPSC for studying CVDs. Recently, several genome-editing systems have been used to modify hPSCs, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9). We focused on the recent advancement of genome editing in hPSCs, which dramatically improved the efficiency of the cell-based mechanism study and therapy for cardiac diseases. Graphical abstract
KCa3.1 channels are expressed in mitochondria isolated from NSCLC cells. (a) KCa3.1 protein expression in isolated mitochondria (Mito) and whole cell lysates (WL) of A549, H1299, and H1975 cells. Mitochondrial isolation was confirmed by detecting the mitochondrial marker protein BAK. An antibody against α-tubulin was used as a positive loading control for whole cell lysates (N = 3). (b) Summary of the densitometric analyses from mitochondrial lysates (normalized to BAK) and (c) from whole cell lysates (normalized to α-tubulin)
Co-localization of KCa3.1 channels and mitochondria in NSCLC cells. (a) Immunofluorescence image of the lung cancer cell line H1975, co-stained for mitochondria (red) and KCa3.1 channels (green). The square represents the enlarged section shown in (b). (c) Intensity profile along the white line shown in (b). A KCa3.1 channel (green dot) colocalizes with a mitochondrium (red). (d) KCa3.1 channel density of 5 regions per cell. Mean values of total numbers from at least 12 different cells are displayed. The statistical analysis was performed using two-way ANOVA, followed by Bonferroni’s multiple comparisons test. (e) Percentage of the total number of KCa3.1 channels localized in mitochondria in different NSCLC cell lines. We analyzed at least 12 different cells per cell line. For the statistical analysis, a two-way ANOVA was performed, followed by Bonferroni’s multiple comparisons tests
The KCa3.1 channel blocker senicapoc hyperpolarizes the mitochondrial membrane potential of NSCLC cells. (a, b) Representative images of NSCLC cells stained with TMRM at different time points in the absence (control) and in the presence of senicapoc (scale bar: 50 µm). (c, d) Mitochondrial membrane potential measurements with A549 and H1299 cells. Left figures, senicapoc (30 µM), its solvent DMSO (1:1000 control) or maurotoxin (MTX; 20 nM) were added at t = 0 h. At the end of the experiments (t = 30 min), 1 µM FCCP was added. Data were normalized to fluorescence intensity at t =  − 10 min, and the curves represent the mean data from three different experiments (N = 3). Right figures, mitochondrial membrane potential determined at t = 25 min. For the statistical analysis, a two-way ANOVA of the mean data was performed, followed by Bonferroni’s multiple comparisons test. A p < 0.05 was defined as a statistically significant
The KCa3.1 channel blockers TRAM-34 or senicapoc increase the production of superoxides (O2⁻) in NSCLC cells. (a) Representative images of A549 cells stained with MitoSOX™ Red and acquired at t = 6 min and t = 24 min (scale bar: 20 µm). (b) Generation of O2⁻ in A549 cells treated with the KCa3.1 channel blockers senicapoc (30 µM), TRAM-34 (10 µM), or the complex III inhibitor Antimycin A (2 µM) was recorded for a time period of 30 min. NSCLC cells were loaded with the mitochondrial superoxide indicator MitoSOX™ Red (10 µM) for 10 min. Antimycin A was used as a positive control for the generation of O2⁻. (c) Analysis of the endpoints (after 30 min) from each curve shown in Fig. 4b. Statistical analysis by using two-way ANOVA followed by Dunnett’s multiple comparisons test reveals a significant difference (*p < 0.05) between control conditions and the treatment with the KCa3.1 channel blockers senicapoc or TRAM-34 (N = 4). (d) Generation of superoxide in H1299 cells exposed to KCa3.1 channel blockers. (e) Analysis of the endpoints (after 30 min) from each curve shown in Fig. 4d. TRAM-34 induces the generation of O2.⁻ in H1299 cells (N = 4; two-way ANOVA, followed by Dunnett’s multiple comparisons tests; *p < 0.05)
Lung cancer is one of the leading causes of cancer-related deaths worldwide. The Ca²⁺-activated K⁺ channel KCa3.1 contributes to the progression of non-small cell lung cancer (NSCLC). Recently, KCa3.1 channels were found in the inner membrane of mitochondria in different cancer cells. Mitochondria are the main sources for the generation of reactive oxygen species (ROS) that affect the progression of cancer cells. Here, we combined Western blotting, immunofluorescence, and fluorescent live-cell imaging to investigate the expression and function of KCa3.1 channels in the mitochondria of NSCLC cells. Western blotting revealed KCa3.1 expression in mitochondrial lysates from different NSCLC cells. Using immunofluorescence, we demonstrate a co-localization of KCa3.1 channels with mitochondria of NSCLC cells. Measurements of the mitochondrial membrane potential with TMRM reveal a hyperpolarization following the inhibition of KCa3.1 channels with the cell-permeable blocker senicapoc. This is not the case when cells are treated with the cell-impermeable peptidic toxin maurotoxin. The hyperpolarization of the mitochondrial membrane potential is accompanied by an increased generation of ROS in NSCLC cells. Collectively, our results provide firm evidence for the functional expression of KCa3.1 channels in the inner membrane of mitochondria of NSCLC cells.
Feedback loops stiff tissue/metabolic syndrome. Persistent mechanosensitive cation channel (MSCC; paradigmatically represented by ENaC) activation (external mainly by “western diet,” sedentary lifestyle, high sodium intake) leads to endothelial cell stiffening by stimulation of F-actin polymerization and contractility. Cytoskeletal stiffness is further enhanced by junctional proteins like JACD and RhoA coupled phospholipase C coupled G-protein (Gαq) (e.g., angiotensin II) receptors and integrins. Elevated cytoskeletal stiffness reduces NO and induces nuclear translocation of dephosphorylated YAP/TAZ and activation of their target genes. Their activity culminates in the pathophysiological picture of activated/dysfunctional (stiff) endothelium with rigid ECM. High ECM stiffness in turn activates via integrins/RhoA further cytoskeletal rigor, thus closing a positive feedback loop. Stiff endothelium-induced and maintained by enhanced activity of ENaC and YAP/TAZ leads via inflammatory and fibrosing mediators to stiff vessel phenotype (characterized by elevated ENaC and YAP/TAZ action in multiple cell types). Reciprocal mechano-metabolic coupling of vessels and organs/tissues induces stiff organ/tissue phenotype, again showing elevated ENaC and YAP/TAZ effects. In the wake of organ/tissue stiffening, the components of metabolic syndrome emerge, closing by further endothelial stiffening another positive feedback loop
Established risk factors for the metabolic syndrome as diabetes and arterial hypertension are believed to be the cause of arteriosclerosis and subsequently following diseases like coronary heart disease, apoplexy, or chronic renal failure. Based on broad evidence from the already available experimental literature and clinical experience, an alternative hypothesis is presented that puts an increased vessel and organ stiffness to the beginning of the pathophysiological scenario. The stiffness itself is caused by a persistent activation of mechano-sensitive cation channels like the epithelial/endothelial sodium channel. A further enhancement takes place by proteins like JACD and RhoA coupled phospholipase C coupled G-protein receptors and integrins. A self-enhancing positive feedback loop by activation of YAP/TAZ signaling is a further central pillar of this theory. Further investigations are necessary to verify this hypothesis. If this hypothesis could be confirmed fundamental changes regarding the pharmacologic therapy of the diseases that are currently summarizes as metabolic syndrome would be the consequence.
Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and β-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (β1 + β2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (β1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (β2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating β1- and α1-ARs in both human and rabbit, with a β2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.
