In this study we performed detailed analysis of the action of Fe ions on cytoplasmic free calcium ion concentration ([Ca2+]i) in astrocytes. Administration of Fe2+ or Fe3+ in μM concentrations evoked [Ca2+]i in astrocytes in vitro and in vivo. Iron ions trigger increase in [Ca2+]i through two distinct molecular cascades. Uptake of Fe2+ by DMT1 inhibits astroglial Na+-K+-ATPase, which leads to elevation in cytoplasmic Na+ concentration, thus reversing Na+/Ca2+ exchanger and thereby generating Ca2+ influx. Uptake of Fe3+ by TF-TFR stimulates phospholipase C to produce inositol 1,4,5-trisphosphate (InsP3), thus triggering InsP3 receptor-mediated Ca2+ release from endoplasmic reticulum. In summary, these findings reveal the mechanisms of iron-induced astrocytic signalling operational in conditions of iron overload.Iron is the fundamental element for numerous physiological functions. Plasmalemmal divalent metal ion transporter 1 (DMT1) is responsible for cellular uptake of ferrous (Fe Wenzheng Guan and Maosheng Xia et al. use a combination of pharmacological, genetic, and calcium imaging approaches to describe two mechanisms of iron-induced astrocytic calcium signaling. Altogether, these results suggest that astrocytes may act as a barrier to excessive iron in the brain, thereby improving our understanding of the role of astrocytes in normal neurodevelopment. In biological systems iron is present in either reduced ferrous (Fe2+) or oxidized ferric (Fe3+) state. The brain has the second (after liver) highest quantity of iron in the human body with total non-haem iron in the brain reaching about 60 mg3. The non-haem iron concentration in the serum ranges between 9 and 30 μM, whereas the iron concentration in cerebrospinal fluid (CSF) is much smaller being around 0.3–0.75 μM5. Transport of iron across the blood–brain barrier (BBB) is mediated either by transferrin receptor (TFR)-mediated endocytosis of Fe3+-bound to transferrin (holo-TF), or, for non-TF-bound iron, by vesicular and non-vesicular pathways6. Membrane transport of Fe2+ is also mediated by divalent metal ion transporter 1 (DMT1/SLC11A2) which underlies Fe2+ uptake through the plasma membrane or from endosomes6. Under physiological conditions, the intracellular cytosolic ionised iron levels fluctuate around 0.5–1.5 μM7.Iron contributes to numerous cellular and biochemical processes and acts as a co-factor in various molecular cascades in the nervous tissue including the synthesis and metabolism of several brain-specific enzymes and neurotransmitters8. Astrocytes in particular are fundamental elements of ionostatic control over CNS environment9. Ionised Fe2+ enters astrocytes through DMT1/SLC11A2 transporters which are particularly concentrated in the endfeet of cerebral and hippocampal astrocytes10. In physiological condition, DMT1 is widely distributed in the nervous system, being expressed in neurones13, astrocytes15 and oligodendrocytes16 in vitro and in the brain tissue. In astrocytes, DMT1 mediates non‐transferrin‐bound iron (NTBI) transport, thus contributing to the brain iron homoeostasis in development and adulthood19. Conditional deletion of DMT1 form oligodendrocyte precursors substantially inhibited both myelination in development and remyelination in pathology16. Evidence for the expression of TFR in astroglial cells remains controversial21, while iron overload may influence the expression or distribution of TFR in astrocytic compartments22. Cellular uptake of Fe3+ requires internalization of TF–TFR complex23. An adaptor protein Disabled-2 (Dab2) plays an essential role in cell signalling, migration and development24. In mice the Dab2 has two isoforms of 96 and 67 kDa (p96 and p6724). In human K562 cells, Dab2 regulates internalization of TFR and uptake of TF25. Dab2 is also widely distributed in immune cells and in neuroglia24, although the functional link between Dab-2 and TFR in astrocytes has not been demonstrated.In the brain, up to three-fourths of total iron is accumulated within neuroglial cells27. Astroglial Ca2+ signalling is mediated by Ca2+ release from the endoplasmic reticulum (ER) following activation of inositol-1,4,5-trisphosphate receptor (InsP3R), or intracellular Ca2+-gated Ca2+ channels known as ryanodine receptors (RyR). Astroglial Ca2+ signals may also be generated by plasmalemmal Ca2+ entry through Ca2+-permeable channels or by sodium-calcium exchanger (NCX) operating in the reverse mode28. Astroglial Na+ signalling is shaped by plasmalemmal Na+ entry through cationic channels and numerous Na+-dependent transporters, of which the major role belongs to Na+-dependent glutamate transporters31; Na+ extrusion is mediated by the sodium-potassium pump (NKA). Both Na+ and Ca2+ signalling systems are closely coordinated, with NKA and NCX accomplishing this coordination at the molecular level32. Astrocytes specifically express α2-subunit containing NKA which is fundamental for astroglial K+ buffering33. Astrocytes express all three isoforms of NCX - NCX1/SLC8A1, NCX2/SLC8A2 and NCX3/SLC8A3, with some evidence indicating higher expression of NCX134. The NKA, the NCX and glutamate transporters are known to be preferentially concentrated in the perisynaptic astroglial membranes indicating intimate relationship between these ion-transporting molecules36. The NCX is also known to localise at caveolae rich in caveolin-3 (Cav-3), the latter isoform being predominantly expressed in astrocytes37.Astrocytes possess a special form of intracellular ionic excitability, mediated by temporal and spatial fluctuations in the intracellular ion concentration2+ and Fe3+) on astrocytic Ca2+ and Na+ dynamics. We found that Fe2+ (through DMT1) and Fe3+-TF (through TFR) evoke [Ca2+]i transients in astrocytes in culture and in vivo. Effects of Fe2+ on [Ca2+]i are mediated mainly by the reversed NCX, whereas Fe3+ triggers Ca2+ release from the endoplasmic reticulum by stimulations of InsP3R. In conclusion, we discovered that iron ions trigger astrocytic Ca2+ signalling by acting through two distinct molecular cascades.In the present paper we performed an in depth analysis of the action of ferrous and ferric  or ferric ammonium citrate-TF (Fe3+) increased [Ca2+]i in concentration-dependent manner, albeit with different kinetics. In the presence of Fe2+ an increase in [Ca2+]i demonstrated prominent plateau, whereas Fe3+ triggered transient, relatively rapidly decaying [Ca2+]i response  whereas Fe3+ increased the peak of fluorescent signal to 308.50% ± 13.01%  of the basal value. In experiments in vitro we characterised concentration-dependence of [Ca2+]i responses: the EC50 was around 0.4–0.6 μM for both Fe2+ and Fe3+  we found that addition of either Fease Fig. . Adminise3+ Fig. .2+ uptake could be mediated by plasmalemmal transporter DMT1, whereas Fe3+ is mainly accumulated in TF-bound form by TFRs , whereas treatment with TFR siRNA decreased TFR levels to 7.84% ± 2.10%  from the control values. Administration of Fe2+ to DMT1-deficient astrocytes failed to induce any changes in [Ca2+]i. At the same time Fe2+ induced robust [Ca2+]i elevation in astrocytes treated with siRNA(−)  Fig. . Similarons Fig. . At the 2+ for 5 min we observed redistribution of DMT1 from the nuclear region to the plasma membrane , whereas the level of cytoplasmic DMT1 increased to 117.37% ± 5.79% of control group   Fig. .2+]i increase in astrocytes are (i) Ca2+ release from the ER following the opening of InsP3Rs or RyRs, or (ii) plasmalemmal Ca2+ influx through either Ca2+ permeable channels (such as L-type Ca2+ channels or TRP channels) or NCX operating in the reverse mode act Fig. . This hi2+ channel nifedipine (10 μM) did not affect [Ca2+]i responses to Fe2+ or to Fe3+ 38, Exposure of cultured astrocytes to 10 μM of XeC effectively suppressed [Ca2+]i response to Fe3+, without much affecting Fe2+-induced [Ca2+]i transient  of the control. Exposure to 100 nM of the specific NKA inhibitor, ouabain, reduced NKA activity to 72.30 ± 5.91%  of the control   Fig. .Fig. 4Fe+]i. When monitoring [Na+]i in cultured astrocytes with Na+-sensitive probe SBFI we found that both Fe2+ (10 μM) and ouabain (100 nM) triggered rapid and substantial elevation of [Na+]i  of the control . In control astrocytes treated with siRNA(−) the amplitude of Fe2+-induced [Ca2+]i increase reached 363.34 ± 11.62%  of the control values. After pre-treatment with Cav3 siRNA duplex chains, Fe2+ increased the level of DMT1 only to 195.77 ± 20.19%  of control group  and 263.66 ± 25.93%  of the controls, after knocking down Cav3, Fe2+ elevated the level of NCX1 and NKA only to 172.96 ± 11.76%  and 200.86 ± 18.14%  of control values , from the control InsP3 level of 24.40 ± 6.35 ng/ml. In cells treated with TF alone the InsP3 level was 28.00 ± 12.62 ng/ml   and to 11.88 ± 6.51%  of the control values ; Dab2 knockdown: 30.60 ± 10.42 ng/ml  Fig. . Subsequins Fig. . After Rues Fig. . The kno3+, no [Ca2+]i increase was recorded  channels42. Expression of TF-Fe3+-transporting TFR has been noted in astrocytes in culture46; it is, nonetheless, generally believed that astrocytes in vivo are not in a possession of TFR and hence cannot accumulate Fe3+ 49. This conclusion, however, has been made on the basis of rather limited investigations46; while expression of TFR-specific mRNA was detected in astrocyte transriptome50. In our study we confirmed expression of DMT1, at mRNA and protein levels as well as by immunostaining, in acutely isolated astrocytes, in astroglial primary culture and in situ in cortical tissue; the DMT1 was particularly enriched in the endfeet , and iron regulatory protein 1 and 2 (IRP1and IRP2) for regulating intracellular iron homoeostasiseet Fig. . We alsoeet Fig. . We furteet Fig. . In the eet Fig. .2+ signalling in the cellular elements of the CNS. In the literature, we found only a single example of Fe3+-induced [Ca2+]i transient in cultured hippocampal neurones51. To the best of our knowledge here we present the first recordings of Fe2+/Fe3+ induced Ca2+ signals in astrocytes. Both ions evoked [Ca2+]i elevation in primary cultured astrocytes and in EGFP-labelled astrocytes in the cortices of alive animals studied with transcranial confocal microscopy. Both ions acted in the low μM range of concentrations, however the kinetics of respective [Ca2+]i transients are different. Exposure of cultured astrocytes to Fe2+ triggered rapid [Ca2+]i increase with long-lasting plateau; the [Ca2+]i barely declined in the presence of Fe2+. In contrast, Fe3+-induced transient elevation of [Ca2+]i recovers to the baseline within ~ 200–300 s in the presence of Fe3+  and by U-73122 (inhibitor of PLC) thus revealing the central role for InsP3-medaited ER Ca2+ release. Initiation of this signalling cascade requires transmembrane transport of Fe3+: in vitro knockdown of TFR eliminates Fe3+-evoked [Ca2+]i dynamics. The internalisation of TF-Fe3+-TFR complex also requires functional Dab2 protein. This protein is a multi-modular scaffold protein with signalling roles in ion homoeostasis, inflammation and receptors internalization53. Ablation of Dab2 in astrocytes with specific siRNA interrupts signalling chain and blocks Fe3+-dependent Ca2+ signalling  environment of the endosome achieved through the action of ATP-dependent H+ pumps54, reduces the affinity of TF to Fe3+ and the latter is released55. Subsequently, an endosomal reductase reduces Fe3+ to Fe2+, and Fe2+ can be transported into the cytosol by DMT1 or by ZIP8 or ZIP14 Zn transporters56. Our data question the DMT1 pathway because siRNA knockout of DMT1 did not affect Fe3+-induced Ca2+ signalling; the detailed mechanism of Fe3+ transport in astrocytes remains to be fully characterised.Import of TF-Fe57. Caveolae and their main structural and regulatory proteins Caveolin-1,2,3 are present in astrocytes (with predominant expression of Cav3); astrocytic caveolae contribute to signal transduction, formation of signalling protein complexes and are involved in action of various neuroactive substances and drugs59. Caveolae are known to form functional