Apical Plasma Membrane Research Articles

AKAP2 plays a pivotal role in renal acid-base homeostasis during metabolic acidosis

Reversible phosphorylation and dephosphorylation of kidney proteins modulate their activities and localization and play a central role in maintaining fluid and acid-base homeostasis. The proximal tubule and the intercalated cells of the nephron are main sites of acid-base balance. It was previously demonstrated that some kidney transporters involved in the pH homeostasis were regulated by the c-AMP/Protein Kinase A (PKA) signaling pathway.The A-Kinase anchoring protein 2 (AKAP2), is a scaffold protein that binds to the Protein Kinase A (PKA) allowing the phosphorylation of target proteins but is also known to interact with Protein Phosphatase 1 and consequently contributes to protein dephosphorylation. In this study, we hypothesize that AKAP2 regulates the activity and localization of renal transporters during metabolic acidosis. An inducible and nephron-specific Akap2Pax8/LC1 knockout mouse model was generated. Fluorescent immunostaining showed that AKAP2 is present in the tubules mainly at the apical plasma membrane. AKAP2 knock-out mice in normal conditions presented an acidic urine pH and polyuria compared to control mice. Ex-vivo data from micro-perfused CCDs demonstrated a higher Na+ reabsorption and K+ secretion in AKAP2 KO mice. This suggests its importance in maintaining acid-base balance even under physiological conditions. Moreover, when treated with an ammonium chloride diet, KO mice were sensitive to the acid load; showed a lower blood pH, hyperchloremia, a lower bicarbonate level and a two-fold higher blood ammonia. The sensitivity of the KO mice further supports the role of AKAP2 in acid-base homeostasis. Although we think this is due to renal acid-base transporters activity deregulation, the consideration of a potential defect in water reabsorption mechanisms contributing to acidosis is also valid. Our study provides valuable insights into the role of AKAP2 in maintaining fluid and acid-base homeostasis in the kidneys. The findings open avenues for further research into the molecular mechanisms underlying AKAP2-mediated regulation of renal transporters and its impact on acid-base balance. Swiss National Science Foundation (SNSF). This is the full abstract presented at the American Physiology Summit 2024 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.

Aldosterone applied mucosally or serosally is equally effective in stimulating ENaC activity in rat distal colon

Background: Aldosterone (ALDO), a mineralocorticoid hormone, is essential for regulating blood volume and electrolyte homeostasis. ALDO acts via intracellular mineralocorticoid receptors to stimulate epithelial Na+ channel (ENaC)-mediated Na+ absorption and large conductance K+ (BK) channel-mediated K+ secretion in both kidney and distal colon. We recently showed that ALDO stimulates iron absorption in rat distal colon and SK-CO15 cells (human colon cancer cell line), raising the prospect that ALDO might be used as adjuvant therapy in iron-deficient patients refractory to oral iron. Since, parenteral ALDO (or Fludrocortisone, a synthetic mineralocorticoid agonist) would adversely affect cardiovascular function, an alternative route of administration is required. Parenteral ALDO gains access to target epithelial cells via their serosal (basolateral) membranes, and in vitro studies have always employed serosally applied ALDO to determine its effects on colonic ion transport, but it is not known if mucosally (apically) applied ALDO has similar effects. Hypothesis: We hypothesized that like serosal ALDO, mucosal ALDO would diffuse across apical cell membranes and induce ENaC activity in normal rat distal colon. Aim: To determine whether mucosally applied ALDO stimulates amiloride-sensitive electrogenic Na+ transport in rat distal colon. Methods: Muscularis stripped normal rat (Sprague-Dawley; 126 – 150 g) distal colon was mounted under voltage clamp condition in Ussing chambers. ALDO (10 mM) was added to either the mucosal or serosal bath. ENaC activity was measured as the change in short-circuit current (Isc) following the mucosal addition of amiloride (10 mM). ALDO in mucosal and serosal bath solutions was measured using mass-spectroscopy. Results: With serosal addition of ALDO, transcriptionally induced ENaC activity began to increase after 4 hr and reached maximum/plateau by 5 hr. By contrast, with mucosal addition of ALDO, ENaC activity began to increase after 8 hr and slowly reached maximum/plateau by 9.5 hr. Maximal amiloride-sensitive ENaC activities were similar irrespective of whether ALDO was applied mucosally or serosally. Following addition of ALDO to the mucosal or serosal bath, a similar amount (40%) of ALDO had moved from mucosa to serosa or serosa to mucosa after 24 hr. Conclusions: We conclude that: (1) ALDO induces ENaC activity to the same extent, whether applied mucosally or serosally, and (2) the delayed effect of mucosally applied ALDO may reflect slower diffusion across apical membranes than across basolateral membranes. We speculate that developing a colon-specific delivery system may avoid the adverse cardiovascular effects of oral mineralocorticoid agonists (e.g. Fludrocortisone) if used to enhance colonic iron absorption in iron-deficient patients refractory to oral iron. Transition GrantSupport; Offce of Research and Graduate Education, WVU Health Sciences Center. This is the full abstract presented at the American Physiology Summit 2024 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.