Molecular characterization of monocyte-derived MΦs. Representative bright-field images of (a) healthy-derived and (b) patient-derived MΦ cultures. Scale bars: 50 µm. Representative scanning electron microscopy images of (c) round- and (d) spindle-shaped MΦs derived from healthy-derived cultures. Scale bars: 10 µm. Flow cytometry analysis of CD11B-FITC immunolabeled MΦ cultures derived from (e) healthy individuals and (f) patients. The values were given as an average of n = 3 for each condition. Gene expression analysis of (g)CD68, (h)CD11B, (i)CD206, (j)IL-10 in control and patient-derived CD14⁺ monocytes and MΦs (n = 4). Confocal fluorescence images of D14 (k) healthy-derived control and (l) patient-derived MΦ cultures immunostained with pan-MΦ marker CD68 and M2 marker CD206. DAPI was used for nuclear staining. Scale bars: 50 µm. Gene expression analysis of (m)IL-1B and (n)CD86 in control and patient-derived CD14⁺ monocytes and MΦs (n = 4). The expression of CD14⁺ monocytes and MΦs were calculated relative to the expression of CD14⁻ population, which was arbitrarily set to 1. Exp: Expression, FITC: Fluorescein isothiocyanate, FSC: Forward scatter, MΦ: Macrophage, Mono: MACS-sorted CD14⁺ Monocytes, n.s.: non-significant. Data were shown as mean ± STD. The p values were categorized as: *p < 0.05, **p < 0.01, ***p < 0.001. The Mann–Whitney U test was performed for non-parametric distributions
The detailed morphological and immunocytochemical characterization of iPSC-CM and MΦ cocultures. (a) The cartoon depicting the generation of coculture groups by combining control- (C) and patient- (P) derived MΦs and iPSC-CMs. (b) Representative bright-field images of Group #1 (left) and Group #4 (right) MΦ and iPSC-CM cocultures. Scale bars: 50 µm. (c) Representative scanning electron microscopy images of Group #1 cocultures demonstrating MΦ and iPSC-CM interaction. The inlet showed 10 times the magnified image from the original image. Scale bar: 10 µm. (d) Imaging of DIC and WGA-AF647 (red) labeled live MΦ and iPSC-CM coculture. (e–f) Confocal fluorescence images of MΦ and iPSC-CM cocultures immunolabeled with WGA-AF647 (red), anti-CTNI (green) and anti-CD68 (orange) antibodies. The inlets showed 6.3 times and 2.5 times the magnified images from the original images in e or f, respectively. Scale bars: 25 µm. (g-h) Representative confocal images of MΦ and iPSC-CM cocultures immunolabeled with WGA-AF647 (red), anti-CTNT (orange), and anti-CX43 (green) antibodies. Representative images were selected from 20 different areas of 2 independent cultures. Scale bar: 50 µm. DAPI (blue) was used to mark the nucleus. CM: iPSC-CM, CTNI: Cardiac troponin-I, CTNT: Cardiac troponin-T, CX43: Connexin 43, MΦ: Macrophage, WGA: Wheat germ agglutinin
Contraction analysis of cocultures of iPSC-CM and MΦs derived from healthy individuals or arrhythmia patients. Representative 15-s contraction and relaxation cycle graphics of (a) C-CM and (b) P-CM that were analyzed by MUSCLEMOTION plugin within ImageJ. (c) Representative mean fluorescence intensity (MFI) comparison of 10-s intracellular Ca²⁺ ion exchange in C-CM and P-CM cultures. (d) Scatter dot plots of average contraction rates, as BPM, of C-CM and P-CM, showing tachycardia phenotype of P-CM cultures. The average BPM rates of C-CMs and P-CMs were calculated from the values of single iPSC-CM cultures. The experiments were independently repeated three times for each sample of both C-CM (green, cyan, dark blue colors represent data collected from each independent replicate) and P-CM (magenta, purple, red colors represent data collected from each independent replicate) on the same differentiation days of the culture. Circles represent C-CM (Control-1) and squares represent P-CM (Patient-1). The Mann–Whitney U test was performed for non-parametric distributions. (e) Scatter dot plots of average BPM values of single iPSC-CM cultures and the coculture groups. Group #1 (Brown circles: Control-1 C-CM + Control-1 C-MΦ). Group #2 (Yellow circles: Control-1 C-CM + Patient-2 P-MΦ; Orange circles: Control-1 C-CM + Patient-3 P-MΦ). Group #3 (Pink squares: Patient-1 P-CM + Control-1 C-MΦ). Group #4 (Blue squares: Patient-1 P-CM + Patient-2 P-MΦ; White squares: Patient-1 P-CM + Patient-3 P-MΦ). The Mann–Whitney U test was performed for the comparison of monoculture and coculture BPM values with a non-parametric distribution. Scatter dot plots of average BPM values of subgroups in (f) Group #1, (g) Group #2, (h) Group #3, and (i) Group #4. The Kruskal–Wallis test was performed for multiple comparisons between the coculture groups. All coculture experiments were independently repeated three times for each sample of both single and coculture groups on the same differentiation days of the culture. a.u.: arbitrary unit. BPM: Beats per minute. C-CM: Control-derived iPSC-Cardiomyocytes. P-CM: Patient-derived iPSC-Cardiomyocytes. C-MΦ: Control-derived macrophages. P-MΦ: Patient-derived macrophages. MFI: Mean fluorescence intensity. n.s.: non-significant. Data were shown as mean ± STD. The p values were categorized as: *p < 0.05, **p < 0.01, ***p < 0.001
Intracellular Ca²⁺ imaging of the MΦ and co-cultures. Representative brightfield and fluorescence imaging of intracellular Ca²⁺ sensitive dye Fluo-4-AM in single (a) C-MΦ or (b) P-MΦ cultures. (c) Average of 15 s intracellular Ca²⁺ signal values detected in C-MΦ and P-MΦ cultures. Representative brightfield and fluorescence microscopy imagings of CM-MΦ membrane connections and MFI of intracellular Ca²⁺ exchange analysis of (d) Group #1, (e) Group #2, (f) Group #3, and (g) Group #4. Experiments were repeated n = 3 for all single and co-culture groups. MFI values were calculated using ROI analysis in ImageJ software from intracellular Ca²⁺ imaging movies recorded for 15 s from at least 20 random contracting areas of three independent wells of each group. C: Control. Ca²⁺: Calcium. CM: Cardiomyocyte. MΦ: Macrophage. MFI: Mean fluorescence intensity. P: Patient. s: second
Rhythmicity analysis of iPSC-CMs in cocultures with MΦ from healthy or SCN5A mutation bearing background. Representative PPI (peak-to-peak interval) profile from (a) C-CM monocultures, (b) P-CM monocultures, (c) Group #1, (d) Group #2, (e) Group #3, and (f) Group #4. (g) Scatter dot plots of average percentages of rhythmicity of single iPSC-CM cultures and the coculture groups. PPI was calculated between each peak of the contraction in all single and cocultures (n = 3). Circles represented healthy-derived iPSC-CMs and squares represented patient-derived iPSC-CMs. C: Control. CM: Cardiomyocyte. P: Patient. s: second. n.s.: non-significant. Data were shown as mean ± STD. The Mann–Whitney U test was performed for detecting the significance difference in average rhythmic values of coculture groups in comparison with single CM cultures. The p values were categorized as: *p < 0.05, **p < 0.01, ***p < 0.001
The electrophysiological regulation of cardiomyocytes (CMs) by the cardiac macrophages (MΦs) has been recently described as an unconventional role of MΦs in the murine heart. Investigating the molecular and physiological modulation of CM by MΦ is critical to understand the novel mechanisms behind cardiac disorders from the systems perspective and to develop new therapeutic approaches. Here, we developed an in vitro direct coculture system to investigate the cellular and functional interaction between human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and monocyte-derived MΦs both in healthy-state and congenital arrhythmia disease model associated with SCN5A ion channel mutations. Congenital arrhythmia patient-derived (P) and healthy individual-derived control (C) monocytes and derived MΦs exhibited distinct M1- and M2-like polarization-related gene expression pattern. The iPSC-CMs and MΦs formed direct membrane contacts in cocultures demonstrated by time-lapse imaging, scanning electron microscopy, and immunolabeling. The intracellular Ca²⁺ transients were observed in iPSC-CMs and MΦs when in contact with each other. Interestingly, the C-MΦs in direct contact with C-CMs significantly accelerated the contraction rates, demonstrating the positive chronotropic effect of MΦs on healthy cardiac cultures. Furthermore, the MΦs carrying the SCN5A gene mutation significantly enhanced the arrhythmic events in both C-CMs and P-CMs, implying that the sodium channel mutation in the MΦ is important for the CM function. Importantly, when C-MΦs were coupled to tachycardic P-CMs, the contraction frequency drastically decreased, and rhythmicity enhanced implicating the amelioration of the disease phenotype in vitro. Consequently, our results indicated the functional regulatory role of MΦs on human iPSC-CM contractility by membrane contacts in a physiologically relevant in vitro coculture model of both steady-state and arrhythmia. Our findings could serve as a valuable source for the development of effective immunoregulatory therapies for cardiac arrhythmia in the future. Graphical abstract
This Commentary highlights the excellent new findings published recently by Jochen Roeper’s group on Science Advances (Shin et al. Sci Adv 8: eabm4560, 2022) about the role that Cav1.3 L-type channels play in acting as full-range linear amplifiers of firing frequencies in lateral dopamine (DA) substantia nigra (SN) neurons. Using in vitro DA SN neurons from Cav1.2DHP−/− mice and variable doses of the L-type selective blocker isradipine (30-300 nM), the group shows that the selective block of Cav1.3 channels by isradipine amplifies by 30% the firing frequency in their full range (2-50 Hz). In addition, clinically relevant doses of isradipine (10 nM) induce the same in vitro linear amplification of firing frequencies in in vivo identified DA SN. This allows concluding that Cav1.3 channels are functional pacemaker channels in midbrain DA neurons and that repurposing isradipine in Parkinson’s disease treatment may be a feasible strategy to selectively modulate the in vivo activity of highly vulnerable DA SN subpopulations.
Leukocyte telomere regulating gene expression changes after exercise in Thoroughbred racehorses. A Thoroughbred racehorse exercise training and heart rate responses (n = 10–13). The average heart rate of the horses progressively increased with the elevated training intensities (p < 0.001). Data are from a mixed-effect model. TERT (B) and shelterin (C–H) gene expression changes immediately after and 24 h after vigorous exercise training in Thoroughbred horses (n = 17–22). Data are mean ± SD relative gene expression. *p < 0.05, **p < 0.01, ***p < 0.001
TERT and POT1 gene expression changes with ageing. Differentially expressed TERT (A) and POT1 (B) gene expression differences in young versus middle-aged Thoroughbred racehorses. Data are mean ± SD relative gene expression. *p < 0.05, **p < 0.01
Leukocyte microRNA expression after exercise in Thoroughbred racehorses. Leukocyte microRNA expression changes caused by exercise training (A–C). Data are expressed as mean ± SD relative miRNA expression. *p < 0.05, **p < 0.01, ***p < 0.001
Leukocyte miRNAs and ageing. Leukocyte miR-143, miR-233 and miR-486-5p miRNAs in young and middle-aged Thoroughbred horses (A, B and C, respectively). Data are expressed as mean ± SD relative miRNA expression. *p < 0.05
Ageing causes a gradual deterioration of bodily functions and telomere degradation. Excessive telomere shortening leads to cellular senescence and decreases tissue vitality. Six proteins, called shelterin, protect telomere integrity and control telomere length through telomerase-dependent mechanisms. Exercise training appears to maintain telomeres in certain somatic cells, although the underlying molecular mechanisms are incompletely understood. Here, we examined the influence of a single bout of vigorous exercise training on leukocyte telomerase reverse transcriptase (TERT) and shelterin gene expression, and the abundance of three microRNAs (miRNAs) implicated in biological ageing (miRNA-143,-223 and-486-5p) in an elite athlete and large animal model, Thoroughbred horses. Gene and miRNA expression were analysed using primer-based and TaqMan Assay qPCR. Leukocyte TRF1, TRF2 and POT1 expression were all significantly increased whilst miR-223 and miR-486-5p were decreased immediately after vigorous exercise (all p < 0.05), and tended to return to baseline levels 24 h after training. Relative to the young horses (~ 3.9 years old), middle-aged horses (~ 14.8 years old) exhibited reduced leukocyte TERT gene expression, and increased POT1 and miR-223 abundance (all p < 0.05). These data demonstrate that genes transcribing key components of the shelterin-telomere complex are influenced by ageing and dynamically regulated by a single bout of vigorous exercise in a large, athletic mammal-Thoroughbred horses. Our findings also implicate TERT and shelterin gene transcripts as potential targets of miR-223 and miR-486-5p, which are modulated by exercise and may have a role in the telomere maintenance and genomic stability associated with long-term aerobic training.
