AQP1 Protein Research Articles

Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration-A New Potential Drug in Sepsis Therapy?

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10-5 M) and furosemide (10-6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts

Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA–protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

Radix Astragali decoction improves liver regeneration by upregulating hepatic expression of aquaporin-9.

The therapeutic efficacy of liver injuries heavily relies on the liver's remarkable regenerative capacity, necessitating the maintenance of glycose/lipids homeostasis and oxidative eustasis during the recovery process. Astragali Radix, an herbal tonic widely used in China and many other countries, is believed to have many positive effects, including immune stimulation, nourishing, antioxidant, liver protection, diuresis, anti-diabetes, anti-cancer and expectorant. Astragali Radix is widely integrated into hepatoprotective formulas as it is believed to facilitate liver regeneration. Nevertheless, the precise molecular pharmacological mechanisms underlying this hepatoprotective effect remain elusive. To investigate the improving effects of Astragali Radix on liver regeneration and the underlying mechanisms. A mouse model of 70% partial hepatectomy (PHx) was employed to investigate the impact of Radix Astragali decoction (HQD) on liver regeneration. HQD was orally administered for 7 days before the PHx procedure and throughout the experiment. N-acetylcysteine (NAC) was used as a positive control for liver regeneration. Liver regeneration was assessed by evaluating the liver-to-body weight ratio (LW/BW) and the expression of representative cell proliferation marker proteins. Oxidative stress and glucose metabolism were analyzed using biochemical assays, Western blotting, dihydroethidium (DHE) fluorescence, and periodic acid-Schiff (PAS) staining methods. To understand the role of AQP9 as a potential molecular target of HQD in promoting liver regeneration, td-Tomato-tagged AQP9 transgenic mice (AQP9-RFP) were employed to determine the expression pattern of AQP9 protein. AQP9 knockout mice (AQP9-/-) were used to assess the specific targeting of AQP9 in the promotion of liver regeneration by HQD. HQD significantly upregulated hepatic AQP9 expression, alleviated liver injury and promoted liver regeneration in wild-type (AQP9+/+) mice after 70% PHx. However, the beneficial impact of HQD on liver regeneration was absent in AQP9 gene knockout (AQP9-/-) mice. Moreover, HQD facilitated the uptake of glycerol by hepatocytes, enhanced gluconeogenesis, and concurrently reduced H2O2 content and oxidative stress levels in AQP9+/+ but not AQP9-/- mouse livers. Additionally, main active substance of Radix Astragali, astragaloside IV (AS-IV) and cycloastragenol (CAG), demonstrated substantial upregulation of AQP9 expression and promoted liver regeneration in AQP9+/+ but not AQP9-/- mice. This study is the first to demonstrate that Radix Astragali and its main active constituents (AS-IV and CAG) improve liver regeneration by upregulating the expression of AQP9 in hepatocytes to increase gluconeogenesis and reduce oxidative stress. The study revealed novel molecular pharmacological mechanisms of Radix Astragali and provided a promising therapeutic target of liver diseases.

Blood-nerve barrier enhances chronic postsurgical pain via the HIF-1α/ aquaporin-1 signaling axis

BackgroundBlood nerve barrier (BNB) participates in the development of neuropathic pain. AQP1 is involved in peripheral pain perception and is negatively correlated with HIF-1α phenotype, which regulates endothelial permeability. However, the role of HIF-1α-AQP1-mediated BNB dysfunction in Chronic Postsurgical Pain (CPSP) has not been reported.MethodsMale Sprague-Dawley rats were randomized into 5 groups: (i) Naive group; (ii) Sham group; (iii) SMIR group: skin/muscle incision and retraction for one hour. Behavioral tests were performed for the three groups, BNB vascular permeability and western blotting were conducted to determine HIF-1α and AQP1 protein expression. (iv) The SMIR + HIF-1α inhibitor group; (v) SMIR + DMSO group. Rats in the two groups were administered with HIF-1α inhibitor (2ME2) or DMSO intraperitoneally on the third day post-SMIR surgery followed by performance of behavioral tests, BNB permeability assessment, and determination of HIF-1α, AQP1 and NF200 protein levels.ResultsThe permeability of BNB was significantly increased and the expression of AQP1 was downregulated on the 3rd and 7th days post-operation. AQP1 is mainly located in neurons and NF200, CGRP-positive nerve fibers. HIF-1α was highly expressed on the third day post-operation. HIF-1α inhibitor reversed the decrease in AQP1 expression and increase in NF200 expression, barrier permeability and hyperalgesia induced by SMIR on the 3rd day post-surgery.ConclusionsEarly dysfunction of BNB mediated by HIF-1α/AQP1 activated by SMIR may be an important mechanism to promote acute postoperative painful transformation of CPSP. Preadaptive protection of endothelial cells around nerve substructures may be an important countermeasure to inhibit CPSP transformation. Early impairment of BNB function mediated by HIF-1α/AQP1 activated by SMIR may be an important mechanism for promoting acute postoperative pain transformation of CPSP.