Inhibition of SGL2 Prevents Albumin Induced Proximal Tubule Inflammation

Albuminuria is an independent risk factor for cardiovascular disease and renal damage in hypertension. It is associated with a higher risk of cardiovascular morbidity and mortality, and with declining kidney function. The Na+-glucose cotransporter-2 (SGLT2) plays a crucial role in glucose and sodium reabsorption in the proximal tubule. Clinical trials have shown that SGLT2 inhibitors can reduce cardiovascular events, lower blood pressure, and decrease the risk of end-stage kidney disease in individuals with type 2 diabetes mellitus. These trials have also demonstrated the potential of SGLT2 inhibitors to provide additional benefits beyond their glucose control, including decreased albuminuria. In this study, we hypothesized that exposure to elevated albumin causes proximal tubule injury and the release of cytokines from the basolateral membrane. We propose that by reducing transport in the proximal tubule, SGLT2 inhibitors may alleviate kidney damage by reducing cytokine production and subsequently renal inflammation. To test our hypothesis, we conducted experiments using Dahl salt-sensitive (SS) rats and immortalized human proximal tubule cells (RPTEC-TERT1). To determine how early albuminuria and proximal injury occur in the development of salt-sensitive hypertension, 24-hour urine was collected from SS rats fed either a control salt (CS, 0.4% NaCl, n=10) or a high salt (HS, 4.0% NaCl, n=11) diet for 7 days. Significant increases in albuminuria were observed after 1 day of HS (CS: 25±7 vs. HS: 59±13 mg/day p=0.016). This was followed by significant increases in urinary kidney injury molecule-1 (Kim-1) after 2 days of HS (CS: 13±2 vs. HS: 23±3 pg/day p=0.021). Preliminary analysis found substantial increases in urinary CCL2 after 4 days of HS (CS: 58±7 vs. HS: 135±25 pg/mg, n=5). To investigate whether the renoprotective effects of SGLT2 inhibitors are in part due to their ability to reduce inflammation in the proximal tubule, we exposed the apical membrane of RPTEC-TERT1 cells to human serum albumin (HSA, 10mg/ml, n=6) with and without Dapagliflozin (Dapa, 15ng/ml, n=5) for 24-hours and measured the levels of CCL2 in the basolateral media. HSA caused a significant increase in CCL2 levels relative to control cells (Control: 42.5±5.6 vs. HSA: 84.1±8.7 pg/mg, p=0.00). This increase was prevented by co-treatment with Dapa and CCL2 levels were equivalent to those in the control cells (HSA+Dapa: 44.0±4.5 pg/mg, p=0.988). In conclusion, our findings support the hypothesis that albuminuria precedes proximal tubule injury and may contribute to the development of renal inflammation in salt-sensitive hypertension. The ability of Dapa to reduce prevent albumin induced CCL2 production in RPTEC-TERT1 cells suggests that SGLT2 inhibitors may reduce renal inflammation in salt-sensitive hypertension and is an area of ongoing studies. Studies funded by R01HL152166 to LE. This is the full abstract presented at the American Physiology Summit 2024 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.