Ca2+ signalling units, establish links between Ca2+ channels and various transports and may contribute to formation of plasmalemmal/ER functional domains operational in astrocytes60. We found that down-regulation of expression of Cav3 in cultured astrocytes substantially reduced the amplitude of Fe2+-evoked [Ca2+]i responses. We suggest therefore that Cav3 and caveolae integrate DMT1, NKA and NCX into a single Ca2+ signalling unit  result in iron deposition in the brain with subsequent neurodegeneration characteristic for aceruloplasminemia62 and neuroferritinopathy63. In aceruloplasminemia patients, iron overload has been observed in astrocytes, particularly in the basal ganglia64. Ceruloplasmin oxidises Fe2+ to Fe3+ in order to generate the oxidised form of iron that can bind to extracellular transferrin; in astrocytes ceruloplasmin is also required for iron export64. Thus, in aceruloplasminemia, iron entering the CNS as ferrous might escape oxidation; hence cells exposed to the excess of Fe2+ may readily become iron overloaded65. Similarly, abnormal iron accumulation has been characterised in neurodegenerative diseases including Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis and Huntington disease to name but a few48. Specific class of neurodegeneration with brain iron accumulation (NBIA) has been also categorised in recent years66.Iron homoeostasis is of fundamental importance for cells, tissues and organisms, as iron contributes to a wide range of vital biological pathways2+ signals, which control astrocytic reactivity. Iron-induced Ca2+ signalling is activated at low pathological iron concentrations . Importantly, two distinct signalling cascades  distinguish between ferric and ferrous. These distinct pathways may define very different outputs: it is known for example that activation of astroglial InsP3 receptors type II is linked to initiation of reactive astrogliosis68. In our recent report, exposure to iron was found to stimulate the over-expression of DMT1 in astrocytes and microglia, but not in neurones, which may result in the neuroprotection by glial uptake of excessive iron13. Astrogliosis plays important, if not defining role in evolution of many neurological diseases69. Our previous experiments have shown that formation of brain deposits of iron up-regulates astroglial expression of TFR and instigates reactive astrogliosis70. What characterises the iron-induced reactive phenotype and what is the role of astroglial reactivity in managing excessive iron in the brain remains to be found. In conclusion, our study presented a phenomenon that iron ions (Fe2+ and Fe3+) directly induce intracellular Ca2+ signalling and stimulate astroglial protective mechanisms against iron overload in broad pathological contexts.Based on our data we propose that astrocytes mount the defence against acute iron overload. This defence includes iron accumulation through both DMT1 and TFR, redistribution of DMT1 from intracellular locations to plasmalemma and generation of Ca+/K+-ATPase alpha1/2 antibody and the secondary antibodies were bought from Santa Cruz Biotechnology . NCX1 antibody, NCX3 antibody and native mouse apo-transferrin  were from Abcam . Donkey serum, xestospongin C (Xe-C), nifedipine, ferrous sulfate heptahydrate (FeSO4), sulforhodamine 101 (SR101) and ferric ammonium citrate were purchase from Sigma-Aldrich . Ryanodine and KB-R7943 were purchased from Calbiochem . Secondary antibody staining with donkey anti-mouse or anti-rabbit Cy-2/3 were from Jackson Immuno-Research . Primary antibody of histone H3 was purchased from EarthOx .The culture medium including DMEM and fetal bovine serum were purchased from Gibco Life Technology Invitrogen . Oligo-fectamine, MEMI, fluo-4 AM, sodium-binding benzofuran isophthalate (SBFI) AM, G-agarose bead, TFR antibody, β-actin antibody, GFAP antibody, DMT1 antibody and DMT1 siRNA duplex chains were from Thermo Fisher Scientific ; siRNA duplex chains of TFR, NCX1-3, Cav-3 and Dab2, NCX2 antibody, Naad libitum. All experiments were performed in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023) and its 1978 revision, and all experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University, No. [2019]059.The C57BL/6 mice, FVB/N-Tg(GFAP-eGFP)14Mes/J and B6.Cg-Tg(Thy1-YFP)HJrs/J transgenic mice were all purchased from the Jackson Laboratory . The animals were raised in standard housing conditions , with water and food available 72. In brief, the cerebral hemispheres were isolated, dissociated and filtered. Isolated astrocytes were grown in Dulbecco’s Minimum Essential Medium (DMEM) with 7.5 mM glucose supplemented with 10% foetal bovine serum. Astrocytes were incubated at 37 °C in a humidified atmosphere of CO2/air (5:95%). The cultures are highly enriched in astrocytes, the purity is >95% as judged by GFAP staining73.Astrocytes were cultured from newborn mice3+-TF solution, ferric ammonium citrate and mouse apo-TF were incubated at a 2:1 ratio in serum-free culture medium for 1 h at 37 °C75. The same concentration of apo-TF in the same volume of culture medium but without Fe3+ was used for control treatments. For the Fe2+ solution, FeSO4 was freshly dissociated in serum-free culture medium at 37 °C and used immediately, the same volume of serum-free culture medium was used as the control for Fe2+ group.For preparing Fe77. A transfection solution containing 2 μl oligo-fectamine , 40 μl MEMI, and 2.5 μl siRNA  was added to the culture medium in every well for 8 h. In the siRNA-negative control cultures, transfection solution without siRNA was added. Thereafter, DMEM with three times serum was added to the cultures. These siRNA duplex chains were purchased from Santa Cruz Biotechnology .The cultured astrocytes were incubated in DMEM without serum for 12 hours before transfection79. In brief, primary cultured astrocytes were collected and homogenised in SET , and centrifuged for 1 h at 1,000 × g. The pellets were re-solubilised in SET and layered on Percoll (30% in SET) followed centrifugation at 1,000 × g. The pellets were re-homogenised and re-layered to three sucrose density gradient solution  with ultra-centrifugation at 175,000 × g. Finally, the purified caveolae were collected and re-suspended in SET.Cell homogenization and the caveolae preparation from astrocytes was made76. After homogenization, protein content was determined by the Bradford method80 using bovine serum albumin as the standard. For immunoprecipitation of NCX1-3, whole cell lysates (500 μg) were incubated with 20 μg of anti-NCX1, anti-NCX2 or anti-NCX3 antibody for overnight at 4 °C. Then, 200 μl of washed protein G-agarose bead slurry was added, and the mixture was incubated for another 2 hours at 4 °C. The agarose beads were washed three times with cold phosphate buffer solution (PBS) and collected by pulsed centrifugation , the supernatant was drained off, and the beads were boiled for 5 min. Thereafter, the supernatant was collected by pulsed centrifugation, and the entire immunoprecipitates were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).We used technologies of co-immunoprecipitation and subsequent western blotting to check the conjunction level between NCXs and DMT181, according to the manufacturer’s protocol. For further western blotting assays, cytoplasmic and nucleic protein extractions were kept under −20 °C.The subcellular fraction was analyzed using the protein extraction Kit TM FC 32-bit software82.For quantifying expressions of DMT1, TFR, NCX1-3, Cav-3 or Dab2, the samples containing 100 μg of protein were added to slab gels. After transferring to PVDF membranes, the samples were blocked by 10% skimmed milk powder for 1 h, and membranes were incubated overnight with the primary antibodies, specific to either DMT1 at a 1:300 dilution, TFR at a 1:200 dilution, NCX1 at a 1:100 dilution, NCX2 at a 1:200 dilution, NCX3 at a 1:150 dilution, Cav-3 at a 1:200 dilution, Dab2 at a 1:100 dilution, Histone H3 at 1:500 dilution or β-actin at a 1:1000 dilution. After washing, specific binding was detected by horseradish peroxidase-conjugated secondary antibodies. Images were analysed with an Electrophoresis Gel Imaging Analysis System . Band density was measured with Window AlphaEase2+]i monitoring and imaging in cultured astrocytes84, after the pre-treatment with or without inhibitors or siRNA duplex chains, the primarily cultured astrocytes were loaded with 5 μM fluo-4-AM, ), for 30 min. Fluo-4 signals were visualised by fluorescent microscopy . The readings from all fluo-4 positive cells in one measured field of view in each culture were included in the statistics, the fluorescence intensity of fluo-4 was normalised to the baseline intensity before stimulation. The measurements were repeated in 10 different cultures. Cultured astrocytes were also stained with specific marker sulforhodamine 101 (SR101) at 1:2000 dilution.For i) in cultured astrocytes, primary cultured astrocytes were loaded with 10 μM of Na+-sensitive indicator SBFI-AM for 30 min in serum-free medium, with subsequent 1 h of washout. SBFI was alternatively excited at 340 nm and 380 nm, and the emission was monitored at 500 nm. The SBFI signals were measured by fluorescent microscopy  and expressed as a ratio (R = F340/F380)52.For monitoring intracellular ionised Na82. The cultured cells were fixed with 100% methanol at −20 °C. Brain slices or cells were permeabilised by incubation for 1 h with donkey serum. Primary antibodies against DMT1 or TFR were used at a 1:100 dilution, against glial fibrillary acidic protein (GFAP) was used at 1:200 dilution. And nuclei were stained with marker 4′, 6′-diamidino-2-phenylindole (DAPI) at 1:1000 dilution. The primary antibodies were incubated overnight at 4 °C and then donkey anti-mouse or anti-rabbit Cy-2/3 conjugated secondary antibody for 2 h at room temperature. Images were captured using a confocal scanning microscope .The brain tissue was fixed by immersion in 4% paraformaldehyde and cut into 100 μm slices2+/Fe3+ or inhibitors, the astrocytes were collected and centrifuged at 10,000 × g for 10 min to remove floating cells and/or cell debris at 4 °C. To assay the NKA activity, a commercial ELISA kit  was used and operated as the protocols, the sensitivity is 0.19 ng/mL, the optical density (OD) was measured at 450 nm and the OD value was normalized by the control group. To assay the InsP3 concentration86, the supernatant was collected and the concentration of InsP3 assayed using a commercial ELISA kit .Astrocytes were incubated at 37 °C in a fresh serum-free culture medium; after the treatment with Fe87. The RNA of the sorted cells and cerebral tissue was extracted by Trizol. Total RNA was reverse transcribed and PCR amplification was performed in a Robo-cycler thermocycler84. The relative quantity of transcripts was assessed using five-folds serial dilutions of RT product (200 ng). RNA quantity was normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and values are expressed as the ratio TFR/GAPDH or DMT1/GAPDH.To measure the mRNA for TFR and DMT1, astrocytes expressing fluorescent marker GFP (GFAP-GFP mice) and neurons expressing fluorescent marker YFP (Thy1-YFP mice) were used; we also extracted the cerebral hemispheres tissues from wild type mice. The cells from transgenic mice were used for specific sorting of astrocytes or neurons with FACSp < 0.05.For statistical analysis, we used one-way analysis of variance (ANOVA) followed by a Tukey’s or Dunnett’s post hoc multiple comparison test for unequal replications using GraphPad Prism 5 software  and SPSS 24 software . One-way ANOVA for comparisons including more than two groups; unpaired two-tailed t-test for two-group comparisons. All statistical data in the text are presented as the mean ± SD, the value of significance was set at Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The interplay among the kidney, bone, intestine, and parathyroid gland in Ca2+ homeostasis is strictly modulated by numerous hormones and signaling pathways. The calcium-sensing receptor (CaSR) is a G