TRPM7 Expression Research Articles

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

MiR-135a Regulates Atrial Fibrillation by Targeting Smad3.

Atrial fibrillation (AF) is the most common arrhythmia in clinical. Atrial fibrosis is a hallmark feature of atrial structural remodeling in AF, which is regulated by the TGF-β1/Smad3 pathway. Recent studies have implicated that miRNAs are involved in the process of AF. However, the regulatory mechanisms of miRNAs remain largely unknown. This study is aimed at investigating the function and regulatory network of miR-135a in AF. In vivo, the plasma was collected from patients with AF and non-AF subjects. Adult SD rats were induced by acetylcholine (ACh) (66 μg/ml)-CaCl2 (10 mg/ml) to establish an AF rat model. In vitro, atrial fibroblasts (AFs), isolated from adult SD rats, were treated with high-frequency electrical stimulation (HES) (12 h) and hypoxia (24 h) to mimic the AF and atrial fibrosis, respectively. miR-135a expression was detected through quantitative real-time polymerase chain reaction (qRT-PCR). The association between miR-135a and Smad3 was speculated by the TargetScan database and confirmed by the luciferase reporter assay. Fibrosis-related genes, Smad3, and TRPM7 were all assessed. The expression of miR-135a was markedly decreased in the plasma of AF patients and AF rats, which was consistent with that in HES-treated and hypoxia-treated AFs. Smad3 was identified as a target of miR-135a. the downregulation of miR-135a was associated with the enhancement of Smad3/TRPM7 expressions in AFs. Additionally, the knockdown of Smad3 significantly reduced the expression of TRPM7 and further inhibited atrial fibrosis. Our study demonstrates that miR-135a regulates AF via Smad3/TRPM7, which is a potential therapeutic target for AF.

Cold bandage contact lens use reduces post-photorefractive keratectomy or corneal collagen-crosslinking pain perception in patients.

To compare post-operative pain perception using bandage contact lens (BCL) stored at 2-8°C (Cold BCL, CL-BCL) or room temperature (23 - 25°C, RT-BCL) after photorefractive keratectomy (PRK) or corneal collagen-crosslinking (CXL) and determine status of nociception associated factors. In this prospective interventional study, 56 patients undergoing PRK for refractive correction and 100 keratoconus (KC) undergoing CXL were recruited following approval from the institutional ethics committee with informed consent. Patients undergoing bilateral PRK received RT-BCL on one eye and CL-BCL on the other. Pain was graded by Wong-Baker scoring on the first post-operative day (PoD1). Expression of transient receptor potential channels (TRPV1, TRPA1, TRPM8), calcitonin gene-related peptide (CGRP) and IL-6 was measured in cellular content from used BCLs collected on PoD1. Equal number of KC patients received RT-BCL or CL-BCL post-CXL. Pain was graded by Wong-Baker scoring on PoD1. Pain scores on PoD1 were significantly (P < 0.0001) reduced in subjects receiving CL-BCL (Mean ± SD: 2.6 ± 2.1) compared to RT-BCL (6.0 ± 2.4) post-PRK. 80.4% of subjects reported reduced pain scores with CL-BCL. 19.6% reported no change or increased pain scores with CL-BCL. TRPM8 expression was significantly (P < 0.05) increased in BCL of subjects reporting reduced pain with CL-BCL compared to those who did not. Pain scores on PoD1 were significantly (P < 0.0001) reduced in subjects receiving CL-BCL (3.2 ± 2.1) compared to RT-BCL (7.2 ± 1.8) post-CXL. The simple approach of using a cold BCL post-operatively substantially reduced pain perception and could overcome post-operative pain-related limited acceptance of PRK/CXL.