Urinary and plasma levels of Pi and Ca²⁺ correlate with dietary Pi content. (a) Plasma concentration of Pi, (b) urinary excretion of Pi, (c) fractional excretion of Pi, (d) plasma concentration of Ca²⁺, (e) urinary excretion of Ca²⁺, (f) fractional excretion of Ca²⁺, (g) plasma creatinine, (h) urinary excretion of creatinine and (i) urinary volume in samples from rats fed chronically (5 days) with low Pi (LL), acutely changed (12 h) from low to high Pi (LH), chronically fed with high Pi (HH) and acutely changed from high to low Pi (HL). Statistical significances were calculated with one-way ANOVA with Bonferroni’s multiple comparison test. n = 5 for each group, * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001
Plasma levels of Pi-regulating hormones differentially adapt to changes in dietary Pi. Plasma levels of (a) intact FGF-23, (b) intact PTH and (c) 1,25(OH)2 vitamin D3 in samples from rats fed chronically (5 days) with low Pi (LL), acutely changed (12 h) from low to high Pi (LH), chronically fed with high Pi (HH) and acutely changed from high to low Pi (HL). Statistical significances were calculated with one-way ANOVA with Bonferroni’s multiple comparison test. n = 5 for each group, **** P < 0.0001
The abundance of the proteolytic fragment of NaPi-IIa in UEV partially correlates with its renal expression. Western blots for NaPi-IIa in renal BBM from rats fed (a) chronically (5 days) low Pi (LL) and acutely changed (12 h) from low to high Pi (LH), (b) chronically fed high Pi (HH) and acutely changed from high to low Pi (HL), (c) chronically fed low (LL) or high Pi (HH), and (d) UEV isolated from all 4 groups. Graphs show the quantifications of the full-length and proteolytic fragment normalized to (a–c) LiCor total protein stain (suppl Fig. 1), (d) urinary creatinine and TSG101. (a’–c’) real-time PCRs on renal RNA samples from the same groups. Statistical significances were calculated by t-test (a–c and a’–c’) or with one-way ANOVA with Bonferroni’s multiple comparison test (d). n = 5 for each group, *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001. Red asterisks indicate changes in UEV similar to those described in renal BBM
The abundance of NaPi-IIc in UEV partially correlates with its renal expression. Western blots for NaPi-IIc in renal BBM from rats fed (a) chronically (5 days) low Pi (LL) and acutely changed (12 h) from low to high Pi (LH), (b) chronically fed high Pi (HH) and acutely changed from high to low Pi (HL), (c) chronically fed low (LL) or high Pi (HH), and (d) UEV isolated from all groups. Graphs show the quantifications normalized to (a–c) LiCor total protein stain (supplementary Fig. 1), (d) urinary creatinine and TSG101. (a’–c’) real time PCRs on renal RNA samples from the same groups. Statistical significances were calculated by t-test (a–c and a’–c’) or with one-way ANOVA with Bonferroni’s multiple comparison test (d). n = 5 for each group, *P < 0.05, ** P < 0.01. Asterisks indicate changes in UEV similar to those described in renal BBM
The abundance of AQP2 in UEV correlates with its renal expression. Western blots for AQP2 in renal BBM from rats fed (a) chronically (5 days) low Pi (LL) and acutely changed (12 h) from low to high Pi (LH), (b) chronically fed high Pi (HH) and acutely changed from high to low Pi (HL), (c) chronically fed low (LL) or high Pi (HH), and (d) UEV isolated from all 4 groups. Graphs show the quantifications of the unglycosylated and glycosylated channel normalized to (a–c) LiCor total protein stain (supplementary Fig. 2), (d) urinary creatinine and TSG101. Statistical significances were calculated by t-test (A–C) or with one-way ANOVA with Bonferroni’s multiple comparison test (D). n = 5 for each group, *P < 0.05
Studies addressing homeostasis of inorganic phosphate (Pi) are mostly restricted to murine models. Data provided by genetically modified mice suggest that renal Pi reabsorption is primarily mediated by the Na ⁺ /Pi cotransporter NaPi-IIa/ Slc34a1 , whereas the contribution of NaPi-IIc/ Slc34a3 in adult animals seems negligible. However, mutations in both cotransporters associate with hypophosphatemic syndromes in humans, suggesting major inter-species heterogeneity. Urinary extracellular vesicles (UEV) have been proposed as an alternative source to analyse the intrinsic expression of renal proteins in vivo. Here, we analyse in rats whether the protein abundance of renal Pi transporters in UEV correlates with their renal content. For that, we compared the abundance of NaPi-IIa and NaPi-IIc in paired samples from kidneys and UEV from rats fed acutely and chronically on diets with low or high Pi. In renal brush border membranes (BBM) NaPi-IIa was detected as two fragments corresponding to the full-length protein and to a proteolytic product, whereas NaPi-IIc migrated as a single full-length band. The expression of NaPi-IIa (both fragments) in BBM adapted to acute as well to chronic changes of dietary Pi, whereas adaptation of NaPi-IIc was only detected in response to chronic administration. Both transporters were detected in UEV as well. UEV reflected the renal adaptation of the NaPi-IIa proteolytic fragment (but not the full-length protein) upon chronic but not acute dietary changes, while also reproducing the chronic regulation of NaPi-IIc. Thus, the composition of UEV reflects only partially changes in the expression of NaPi-IIa and NaPi-IIc at the BBM triggered by dietary Pi.
Guanosine (GUO), widely considered a key signaling mediator, is implicated in the regulation of several cellular processes. While its interaction with neural membranes has been described, GUO still is an orphan neuromodulator. It has been postulated that GUO may eventually interact with potassium channels and adenosine (ADO) receptors (ARs), both particularly important for the control of cellular excitability. Accordingly, here, we investigated the effects of GUO on the bioelectric activity of human neuroblastoma SH-SY5Y cells by whole-cell patch-clamp recordings. We first explored the contribution of voltage-dependent K + channels and, besides this, the role of ARs in the regulation of GUO-dependent cellular electro-physiology. Our data support that GUO is able to specifically modulate K +-dependent outward currents over cell membranes. Importantly, administering ADO along with GUO potentiates its effects. Overall, these results suggested that K + outward membrane channels may be targeted by GUO with an implication of ADO receptors in SH-SY5Y cells, but also support the hypothesis of a functional interaction of the two ligands. The present research runs through the leitmotif of the deorphani-zation of GUO, adding insight on the interplay with adenosinergic signaling and suggesting GUO as a powerful modulator of SH-SY5Y excitability.
We studied the efficacy of a near-infrared laser (1475 nm) to activate rat dorsal root ganglion (DRG) neurons with short punctate radiant heat pulses (55 µm diameter) and investigated temporal and spatial summation properties for the transduction process for noxious heat at a subcellular level. Strength-duration curves (10–80 ms range) indicated a minimum power of 30.2mW for the induction of laser-induced calcium transients and a chronaxia of 13.9 ms. However, threshold energy increased with increasing stimulus duration suggesting substantial radial cooling of the laser spot. Increasing stimulus duration demonstrated suprathreshold intensity coding of calcium transients with less than linear gains (Stevens exponents 0.29/35mW, 0.38/60mW, 0.46/70mW). The competitive TRPV1 antagonist capsazepine blocked responses to short near-threshold stimuli and significantly reduced responses to longer duration suprathreshold heat. Heating 1/3 of the soma of a neuron was sufficient to induce calcium transients significantly above baseline ( p < 0.05), but maximum amplitude was only achieved by centering the laser over the entire neuron. Heat-induced calcium increase was highest in heated cell parts but rapidly reached unstimulated areas reminiscent of spreading depolarization and opening of voltage-gated calcium channels. Full intracellular equilibrium took about 3 s, consistent with a diffusion process. In summary, we investigated transduction mechanisms for noxious laser heat pulses in native sensory neurons at milliseconds temporal and subcellular spatial resolution and characterized strength duration properties, intensity coding, and spatial summation within single neurons. Thermal excitation of parts of a nociceptor spread via both membrane depolarization and intracellular calcium diffusion.
The mechanism for limb ischemic precondition (RLIPC)-induced suppression of reperfusion arrhythmia remains unknown. The purpose of this study was to examine the roles of the pro-survival reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways in this RLIPC-mediated antiarrhythmic activity. Male Sprague Dawley rats were assigned to sham-operated, control, or RLIPC groups. All rats except for the sham rats had 5 min of left main coronary artery occlusion with another 20 min of reperfusion. RLIPC was initiated by four cycles of limb ischemia (5 min) and reperfusion (5 min) on the bilateral femoral arteries. Hearts in every group were taken for protein phosphorylation analysis. RLIPC ameliorated reperfusion-induced arrhythmogenesis and reduced the incidence of sudden cardiac death during the entire 20-min reperfusion period (66.7% of control rats had SCD vs. only 16.7% of RLIPC-treated rats). RLIPC enhances ventricular ERK1/2 phosphorylation after reperfusion. RLIPC-induced antiarrhythmic action and ERK1/2 phosphorylation are abolished in the presence of the ERK1/2 inhibitor U0126. Limb ischemic preconditioning protects the heart against myocardial reperfusion injury-induced lethal arrhythmia. These beneficial effects may involve the activation of ERK1/2 in the RISK signaling pathway.
Polyunsaturated fatty acids (PUFAs) are used as traditional remedies to treat hair loss, but the mechanisms underlying their beneficial effects are not well understood. Here, we explored the role of PUFA metabolites generated by the cytochrome P450/soluble epoxide hydrolase (sEH) pathway in the regulation of the hair follicle cycle. Histological analysis of the skin from wild-type and sEH−/− mice revealed that sEH deletion delayed telogen to anagen transition, and the associated activation of hair follicle stem cells. Interestingly, EdU labeling during the late anagen stage revealed that hair matrix cells from sEH−/− mice proliferated at a greater rate which translated into increased hair growth. Similar effects were observed in in vitro studies using hair follicle explants, where a sEH inhibitor was also able to augment whisker growth in follicles from wild-type mice. sEH activity in the dorsal skin was not constant but altered with the cell cycle, having the most prominent effects on levels of the linoleic acid derivatives 12,13-epoxyoctadecenoic acid (12,13-EpOME), and 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Fitting with this, the sEH substrate 12,13-EpOME significantly increased hair shaft growth in isolated anagen stage hair follicles, while its diol; 12,13-DiHOME, had no effect. RNA sequencing of isolated hair matrix cells implicated altered Wnt signaling in the changes associated with sEH deletion. Taken together, our data indicate that the activity of the sEH in hair follicle changes during the hair follicle cycle and impacts on two stem cell populations, i.e., hair follicle stem cells and matrix cells to affect telogen to anagen transition and hair growth.