Synergistic effect of Euphorbia kansui stir-fried with vinegar and bile acids on malignant ascites effusion through modulation of gut microbiota.

Background: Toxic Euphorbia kansui (EK) is employed to treat malignant ascites effusion (MAE). EK stir-fried with vinegar (VEK) has been demonstrated to reduce toxicity due to its preserved water-expelling effect. This was demonstrated to be correlated with gut microbiota. Therein, bile acids (BAs) have a bidirectional relationship with the gut microbiota. Therefore, the aim of this study is to explore whether BA-mediated gut microbiota influences the water-expelling effect of VEK against MAE. Methods: The MAE rat model was established by intraperitoneal injection of Walker-256 tumor cells. A reliable simultaneous method for the determination of 15 bile acids in rat feces using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established and applied to analyze the fecal BAs in rats treated with VEK. The screened BA was then administered to VEK-treated MAE rats. The water-expelling effect was evaluated using histopathological analysis, biochemical examination, inflammatory factors in ascites, urine volume, ascites amount, and intestinal aquaporin expression. The microbial composition was determined using 16S rRNA sequencing, and the contents of bile acids were finally measured. Results: VEK decreased the content of fecal deoxycholic acid (DCA), lithocholic acid (LCA), and taurocholic acid (TCA) while increasing the content of ursodeoxycholic acid (UDCA). VEK alleviated liver, stomach, and intestinal injuries; oxidative damage; and inflammation, which were further ameliorated with UDCA intervention. VEK alleviated MAE by increasing the fecal water content, urine volume, and AQP3 protein expression and decreasing the urine levels of Na+, K+, and Cl-. This was retained with the intervention of UDCA. UDCA and VEK regulated the BA metabolism disorder to a certain extent. Analysis of gut microbiota showed that VEK increased the abundance of Lactobacillus and decreased that of Prevotella_9 in MAE rats. The combined administration of UDCA and VEK showed a better modulation of the microbiota structure than that of VEK alone, and the effect of this administration reached closer to the reference state. Conclusion: The water-expelling effect of VEK did not directly depend on the BA-mediated gut microbiota. However, VEK and BAs had a synergistic effect on malignant ascites effusion through the regulation of the gut microbiota. These results provided a scientific basis for the reasonable usage of VEK and the novel combination treatment strategy of VEK and UDCA.

Distributions and expressions of Aquaporin-5 and 7 in the testes of developing male chicks.

Aquaporins (AQPs) are integral membrane proteins that act as water channels for which a total of 13 orthologs of AQP genes in birds have been reported. Tissue expression and cellular or subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. We aimed to determine the distribution and localization of AQP5 and AQP7 proteins by immunocytochemistry in testicular tissues obtained from developing chicks (14, 21, 28, 35 and 42 days old). Totally 175 male chicks (Ross308) were used in the study from which testicular tissue was removed, fixed in 10% formaldehyde solution, then embedded in paraffin blocks. Five μm sections were cut, mounted on poly-L-lysine slides, dried in an oven, then dehydrated using standard immunohistochemistry staining protocol. The sections were imaged with a Nikon Eclipse 50i trinocular light microscope. Immunohistochemical evaluation of the immune reactivity of AQP5 revealed a positive immune reaction in spermatocytes and interstitial areas of the testes in 14-day-old chicks. Testicular tissue AQP5 immune reactivity was observed in the tubule and the interstitial regions of 21-, 28-, 35- and 42-day-old chicks. AQP7 immune reactions were determined in the tubule and interstitial areas testes of developing chicks' testis tissue, with increasing positivity corresponding to older age. The expression of AQP5 and AQP7 appears to be species-specific due to differences in localization and expression in male chicks compared with studies of other mammals, which is likely to play an important role in regulating fluid and sperm volume. This research can serve as a base for future studies that will contribute to the understanding of the male genital system of AQPs.