TRPM8 channels do not regulate endothelium-dependent and -independent vascular reactivity in mice

Foremost among the ion channels that control vascular tone is the transient receptor potential (TRP) cation channels. The TRPM subfamily of the TRP channels includes eight members, expressed in various excitable and non-excitable cells and involved in diverse functions, such as sensing membrane potential, cold, taste, redox, osmolarity, and cell survival. Previous analysis of TRPM gene expression showed the highest expression of TRPM8 in intact blood vessels compared with other isoforms (TRPM1-7). Studies have also revealed that activating TRPM8 by menthol and icilin modulates endothelium-dependent and independent vascular reactivity. However, the non-selectivity of pharmacological TRPM8 modulators constitutes a significant limitation in the prior studies aimed at delineating TRPM8-dependent vasoregulation. In this study, we demonstrate that acetylcholine (ACH)- and sodium nitroprusside (SNP)-induced reduction in arterial pressure was unaltered in mice lacking TRPM8. We generated endothelial-specific knockout (KO) of TRPM8 (ecTRPM8-/-) by breeding TRPM8 floxed (TRPM8fl/fl) with Cdh5-Cre (VE-Cadherin-Cre) mice. Unlike global TRPM8 KO, ecTRPM8-/- mice were not resistant to cold temperatures. Phenylephrine-induced contraction of renal and mesenteric arteries isolated from TRPM8fl/fl and ecTRPM8-/- mice were similar. Also, ACH- and SNP-induced relaxation of phenylephrine-precontracted renal and mesenteric arteries from TRPM8fl/fl and ecTRPM8-/- mice were comparable. These findings suggest that TRPM8 channels do not regulate endothelium-dependent and -independent vasorelaxation in mice. Disclosure of Funding: NHLBI: R01 HL151735-01 and R01HL151735-S This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Profile of MUC5AC and MUC5B mucins expression in asthma patients under cold exposure

Introduction. Cold airway hyperresponsiveness (CAH) is a common condition in patients with asthma, which worsens the clinical course of the disease and the patients’ quality of life. MUC5AC and MUC5B are the main secreted mucins in the respiratory tract, which are involved in normal mucociliary clearance, but also capable of provoking the development of pathological changes in case of dysregulation of their balanced production.Aim. The aim of this study was to determine the dynamics of MUC5AC and MUC5B expression during experimental cooling in patients with asthma depending on the status of CAH.Materials and methods. The study enrolled 98 subjects including 26 patients with chronic non-obstructive bronchitis without exacerbation (control group) and 72 patients with asthma. The expression of MUC5AC, MUC5B and TRPM8 was determined in the upper respiratory tract by quantitative reverse transcription PCR. The production of MUC5AC and MUC5B was also measured in sputum by ELISA. All patients underwent a bronchoprovocation test with isocapnic cold air hyperventilation to detect CAH, and a similar nasal challenge was performed to assess the effect of cooling on the expression of the studied genes.Results. Patients with asthma had 4.22-fold increase in the expression of MUC5AC (p=0.02) in the nasal epithelium as compared with the control group. CAH was associated with an initial 7.33-fold upregulation of MUC5AC (p=0.008) as well as with further increase in MUC5AC expression but a decrease in MUC5B in response to cooling, which was not observed in asthma patients without CAH. Basal TRPM8 expression was associated with baseline level of MUC5AC (ρ=0.41, p=0.04), MUC5B (ρ=0.55, p&lt;0.001) and amount of sputum produced after the cold bronchoprovocation.Conclusion. Asthma patients with CAH demonstrate a more pronounced imbalance in the production of mucins, which is aggravated by cold exposure. This, in turn, can lead to a number of pathological disorders associated with a more severe course of the disease.

Ion channels in dry eye disease.