Growth of WD-fed mice in dependency of the PCSK9 viral load. A development of body weight within the 12-week treatment period; means ± SDs (n = 5–6), 2-way ANOVA followed by Tukey’s multiple comparisons test [time: F = 277, P < 0.0001, treatment: F = 2.9, P = 0.068, interaction (time × treatment): F = 216, P < 0.0001]; B gain in body weight (1-way ANOVA [F = 5.568, P = 0.0065) followed by Tukey’s multiple comparisons test); C mass distribution after 12 weeks (Kruskal Wallis Test (fat mass P = 0.0028; free body fluid P = 0.0906; lean mass P = 0.359) followed by Dunn’s multiple comparisons test was calculated as Gaussian distribution of the values was not given; the median is depicted in box blots; the box extends from the 25th to 75th percentiles and the whiskers go down to the smallest value and up to the largest; n = 5–6; *P < 0.05 vs 0
Plasma concentrations of cholesterol (A) and triglycerides (B) after 6 and 12 weeks in WD-fed mice in dependency of the PCSK9DY viral load. Plasma cholesterol and triglycerides were calculated using by using 2-way ANOVA considering the factors time and viral load (cholesterol F = 17.75, P < 0.0001; TG F = 11.36, P = 0.0002) followed by Sidak’s multiple comparisons test. n = 5–6; the median is depicted in box blots; the box extends from the 25th to 75th percentiles and the whiskers go down to the smallest value and up to the largest. *P < 0.05 vs 0; †P = 0.05 vs. 0.5 × 10¹¹
Histologic evaluation of aortic root of WD-fed mice in dependency of the PCSK9.DY viral load. A depicts exemplary aortic segments of each treatment group upon Oil Red O staining (the brownish colorations marked with an arrow are blood residues in the samples and are evaluated as artifacts; scale bar: 100 µm); B quantitative evaluation of plaque content; C quantitative evaluation of fat content. A Kruskal Wallis test (plaque content P = 0.0044; fat content P = 0.0017) followed by Dunn’s multiple comparisons test was calculated; correlation analyses were performed by 2-tailed Pearson test; the median is depicted in box blots; the box extends from the 25th to 75th percentiles and the whiskers go down to the smallest value and up to the largest
Cortical stiffness in WD-fed C57BL/6 N mice which were treated with AAV-PCSK9 (0, 0.5, 1 or, 5 × 10.¹¹ VG). A Stiffness values were calculated in 44–62 cells of each mouse from force-distance curves in a blinded manner by using the PUNIAS 3D version 1.0 release 1.8 (; to detect statistical differences between groups, a Kruskal Wallis test (P < 0.0001) was calculated followed by Dunn’s multiple comparisons test; B evaluation considering aortic stiffness in individual mice; statistical differences between groups were detected by 1-way ANOVA testing (F = 12.24, P = 0.0001) followed by Tukey’s multiple comparisons test; values are depicted as box plots showing 25th and 75th percentile (box) and maximum/minimum values (whiskers). The horizontal black line depicts the median in each group; C–E Correlation analysis between cortical stiffness and cholesterol, triglycerides, or plaque content. Correlations between 2 factors were calculated by Pearson
Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE−/− or LDL-R−/− mice transgenic models. A new AAV-PCSK9DYDY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9DY (0.5, 1 or 5 × 10¹¹ VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 10¹¹ VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.
Elevated levels of the intracellular second messenger cAMP can stimulate intestinal oxalate secretion however the membrane transporters responsible are unclear. Oxalate transport by the chloride/bicarbonate (Cl−/HCO3−) exchanger Slc26a6 or PAT-1 (Putative Anion Transporter 1), is regulated via cAMP when expressed in Xenopus oocytes and cultured cells but whether this translates to the native epithelia is unknown. This study investigated the regulation of oxalate transport by the mouse intestine focusing on transport at the apical membrane hypothesizing PAT-1 is the target of a cAMP-dependent signaling pathway. Adopting the Ussing chamber technique we measured unidirectional 14C-oxalate and 36Cl− flux (\({J}_{ms}^{ion}\) and \({J}_{sm}^{ion}\)) across distal ileum, cecum and distal colon, employing forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) to trigger cAMP production. FSK/IBMX initiated a robust secretory response by all segments but the stimulation of net oxalate secretion was confined to the cecum only involving activation of \({J}_{sm}^{Ox}\) and distinct from net Cl− secretion produced by inhibiting \({J}_{ms}^{Cl}\). Using the PAT-1 knockout (KO) mouse we determined cAMP-stimulated \({J}_{sm}^{Ox}\) was not directly dependent on PAT-1, but it was sensitive to mucosal DIDS (4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid), although unlikely to be another Cl−/HCO3− exchanger given the lack of trans-stimulation or cis-inhibition by luminal Cl− or HCO3−. The cAMP-activated oxalate efflux was reliant on CFTR (Cystic Fibrosis Transmembrane conductance Regulator) activity, but only in the presence of PAT-1, leading to speculation on the involvement of a multi-transporter regulatory complex. Further investigations at the cellular and molecular level are necessary to define the mechanism and transporter(s) responsible.
Parietal cortex unit modulated by fast respiration during active waking. A: Raw respiration (Resp) signal, local field potential (LFP) from the parietal cortex, accelerometer activity indicating movement (Mov), multi-unit activity (MUA) from one tetrode wire, and the time stamps of a sorted and quality tested unit (unit 3) during active waking. B: Power spectra of the parietal cortex LFP (black) and Resp (blue) during active waking. θ and Resp are partly overlapping, yet with different frequencies of maximal power (Resp > θ). C: Averaged unit waveform (black) on the background of superimposed 353 single spikes (gray). D: Phase histogram of unit 3 firing probability based on Resp cycles (frequency range 4–14 Hz; “fast respiration”) showing significant modulation of unit discharges. R indicates coupling strength. E: Polar plot showing Resp phase-dependent activation of unit 3. The firing probability is maximal between 90 (maximum of expiration, Ex) and 270 degrees (maximum of inspiration, In). F: Phase histogram of unit 3 based on θ cycles; the unit was not significantly modulated by θ (5–12 Hz)
Modulation of parietal cortex units by θ and respiration in sleep and wakefulness. A: Mean power of slow rhythms (Resp, blue; θ, black) during REM sleep, NREM sleep, waking immobility (WI) and active waking (AW). Note absence of θ activity in NREM sleep and WI. B: Proportion of units modulated by θ (black), slow Resp (blue), fast Resp (magenta) or θ and Resp (slow and fast) simultaneously (“Both,” green). Orange indicates non-modulated units. Note that θ-modulation dominates in REM sleep whereas modulation by fast Resp prevails in AW. C: Relation between firing rate and unit coupling to θ or Resp. Coupling to θ increases significantly with firing rate in REM but not in AW. Unit modulation by Resp increases significantly with firing rate in REM, NREM and AW but does not change in WI (see Table 2 for details). D: Coupling strength of units to θ (black dots and errors: 25% and 75% percentiles) and respiration (blue: slow Resp, magenta: fast Resp). Differences in coupling strength for the respective states are indicated by brackets and p values
State-dependent phase preference of parietal cortex. A: Units modulated by θ have significantly different θ phase preferences in REM (169 deg, after peak) compared to AW (46 deg, before peak). B: Units coupled to slow Resp are entrained to different phases of respiration in REM compared to NREM and in NREM compared to WI; units in NREM preferentially fire during the rising flank of inspiration (In, 180 to 270 deg), whereas units in WI fire mostly during expiration (Ex, 0 to 180 deg). In AW, units modulated by fast Resp (fR) preferentially fire during the rising flank of In and the falling flank of Ex. p values are based on the Watson U2 test
State- and depth-dependent interference of Resp and θ on spike phase preference. A: θ-only modulated units have a significantly different θ phase preference than units co-modulated by Resp during AW. B: θ-only modulated units have a significantly different θ phase preference than units co-modulated by Resp in deep layers (0.4–0.8 mm depth) during REM. C: Fast Resp-only modulated units have a significantly different Resp phase preference than units co-modulated by θ in superficial layers (< 0.4 mm depth) during AW. p values are based on the Watson U2 test
Synchronous oscillations are essential for coordinated activity in neuronal networks and, hence, for behavior and cognition. While most network oscillations are generated within the central nervous system, recent evidence shows that rhythmic body processes strongly influence activity patterns throughout the brain. A major factor is respiration (Resp), which entrains multiple brain regions at the mesoscopic (local field potential) and single-cell levels. However, it is largely unknown how such Resp-driven rhythms interact or compete with internal brain oscillations, especially those with similar frequency domains. In mice, Resp and theta (θ) oscillations have overlapping frequencies and co-occur in various brain regions. Here, we investigated the effects of Resp and θ on neuronal discharges in the mouse parietal cortex during four behavioral states which either show prominent θ (REM sleep and active waking (AW)) or lack significant θ (NREM sleep and waking immobility (WI)). We report a pronounced state-dependence of spike modulation by both rhythms. During REM sleep, θ effects on unit discharges dominate, while during AW, Resp has a larger influence, despite the concomitant presence of θ oscillations. In most states, unit modulation by θ or Resp increases with mean firing rate. The preferred timing of Resp-entrained discharges (inspiration versus expiration) varies between states, indicating state-specific and different underlying mechanisms. Our findings show that neurons in an associative cortex area are differentially and state-dependently modulated by two fundamentally different processes: brain-endogenous θ oscillations and rhythmic somatic feedback signals from Resp.
