Pflügers Archiv - European Journal of Physiology

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Online ISSN: 1432-2013
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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.
Genetic architecture of disease: spectrum of allele frequency and effect size. Variants in important genes may be involved in a continuum between rare and complex diseases, according to the risk allele frequency in the population (x-axis) and the strength of the effect size (odds ratio, y-axis). (Ultra)-rare alleles can be identified by next-generation sequencing (NGS), whereas common variants can be identified using genome-wide association studies (GWAS). Intermediate-size effect variants are predicted to complete the dichotomy, manifesting as a non-fully penetrant Mendelian disease or an oligo/polygenic model modifying disease expressivity. Figure adapted from Manolio et al. [34]
Infographics summarizing the design of the CoLaus and SKIPOGH population-based studies
Site of production and structure of uromodulin. Uromodulin is mainly produced by the cells that line the thick ascending limb (TAL), a segment involved in the reabsorption of NaCl and divalent cations, while being not permeable to water. Uromodulin is a glycosylphosphatidylinositol (GPI)-anchored protein which traffics to the apical membrane of the cells, where it is cleaved by the serine protease hepsin and released in the urine where it forms large polymers. These polymers form the matrix of the urinary casts. The predicted structure of uromodulin contains a leader peptide (L); four EGF-like domains (I to IV); a cysteine-rich D8C domain; a bipartite C‑terminal Zona Pellucida domain (ZP_N and ZP_C) connected by a linker; and a GPI-anchoring site at position 614. The seven N‑glycosylation sites are indicated by triangles. Figure adapted from Devuyst et al. [11, 12]
Mendelian randomization analyses supporting the association of higher levels of urinary uromodulin with lower kidney function and higher blood pressure. a Two-sample MR to assess the bidirectional causal effects between urinary uromodulin (uUMOD) and eGFR and between CKD and blood pressure (BP). The analyses were performed in the meta–GWAS for uUMOD involving 10,884 individuals; the CKD Genetics (CKDGen) consortium, a meta-analysis of 121 GWASs including 567,460 individuals of European ancestry; and a combined analysis of the UK Biobank (UKB) and the International Consortium of Blood Pressure (ICBP) GWAS, which amounted to 757,601 individuals. b Multivariable MR to assess direct and indirect effects of uUMOD on BP through eGFR and of uUMOD on eGFR through BP based on MR causal effects between the exposure, mediator, and outcome in the 2-sample MR analyses. The analysis suggests that the association of uUMOD with higher BP is partially through decreased kidney function, whereas BP does not appear to mediate the association of uUMOD with low kidney function.
Modified from Ponte et al. [48] and Turner and Staplin [66]
The identification of genetic factors associated with the risk, onset, and progression of kidney disease has the potential to provide mechanistic insights and therapeutic perspectives. In less than two decades, technological advances yielded a trove of information on the genetic architecture of chronic kidney disease. The spectrum of genetic influence ranges from (ultra)rare variants with large effect size, involved in Mendelian diseases, to common variants, often non-coding and with small effect size, which contribute to polygenic diseases. Here, we review the paradigm of UMOD , the gene coding for uromodulin, to illustrate how a kidney-specific protein of major physiological importance is involved in a spectrum of kidney disorders. This new field of investigation illustrates the importance of genetic variation in the pathogenesis and prognosis of disease, with therapeutic implications.
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.
Dietary phosphate and phosphate homeostasis in humans. Milk and dairy products, followed by grain-based dishes and bread, are the major contributors of dietary phosphate, when food categories are considered [14]. Phosphate in the diet has different bioavailability depending on the chemical structure and source. Inorganic phosphate from ultrapocessed foods such as beverages and canned food has a high bioavailability followed by organic phosphate from animal origin. Phosphate in plants is mostly present as phytates and has the lowest bioavailability [108]. When phosphate enters the gastrointestinal tract, phosphate permeability occurs already in the stomach as assessed in intestinal cell models [55]. This paracellular transport occurs along the whole intestine. The active sodium-dependent transports occur predominantly in the small intestine, and NaPi-IIb is probably the most predominant phosphate transporter and it is regulated by calcitriol, an hormone secreted by the kidneys [66]. The mechanisms how phosphate leaves the enterocytes are still unclear, but several studies suggest it may be mediated by Xpr1, although the basolateral localization has not been confirmed yet [38]. Phosphate is maintained in the blood at concentrations around 0.8 to 1.5 mM. The kidneys play the major role in excreting excess phosphate from the diet [38]. When phosphate levels in plasma raise, PTH and FGF23 secreted from parathyroid glands and osteocytes, respectively, decrease the expression and translocation especially of NaPi-IIa and NaPi-IIc to the apical membrane which results in lower reabsorption and higher phosphate excretion in the urine. These sodium-dependent phosphate transporters are localized in the proximal cells in the kidney [38]. In these cells, Xpr1 may also mediate efflux from the epithelial cells. High FGF23 concentrations inhibit calcitriol synthesis, whereas high PTH concentrations in the blood promote calcitriol synthesis in the kidney [10]
Impact of a chronic high phosphate diet on renal function and associated risks. A chronic high dietary phosphate intake provokes phosphaturia, which leads to the accumulation of calcium phosphate particles and renal inflammation. This further leads to decline of kidney function. A chronic high phosphate intake may also lead to higher phosphate levels than are associated with kidney function decline at values higher than 1.3 mM. Both kidney function decline and hyperphosphatemia (> 1.5. mM) lead to a higher CVD and mortality risk
Phosphate is essential in living organisms and its blood levels are regulated by a complex network involving the kidneys, intestine, parathyroid glands, and the skeleton. The crosstalk between these organs is executed primarily by three hormones, calcitriol, parathyroid hormone, and fibroblast growth factor 23. Largely due to a higher intake of ultraprocessed foods, dietary phosphate intake has increased in the last decades. The average intake is now about twice the recommended dietary allowance. Studies investigating the side effect of chronic high dietary phosphate intake suffer from incomplete dietary phosphate assessment and, therefore, often make data interpretation difficult. Renal excretion is quickly adapted to acute and chronic phosphate intake. However, at the high ends of dietary intake, renal adaptation, even in pre-existing normal kidney function, apparently is not perfect. Experimental intervention studies suggest that chronic excess of dietary phosphate can result in sustained higher blood phosphate leading to hyperphosphatemia. Evidence exists that the price of the homeostatic response (phosphaturia in response to phosphate loading/hyperphosphatemia) is an increased risk for declining kidney function, partly due by intraluminal/tubular calcium phosphate particles that provoke renal inflammation. High dietary phosphate intake and hyperphosphatemia are progression factors for declining kidney function and are associated with higher cardiovascular disease and mortality risk. This is best established for pre-existing chronic kidney disease, but epidemiological and experimental data strongly suggest that this holds true for subjects with normal renal function as well. Here, we review the latest advances in phosphate intake and kidney function decline.
Schematic overview of different aspects of renin-lineage cell plasticity. Renin-lineage cells show a high plasticity and can fulfill different functions in order to maintain renal function, blood pressure, and water and salt balance in the body. Topics highlighted in this review are indicated in bold font
Interstitial renin-expressing cells on kidney sections of wild-type mice. A Spatial distribution pattern of interstitial renin-expressing cells was visualized with RNAscope, a high resolution in situ hybridization technology. Renin mRNA-expressing cells were highlighted with yellow dots on a kidney section of a wild-type mouse. Interstitial renin mRNA-expressing cells are mainly distributed in the outer medulla and to a lesser extent in the renal cortex. Nuclei were counterstained with DAPI (gray). Scale bar 500 μm. B Medullary detail of a co-RNAscope for renin (green) and PDGFR-β (red) mRNA on a wild-type kidney section. Colocalization of both mRNAs identifies interstitial renin⁺ cells as fibroblast-like cells. Nuclei were counterstained with DAPI (gray). Scale bar 20 μm
Schematic overview of distinct markers conveying the identity of juxtaglomerular renin cells and of signaling pathways involved in recruitment of extraglomerular (EGM) cells and vascular smooth muscle cells (VSMCs) for renin production. Phenotypic changes accompanying recruitment are also depicted
Schematic overview illustrating the transformation of juxtaglomerular or interstitial renin⁺ cells into EPO-producing cells
The protease renin, the key enzyme of the renin–angiotensin–aldosterone system, is mainly produced and secreted by juxtaglomerular cells in the kidney, which are located in the walls of the afferent arterioles at their entrance into the glomeruli. When the body’s demand for renin rises, the renin production capacity of the kidneys commonly increases by induction of renin expression in vascular smooth muscle cells and in extraglomerular mesangial cells. These cells undergo a reversible metaplastic cellular transformation in order to produce renin. Juxtaglomerular cells of the renin lineage have also been described to migrate into the glomerulus and differentiate into podocytes, epithelial cells or mesangial cells to restore damaged cells in states of glomerular disease. More recently, it could be shown that renin cells can also undergo an endocrine and metaplastic switch to erythropoietin-producing cells. This review aims to describe the high degree of plasticity of renin-producing cells of the kidneys and to analyze the underlying mechanisms.
Key factors determining oxygen partial pressure in tissue. Top panel: Tissue oxygenation is a function of O2 delivery (DO2), consumption (QO2), and removal (RO2). About 10–15% of O2 delivered to the kidney is consumed under normal physiologic conditions. Not all renal tissues are supplied equally, which is, in part, due to arterial-to-venous oxygen shunting (XO2). O2 not consumed by the kidney is removed by venous efflux. Bottom panel: Tissue partial pressure of oxygen (ptO2) is dependent on factors that influence O2 delivery, consumption, and removal. Circled + and − signs indicate the effect of an increase in the factor upstream of the corresponding arrow on the parameter pointed by the arrowhead under the assumption that everything else remains the same. Circled i indicates that an increase in the respective factor will influence the indicated parameter. Only selected factors are shown. An increase in renal blood flow (RBF) increases DO2 and glomerular filtration rate (GFR), and may influence XO2. Whether XO2 increases is dependent on the location of the tissue under observation, among other factors. XO2 is also influenced by QO2. Since QO2 produces the arterial-to-venous pO2 gradients necessary for XO2, an increase in consumption will likely, but not necessarily, increase shunting; the local arrangement of O2 sinks and sources plays a role as well. More shunting leads to reduced ptO2. Increased GFR leads to higher QO2, as O2 demand for Na⁺ reabsorption increases, and thereby to reduced ptO2. DO2 increases with increased arterial blood oxygen concentration (caO2) and partial pressure. Capillary rarefaction and fibrosis reduce oxygen delivery to tissue due to increased diffusion distance and reduced diffusivity, respectively
Regulation of tissue pO2 (ptO2) by neuro-hormonal agents. The vasoconstrictor factors that reduce RBF and O2 delivery (DO2) generally also stimulate sodium reabsorption (TNa) and increase O2 consumption (QO2). Conversely, the vasodilator factors that increase RBF and DO2 may also act to reduce TNa and QO2. However, these effects are blunted by two mechanisms: increasing RBF also raises GFR and therefore TNa, whereas increases in TNa in the proximal tubule and the ascending limb may reduce NaCl delivery to the macula densa and raise RBF via tubulo-glomerular feedback (TGF). Hence, the effectiveness of ptO2 regulation also depends on how neuro-hormonal agents modulate the coupling between RBF and GFR (i.e., by changing the filtration fraction), or between TNa and QO2 (i.e., by changing the metabolic efficiency of Na⁺ transport). These effects are not explicitly shown in the figure (see text). Not shown either are the synergistic and antagonistic effects between various neuro-hormonal agents
Our kidneys receive about one-fifth of the cardiac output at rest and have a low oxygen extraction ratio, but may sustain, under some conditions, hypoxic injuries that might lead to chronic kidney disease. This is due to large regional variations in renal blood flow and oxygenation, which are the prerequisite for some and the consequence of other kidney functions. The concurrent operation of these functions is reliant on a multitude of neuro-hormonal signaling cascades and feedback loops that also include the regulation of renal blood flow and tissue oxygenation. Starting with open questions on regulatory processes and disease mechanisms, we review herein the literature on renal blood flow and oxygenation. We assess the current understanding of renal blood flow regulation, reasons for disparities in oxygen delivery and consumption, and the consequences of disbalance between O 2 delivery, consumption, and removal. We further consider methods for measuring and computing blood velocity, flow rate, oxygen partial pressure, and related parameters and point out how limitations of these methods constitute important hurdles in this area of research. We conclude that to obtain an integrated understanding of the relation between renal function and renal blood flow and oxygenation, combined experimental and computational modeling studies will be needed.