Dry eye disease (DED) which affects millions of people worldwide is an ocular surface disease that is strongly associated with pain, discomfort, and visual disturbances. Altered tear film dynamics, hyperosmolarity, ocular surface inflammation, and neurosensory abnormalities are the key contributors to DED pathogenesis. The presence of discordance between signs and symptoms of DED in patients and refractoriness to current therapies in some patients underpin the need for studying additional contributors that can be modulated. The presence of electrolytes or ions including sodium, potassium, chloride, bicarbonate, calcium, and magnesium in the tear fluid and ocular surface cells contribute to ocular surface homeostasis. Ionic or electrolyte imbalance and osmotic imbalance have been observed in DED and feed-forward interaction between ionic imbalances and inflammation alter cellular processes in the ocular surface resulting in DED. Ionic balances in various cellular and intercellular compartments are maintained by dynamic transport via ion channel proteins present in cell membranes. Hence, alterations in the expression and/or activity of about 33 types of ion channels that belong to voltage-gated channels, ligand-gated channels, mechanosensitive ion channel, aquaporins, chloride ion channel, sodium-potassium-chloride pumps or cotransporters have been investigated in the context of ocular surface health and DED in animal and/or human subjects. An increase in the expression or activity of TRPA1, TRPV1, Nav1.8, KCNJ6, ASIC1, ASIC3, P2X, P2Y, and NMDA receptor have been implicated in DED pathogenesis, whereas an increase in the expression or activity of TRPM8, GABAA receptor, CFTR, and NKA have been associated with resolution of DED.

Prepubertal to adulthood exposure to low doses of glyphosate-based herbicide increases the expression of the Havcr1 (Kim1) biomarker and causes mild kidney alterations

Glyphosate is a nonselective and postemergent herbicide used to combat weeds in several crops, which raises concerns about risks to human health since residues are detected in urine, human milk, surface water and several types of food. Feces and urine are the major routes of elimination of glyphosate, making the kidney a sensitive target for the development of toxicity. In fact, farmers are at high risk of developing chronic kidney disease. In this sense, this study aims to investigate kidney function by measuring the serum levels of urea and creatinine, examining the histological morphology, and analyzing the mRNA expression of genes related to tubular transport of ions, urea and urates and the biomarker of kidney disease Kim1, and the levels of lead in the kidney in male Wistar rats orally exposed to low levels of glyphosate-based herbicide (GBH: 0, 0.5 or 5 mg/kg) from weaning to adult life by gavage. GBH0.5 showed reduced serum urea concentration, presence of tubulointerstitial swelling and mononuclear cell infiltration into the interstitium, increased gene expression of Kim1 and reduced gene expression of Slc14a1. GBH5 showed reduced serum urea and increased serum creatinine concentrations, tubulointerstitial swelling, interstitial fibrosis, and reduced expression of Trpm6 and Trpv5. Exposure to GBH did not affect the levels of Pb in the kidneys of animals. In conclusion, glyphosate at low doses may cause mild kidney damage. It is necessary to evaluate whether the long-term effects of this constant injury may contribute to the development of chronic kidney disease of uncertain etiology.

Neuroimmune Mediators of Pruritus in Hispanic Scalp Psoriatic Itch.

Scalp psoriatic itch is a common, bothersome, yet understudied, condition with numerous associated treatment challenges. The aim of this study was to enhance our understanding of the pathophysiology of scalp psoriatic itch. Immunohistochemical analysis of known neuroimmune mediators of pruritus was conducted using scalp biopsies from 27 Hispanic psoriatic patients. Patients were categorized into mild/moderate or severe itch groups according to their itch intensity rating of scalp itch. Protease activated receptor (PAR2), substance P, transient receptor potential (TRP)V3, TRPM8 and interleukin-23 expression all correlated significantly with itch intensity. The pathophysiology of scalp psoriasis is largely non-histaminergic, mediated by PAR2, interleukin-23, transient receptor potential channels, and substance P.

Ginkgolide A downregulates transient receptor potential (melastatin) 2 to protect cisplatin-induced acute kidney injury in rats through the TWEAK/Fn14 pathway: Ginkgolide A improve acute renal injury.