Uncovering the role of GPR39 in the kidney

G protein-coupled receptors (GPCRs) constitute the largest class of proteins in the mammalian genome. Upon activation by a specific ligand, these receptors initiate downstream cellular responses that regulate various physiological processes. One such receptor under investigation in our lab is the ghrelin family receptor GPR39. Unlike its relatives, GPR39 does not respond to peptide hormones or neuropeptides. Initial studies suggested zinc as the endogenous ligand, but recent research indicates that zinc functions as an allosteric potentiator for another unidentified endogenous ligand. To date, synthetic agonists for GPR39 have revealed functions for GPR39 in the heart, bone, skin, pancreas, and gastrointestinal tract. However, GPR39’s role in renal physiology is currently unknown, despite relatively high kidney expression. To address this gap in knowledge, we first worked to localize GPR39 within the kidney. As a reliable antibody for GPR39 is not available, we utilized a combination of RNAScope (Gpr39) and immunofluorescence (AQP2). We find that GPR39 is expressed in the renal collecting duct, with the highest expression in AQP2-positive cells (principal cells) in the inner medullary collecting duct, and lesser expression in the cortical collecting duct. To determine if GPR39 is expressed apically or basolaterally, we then cloned GPR39 with a C-terminal EGFP tag and observed basolateral targeting of GPR39 in polarized MDCK cells grown on filters. In order to query the function of GPR39 in principal cells, we used murine principal kidney cortical collecting duct cells (mpkCCD). As others have shown, we find that mpkCCD express and traffc AQP2 to the apical plasma membrane only in the presence of the vasopressin analog dDAVP. Furthermore, we observe that treatment of mpkCCD with the GPR39-specific agonist cpd1324 induces internalization of AQP2 into cytoplasmic vesicles, even in the continued presence of dDAVP. Under vehicle conditions (dDAVP + vehicle), 80.9±7.7% (mean±SD) of AQP2 stain colocalized with an apical membrane marker (WGA staining). In contrast, when mpkCCD cells were co-treated ddAVP+cpd1324, only 58.3±6.02% of AQP2 stain colocalized with the apical membrane (p < 0.001, t-test). These results indicate that GPR39 activation may act to antagonize vasopressin-induced AQP2 traffcking in mpkCCD cells. NHLBIT32HL007534 (MK) and American Heart Association Established Investigator Award (JLP). This is the full abstract presented at the American Physiology Summit 2024 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.

Ligand-based virtual-screening identified a novel CFTR ligand which improves the defective cell surface expression of misfolded ABC transporters.

Cystic fibrosis (CF) is a monogenetic disease caused by the mutation of CFTR, a cAMP-regulated Cl- channel expressing at the apical plasma membrane (PM) of epithelia. ∆F508-CFTR, the most common mutant in CF, fails to reach the PM due to its misfolding and premature degradation at the endoplasmic reticulum (ER). Recently, CFTR modulators have been developed to correct CFTR abnormalities, with some being used as therapeutic agents for CF treatment. One notable example is Trikafta, a triple combination of CFTR modulators (TEZ/ELX/IVA), which significantly enhances the functionality of ΔF508-CFTR on the PM. However, there's room for improvement in its therapeutic effectiveness since TEZ/ELX/IVA doesn't fully stabilize ΔF508-CFTR on the PM. To discover new CFTR modulators, we conducted a virtual screening of approximately 4.3 million compounds based on the chemical structures of existing CFTR modulators. This effort led us to identify a novel CFTR ligand named FR3. Unlike clinically available CFTR modulators, FR3 appears to operate through a distinct mechanism of action. FR3 enhances the functional expression of ΔF508-CFTR on the apical PM in airway epithelial cell lines by stabilizing NBD1. Notably, FR3 counteracted the degradation of mature ΔF508-CFTR, which still occurs despite the presence of TEZ/ELX/IVA. Furthermore, FR3 corrected the defective PM expression of a misfolded ABCB1 mutant. Therefore, FR3 may be a potential lead compound for addressing diseases resulting from the misfolding of ABC transporters.

Role of Organic Anion Transporter NPT4 in Renal Handling of Uremic Toxin 3-indoxyl Sulfate

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3−/− mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3−/− mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3−/− mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3−/− mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.

Regulation of renal aquaporin water channels in acute pyelonephritis.