Molecular model of fetuin-A. The model contains the amino-terminal cystatin-like domain 1 CY1 (blue-teal), CY2 (green-yellow), and the carboxyl-terminal region CTR (orange-red) of mouse fetuin-A (UniProtKB—P29699). Acidic residues Asp and Glu are depicted with ball and stick side chains; putative Ser/Thr phosphorylation sites 135, 138, 305, 309, 312, 314, 317, and 320 were replaced by Glu residues in this model. Model generated by AlphaFold2 and depicted by Chimera software [53, 90]
Hierarchical model of mineral-induced stress. The model illustrates the interdependence of the degree of mineral stress and fetuin-A levels, the predominant entities of calcium phosphate mineral particles, their target cells, elicited cellular responses, and possible therapeutic measures. Mineral stress is generally low and reversible under physiological conditions of fetuin-A abundance, when larger, pathological mineral complexes (CPP1 and CPP2) are mostly absent. With decreasing fetuin-A levels, the degree of mineral stress increases due to enhanced formation of CPP. A chronic mineral disbalance eventually leads to irreversible tissue remodeling, inflammation, and calcification
Local tissue protective role of fetuin-A. The model depicts the crucial importance of fetuin-A to safeguard tissue integrity from hypoxia-induced damage in the kidney, through the clearance of calcifying protein-mineral particles, mitigation of inflammation, attenuation of fibrotic tissue remodeling, and polarization of macrophages. (A–D) Clockwise depiction of the 4 different scenarios combining wildtype (WT) or fetuin-A (Ahsg) KO mice with normoxic or hypoxic conditions based on tissue damage intensity: no damage in normoxic WT (A), low damage in normoxic KO (B) and hypoxic WT (C), and strong damage in hypoxic KO (D). In normoxia (A and B) mineral stress is generally low and the absence of liver-derived fetuin-A (green) in B results in slightly elevated fibrotic remodeling. In hypoxia (C and D), mineral stress is generally high due to calcium overload, but extensive tissue damage can be prevented in C by the concerted action of systemic and locally produced fetuin-A (yellow), counteracting calcification and polarization of pro-inflammatory M1 macrophages (M1 MΦ, light blue). Conversely, the absence of fetuin-A in D leads to enhanced calcification, inflammation, and fibrosis
Traditionally, fetuin-A embodies the prototype anti-calcification protein in the blood, preventing cardiovascular calcification. Low serum fetuin-A is generally associated with mineralization dysbalance and enhanced mortality in end stage renal disease. Recent evidence indicates that fetuin-A is a crucial factor moderating tissue inflammation and fibrosis, as well as a systemic indicator of acute inflammatory disease. Here, the expanded function of fetuin-A is discussed in the context of mineralization and inflammation biology. Unbalanced depletion of fetuin-A in this context may be the critical event, triggering a vicious cycle of progressive calcification, inflammation, and tissue injury. Hence, we designate fetuin-A as tissue chaperone and propose the potential use of exogenous fetuin-A as prophylactic agent or emergency treatment in conditions that are associated with acute depletion of endogenous protein.
Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) as the bladder fills acts as an endogenous DSM relaxant to facilitate bladder storage function. Here, the effects of exogenous PTHrP on transient pressure rises (TPRs) in the bladder and associated afferent nerve activity during bladder filling were investigated. In anaesthetized rats, changes in the intravesical pressure were measured while the bladder was gradually filled with saline. Afferent nerve activity was simultaneously recorded from their centrally disconnected left pelvic nerves. In DSM strips, spontaneous and nerve-evoked contractions were isometrically recorded. The distribution of PTHrP receptors (PTHrPRs) in the bladder wall was also examined by fluorescence immunostaining. The bladders in which the contralateral pelvic nerve was also centrally disconnected developed nifedipine, an L-type voltage-dependent Ca²⁺ channel blocker-sensitive TPRs (< 3 mmHg). Intravenous administration of PTHrP suppressed these TPRs and associated bursts of afferent nerve activity. In the bladders with centrally connected contralateral pelvic nerves, atropine, a muscarinic receptor antagonist-sensitive large TPRs (> 3 mmHg) developed in the late filling phase. PTHrP diminished the large TPRs and corresponding surges of afferent nerve activity. In DSM strips, bath-applied PTHrP (10 nM) suppressed spontaneous phasic contractions, while less affecting nerve-evoked contractions. PTHrPRs were expressed in DSM cells but not in intramural nerve fibers. Thus, PTHrP appears to suppress bladder TPRs and associated afferent nerve activity even under the influence of low degree of parasympathetic neural input during storage phases. Endogenous PTHrP may indirectly attenuate afferent nerve activity by suppressing TPRs to facilitate urinary accommodation.
Anatomical substrates for the mixing of information in the cerebral cortex. a Drawing of cortical pyramidal neurons by Ramon y Cajal showing horizontal axon collaterals forming connections to neighboring neurons and long-range projections of the same axons leaving the local network to connect to other cortical areas or subcortical structures [40]. b Silver stain of axons in the cerebral cortex forming a dense network with projections running equally in all directions [11, 12]. c Schematic illustration of some of the long-range axonal pathways forming connections between distant brain areas (yellow: uncinate fasciculus; green: arcuate fasciculus: blue: superior longitudinal fasciculus). d Schematic illustration of how neuronal activity propagates between cortical areas, resulting in the convergence, divergence, and integration of information via brain-wide networks
It is common to distinguish between “holist” and “reductionist” views of brain function, where the former envisions the brain as functioning as an indivisible unit and the latter as a collection of distinct units that serve different functions. Opposing reductionism, a number of researchers have pointed out that cortical network architecture does not respect functional boundaries, and the neuroanatomist V. Braitenberg proposed to understand the cerebral cortex as a “great mixing machine” of neuronal activity from sensory inputs, motor commands, and intrinsically generated processes. In this paper, we offer a contextualization of Braitenberg’s point, and we review evidence for the interactions of neuronal activity from multiple sensory inputs and intrinsic neuronal processes in the cerebral cortex. We focus on new insights from studies on audiovisual interactions and on the influence of respiration on brain functions, which do not seem to align well with “reductionist” views of areal functional boundaries. Instead, they indicate that functional boundaries are fuzzy and context dependent. In addition, we discuss the relevance of the influence of sensory, proprioceptive, and interoceptive signals on cortical activity for understanding brain-body interactions, highlight some of the consequences of these new insights for debates on embodied cognition, and offer some suggestions for future studies.
Diazepam affects the variability of the chewing reflex at P0–1. Effects of prenatal diazepam exposure (DZP) or vehicle (CTRL) on chewing (a) and righting (b) reflexes in female (red) and male (blue) newborn rats (P0). Values are expressed as mean ± SEM. The dots represent the values for each animal
Diazepam-treated females and males had a higher body mass at P21-22. Body mass in grams (g) of control (CTRL) and diazepam (DZP) exposed females (red, a) and males (blue, b), at P0–1, P12–13, and P21–22. Values are expressed as mean ± SEM. The dots represent the values for each animal. A single asterisk (*) indicates a statistical difference between CTRL and DZP groups at the same age and sex
At normoxic normocapnia, P0–1 DZP-treated females show an increase in fR and V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}, whereas males hypoventilate at P12–13 and P21–22. Ventilation (V˙E\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{V}}_{E}$$\end{document}), tidal volume (VT), respiratory frequency (fR), oxygen consumption (V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}) and air convection requirements (V˙E\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{V}}_{E}$$\end{document}/V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}) of control (CTRL, full bars) and diazepam-treated (DZP, empty bars) female (red, a, b, c, d, e) and male (blue, f, g, h, i, j) rats during normoxic normocapnia (21% O2 and 0% CO2) at P0–1, P12–13, and P21–22. Values are expressed as mean ± SEM. The dots represent the values for each animal. A single asterisk (*) indicates a statistical difference between the CTRL and DZP groups at the same age and sex
Diazepam exposure decreased ventilation due to a reduction of VT in females (P0–1 and P12–13), reduced the fR in males (P0–1 and P12–13) and their metabolism (P12–13) under hypercapnia. Ventilation (V˙E\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{V}}_{E}$$\end{document}), tidal volume (VT), respiratory frequency (fR), oxygen consumption (V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}) and air convection requirements (V˙E\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{V}}_{E}$$\end{document}/V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}) of control (CTRL, full bars) and diazepam-treated (DZP, empty bars) female (red, a, b, c, d, e) and male (blue, f, g, h, i, j) rats during hypercapnia (7% CO2) at P0–1, P12–13, and P21–22. Values are expressed as percentage from baseline with mean ± SEM. The dots represent the values for each animal. A single asterisk (*) indicates a statistical difference between the CTRL and DZP groups at the same age and sex
DZP-treated females had a lower VT at P12–13 during hypoxic challenge, and P12–13 males had an attenuation of the hypoxic ventilatory response. Ventilation (V˙E\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{V}}_{E}$$\end{document}), tidal volume (VT), respiratory frequency (fR), oxygen consumption (V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}) and air convection requirements (V˙E\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{V}}_{E}$$\end{document}/V˙O2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}{\mathrm{O}}_{2}$$\end{document}) of control (CTRL, full bars) and diazepam-treated (DZP, empty bars) female (red, a, b, c, d, e) and male (blue, f, g, h, i, j) rats during hypoxia (10% O2) at P0–1, P12–13, and P21–22. Values are expressed as percentage from baseline with mean ± SEM. The dots represent the values for each animal. A single asterisk (*) indicates a statistical difference between the CTRL and DZP groups at the same age and sex
Pregnancy is highly affected by anxiety disorders, which may be treated with benzodiazepines, especially diazepam (DZP), that can cross the placental barrier and interact with the fetal GABAergic system. We tested whether prenatal exposure to DZP promotes sex-specific postnatal changes in the respiratory control of rats. We evaluated ventilation ([Formula: see text]) and oxygen consumption ([Formula: see text] O2) in resting conditions and under hypercapnia (7% CO2) and hypoxia (10% O2) in newborn [postnatal day (P) 0-1 and P12-13)] and young (P21-22) rats from mothers treated with DZP during pregnancy. We also analyzed brainstem monoamines at the same ages. DZP exposure had minimal effects on room air-breathing variables in females, but caused hypoventilation (drop in [Formula: see text]/[Formula: see text] O2) in P12-13 males, lasting until P21-22. The hypercapnic ventilatory response was attenuated in P0-1 and P12-13 DZP-treated females mainly by a decrease in tidal volume (VT), whereas males had a reduction in respiratory frequency (fR) at P12-13. Minor changes were observed in hypoxia, but an attenuation in [Formula: see text] was seen in P12-13 males. In the female brainstem, DZP increased dopamine concentration and decreased 5-hydroxyindole-3-acetic acid (5-HIAA) and the 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine ratio at P0-1, and reduced DOPAC concentration at P12-13. In males, DZP decreased brainstem noradrenaline at P0-1. Our results demonstrate that prenatal DZP exposure reduces CO2 chemoreflex only in postnatal females and does not affect hypoxia-induced hyperventilation in both sexes. In addition, prenatal DZP alters brainstem monoamine concentrations throughout development differently in male and female rats.