In order to seek effective drugs for treating cisplatin-induced acute renal injury and explore the corresponding potential mechanism. Mouse kidney injury model was established by intraperitoneal injection of 20mg/kg cisplatin. The temporal expression of TRPM2 and the regulation of Ginkgolide A on its expression were analyzed by western blot. In order to perform the mechanical analysis, we used TRPM2-KO knockout mice. In this study, we evaluated the repair effect of GA on acute kidney injury through renal function factors, inflammatory factors and calcium and potassium content. Pathological injury and cell apoptosis were detected by H&E and TUNEL, respectively. Ginkgolide A inhibited inflammatory reaction and excessive oxidative stress, reduced renal function parameters, and improved pathological injury. Meanwhile, we also found that the repair effect of Ginkgolide A on renal injury is related to TRPM2, and Ginkgolide A downregulated TRPM2 expression and inactivated TWEAK/Fn14 pathway in cisplatin-induced renal injury model. We also found that inhibition of TWEAK/Fn14 pathway was more effective in TRPM2-KO mice than TRPM2-WT mice. Ginkgolide A was the effective therapeutic drug for cisplatin-induced renal injury through acting on TRPM2, and TWEAK/Fn14 pathway was the downstream pathway of Ginkgolide A in acute renal injury, and Ginkgolide A inhibited TWEAK/Fn14 pathway in cisplatin-induced renal injury model.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

SHANK3 in vagal sensory neurons regulates body temperature, systemic inflammation, and sepsis.

Excessive inflammation has been implicated in autism spectrum disorder (ASD), but the underlying mechanisms have not been fully studied. SHANK3 is a synaptic scaffolding protein and mutations of SHANK3 are involved in ASD. Shank3 expression in dorsal root ganglion sensory neurons also regulates heat pain and touch. However, the role of Shank3 in the vagus system remains unknown. We induced systemic inflammation by lipopolysaccharide (LPS) and measured body temperature and serum IL-6 levels in mice. We found that homozygous and heterozygous Shank3 deficiency, but not Shank2 and Trpv1 deficiency, aggravates hypothermia, systemic inflammation (serum IL-6 levels), and sepsis mortality in mice, induced by lipopolysaccharide (LPS). Furthermore, these deficits can be recapitulated by specific deletion of Shank3 in Nav1.8-expressing sensory neurons in conditional knockout (CKO) mice or by selective knockdown of Shank3 or Trpm2 in vagal sensory neurons in nodose ganglion (NG). Mice with Shank3 deficiency have normal basal core temperature but fail to adjust body temperature after perturbations with lower or higher body temperatures or auricular vagus nerve stimulation. In situ hybridization with RNAscope revealed that Shank3 is broadly expressed by vagal sensory neurons and this expression was largely lost in Shank3 cKO mice. Mechanistically, Shank3 regulates the expression of Trpm2 in NG, as Trpm2 but not Trpv1 mRNA levels in NG were significantly reduced in Shank3 KO mice. Our findings demonstrated a novel molecular mechanism by which Shank3 in vagal sensory neurons regulates body temperature, inflammation, and sepsis. We also provided new insights into inflammation dysregulation in ASD.

Comparative transcriptome analysis of rainbow trout gonadal cells (RTG-2) infected with U and J genogroup infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is the causative pathogen of infectious hematopoietic necrosis, outbreaks of which are responsible for significant losses in rainbow trout aquaculture. Strains of IHNV isolated worldwide have been classified into five major genogroups, J, E, L, M, and U. To date, comparative transcriptomic analysis has only been conducted individually for the J and M genogroups. In this study, we compared the transcriptome profiles in U genogroup and J genogroup IHNV-infected RTG-2 cells with mock-infected RTG-2 cells. The RNA-seq results revealed 17,064 new genes, of which 7,390 genes were functionally annotated. Differentially expressed gene (DEG) analysis between U and J IHNV-infected cells revealed 2,238 DEGs, including 1,011 downregulated genes and 1,227 upregulated genes. Among the 2,238 DEGs, 345 new genes were discovered. The DEGs related to immune responses, cellular signal transduction, and viral diseases were further analyzed. RT-qPCR validation confirmed that the changes in expression of the immune response-related genes trpm2, sting, itgb7, ripk2, and irf1, cellular signal transduction-related genes irl, cacnb2, bmp2l, gadd45α, and plk2, and viral disease-related genes mlf1, mtor, armc5, pik3r1, and c-myc were consistent with the results of transcriptome analysis. Taken together, our findings provide a comprehensive transcriptional analysis of the differential virulence of the U and J genogroups of IHNV, and shed new light on the pathogenic mechanisms of IHNV strains.