Acute pyelonephritis (APN) is most frequently caused by uropathogenic Escherichia coli (UPEC), which ascends from the bladder to the kidneys during a urinary tract infection. Patients with APN have been reported to have reduced renal concentration capacity under challenged conditions, polyuria, and increased aquaporin-2 (AQP2) excretion in the urine. We have recently shown increased AQP2 accumulation in the plasma membrane in cell cultures exposed to E. coli lysates and in the apical plasma membrane of inner medullary collecting ducts in a 5-day APN mouse model. This study aimed to investigate if AQP2 expression in host cells increases UPEC infection efficiency and to identify specific bacterial components that mediate AQP2 plasma membrane insertion. As the transepithelial water permeability in the collecting duct is codetermined by AQP3 and AQP4, we also investigated whether AQP3 and AQP4 localization is altered in the APN mouse model. We show that AQP2 expression does not increase UPEC infection efficiency and that AQP2 was targeted to the plasma membrane in AQP2-expressing cells in response to the two pathogen-associated molecular patterns (PAMPs), lipopolysaccharide and peptidoglycan. In contrast to AQP2, the subcellular localizations of AQP1, AQP3, and AQP4 were unaffected both in lysate-incubated cell cultures and in the APN mouse model. Our finding demonstrated that cellular exposure to lipopolysaccharide and peptidoglycan can trigger the insertion of AQP2 in the plasma membrane revealing a new regulatory pathway for AQP2 plasma membrane translocation, which may potentially be exploited in intervention strategies.NEW & NOTEWORTHY Acute pyelonephritis (APN) is associated with reduced renal concentration capacity and increased aquaporin-2 (AQP2) excretion. Uropathogenic Escherichia coli (UPEC) mediates changes in the subcellular localization of AQP2 and we show that in vitro, these changes could be elicited by two pathogen-associated molecular patterns (PAMPs), namely, lipopolysaccharide and peptidoglycan. UPEC infection was unaltered by AQP2 expression and the other renal AQPs (AQP1, AQP3, and AQP4) were unaltered in APN.

Mining phase separation-related diagnostic biomarkers for endometriosis through WGCNA and multiple machine learning techniques: a retrospective and nomogram study.

The objective of this study was to investigate the role of phase separation-related genes in the development of endometriosis (EMs) and to identify potential characteristic genes associated with the condition. We used GEO database data, including 74 non-endometriosis and 74 varying-degree EMs patients. Our approach involved identifying significant gene modules, exploring gene intersections, identifying core genes, and screening for potential EMs biomarkers using weighted gene co-expression network analysis (WGCNA) and various machine learning approaches. We also performed gene set enrichment analysis (GSEA) to understand relevant pathways. This comprehensive approach helps investigate EMs genetics and potential biomarkers. Nine genes were identified at the intersection, suggesting their involvement in EMs. GSEA linked DEGs to pathways like complement and coagulation cascades, DNA replication, chemokines, apical plasma membrane processes, and diseases such as Hepatitis B, Human T-cell leukemia virus 1 infection, and COVID-19. Five feature genes (FOS, CFD, CCNA1, CA4, CST1) were selected by machine learning for an effective EMs diagnostic nomogram. GSEA indicated their roles in mismatch repair, cell cycle regulation, complement and coagulation cascades, and IL-17 inflammation. Notable differences in immune cell proportions (CD4 T cells, CD8 T cells, DCs, macrophages) were observed between normal and disease groups, suggesting immune involvement. This study suggests the potential involvement of phase separation-related genes in the pathogenesis of endometriosis (EMs) and identifies promising biomarkers for diagnosis. These findings have implications for further research and the development of new therapeutic strategies for EMs.

Identification of Hub Genes and Potential Pathogenesis in Gastric Cancer Based on Integrated Gene Expression Profile Analysis.