Comparative study of purgative pharmacological effects and mechanisms of Moringa oleifera leaves and Rhei Radix et Rhizoma

Moringa oleifera leaves are known for their "Virechana"(purgative) effect in Ayurvedic medicine in India. This study compared the purgative effects and mechanisms of M. oleifera leaves with the reference Rhei Radix et Rhizoma to establish a foundation for the further application of M. oleifera leaves in traditional Chinese medicine(TCM). Using network pharmacology and molecular docking methods, this study identified the material basis, common targets, and signaling pathways through which Rhei Radix et Rhizoma and M. oleifera leaves exerted their purgative pharmacological effects. A low-fiber diet-induced constipation mouse model was established to measure fecal parameters and small intestinal propulsion rate, and histological changes in the colon were observed using HE staining. Relative expression levels of relevant genes and target proteins were assessed using RT-qPCR and immunohistochemistry, respectively. The results showed that mapping the targets of Rhei Radix et Rhizoma and M. oleifera leaves onto the biological process network of constipation revealed close proximity, indicating that they may exert their therapeutic effects on constipation through similar biological processes. Molecular docking results indicated that compounds such as sennoside C and isoquercitrin could target serine/threonine protein kinases(AKT1) and mitogen-activated protein kinase 3(MAPK3), thereby affecting MAPK and calcium signaling pathways to promote defecation. Animal experiments demonstrated that both M. oleifera leaves and Rhei Radix et Rhizoma increased the number of fecal pellets and water content in constipated mice, improved small intestine motility, colon mucosal thickness, and muscle layer thickness, upregulated the gene expression levels of AKT1 and MAPK3 in the colon, and downregulated the expression of AQP3 protein. These findings suggest that M. oleifera leaves and Rhei Radix et Rhizoma share similarities in their therapeutic efficacy and mechanisms for treating constipation. Using Rhei Radix et Rhizoma as a reference can provide a better understanding of the characteristics of the "Virechana"(purgative) effect of M. oleifera leaves in TCM.

Salinity effects on expression and localization of aquaporin 3 in gills of the euryhaline milkfish (Chanos chanos).

Milkfish (Chanos chanos) are important euryhaline fish in Southeast Asian countries that can tolerate a wide range of salinity changes. Previous studies have revealed that milkfish have strong ion regulation and survival abilities under osmotic stress. In addition to ion regulation, water homeostasis in euryhaline teleosts is important during environmental salinity shifts. Aquaporins (AQP) are vital water channels in fish, and different AQPs can transport water influx or outflux from the body. AQP3 is one of the AQP channels, and the function of AQP3 in the gills of euryhaline milkfish is still unknown. The aim of this study was to investigate the expression and localization of AQP3 in the gills of euryhaline milkfish to contribute to our understanding of the physiological role and localization of AQP3 in fish. The AQP3 sequence was found in the milkfish next-generation sequencing (NGS) database and is mainly distributed in the gills of freshwater (FW)-acclimated milkfish. Under hypoosmotic and hyperosmotic stress, the osmolality of milkfish immediately shifted, similar to the aqp3 gene expression. Moreover, the abundance of AQP3 protein significantly decreased 3 h after transferring milkfish from FW to seawater (SW). However, there was no change within 7 days when the milkfish experienced hypoosmotic stress. Moreover, double immunofluorescence staining of milkfish gills showed that AQP3 colocalized with Na+ /K+ ATPase at the basolateral membrane of ionocytes. These results combined indicate that milkfish have a strong osmoregulation ability under acute osmotic stress because of the quick shift in the gene and protein expression of AQP3 in their gills.

Aquaporin 1 is a prognostic marker and inhibits tumour progression through downregulation of Snail expression in intrahepatic cholangiocarcinoma.

Recently, some studies have suggested a link between AQP1 and cancer progression. The aim of the present study was to investigate the influence of AQP1 on the clinicopathology and prognosis of intrahepatic cholangiocarcinoma (ICC) patients. We retrospectively detected the expression of AQP1 protein in 307 patients with ICC who underwent partial hepatectomy. Western blot analysis was used to detect AQP1 protein levels in stable AQP1 overexpression and knockdown cell lines. The influence of AQP1 on the invasion and metastasis ability of ICC cells was assessed by wound-healing and Transwell assays in vitro as well as by a splenic liver metastasis model in vivo. Positive membranous AQP1 expression was identified in 34.2% (105/307) of the ICC specimens. Survival data revealed that positive AQP1 expression was significantly associated with favourable disease-free survival (DFS) and overall survival (OS) (p=0.0290 and p=0003, respectively). Moreover, high AQP1 expression inhibited the invasion and migration of ICC cells in vitro as well as inhibited liver metastasis in nude mice. Mechanistically, high AQP1 expression in ICC cells increased the levels of E-cadherin but decreased the levels of the Snail transcription factor. AQP1 expression is associated with a favourable prognosis in ICC patients. AQP1 inhibits ICC cell invasion, metastasis, and epithelial-mesenchymal transition (EMT) through downregulation of Snail expression.