More than 50 years ago, it was proposed that breathing shapes pupil dynamics. This widespread idea is also the general understanding currently. However, there has been no attempt at synthesizing the progress on this topic since. We therefore conducted a systematic review of the literature on how breathing affects pupil dynamics in humans. We assessed the effect of breathing phase, depth, rate, and route (nose/mouth). We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and conducted a systematic search of the scientific literature databases MEDLINE, Web of Science, and PsycInfo in November 2021. Thirty-one studies were included in the final analyses, and their quality was assessed with QualSyst. The study findings were summarized in a descriptive manner, and the strength of the evidence for each parameter was estimated following the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. The effect of breathing phase on pupil dynamics was rated as “low” (6 studies). The effect of breathing depth and breathing rate (6 and 20 studies respectively) were rated as “very low”. Breathing route was not investigated by any of the included studies. Overall, we show that there is, at best, inconclusive evidence for an effect of breathing on pupil dynamics in humans. Finally, we suggest some possible confounders to be considered, and outstanding questions that need to be addressed, to answer this fundamental question. Trial registration: This systematic review has been registered in the international prospective register of systematic reviews (PROSPERO) under the registration number: CRD42022285044.
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.
COVID-19 (C19) sera treatment damages the endothelial glycocalyx (eGC). A Endothelial cells were incubated with 10% sera from C19 patients with mild symptoms (patients 21–59) for 24 h. Analysis of the eGC by atomic force microscopy showed eGC damage with a reduced eGC height in a range of 81.6 nm to 130.1 nm compared to 220.7 nm of the control group (N = 3, n = 5–6; **** (all patients) p < 0.0001 vs. control). B Sera from a second patient cohort with severe SARS-CoV-2 infection and mandatory intensive care (patients 14–37) were incubated with endothelial cells for 24 h. Here, eGC height was further damaged, reducing eGC height in a range of 57.7 nm to 90.1 nm compared to 175.6 nm under control conditions (N = 3, n = 3–5; ** (all patients) p < 0.01 vs. control). C Average eGC height of control, C19 mild, and C19 severe are shown. C19 treatment leads to a reduction in eGC height by 48.9 and 60.8% compared to control in C19 mild and C19 severe, respectively (N = 3, n = 9–12, ****p < 0.0001). D IL-6 levels in mild and severe C19 patients were analyzed. In C19 patients, IL-6 increases compared to the healthy control reference level (7 ng/L). IL-6 was significantly higher in severe than in mild C19 samples
Treatment with spironolactone attenuates COVID-19 (C19)-induced endothelial glycocalyx (eGC) damage. A Sera from COVID-19 patients (mild course) were pooled and used for 24 h stimulation (10%) on endothelial cells. Treatment with C19 sera damaged the eGC and reduced height by 46% compared to treatment with healthy control sera. Coincubation with spironolactone strongly diminished the detrimental COVID-19 effect on eGC height within the C19-treated group (N = 3, n = 4–8; ****p < 0.0001). B Exemplary WGA stainings of HUVECs treated with 10% control or C19 sera with and without additional spironolactone incubation (100 nM). C WGA stainings were used to validate atomic force microscope measurements. Reduced eGC height in C19-treated cells was confirmed by decreased WGA fluorescence intensity compared to the control group. Spironolactone treatment strongly attenuated this effect (N = 3, n = 3–4, *p < 0.05, ****p < 0.0001)
Proinflammatory cytokines target vascular endothelial cells during COVID-19 infections. In particular, the endothelial gly-cocalyx (eGC), a proteoglycan-rich layer on top of endothelial cells, was identified as a vulnerable, vasoprotective structure during infections. Thus, eGC damage can be seen as a hallmark in the development of endothelial dysfunction and inflam-matory processes. Using sera derived from patients suffering from COVID-19, we could demonstrate that the eGC became progressively worse in relation to disease severity (mild vs severe course) and in correlation to IL-6 levels. This could be prevented by administering low doses of spironolactone, a well-known and highly specific aldosterone receptor antagonist. Our results confirm that SARS-CoV-2 infections cause eGC damage and endothelial dysfunction and we outline the underlying mechanisms and suggest potential therapeutic options.
Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca2+ concentration ([Ca2+]i). The mechanisms enhancing airway ciliary beating by nAChRs have remained largely unknown, although those by mAChRs are well understood. In this study, we focused on the effects of α7-nAChRs and voltage-gated Ca2+ channels (CaVs) on the airway ciliary beating. The activities of ciliary beating were assessed by frequency (CBF, ciliary beat frequency) and amplitude (CBD, ciliary bend distance) measured by high-speed video microscopy. ACh enhanced CBF and CBD by 25% mediated by an [Ca2+]i increase stimulated by mAChRs and α7-nAChRs (a subunit of nAChR) in airway ciliary cells of mice. Experiments using PNU282987 (an agonist of α7-nAChR) and MLA (an inhibitor of α7-nAChR) revealed that CBF and CBD enhanced by α7-nAChR are approximately 50% of those enhanced by ACh. CBF, CBD, and [Ca2+]i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of CaVs by α7-nAChRs. Experiments using a high K+ solution with/without nifedipine (155.5 mM K+) showed that the activation of CaVs enhances CBF and CBD via an [Ca2+]i increase. Immunofluorescence and immunoblotting studies demonstrated that Cav1.2 and α7-nAChR are expressed in airway cilia. Moreover, IL-13 stimulated MLA-sensitive increases in CBF and CBD in airway ciliary cells, suggesting an autocrine regulation of ciliary beating by CaV1.2/α7-nAChR/ACh. In conclusion, a novel Ca2+ signalling pathway in airway cilia, CaV1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways.
Electron transport chain transfer protons H⁺ across a membrane to synthesize ATP. Created with
Mitochondrial coupling and uncoupling. A Mitochondrial coupling: proton pumps of the electron transport chain uses redox energy to generate proton motive force. This force will regenerate ATP by ATP synthase. B Mitochondrial uncoupling inhibits the coupling between electron transport and ATP-synthetic reactions. This in turn causes loss of the energy as heat. Created with
Measuring mitochondrial temperature by fluorescent thermosensors. a Cells are stained by fluorescent thermosensors and gradually heated. b Fluorescence intensity, measured by means of fluorescent microscopy at different temperatures will be recorded. c A plot of fluorescence intensity versus temperature will be drawn “Calibration plot.” d Uncoupler reagent (such as FCCP) will be added to induce mitochondrial uncoupling. e By means of fluorescence microscope, the fluorescence intensity of the cells will be measured by fluorescence microscopy and plotted (c) to conclude the cell temperature
Mitochondrial temperature fluctuation response to FCCP inhibition in HeLa cells. Live HeLa cells were prestained with the T sensing probe (0.5 mg mL⁻¹, 20 min) and the ATP sensing probe (5.0 μM, 20 min). The intensity data was obtained from 25 live HeLa cells. Reprinted with permission from “Qiao, J., Chen, C., Shangguan, D., Mu, X., Wang, S., Jiang, L., & Qi, L. (2018). Simultaneous monitoring of mitochondrial temperature and ATP fluctuation using fluorescent probes in living cells. Analytical chemistry, 90(21), 12,553–12,558”, Fig. 6A. Copyright 2022 American Chemical Society
Visualization of mitochondrial thermal dynamics in HeLa cells response to glucose stimulations. (Left) Upconversion nanoparticles (UCNPs) at (3carboxypropyl) triphenylphosphonium bromide (TPP) images. MitoTracker (red) and UCNPs@TPP (green) from different treatments. (Middle) Mitochondrial temperature dynamics in the presence of 5 mg/mL glucose within 30 min. (Right) Student’s t test of both no glucose and glucose at 10 min (p < 0.0001). Reprinted with permission from “Di, X., Wang, D., Zhou, J., Zhang, L., Stenzel, M. H., Su, Q. P., & Jin, D. (2021). Quantitatively monitoring in situ mitochondrial thermal dynamics by upconversion nanoparticles. Nano letters, 21(4), 1651–1658”, Fig. 4A. Copyright 2022 American Chemical Society
Mitochondrial temperature is produced by various metabolic processes inside the mitochondria, particularly oxidative phos-phorylation. It was recently reported that mitochondria could normally operate at high temperatures that can reach 50℃. The aim of this review is to identify mitochondrial temperature differences between normal cells and cancer cells. Herein, we discussed the different types of mitochondrial thermosensors and their advantages and disadvantages. We reviewed the studies assessing the mitochondrial temperature in cancer cells and normal cells. We shed the light on the factors involved in maintaining the mitochondrial temperature of normal cells compared to cancer cells.