Obesity is linked to reproductive disorders. Novel neuropeptide phoenixin demonstrated many therapeutic actions. In this study, we aim to evaluate phoenixin's potential effect in obesity-induced infertility through modulating mitochondrial dynamics. Ninety adult female rats were divided to 4 groups: (I), fed with normal pellet diet; (II), given phoenixin; (III), fed with high-fat diet. Rats that developed obesity and infertility were divided to 2 groups: (III-A), received no further treatment; (III-B), given phoenixin. Our results showed that phoenixin treatment in obese infertile rats significantly decreased serum levels of insulin and testosterone and ovarian levels of dynamin-related protein1(Drp1),reactive oxygen species ROS, TNF-α, MDA, and caspase-3. Phoenixin treatment also significantly increased serum estrogen progesterone, LH, and FSH together with ovarian levels of GnRH receptor (GnRHR), mitofusin2(Mfn2), mitochondrial transmembrane potential (ΔΨm), and electron transport chain (ETC) complex-I significantly when compared with obese group. Ovarian histopathological changes were similarly improved by phoenixin. Our data demonstrate phoenixin's role in improving obesity-induced infertility.
Specific passive tension of rat soleus muscle after 7-day HS (a). Specific passive tension of rat soleus muscle after 14-day HS (b). Data shown as % of the C group (mean ± SD), n = 8 per group. *Significant difference from the C group, p < 0.05; $significant difference from the 7HS group (p < 0.05); #significant difference of blebbistatin-treated muscles from blebbistatin-untreated muscles, p < 0.05. C, control group; HS, hindlimb suspension group; HS + PD, hindlimb suspension + treatment with calpain inhibitor (PD150606). Circles represent individual data points
Quantification of desmin, α-actinin-2, α-actinin-3, and telethonin in rat soleus after 7-day HS (a). Quantification of desmin, α-actinin-2, α-actinin-3, and telethonin in rat soleus after 14-day HS (b). Representative immunoblots for the studied proteins in the 7-day experiment (c). Representative immunoblots for the studied proteins in the 14-day experiment (d). Representative Ponceau-stained membranes (e). Data shown as % of the C group (mean ± SD), n = 8 per group. *Significant difference from the C group, p < 0.05; $significant differences from the 7HS group (p < 0.05); T, downward trend compared to the C group, p < 0.07. C, control group; HS, hindlimb suspension group; HS + PD, hindlimb suspension + treatment with calpain inhibitor (PD150606). Circles represent individual data points
Quantification of intact titin (T1), proteolytic fragment of titin (T2), and nebulin in rat soleus muscle after 7-day HS (a). Quantification of intact titin (T1), proteolytic fragment of titin (T2), and nebulin in rat soleus after 14-day HS (b). Representative images for the studied proteins in the 7-day experiment (c). Representative images for the studied proteins in the 14-day experiment (d). Data shown as % of the C group (mean ± SD), n = 8 per group. *Significant difference from the C group, p < 0.05; $significant difference from the 7HS group (p < 0.05). C, control group; HS, hindlimb suspension group; HS + PD, hindlimb suspension + treatment with calpain inhibitor (PD150606). Circles represent individual data points
Collagen Ia, IIIa, IV, and VIa2 mRNA content after 7-day HS (a). Collagen Ia, IIIa, IV, and VIa2 mRNA content after 14-day HS (b). Fibronectin, ubiquitin, and LC3B mRNA content after 7-day HS (c). Fibronectin, ubiquitin, and LC3B mRNA content after 14-day HS (d). Data are shown as % of the C group (mean ± SD), n = 8 per group. *Significant difference from the C group, p < 0.05; $significant difference from the 7HS group (p < 0.05). C, control group; HS, hindlimb suspension group; HS + PD, hindlimb suspension + treatment with calpain inhibitor (PD150606). Circles represent individual data points
In mammals, prolonged mechanical unloading results in a significant decrease in passive stiffness of postural muscles. The nature of this phenomenon remains unclear. The aim of the present study was to investigate possible causes for a reduction in rat soleus passive stiffness after 7 and 14 days of unloading (hindlimb suspension, HS). We hypothesized that HS-induced decrease in passive stiffness would be associated with calpain-dependent degradation of cytoskeletal proteins or a decrease in actomyosin interaction. Wistar rats were subjected to HS for 7 and 14 days with or without PD150606 (calpain inhibitor) treatment. Soleus muscles were subjected to biochemical analysis and ex vivo measurements of passive tension with or without blebbistatin treatment (an inhibitor of actomyosin interactions). Passive tension of isolated soleus muscle was significantly reduced after 7- and 14-day HS compared to the control values. PD150606 treatment during 7- and 14-day HS induced an increase in alpha-actinin-2 and -3, desmin contents compared to control, partly prevented a decrease in intact titin (T1) content, and prevented a decrease in soleus passive tension. Incubation of soleus muscle with blebbistatin did not affect HS-induced reductions in specific passive tension in soleus muscle. Our study suggests that calpain-dependent breakdown of cytoskeletal proteins, but not a change in actomyosin interaction, significantly contributes to unloading-induced reductions in intrinsic passive stiffness of rat soleus muscle.
Segment-specific sodium transport mechanisms in the distal nephron. a Schematic representation of a single nephron highlighting different segments of the distal nephron, i.e., the distal convoluted tubule with its early (DCT1) and late (DCT2) portion, the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct (OMCD). b Tubule epithelial cell models illustrating segment-specific apical sodium uptake mechanisms. Basolateral sodium extrusion in exchange for potassium (3Na⁺/2 K⁺) is accomplished by the basolateral Na⁺-K⁺-ATPase in all cell types. A defining feature of both DCT1 and DCT2 is the apical Na⁺-Cl⁻ cotransporter (NCC); DCT2, but not DCT1, also expresses the epithelial sodium channel (ENaC). ENaC is the sole apical sodium uptake mechanism in CNT and CCD principal cells. In addition to playing a decisive role in fine tuning renal sodium absorption, ENaC also generates the electrical driving force necessary for K⁺ secretion meditated primarily by the apical renal outer medullary K⁺ channel (ROMK). In the late CNT and entire CCD (CNT/CCD), aldosterone (A) is the key hormonal activator of ENaC through the mineralocorticoid receptor (MR) which is protected from glucocorticoid action by 11ß-hydroxysteroid dehydrogenase type 2 (11βHSD2). In the DCT2 and early CNT (DCT2/CNT), MR appears to have constitutive activity, possibly due to low levels of 11βHSD2, allowing glucocorticoids (G) to activate the receptor. This provides a potential explanation for the aldosterone-independent but MR-dependent ENaC activity in the latter region, which is probably important for Na⁺ homeostasis and blood pressure control, as well as aldosterone-independent K⁺ secretion
Coordinated regulation of ENaC and NCC by interstitial potassium. The effects of increased interstitial K⁺ on Na⁺ transport are shown for a DCT1 cell (top) and CNT/CCD cell (bottom). Baseline membrane potential is controlled primarily by Kir4.1/5.1. Increased interstitial K⁺ concentration ([K⁺]↑) depolarizes the basolateral membrane potential (Vbl↓), thus altering the electrochemical gradient for Cl⁻ across the basolateral membrane equipped with Cl⁻ channels (in particular ClC-K2 in DCT1), and eventually causes an increase in intracellular Cl⁻ concentration ([Cl⁻]↑) in both the DCT1 and CCD. Chloride can then bind to WNK1/4, which inhibits its kinase activity and prevents NCC activation in the DCT1. In the CCD, chloride-bound WNK1/4 interacts with both mTORC2 and SGK1 to increase SGK1 phosphorylation and subsequent ENaC activation. Increased electrogenic ENaC activity depolarizes the apical membrane potential (Vap↓), thereby stimulating ROMK-mediated K⁺ secretion. Aldosterone (A) contributes to ENaC regulation in the CCD by binding to the mineralocorticoid receptor (MR) and increasing SGK1 transcription. Purple arrows indicate effects due to an increase in interstitial K.⁺ and red arrows depict the effects of aldosterone (A)
Regulated Na⁺ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na⁺ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na⁺ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K⁺, Ca⁺⁺, Mg⁺⁺, Cl⁻, and HCO3⁻, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na⁺ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na⁺ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components—kinases, ubiquitin ligases, phosphatases, transcription factors, and others—have also been identified and many of their actions elucidated. This review will touch on selected aspects of ion transport regulation, and its impact on fluid and electrolyte homeostasis. A particular focus will be on emerging evidence for site-specific regulation of the epithelial sodium channel (ENaC) and its role in both Na⁺ and K⁺ homeostasis. In this context, the critical regulatory roles of aldosterone, the mineralocorticoid receptor (MR), and the kinases SGK1 and mTORC2 will be highlighted. This includes a discussion of the newly established concept that local K⁺ concentrations are involved in the reciprocal regulation of Na⁺-Cl⁻ cotransporter (NCC) and ENaC activity to adjust renal K⁺ secretion to dietary intake.
