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American Journal of Physiology. Renal... Jul 2024The pore-forming α-subunit of the large-conductance K (BK) channel is encoded by a single gene, BK channel-mediated K secretion in the kidney is crucial for overall...
The pore-forming α-subunit of the large-conductance K (BK) channel is encoded by a single gene, BK channel-mediated K secretion in the kidney is crucial for overall renal K homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of alternative exon splicing within these two sites by dietary K loading is both site and sex specific. In microdissected distal tubules, the alternative splicing profile, as well as its regulation by dietary K, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in splicing events. Overall, our data provide evidence that alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K loading in mouse kidney. We identified the major splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that alternative splicing is developmentally regulated and subject to changes in dietary K.
Topics: Animals; Alternative Splicing; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Potassium, Dietary; Kidney; Mice, Inbred C57BL; Mice; Male; Gene Expression Regulation, Developmental; Exons; Female
PubMed: 38779757
DOI: 10.1152/ajprenal.00100.2024 -
The World Journal of Men's Health Apr 2024The study aimed to comprehensively analyze testosterone and precursor concentrations in the testicular interstitial fluid (TIF) of men with azoospermia, exploring their...
PURPOSE
The study aimed to comprehensively analyze testosterone and precursor concentrations in the testicular interstitial fluid (TIF) of men with azoospermia, exploring their significance in the testicular microenvironment and their correlation with testicular sperm retrieval outcomes.
MATERIALS AND METHODS
We analyzed 37 TIF samples, including 5 from men with obstructive azoospermia (OA) and 32 from men with non-obstructive azoospermia (NOA). Liquid chromatography with tandem mass spectrometry quantified testosterone and precursor levels. Comparative assessments of the outcomes of testicular sperm retrieval were performed between the OA and NOA groups as well as among men with NOA.
RESULTS
Men with NOA who had not undergone hormone treatment exhibited significantly higher intratesticular concentrations of testosterone (median 1,528.1 207.5 ng/mL), androstenedione (median 10.6 1.9 ng/mL), and 17-OH progesterone (median 13.0 1.8 ng/mL) than men diagnosed with OA. Notably, in the subgroup of patients with NOA subjected to medical treatment, men with successful sperm retrieval had significantly reduced levels of androstenedione (median androstenedione 5.7 18.5 ng/mL, p=0.004). Upon a more detailed analysis of these men who underwent hormone manipulation treatment, the testosterone/androstenedione ratio (indicative of HSD17B3 enzyme activity) was markedly increased in men with successful sperm retrieval (median: 365.8 165.0, p=0.008) compared with individuals with NOA who had unsuccessful sperm recovery. Furthermore, within the subset of men with NOA who did not undergo medical treatment before microdissection testicular sperm extraction but achieved successful sperm retrieval, the ratio of 17-OH progesterone/progesterone (indicative of CYP17A1 activity) was substantially higher.
CONCLUSIONS
The study suggests distinct testosterone biosynthesis pathways in men with compromised spermatogenesis and those with normal spermatogenesis. Among NOA men with successful retrieval after hormone optimization therapy, there was decreased androstenedione and increased HSD17B3 enzyme activity. These findings have diagnostic and therapeutic implications for the future.
PubMed: 38772536
DOI: 10.5534/wjmh.230265 -
BMC Nephrology May 2024Lipoprotein glomerulopathy (LPG) is a apolipoprotein E (ApoE)-related glomerular disease and has been associated with type III hyperlipidemia. Without appropriate...
BACKGROUND
Lipoprotein glomerulopathy (LPG) is a apolipoprotein E (ApoE)-related glomerular disease and has been associated with type III hyperlipidemia. Without appropriate treatment, chronic kidney disease (CKD) caused by LPG progresses, and approximately half of the patients develop end-stage kidney disease within 1-27 years of disease onset. However, few studies have highlighted the clinical course of cardiovascular diseases (CVDs) in patients with LPG. Herein, we report the first case of LPG in which the CVD risk was assessed using arterial stiffness.