Hemodynamic results. LVSP, left ventricular systolic pressure (n = 7–8); LV dP/dtmax, left ventricular contractility (n = 7–8); RVSP, right ventricular systolic pressure (n = 7–9); RV dP/dtmax,right ventricular contractility (n = 7–9); CI, cardiac index (n = 6–8); TPR, total peripheral resistance (n = 6–7). Data is given as mean ± SEM. Significant differences vs. NCtrl: * p < 0.05; ** p < 0.01; *** p < 0.001; significant differences vs NRLX-L: # p < 0.05
Lung histology. A NCtrl, normoxic control: normal lung tissue without edema. B HCtrl, hypoxic control: moderate interstitial edema. C HRLX-L, hypoxia + 15 μg RLX kg⁻¹ day⁻¹: moderate interstitial edema. D HRLX-H, hypoxia + 75 μg RLX kg⁻¹ day⁻¹: severe interstitial edema. All slices (A, B C, D) are stained with hematoxylin–eosin; original magnification 5 × . Histologic examples for the normoxic RLX-L group are given in Fig. 3. E Pulmonary edema index (total lung). Data is given as mean ± SEM, n = 8–10. Significant differences vs. NCtrl: * p = 0.017; ** p = 0.002; *** p = 0.001
Lung histology, comparison basal lobe (BL) vs. apical lobe (AL). A NRLX-L, normoxia + 15 μg RLX kg⁻¹ day⁻.¹), basal lobe: moderate interstitial edema. B NRLX-L (same animal as in (A)), apical lobe: severe interstitial edema. The slices (A, B) are stained with hematoxylin–eosin; original magnification 5 × . C Pulmonary edema index determined in basal lobe (BL ) and apical lobe (AL ). Data is given as mean ± SEM, n = 8–10. Significant differences vs. NCtrl: * p = 0.025; *** p < 0.001; significant differences AL vs. BL: p < 0.05
TNFα expression in lung tissue. Upper panels, left: NCtrl, normoxic control: normal lung tissue without inflammation; right: HRLX-H, hypoxia + 75 μg RLX kg⁻¹ day⁻¹: marked peribronchial expression of TNFα. Middle panels, left: HCtrl, hypoxic control: mild-to-moderate expression of TNFα; mid: NRLX-L, normoxia + 15 μg RLX kg⁻¹ day⁻¹: mild expression of TNFα; right: HRLX-L, hypoxia + 15 μg RLX kg⁻¹ day⁻¹: mild-to-moderate expression of TNFα. All slices: original magnification 10 × . Lower panel: TNFα expression (in a.u./µm²). Data is given as medians (lines in the boxes) with 25th/75th percentiles (boxes), 10th/ percentiles (whiskers), and outliers (circles); n = 7–10. Significant differences vs. NCtrl: * p = 0.014; ** p = 0.002
Acute hypoxia impairs left ventricular (LV) inotropic function and induces development of pulmonary edema (PE). Enhanced and uneven hypoxic pulmonary vasoconstriction is an important pathogenic factor of hypoxic PE. We hypothesized that the potent vasodilator relaxin might reduce hypoxic pulmonary vasoconstriction and prevent PE formation. Furthermore, as relaxin has shown beneficial effects in acute heart failure, we expected that relaxin might also improve LV inotropic function in hypoxia. Forty-two rats were exposed over 24 h to normoxia or hypoxia (10% N 2 in O 2 ). They were infused with either 0.9% NaCl solution (normoxic/hypoxic controls) or relaxin at two doses (15 and 75 μg kg ⁻¹ day ⁻¹ ). After 24 h, hemodynamic measurements and bronchoalveolar lavage were performed. Lung tissue was obtained for histological and immunohistochemical analyses. Hypoxic control rats presented significant depression of LV systolic pressure by 19% and of left and right ventricular contractility by about 40%. Relaxin did not prevent the hypoxic decrease in LV inotropic function, but re-increased right ventricular contractility. Moreover, hypoxia induced moderate interstitial PE and inflammation in the lung. Contrasting to our hypothesis, relaxin did not prevent hypoxia-induced pulmonary edema and inflammation. In hypoxic control rats, PE was similarly distributed in the apical and basal lung lobes. In relaxin-treated rats, PE index was 35–40% higher in the apical than in the basal lobe, which is probably due to gravity effects. We suggest that relaxin induced exaggerated vasodilation, and hence pulmonary overperfusion. In conclusion, the results show that relaxin does not prevent but rather may aggravate PE formation.
Blood pressure measurements in mice receiving continuous i.v. IL-17A for 8 days. Blood pressure measurements (n = 11) are presented as circadian rhythm average day/night blood pressure measurements (left side) and average blood pressure measurements at baseline and during doses 1 (3.2), 2 (16 or 32), and 3 (32 or 320 ng/kg/min) (right side). A Mean arterial pressure (MAP) was significantly decreased at all 3 doses of IL-17A infusion. B, C Systolic and diastolic blood pressures (SBP, DBP) were significantly decreased during high-dose IL-17A infusion. D Heart rate (HR) was unchanged. E Plasma IL-17A and IL-6 concentrations were significantly elevated above baseline levels. Red and black circles represent mice that received lower and higher concentrations of IL-17A respectively. For statistical comparison between baseline and dose 1, 2, and 3 blood pressure and heart rate, one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison test was used (MAP, heart rate) or Friedmann’s test followed by Dunn’s multiple comparison test was performed (SBP, DBP). Differences in plasma cytokine concentrations were analyzed with paired t-tests. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). Data are presented as mean ± SEM or median with interquartile range
Blood pressure measurements in mice receiving i.v. IL-17A bolus infusions (n = 5). A, B Mean arterial pressure (MAP) and heart rate (HR) recordings after bolus infusion with four different concentrations (455, 910, 1820, and 3640 mg/kg) with 10-min interval. No blood pressure changes were observed during IL-17A infusion. C Bolus infusion (1–4) did not affect MAP when compared to baseline or glucose/heparin infusion. During ANGII and ACh infusion, MAP increased with 15 mmHg and decreased with 10 mmHg respectively when compared to baseline levels, and heart rate (HR) was unchanged. D Plasma IL-17A concentrations were significantly elevated after IL-17A infusion. Red and black circles represent mice that received 2 and 4 bolus infusions of IL-17A respectively. For statistical comparison between baseline or glu/hep bolus and after IL-17A infusion MAP or HR measurements, one-way ANOVA with repeated measures followed by Dunnett’s multiple comparison test was performed. For baseline and after IL-17A infusion comparison of plasma IL-17A levels, a paired t-test was performed. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001). Data are presented as mean ± SEM
Blood pressure measurements in mice receiving i.v. ANGII with IL-17A (n = 5) or saline (n = 5). Blood pressure measurements are presented as average 12-h day/night measurements (left side). Blood pressure measurements at baseline, during IL-17A/saline infusion and ANGII infusion, are presented as average measurements over 3, 2, and 7 days respectively from 5 mice (right side). A Mean arterial pressure (MAP), B systolic blood pressure (SBP), and C diastolic blood pressure (DBP) all increased upon ANGII infusion in both groups. D Heart rate decreased upon ANGII infusion in both groups. Blood pressure changes between groups during IL-17A/saline infusion alone were not different. MAP, SBP, and DBP were all significantly attenuated during ANGII co-infusion with IL-17A when compared to vehicle mice. Data are presented as median with interquartile range. For statistical comparison, a two-way ANOVA with mixed analysis was performed followed by Tukey’s multiple comparison test. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)
Plasma IL-17A and IL-6 concentration. A Plasma IL-17A was unchanged in mice that were given ANGII infusion with saline, B but significantly increased in those given IL-17A. C-D Plasma IL-6 increased in 3 of the mice that were given ANGII with saline or IL-17A, where IL-17A addition increased IL-6 by approximately 3-fold, however not statistically significant (n = 4 in each group). For statistical comparison, paired t-test or Mann-Whitney test was carried out. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001)
Interleukin 17A (IL-17A) is a candidate mediator of inflammation-driven hypertension, but its direct effect on blood pressure is obscure. The present study was designed to test the hypothesis that systemic IL-17A concentration-dependently increases blood pressure and amplifies ANGII-induced hypertension in mice. Blood pressure was measured by indwelling chronic femoral catheters before and during IL-17A infusion w/wo angiotensin II (ANGII, 60ng/kg/min) in male FVB/n mice. Baseline blood pressure was recorded, and three experimental series were conducted: (1) IL-17A infusion with increasing concentrations over 6 days (two series with IL-17A from two vendors, n = 11); (2) ANGII infusion with IL-17A or vehicle for 9 days (n = 11); and (3) acute bolus infusions with four different concentrations (n = 5). Plasma IL-17A and IL-6 concentrations were determined by ELISA. Mean arterial and systolic blood pressures (MAP, SBP) decreased significantly after IL-17A infusion while heart rate was unchanged. In these mice, plasma IL-17A and IL-6 concentrations increased up to 3500- and 2.4-fold, respectively, above baseline. ANGII infusion increased MAP (~ 25 mmHg) and co-infusion of IL-17A attenuated ANGII-induced hypertension by 4.0 mmHg. Here, plasma IL-17A increased 350-fold above baseline. Acute IL-17A bolus infusion did not change blood pressure or heart rate. IL-17A receptor and IL-6 mRNAs were detected in aorta, heart, and kidneys of mice after IL-17A infusion. Nonphysiologically high concentrations of IL-17A reduce baseline blood pressure and increase IL-6 formation in male FVB/n mice. It is concluded that IL-17A is less likely to drive hypertension as the sole cytokine mediator during inflammation in vivo.