Schematic representation of the main mechanisms ensuring the stability of extracellular fluid calcium under normal conditions. The regulation of calcium concentration involves parathyroid hormone (PTH), 1,25dihydroxyvitamin D and extracellular fluid calcium itself via the calcium-sensing receptor (CaSR) in the kidneys and the parathyroid glands. A decrease in serum calcium concentration stimulates PTH secretion by parathyroid glands, which increases calcium release from bone tissue and calcium reabsorption by the kidney both in the TAL and the DCT/CNT (see the “Parathyroid hormone” section for further details). Moreover, PTH and a decrease in serum calcium concentration stimulate 1,25(OH)2vitamin D secretion by the kidney, which increases calcium release from bone tissue and calcium reabsorption by the kidney in the DCT (see the “Vitamin D metabolites” section). In return, 1,25(OH)2vitamin D inhibits PTH secretion. Finally, a decrease in ECF calcium itself inactivates CaSR in the TAL and enhances calcium reabsorption by the kidney, by increasing paracellular permeability to calcium in the TAL. Thus, a decrease in serum calcium concentration stimulates calcium reabsorption by the kidney and net bone calcium release, allowing serum calcium to increase back to its reference value. Green arrow: increase; red arrow: decrease
Model of calcium reabsorption in the proximal tubule (PT). In the PT, calcium reabsorption takes place mainly along the paracellular pathway, and depends on (1) a paracellular permeability to cations owing to the expression of claudin (CLDN)-2 and CLDN-12 at the tight junction, (2) a transepithelial chemical gradient generated by water reabsorption, itself linked to a transepithelial reabsorption of Na⁺ and anions, and (3) a small lumen-positive transepithelial potential difference in mid and late PT. Water reabsorption occurs either transcellularly via aquaporin 1 (AQP1) expressed at both apical and basolateral membrane or paracellularly. Na⁺ is reabsorbed actively, largely via the apical Na⁺/H⁺ exchanger 3 (NHE3) and to a lesser extent by various apical Na⁺ cotransporters (with glucose, phosphate, amino acids…). HCO3⁻ ions are produced by dissociation of H2CO3 in the cell mediated by the carbonic anhydrase and exit the cell via the Na⁺/HCO3⁻ cotransporter (NBCe1). The expression of CLDN10a at the tight junction increases the paracellular permeability to anions (Cl.⁻). The proximal tubule is also a main site of 1,25(OH)2vitamin D synthesis, where it is activated by parathyroid hormone acting on the mitochondrial 1alpha hydroxylase (CYP27B2)
Model of calcium reabsorption in the C-TAL of Henle’s loop. In the C-TAL of Henle’s loop, calcium reabsorption occurs paracellularly and depends on (1) a selective paracellular permeability to divalent cations (Ca²⁺ and Mg²⁺) owing to the expression of the claudin (CLDN)-16 and CLDN-19 heterodimeric complex at the tight junction and (2) the lumen positive transepithelial voltage generated by the active transcellular transport of NaCl via the apical Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2). A large amount of K⁺ that enters the cell recycles back to the lumen via the apical potassium channel (ROMK), thereby hyperpolarizing the apical membrane. Most of the chloride leaves the cell via the basolateral chloride channel (ClC-Kb), resulting in membrane depolarization. Calcium reabsorption is controlled by the activation of calcium-sensing receptor (CaSR) and parathyroid hormone receptor type 1 (PTH1R) both expressed at the basolateral membrane. CaSR activation decreases the paracellular permeability to Ca.²⁺ while PTH1R activation increases it. PTH1R activation also increases the lumen positive transepithelial voltage by increasing NaCl reabsorption via NKCC2
Calcium reabsorption in the distal tubule (DT). In the DT, calcium reabsorption occurs transcellularly both in the distal convoluted tubule (DCT) and the connecting tubule (CNT). Ca²⁺ enters the cell thanks to (1) the expression of the apical transient receptor potential vanilloid 5 (TRPV5) and (2) an electrochemical free calcium gradient between the lumen and the cytosol, due to Ca²⁺ binding to calbindin-D28K and D9K (calcium-binding protein Ca-BP). Then, Ca²⁺ is ferried to the basolateral membrane and exits the cell via two transporters: the Na⁺/Ca²⁺ exchanger (NCX1) and the plasma membrane Ca²⁺-ATPase (PMCA4). Na⁺ enters the cell via the apical Na⁺-Cl⁻ cotransporter (NCC) in the DCT and the epithelial Na⁺ channel (ENaC) in the CNT. In the DCT, Cl⁻ leaves the cell via the basolateral chloride channel (ClC-Kb) while in the CNT, Cl⁻ is reabsorbed along the paracellular pathway. In both DCT and CNT, PTH1R activation and Klotho increase calcium reabsorption by enhancing TRPV5 activity; 1,25 (OH)2 vitamin D increases TRPV5 and Ca-BP expression. A drop in urine pH decreases calcium reabsorption in the DT by decreasing TRPV5 activity
Extracellular fluid calcium concentration must be maintained within a narrow range in order to sustain many biological functions, encompassing muscle contraction, blood coagulation, and bone and tooth mineralization. Blood calcium value is critically dependent on the ability of the renal tubule to reabsorb the adequate amount of filtered calcium. Tubular calcium reabsorption is carried out by various and complex mechanisms in 3 distinct segments: the proximal tubule, the cortical thick ascending limb of the loop of Henle, and the late distal convoluted/connecting tubule. In addition, calcium reabsorption is tightly controlled by many endocrine, paracrine, and autocrine factors, as well as by non-hormonal factors, in order to adapt the tubular handling of calcium to the metabolic requirements. The present review summarizes the current knowledge of the mechanisms and factors involved in calcium handling by the kidney and, ultimately, in extracellular calcium homeostasis. The review also highlights some of our gaps in understanding that need to be addressed in the future.
Oxygen sensing and signalling. The prolyl-4-hydroxylase domain (PHD) enzymes PHD1, PHD2, and PHD3, and the asparaginyl hydroxylase factor inhibiting HIF (FIH) utilize the co-substrates molecular oxygen and 2-oxoglutarate (2-OG) to hydroxylate the hypoxia-inducible factor (HIF) α subunits, along with the conversion of 2-OG to succinate by oxidative decarboxylation. Ferrous iron and reducing agents such as ascorbate (vitamin C) serve as co-factors required for enzymatic function. Hydroxylase activity is inhibited by hypoxia, several Krebs cycle intermediates and agents that interfere with ferrous iron, including transition metals, iron chelators, nitric oxide, and other oxidative reactive oxygen species (ROS). Hydroxylated HIFα is recognized by the von Hippel-Lindau (VHL) ubiquitin E3 ligase adaptor protein, and subsequently subjected to proteasomal degradation. Non-hydroxylated HIFα heterodimerizes with the common HIF-β subunit and forms a transcriptional enhancer complex at hypoxia response elements (HREs) of HIF target genes. PHD2 and PHD3 are among these genes, establishing a negative feedback loop that limits HIF activity and adapts the hypoxic set point to the microenvironmental oxygen partial pressure
Regulatory elements of the human EPO gene. Hypoxia response elements (HREs) have been identified in the distal 5′ and 3′ enhancer regions as well as in the proximal promoter region of the EPO gene. Hypoxia-inducible transcription factor complexes interact with these HREs in a cell-type–specific manner to govern EPO gene expression as indicated. kb, kilobases
Renal Epo-producing (REP) cells. Epo-CreERT2xtdTomato reporter mice were treated with tamoxifen and exposed for 4 h to 0.1% carbon monoxide (CO) resulting in ~ 50% CO saturation of hemoglobin (hypoxemia), a strong but short-lived stimulus for Epo expression. a Fluorescence microscopy of a REP cell 3 weeks after the permanent labelling with fluorescent tdTomato protein (red). b Reporter mice were treated with a second identical hypoxic stimulus 1 week after the initial REP tagging, and analyzed immediately by Epo mRNA fluorescent in situ hybridization (FISH; white). Because the FISH procedure destroyed its fluorescence, tdTomato protein was detected by anti-RFP immunofluorescence (αRFP IF; red). a, b Tubuli were visualized by their autofluorescence (green) and nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; blue)
Loss of tubular function during chronic kidney disease (CKD). In this hypothetical model, renal “microenvironmental relative hyperoxia” is caused by decreased oxygen consumption of damaged tubules during the course of the disease, which leads to an attenuated hemoglobin oxygen desaturation of the capillary blood in the vicinity of the pericytic Epo-producing (REP) cells. Consequently, intracellular pO2 levels in REP cells exceed the normal hypoxic set point required for Epo production, leading to renal anemia
Proposed three-dimensional model for tissue oxygen partial pressure “sensing” of arterial blood oxygen content by renal Epo-producing (REP) cells. Pericytic REP cells are longitudinally aligned along post-glomerular blood vessels and are depicted within the calculated isobaric oxygen partial pressure (pO2) values forming the Krogh tissue cylinder [117]. Strong transversal O2 fluxes through REP cells, especially around the arterial end of the blood vessel, are generated by high O2 consumption of tubular epithelial cells located in the outer parts of the tissue cylinder (not shown). In the “radial proportional” mode (upper part), REP cells sense the mean cellular pO2 levels and regulate Epo in inverse proportion to this value. In the “longitudinal differential” mode (lower part), REP cells integrate the information obtained from distal pO2 values along the blood vessel and regulate Epo based on this differential value that provides information about the longitudinal localization. Under anemic conditions, the venous (but not the arterial) pO2 is lowered, and the critical tissue pO2 required for Epo induction is shifted towards the “arterial end” of the capillary
Renal erythropoietin (Epo)-producing (REP) cells represent a rare and incompletely understood cell type. REP cells are fibroblast-like cells located in close proximity to blood vessels and tubules of the corticomedullary border region. Epo mRNA in REP cells is produced in a pronounced “on–off” mode, showing transient transcriptional bursts upon exposure to hypoxia. In contrast to “ordinary” fibroblasts, REP cells do not proliferate ex vivo, cease to produce Epo, and lose their identity following immortalization and prolonged in vitro culture, consistent with the loss of Epo production following REP cell proliferation during tissue remodelling in chronic kidney disease. Because Epo protein is usually not detectable in kidney tissue, and Epo mRNA is only transiently induced under hypoxic conditions, transgenic mouse models have been developed to permanently label REP cell precursors, active Epo producers, and inactive descendants. Future single-cell analyses of the renal stromal compartment will identify novel characteristic markers of tagged REP cells, which will provide novel insights into the regulation of Epo expression in this unique cell type.