protein–coupled receptor, that is expressed in calcitropic tissues such as the parathyroid gland and the kidney, plays a pivotal role in Ca2+ regulation. CaSR is important for renal Ca2+, as a mutation in this receptor leads to hypercalciuria and calcium nephrolithiasis. In addition, CaSR is also widely expressed in the vascular system, including vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) and participates in the process of vascular calcification. Aberrant Ca2+ sensing by the kidney and VSMCs, owing to altered CaSR expression or function, is associated with the formation of nephrolithiasis and vascular calcification. Based on emerging epidemiological evidence, patients with nephrolithiasis have a higher risk of vascular calcification, but the exact mechanism linking the two conditions is unclear. However, a dysregulation in Ca2+ homeostasis and dysfunction in CaSR might be the connection between the two. This review summarizes renal calcium handling and calcium signaling in the vascular system, with a special focus on the link between nephrolithiasis and vascular calcification.Calcium (Ca As a result, it participates in several physiological and pathological processes. More than 99% of Ca2+ in the entire body and 85% of PO43− are combined as hydroxyapatite and deposited in bone under tight regulation [2+ can be found in the blood, the extracellular fluid (ECF), and soft tissues. Serum Ca2+ concentration is strictly maintained in the range between 8.5 and 10.5 mg/dL under the controls of parathyroid hormone (PTH), calcitonin, and 1,25-dihydroxyvitamin D 2D3) [2D3 are regulated by the calcium-sensing receptor (CaSR), located in the parathyroid gland and kidney. CaSR can specifically detect a slight increase in serum Ca2+ and it can be activated by Ca2+ with low affinity [2+ reabsorption will significantly decrease. Decreased PTH level directly results in decreased Ca2+ reabsorption by the kidney, decreased Ca2+ resorption from bone, and decreased Ca2+ absorption in the intestine. As a result, this complex and precise modulatory mechanism is the interplay among the kidney, bone, intestine, and parathyroid gland. Ca2+ homeostasis is associated with multicellular dysfunctions, including cellular apoptosis, mitochondrial dysfunction, and oxidative stress [2+ may also contribute to the pathogenesis of numerous diseases, including malignancy, diabetes mellitus (DM), and hypertension (HTN) [2+ dysregulation that can occur in vessels, kidneys, and many other organs. Vascular calcification is associated with a high cardiovascular-related morbidity and mortality, and can be a consequence of aging, chronic kidney disease (CKD), and HTN [2+ in the kidney parenchyma). The most common composition of renal stone is calcium oxalate (CaOx), followed by calcium phosphate (CaP). Although the exact mechanism of kidney stone formation is still unclear, Randall’s plaque theory is one of the most accepted hypotheses [2+ handling and the renal CaSR [2+ signaling could be considered as the connection between vascular calcification and nephrolithiasis. Thus, we have conducted this narrative review, which is based on the articles retrieved from the databases Medline and PubMed using the search terms “calcium signaling”, “calcium sensing receptor”, “vascular calcification”, “kidney stone” and “nephrolithiasis” for the topic.Calcium (Cagulation . The reme stress . Furtheron (HTN) ,8,9. Patpotheses . Randallpotheses . These pnal CaSR . In recenal CaSR . As the 2+ signaling pathway in the kidney and the role of renal CaSR. Owing to the critical role of CaSR in controlling physiological and pathological calcifications, we also addressed the impact of Ca signaling on vascular calcification and nephrolithiasis. Finally, we summarized the extensive literature on the link between vascular calcification and nephrolithiasis, and reviewed the possible hypothesis that may explain this link and the role played by the CaSR in this connection.In this review, we first briefly review the Ca2+ [2+ will directly lead to increased urinary Ca2+ independent of PTH levels [2+ in the ECF is not protein-bound, and is thus freely filtered by the renal glomeruli [2+ will be reabsorbed via various transepithelial transport routes. As a result, urinary Ca2+ excretion will be less than 2% of the daily filtered load of Ca2+. There are two major types of transepithelial Ca2+ transport pathways—paracellular and transcellular [2+ ATPase. More details are provided in the subsequent section.Owing to the high expression of the CaSR, the kidney is considered to be a calcium-sensing organ that can monitor the urine and serum levels of Ca2+ . ElevateH levels . The 60%lomeruli . In the cellular . Paracel2+, a general concept of Ca2+ homeostasis must first be established first. Increased serum Ca2+ can activate the CaSR, which is responsible for a physiological brake on PTH secretion. The decline in PTH results in decreased reabsorption of Ca2+ in the kidney, Ca2+ absorption in the intestine, and Ca2+ resorption from bone. The CaSR is expressed throughout the entire renal tubule system, and elevated extracellular Ca2+ can also stimulate renal CaSR, independent of the PTH effects. The activation of renal CaSR in different segments could result in different effects on Ca2+ reabsorption [2+ signaling is more distinctive in the glomerulus. Among the other tubular systems, the CaSR is accepted to possess the following functions—1. Ca2+ and phosphate (Pi) homeostasis; 2. Cation transport; 3. Fluid/NaCl reabsorption; and 4. Urinary acidification. As the individual functions are dependent on the different segments of the tubular system, we opted to discuss the different segments individually to shed light on their functions.Before exploring the renal handling of Casorption . In the sorption . However2+ entry [2+ is maintained at a low concentration; however, under a disease status, its concentration is increased. A continuous increase in intracellular calcium has harmful effects on glomerular cells as it triggers an acute reorganization of the actin cytoskeleton, resulting in glomerulosclerosis and nephropathy [2+ handling [2+ influx in glomerular cells. In fact, gain-of-function mutations in TRPC6 can cause focal segmental glomerulosclerosis (FSGS) [2+ or calcimimetics (CaSR agonist) can induce an increase in intracellular Ca2+ in a dose-dependent manner, and the activation exerts a stable effect on the actin cytoskeleton and attenuates glomerular damage via an unclear mechanism. A recent study has revealed that the renoprotective effect of the CaSR could originate from AMP-activated protein kinase (AMPK) activation [The mature glomerulus contains four cell types—podocytes, endothelial cells, epithelial cells, and mesangial cells. Besides other parts of the tubular system, the expression of the CaSR is found to be relatively low in all cell types of the glomerulus . Despite2+ entry ,19. The hropathy . In the handling . Among ts (FSGS) . Howevers (FSGS) . Howevers (FSGS) ,24,25,262+ filtered by the glomerulus [+-H+ cotransporter (SLC9A3) and Na+-Pi cotransporter (SLC34A3). This inhibition leads to an increase in Pi excretion. However, when the CaSR is activated, the PTH effect will be blunt, causing decreased phosphaturia and increased urine acidification.The CaSR is expressed apically in the PT and is responsible for the reabsorption of 60–70% of the Caomerulus . The reg2D3, which is the physiologically active form of vitamin D, is another major function associated with renal CaSR. The inactive form of vitamin D, that is 25-hydroxyvitamin D (25(OH)D), is converted to 1,25(OH)2D3 via 1α-hydroxylase (CYP27B1). Of note, the CYP27B1 is only located in the PT. Although PTH could activate CYP27B1, the activation of the CaSR could inversely inhibit CYP27B1. Thus, renal CaSR in the PT will reduce the conversion of 1,25(OH)2D3. The major function of 1,25(OH)2D3 is to promote intestinal Ca2+ absorption and bone Ca2+ resorption [2+ by indirectly affecting organs besides the kidneys.In the PT, the regulation of serum 1,25(OH)sorption . The inh2+ is mainly reabsorbed through the paracellular transepithelial routes in the PT. Activation of the CaSR leads to Na+ reabsorption and proton excretion. Increased intracellular Na+ causes increased osmolarity and provides a paracellular driving force to strengthen Ca2+ and H2O reabsorption.Ultimately, Ca2+ permeability [2+ is reabsorbed in the TAL through the paracellular transepithelial routes [2+ reabsorption in TAL, the role of Na+ and K+ should be considered. There are two important transporters on the apical membrane of TAL—renal-outer-medullary-potassium (ROMK) and Na+-K+-Cl− cotransporters 2 (NKCC2) [+ into urine and is the rate-limiting step of NKCC2 (encoded by the SLC12A1 gene). NKCC2 mediates the transepithelial NaCl reabsorption in the TAL and plays an essential role in the urinary concentration. These two transporters are activated by intracellular cAMP through the GPCR pathway. The GPCR pathway can be activated by PTH, calcitonin, and arginine vasopressin (AVP). Thus, PTH can lead to a voltage difference via K+ recycling and promote Ca2+ reabsorption paracellularly. In contrast, CaSR activation can concurrently inhibit cAMP and ROMK. This effect gives rise to a reduction in luminal transepithelial potential difference and decreases the paracellular driving force of Ca2+. As a result, typical clinical manifestations of Bartter syndrome, including hypokalemia, hypochloridemia and metabolic alkalosis, will develop [After the PT, the thin descending, and ascending limbs of the loop of Henle all have limited Caeability . In bothl routes . To unde (NKCC2) . ROMK is develop .2+ flow is strictly regulated by claudins (CLDNs), including CLDN14, CLDN16, and CLDN19. CLDNs are tight-junction proteins consisting of a family of 27 members that function as ion channels to allow selective permeation. CLDN16 and CLDN19 are fully expressed in the TAL and form a heteromeric cation channel. The functions of these cation channels include—(1) facilitating the permeation of Ca2+ and Mg2+; and (2) creating a lumen-positive voltage potential in the late TAL and inducing the electrical driving force for Ca2+ and Mg2+ reabsorption [2+ influx. CLDN14 is exclusively expressed in the TAL and is undetectable at baseline. However, hypercalcemia activates the CaSR expressed in the basolateral membrane and increases CLDN14 expression [2+ flux, which promotes urinary Ca2+ excretion. Based on accumulating evidence, a dysfunction in CLDN14 is highly associated with kidney stone formation [Paracellular Casorption . Howeverpression . Accordiormation .2+ is reabsorbed through the transcellular transepithelial routes. The CaSR is expressed both on the apical membrane and on the basolateral membrane, and its functions vary between the two locations. First, after activation of the apical CaSR, Ca2+ uptake occurs via the TRPV5 transporter. TRPV5 can also be activated by increased cAMP, which is caused by PTH activation. Second, Ca2+ will be bound to calbindin-d28k (CALB1) and transported from the apical side to the basolateral membrane. Third, there are two Ca2+ transport systems located on the basolateral membrane—plasma membrane calcium ATPase 4 (PMCA4) and the calcium/sodium exchanger (NCX1). Recent evidence has revealed that NCX1, not PMCA4, is the main regulating transporter [+ level, which is closely associated with apical SLC12A3 (NCC) and the basolateral Na+/K+ ATPase. The former is responsible for NaCl reabsorption while the latter pumps sodium into the basolateral space. The activation of the basolateral CaSR will inhibit the KCNJ10 and PMCA4 transporters. KCNJ10 and PMCA4 allow K+ and Ca2+ to flow into the basolateral space, respectively. The activation of CaSR will thus directly promote Ca2+ reabsorption and indirectly inhibit NaCl reabsorption.In the DCT, approximately 10% of filtered Cansporter . The act+ and water regulation while the ICs mainly regulate acid–base homeostasis. In the principal cells, the activation of CaSR expressed apically