Effect of CoCl2 added at the beginning of reperfusion on heart mechanical and energetic parameters during I/R. Changes in end diastolic pressure (ΔLVEDP) (A), percentage of intraventricular pressure developed during contraction (P) (B), percentage of total heat flow (Ht) (C), and percentage of total muscle economy (P/Ht) (D) measured in rat hearts exposed to I/R in the presence (O) or absence of CoCl2 (●) during reperfusion, or in the presence of CoCl2 during reperfusion for 20 min and subsequent withdrawal (▲). Black arrow indicates the time of CoCl2 withdrawal. B: basal, I: ischemia, R: reperfusion. Data are expressed as the mean ± SEM. Control: n = 5, CoCl2: n = 6, CoCl2 withdrawal: n = 6. Two-way ANOVA for repeated measurements: * p < 0.05 CoCl2 vs. control, § p < 0.05 CoCl2 withdrawal vs. control
Arrhythmias. A Representative software image where dark bars indicate the presence of arrhythmia. B: Quantification of cardiac arrhythmias produced between 10 and 20 min of reperfusion period in the presence or absence of CoCl2. Bars represent the mean value of each group. Control: n = 5, CoCl2: n = 7. Mann–Whitney test: * p < 0.05 vs. control
Myocardial Infarction Area. A Representative images of the heart sections dyed with TTC. B Quantification of the infarct size area. Bars represent the mean value of each group. Mann–Whitney test *p < 0.05 vs control
Contracture induced by caffeine-low Na⁺ media. Changes in contracture pressure (ΔCP) above the LVEDP of immediately previous beating heart induced by caffeine and low Na⁺ media in hearts perfused for 20 min in the presence (O) or absence of CoCl2 (●) during reperfusion (A). Area under the curve of contracture (B) and the rate of contracture tension relaxation (C). Bars represent the mean value of each group. Control: n = 7, CoCl2: n = 8. T test: *p < 0.05 vs. control
Effect of CoCl2 added later during reperfusion on heart mechanical and energetic parameters during I/R. Changes in diastolic pressure (ΔLVEDP) (A), percentage of intraventricular pressure developed during contraction (P) (B), percentage of total heat flow (Ht) (C) and percentage of total muscle economy (P/Ht) (D) measured in rat hearts exposed to I/R in the absence of CoCl2 (●) or in the presence of CoCl2 added at 20 min of reperfusion. Black arrow indicates the time of CoCl2 addition. B: basal, I: ischemia, R: reperfusion. Data are expressed as the mean ± SEM. Control: n = 5, CoCl2: n = 7. Two-way ANOVA for repeated measurements. No significant differences vs control
Since damage induced by ischemia–reperfusion (I/R) involves alterations in Ca²⁺ homeostasis and is reduced by ischemic postconditioning (IP) and that CoCl2 can trigger changes resembling the response to a hypoxic event in normoxia and its blockade on Ca²⁺ current in heart muscle, our aim was to evaluate CoCl2 as an IP therapeutic tool. Mechanic and energetic parameters of isolated and arterially perfused male Wistar rat heart ventricles were simultaneously analyzed in a model of I/R in which 0.23 mmol/L CoCl2 was introduced upon reperfusion and kept or withdrawn after 20 min or introduced after 20 min of reperfusion. The presence of CoCl2 did not affect diastolic pressure but increased post-ischemic contractile recovery, which peaked at 20 min and decreased at the end of reperfusion. This decrease was prevented when CoCl2 was removed at 20 min of reperfusion. Total heat release increased throughout reperfusion, while economy increased between 15 and 25 min. No effect was observed when CoCl2 was introduced at 20 min of reperfusion. In addition, both the area under the contracture curve evoked by 10 mmol/L caffeine–36 mmol/L Na⁺ and the contracture tension relaxation rate were higher with CoCl2. Furthermore, CoCl2 decreased the number of arrhythmias during reperfusion and the ventricular damaged area. The presence of CoCl2 in reperfusion induces cardioprotection consistent with the improvement in cellular calcium handling. The use of CoCl2 constitutes a potential cardioprotective tool of clinical relevance.
Mild to moderate-intensity endurance exercise training combined with hind-limb blood flow restriction (BFR) induces elderly heart rejuvenation and improves cardiac inotropy and resistance to ischemia. However, the mediators of these beneficial effects are still not well known. The present study investigated the possible role of some important molecules in the mediatory of this model of exercise training in the promotion of heart health in aged rats. Male old Wistar rats randomly were divided into control-sham (CTL), hind limbs blood flow restriction (BFR), sham-operated plus 10 weeks' treadmill exercise training (Ex), and BFR plus exercise (BFR + Ex) groups. Left ventricular end-diastolic pressure (LVEDP), contractility, and Tau indices were measured. ELISA and western blot tests were used for measuring determined cardiac biochemical factors. BFR + Ex displayed significantly lower LVEDP (P < 0.05 and P < 0.01 vs. Ex, and other groups, respectively), improved heart cardiac contractility (P < 0.01), and significantly reduced Tau index in comparison with other groups. BFR + Ex significantly reduced both BAX and BAX to BCL2 ratio (P < 0.05) and as well MDA to TAC ratio (P < 0.05, compared to the CTL group). Also, BFR + Ex significantly increased the level of klotho (P < 0.05) and PGC1-α (P < 0.001) proteins compared to the CTL group but had no significant effect on P-STAT3 expression. Exercise training alone increased Apelin protein (P < 0.05). Our findings suggest that mild to moderate BFR endurance training improves heart performance in the aging rat partly through ameliorating apoptosis, recovering redox balance, improving the longevity factor klotho, and increasing the key energy metabolism regulator PGC1-α.
Schematic illustrating the presence of different ion channel subunits that may be expressed in native HEK-293 cells endogenously. These include voltage-gated Na⁺ channels (which may be encoded by NaV1.7), the β1A subunit, Ca²⁺ channels (which may be encoded by CaV1.2 and CaV1.3), outward rectifier K⁺ channels (which may be encoded by KV1.1, KV1.2, KV1.3, KV1.6, or KV3.1), transient outward K⁺ channels (which may be encoded by KV1.4, KV3.3, KV3.4, or KV4.1), Kvβ, Ca²⁺-activated K⁺ channels, voltage-gated Cl⁻ channels (which may be encoded by CLC-2), TMEM16A-mediated Ca²⁺-activated Cl⁻ channels, TMEM206-mediated proton-activated Cl⁻ channels, β3 subunit for GABAA receptors, LRRC8A/C swelling-activated Cl⁻ channels, transient receptor potential (TRP) channels, nonselective cation channels, acid-sensitive ion channels (ASIC1a), store-operated Ca²⁺ channels (Orail 1), and mechanical activated channels (Piezo1). Note that Ca²⁺-activated K⁺ channels and Cl⁻ channels can be activated by Ca²⁺ influx via voltage-gated Ca²⁺ channels, TRP channels, nonselective cation channels, Orail 1 channels, and Piezo1 channels
Mammalian expression systems, particularly the human embryonic kidney (HEK-293) cells, combined with electrophysiological studies, have greatly benefited our understanding of the function, characteristic, and regulation of various ion channels. It was previously assumed that the existence of endogenous ion channels in native HEK-293 cells could be negligible. Still, more and more ion channels are gradually reported in native HEK-293 cells, which should draw our attention. In this regard, we summarize the different ion channels that are endogenously expressed in HEK-293 cells, including voltage-gated Na⁺ channels, Ca²⁺ channels, K⁺ channels, Cl⁻ channels, nonselective cation channels, TRP channels, acid-sensitive ion channels, and Piezo channels, which may complicate the recording of the heterogeneously expressed ion channels to a certain degree. We noted that the expression patterns and channel profiles varied with different studies, which may be due to the distinct originality of the cells, cell culture conditions, passage numbers, and different recording protocols. Therefore, a better knowledge of endogenous ion channels may help minimize potential problems in characterizing heterologously expressed ion channels. Based on this, it is recommended that HEK-293 cells from unknown sources should be examined before transfection for the characterization of their functional profile, especially when the expression level of exogenous ion channels does not overwhelm the endogenous ion channels largely, or the current amplitude is not significantly higher than the native currents.
HCN4 is expressed in the sinoatrial node. (A) Right, schematic diagram of the cardiac conduction system (green). The primary pacemaker site is the sinoatrial node (SAN). The atrioventricular node (AVN) is the only electrically conductive connection between the atria and the ventricles. The bundle of His (His) splits into a left and right bundle branch (LBB/RBB, left/right bundle branch), which spread out to the left and right Purkinje fiber (PF) network. Abbreviations: LA, left atrium; RA, right atrium; PV, pulmonary veins; VCS, superior vena cava; VCI, inferior vena cava; CS, coronary sinus; RV, right ventricle; LV, left ventricle; VS, ventricular septum. (A) Left, upper panel: distribution of HCN4 (green) in a transverse section of the murine SAN. HCN4 is expressed across the entire SAN region. Abbreviations: CT, crista terminalis; IAS, interatrial septum. Scale bar: 100 µm. Lower panel: schematic illustration of the original image shown in the upper panel. (B) Action potential recordings of isolated SAN cells demonstrating the chronotropic effect at the single cell level. Input from the sympathetic nervous system accelerates SDD and increases the firing rate of pacemaker cells, whereas input from the parasympathetic nervous system slows down SDD and decelerates the firing rate. Abbreviations: SDD, slow diastolic depolarization; TP, threshold potential; NS, nervous system
CDR and hysteresis of HCN4 control the firing mode of SAN cells. (A) Action potential recording of an isolated pacemaker cell showing the typical alternation between firing and nonfiring. The mean membrane potential is more depolarized during firing (~ − 55 mV, green line) and more hyperpolarized during nonfiring (~ − 75 mV, red line). At the same time, slow drifts in membrane potential occur. The firing mode is characterized by a slow, progressive hyperpolarization (Δ =  − 7 mV) until firing stops (1), leading to an abrupt drop to significantly more hyperpolarized potentials. Conversely, during nonfiring, a slow and progressive depolarization occurs until the threshold for firing is reached (2), and the membrane potential abruptly jumps to substantially more depolarized values. Due to hysteresis of HCN4, the changes in membrane potential have important consequences for the voltage-dependent activation of the channel. (B) Original steady-state activation curves recorded from HCN4 channels heterologously expressed in HEK239 cells without cAMP in the intracellular solution. The long-lasting, mean membrane potentials during firing (− 55 mV) and nonfiring (− 75 mV) are mimicked by the holding potential (HP). At a relatively depolarized holding potential of − 55 mV, the activation curve is positioned at extremely hyperpolarized voltages (left curve). Conversely, at a relatively hyperpolarized holding potential of − 75 mV, the activation curve is positioned at extremely depolarized voltages (right curve). Points (1) and (2) on the activation curves reflect the time points (1) and (2) of the action potential measurements shown in panel (A). At the end of firing (1), the activation curve is shifted to the left and the membrane potential is relatively positive, resulting in a small number of open HCN4 channels. The consequent lack of a sufficiently depolarizing If current causes or supports the transition of pacemaker cells to the nonfiring mode. At the end of nonfiring (2), the activation curve is shifted to the right and the membrane potential is relatively negative, leading to a substantial increase in the number of open HCN4 channels, thereby causing or supporting the return of pacemaker cells to the firing mode. (C) Original activation curves of HCN4 channels recorded in the presence of 100 µM cAMP in the intracellular solution. Compared to panel (B), both curves are markedly shifted to the right. Under comparable conditions, SAN cells do not switch into the nonfiring mode, and the mean membrane potential permanently remains at depolarized values (− 55 mV), leading to a sufficient number of open HCN4 channels to maintain continuous firing
Tonic entrainment in the SAN. (A) Scheme of a SAN cell visualizing the signal transduction pathway following stimulation by the ANS. Gs protein-coupled beta-1-adrenergic receptors (green) are activated by norepinephrine (NE) released from sympathetic nerve terminals. Subsequent Gαs signaling stimulates adenylyl cyclases (ACs, gray) to synthetize cAMP, which directly activates HCN4 channels. Conversely, Gi protein-coupled M2 muscarinic receptors (red) are activated by acetylcholine (ACh) released from vagal nerve terminals. Gαi signaling inhibits ACs and thereby reduces the intracellular cAMP level, leading to a decrease in HCN4 activity. Similar effects are evoked by TRIP8bnano, a synthetic peptide that binds to the CNBD of HCN4 and inhibits cAMP-dependent activation of the channel. (B) cAMP-dependent activation of HCN4 reduces the number of nonfiring cells in the SAN, which stabilizes the network rhythm during HR acceleration. (C) Reduction in cAMP-dependent activation of HCN4 increases the number of nonfiring cells in the SAN. Overshooting inhibition leads to bradycardia and SAN dysrhythmia due to destabilization of the network rhythm. (D) The tonic entrainment process takes place between firing cells (left) and neighboring nonfiring cells (right). Pacemaker cells in the nonfiring mode are more hyperpolarized and electrotonically draw the flow of cations from more depolarized neighboring cells in the firing mode via gap junctions. This slightly depolarizes the nonfiring cells (green arrow) and hyperpolarizes the firing cells to the same extent (red arrow)
Cardiac phenotype of HCN4FEA mice. (A) Telemetric ECG trace of an HCN4FEA mouse showing severe sinus dysrhythmia. (B) Mean (left), minimum (middle), and maximum (right) heart rate of WT (black) and HCN4FEA mice (green) calculated from 72-h telemetric ECG recordings. (C) Heart rate histograms determined from 72-h recordings. In HCN4FEA mice, the average HR and full HR range is shifted towards lower HR values, demonstrating intrinsic bradycardia. (D) Comet-shaped Poincaré plots display high beat-to-beat dispersion in HCN4FEA mice (green). (E) Tachograms of WT (black) and HCN4FEA mice (green) before and after consecutive injections of propranolol and atropine. (F) Optical imaging measurements of biatrial SAN explants reveal prolonged sinoatrial conduction time (SACT) in HCN4FEA mice. (G) Quantification of SACT determined from optical measurements as shown in panel (F). (H) Combined telemetric blood pressure (upper panel) and ECG recordings (RR intervals, lower panel) used to determine baroreflex sensitivity in vivo. (I) Plot of systolic blood pressure (SBP) and corresponding RR intervals (upper panel) demonstrates a steeper slope of the RR/SBP relationship in HCN4FEA mice (green), reflecting inappropriately enhanced HR responses of the SAN to vagal nerve activity in HCN4FEA mice. Lower panel: quantification of the slope of RR/SBP relations in WT (black) and HCN4FEA mice (green). (J) Telemetric ECG trace of an HCN4FEA mouse during episodes with junctional escape rhythm (JER). (K) Telemetric ECG trace of an HCN4FEA mouse during episodes with isorhythmic AV dissociation (IAVD). Figure is
modified from Fenske et al. [22]
Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are the molecular correlate of the If current and are critically involved in controlling neuronal excitability and the autonomous rhythm of the heart. The HCN4 isoform is the main HCN channel subtype expressed in the sinoatrial node (SAN), a tissue composed of specialized pacemaker cells responsible for generating the intrinsic heartbeat. More than 40 years ago, the If current was first discovered in rabbit SAN tissue. Along with this discovery, a theory was proposed that cyclic adenosine monophosphate–dependent modulation of If mediates heart rate regulation by the autonomic nervous system—a process called chronotropic effect. However, up to the present day, this classical theory could not be reliably validated. Recently, new concepts emerged confirming that HCN4 channels indeed play an important role in heart rate regulation. However, the cellular mechanism by which HCN4 controls heart rate turned out to be completely different than originally postulated. Here, we review the latest findings regarding the physiological role of HCN4 in the SAN. We describe a newly discovered mechanism underlying heart rate regulation by HCN4 at the tissue and single cell levels, and we discuss these observations in the context of results from previously studied HCN4 mouse models.