CASE PRESENTATION
A 32-year-old Japanese man was referred to our hospital due to persistent proteinuria. Kidney biopsy showed markedly dilated capillary lumens containing pale-stained thrombi, which stained positively with Oil Red O. Electron microscopy revealed the presence of thrombi in the capillary lumen with low electron density and vacuoles of various sizes in part of the thrombi. Toluidine blue and Sudan IV stains were used to stain the thin sections of Epon-embedded tissue samples for electron microscopy. Sudan IV-positive droplets were observed in the capillary lumens, vascular walls, and cytoplasm of tubular cells. Increased serum ApoE concentration was observed. Liquid chromatography-tandem mass spectrometry of laser-microdissected glomeruli from paraffin sections revealed an increase in ApoE. Direct deoxyribonucleic acid sequencing of ApoE revealed a heterozygous ApoE Sendai mutation (Arg145Pro). The patient was finally diagnosed with LPG with heterozygosity for ApoE-Sendai mutation (Arg145Pro). Notably, at the time of diagnosis, he had markedly increased arterial stiffness for his age. Arterial stiffness was measured using brachial-ankle pulse wave velocity (baPWV), which was equivalent to that of a 56-year-old man. After three months of treatment with fenofibrate and losartan, a significant reduction in proteinuria was achieved along with an improvement in baPWV. Furthermore, these effects were maintained despite the lack of decrease in serum ApoE levels.
CONCLUSION
Herein, we report the case of a patient with LPG with markedly increased arterial stiffness at the time of diagnosis, in whom combination therapy with fenofibrate and losartan successfully improved proteinuria and arterial stiffness. To the best of our knowledge, this is the first case report of LPG in which CVD risk was assessed using arterial stiffness.
Topics: Humans; Male; Adult; Losartan; Vascular Stiffness; Fenofibrate; Drug Therapy, Combination; Hypolipidemic Agents; Kidney Diseases; Apolipoproteins E
PubMed: 38769490
DOI: 10.1186/s12882-024-03612-z -
STAR Protocols Jun 2024Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon...
Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al..
Topics: Animals; Neuroglia; Swine; Myenteric Plexus; Colon; Cell Culture Techniques; Animals, Newborn; Submucous Plexus; Cells, Cultured
PubMed: 38762883
DOI: 10.1016/j.xpro.2024.103057 -
Plant Methods May 2024Single-cell analysis, a rapidly evolving field, encounters significant challenges in detecting individual cells within complex plant tissues, particularly oil cells...
BACKGROUND
Single-cell analysis, a rapidly evolving field, encounters significant challenges in detecting individual cells within complex plant tissues, particularly oil cells (OCs). The intricate process of single-cell isolation, coupled with the inherent chemical volatility of oil cells, necessitates a comprehensive methodology.
RESULTS
This study presents a method for obtaining intact OC from Asari Radix et Rhizoma (ARR), a traditional herbal medicine. The developed approach facilitates both qualitative and quantitative analysis of diverse OCs. To determine the most reliable approach, four practical methods-laser capture microdissection, micromanipulation capturing, micromanipulation piping, and cell picking-were systematically compared and evaluated, unequivocally establishing cell picking as the most effective method for OC isolation and chemical analysis. Microscopic observations showed that OCs predominantly distribute in the cortex of adventitious and fibrous roots, as well as the pith and cortex of the rhizome, with distinct morphologies-oblong in roots and circular in rhizomes. Sixty-three volatile constituents were identified in OCs, with eighteen compounds exhibiting significant differences. Safrole, methyleugenol, and asaricin emerged as the most abundant constituents in OCs. Notably, cis-4-thujanol and tetramethylpyrazine were exclusive to rhizome OCs, while isoeugenol methyl ether was specific to fibrous root OCs based on the detections. ARR roots and rhizomes displayed marked disparities in OC distribution, morphology, and constituents.
CONCLUSION
The study highlights the efficacy of cell picking coupled with HS-SPME-GC-MS as a flexible, reliable, and sensitive method for OC isolation and chemical analysis, providing a robust methodology for future endeavors in single-cell analyses.
PubMed: 38760854
DOI: 10.1186/s13007-024-01184-5 -
Methods in Molecular Biology (Clifton,... 2024Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic...
Advances in understanding cellular aging research have been possible due to the analysis of the replicative lifespan of yeast cells. Studying longevity in the pathogenic yeast Cryptococcus neoformans is essential because old yeast cells with age-related phenotypes accumulate during infection and are associated with increased virulence and antifungal tolerance. Microdissection and microfluidic devices are valuable tools for continuously tracking cells at the single-cell level. In this chapter, we describe the features of these two platforms and outline technical limitations and information to study aging mechanisms while assessing the lifespan of yeast cells.