inhibits AQP2, which is responsible for water reabsorption from the urine. The mechanism of AQP2 begins with vasopressin bound to the type 2 vasopressin receptor (V2R), located on the basolateral membrane of the principal cells. After vasopressin binds to AVP, the cAMP/PKA signal is activated, causing the AQP2-containing vesicles to fuse with the plasma membrane. AQP2 is then apically inserted into the cell. As a result, there is an increase in water permeability, which allows water reabsorption from urine. Ultimately, the activation of the CaSR reduces the water reabsorption and promotes urinary dilution.The collecting duct is the final part of the renal tubular system. This structure generally allows water reabsorption and proton excretion . The ent3− secretion. ICs can excrete H+ into urine via the H+-ATPase on the apical membrane and the activation of CaSR can promote the activity of the H+-ATPase and induce urinary acidification.Three types of ICs are found in the collecting duct—type A (A-ICs), type B (B-ICs), and the non-A, non-B ICs . The A-I2+/Mg2+ by inhibiting NKCC2 and ROMK, and then reduce Ca2+ transport via the paracellular routes by inhibiting CLDN16. These effects are related to the reduced reabsorption of Ca2+. In the DCT, the apical CaSR increases Ca2+ reabsorption via TRPV5 independent of NaCl reabsorption. Finally, in the collecting duct, the activation of CaSR will cause urinary dilution and induce urinary acidification through the promotion of the H+-ATPase , VSMCs, and peri-vascular nerves to regulate vessel elasticity. The activation of the CaSR in these cells mainly results in vascular relaxation under complex pathways, which is closely related to blood pressure control. However, in vascular calcification, the CaSR in mainly involved in the dysfunction of Cafication ,46. The and Ca2+ . These ffication ,37. Thisfication . This fification ,50. The fication ,52. The fication . MGP is fication  and is afication . The actfication .2+ signaling dysregulation is always considered to be the most critical factor for the induction of nephrolithiasis formation. Although the mechanism of stone formation has been studied for years, it remains incompletely understood. Kidney stone formation is a process involving physicochemical changes and supersaturation of urine. The physicochemical changes imply that an imbalance exists between urinary promoters  and urinary inhibitors ). Hypercalciuria was found to be the most common metabolic risk factor in stone formers [Nephrolithiasis is a major urological disease with a lifetime prevalence of approximately 10% . Calcium formers . Of note formers . Crystal2+ handling pathway, it is easier to understand the genetic influences underlying nephrolithiasis formation. First, the CaSR has the most important and complex role in renal Ca2+ handling. In fact, the activation of the CaSR can result in reduced calcium reabsorption in the TAL and increased calcium reabsorption in the DCT. Approximately 200 nonsynonymous single-nucleotide polymorphisms have been found in the human CaSR gene, and some of these are associated with idiopathic calcium kidney stones [2+ in the TAL and to dilute urine may thus be the causes; however, the exact mechanism is still unclear. Interestingly, the protective role of the CaSR in calcium nephrolithiasis is akin to its role in vascular calcification. Second, the mutation of genes associated with calcium homeostasis in the TAL results in a syndrome (Bartter syndrome) that presents with hypokalemic alkalosis, hyperreninemic hyperaldosteronism, and hypercalciuria [2+ reabsorption during elevated serum Ca2+ levels, and dysregulation of CLDN14 contributes to the development of nephrolithiasis [2+. Patients with mutations in the CLDN16 or CLDN19 genes, which are characterized by excessive renal magnesium and calcium excretion, are diagnosed with a syndrome of familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) [With advance in genome sequencing technologies in recent years, studies have emphasized the importance of genetic causes of kidney stone disease. Past literature has reported that a monogenic cause could be detected in 10–15% of individuals with adult-onset nephrolithiasis and in 15–20% of individuals <18 years old ,62,63,64y stones . Decreasy stones . The inaalciuria . These malciuria . In addiithiasis . CLDN16  (FHHNC) . Recent  (FHHNC) . CLDN2-kIn recent decades, growing evidence has linked nephrolithiasis to various metabolic diseases, including DM, obesity, cardiovascular disease, CKD, and metabolic syndrome ,73,74. AAlthough a consistent association between nephrolithiasis and vascular calcification has been established, the underlying mechanism remains uncertain. Nonetheless, several theories can be considered . First, 2+ signaling has become clearer over the years. In addition, the pathogenic link between vascular calcification and nephrolithiasis has been further corroborated by a growing body of evidence. As calcium is the main component in these two distinct conditions, a dysfunction in calcium homeostasis might be the mechanism connecting the two. Although calcium dysregulation cannot be used to explain the mechanisms in all patients, knowledge of the Ca2+ signaling can aid in the discovery of the optimal drug targets. Currently, vascular calcification or nephrolithiasis occurs due to a lack of effective medications. Consequently, therapeutic strategies targeting the CaSR might reduce the progression of vascular calcification and nephrolithiasis.Owing to more advanced animal and molecular studies, the process of renal Ca


This translational study aimed at gaining insight into the effects of lenalidomide in acute myeloid leukemia (AML). Forty-one AML patients aged 66 or older of the Swiss cohort of the HOVON-103 AML/SAKK30/10 study were included. After randomization, they received standard induction chemotherapy with or without lenalidomide. Bone marrow biopsies at diagnosis and before the 2nd induction cycle were obtained to assess the therapeutic impact on leukemic blasts and microenvironment. Increased bone marrow angiogenesis, as assessed by microvessel density (MVD), was found at AML diagnosis and differed significantly between the WHO categories. Morphological analysis revealed a higher initial MVD in AML with myelodysplasia-related changes (AML-MRC) and a more substantial decrease of microvascularization after lenalidomide exposure. A slight increase of T-bet-positive TH1-equivalents was identifiable under lenalidomide. In the subgroup of patients with AML-MRC, the progression-free survival differed between the two treatment regimens, showing a potential but not significant benefit of lenalidomide. We found no correlation between the cereblon genotype  and treatment response or prognosis. In conclusion, addition of lenalidomide may be beneficial to elderly patients suffering from AML-MRC, where it leads to a reduction of microvascularization and, probably, to an intensified specific T cell-driven anti-leukemic response.The online version contains supplementary material available at 10.1007/s00277-021-04467-2. Most individuals with acute myeloid leukemia (AML) are older than 65 years upon diagnosis . As the CSNK1A1 gene, which can be deleted or mutated in del(5q) MDS. In murine models, haploinsufficiency of this gene leads to hematopoietic stem cell expansion, whereas a complete loss induces stem cell apoptosis by activation of p53, explaining at least partially the effect of lenalidomide in del(5q) MDS , who were assigned to the category AML with defining translocations; both patients underwent standard treatment. The AML category with myelodysplasia-related changes (AML-MRC), either histomorphologically or genetically, applied to 8 patients; 7 of them were treated with additional lenalidomide and only one with the standard regimen  . Of these, ten received standard treatment and eleven additional lenalidomide. AML with defining mutations , , while in all other subgroups it was comparable between both treatment arms.Irrespectively of the therapeutic regimes, the amount of blasts significantly decreased after treatment: from 45.4% (± 26.7) to 9.2% (± 21.7) in the standard arm vs. 51.9% (± 25.0) to 17.0% (± 20.8) in the lenalidomide arm (2), is significantly higher in newly diagnosed AML compared to healthy control individuals  and being more frequently encountered in AML-MRC, as well as AML-MRC per se were found to be associated with higher T cell induced cytolytic activity [The presence of cytotoxic, granzyme-B-positive T cells differed between various AML categories of our study collective, being highest in AML-MRC. This observation is supported by recently published data, demonstrating an association between cell-intrinsic genetic alterations in AML and the amount of cytotoxic lymphocytes, suggesting that AML-MRC may be more immunogenic. Indeed, genetic alterations linked with poor prognosis [activity .FLT3 and NPM1 has been linked to low cytolytic activity of the tumor microenvironment [Significant decrease of leukemic blasts under treatment was observed in both arms. With respect to the AML-categories, a more substantial blast drop under lenalidomide was noticed in AML with defining mutations, despite the fact that the presence of driver mutations such as ironment . If thisironment .CRBN), which is in line with data from other IMiDs [Strong expression of lenalidomide’s target cereblon in the leukemic blasts was rather associated with an unfavorable OS, which has also been documented for gastric marginal zone lymphomas , but haser IMiDs .Our study has several shortcomings. We were not able to investigate the bone marrow samples of all patients included in the HOVON103 AML/SAKK 30/10 study due to lacking material and therefore the sample size is very limited. Due to the random distribution of cases, there was an imbalance of the assigned treatment arms among the AML categories. Finally, the observed beneficial effect of the addition of lenalidomide was present only in a subgroup of patients, i.e., AML-MRC, which although being a WHO category, is yet a post-hoc subcohort from the perspective of the initial clinical trial design .Observable on a small number of patients, addition of lenalidomide led to a perceptible but not significant increase of PFS in patients with AML-MRC, a category characterized by a poor prognosis and often complex karyotypes. Our findings are in keeping with encouraging results in the literature, showing on the one hand a direct anti-leukemic effect of lenalidomide, and, on the other hand, an important immune-activating impact on the tumor microenvironment. We think that our observations also highlight the importance of taking the WHO defined subentities into consideration when designing clinical trials in AML.Supplementary Figure 1A: Conventional morphology at initial diagnosis, H&E. B: Conventional morphology before the second induction cycle, H&E. C: CD34 staining revealing initial microvessel density and distinct positivity of the leukemic blasts before treatment, immunoperoxidase. D: CD34 staining of the post-treatment biopsy before the second induction cycle illustrating a profound decrease of CD34-positive blasts and constant microvessel density, immunoperoxidase. E and F: Pre-treatment amount of CD8-positive T-cells (8%) (E), which slightly decreased after chemotherapy to 5% (F), immunoperoxidase. G and H: Increasing amount of T-bet-positive T-helper cells from 1% pre-treatment (G) to 4% post-treatment (H), immunoperoxidase. (JPG 6819 kb)Pre- (left side) and post-treatment (right side) bone marrow biopsies of a female patient with acute myeloid leukemia, not otherwise specified who has been treated with chemotherapy only. ESM 2(XLSX 18 kb)