Gastric cancer (GC) is one of the most common malignancies and ranks third in terms of cancer-related mortality. This study aims to identify the hub genes and potential mechanisms in GC using a bioinformatics approach. Microarray data GSE54129, GSE79973, GSE55696 were extracted from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) was identified using Benjamini-Hochberg method in the limma package. GO and KEGG pathway enrichment analyses of the DEGs were conducted. Furthermore, protein-protein interaction network was constructed the STRING platform, and the hub genes were discovered using Maximal Clique Centrality method via cytoHubba. The predictive significance of hub genes was evaluated through GSE15459 dataset. A total of 73 genes was identified as DEGs in GC. Volcano plots and heatmaps of DEGs were visualized. Functional enrichment analysis revealed that the genes were mostly enriched in response to xenobiotic stimulus, digestion, cellular hormone metabolic process, extracellular matrix structural constituent, calcium-dependent cysteine-type endopeptidase activity, aromatase activity, apical part of cell, basal part of cell, and apical plasma membrane. Regarding KEGG pathway-enrichment, the genes were mainly involved in Drug metabolism-cytochrome P450, Retinol metabolism, Chemical carcinogenesis-DNA adducts, Gastric acid secretion, and Metabolism of xenobiotics by cytochrome P450. By combining the results of Cytohubba, the top five intersecting genes identified were SPP1, INHBA, MMP7, THBS2 and FAP. Kapplan-Meier analysis results showed that these 5 hub genes were highly related to the overall survival of patients. SPP1, INHBA, MMP7, THBS2, and FAP were identified as prospective biomarkers and therapeutic targets for GC that might be utilized for prognostic evaluation and scheme selection.

Organic Synthesis and Current Understanding of the Mechanisms of CFTR Modulator Drugs Ivacaftor, Tezacaftor, and Elexacaftor.

The monogenic rare disease Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance (CFTR) protein, an anion channel expressed at the apical plasma membrane of epithelial cells. The discovery and subsequent development of CFTR modulators-small molecules acting on the basic molecular defect in CF-have revolutionized the standard of care for people with CF (PwCF), thus drastically improving their clinical features, prognosis, and quality of life. Currently, four of these drugs are approved for clinical use: potentiator ivacaftor (VX-770) alone or in combination with correctors lumacaftor, (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445). Noteworthily, the triple combinatorial therapy composed of ivacaftor, tezacaftor, and elexacaftor constitutes the most effective modulator therapy nowadays for the majority of PwCF. In this review, we exploit the organic synthesis of ivacaftor, tezacaftor, and elexacaftor by providing a retrosynthetic drug analysis for these CFTR modulators. Furthermore, we describe the current understanding of the mechanisms of action (MoA's) of these compounds by discussing several studies that report the key findings on the molecular mechanisms underlying their action on the CFTR protein.

Renal Endocytic Regulation of Vitamin D Metabolism during Maturation and Aging in Laying Hens

Egg-laying hens undergo a specific and dramatic calcium metabolism to lay eggs with eggshells composed of calcium carbonate. Calcium metabolism is mainly regulated by vitamin D3. Although vitamin D3 metabolism is closely related to the deterioration of eggshell quality associated with aging and heat stress, the details of the mechanisms regulating vitamin D3 metabolism are not clear. In mammals, the vitamin D3 metabolite (25(OH)D3) produced in the liver binds to the vitamin binding protein (DBP), is subsequently taken up by renal proximal tubular cells via the endocytic receptors megalin (Meg) and cubilin (CUB), and is metabolized to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Therefore, the present study aimed to examine the expression and localization of Meg and CUB in the kidneys of immature chicks and mature and aged laying hens to prevent eggshell quality deterioration. As a result, we showed that as circulating 1,25(OH)2D3 concentrations increased from 156.0 ± 13.5 pg/mL to 815.5 ± 61.4 pg/mL with maturation in immature chicks, relative expression levels (arbitrary units; AU) of Meg and CUB mRNA in the kidneys of mature hens significantly increased 1.92- and 2.75-fold, respectively, compared to those in immature chicks. On the other hand, the Meg mRNA expression levels of mature hens did not change with age, while CUB mRNA expression levels (1.03 ± 0.11 AU) were significantly decreased compared to mature hens (2.75 ± 0.24 AU). Immunohistochemical observations showed that Meg and CUB proteins were localized to the apical membrane of renal proximal tubular epithelial cells in immature chicks, mature hens, and aged hens, and that DBP protein was observed as granular endosomes in the cytoplasm of proximal tubular cells from the apical membrane to the cell nucleus. Especially in mature hens, the endosomes were larger and more numerous than those in immature chicks. In contrast, in aged hens, DBP-containing endosomes were smaller and limited to the apical cytoplasm. These results indicate that with maturation, the expression of Meg and CUB is promoted in the renal proximal tubules of laying hens, facilitating the uptake of the 25(OH)D3-DBP complex and its conversion to 1,25(OH)2D3, and regulating calcium metabolism in eggshell formation. On the other hand, it is suggested that the age-related decrease in CUB expression suppresses the uptake of the 25(OH)D3-DBP complex in the kidney, resulting in a deterioration of eggshell quality.