How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel’s α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to invitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 β (GSK3β). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3β had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3β but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3β resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3β inhibits the currents.
KCNQ channels participate in the physiology of several cell types. In neurons of the central nervous system, the primary subunits are KCNQ2, 3, and 5. Activation of these channels silence the neurons, limiting action potential duration and preventing high-frequency action potential burst. Loss-of-function mutations of the KCNQ channels are associated with a wide spectrum of phenotypes characterized by hyperexcitability. Hence, pharmacological activation of these channels is an attractive strategy to treat epilepsy and other hyperexcitability conditions as are the evolution of stroke and traumatic brain injury. In this work we show that triclosan, a bactericide widely used in personal care products, activates the KCNQ3 channels but not the KCNQ2. Triclosan induces a voltage shift in the activation, increases the conductance, and slows the closing of the channel. The response is independent of PIP2. Molecular docking simulations together with site-directed mutagenesis suggest that the putative binding site is in the voltage sensor domain. Our results indicate that triclosan is a new activator for KCNQ channels.
Linescan method for in vivo SNGFR measurements. Panels a and b show the glomerular filtration of low-molecular weight FITC-dextran (3–5 kDa) during the linescan acquisition. Multiple crossings are hand-drawn perpendicularly to the tubular lumen and acquired while the fluorescent dextran is injected. FITC 3–5 kDa (green) is freely filtered through the glomerulus (G) and streams along the tubular lumen of early proximal tubule (S1). t represents the time in seconds after bolus injection. Scale bar is 50 μm. In panel c, each fluorescent line corresponds to a tubular crossing. Red selected areas indicate the two tubular crosses used for the analysis. In panel d, FITC 3–5-kDa fluorescence intensity as arbitrary unit (AU) acquired at red selected areas is plotted over time. The blue curves represent the original intensity plots, while the fitted curves are shown in red. Panel e shows SNGFR as calculated in the same tubules (individual colors) at different distances between the two crosses (45 measurements from 12 different tubules)
SNGFR measurement with linescan method. In panel a, SNGFR values evaluated by linescan method (black square) were compared with data obtained by full-frame MPM (gray square) [15] and micropuncture studies [23, 32] (white square). SNGFR measurements in mice were compared with micropuncture data [19]. *** is for p-value < 0.001 (unpaired t-test). For male rats one-way ANOVA followed by Tukey’s multiple comparison test was used. In panel b, the average number of glomeruli-S1 complex per rat acquired with linescan or micropuncture ([4, 11, 24, 27, 28, 35]) is reported (15 ± 0.6 from 10 rats versus 4.7 ± 0.72 from 6 different experimental studies). **** is for p-value < 0.0001 (unpaired t test). In panel c, the distribution of the SNGFR values per single glomerulus evaluated by linescan tool is measured at control, low dose dopamine infusion and after IRI. * is for p-value < 0.05 and ** is for p-value < 0.01 versus the control group (one-way ANOVA, followed by Tukey’s multiple comparison test). In panel d, the mean SNGFR values evaluated at control, low-dose dopamine infusion and IRI with linescan method or micropuncture were compared. * is for p-value < 0.05 (unpaired t test). All the data are expressed as mean ± standard error
Visualization of healthy and ischemic rat kidney. Representative images from control MWF rats (panels a and b) and IRI-treated rats (panels c and d). Renal vasculature is labeled with TRITC-dextran 500 kDa (red), while kidney autofluorescence appears in blue. Tubular damage occurs after 30 min from IRI. Altered tubular morphology (asterisks) and intraluminal debris (arrows) appear during the reperfusion phase. Panels b and d include the vasculature. Scale bar is 50 μm
Renal micropuncture, which requires the direct access to the renal tubules, has for long time been the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models. This approach is challenging by virtue of complex animal preparation and numerous technically difficult steps. The introduction of intravital multiphoton microscopy (MPM) offers another approach to the measure of the SNGFR by mean of the high laser-tissue penetration and the optical sectioning capacity. Previous MPM studies measuring SNGFR in vivo relied on fast full-frame acquisition during the filtration process obtainable with high performance resonant scanners. In this study, we describe an innovative linescan–based MPM method. The new method can discriminate SNGFR variations both in conditions of low and high glomerular filtration, and shows results comparable to conventional micropuncture both for rats and mice. Moreover, this novel approach has improved spatial and time resolution and is faster than previous methods, thus enabling the investigation of SNGFR from more tubules and improving options for data-analysis.
Respiratory rhythm can bind most areas in low and high frequency bands during quiet waking. (A) Raw LFP traces from aPCX (black) and mPFC (gray), and the respiratory signal (green). This dataset collects simultaneous LFP recordings from 6 brain areas (medial prefrontal cortex (mPFC), anterior piriform cortex (aPCX), primary somatosensory cortex (S1), primary visual cortex (V1), CA1 and dentate gyrus (DG)), and respiration. (B) Summarized methods for constructing graphs in C. (B Left) Step-by-step method for quantification of RRo co-occurrence in low frequency (0.3–15 Hz) LFP. ① LFP low pass (0.3–15 Hz) filtering. ② RRo detection respiratory cycle by cycle in each area individually. ③ Quantification of RRo co-occurrence for each pair of areas and each brain state. ④ Probabilities averaging (n = 4 rats). (B Right) Step by step method for quantification of gamma (55–100 Hz) synchronization during RRo co-occurrence. Only periods associated with RRo were analyzed. ① LFP band-pass (55–100 Hz) filtering. ② Extraction of instantaneous LFP phase. Phase difference was obtained by subtraction. Stability of the phase difference (blue trace) was assessed as follows: mean vector of the phase difference signal was computed using sliding windows of 37.5 ms. The longest the vector, the more stable the phase difference (blue trace). ③ Stability of the phase difference averaged across respiratory cycles. ④ Quantification of the respiratory modulation: the mean vector of the respiratory cycle-averaged stability was computed. Length of this vector represents a respiratory modulation index; the longest, the strongest the modulation is. Length was computed and was statistically compared with surrogates. ⑤ Respiratory modulation index averaging. (C) Binding between the 6 areas network across brain states. (B Left) Width of black links between areas represents the mean RRo co-occurrence probability ± sem (gray). (B Right) Width of black links between areas represents the mean respiratory modulation index ± sem (gray). Number of averaged animals is indicated on the link
Respiration-driven networks are favored by respiratory pattern characteristics of quiet waking state. The brain state-dependent variability of respiratory pattern was tracked with inspiratory peak flowrate and duration; these two respiratory parameters being the best suited to differentiate the respiratory regimes [43]. For each network size (from one to six areas), the probability to observe a respiration-driven network is represented as a function of the inspiratory peak flowrate and duration. For a given bin of inspiratory peak flowrate and duration, this probability represents the number of respiratory cycles during which N areas displayed simultaneously respiration-related oscillations (RRo divided by the number of respiratory cycles in the bin). Each map is the average of data from 4 animals. In all animals, the 6 recorded areas were medial prefrontal cortex (mPFC), anterior piriform cortex (aPCX), primary somatosensory cortex (barrel field, S1), primary visual cortex (V1), CA1, and dentate gyrus (DG). Probabilities are color-coded. Each map has its own scale. Orange, green, red, and cyan curves represent the median distribution of respiratory cycles associated to quiet waking (QW), exploration (EX), slow wave sleep (SWS), and rapid-eye movements sleep (REM) respectively
As a possible body signal influencing brain dynamics, respiration is fundamental for perception, cognition, and emotion. The olfactory system has recently acquired its credentials by proving to be crucial in the transmission of respiratory influence on the brain via the sensitivity to nasal airflow of its receptor cells. Here, we present recent findings evidencing respiration-related activities in the brain. Then, we review the data explaining the fact that breathing is (i) nasal and (ii) being slow and deep is crucial in its ability to stimulate the olfactory system and consequently influence the brain. In conclusion, we propose a possible scenario explaining how this optimal respiratory regime can promote changes in brain dynamics of an olfacto-limbic-respiratory circuit, providing a possibility to induce calm and relaxation by coordinating breathing regime and brain state.