About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence. Glioblastoma (GBM) is the most common and lethal brain tumor with a median survival of around 15 months after diagnosis . Despite+) [GBM is characterized by a high degree of heterogeneity on phenotypic, genetic, and cellular levels . Like ot+) . This su+) . i value of 3 nM for the full-length enzyme, we utilized brinzolamide (BRZ) in cell viability assays in comparison to the well-established pan-CA inhibitor acetazolamide (ACZ) and analyzed the potential and the mechanism of CA2 inhibition on TMZ sensitivity. We show that the therapeutic effect of brinzolamide is advantageous compared to acetazolamide. As no functional data on CA2 in GBM cells are available so far, we analyzed the functional effects of CA2 expression in GBM cells by assessing metabolic parameters, such as the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) in cells expressing CA2. Moreover, we investigated how CA2 affects proliferation, invasion, and TMZ resistance. As a potent inhibitor of CA2 with a Kp < 0.01). Glioblastoma stem-like cells (GSCs) are the major tumor cell type exerting chemoresistance with a strong capacity for self-renewal, one of the main reasons for GBM recurrence. For this reason, we analyzed patient-matched GBM samples from initial and from recurrent GBMs (20 samples from 10 patients). In this particular patient cohort, we observed that CA2 is significantly up-regulated , the localization of CA2 was determined by immunofluorescence in paraffin slides of rGBM patients D. In comNotably, higher expression levels of CA2 mRNA were detected in all GSCs compared to the GBM cell lines, whereas mRNA expression of CA9 was lower in all GSCs, and only one GSC line showed a higher expression level of CA12 while two of them were lower compared to GBM cell lines G. From aTo characterize the function of CA2 in GBM cells by gain-of-function analyses, stable cell clones of U87 and U251 GBM cell lines were generated by transfection with either a control vector (U87_Ctrl and U251_Ctrl) or with a CA2 plasmid (U87_CA2 and U251_CA2). After selection with G418, stable cell clones were generated and analyzed for CA2 expression by Western Blot and qPCR A,C, and ® assay . In addition, the invasive GBM cell line U251 shows higher rates of cell invasion into Matrigel when expressing CA2 (p < 0.001).Cell proliferation of these cell clones was determined using a CellTiter-Glo® assay B,D. BothTo analyze whether CA2 expression affects the cellular metabolism of U87 and U251 cells, we used a Seahorse Analyzer that continuously measures oxygen concentration as oxygen consumption rate (OCR) and the proton flux into the cell supernatant determined as extracellular acidification rate (ECAR) enabling quantification of mitochondrial respiration, ATP production, and glycolysis. Indicative for CA2 function, a notable increase in OCR G,I and Ehttps://www.selleckchem.com/carbonic-anhydrase.html; accessed on 12 December 2021). OCR and ECAR rates were determined after adding either ACZ or BRZ. Metabolic changes were hardly observed in U251_Ctrl cells  significantly augments TMZ cytotoxicity in all GSC lines. These results indicate that co-treatment with the CA inhibitor BRZ and TMZ leads to a better re-sensitization of GBM stem cells than ACZ applied in combination with TMZ E–G. 50 value of TMZ as shown previously [Next, we sought to explore the cytotoxic effects of single TMZ treatment and co-treatment with ACZ or BRZ in a GSC line with a high TMZ resistance (GSC_TMZ), resembling a GSC type present in recurrent GBM in comparison to the naïve GSC line (GSC_DMSO) . Firstlyeviously . SimilarAs established previously, autophagy is altered in GBM cells and can be induced by TMZ, so that many resistance mechanisms exist to prevent chemotherapy-induced autophagy. Autophagy itself might exert either a pro-tumor or an anti-tumor effect. We, therefore, asked whether this phenomenon that we observed above might be due to co-treatment with CA inhibitors promoting cell death via enhanced activation of autophagy. To test our hypothesis, LC3II, the lipidated autophagosome-associated form of LC3, and p62, a marker of autophagic flux were measured by Western Blot and as LC3-II puncta formation by immunocytochemistry. We noticed that BRZ/TMZ but not ACZ or BRZ enhanced autophagy of U251_CA2 cells (ratio of LC3II to LC3I) A,B. We also examined the effects of the combined treatment with TMZ/ACZ or TMZ/BRZ on CA2, LC3, p62 expression in U87_CA2 and U251_CA2 cells . Our resThese results indicate that BRZ might be involved in mediating TMZ chemosensitivity of CA2 overexpressing GBM cell lines through enhanced activation of autophagy. This mechanism is similar in GSCs, as the combination of BRZ and TMZ also increased cell death of GSCs. For a precise evaluation of the autophagic progress in GSCs, CA2, LC3, p62 were measured by Western Blot after treatment with TMZ alone, ACZ/BRZ alone, or co-treatment for 24 h and compared to controls. All three GSCs showed, in the presence of BRZ, a significant increase in LC3II that was accompanied by a decrease in LC3I in Western Blots E–G. ThisThe current standard strategies for patients with newly diagnosed GBM glioma include surgical resection, radiation therapy, and chemotherapy . HoweverIn our study, we identified a strategy to overcome GBM chemoresistance by pharmacological targeting of carbonic anhydrase 2 (CA2) for the improvement of TMZ efficacy which could have implications for further clinical strategies in GBM treatment. Transcriptomic data from recurrent tumor tissues and TMZ-resistant GSCs previously revealed consistently enhanced levels of CA2, however, no functional data on CA2 in GBM cells and GSCs were available. Firstly, we found higher expression levels of CA2 in GSCs compared to GBM cell lines, U87 and U251. To investigate the functional consequences of CA2 expression in these cells, we established stable CA2 overexpressing cell clones of U87 and U251 cells and determined their cellular behavior. 2 and H2O into bicarbonate. Hence, CAs are involved in cellular processes, such as mitochondrial respiration and acid-base regulation, electrolyte secretion, bone reabsorption, and calcification, also in lipogenesis and gluconeogenesis [As evolutionary highly conserved zinc-dependent proteins with one of the highest substrate turnover rates, CAs catalyze the conversion of COogenesis . As a hiogenesis ,35. It wogenesis . As a meogenesis  to contrhttps:clinicaltrials.gov; Study Number NCT03011671; accessed on 9 November 2021) in a dose of 500 mg per day and escalated to 1000 mg per day between cycle day 1–21. However, due to its pan-CA characteristics, ACZ could inhibit carbonic anhydrases which could have beneficial effects, i.e., by sensitizing GBM cells to TMZ, so that the ACZ effect might be less significant as more specific CA2 inhibition. Indeed, depending on the cell type investigated, there is a difference in the efficacy of BRZ/TMZ treatment compared to ACZ/TMZ treatment, which is highly significant in U251_CA2 cells and two of the three patient-derived GSC lines. Given these results, we hypothesize that a combination of the CA2 inhibitor brinzolamide (BRZ) in conjunction with TMZ might provide an improved patient benefit by acting on several cellular processes in which carbonic anhydrases are involved. Acetazolamide (ACZ) has been reported to have synergistic effects when combined with TMZ ,37,38 ani value of 3 nM for the full-length enzyme. However, brinzolamide is also able to inhibit CA12 with a Ki value of 3 nM for the catalytic domain. As an argument for higher specificity of brinzolamide for CA2 over CA12, the Ki value for the full-length enzyme of CA12 is unknown and Ki values for the full-length enzyme are likely by an order of magnitude higher than for the isolated catalytic domain [Concerning carbonic anhydrases known to be physiologically relevant, the specificity of CA-inhibitors needs to be evaluated . In contc domain ,24. NeveIn this respect, our study demonstrates the differences between the application of a pan-CA and a more specific CA2 inhibitor. Metabolic processes in GSCs were more severely impaired by ACZ as compared to BRZ. This can be explained by the fact that all CAs present in GBM cells  are part of the inside/out regulation of protons and the monocarboxylate transport mechanism. Thus, as observed in GBM cells, ACZ reduces OCR and ECAE in CA2 overexpressing cells, but the effects of ACZ are significantly stronger than those of BRZ.However, there might be a certain “hierarchy” in CAs, in which CA2 might have stronger effects on cellular processes apart from metabolism, and this is the induced invasion seen under CA2 expression. It is, therefore, interesting to notice that in this particular cellular process, the effect of BRZ is stronger than that of ACZ. Both CA inhibitors, ACZ and BRZ, are not cytotoxic as our data suggest in all cell lines investigated. However, both inhibitors can increase TMZ efficacy. The highest TMZ efficacy is achieved at physiological pH. However, tumor cells often exhibit an alkaline pH, so that acidification as a result of CA2 inhibition leads to a physiological pH, thereby enhancing the uptake of TMZ into tumor cells. The described alterations of metabolism in vivo often causally correlate with hypoxia ,38. HypoIn our study, we confirmed that in GBM cells expressing CA2 and in GSCs, CA2 can suppress autophagy, as autophagy is activated after treatment with the CA inhibitor BRZ ,42. SimiIn addition to autophagy regulation, we show in our study that inhibition of CA2 by brinzolamide has a strong effect on the invasive behavior of GBM cells which is superior to ACZ. Whether this effect is solely based on metabolic effects remains to be determined.These findings have implications for clinical studies. Firstly, we show that ACZ is only effective in higher doses of 400 μM. As an estimate, the ACZ concentration of current clinical studies is in the range of 100 μM so that autophagy induction is most likely not sufficient to exert a significant clinical benefit to patients, as these ACZ doses used for co-treatment with TMZ are shown to be inefficient in our cell-based studies as demonstrated here. Based on these data, a TMZ/BRZ co-treatment would be a more promising approach. However, so far, pharmacological use of BRZ is only approved for ophthalmologic applications  and it rAll tumor tissue samples from patients obtained approval from the ethics committee of the Medical Faculty, Philipps University of Marburg .http://timer.cistrome.org/; 12 December 2021), data are not shown. For GBM only 5 normal tissues, we used the “Expression analysis Box Plots” part of the GEPIA2 website  to obtain the CA2 expression difference between GBM tumor tissues and the normal tissues of the GETx (Genotype-Tissue Expression) database.We checked the CA2 gene expression in the “Gene-DE” part of the TIMER2 website and observed the CA2 difference expression between different tumor tissues and adjacent normal tissues in the TCGA database (2 humidified incubator.U87 and U251 glioblastoma cell lines (identity of cell lines was verified by karyotyping and STR profiling) were obtained from ECACC and were cultured in DMEM cell medium  supplemented with 10% fetal bovine serum (FBS) , 1% penicillin/streptomycin , 1% Non-essential amino acids (NEAA)  and 1% Sodium pyruvate . Glioblastoma stem-like cells were isolated as described previously . Cells wU87 and U251 CA2 expressing cells were generated by transfection with CA2 plasmid  using 2.5 μg of plasmid DNA mix Lipofectamine LTX Reagent . After 48 h, the medium was changed to complete DMEM with G418  (400 μg/mL for U87 cells and 600 μg/mL for U251 cells) for 3 weeks. Positive clones were picked and further selected for stable integration. The plasmid pCMV6-LacZ-Bbsl  was used for vector control cell lines.3 cells in 50 μL medium were seeded on the other side of the insert, turn the transwell right side up again after 4 h adherence. Around 250 μL medium containing 20% FBS was supplied to the transwell on the upper chamber whereas 750 μL medium containing 0.5% FBS was added into the 24-well plate to create a concentration gradient. Cells invaded along the FBS gradient into the matrigel for 24 h, and cells were fixed with 4% PFA, permeabilized with 0.3% Triton , and nuclei were