Cell-wide arrangement of Golgi/RE units depends on the microtubule organization.

We have previously shown that Golgi stacks and recycling endosomes (REs) exist as Golgi/RE units in sea urchin embryos. In this study, we showed that Golgi/RE units were scattered throughout the cytoplasm at early developmental stages but gathered to form a "Golgi ring" surrounding the centric REs at the blastula stage. This change in the cell-wide arrangement of Golgi/RE units coincided with a dramatic change in microtubule organization from a randomly oriented cortical pattern to radial arrays under the apical plasma membrane. A single gigantic Golgi apparatus surrounding centric RE is clearly associated with the center of the radial microtubule arrays. Furthermore, we found that in some animal species belonging to different clades, Golgi stacks lack lateral connections but are likely centralized by microtubule motors. These results suggest that Golgi centralization depends on the organization of the microtubule array in addition to the lateral linking between Golgi stacks. Key words: Golgi stack, recycling endosome, Golgi-ribbon, microtubule, cilium, sea urchin, ascidian.

Prognostic Significance of Iron Metabolism and Immune-Related Genes as Risk Markers in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with significant heterogeneity, which poses challenges in predicting prognosis and treatment outcomes. The impact of iron metabolism and immune-related genes (IMRGs) on HCC patient prognoses remains elusive. We utilized The Cancer Genome Atlas (TCGA) dataset to obtain mRNA expression data and clinical information from HCC patients. Through the application of LASSO regression and univariate/multivariate Cox regression analyses, we identified five IMRGs significantly associated with survival of HCC patients. We constructed a prognostic model comprising these five genes. The model demonstrated excellent predictive performance, not only within TCGA dataset but also when validated using the Gene Expression Omnibus (GEO) dataset. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses presented significant variations in functional categories, such as apical plasma membrane and collagen-containing extracellular matrix. Several pathways, including the PI3K-AKT signaling pathway and the calcium signaling pathway, exhibited significant variations among HCC patients with varying prognoses (P < 0.05). Immune infiltration analysis indicated significantly lower levels of various immune cells, immune functions, and immune checkpoints, such as B cells, CD8+ T cells, and TILs, in the high-risk group (P < 0.05). Immunophenoscore results suggested that the low-risk group may exhibit a more favorable response to immune therapy. Furthermore, the CellMiner database predicted anti-tumor drugs significantly associated with prognostic genes (P < 0.001). In conclusion, our findings highlight the predictive role of IMRGs in prognosis and immune treatment of HCC, indicating that ADAMTS13, CRHBP, VIPR1, FCN3, and CLEC1B may serve as potential prognostic biomarkers for HCC.

Double-Barrel Perfusion System for Modification of Luminal Contents of Intestinal Organoids.

Organoids are 3D cultures of self-organized adult or pluripotent stem cells with an epithelial membrane enclosing a defined fluid-filled lumen. These organoids have been demonstrated with a wide range of organotypic tissue types, but the enclosed nature of the structure restricts access to the lumen and apical surface of the cell membrane. To increase the potential applications of organoids, new technologies are required to provide access to the lumen of the organoid and apical surface of the epithelial cell membrane to enable new biomedical studies. This chapter details a method to access the lumen and apical surface of an organoid utilizing a double-barrel pulled glass capillary and pressure-based pump. The organoid perfusion system uses a three-axis micromanipulator to position the double-barrel capillary to pierce the organoid with the tip of the capillary. Each barrel of the double-barrel capillary is controlled independently with the pressure-based pump to allow injection and removal of material into and from the lumen. Additionally, the organoid is immobilized with a custom-designed PDMS organoid holder. The design of the components for the organoid perfusion system and details on their use are presented here and can be utilized as the basis to enable a wide range of organoid studies including but not limited to modifying luminal contents and apical cell membrane interactions during organoid cultures, recapitulation of physiological flow within the normally static organoid lumen, and effects of mechanical strain on organoid cell development.