Topics: Cryptococcus neoformans; Microdissection; Cellular Senescence; Lab-On-A-Chip Devices; Single-Cell Analysis; Cryptococcosis
PubMed: 38758331
DOI: 10.1007/978-1-0716-3722-7_25 -
ACS Chemical Neuroscience Jun 2024Growth hormone-releasing hormone (Ghrh) neurons in the dorsomedial ventromedial hypothalamic nucleus (VMNdm) express the metabolic transcription factor steroidogenic...
Sex-Dimorphic Effects of Hypoglycemia on Metabolic Sensor mRNA Expression in Ventromedial Hypothalamic Nucleus-Dorsomedial Division (VMNdm) Growth Hormone-Releasing Hormone Neurons.
Growth hormone-releasing hormone (Ghrh) neurons in the dorsomedial ventromedial hypothalamic nucleus (VMNdm) express the metabolic transcription factor steroidogenic factor-1 and hypoglycemia-sensitive neurochemicals of diverse chemical structures, transmission modes, and temporal signaling profiles. Ghrh imposes neuromodulatory control of coexpressed transmitters. Multiple metabolic sensory mechanisms are employed in the brain, including screening of the critical nutrient glucose or the energy currency ATP. Here, combinatory laser-catapult-microdissection/single-cell multiplex qPCR tools were used to investigate whether these neurons possess molecular machinery for monitoring cellular metabolic status and if these biomarkers exhibit sex-specific sensitivity to insulin-induced hypoglycemia. Data show that hypoglycemia up- (male) or downregulated (female) Ghrh neuron glucokinase (Gck) mRNA; Ghrh gene silencing decreased baseline and hypoglycemic patterns of Gck gene expression in each sex. Ghrh neuron glucokinase regulatory protein (Gckr) transcript levels were respectively diminished or augmented in hypoglycemic male vs female rats; this mRNA profile was decreased by Ghrh siRNA in both sexes. Gene transcripts encoding catalytic alpha subunits of the energy monitor 5-AMP-activated protein kinase (AMPK), i.e., Prkaa1 and 2, were increased by hypoglycemia in males, yet only the former mRNA was hypoglycemia-sensitive in females. Ghrh siRNA downregulated baseline and hypoglycemia-associated Prkaa subunit mRNAs in males but elicited divergent changes in Prkaa2 transcripts in eu- vs hypoglycemic females. Results provide unique evidence that VMNdm Ghrh neurons express the characterized metabolic sensor biomarkers glucokinase and AMPK and that the corresponding gene profiles exhibit distinctive sex-dimorphic transcriptional responses to hypoglycemia. Data further document Ghrh neuromodulation of baseline and hypoglycemic transcription patterns of these metabolic gene profiles.
Topics: Animals; Growth Hormone-Releasing Hormone; Hypoglycemia; Ventromedial Hypothalamic Nucleus; Male; Female; Neurons; Rats; Sex Characteristics; RNA, Messenger; Rats, Sprague-Dawley; Glucokinase; Dorsomedial Hypothalamic Nucleus
PubMed: 38757688
DOI: 10.1021/acschemneuro.4c00206 -
Reproductive Medicine and Biology 2024This study compared the clinical outcomes of men with Klinfelter syndrome based on karyotype.
PURPOSE
This study compared the clinical outcomes of men with Klinfelter syndrome based on karyotype.
METHODS
The authors analyzed the outcomes of microdissection testicular sperm extraction (micro-TESE) performed on 57 patients with Klinfelter syndrome (KS) at our clinic.
RESULTS
The average ages of the non-mosaic and mosaic KS groups were 32.2 ± 4.8 and 45.9 ± 13.1 years, respectively. The sperm retrieval rates of the non-mosaic and mosaic KS groups were 46.5% (20/43) and 50.0% (7/14), respectively. The fertilization rates after intracytoplasmic sperm injection did not significantly differ between the non-mosaic and mosaic KS groups. The mosaic KS group had higher cleavage and blastocyst development rates than the non-mosaic KS group (72.2% vs. 96.2% and 30.5% vs. 44.7%, respectively). The group using motile sperm had better outcomes than the group using immotile sperm. The embryo transfer outcomes of the non-mosaic and mosaic KS groups did not significantly differ (clinical pregnancy rate: 28.0% vs. 20.7%, miscarriage rate: 14.3% vs. 33.3%, production rate per transfer: 22.0% vs. 13.8%, and production rate per case: 58.8% vs. 57.1%).