NEUROMYELITIS OPTICA. CASE REPORT

Introduction. Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease that causes severe demyelination, especially in the optic nerve and spinal cord with typical clinical manifestations of acute optic neuritis and transverse myelitis. The frequency of occurrence worldwide is estimated at 1-2 people out of every 100,000 people. Case report. Our patient was a 37-year-old woman. She felt a sudden vision loss since May 2022. She related this condition to another case of unknown etiology that she encountered in January of that year - numerous vesicular-type rashes appeared on the abdomen and back and lasted for 3 days, but the traces of those rashes remained for some time in the form of hypopigmentation. In November 2022, she has a sudden vision loss again. "Vitiligo-type" white spots were visible around the eyes and on the hands. In neurological examination: eyeball movements are complete in all directions, photoreaction is preserved (D=S), nose-lip folds are symmetrical (D=S), tongue is on the midline, swallowing is not disturbed. Muscle tone and muscle strength in upper and lower limbs are normal, reflexes have increased (D=S). No changes in gait and sensory disturbances have been observed. The functions of the pelvic organs have not been disturbed. Intellect appears appropriate for age, speech has not changed. Ophthalmologist's examination, both eyes have high degree of myopia, myopic astigmatism. Contrasted Cranial MRI- Angiography revealed increased signal in the intracranial segments of the bilateral optic nerve, chiasm, and bilateral optic tract level. The MRI image was initially evaluated in favor of bilateral optic neuritis. USG of the thyroid gland showed sonographic changes in favor of Hashimoto's thyroiditis, and autoimmune thyroiditis was detected. In chest MRI, a demyelinating lesion of 3 mm at C4 level, a 2 mm broad-based protrusion at C4-5 disc that minimally compresses the spinal cord, an annular swelling at C5-6 disc that compresses the anterior subarachnoid area, Th1 – 6 mm, Th5 – 12 mm, Th9 – 10-38 mm long demyelinating lesions were seen. Discussion. According to the available literature, the detection of AQP-4 protein antibodies in serum, which has a role in the pathogenesis of the disease, can play a decisive role in differential diagnosis. In clinical practice, despite the phenotypic and serological characteristics of this disease, the diagnosis is often difficult. Because there are various autoimmune, infectious and neoplastic etiologies phenotypic, manifestations of diseases can mimic neuromyelitis optica. However, based on the clinical symptoms and the results of instrumental examinations, our patient was diagnosed with neuromyelitis optica. She was treated with hormonal therapy and plasmapheresis, and a significant positive change in neurological symptoms was observed. Conclusion. Despite the difficulties in the differential diagnosis of neuromyelitis optica with other demyelinating diseases, especially with multiple sclerosis, it is possible to correctly assess the diagnostic criteria by understanding the detailed clinical, radiological and prognostic differences.

Genome-Wide Identification of Aqp Family Related to Spermatogenesis in Turbot (Scophthalmus maximus).

The development and maturation of sperm entails intricate metabolic processes involving water molecules, amino acids, hormones, and various substances. Among these processes, the role of aquaporins (aqps) in the testis is crucial. Turbot (Scophthalmus maximus) is a significant marine flatfish species in China; however, natural egg laying in females is not feasible under cultured conditions. Consequently, artificial insemination becomes necessary, requiring the retrieval of sperm and eggs through artificial methods. In this study, we combined genomic, transcriptomics, RT-qPCR, computer-assisted sperm analysis (CASA), and immunohistochemistry to investigate the involvement of the aqp family in spermatogenesis in turbot. Through genomic data analysis, we identified 16 aqps genes dispersed across 13 chromosomes, each exhibiting the characteristic major intrinsic protein (MIP) domain associated with AQPs. The results from RNA-seq and RT-qPCR analysis revealed prominent expression of aqp4, 10, and 12 during the proliferative stage, whereas aqp1 showed primary expression during the mature stage. aqp11 displayed high expression levels during both MSII and MSV stages, potentially contributing significantly to the proliferation and maturation of male germ cells. Conversely, aqp8 showed elevated expression levels during the MSIII, MSIII-IV, and MSIV stages, suggesting its direct involvement in spermiogenesis. Immunohistochemical analysis unveiled the predominant localization of AQP1 protein in male germ cells rather than Sertoli cells, specifically concentrated in the head of sperm within cysts. Furthermore, a noteworthy decline in sperm motility was observed when sperm were subjected to treatment with either the AQP1-specific inhibitor (HgCl2) or the AQP1 antibody. However, no direct correlation was found between the expression of Smaqp1 and sperm quality. Overall, these findings provide new insights into the involvement of aqps in teleost spermatogenesis. Moreover, they hold potential for improving techniques related to sperm activation and cryopreservation, offering valuable knowledge for future advancements in this field.