Physiology and Mechanism of Milk Fat Globules Secretion in Dairy Animals: A Review

Milk fat globules (MFGs) are a complex compound secreted in the mammary gland, composed of triglycerides; phosphatidylethanolamine (PE) phosphatidylcholine (PC) with unclear mechanism and the fused lipid droplets enclosed by inner layer derived from the endoplasmic reticulum and outer bilayer is directly derived from the apical plasma membrane as the lipid droplets bud out from the cell. The core components of MFGs, triglycerides are synthesized via the de novo fatty acid pathway and LCFA was directly taken from serum. The de novo fatty acid pathway was significantly dependent on the enzymes acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and acyl-CoA synthetase short-chain family member 2 (ACSS2) to synthesize short and medium-chain fatty acids, whereas the bio-hydrogenation pathway is a principal pathway for the synthesis of long-chain fatty acids. Following this, the milk fat globules coated by the membrane are released from the MEC to the lumen with or without other milk components and the membrane that surrounds the fat globule stabilize the milk fat in its dispersed state, prevent flocculation and globule coalescence, and protects against the deleterious effects of lipases. Even though the secretion mechanism of lipid droplets is controversial hormonal and molecular mechanisms are involved. Moreover, the fat globule size and composition are determined by factors such as stage of lactation, physiological state of the animal, Diacylglycerol Acyltransferase1 (DGAT1) activity, and PC/PE ratio along with breed and animal species. While some studies were conducted on milk synthesis and secretion, the molecular mechanism behind lipid droplet fusion, secretion in the apical membrane and how MFGM is formed and the mechanism of MFG synthesis regulation are undiscovered. Therefore, it needs advanced investigation.

Comparative proteomic analysis of human milk fat globules and paired membranes and mouse milk fat globules identifies core cellular systems contributing to mammary lipid trafficking and secretion.

Introduction: Human milk delivers critical nutritional and immunological support to human infants. Milk fat globules (MFGs) and their associated membranes (MFGMs) contain the majority of milk lipids and many bioactive components that contribute to neonatal development and health, yet their compositions have not been fully defined, and the mechanisms responsible for formation of these structures remain incompletely understood. Methods: In this study, we used untargeted mass spectrometry to quantitatively profile the protein compositions of freshly obtained MFGs and their paired, physically separated MFGM fractions from 13 human milk samples. We also quantitatively profiled the MFG protein compositions of 9 pooled milk samples from 18 lactating mouse dams. Results: We identified 2,453 proteins and 2,795 proteins in the majority of human MFG and MFGM samples, respectively, and 1,577 proteins in mouse MFGs. Using paired analyses of protein abundance in MFGMs compared to MFGs (MFGM-MFG; 1% FDR), we identified 699 proteins that were more highly abundant in MFGMs (MFGM-enriched), and 201 proteins that were less abundant in MFGMs (cytoplasmic). MFGM-enriched proteins comprised membrane systems (apical plasma membrane and multiple vesicular membranes) hypothesized to be responsible for lipid and protein secretion and components of membrane transport and signaling systems. Cytoplasmic proteins included ribosomal and proteasomal systems. Comparing abundance between human and mouse MFGs, we found a positive correlation (R 2 = 0.44, p < 0.0001) in the relative abundances of 1,279 proteins that were found in common across species. Discussion: Comparative pathway enrichment analyses between human and mouse samples reveal similarities in membrane trafficking and signaling pathways involved in milk fat secretion and identify potentially novel immunological components of MFGs. Our results advance knowledge of the composition and relative quantities of proteins in human and mouse MFGs in greater detail, provide a quantitative profile of specifically enriched human MFGM proteins, and identify core cellular systems involved in milk lipid secretion.

The V-type H+-ATPase is targeted in antidiuretic hormone control of the Malpighian "renal" tubules.

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti, the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs, whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. V1 immunolocalization was observed strictly in the apical membrane of DH31-treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.

Inhibition of Amino Acids Influx into Proximal Tubular Cells Improves Lysosome Function in Diabetes.