The LHX2-OTX2 transcriptional regulatory module controls retinal pigmented epithelium differentiation and underlies genetic risk for age-related macular degeneration.

Tissue-specific transcription factors (TFs) control the transcriptome through an association with noncoding regulatory regions (cistromes). Identifying the combination of TFs that dictate specific cell fate, their specific cistromes and examining their involvement in complex human traits remain a major challenge. Here, we focus on the retinal pigmented epithelium (RPE), an essential lineage for retinal development and function and the primary tissue affected in age-related macular degeneration (AMD), a leading cause of blindness. By combining mechanistic findings in stem-cell-derived human RPE, in vivo functional studies in mice and global transcriptomic and proteomic analyses, we revealed that the key developmental TFs LHX2 and OTX2 function together in transcriptional module containing LDB1 and SWI/SNF (BAF) to regulate the RPE transcriptome. Importantly, the intersection between the identified LHX2-OTX2 cistrome with published expression quantitative trait loci, ATAC-seq data from human RPE, and AMD genome-wide association study (GWAS) data, followed by functional validation using a reporter assay, revealed a causal genetic variant that affects AMD risk by altering TRPM1 expression in the RPE through modulation of LHX2 transcriptional activity on its promoter. Taken together, the reported cistrome of LHX2 and OTX2, the identified downstream genes and interacting co-factors reveal the RPE transcription module and uncover a causal regulatory risk single-nucleotide polymorphism (SNP) in the multifactorial common blinding disease AMD.

NMDA Receptor Activation Stimulates Hypoxia-Induced TRPM2 Channel Activation, Mitochondrial Oxidative Stress, and Apoptosis in Neuronal Cell Line: Modular Role of Memantine

TRPM2 channel is activated by the increase of hypoxia (HYP)-mediated excessive mitochondrial (mROS) and cytosolic (cROS) free reactive oxygen species generation and intracellular free Ca2+ ([Ca2+]i) influx. The stimulations of the N-methyl-d-aspartate(NMDA) receptor and TRPM2 channel induce mROS and apoptosis in the neurons, although their inhibitions via the treatments of memantine (MEM) and MK-801 decrease mROS and apoptosis. However, the molecular mechanisms underlying MEM treatment and NMDA inhibition’ neuroprotection via TRPM2 inhibition in the HYP remain elusive. We investigated the modulator role of MEM and NMDA via the modulation of TRPM2 on oxidative neurodegeneration and apoptosis in SH-SY5Y neuronal cells. Six groups were induced in the SH-SY5Y and HEK293 cells as follows: Control, MEM, NMDA blocker (MK-801), HYP (CoCl2), HYP + MEM, and HYP + MK-801. The HYP caused to the increases of TRPM2 and PARP-1 expressions, and TRPM2 agonist (H2O2 and ADP-ribose)-induced TRPM2 current density and [Ca2+]i concentration via the upregulation of mitochondrial membrane potential, cROS, and mROS generations. The alterations were not observed in the absence of TRPM2 in the HEK293 cells. The increase of cROS, mROS, lipid peroxidation, cell death (propidium iodide/Hoechst) rate, apoptosis, caspase −3, caspase −8, and caspase −9 were restored via upregulation of glutathione and glutathione peroxidase by the treatments of TRPM2 antagonists (ACA or 2-APB), MEM, and MK-801. In conclusion, the inhibition of NMDA receptor via MEM treatment modulated HYP-mediated mROS, apoptosis, and TRPM2-induced excessive [Ca2+]i and may provide an avenue for protecting HYP-mediated neurodegenerative diseases associated with the increase of mROS, [Ca2+]i, and apoptosis.

Effects of Vitamin D and Memantine on Repetitive Mild Traumatic Brain Injury via mTOR, TRPM2, and GABA Expression Levels on Juvenile Rats.