Tanshinone IIA changed the amniotic fluid volume and regulated expression of AQP1 and AQP3 in amniotic epithelium cells: a promising drug treating abnormal amniotic fluid volume

BackgroundMany studies have confirmed the association of aquaporins (AQPs) with abnormal amniotic fluid volume (AFV). In our previous experiments, we found that Tanshinone IIA was able to regulate the expression of AQP1 and AQP3. However, the exact mechanism by which Tanshinone IIA regulates AQPs protein expression and its effect on AFV remains unclear. The purpose of this study was to investigate the effects of Tanshinone IIA on AFV and the possible molecular mechanism of regulation of AQP1 and AQP3.MethodsThe expression of AQPs protein in the amniotic membranes was compared between pregnant women with normal pregnancy and those with isolated oligohydramnios. The AQP1 knockout (AQP1-KO) mice and wild-type (WT) mice were treated with saline or Tanshinone IIA (10 mg/kg) at 13.5GD and 16.5GD. Human amniotic epithelium cells (hAECs) from pregnant women with normal AFV and isolated oligohydramnios were incubated with 35 μmmol/L Tanshinone IIA or 25 mmol/L LiCl [inhibitor of glycogen synthetic kinase 3β (GSK-3β)]. The protein expressions of AQPs, GSK-3β, phospho-GSK-3β (Ser9) in fetal membranes of mice and human amniotic epithelium cells were detected by western blotting.ResultsThe expression of AQP1 protein in the amniotic membrane of isolated oligohydramnios was increased compared with normal pregnancy. The AFV in AQP1-KO mice is higher than that in WT mice. In wild-type mice, AFV in Tanshinone IIA group was significantly higher than that in control group, and AQP1 protein expression was significantly lower than that in control group, but in AQP1 knockout mice, Tanshinone IIA reduced amniotic fluid volume and AQP3 protein expression at 16.5GD. Tanshinone IIA reduced AQP1, AQP3 and p-GSK-3β (Ser9) protein expression in normal hAECs, and this effect was inhibited by LiCl. In hAECs with oligohydramnios, the down-regulation of AQP1 and up-regulation of AQP3 by Tanshinone IIA was independent of GSK-3β signaling pathway.ConclusionsTanshinone IIA may increase AFV in normal pregnancy by downregulating AQP1 protein expression in the fetal membranes, which may be associated with p-GSK-3β signaling pathway. But a larger AFV in AQP1-KO mice was significantly attenuated by Tanshinone IIA, which may be related to AQP3. Tanshinone IIA is a promising drug for the treatment of amniotic fluid abnormality.

#4082 TAZ-KNOCKDOWN AFFECTS THE VASOPRESSIN-INDUCED AQUAPORIN-2 (AQP2) TRAFFICKING AND PROTEIN ABUNDANCE IN KIDNEY COLLECTING DUCT CELLS

Abstract Background and Aims Transcriptional coactivator with PDZ-binding motif (TAZ), a downstream effector of the hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ KO mice were known to display multicystic kidneys with polyuria. We aimed to study the role of TAZ in vasopressin-induced AQP2 regulation. Method 1) siRNA-mediated knockdown of TAZ in mpkCCDc11 cells; 2) RT-qPCR, semiquantitative immunoblotting, immunocytochemistry of AQP2; 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, the mouse collecting duct cell line. Results Endogenous AQP2 expression was induced in mpkCCDc11 cells by dDAVP (10−9M) treatment. After starvation for 24 h, dDAVP (10−9M) stimulation for 15 and 30 min induced a significant AQP2 translocation to the cell membrane. In contrast, the dDAVP-induced AQP2 translocation was markedly attenuated in the cells with siRNA-mediated TAZ knockdown (TAZ-KD), despite no changes in cAMP production. Phalloidin staining demonstrated the excessive stress fiber formation in the TAZ-KD. dDAVP (10−9 M) treatment for 24 h induced AQP2 mRNA (12,608 ± 177% of the control) and AQP2 protein abundance (270 ± 18%). In contrast, dDAVP-induced increase of AQP2 mRNA (273 ± 14% of the control) and protein abundance (99 ± 17%) was significantly attenuated in TAZ-KD. Unchanged TonEBP protein abundance was observed in TAZ-KD. NGS identified several potential AQP2 transcription factors (TF), and Klf6, Irf3, Cebpb, and Nr4a1 were selected based on previous in silico database. Among them, Nr4a1 was chosen for further studies due to a significant decrease in the mRNA expression levels in TAZ-KD, as demonstrated by RT-qPCR. Conclusion TAZ is likely to affect dDAVP-induced AQP2 trafficking. This is not mediated by the changes in cAMP/PKA pathway, but other non-canonical pathways are involved. TAZ could regulate AQP2 abundance, possibly via an interaction with several TF.