Key Points Collectrin serves as a chaperone for the trafficking of neutral amino acid (AA) transporters in the apical membranes of proximal tubular cells (PTCs). Cltrn knockout reduced AAs influx into PTCs, inactivated mTOR, activated transcription factor EB, improved lysosome function, and ameliorated vacuolar formation of PTCs in diabetic mice treated with streptozotocin and high-fat diet.The inhibition of neutral AA transporter, such as B0AT1 (SLC6A19), and transcription factor EB activator is a new therapeutic strategy against diabetic kidney disease. Background Inhibition of glucose influx into proximal tubular cells (PTCs) by sodium–glucose cotransporter 2 inhibitors revealed prominent therapeutic effects on diabetic kidney disease. Collectrin (CLTRN) serves as a chaperone for the trafficking of neutral amino acid (AA) transporters in the apical membranes of PTCs. We investigated the beneficial effects of reduced influx of AAs into PTCs in diabetes and obesity model of Cltrn−/y mice. Methods Cltrn+/y and Cltrn−/y mice at age 5 weeks were assigned to standard diet and streptozotocin and high-fat diet (STZ-HFD)–treated groups. Results At age 22–23 weeks, body weight and HbA1c levels significantly increased in STZ-HFD-Cltrn+/y compared with standard diet-Cltrn+/y; however, they were not altered in STZ-HFD-Cltrn−/y compared with STZ-HFD-Cltrn+/y. At age 20 weeks, urinary albumin creatinine ratio was significantly reduced in STZ-HFD-Cltrn−/y compared with STZ-HFD-Cltrn+/y. Under the treatments with STZ and HFD, the Cltrn gene deficiency caused significant increase in urinary concentration of AAs such as Gln, His, Gly, Thr, Tyr, Val, Trp, Phe, Ile, Leu, and Pro. In PTCs in STZ-HFD-Cltrn+/y, the enlarged lysosomes with diameter of 10 μm or more were associated with reduced autolysosomes, and the formation of giant lysosomes was prominently suppressed in STZ-HFD-Cltrn−/y. Phospho-mTOR and inactive form of phospho-transcription factor EB were reduced in STZ-HFD-Cltrn−/y compared with STZ-HFD-Cltrn+/y. Conclusions The reduction of AAs influx into PTCs inactivated mTOR, activated transcription factor EB, improved lysosome function, and ameliorated vacuolar formation of PTCs in STZ-HFD-Cltrn−/y mice.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Following the cellular itinerary of renal aquaporin-2 shuttling with 4.5x expansion microscopy.

The shuttling of renal collecting duct aquaporin-2 (AQP2) between intracellular vesicles and the apical plasma membrane is paramount for regulation of renal water reabsorption. The binding of the circulating antidiuretic hormone arginine vasopressin (AVP) to the basolateral AVP receptor increases intracellular cAMP, which ultimately leads to AQP2 plasma membrane accumulation via a dual effect on AQP2 vesicle fusion with the apical plasma membrane and reduced AQP2 endocytosis. This AQP2 plasma membrane accumulation increases water reabsorption and consequently urine concentration. Conventional fluorescent microscopy provides a lateral resolution of ∼250 nm, which is insufficient to resolve the AQP2-containing endosomes/vesicles. Therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x ExM, we detected AQP2 vesicles/endosomes as small as 79 nm considering an average expansion factor of 4.3 for endosomes. Using different markers of the endosomal system provided detailed information of the cellular AQP2 itinerary upon changes in endogenous cAMP levels. Before cAMP elevation, AQP2 colocalized with early and recycling, but not late endosomes. Forskolin-induced cAMP increase was characterized by AQP2 insertion into the plasma membrane and AQP2 withdrawal from large perinuclear endosomes as well as some localization to lysosomal compartments. Forskolin washout promoted AQP2 endocytosis where AQP2 localized to not only early and recycling endosomes but also late endosomes and lysosomes indicating increased AQP2 degradation. Thus, our results show that 4.5 ExM is an attractive approach to obtain detailed information regarding AQP2 shuttling.NEW & NOTEWORTHY Renal aquaporin-2 (AQP2) imaged by expansion microscopy provides unprecedented 3-D information regarding the AQP2 itinerary in response to changes in cellular cAMP.