Schematic showing different pathways by which oxygen can modulate neural excitability. Top, oxygen levels in major supply arteries oscillate on a breath-by-breath basis, as well as showing an overall increase with respiration rate. Scale is for expected values in a mouse. Respiration shows idealized measurement from a thermocouple, with upswings representing exhalation. Bottom left, oxygen modulates K + channels and TASK activity in neurons. Bottom middle, oxygen modulates tryptophan hydroxylase (TPH) synthesis of serotonin (5-HT) and tyrosine hydroxylase (TyrH) synthesis of dopamine (D) and norepinephrine (NE). Coloration of neurons is aesthetic. Bottom right, oxygen decreases nitric oxide (NO) concentrations which modulates neural activity
a–b Respiration drives changes in cerebral and blood oxygenation. a Measuring respiration using a thermocouple. Top, example data showing tissue oxygenation in the somatosensory cortex of an awake, headfixed mouse measured using an oxygen sensitive microelectrode (black trace) and respiratory rate (orange trace), during locomotion. Middle, signal from a thermocouple placed near the nostril of the mouse. The thermocouple voltage tracks inhalation and exhalation due to the higher temperature of exhaled air, which causes increase in the thermocouple signal. Bottom left, expanded thermocouple signal showing of the detection of the onset of inspiratory (magenta dot) and expiratory phase (blue dot). Bottom right, schematic showing respiration measurement using a thermocouple. b Example data showing the temporal relation between respiratory rate (black) and oxygen tension (PaO2, blue) in the center of one artery in somatosensory cortex of a mouse during periods of rest. The phase shift is caused by transit time from lungs to brain. c PaO2 fluctuates within the respiratory cycle. The PaO2 change in one artery of a headfixed, un-anesthetized mouse during the respiratory cycle at rest was measured using an intravascularly injected phosphorescent oxygen dye using a two-photon microscope. This technique allows measurement of the concentration of oxygen in the blood plasma from a single location in the vasculature. PaO2 data (15 recordings with each of 50 s in duration) were aligned to the offset of inspiration. Each circle denotes averaged PaO2 over a short window (20 ms) and over the 15 recordings. The solid curve denotes filtering of data (first order binomial filter, 5 repetitions). Tmin denotes the time period (40 ms) PaO2 reaches minimum. Tmax denotes the time period (40 ms) PaO2 reaches maximum. d Example data showing the temporal relation between respiratory rate (black) and pupil diameter (blue, an indicator of noradrenergic activity) during periods of rest in an awake, headfixed mouse. e Cross-correlation between respiratory rate and pupil diameter during periods of rest. Gray shaded area indicates 95% confidence interval. f–g Nasal inhalation at visuospatial task onset is associated with improved performance in humans. f Mean event-related nasal respiratory signal used to trigger trial-onset time-locked to inhalation (orange) or exhalation (blue). Time 0 denotes task initiation. The gray rectangle along the x axis represents the stimulus (1,200 ms). Inset: a polar plot of the respiratory phase (in degrees) at trial onset is shown. The orange and blue bins are trials triggered by inhalation and exhalation, respectively (n = 28). g Scatter plot of performance in the EEG visuospatial task in inhalation and exhalation. Each point is a participant (n = 28). The diagonal line is the unit slope line (x = y). Thus, if points accumulate below the line, this means performance was better during inhalation. In the inlay, the mean group performance is shown. Error bars are SEM. a–c adapted from [123], f–g adapted from [75]
Oxygen is critical for neural metabolism, but under most physiological conditions, oxygen levels in the brain are far more than are required. Oxygen levels can be dynamically increased by increases in respiration rate that are tied to the arousal state of the brain and cognition, and not necessarily linked to exertion by the body. Why these changes in respiration occur when oxygen is already adequate has been a long-standing puzzle. In humans, performance on cognitive tasks can be affected by very high or very low oxygen levels, but whether the physiological changes in blood oxygenation produced by respiration have an appreciable effect is an open question. Oxygen has direct effects on potassium channels, increases the degradation rate of nitric oxide, and is rate limiting for the synthesis of some neuromodulators. We discuss whether oxygenation changes due to respiration contribute to neural dynamics associated with attention and arousal.
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.
We recently demonstrated that the hypoxic ventilatory response (HVR) is facilitated by the AMP-activated protein kinase (AMPK) in catecholaminergic neural networks that likely lie downstream of the carotid bodies within the caudal brainstem. Here, we further subcategorise the neurons involved, by cross-comparison of mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic (TH-Cre) or adrenergic (PNMT-Cre) neurons. As expected, the HVR was markedly attenuated in mice with AMPK-α1/α2 deletion in catecholaminergic neurons, but surprisingly was modestly augmented in mice with AMPK-α1/α2 deletion in adrenergic neurons when compared against a variety of controls (TH-Cre, PNMT-Cre, AMPK-α1/α2 floxed). Moreover, AMPK-α1/α2 deletion in catecholaminergic neurons precipitated marked hypoventilation and apnoea during poikilocapnic hypoxia, relative to controls, while mice with AMPK-α1/α2 deletion in adrenergic neurons entered relative hyperventilation with reduced apnoea frequency and duration. We conclude, therefore, that AMPK-dependent modulation of non-adrenergic networks may facilitate increases in ventilatory drive that shape the classical HVR, whereas AMPK-dependent modulation of adrenergic networks may provide some form of negative feedback or inhibitory input to moderate HVR, which could, for example, protect against hyperventilation-induced hypocapnia and respiratory alkalosis.
A Tree view of hDRG sensory neuron subpopulations, derived from Tavares-Ferreira et al. [52]. B Mapping of iPSC-derived sensory neuron equivalents to hDRG single-nuclei data derived from Nguyens (2021)
Nociceptors and associated cells in the target tissue (skin, left), DRG, and the spinal cord (right) to be implemented in complex model systems (generated with BioRender®)
Despite numerous studies which have explored the pathogenesis of pain disorders in preclinical models, there is a pronounced translational gap, which is at least partially caused by differences between the human and rodent nociceptive system. An elegant way to bridge this divide is the exploitation of human-induced pluripotent stem cell (iPSC) reprogramming into human iPSC-derived nociceptors (iDNs). Several protocols were developed and optimized to model nociceptive processes in health and disease. Here we provide an overview of the different approaches and summarize the knowledge obtained from such models on pain pathologies associated with monogenetic sensory disorders so far. In addition, novel perspectives offered by increasing the complexity of the model systems further to better reflect the natural environment of nociceptive neurons by involving other cell types in 3D model systems are described.
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.
Schematic illustration of ROP disease manifestation. Physiological retinal vessel growth in humans starts at the beginning of the fourth month of gestational age (“natural hypoxia”) and is normally finished shortly before full-term birth. Accordingly, premature infants have incompletely vascularized retinas. After birth, loss of nutrients and physiological growth factors provided at the maternal–fetal interface together with the increased oxygen pressure (oxygen pressure of ambient air ~ 160 mmHg; plus additional oxygen supplementation) result in a persistently undervascularized (vasoobliterative phase) and later hypoxic retina. This causes excess production of VEGF and other oxygen-regulated vascular growth factors, resulting in pathological retinal neovascularization (neovascularization phase)
of the main pathogenic events causing pathological neovascularization in the retina of patients with DR. In healthy retinal capillaries, adequate pericyte coverage supports endothelial cell survival and integrity of blood retina barrier. Long-term diabetes induces alterations and damage in several cell types resulting in progressive vasoregression. Occluded remnants of capillaries are no longer perfused, leading to tissue hypoxia and a subsequent upregulation of survival/growth factors such as VEGF. As a consequence, ischemia/hypoxia-induced, pathological neovascularization is triggered. EC endothelial cells, BRB blood retina barrier, ECM extracellular matrix, AGE advanced glycosylation end products, VEGF vascular endothelial growth factor, Epo erythropoietin
Schematic illustration of the mouse OIR model. Neonatal mice are kept in ambient air (21% oxygen) from birth until postnatal day 7 (P7); meanwhile, normal vascular development starts. At P7, mice are then exposed to 75% oxygen, resulting in the inhibition and regression of retinal vessel growth (vaso-obliteration). At P12, mice are returned to ambient air. The drop in oxygen pressure leads to the development of hypoxia in avascular retinal areas, triggering both normal vessel regrowth and pathological neovascularization. Pathological vessel growth reaches its maximum at P17 and spontaneously regresses thereafter until the retinal vasculature is completely normalized by P25
of the action of MPs causing pathological neovascularization in the retina. Two immune cell populations are described to affect pathological neovascularization. These are either macrophages derived from the circulation or resident microglia. In the stressed retina, ramified microglia become activated and differentiate into an M1 or M2 phenotype, which goes along with respective changes in cell morphology, proliferation, migration, phagocytosis, and alterations in cytokine/growth factor/protease production. MP mononuclear phagocyte, EC endothelial cells, BRB blood retina barrier, ECM extracellular matrix, GF growth factor, AGE advanced glycosylation end products VEGF vascular endothelial growth factor, Epo erythropoietin
Ischemic retinopathies (IR) are vision-threatening diseases that affect a substantial amount of people across all age groups worldwide. The current treatment options of photocoagulation and anti-VEGF therapy have side effects and are occasionally unable to prevent disease progression. It is therefore worthwhile to consider other molecular targets for the development of novel treatment strategies that could be safer and more efficient. During the manifestation of IR, the retina, normally an immune privileged tissue, encounters enhanced levels of cellular stress and inflammation that attract mononuclear phagocytes (MPs) from the blood stream and activate resident MPs (microglia). Activated MPs have a multitude of effects within the retinal tissue and have the potential to both counter and exacerbate the harmful tissue microenvironment. The present review discusses the current knowledge about the role of inflammation and activated retinal MPs in the major IRs: retinopathy of prematurity and diabetic retinopathy. We focus particularly on MPs and their secreted factors and cell–cell-based interactions between MPs and endothelial cells. We conclude that activated MPs play a major role in the manifestation and progression of IRs and could therefore become a promising new target for novel pharmacological intervention strategies in these diseases.