Human Platelet-Rich Plasma Regulates Canine Mesenchymal Stem Cell Migration through Aquaporins.

Platelet products are commonly used in regenerative medicine due to their effects on the acceleration and promotion of wound healing, reduction of bleeding, synthesis of new connective tissue, and revascularization. Furthermore, a novel approach for the treatment of damaged tissues, following trauma or other pathological damages, is represented by the use of mesenchymal stem cells (MSCs). In dogs, both platelet-rich plasma (PRP) and MSCs have been suggested to be promising options for subacute skin wounds. However, the collection of canine PRP is not always feasible. In this study, we investigated the effect of human PRP (hPRP) on canine MSCs (cMSCs). We isolated cMSCs and observed that hPRP did not modify the expression levels of the primary class of major histocompatibility complex genes. However, hPRP was able to increase cMSC viability and migration by at least 1.5-fold. hPRP treatment enhanced both Aquaporin (AQP) 1 and AQP5 protein levels, and their inhibition by tetraethylammonium chloride led to a reduction of PRP-induced migration of cMSCs. In conclusion, we have provided evidence that hPRP supports cMSC survival and may promote cell migration, at least through AQP activation. Thus, hPRP may be useful in canine tissue regeneration and repair, placing as a promising tool for veterinary therapeutic approaches.

Aquaporin 1 (AQP1)/ GSK3β interactions control pulmonary arterial smooth muscle cell (PASMC) function

Pulmonary hypertension (PH) is a complex, incurable, devastating condition, due to pathology of the pulmonary circulation. PH can be induced by hypoxia and is notable for severe wall thickening in the distal vasculature, characterized by enhanced PASMC migration and proliferation. Previously our lab discovered AQP1, a water channel, was highly expressed in PASMCs and mediated hypoxia-induced migration and proliferation. Moreover, increasing AQP1 protein was sufficient to induce PASMC migration and proliferation without hypoxia. We showed the C-terminal tail was required for regulating cell function via accumulation of β-catenin independent of water transport.β-catenin protein is tightly controlled by glycogen synthase kinase 3 β (GSK3β). Binding to GSK3β results in β-catenin degradation; when stabilized, β-catenin mediates transcription of pro-growth genes. Through in silico analysis, we identified a putative binding site for GSK3β in the AQP1 C-terminal tail, allowing us to hypothesize a potential mechanism whereby AQP1 binds GSK3β, releasing β-catenin from degradation, and thereby increases PASMC migration and proliferation.First, we used BioID, a proximity-based assay, to identify GSK3β and AQP1 interactions. We designed BioID constructs with wild-type AQP1 (BioID-AQP1) and AQP1 with mutations in the putative GSK3β binding site (BioID-AQP1M) fused to biotin ligase and compared results to wild-type AQP1 (AQP1), as a control for transfection. We used HEK293 cells which do not express endogenous AQP1. In cells transfected with AQP1 and incubated with cell permeable biotin, following biotin-affinity capture of streptavidin, no biotinylated GSK3b was measured. In cells transfected with BioID-AQP1, a significant amount of GSK3β protein was biotinylated, suggesting AQP1/GSK3β protein-protein interactions. When the putative GSK3b binding site was mutated, very low levels of GSK3b were biotinylated.Next, primary cultured rat distal PASMCs were infected with adenoviral constructs expressing wild-type AQP1 (AdAQP1), AQP1 with the putative GSK3β binding site mutated (AdAQP1M), or green fluorescent protein (AdGFP; control for infection). Compared to AdGFP, infection with AdAQP1 and AdAQP1M increased PASMC total and surface AQP1 protein levels. Using calcein self-quenching, the rate change of water influx was similar for both AQP1 constructs, indicating the mutation did not impair function as a water channel. Infection with AdAQP1 significantly increased both PASMC migration and proliferation, measured by BrdU incorporation and transwell assays, respectively, whereas infection with AdAQP1M had no effect.Our results suggest AQP1 controls PASMC migration and proliferation via a mechanism that requires GSK3β interactions. We speculate high levels of AQP1, as occur during PH, act as a sink for GSK3β, allowing β-catenin to escape from the destruction complex and facilitate cell migration and initiate gene transcription involved in cell proliferation. HL126514 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.