Pathogenesis of glucocorticoid resistance. A Consequences of GR mutations on the hypothalamic–pituitary axis causing hypercortisolism. ACTH adenocorticotropic hormone; CRH corticotropin-releasing hormone B Linear model of the human GR structure and localization of identified mutations. C Linear structure of rodent GRs carrying the mutant em2, em4 and β geo allele; NTD N-terminus domain, DBD DNA binding domain, HR hinge region, LBD ligand binding domain
Graphic representing percentages of all clinical features observed in patients carrying 38 GR mutations (see Table 1) causing glucocorticoid resistance
Hypothetical scheme of mechanisms implicated in the generation of salt -sensitive hypertension and adrenal hyperplasia in human and animal models
Overview on documented NR3C1 mutations with their phenotype
Hypertension is one of the leading causes of premature death in humans and exhibits a complex aetiology including environmental and genetic factors. Mutations within the glucocorticoid receptor (GR) can cause glucocorticoid resistance, which is characterized by several clinical features like hypercortisolism, hypokalaemia, adrenal hyperplasia and hypertension. Altered glucocorticoid receptor signalling further affects sodium and potassium homeostasis as well as blood pressure regulation and cell proliferation and differentiation that influence organ development and function. In salt-sensitive hypertension, excessive renal salt transport and sympathetic nervous system stimulation may occur simultaneously, and, thus, both the mineralocorticoid receptor (MR) and the GR-signalling may be implicated or even act interdependently. This review focuses on identified GR mutations in human primary generalized glucocorticoid resistance (PGGR) patients and their related clinical phenotype with specific emphasis on adrenal gland hyperplasia and hypertension. We compare these findings to mouse and rat mutants harbouring genetically engineered mutations to further dissect the cause and/or the consequence of clinical features which are common or different.
Mechanisms to maintain potassium (K) homeostasis after a K-rich meal. A K-rich meal stimulates the intestine to release an unidentified “gut factor” that potentially targets the pituitary to release a mediator that targets skeletal muscle and liver to take up K from ECF to ICF, in addition to stimulating the renal nephron to excrete K before a rise in plasma [K]. Additionally, the meal stimulates the pancreas to release insulin, a mediator that activates muscle Na,K-ATPase independent of a rise in plasma [K]. These feedforward mechanisms are the initial homeostatic response to buffering ECF [K] in response to K intake. As the meal is absorbed, the rise in plasma [K] is a power signal that targets (1) Skeletal muscle Na,K-ATPase alpha 2 isoform, which is kinetically activated by rising [K], (2) Kidney basolateral membrane K-channel sensors Kir4.1/5.1 and potentially Kir4.2/5.1, which rapidly stimulate signaling cascades that mediate increased K secretion and excretion and depress ammoniagenesis, and (3) Adrenal glomerulosa cell K channels GIRK-4, TASK-1, and TASK-3 which change membrane potential and cell [Ca] and stimulate biosynthesis and release of the mediator aldosterone. Aldosterone targets mineralocorticoid receptors in the renal distal nephron, colon, sweat and salivary glands to secrete and excrete K. Together, stimulation of K secretion and excretion match K output to input, forming complex feedforward plus feedback loops that are needed to tightly maintain ECF [K]. Figure created with
Basics of renal K handling. The different segments of the renal tubule involved in K reabsorption and secretion are highlighted along with some of the main apical plasma membrane transport pathways. Some of the mediators of K transport are also shown (? indicates potential role). Reabsorption of K in the proximal tubule occurs passively via a paracellular pathway secondary to the reabsorption of Na. In the TAL, secondary active transport provides a transcellular route for K reabsorption and a lumen-positive potential difference drives paracellular K reabsorption. Cells of the late DCT, CNT, and CCD are important for K secretion by K chloride cotransporters (KCC), the renal outer medullary K channel (ROMK), and “Big” K (BK) channels. Distal delivery of Na alters electrogenic Na reabsorption by the epithelial Na⁺ channel (ENaC) and subsequent K secretion by ROMK, whereas increased tubular flow stimulates K secretion by BK channels. Reabsorption of K through the HK-ATPase in type A intercalated cells can also occur. Abbreviations: DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; NHE3, sodium-hydrogen exchanger 3; NKCC2, Na⁺-K⁺-Cl.⁻ cotransporter 2. Figure created with
Effects of low and high dietary K intake on NCC-mediated Na reabsorption in the DCT. During hypokalemia, Kir4.1/5.1 is activated leading to a reduction in intracellular [K] and hyperpolarization of the basolateral membrane, thus creating a driving force for Cl⁻ efflux via ClC-Kb. The subsequent reduction in intracellular Cl⁻ relieves the inhibition of WNK4 autophosphorylation and allows the WNK-SPAK pathway to phosphorylate and activate NCC. This leads to greater NaCl reabsorption in the DCT, less Na delivery to the distal tubule and reduced K secretion. During high K intake, high ECF [K] inhibits Kir4.1/5.1 leading to membrane depolarization and inhibition of Cl⁻ efflux via ClC-Kb. The increase in intracellular [Cl⁻] inhibits WNK4 resulting in reduced SPAK and NCC phosphorylation. The influence of the SPAK pathway is also reduced by enhanced proteasomal degradation of WNK4. High ECF [K] also causes NCC dephosphorylation and degradation. The reduction in NCC activity increases distal Na delivery and flow which promotes kaliuresis. Figure created with
Transmembrane potassium (K) gradients are key determinants of membrane potential that can modulate action potentials, control muscle contractility, and influence ion channel and transporter activity. Daily K intake is normally equal to the amount of K in the entire extracellular fluid (ECF) creating a critical challenge — how to maintain ECF [K] and membrane potential in a narrow range during feast and famine. Adaptations to maintain ECF [K] include sensing the K intake, sensing ECF [K] vs. desired set-point and activating mediators that regulate K distribution between ECF and ICF, and regulate renal K excretion. In this focused review, we discuss the basis of these adaptions, including (1) potential mechanisms for rapid feedforward signaling to kidney and muscle after a meal (before a rise in ECF [K]), (2) how skeletal muscles sense and respond to changes in ECF [K], (3) effects of K on aldosterone biosynthesis, and (4) how the kidney responds to changes in ECF [K] to modify K excretion. The concepts of sexual dimorphisms in renal K handling adaptation are introduced, and the molecular mechanisms that can account for the benefits of a K-rich diet to maintain cardiovascular health are discussed. Although the big picture of K homeostasis is becoming more clear, we also highlight significant pieces of the puzzle that remain to be solved, including knowledge gaps in our understanding of initiating signals, sensors and their connection to homeostatic adjustments of ECF [K].
Hormonal control of water transport by collecting duct. Arginine vasopressin (AVP) is synthesized in the hypothalamus and released by the posterior pituitary gland. In the renal collecting duct principal cells, AVP binds to the vasopressin receptor type 2 (V2R), which leads to the activation of the protein kinase A (PKA) and increases aquaporin-2 (AQP2) abundance in the apical plasma membrane by stimulating its translocation from intracellular storage vesicles. Water is reabsorbed via AQP2 at the apical membrane and then exits the cell through aquaporin-3 (AQP3) and aquaporin-4 (AQP4) at the basolateral membrane
Aquaporin (AQP) distribution along the nephron. AQP1 is located in both the apical and basolateral membrane of proximal tubule cells, which is highly permeable to water and accounts for 70% of total water reabsorption. AQP7 is additionally found along the brush border of proximal tubule cells. The thin descending limb of Henle accounts for 20% of water reabsorption via the AQP1 located at both apical and basolateral sides of the plasma membrane. The collecting duct is less permeable to water (0 to 9%) and expresses three aquaporins in principal cells: AQP2 at the apical membrane, and AQP3 and AQP4 at the basolateral membrane
Major biological and clinical characteristics of the various types of hyponatremias
Saving body water by optimal reabsorption of water filtered by the kidney leading to excretion of urine with concentrations of solutes largely above that of plasma allowed vertebrate species to leave the aquatic environment to live on solid ground. Filtered water is reabsorbed for 70% and 20% by proximal tubules and thin descending limbs of Henle, respectively. These two nephron segments express the water channel aquaporin-1 located along both apical and basolateral membranes. In the proximal tubule, the paracellular pathway accounts for at least 30% of water reabsorption, and the tight-junction core protein claudin-2 plays a key role in this permeability. The ascending limb of Henle and the distal convoluted tubule are impermeant to water and are responsible for urine dilution. The water balance is adjusted along the collecting system, i.e. connecting tubule and the collecting duct, under the control of arginine-vasopressin (AVP). AVP is synthesized by the hypothalamus and released in response to an increase in extracellular osmolality or stimulation of baroreceptors by decreased blood pressure. In response to AVP, aquaporin-2 water channels stored in subapical intracellular vesicles are translocated to the apical plasma membrane and raise the water permeability of the collecting system. The basolateral step of water reabsorption is mediated by aquaporin-3 and -4, which are constitutively expressed. Drugs targeting water transport include classical diuretics, which primarily inhibit sodium transport; the new class of SGLT2 inhibitors, which promotes osmotic diuresis and the non-peptidic antagonists of the V2 receptor, which are pure aquaretic drugs. Disturbed water balance includes diabetes insipidus and hyponatremias. Diabetes insipidus is characterized by polyuria and polydipsia. It is either related to a deficit in AVP secretion called central diabetes insipidus that can be treated by AVP analogs or to a peripheral defect in AVP response called nephrogenic diabetes insipidus. Diabetes insipidus can be either of genetic origin or acquired. Hyponatremia is a common disorder most often related to free water excess relying on overstimulated or inappropriate AVP secretion. The assessment of blood volume is key for the diagnosis and treatment of hyponatremia, which can be classified as hypo-, eu-, or hypervolemic.