stained with Hoechst 33342  for 15 min at RT in the dark. Images were acquired using Leica confocal microscope. Cells were counted in six random areas and were calculated as the percentage of cells that passed across the membrane.Acetazolamide  and Brinzolamide  were used for cell treatment 24 h before cell invasiveness analysis. An 8 μm pore transwell insert  in conjunction with a 24-well plate was used. Transwells were coated with 50 μL matrigel Mix (matrigel mixed 1:1 with cold DMEM) , transwell was turned upside down after 1 h after solidification, and 2.5 × 103 cells per well, GSCs were seeded at a density of 1.0 × 104 cells per well, TMZ-resistant GSCs were seeded as described previously [The survival effect of cells was determined using the CellTiter-Glo 3D cell viability assay . GBM cell lines were seeded at a density of 2.0 × 10eviously . All celThe seahorse system XF96-Analyzer  was used to measure alterations in the metabolism of cells to analyze mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), 9000 cells were seeded in micro-plate overnight, after inhibitor treatment 24 h, the conditional medium was replaced by seahorse medium and incubated for 1 h before measurement. The whole detailed steps were executed as described before −ΔΔCT. Total RNA was isolated by QIAzol reagent  and absorbance was measured with OD 260/280 ratio between 1.8 and 2.1. For qPCR analysis, 2 μg of RNA was subjected to the synthesis of cDNA by using RNA to cDNA EcoDry Premix  according to the manufacturer’s instructions. Total reaction volume (20 μL) includes 10 μL SYBR Green/Rox Master Mix , 2 μL primers synthesized by Qiagen GmbH , 6 μL nuclease-free water, and 2 μL cDNA. The qPCR protocol set initial denaturation at 95 °C for 10 min, then 40 amplification cycles including 95 °C for 15 s and 60 °C for 1 min. The XS-13 was used as an internal reference for all reactions. The fold changes in gene expression relative to control were calculated by 2After cells were washed 3 times with ice-cold PBS, total proteins were extracted with RIPA buffer including a protease inhibitor  and phosphatase inhibitor . Protein lysates were boiled in Laemmli and sample reducing buffer  for 5 min. An equal amount of protein samples were separated by 12.5% SDS polyacrylamide gel electrophoresis and transferred onto nitrocellulose (NC) membranes . After blocking with 5% non-fat milk  for 1 h at RT, and membrane were incubated overnight at 4 °C with primary antibody, the following antibodies were used: anti-CA2 , anti-MAP LC3 , anti-p62 , and anti-β-Tubulin  in 5% milk in TBST. Subsequently, the NC membranes were incubated with secondary antibody Donkey (Dnk) pAb to Mouse (Ms) IgG (HRP)  and Dnk pAb to Rabbit (Rb) IgG (HRP)  for 1 h at RT. The membranes were washed with TBS-T and scanned with Chemostar Imager .Paraffin sections (5 μm thick) were obtained from GBM patients. All slides were blocked to avoid non-specific binding with 1% BSA , and incubated with Rabbit Anti-CA2 antibody  and Mouse Anti-Sox2 antibody  at 4 °C overnight, washed in PBS 3 times, and incubated with secondary antibody Donkey anti-Rabbit 550 , Donkey anti-Mouse 488  at least 1 h at RT avoid night. Nuclei were stained with Hoechst 33342  15 min at RT avoid night before cover with anti-fade mounting medium  was done. Images were acquired using a Leica DM 5500 microscope.For IF, cells were cultured on collagen I  coated coverslips in a 24-well plate overnight, cells were washed in PBS 3X, fixed with 4% paraformaldehyde 15 min at RT, permeabilized with 0.3% Triton X100  15 min at RT, blocked to avoid non-specific binding with 5% BSA 1 h, and incubated with Mouse anti-MAP LC3  at 4 °C overnight, washed, and incubated with secondary antibody donkey anti-Mouse 488  at least 1 h at RT avoid night. Nuclei were stained with Hoechst 33342 15 min at RT in the dark before covering with the anti-fade mounting medium was done.t-tests were used for statistical comparison among two groups. Analysis of variance (ANOVA) test was performed for multicomponent comparisons (one-way or two-way ANOVA), all statistical tests have been indicated in the figure legends. p-value < 0.05 was considered statistically significant.All data were shown as the mean ± SD or SEM and analyzed using GraphPad Prism software . Unpaired Student’s As recurrence caused by TMZ resistance is almost unavoidable for GBM patients, there is an urgent need for improved treatment options. Here we show the functional consequences of CA2 in GBM cell lines and GSCs and provide a rationale for inhibition of CA2 bearingf a novel therapeutic potential by sensitizing GBM cell lines and GSCs for TMZ treatment more efficiently as pan-CA inhibition using acetazolamide.


A mechanistic model from radiobiology has emerged by pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition, the repair of DNA double-strand breaks (DSB), and the final response to genotoxic stress. More recently, we provided evidence in this journal that the RIANS model is also relevant for exposure to metal ions. To document the role of the ATM-dependent DSB repair and signaling after pesticide exposure, we applied six current pesticides of domestic and environmental interest  to human skin fibroblast and brain cells. Our findings suggest that each pesticide tested may induce DSB at a rate that depends on the pesticide concentration and the RIANS status of cells. At specific concentration ranges, the nucleo-shuttling of ATM can be delayed, which impairs DSB recognition and repair, and contributes to toxicity. Interestingly, the combination of copper sulfate and thiabendazole or glyphosate was found to have additive or supra-additive effects on DSB recognition and/or repair. A general mechanistic model of the biological response to metal and/or pesticide is proposed to define quantitative endpoints for toxicity. While pesticides are abundantly used in agriculture, there is increasing evidence that their contact and handling may be associated with various cancer, neurodegenerative, respiratory, metabolic, and developmental diseases ,4,5,6,7.Pesticides represent an actual issue of public health through two essential clinical features, at least: toxicity and cancer risk . By consThe non-homologous end-joining (NHEJ) pathway is the predominant DSB recognition and repair pathway in human cells ,20. NotaHere, we examined whether a model based on the nucleo-shuttling of the ATM protein is also relevant for the exposure to pesticide. To this aim, human skin fibroblast and brain astrocyte cells from different RIANS statuses were exposed to six current pesticides of domestic and environmental interest, namely gamma-hexachlorocyclohexane  ,31, atraTwo types of human cells were used in this study: skin fibroblasts and brain astrocytes. All the cell lines tested were untransformed to avoid any bias linked to genomic stability. Furthermore, all the experiments were performed with cells in the plateau phase of growth (95–99% in G0/G1) to overcome any cell cycle effects, and to focus on the potential perturbations of the NHEJ pathway, the DSB repair and signaling pathway the most predominant in humans, which may be caused by exposure to pesticides. 2 humid conditions as monolayers with Dulbecco’s modified Eagle’s minimum medium (DMEM) , supplemented with 20% fetal calf serum, penicillin, and streptomycin. The origin and the radiobiological features of the RIANS-normal radioresistant 1BR3, 149BR controls were published elsewhere [Skin fibroblasts were routinely cultured at 37 °C in 5% COlsewhere ,39. It ilsewhere ,38,39. Tlsewhere .2 humid conditions, with a specific culture medium provided by the same manufacturer (#1801) supplemented with 20% fetal bovine serum (#0010), penicillin/streptomycin solution (#0503), and growth supplement (#1852). The radiobiological features of these brain astrocytes were published elsewhere [The human cortex , hippocampus , and spinal cord  astrocyte cells were purchased from ScienCell Research Laboratories  and routinely cultured as monolayers at 37 °C in 5% COlsewhere .All the pesticides tested here were purchased from Sigma-Aldrich France, Saint-Quentin-Fallavier, France): gamma-hexachlorocyclohexane , atrazin , glyphosate , permethrin , pentachlorophenol , and thiabendazole  . Pestici2 and CuSO4 metal species were purchased from Sigma-Aldrich (#751944 and #451657), respectively. Metals were diluted into culture medium for 24 h at the indicated concentrations. The 99% purified metal-pesticides complexes solution was prepared according to a protocol detailed elsewhere [The CuCllsewhere .−1) at the Anti-Cancer Centre Léon-Bérard  [Irradiations were performed with a 6 MeV X-rays medical SL 15 irradiator  (dose-rate: 6 Gy.min France) ,39. In aser139γH2AX antibody  was used at 1:800. The monoclonal anti-mouse anti-pATMser1981 (#ab2888) from Abcam  was used at 1:100. Incubations with anti-mouse fluorescein (FITC) and rhodamine (TRITC) secondary antibodies were performed at 1:100 at 37 °C for 20 min. Slides were mounted in 4′,6′ Diamidino-2-Phenyl-indole (DAPI)-stained Vectashield  for scoring micronuclei and mitoses, and examined with an Olympus fluorescence microscope. DAPI staining also indirectly permitted the evaluation of the yield of G1 cells (nuclei with homogeneous DAPI staining), S cells (nuclei showing numerous γH2AX foci), G2 cells (nuclei with heterogeneous DAPI staining), and metaphase (visible chromosomes). The immunofluorescence protocol was described elsewhere ,43. BrieThe foci scoring procedure applied here has received the certification agreement of CE mark and ISO-13485 quality management system norms. Our foci scoring procedure also developed some features that are protected in the frame of the Soleau Envelop and patents  . More thThe response curves data were fitted to the sigmoidal function, as defined in The number of the nuclear γH2AX foci was assessed in the human radioresistant and RIANS-normal 1BR3 fibroblasts 24 h after the introduction of pesticide molecules in the culture medium. The exposure of the 1BR3 cells to pesticides resulted in the appearance of γH2AX foci, suggesting the induction of DSB managed by the NHEJ pathway. All the response curves obeyed the similar sigmoidal functions of the pesticide concentration, but the rate of DSB production appeared to be specific to each pesticide tested . Three s4), or chloride (CuCl2) (p < 0.01) and ATR (p < 0.05), suggesting that the RIANS status may condition the rate of the production of DSB induced in a specific pesticide concentration-dependent manner  B. Simila (CuCl2) C. Convert manner D.In our hands, the average background level of untransformed human fibroblasts was found to be lower than 2 γH2AX foci per cell ,29. Furt>2, was deduced from data and analyzed. Each pesticide tested was found to be characterized by a specific TMC>2 value of some µM, reflecting the capacity of the pesticide to induce DSB , a quantitative correlation appeared , revealing that the TMC>2 from the RIANS-delayed 08HNG cells were 4.2 (=1/0.238) times higher than those of the RIANS-normal control cells without any change in the rank order of pesticides , a quantitative correlation appeared, suggesting that, whatever the pesticide tested, the higher the capacity to induce DSB managed by NHEJ, the higher the yield of micronuclei , suggesting that their presence during irradiation influences DSB recognition. Conversely, the number of γH2AX foci assessed 24 h post-irradiation was not found to be significantly influenced by the presence of pesticides , suggesting that the repair of recognized DSB was not affected by the presence of pesticides (data not shown).Cells were exposed to pesticides for 24 h, then exposed to 2 Gy X-rays. After culture medium renewal immediately after irradiation, a repair time ranging from 10 min to 24 h was applied . The γH2>2 values: a quantitative correlation appeared for the RIANS-normal 1BR3 cells, suggesting that the TMC>2 values the lower, and the inhibition power of DSB recognition the stronger . We examined, therefore, the nuclear relocalization