Role of aquaporin 3 in reproductive performance of dairy goats after repeated estrus synchronization stimulation.

This study explored the specific molecular mechanisms through which repeated estrus synchronization (ES) treatments reduce the reproductive performance of dairy goats. Ninety-six goats (n=24/group) were randomly assigned to two groups receiving ES treatments thrice every fortnight (3- equine chorionic gonadotropin [eCG] and 3-follicle stimulating hormone [FSH] groups) and two groups receiving one ES treatment (1-eCG and 1-FSH groups). ES treatments of 1- and 3-eCG goats were performed via the intravaginal insertion of a controlled internal drug release (CIDR) device containing 300 mg progesterone (P4), followed by 300 IU eCG injections 48 h before CIDR withdrawal. The 1- and 3-FSH goats received CIDR for 10 d, followed by 50 IU FSH and 100 μg PGF2α within 12 h of CIDR withdrawal. Ovaries of three goats in estrus from both groups were harvested for analysis. Subsequently, all the goats in estrus were artificially inseminated twice. Consequently, 3-eCG and 3-FSH goats showed a considerably reduced estrus rate and litter size than 1-eCG and 1-FSH goats. AQP3 mRNA and protein expression were significantly higher in the 3-eCG and 3-FSH groups than in the 1-eCG and 1-FSH groups. AQP3 overexpression led to cell apoptosis and decreased steroid hormone secretion ability of ovarian granulosa cells. Moreover, it resulted in a decrease in maturation and cleavage rates after parthenogenetic activation and in vitro fertilization, respectively. AQP3 gene was involved in reducing the reproductive performance of repeated ES-treated dairy goats. These findings provide a theoretical foundation for the effective use of reproductive hormones in breeding techniques for livestock.

Alteration of Aquaporins 1 and 4 immunohistochemical and gene expression in the cerebellum of diabetic albino rat

Aquaporins (AQPs) are a family of transmembrane channel proteins. AQP1 and AQP4 are expressed in cerebellum amongst others. This study was designed to assess the effect of diabetes on AQP1 and AQP4 expression in cerebellum of rats. Diabetes was induced by a single intraperitoneal injection of Streptozotocin 45 mg/kg in 24 adult male Sprague Dawley rats. Six rats from control and diabetic groups were sacrificed at one, four, and eight weeks post diabetic confirmation. After eight weeks, measurement of malondialdehyde (MDA), reduced glutathione (GSH) concentrations, and cerebellar mRNA expression for AQP1 and AQP4 genes were performed. Immunohistochemical evaluation of AQP1, AQP4, and glial fibrillary acidic protein (GFAP) for cerebellar sections was performed for all groups. Diabetes caused degenerative changes in Purkinje cells with a significant increase in the cerebellar level of MDA and AQP1 immunoreactivity and a significant decrease in GSH level and AQP4 expression levels. However, the alteration in the AQP1 mRNA level was not statistically significant. GFAP immunoreactivity was increased in 8 W diabetic rats following its decrease in 1 W diabetic rats. Diabetes caused some alteration in the AQPs 1 and 4 expression in the cerebellum of diabetic rats which may contribute to diabetes-induced cerebellar complications.

Alleviating effects and mechanisms of action of large-leaf yellow tea drinking on diabetes and diabetic nephropathy in mice

Our previous study found that large-leaf yellow tea (LYT) had interesting hypoglycemic activity in high-fat diet-induced obese mice and highly safety in healthy mice. To study the anti-diabetic potential of LYT, the present study further investigated the preventive effects and mechanisms of action of LYT administration on diabetes and diabetic nephropathy in high-fat diet plus streptozotocin-induced diabetic mice. Results showed that LYT infusions (1/100 and 1/50, m/V) as drinking fluid for 4 weeks reduced diabetic polydipsia and polyuria, enhanced glucose tolerance and insulin sensitivity, and lowered fasting blood glucose level. The underlying mechanisms involve downregulation of gluconeogenesis (lower protein levels of TXNIP and FBP and enzyme activity of FBP), upregulation of lipid catabolism (higher protein levels of CPT-1α and PPARα), downregulation of lipogenesis (lower protein level of SREBP-1), and modification of the structure and abundance of gut microbiota to modulate metabolic homeostasis. Moreover, LYT administration prevented diabetic nephropathy, possibly due to reduced glucose-caused osmotic diuresis and lowered levels of renal PKC-β2, NLRP3 as well as membrane PKC-α, AQP2 and glycosylated AQP2 proteins. Taken together, LYT exhibits the activities in alleviating diabetic symptoms, ameliorating glucose and lipid dysmetabolism and fatty liver, and preventing diabetic nephropathy in diabetic mice. These activities may be explored for the prevention and treatment of diabetes in humans.