Schematic of metabolic substrate usage along the proximal tubule. The convoluted part of the proximal tubule (PT) comprises of 2 distinct segments (S1 and S2), which display differences in expression levels of membrane transporters for metabolic substrates. Metabolites filtered by the glomerulus are reabsorbed from the primary urine by S1 cells, across the apical membrane. Conversely, cells in S2 have a high abundance of basolateral fatty acid and organic anion transporters, which can import substrates directly from the blood. Moreover, they are more densely packed with peroxisomes that can generate lipid substrates by beta oxidation of long chain fatty acids. Meanwhile, excess free fatty acids within S2 cells can be stored in specialized multi-lamellar bodies found in this region, to prevent potentially harmful lipotoxicity. LoH, loop of Henle; DT, distal tubule; CD, collecting duct
Examples of recently discovered metabolic pathways in acute kidney injury. (1) Uptake of filtered, non-degradable immunoglobulin light chains in proximal tubular cells and accumulation in lysosomes induces reactive oxygen species (ROS) production, which then activates the redox-sensitive JAK2/STAT1 pathway and interstitial inflammation and fibrosis. (2) During acute metabolic acidosis, changes in redox state of the vital metabolic co-factor NADH (towards oxidation) and inhibition of fatty acid oxidation lead to the accumulation of intracellular lipids. (3) The nephrotoxic iron chelator deferasirox (DFX) is highly lipophilic and interacts with the mitochondrial inner membrane, causing severe swelling in these organelles and a decrease in cellular ATP, probably due to partial uncoupling of the respiratory chain
Alterations in systemic glucose homeostasis in acute kidney injury. Under normal physiological settings, the kidney contributes up to 40% of body gluconeogenesis after fasting, by converting lactate to glucose in the proximal tubule. During acute kidney injury, this process is dramatically downregulated, leading to decreased lactate clearance and increased risk of hypoglycemia
Damage to the proximal tubule (PT) is the most frequent cause of acute kidney injury (AKI) in humans. Diagnostic and treatment options for AKI are currently limited, and a deeper understanding of pathogenic mechanisms at a cellular level is required to rectify this situation. Metabolism in the PT is complex and closely coupled to solute transport function. Recent studies have shown that major changes in PT metabolism occur during AKI and have highlighted some potential targets for intervention. However, translating these insights into effective new therapies still represents a substantial challenge. In this article, in addition to providing a brief overview of the current state of the field, we will highlight three emerging areas that we feel are worthy of greater attention. First, we will discuss the role of axial heterogeneity in cellular function along the PT in determining baseline susceptibility to different metabolic hits. Second, we will emphasize that elucidating insult specific pathogenic mechanisms will likely be critical in devising more personalized treatments for AKI. Finally, we will argue that uncovering links between tubular metabolism and whole-body homeostasis will identify new strategies to try to reduce the considerable morbidity and mortality associated with AKI. These concepts will be illustrated by examples of recent studies emanating from the authors’ laboratories and performed under the auspices of the Swiss National Competence Center for Kidney Research (NCCR
HNF1β regulates expression of channels, and transporters in all segments of the nephron. HNF1β regulates target genes involved in electrolyte handling in the PT including TMEM27 encoding the amino acid transport regulator (Collectrin); SLC17A1 encoding the Na-phosphate transporter 1 (NPT1); SLC22A6, SLC22A8, and SLC22A11 encoding the organic anion transporters (OAT1, OAT3, OAT4); and SLC22A12 encoding the renal urate transporter (URAT1); in the TAL including SLC12A1 encoding the Na⁺-K⁺-2Cl⁻ co-transporter (NKCC2); UMOD encoding uromodulin (UMOD); CASR encoding the calcium sensing receptor (CaSR); and CLDN16 encoding Claudin 16; in the DCT including KCNJ16 encoding the subunit of the inward rectifier K⁺ channel (Kir5.1) and FXYD2 encoding the Na⁺-K⁺-ATPase subunit gamma; in the CD including TMEM27 and NR1H4 encoding the farnesoid X nuclear receptor (FXR). In return, transcription factor FXR regulates expression of AQP2 in the CD. PT proximal tubules, DCT distal convoluted tubule, TAL thick ascending loop of Henle, CD collecting duct, OA⁻ organic anion, DC⁻ dicarboxylate
HNF1β is required for UB branching and nephron segmentation. Schematic representation of different stages of mouse metanephric nephron development. At E10.5, kidney development starts with the outgrowth of the UB into the MM. HNF1β is essential for normal branching of the UB that eventually will form the collecting duct system. Around E12.5, cells of the cap mesenchyme polarize into pretubular aggregates that will form renal vesicles which require MET. Whether HNF1β is involved in this early stage of nephrogenesis is not yet conclusive. Subsequently, renal vesicles differentiate into comma and S-shaped bodies. Hnf1b KO mice develop S-shaped bodies that lack the epithelial bulge that will give rise to the proximal and Henle’s loop tubule in the WT situation. Eventually at E17.5, part of the S-shaped body will associate with capillaries to form the glomerulus and other parts will form the nephron tubule. WD Wolffian duct, UB ureteric bud, MM metanephric mesenchyme, MET mesenchymal-epithelial transition
Hepatocyte nuclear factor 1β (HNF1β) is a transcription factor essential for the development and function of the kidney. Mutations in and deletions of HNF1β cause autosomal dominant tubule interstitial kidney disease (ADTKD) subtype HNF1β, which is characterized by renal cysts, diabetes, genital tract malformations, and neurodevelopmental disorders. Electrolyte disturbances including hypomagnesemia, hyperuricemia, and hypocalciuria are common in patients with ADTKD-HNF1β. Traditionally, these electrolyte disturbances have been attributed to HNF1β-mediated transcriptional regulation of gene networks involved in ion transport in the distal part of the nephron including FXYD2 , CASR , KCNJ16 , and FXR . In this review, we propose additional mechanisms that may contribute to the electrolyte disturbances observed in ADTKD-HNF1β patients. Firstly, kidney development is severely affected in Hnf1b -deficient mice. HNF1β is required for nephron segmentation, and the absence of the transcription factor results in rudimentary nephrons lacking mature proximal tubule, loop of Henle, and distal convoluted tubule cluster. In addition, HNF1β is proposed to be important for apical-basolateral polarity and tight junction integrity in the kidney. Interestingly, cilia formation is unaffected by Hnf1b defects in several models, despite the HNF1β-mediated transcriptional regulation of many ciliary genes. To what extent impaired nephron segmentation, apical-basolateral polarity, and cilia function contribute to electrolyte disturbances in HNF1β patients remains elusive. Systematic phenotyping of Hnf1b mouse models and the development of patient-specific kidney organoid models will be essential to advance future HNF1β research.
Bicarbonate reabsorption and formation of new bicarbonate via ammoniagenesis in coordination with glutamine metabolism, gluconeogenesis, and activity of potassium channels in the proximal tubule. Secretion of H⁺ via NHE3 or H + -ATPase (not shown) leads to reabsorption of HCO3⁻ via NBCe1 (and AE2 in the segment 3). Ammonia and HCO3⁻ are formed from the metabolization of glutamine in the mitochondria, which provides precursors for gluconeogenesis. Glycerol and lactate are additional substrates of gluconeogenesis, but they have a minor role in response to metabolic acidosis in healthy kidneys. The transcription factor NRF2 regulates the expression of the main importer of glutamine into proximal tubular cells during acidosis, SNAT3. Potassium channels in the basolateral membrane control membrane potential impacting NBCe1 activity and ammoniagenesis. NHE3 (SLC9A3) sodium hydrogen exchanger 3, NBCe1 (SLC4A4) electrogenic sodium bicarbonate cotransporter 1, SNAT3 (Slc38a3) sodium-coupled neutral amino acid transporter 3, NRF2 (NFE2L2) nuclear factor-erythroid factor 2-related factor 2, TASK2 (KCNK5) TWIK-related acid-sensitive K( +) channel 2, KIR4.2 (KCNJ15) inward rectifier K⁺ channel KIR4.2, AQP7 aquaporin 7, CAII and CAIV carbonic anhydrase 2 and 4, respectively; SMCTs represent sodium-coupled monocarboxylate transporters 1 and 2 (SLC58 and SLC5A12); MCTs represent different monocarboxylate transporter members, most probably SLC16A1 and SLC16A; PDG (GLS) phosphate-dependent glutaminase, GDH (GLUD1) glutamate dehydrogenase, PEPCK (PCK1) phosphoenolpyruvate carboxykinase
of main renal metabolic pathways altered between kidney transplant recipients (KTRs) with or without acidosis. Bulk RNA sequencing data using RNA from kidney biopsies of KTRs identified genes altered between patients with or without acidosis, but with comparable eGFR. These genes participate in metabolic activities shown in this figure in black. Red lines show molecular pathways that had genes restored by alkali therapy. Blue arrows show direct biochemical reactions, and blue dashed lines show indirect biochemical reactions. Black arrows show movement of molecules. Data originally published in [59]. TCA cycle tricarboxylic acid cycle (also citric acid cycle or Krebs cycle), P5P pyridoxal-5′-phosphate, GSH glutathione, THF tetrahydrofolate
Conceptual framework how chronic kidney disease, inflammation, and deranged metabolism form a vicious cycle involving metabolic acidosis as an engine. Nephron loss and impaired renal function reduce kidney capacity of eliminating acids and generating new bicarbonate which leads to accumulation of acids in the organism. Renal responses to acidosis exacerbate inflammation and deranged metabolism that ultimately reduce kidney function and kidney capacity of keeping pH homeostasis. Steps of this network are shown in continuous black boxes, and open questions related to each of these steps are shown next to it in dashed black boxes. Inflammation and metabolism domains are artificially delimited in different colors as some of these steps may belong to both domains
Kidneys are central in the regulation of multiple physiological functions, such as removal of metabolic wastes and toxins, maintenance of electrolyte and fluid balance, and control of pH homeostasis. In addition, kidneys participate in systemic gluconeogenesis and in the production or activation of hormones. Acid–base conditions influence all these functions concomitantly. Healthy kidneys properly coordinate a series of physiological responses in the face of acute and chronic acid–base disorders. However, injured kidneys have a reduced capacity to adapt to such challenges. Chronic kidney disease patients are an example of individuals typically exposed to chronic and progressive metabolic acidosis. Their organisms undergo a series of alterations that brake large detrimental changes in the homeostasis of several parameters, but these alterations may also operate as further drivers of kidney damage. Acid–base disorders lead not only to changes in mechanisms involved in acid–base balance maintenance, but they also affect multiple other mechanisms tightly wired to it. In this review article, we explore the basic renal activities involved in the maintenance of acid–base balance and show how they are interconnected to cell energy metabolism and other important intracellular activities. These intertwined relationships have been investigated for more than a century, but a modern conceptual organization of these events is lacking. We propose that pH homeostasis indissociably interacts with central pathways that drive progression of chronic kidney disease, such as inflammation and metabolism, independent of etiology.
Top-cited authors
Karl Kunzelmann
  • Universität Regensburg
Rainer Schreiber
  • Universität Regensburg
Tanja Lange
  • Universität zu Lübeck
Carsten Wagner
  • University of Zurich
Ulrich Forstermann
  • Universitätsmedizin der Johannes Gutenberg-Universität Mainz