CONCLUSIONS
Compared with the non-mosaic KS group, the mosaic KS group had significantly better intracytoplasmic sperm injection outcomes because of the higher utilization rate of motile sperm.
PubMed: 38756694
DOI: 10.1002/rmb2.12579 -
Journal of Neuroinflammation May 2024Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop...
Overexpression of pathogenic tau in astrocytes causes a reduction in AQP4 and GLT1, an immunosuppressed phenotype and unique transcriptional responses to repetitive mild TBI without appreciable changes in tauopathy.
Epidemiological studies have unveiled a robust link between exposure to repetitive mild traumatic brain injury (r-mTBI) and elevated susceptibility to develop neurodegenerative disorders, notably chronic traumatic encephalopathy (CTE). The pathogenic lesion in CTE cases is characterized by the accumulation of hyperphosphorylated tau in neurons around small cerebral blood vessels which can be accompanied by astrocytes that contain phosphorylated tau, the latter termed tau astrogliopathy. However, the contribution of tau astrogliopathy to the pathobiology and functional consequences of r-mTBI/CTE or whether it is merely a consequence of aging remains unclear. We addressed these pivotal questions by utilizing a mouse model harboring tau-bearing astrocytes, GFAP mice, subjected to our r-mTBI paradigm. Despite the fact that r-mTBI did not exacerbate tau astrogliopathy or general tauopathy, it increased phosphorylated tau in the area underneath the impact site. Additionally, gene ontology analysis of tau-bearing astrocytes following r-mTBI revealed profound alterations in key biological processes including immunological and mitochondrial bioenergetics. Moreover, gene array analysis of microdissected astrocytes accrued from stage IV CTE human brains revealed an immunosuppressed astroglial phenotype similar to tau-bearing astrocytes in the GFAP model. Additionally, hippocampal reduction of proteins involved in water transport (AQP4) and glutamate homeostasis (GLT1) was found in the mouse model of tau astrogliopathy. Collectively, these findings reveal the importance of understanding tau astrogliopathy and its role in astroglial pathobiology under normal circumstances and following r-mTBI. The identified mechanisms using this GFAP model may suggest targets for therapeutic interventions in r-mTBI pathogenesis in the context of CTE.
Topics: Astrocytes; Animals; Mice; tau Proteins; Aquaporin 4; Tauopathies; Humans; Excitatory Amino Acid Transporter 2; Mice, Transgenic; Brain Concussion; Male; Phenotype; Mice, Inbred C57BL
PubMed: 38750510
DOI: 10.1186/s12974-024-03117-4 -
Asian Journal of Andrology May 2024Microdissection testicular sperm extraction (mTESE) is commonly performed to retrieve sperm in the testes for assisted reproductive techniques in patients with...
Association between anti-Müllerian hormone concentrations and sperm retrieval outcomes in patients with idiopathic nonobstructive azoospermia: a systematic review and meta-analysis.
Microdissection testicular sperm extraction (mTESE) is commonly performed to retrieve sperm in the testes for assisted reproductive techniques in patients with idiopathic nonobstructive azoospermia (iNOA). However, the success rate of sperm retrieval varies among individuals. We aim to investigate the association between clinical parameters and sperm retrieval outcomes in patients with iNOA. We searched PubMed, EMBASE, and Web of Science from database inception to August 2, 2023. The main measure was whether sperm retrieval was successful in patients with iNOA who underwent mTESE. Pooled estimates of the sperm retrieval rate and weighted mean differences were calculated using random-effects models. The overall sperm retrieval rate was 36.8% (95% confidence interval [CI]: 27.5%-46.0%, I2 = 95.0%) in nine studies comprising 1892 patients with iNOA. No significant differences were found in age, testicular volume, serum total testosterone concentrations, or inhibin B concentrations between positive and negative sperm retrieval outcomes. Lower anti-Müllerian hormone concentrations in patients with iNOA were associated with a positive outcome of mTESE (weighted mean differences: -2.70; 95% CI: -3.94--1.46, I2 = 79.0%). In conclusion, this study shows a significant relationship between anti-Müllerian hormone and sperm retrieval outcomes in patients with iNOA, while age, testicular volume, total testosterone, and inhibin B show no significant association. These findings have important implications for assessing the potential success of sperm retrieval and selecting appropriate treatment strategies in patients with iNOA.
PubMed: 38748861
DOI: 10.4103/aja202419