of the auto-phosphorylated form of the ATM protein, reflecting its kinase activity in the nucleus . By applassessed .p < 0.001 for all the pesticides), suggesting that their presence during irradiation influences the DSB recognition. This last conclusion was consolidated by the quantitative correlation between γH2AX and pATM foci data  complexes to the culture medium, and applied it for 24 h to the cells before irradiation. Interestingly, the 99% purified (CuSO4 + TBZ) complexes resulted in less γH2AX foci induced than CuSO4 + TBZ, suggesting that free CuSO4 and/or TBZ molecules were responsible for a large contribution of DSB induction (4 + TBZ) complexes in the inhibition power of the DSB recognition step (4 + TBZ) complexes, their numbers were not found to be significantly different from those obtained with CuSO4 + TBZ, suggesting that the inhibition of the DSB repair process was essentially due to the metal + pesticides complexes. The corresponding pATM data were in quantitative agreement with the γH2AX foci data  complexes were also less marked , hippocampus (Hah), and spinal cord (Hasp) astrocytes .The concomitant application of metal and pesticide to human brain astrocytes led to a similar inhibition power of DSB recognition than that observed with pesticide alone, but a significantly larger number of unrepaired DSB, suggesting a strong toxicity . No specThere is highly documented evidence that exposure to pesticide causes the significant production of oxidative species, notably like superoxide anions, of which the interactions may result in hydrogen peroxide, a very efficient DNA-breaking agent. This is particularly true for the six pesticides tested here, and especially in human cells ,52,53,54In this study, we have deliberately chosen an experimental approach testing several pesticides rather than a single one, in order to identify and quantify a large spectrum of molecular impairments that will lead to different forms of cellular deaths. Hence, while cellular death and clinical consequences may be strongly dependent on the tissue and the organ exposed to pesticides, our approach deliberately consisted in investigating upstream cellular response to genotoxic stress. Such an approach has already been applied in our recent work on metals . FurtherThe choice of the pesticide concentration range was another limit of this study, since the yield of DNA breaks is generally not measurable for nanomolar concentrations in cells exposed in vitro, while occupational and environmental conditions concern this range. Conversely, the preclinical studies involving rodent models or rabbits allow the application of chronic exposure to pesticide at very low concentrations, but may present some biases vis-à-vis the extrapolation to human cells. Besides, there are few preclinical studies involving many pesticides to allow data intercomparison. Hence, we took particular care when analyzing quantitative correlations between each of the molecular endpoints tested, such as micronuclei and γH2AX and pATM foci , to propose a mechanistic model that would be coherent mathematically. In future experiments, we have to foresee investigations with chronic exposure. However, the application of low pesticides concentrations for a very long time  may raise practical problems regarding the maintenance of cultured cells for long periods. Furthermore, chronic exposures also raise the question that the induction and repair of DNA damage occur concomitantly, and therefore, mathematical models should be developed to predict, from single exposures data, the behavior of cells exposed chronically. This specific item raises the question of the dependence of the DSB repair rate on the initial dose. These issues are similar to those of the radiobiology of low doses of IR.Finally, in this study, we have stressed the fact that the response to pesticide exposure may strongly depend on individuals, but also on the nature of the tissue tested: three skin fibroblast cell lines of different RIANS status and three astrocyte cell lines derived from the same donor, but originated from different regions of the brain were tested. To our knowledge, this is the first time that the question of individual and tissue factors is raised with human untransformed skin fibroblasts and brain astrocytes exposed to pesticides. Obviously, further investigations are needed to consolidate our findings with a larger number of other cellular models and molecular endpoints. To date, there is highly documented evidence that the number of unrepairable DNA damage and chromosome breaks may serve as a measure of toxicity . In thisRegarding the γH2AX and pATM foci data assessed early after the exposure to pesticide, it appeared obvious that the presence of pesticide may influence the ATM-dependent phosphorylation of the H2AX at the DSB sites that conditions DSB recognition and triggers DSB repair by NHEJ. As specified above, a similar situation has already been documented in human radiosensitive cells , and in >2, H2AX/micronuclei ratio and DSB recognition inhibition power are plotted against IARC carcinogenicity group classification, it appears that these parameters cannot predict carcinogenicity, and suggest that biomarkers reflecting cancer proneness are also needed (The consequences of a delayed nucleo-shuttling of the ATM monomers may be double: (1) some DSB that are not recognized by the early step of the NHEJ pathway may be unrepaired and participate in the letal effect. These DSB may represent a significant subset of DNA damage that cumulates with the recognized, but unrepairable, DSB, that were revealed by the residual γH2AX foci at 24 h post-stress. Both these DSB subsets may be responsible for micronuclei, cellular death, and toxicity; (2) some DSB that are not recognized by the early step of the NHEJ pathway may be managed by an alternative DSB repair pathway, such as the recombination-like one, and become mis-repaired DSB. These DSB may participate in the carcinogenic process, but further investigations are, however, needed to follow these two categories of DSB after the stress by specific biomarkers that should be independent of the toxic  effect. Particularly in the case of an uncontrolled error-prone recombination-like process, a phenomenon producing a very large number of additional DNA breaks has been observed: this is the hyper-recombination phenomenon ,58,59,60o needed .Our findings related to the copper-pesticide complexes reveal that pesticide and copper may be characterized by specific DSB induction, recognition, and repair rates. The affinity of pesticides for copper and TBZ and GBH has been documented abundantly ,62. FromTogether with our previous report published in this journal , our finWO2017029450—Individual method predictive of the DNA-breaking genotoxic effects of chemical or biochemical agents.


In mesenteric artery smooth muscle cells (MASMCs), Ca2+ influx through Cav1.2 is the indirect mechanism for triggering Ca2+ sparks. This process is facilitated by plasma membrane-sarcoplasmic reticulum (PM-SR) nanojunctions that drive Ca2+ from the extracellular space into the SR via Sarco/Endoplasmic Reticulum Ca2+ (SERCA) pump. Ca2+ sparks produced by clusters of Ryanodine receptors (RyRs) at PM-SR nanodomains, decrease contractility by activating large-conductance Ca2+-activated K+ channels (BKCa channels), which generate spontaneous transient outward currents (STOCs). Altogether, Cav1.2, SERCA pump, RyRs, and BKCa channels work as a functional unit at the PM-SR nanodomain, regulating intracellular Ca2+ and vascular function. However, the effect of the ALDO/MR signaling pathway on this functional unit has not been completely explored. Our results show that short-term exposure to ALDO  increased the expression of Cav1.2 in rat MAs. The depolarization-induced Ca2+ entry increased SR Ca2+ load, and the frequencies of both Ca2+ sparks and STOCs, while [Ca2+]cyt and vasoconstriction remained unaltered in Aldo-treated MAs. ALDO treatment significantly increased the mRNA and protein expression levels of the SERCA pump, which counterbalanced the augmented Cav1.2-mediated Ca2+ influx at the PM-SR nanodomain, increasing SR Ca2+ content, Ca2+ spark and STOC frequencies, and opposing to hyperpolarization-induced vasoconstriction while enhancing Acetylcholine-mediated vasorelaxation. This work provides novel evidence for short-term ALDO-induced upregulation of the functional unit comprising Cav1.2, SERCA2 pump, RyRs, and BKCa channels; in which the SERCA pump buffers ALDO-induced upregulation of Ca2+ entry at the superficial SR-PM nanodomain of MASMCs, preventing ALDO-triggered depolarization-induced vasoconstriction and enhancing vasodilation. Pathological conditions that lead to SERCA pump downregulation, for instance, chronic exposure to ALDO, might favor the development of ALDO/MR-mediated augmented vasoconstriction of mesenteric arteries.In mesenteric arteries (MAs), aldosterone (ALDO) binds to the endogenous mineralocorticoid receptor (MR) and increases the expression of the voltage-gated L-type Ca Clinicav1.2 channel (LTCCs) expression  Ca2+ refilling, Ca2+ spark generation and blood pressure in resistance-sized MAs  junctions in vascular smooth muscle cells (VSMCs). In fact, LTCC-mediated subcellular Ca2+ signals, named Ca2+ sparklets, coincide with junctional SR expressing SERCA pumps and Ryanodine receptors (RyRs), demonstrating the close association of these proteins in the subplasmalemmal nanodomain , are involved in vasorelaxation (2+ sparks activate large-conductance Ca2+-activated K+ channels (BKCa channels) that generate spontaneous transient outward currents (STOCs) (2+ influx through LTCCs), diminishing global cytoplasmic Ca2+ concentration ([Ca2+]cyt), and opposing vasoconstriction  . STOCs htriction . Therefolaxation .Accessory proteins of RyRs  , might av1.2, SERCA2 pump, RyR, and BKCa channels, proteins that regulate intracellular Ca2+ handling and vascular function of mesenteric arteries.In this work, we determined the effect of a short-term (24 h) exposure to aldosterone (ALDO) in the expression and activity of CaAll procedures were performed according to the ethical guidelines of the Mexican Official Norm (NOM-062-ZOO-1999) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication updated in 2011). The animal protocol was approved by the Institutional Bioethical Committee for Care and Handling of Laboratory Animals at the Cinvestav-IPN (approved CICUAL Protocol No. 0100-14). Unless specified, all reagents were purchased from Sigma–Aldrich Quimica, S. de RL. de C.V., Toluca, Mexico.