To investigate the effects of vitamin D and memantine on the healing process in juvenile rats with repetitive brain injury (rTBI) and to elucidate the mechanisms underlying these potential therapeutic effects. Juvenile rats were randomly allocated into seven groups, with eight rats per group: sham-operated (Group I), trauma (Group II), memantine supplementation (10 mg/kg) pre-trauma (Group III), vitamin D supplementation (5 µg/kg) pre-trauma (Group IV), vitamin D supplementation post-trauma (Group V), memantine and vitamin D supplementation post-trauma (Group VI), and vitamin D supplementation pre- and post-trauma with post-trauma memantine supplementation (Group VII). A modified repeated weight drop model was employed to induce rTBI. Brain tissues and blood samples were collected for analysis. Expressions of the mammalian target of rapamycin (mTOR), temporary receptor potential (TRPM2), and GABA receptors were assessed via immunohistochemistry. Levels of 8-hydroxy-2-deoxyguanine (8-OHdG) were determined using high-performance liquid chromatography (HPLC). Matrix metalloproteinases -2 and -9, tissue inhibitors of metalloproteinases-1 and-2, and NADPH oxidation-4 levels were determined using commercially available enzyme-linked immunosorbent Test kits. Immunohistochemistry analyses were performed on the brain cortex and hippocampus. The levels of 8OHdG/106dG, MMP-2, MMP-9, TIMP-1, -TIMP2, and NOX-4 were significantly higher in the trauma group than in the other groups. No difference was found between the control and Pre Vit D+Mem+Post Vit D groups regarding 8OHdG/106dG, MMP-2, -9 and NOX-4 levels. Normalized expressions of mTOR and TRPM2 were observed in Groups VI and VII. Conversely, GABA expression levels decreased in Group II, with the most pronounced therapeutic effects observed in Group VII. Memantine and vitamin D positively affected rTBI when used alone. Their combined use exhibited greater therapeutic outcomes. These effects are mediated by mTOR mRNA, TRPM2 mRNA, and GABA mRNA expressions.

PPAR gamma negatively regulates the expression of TRPM8 in normal epidermal cells but mutually regulate their expressions with TRPM8 by feed-back loop regulation in squamous carcinoma cells

PPAR gamma negatively regulates the expression of TRPM8 in normal epidermal cells but mutually regulate their expressions with TRPM8 by feed-back loop regulation in squamous carcinoma cells

Sevoflurane participates in the protection of rat renal ischemia-reperfusion injury by down-regulating the expression of TRPM7.

To investigate the protective effect of sevoflurane preconditioning on renal ischemia-reperfusion injury (renalischemiareperfusionmodel, RIRI) and its related mechanism. Eighty healthy adult male SD rats were randomly divided into control group (Sham group), model group (RIRI group), sevoflurane pretreatment group (Sev group) and TRPM7 inhibitor combined with sevoflurane pretreatment group (T + Sev group), 20 animals in each group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, and the levels of creatinine and urea nitrogen in each group were detected. Deoxyribonucleic acid terminal transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect renal cell apoptosis, and Western blottingwas used to detect the expression of apoptotic proteins cleaved-caspase-3, bax, Bcl-2, and TRPM7 in renal tissue; Detection of oxidative stress-related index levels in renal tissue and levels of inflammatory factors in renal tissue and serum. Compared with the Sham group, the renal tissue pathological damage was aggravated, the levels of creatinine and blood urea nitrogen were increased, and the apoptosis was increased in the RIR group and the Sev group. Death, malondialdehyde (MDA) levels and inflammatory factors were increased, and superoxide dismutase (SOD) levels were decreased (all p < .05); The scores, apoptosis rate, MDA level, and relative expression of inflammatory factor levels were decreased, and SOD levels were increased (all p < .05). Compared with the Sev group, the renal tissue pathological damage in the T + Sev group was aggravated, creatinine, blood urea nitrogen levels increased, apoptosis increased, apoptosis-related proteins cleaved-caspase-3, bax, Bcl-2 showed increased apoptosis, malondialdehyde (MDA) levels, inflammatory factor levels increased, ultrahigh The levels of oxide dismutase (SOD) were decreased (all p < .05). Therefore, we believe that sevoflurane is involved in the protection of rat renal ischemia-reperfusion injury by downregulating the expression of TRPM7.