Domain structures of HIF-1α/β and its target genes related to metabolism. The HIF-1α subunit is shown in yellow to distinguish it from the HIF-1β subunit (shown in brown). bHLH, basic helix-loop-helix; PAS, Per-Arnt-SIM; ODDD, oxygen-dependent degradation domain; N-TAD, N-terminal transactivation domain; C-TAD, C-terminal transactivation domain; SLC2A1, solute carrier family 2 member 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3; PFKL, phosphofructokinase L; PGK1, phosphoglycerate kinase 1; PDK1, pyruvate dehydrogenase kinase 1, PKM, pyruvate kinase M; TPI, triosephosphate isomerase; HK1, hexokinase 1; HK2, hexokinase 2; GPI, glucose-6-phosphate isomerase; ENO1, enolase 1; LDHA, lactate dehydrogenase A; AK3, adenylate kinase 3; NT5E, 5'-nucleotidase ecto; CP, ceruloplasmin; TF, transferrin; TFRC, transferrin receptor; FABP4, fatty acid binding protein 4; FABP5, fatty acid binding protein 5; FASN, fatty acid synthase; ACSL1, acyl-CoA synthetase long chain family member 1; AGPAT2, 1-acylglycerol-3-phosphate O-acyltransferase 2; LPIN1, lipin 1; PNPLA2, patatin-like phospholipase domain containing 2
Pathways and implications of HIF-1 regulation of fatty acid metabolism. LPA, lysophosphatidic acid; PA, phosphatidic acid; TG, triglyceride; DAG, diacylglycerol; MAG, monoacylglycerol; FA, fatty acid; FATP, fatty acid transport protein; FABP, fatty acid binding protein; pmFABP, plasma membrane fatty acid binding protein; ACSL, acyl-CoA synthetase long chain family member; GPAT, glycerol-3-phosphate acyltransferase; AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; LPIN, lipin, DGAT, diacylglycerol O-acyltransferase; FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; ACLY, ATP citrate lyase; CAT, carnitine translocase; CPT, carnitine palmitoyltransferase; LCAD, long chain acyl-CoA dehydrogenase; MCAD, medium chain acyl-CoA dehydrogenase; FACS, fatty acyl-CoA synthase; ATGL, adipose triglyceride lipase; HSL, hormone sensitive lipase; MGL, monoacylglycerol lipase
HIF-1-targeting agents for anticancer therapy: Hsp90, heat shock protein 90; FIH, factor inhibiting HIF; HDACs, histone deacetylases; 17-AAG, 17-N-allylamino-17-demethoxygeldanamycin; 17-DMAG, 17-dimethylaminoetylamino-17-demethoxygeldanamycin; SAHA, suberoylanilide hydroxamic acid
Cancer cells rewire metabolic processes to adapt to the nutrient- and oxygen-deprived tumour microenvironment, thereby promoting their proliferation and metastasis. Previous research has shown that modifying glucose metabolism, the Warburg effect, makes glycolytic cancer cells more invasive and aggressive. Lipid metabolism has also been receiving attention because lipids function as energy sources and signalling molecules. Because obesity is a risk factor for various cancer types, targeting lipid metabolism may be a promising cancer therapy. Here, we review the lipid metabolic reprogramming in cancer cells mediated by hypoxia-inducible factor-1 (HIF-1). HIF-1 is the master transcription factor for tumour growth and metastasis by transactivating genes related to proliferation, survival, angiogenesis, invasion, and metabolism. The glucose metabolic shift (the Warburg effect) is mediated by HIF-1. Recent research on HIF-1-related lipid metabolic reprogramming in cancer has confirmed that HIF-1 also modifies lipid accumulation, β-oxidation, and lipolysis in cancer, triggering its progression. Therefore, targeting lipid metabolic alterations by HIF-1 has therapeutic potential for cancer. We summarize the role of the lipid metabolic shift mediated by HIF-1 in cancer and its putative applications for cancer therapy.
Induction of nephrotic syndrome and expression of prostasin in Prss8-wt, Prss8-S238A, and Prss8-R44Q mice. a Course of proteinuria after injection of doxorubicin at day 0. b Course of the urinary excretion of prostasin, measured with ELISA. c Western blot for expression of wild-type and mutant prostasin in the plasma with the loading control below. Recombinant truncated murine prostasin (amino acids 30–289, predicted mass 28 kDa) served as positive control. d–e Western blot for expression of wild-type and mutant prostasin in the urine (d) and in kidney lysates (e). The dashed white line is only for optical discrimination, it is one blot each, without cutting. f–g. Densitometric analysis of prostasin expression in urine (n = 4) and kidney (n = 8). h–i Total protein stain for loading control of the blots shown in d (h) and e (i). # indicates significant difference between healthy and nephrotic state, * indicates significant difference to the wildtype
Urinary protease activity against a peptide substrate containing the prostasin cleavage site of γ-ENaC. Relative fluorescence signal reflecting amidolytic activity after 4 h incubation against Ac-FTGR-AMC (a), Ac-FTGRK-AMC (b), Ac-FTGRKR-AMC (c), and Ac-FTGRKRK-AMC (d) in urine samples from healthy or nephrotic Prss8-wt, Prss8-R44Q, and Prss8-S238A mice. Trypsin was used in two concentrations (0.025 mg/mL and 0.1 mg/mL, respectively) to determine the dynamic range of the assay. # indicates significant difference between healthy and nephrotic state. Abbreviations: Bl blank
Activation of ENaC in Prss8-wt, Prss8-S238A, and Prss8-R44Q mice before and after induction of nephrotic syndrome. a Natriuretic response to the acute administration of the ENaC inhibitor triamterene (T, 10 µg/g) or vehicle injection (V, injectable water, 5 µL/g). b Fold-increase of the natriuretic response after triamterene administration. c–f Course of food and fluid intake, urinary sodium excretion in spot urine samples, and body weight taken in the morning after induction of nephrotic syndrome. Inset in g and f depict the minimal urinary sodium excretion and maximal body weight gain, both reflecting maximal ENaC activation. Abbreviations: V vehicle T triamterene. # indicates significant difference between healthy and nephrotic state, * indicates significant difference between the genotypes
Expression of ENaC subunits and proteolytic processing in Prss8-wt, Prss8-S238A, and Prss8-R44Q mice before and after induction of nephrotic syndrome. a Localization of the immunogenic sequences of the used antibodies against murine α-, β- and γ-ENaC. In α- and γ-ENaC, the proximal and distal cleavage sites (designated from the N-terminus, respectively) are depicted. The antibody against N-terminal α-ENaC is supposed to detect full-length α-ENaC at 79 kDa (699 aa) and two N-terminal fragments with a mass of 27 kDa (231 aa), and 24 kDa (205 aa). The antibody against C-terminal β-ENaC is supposed to detect full-length β-ENaC at 72 kDa (638 aa). The antibody against C-terminal γ-ENaC is supposed to detect full-length γ-ENaC at 74 kDa (655 aa) and C-terminal fragments with a mass of 58 kDa (512 aa) after proximal cleavage and at 53 kDa (469 aa) after distal cleavage, respectively. Mass values are calculated from the amino acid sequences (omitting any N-glycosylations). b Representative Western blots showing the expression of α-, β- and γ-ENaC in a plasma membrane preparation of kidney cortex lysates before (healthy) and after induction (nephrotic) of nephrotic syndrome. Note that the samples were deglycosylated before analyzing expression of γ-ENaC and its cleavage products [19]. The white line is only for optical discrimination, it is one blot each, no vertical cutting. c Total protein stain as a loading control. d–i Densitometry of the obtained bands normalized for total protein content of each lane (n = 5–6 each). # indicates significant difference between healthy and nephrotic state
Expression of ENaC subunits and proteolytic processing in Prss8-wt, Prss8-S238A, and Prss8-R44Q mice before and after exposure to a low sodium diet. a Representative Western blots showing the expression of α-, β- and γ-ENaC in a plasma membrane preparation of kidney cortex lysates under a control and low sodium (LS) diet. Note that the samples were deglycosylated before analyzing expression of γ-ENaC and its cleavage products [19]. The white line is only for optical discrimination, it is one blot each, no cutting, no cropping. b Total protein stain as a loading control. c–h Densitometry of the obtained bands normalized for total protein content of each lane (n = 5–6 each). # indicates significant difference between healthy and nephrotic state, * indicates significant difference between the genotypes (tested using two-way ANOVA)
Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice ( p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria.
[K⁺]p vs. blood pH relationship between control and 30 min following hypertonic NaHCO3 infusion (A–D) and boxplot of changes in [K⁺]p throughout the observation period (E). In A–D, filled circles represent control values and filled triangles denote values at 30 min. A normal; B chronic respiratory acidosis; C chronic respiratory alkalosis; D chronic metabolic alkalosis. No significant linear regression was obtained for the [K⁺]p vs. blood pH relationship in chronic metabolic acidosis. E shows that [K⁺]p decreased at 30 min in chronic metabolic acidosis (p < 0.01) and remained essentially unchanged thereafter *p < 0.01 vs control
Diagram depicting processes following NaHCO3 infusion. The alkalinization of ECF following NaHCO3 infusion (A) induces an immediate H⁺ release from cells in exchange for Na⁺ entering cells via the Na⁺-H⁺ antiporter (B). This process constitutes the bulk of the cellular contribution to bicarbonate buffering. The resulting increases in [Na⁺]i and cell pH stimulate the cell membrane Na⁺-K⁺ pump (Na⁺-K⁺ ATPase) causing Na⁺ exit and K⁺ entry that induces prompt hypokalemia in all study groups (C). Variable amounts of Na⁺, K⁺, and HCO3⁻ are excreted in the urine (D). The K⁺ entry mechanism described operates in concert with a K⁺ exit mechanism via K⁺ channels (E)
The hypokalemic response to alkali infusion has been attributed to the resulting extracellular fluid (ECF) expansion, urinary potassium excretion, and internal potassium shifts, but the dominant mechanism remains uncertain. Hypertonic NaHCO3 infusion (1 N, 5 mmol/kg) to unanesthetized dogs with normal acid-base status or one of the four chronic acid-base disorders decreased plasma potassium concentration ([K⁺]p) at 30 min in all study groups (Δ[K⁺]p, − 0.16 to − 0.73 mmol/L), which remained essentially unaltered up to 90-min postinfusion. ECF expansion accounted for only a small fraction of the decrease in ECF potassium content, (K⁺)e. Urinary potassium losses were large in normals and chronic respiratory acid-base disorders, limited in chronic metabolic alkalosis, and minimal in chronic metabolic acidosis, yet, ongoing kaliuresis did not impact the stability of [K⁺]p. All five groups experienced a reduction in (K⁺)e at 30-min postinfusion, Δ(K⁺)e remaining unchanged thereafter. Intracellular fluid (ICF) potassium content, (K⁺)i, decreased progressively postinfusion in all groups excluding chronic metabolic acidosis, in which a reduction in (K⁺)e was accompanied by an increase in (K⁺)i. We demonstrate that hypokalemia following hypertonic NaHCO3 infusion in intact animals with acidemia, alkalemia, or normal acid-base status and intact or depleted potassium stores is critically dependent on mechanisms of internal potassium balance and not ECF volume expansion or kaliuresis. We envision that the acute NaHCO3 infusion elicits immediate ionic shifts between ECF and ICF leading to hypokalemia. Thereafter, maintenance of a relatively stable, although depressed, [K⁺]e requires that cells release potassium to counterbalance ongoing urinary potassium losses.
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