Experimental study on the effect of chlorhexidine gluconate (CG)-induced atrial fibrillation on renal water and sodium metabolism

To construct an animal model of atrial fibrillation and observe the effect of acute atrial fibrillation on renal water and sodium metabolism in mice. A total of 20 C57 mice were randomly assigned to 2 groups (n = 10/group): control group (CON) and atrial fibrillation group (AF). The mice model of atrial fibrillation was induced by chlorhexidine gluconate (CG) in combination with transesophageal atrial spacing. The urine of the two groups of mice was collected, and then we calculate the urine volume and urine sodium content. The expression of TGF-β and type III collagen in the atrial myocardium of the two groups was detected by immunohistochemistry and Western Blot. The levels of CRP and IL-6 in blood were observed by ELISA, and the NF-κB, TGF-β, collagen type III, AQP2, AQP3, AQP4, ENaC-β, ENaC-γ, SGK1 and NKCC proteins in the kidneys of the two groups of mice was observed by Western Blot. Compared with CON, the expression of TGF-β and type III collagen in the atrial myocardium of the mice in AF were increased, the levels of CRP and IL-6 in the blood in AF were increased, and the renal NF-κB, TGF-β, type III collagen AQP2, AQP3, ENaC-β, ENaC-γ, SGK1 and NKCC protein expression in AF were up-regulated. The level of urine volume and urine sodium content in AF were significantly reduced. In the acute attack of atrial fibrillation, the formation of renal inflammatory response and fibrosis is activated, and the renal water and sodium metabolism is hindered, which is related to the up-regulated of the expressions of renal NKCC, ENaC and AQPs.

Modulation of vulvovaginal atrophy (VVA) by Gelam honey in bilateral oophorectomized rats.

Vulvovaginal atrophy (VVA) is a common condition in post-menopausal women. Symptoms of VVA include dyspareunia, vaginal dryness, vaginal and/or vulvar itching, burning and soreness, dysuria and vaginal bleeding accompanying sexual activity. These symptoms are physiological responses to hypoestrogenicity, inducing atrophy of the vagina epithelia and sudden reduction in mucous production. Prevailing therapy for VVA is hormone replacement therapy (HRT), notably estrogen, progesterone or a combination of the two. However, using HRT is associated with an increased incidence of breast and endometrial cancer, venous thromboembolism in the lungs and legs, stroke and cardiovascular complications. This study evaluated Malaysian Gelam honey as a nutraceutical alternative to estrogen HRT (ERT) in alleviating VVA. A total of 24 female 8-weekold Sprague Dawley rats underwent bilateral oophorectomy. A minimum of 14 days elapsed from the time of surgery and administration of the first dose of Gelam honey to allow the female hormones to subside to a stable baseline and complete recovery from surgery. Vaginal tissues were harvested following a 2-week administration of Gelam honey, the harvested vagina tissue underwent immunohistochemistry (IHC) analysis for protein localization and qPCR for mRNA expression analysis. Results indicated that Gelam honey administration had increased the localization of Aqp1, Aqp5, CFTR and Muc1 proteins in vaginal tissue compared to the menopause group. The effect of Gelam honey on the protein expressions is summarized as Aqp1>CFTR>Aqp5>Muc1. Gene expression analysis reveals Gelam honey had no effect on Aqp1 and CFTR genes. Gelam honey had up-regulated Aqp5 gene expression. However, its expression was lower than in the ERT+Ovx group. Additionally, Gelam honey up-regulated Muc1 in the vagina, with an expression level higher than those observed either in the ERT+Ovx or SC groups. Gelam honey exhibits a weak estrogenic effect on the genes and proteins responsible for regulating water in the vaginal tissue (Aqp1, Aqp5 and CFTR). In contrast, Gelam honey exhibits a strong estrogenic ability in influencing gene and protein expression for the sialic acid Muc1. Muc1 is associated with mucous production at the vaginal epithelial layer. In conclusion, the protein and gene expression changes in the vagina by Gelam honey had reduced the occurrence of vaginal atrophy in surgically-induced menopause models.