Temperature-dependent contractility of rat tunica dartos muscle: Contribution of cold, menthol-sensitive TRPM8

Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4‑tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca2+ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.

Prognostic and immunological significance of calcium-related gene signatures in renal clear cell carcinoma.

Background: Calcium signaling is implicated in multiple processes including immune response that important in tumor progression. Kidney renal clear cell carcinoma (KIRC) is the most frequent histological type of renal cell carcinoma with up to a third of cases develop metastases. As a result of a lack of in-depth understanding of the mechanisms underlying KIRC, treatment options have been limited. Here, we aim to comprehensively investigate the landscape of Ca2+ channels, pumps and exchangers in KIRC patients. Methods: The mRNA expression profiles and gene variations of 58 calcium-related genes (CRGs) in KIRC patients and normal control cases were downloaded from TCGA database. CRGs-related risk score was constructed to quantify calcium patterns by using least absolute shrinkage and selection operator (LASSO) regression. The prognostic value, biological functions, immune landscape and therapeutic sensitivities based on CRGs-related risk score were then evaluated using multiple methods. Finally, key gene of CRGs was identified by weighted gene co-expression network analysis (WGCNA). TCGA-CPTAC, GSE53757 datasets, as well as human tissues were used for validation. Results: KIRC patients had significant differences in CRG expression, prognosis, and biological functions between two CRG clusters. CRGs-related risk score was then determined. The prognosis, tumor mutation burden, immune cell infiltration, immune checkpoints, and the response of targeted inhibitors were remarkably different between high and low CRGs-related risk subtypes. CRGs-related high-risk subtype was characterized by immunosuppressive microenvironment with poor prognosis. Meanwhile, several targeted drugs showed distinct sensitivity between CRGs-related risk subtypes. Finally, TRPM3 was identified as a key CRG based on risk score in KIRC patients. TRPM3 mRNA and protein expression were significantly lower in KIRC tumors than in normal controls. Low TRPM3 expression was associated with poor prognosis in KIRC patients. Conclusion: Our study highlighted the promising prognostic value of CRGs in KIRC tumors. The evaluation of CRGs-related risk score will contribute to predicting prognosis and clinical therapy in KIRC patients.

The human ion channel TRPM2 modulates migration and invasion in neuroblastoma through regulation of integrin expression

Transient receptor potential channel TRPM2 is highly expressed in many cancers and involved in regulation of key physiological processes including mitochondrial function, bioenergetics, and oxidative stress. In Stage 4 non-MYCN amplified neuroblastoma patients, high TRPM2 expression is associated with worse outcome. Here, neuroblastoma cells with high TRPM2 expression demonstrated increased migration and invasion capability. RNA sequencing, RT-qPCR, and Western blotting demonstrated that the mechanism involved significantly greater expression of integrins α1, αv, β1, and β5 in cells with high TRPM2 expression. Transcription factors HIF-1α, E2F1, and FOXM1, which bind promoter/enhancer regions of these integrins, were increased in cells with high TRPM2 expression. Subcellular fractionation confirmed high levels of α1, αv, and β1 membrane localization and co-immunoprecipitation confirmed the presence of α1β1, αvβ1, and αvβ5 complexes. Inhibitors of α1β1, αvβ1, and αvβ5 complexes significantly reduced migration and invasion in cells highly expressing TRPM2, confirming their functional role. Increased pAktSer473 and pERKThr202/Tyr204, which promote migration through mechanisms including integrin activation, were found in cells highly expressing TRPM2. TRPM2 promotes migration and invasion in neuroblastoma cells with high TRPM2 expression through modulation of integrins together with enhancing cell survival, negatively affecting patient outcome and providing rationale for TRPM2 inhibition in anti-neoplastic therapy.