® PISA agropecuaria S.A. de C.V. Tula, Hidalgo, Mexico). To rule out the effects of chronic ALDO-mediated maladaptive vessel changes, and to avoid unsought Angiotensin II-induced vascular MR activation . Arteries were cleaned of fat and connective tissue and transferred to free-serum Dulbecco’s Modified Eagle Medium  supplemented with penicillin and streptomycin as previously described  or in its absence (control group). After incubation, MAs were used for vascular reactivity experiments or stored at −80°C for Western Blots and real-time qPCR experiments.Twelve-week-old male Wistar rats (250–300 g of body weight) were anesthetized by intraperitoneal injection of sodium pentobarbital solution  to maintain pH at 7.4. The MA rings were stabilized at a tension equivalent to that generated at 0.9× the diameter of the vessel at 100 mmHg for 45 min before experimentation. To determine the ring viability and achieve the maximal contractile response, MA rings were challenged with 60 mM KCl-containing PSS solution (equimolar substitution with NaCl to maintain constant ionic strength) twice before the beginning of concentration-response curves . Endothelium integrity was tested using an endothelium-dependent agonist, Acetylcholine (ACh). The vasorelaxation response was determined by concentration-response curves  in pre-contracted MA rings with 60 mM KCl. Each vasoconstriction or vasorelaxation experiment was performed in duplicate, with the mean used as a single experimental value. Data are shown as the percentage of maximal contractile response elicited by 60 mM KCl-containing solution, which was considered 100%.Vascular reactivity was assessed in third-order, resistance-sized MAs using a wire myograph for small vessels  as previously described  with som2 and homogenized on ice with a glass tissue grinder (Potter-Elvehjem) containing 200 μl cold homogenization buffer  supplemented with protease inhibitors . Homogenates were centrifuged at 2,000 × g for 10 min at 4°C and the supernatant was collected. Protein concentration was determined by the Bradford method. Supernatants were fractionated on 4–12% discontinuous gradient SDS-PAGE gel . Separated proteins were transferred onto nitrocellulose or PVDF membrane for 2 h, 100 V at 4°C and blocked from non-specific binding with 5% non-fat dried milk in phosphate buffered saline-Tween 20 (0.1%) (PBS-T) for 1 h, before the incubation with commercial primary antibodies previously used at indicated publications, against Cav1.2     (Protein expression levels were assessed by immunoblotting as previously reported  with somGermany) ; SERCA2 Germany) ; RyanodiGermany) ; calsequGermany) ; sorcin Germany) ; FKBP12.Germany) ; MR (1:2Germany) ; BKCa α  Israel) , BKCa β1 Israel) , Orai1 ( Israel)  and GAPD States)  for 2 h ® according to the manufacturer’s protocol. Total RNA was digested for 20 min at 37°C with 0.5 μL DNase . The reverse transcription was performed with Superscript II Reverse Transcriptase KIT (Invitrogen). The resulting DNA samples were amplified by real-time qPCR using the QuantiTect SYBR ®Green PCR Kits (QIAGEN) in a Rotor-Gene cycler . mRNA levels were normalized to the geometric mean of three housekeeping genes, 60S ribosomal protein L32 (Rpl32), 14-3-3 protein zeta/delta (Ywhaz), and transgelin (Sm22)  . All samn (Sm22) . The spe2 for 8 min at 37°C. The digestion was stopped by three washes with cold PSS. Individual cells were dissociated from vessels by gentle trituration with a fire polish glass pipette. The resulting cell suspension was stored at 4°C for up 1 h and was used on the same day. Only long, smooth, and optically refractive cells were used.Single smooth muscle cells from mesenteric arteries (MASMCs) were enzymatically isolated following as previously described  with som2+ concentration ([Ca2+]cyt) and the amplitude of caffeine-induced Ca2+ transients were estimated as previously described  for 25 min at RT and washed. An aliquot of 20 μL of Fura 2-loaded cells was placed in a 1-mL-volume recording chamber (homemade) filled with PSS-20K at RT. Cells were allowed to adhere to the chamber’s glass bottom for at least 5 min. Fura-2 fluorescence excitation ratios (F340/F380) were determined in individual cells each 50 ms within a 100-μm2 recording window in an inverted microscope  coupled to a PTI microfluorometry system . A single and brief pulse (10 s) of 10 mM caffeine (Caff) was locally applied by a pneumatic pump  using a TW100F-4 borosilicate micropipette placed above the cell. Data acquisition and analyses were performed in FeliX32 software . The Fura-2 fluorescence ratio values (F340/F380) were converted to [Ca2+] with the Grynkiewicz equation: 2+]cyt was similar in ALDO-treated and untreated, controls cells, even under mild depolarizing conditions (bath solution containing 20 mM K+). Therefore, Ca2+ influx due to depolarization-induced Cav1.2 activation did not increase global [Ca2+]cyt significantly. Then, we evaluated the SR Ca2+ content by a single caffeine challenge. 2+ transients in MASMCs treated or not with ALDO. Summarized data show a significantly higher amplitude of the caffeine-induced Ca2+ transient in MASMCs treated with ALDO with respect to control cells . These results suggest that mild depolarizing conditions activate Cav1.2-mediated Ca2+ influx, which mainly contributes to increasing SR Ca2+ stores instead of increasing global [Ca2+]cyt. In the presence of the SERCA pump inhibitor thapsigargin  no increase in [Ca2+]cyt was produced by an additional caffeine challenge  channels involved in mechanosensitive-mediated Ca2+ entry   .2+ pump transports Ca2+ ions from the cytoplasm into the SR and contributes to maintaining luminal SR Ca2+ levels  or in its absence. 2+ entry in Fluo 4-loaded MASMCs under all experimental conditions. After maintaining cells in the Na+ and Ca2+ free solution, the reintroduction of extracellular Ca2+ increased cytoplasmic Ca2+ in both CTRL and ALDO-treated cells with respect to basal Ca2+ levels. Importantly, the increase in cytoplasmic Ca2+ level was larger in ALDO-treated cells preincubated with TGN (5 min) than in cells without the SERCA inhibitor  . These results do not rule out the participation of additional mechanisms of Ca2+ entry  , a key SR Ca2+ buffering protein  ; and alsy (SOCE) ; we measy (SOCE) . Considey (SOCE) , we detehannels)  which mi protein , we meas2+ sparks reflecting localized Ca2+ release through type 2 RyR, the predominant isoform of RyRs in these cells  to block Cav1.2-mediated Ca2+ entry. We have found a significant increase in Ca2+ spark frequency in MASMCs treated with ALDO  in control MASMCs, an effect that has been previously observed with diltiazem, a benzothiazepine class of LTCC channel blocker . These data suggest a direct link between the MR signaling pathway activation, SR Ca2+ load and RyR activity in MASMCs. Changes in RyR protein expression or the expression of RyR accessory proteins might alter Ca2+ spark frequency and properties in VSMCs (2+ sparks were associated with downregulation of immunophilin FK506-binding proteins (FKBP12 and 12.6), which regulate RyR activity . Thus, we studied whether the increase in Ca2+ spark frequency in ALDO-treated MASMCs could have an impact on BKCa channel activity by analyzing STOC frequency and amplitude at near resting membrane potential (−40 mV) in PSS. Ca channel activation . However, the maximal relaxation response to ACh showed a tendency to be higher in ALDO-treated MA rings with respect to control arteries; though, this tendency was not significant as illustrated by the P value ; These data suggest that ALDO treatment increased the sensitivity to ACh, but not the maximal relaxation response to this agonist.Large-conductance Calaxation . The funv1.2-mediated Ca2+ entry in ALDO-treated MASMCs. Our work shows that a short-term (24 h) ex vivo exposure to 10 nM ALDO of resistance MAs: (1) increased both Cav1.2 and SERCA pump protein expressions; (2) increased depolarization-induced Ca2+ influx, luminal SR Ca2+ load, and Ca2+ spark and STOC frequencies; in consequence, (3) depolarization-induced vasoconstriction was unaffected as well global [Ca2+]cyt; and (4) ACh-mediated vasorelaxation was enhanced. In the presence of TGN, the reintroduction of extracellular Ca2+ induced a significant increase in cytoplasmic Ca2+ levels, unmasking the key participation of the SERCA pump in counterbalancing Ca2+ influx and avoiding undesired increments of global [Ca2+]cyt. All these data allow us to propose the model depicted in left side), Cav1.2-mediated Ca2+ influx is directed by SERCA pump to the SR Ca2+ stores; this, in turn, promotes the ignition of Ca2+ sparks (via clusters of RyRs) and STOCs (through BKCa channel activation). Therefore, Cav1.2, SERCA pump, RyRs and BKCa channels are actively working as a functional unit at the PM-SR nanodomain, regulating [Ca2+]cyt, luminal SR Ca2+ levels, and opposing to vasoconstriction. The exposure to 10 nM aldosterone  increases Cav1.2 protein expression and induces higher Cav1.2-mediated Ca2+ influx in MASMCs. However, the depolarization-induced vascular contraction is not enhanced because of SERCA pump upregulation, which efficiently counterbalances Ca2+ entry at the PM-SR nanodomain, increasing SR Ca2+ content, Ca2+ spark and STOC frequencies, and enhancing ACh-mediated vasorelaxation. The net result of this new steady-state is higher Ca2+ cycling at the PM-SR nanodomain, dampening unsought elevations of [Ca2+]cyt. However, higher luminal SR Ca2+ levels might also participate in enhancing receptor-mediated Ca2+ release and promote abnormal vasoconstriction, a hallmark feature of overactive ALDO/MR signaling pathway in chronic pathological conditions.In this study, we describe the crucial role of the SERCA pump in buffering Ca3H]-aldosterone has been observed in rat mesenteric vascular arcade  but probably not in others . This idea is further supported by reports showing that when LTCCs are activated through K+-mediated membrane depolarization maneuvers, vasoconstriction responses of mesenteric and cerebral arteries are unaffected even in conditions of ALDO/MR signaling pathway over-activation  negative feedback mechanisms that decrease Ca2+ influx; for instance, Ca2+-dependent inactivation of LTTCs, and BKCa channel activation which induces membrane hyperpolarization and decreases the open probability of LTCCs; (2) cytosolic Ca2+ buffering by proteins such as sorcin and calmodulin; and (3) Ca2+ removal mechanisms at the PM-SR nanodomain which include the SERCA pump, the Na+/Ca2+ exchanger, and the plasma membrane Ca2+ ATPase  in aortic SMCs of ALDO-salt hypertensive rats  .Regarding ALDO effects on RyR expression and activity, this work is the first to report that short-term treatment with ALDO increases Ca complex . Likely,ications . Diltiazy effect . This at6 mM K+) . Nifedipblockers . Consist2+ sparks from Ca2+ discharge regions (v1.2 channel gene inactivation (SMAKO mouse), both frequency and amplitude of STOCs were reduced, together with decreased cytosolic Ca2+ levels and SR Ca2+ load  in MASMCs; an additional Ca2+ source is provided by Cav3.2 channels (20–30%) (2+ channel activity (v1.2 and Cav3.2 to Ca2+ spark generation in ALDO-treated MASMCs.Depolarization of MASMCs increased the frequency and amplitude of STOCs and elicited Ca regions . Moreovea2+ load . Accordi(20–30%) . Besidesactivity . TherefoCa channel expression, and activity. In a mouse model with cardiomyocyte-specific overexpression of the aldosterone synthase gene (MAS mice), an impairment in ACh-induced vasorelaxation, associated with decreased mRNA expression of α and β1 subunits of BKCa channels in coronary arteries has been reported , but none of these studies had evaluated the effect of ALDO/MR signaling pathway on the functional unit that regulates vascular function, comprising Cav1.2, SERCA pump, Ryanodine receptors, and BKCa channels, and we aimed to study it comprehensively. Our work provides novel evidence about ALDO-induced upregulation of this functional unit unveiling the crucial role of the SERCA pump in counterbalancing Cav1.2-mediated Ca2+ influx at the subplasmalemmal space of mesenteric artery SMCs. This work highlights the intricacies of tissue-specific MR signaling and the differential actors of the ALDO/MR signaling pathway across vascular beds, contributing to ALDO-associated impairments of vascular functions. Initial functional changes induced by ALDO are adaptative, but in chronic pathological conditions, they would become maladaptive leading to vascular dysfunction, vascular remodeling, and poor compliant arteries as observed in hypertension.Cumulative evidence has shown that the mineralocorticoid receptor (MR) is found in vascular tissues where regulates expression and activity of ion channels that participate in CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by COMITÉ INTERNO PARA EL CUIDADO Y USO DE LOS ANIMALES DE LABORATORIO (CICUAL) Cinvestav.RS-E contributed to the conceptualization, methodology, data collecting, data analysis, and writing. AG-H contributed to the conceptualization, resources, review and editing. AG contributed to the conceptualization, resources, funding acquisition, and review and editing. J-PB contributed to the conceptualization, methodology, resources, funding acquisition, and review and editing. AR contributed to the conceptualization, methodology, data analysis, writing, resources, funding acquisition, and review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.