Expression Of Aquaporin Research Articles

Histone Deacetylase Inhibition Increases Glycerol Uptake in Normal and Hyperglycemic Conditions in Human Corneal Epithelial Cells

Delayed corneal wound healing, such as is seen in diabetes, can lead to visual impairment. Diabetes afflicts over 30 million Americans and leads to multiple comorbidities. Histone deacetylase (HDAC) expression and activity are increased in diabetes in correlation with an upregulation of inflammatory molecules and reactive oxygen species (ROS). HDACs have been previously found to suppress the expression of the glycerol channel aquaporin-3 (AQP3) in epithelial cells of the skin, the epidermal keratinocytes. Global deletion of AQP3 in mice has been found to delay corneal wound healing. We hypothesized that hyperglycemic conditions would decrease AQP3 levels and function while increasing the expression of HDAC(s), ROS scavenger proteins, and inflammatory mediators in human corneal epithelial cells (HCECs). HCECs were incubated for 48-hours in high glucose (25.5 mM) or normal glucose (~5.0 mM osmotically matched by mannitol) conditions. Under hyperglycemic conditions, the mRNA expression of interleukin-1α and -1β was significantly elevated, providing evidence for a pro-inflammatory effect of acute hyperglycemia in HCECs. Under these conditions, there was a significant increase in the mRNA expression of the ROS scavenger genes, superoxide dismutase-1 and -2 and peroxiredoxin-3 and -6, suggesting a compensatory response to an increase in ROS generation. Under acute hyperglycemic conditions, HDAC3 and HDAC8 mRNA expression also tended to increase while HDAC1 and HDAC2 mRNA levels remained unchanged. Although an upregulation of HDAC expression would be predicted to decrease AQP3 expression, AQP3 mRNA and protein expression levels were also unchanged. In addition, acute hyperglycemia (48 hours) did not alter glycerol transport into the HCECs, as measured by their uptake of radiolabeled [14C]-glycerol. However, HDAC inhibition using the broad-spectrum inhibitor SAHA for 24 hours increased [14C]-glycerol uptake under both normoglycemic and hyperglycemic conditions, suggesting that HDAC activity plays a role in AQP3 function. Overall, acute hyperglycemic conditions were pro-inflammatory and led to increased expression of ROS-scavenging genes; hyperglycemia also tended to upregulate HDAC expression, although without affecting AQP3 expression. On the other hand, HDAC inhibition increased glycerol uptake, an indicator of AQP3 function, suggesting an ability of HDAC activity to regulate AQP3 glycerol transport activity. National Institutes of Health/National Eye Institute award #R01EY030576 (to MW and WBB) VA Merit #BX005055 (to WBB). 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.

TRPV4 Antagonist Treatment of Hydrocephalus Causes Cell and Molecular Changes in Multiple Brain Cell Types

Hydrocephalus is defined by cerebrospinal fluid (CSF) accumulation in the brain’s ventricles causing ventriculomegaly, an imbalance in CSF secretion, flow, and/or absorption. Treatments for hydrocephalus include invasive surgical procedures, which commonly fail throughout a patient’s life. Transient receptor potential vanilloid member 4 (TRPV4) is a mechanosensitive cation channel implicated in osmotic regulation. Our laboratory found that a TRPV4 antagonist, RN1734, ameliorates hydrocephalus in a rat model of congenital hydrocephalus. It was hypothesized that treatment slowed production of CSF by targeting choroid plexus (CP), a contiguous layer of tightly regulated epithelial cells, that is responsible for CSF production. However, hydrocephalus pathology can have various effects on the brain. This study focuses on changes occurring in CP epithelial cells (CPe) and glia, which have roles in brain-barrier maintenance and brain fluid/electrolyte regulation. Interestingly, CPe and glia contain many of the same channels and transporters, including TRPV4, that are important in regulating both CSF and brain interstitial fluid. These cells contain aquaporins (AQP), AQP1 in CPe, and AQP4 in glia. AQPs have been implicated in diseases associated with fluid regulation and may interact with TRPV4. We hypothesize that TRPV4 and AQPs are important in hydrocephalus pathology, and that changes may be attenuated with RN1734 treatment. A point mutation in TMEM67 results in hydrocephalus (hydro). Changes in cell morphology, gene transcription, protein expression, localization of TRPV4, AQP1, and AQP4 were observed. Postnatal day 15 (P15) untreated (unt) and RN1734-treated (RN;2mg/ml DMSO; 4mg/kg intraperitoneal injection daily P7-P14) wildtype and mutant ( Tmem67−/−) rats were utilized in this study. CPe and periventricular (PV) glia were visualized using fluorescent immunohistochemistry (IHC) of GFAP and a plasma membrane marker. Cell volume of CPe’s and process morphology of glia were altered in hydro rats. RN returned these changes to relatively normal in CPe, but not glia. Next, we used IHC to examine localization of TRPV4 and the AQPs. Increased immunoreactivity of TRPV4 and AQP1 was observed in CPe of unt and RN Tmem67−/− rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and PV white matter of hydro rats. TRPV4 immunoreactivity, but not AQP4, was normalized with RN. We then utilized RTqPCR to inspect transcriptional changes. The data showed increased AQP1 and decreased TRPV4 mRNA in PV cortex and CP of unt and RN Tmem67−/− rats. Previous data from our laboratory found no change in TRPV4 protein in unt Tmem67−/− CPe, but we were curious about AQP expression. The amount of AQP1 protein was increased in the CP of unt and RN Tmem67−/− rats. In PV cortex, AQP4, but not TRPV4, protein was increased in hydro rats. With RN, AQP4 protein was not normalized. In summary, TRPV4 changes in the Tmem67−/− rats returned to normal with RN, glial morphology and AQP changes did not. This may indicate residual inflammation in the Tmem67−/− rats, which we aim to address in future studies. To further elucidate TRPV4’s role in hydrocephalus pathology, future studies will explore post-translational and membrane localization changes. Overall, investigating cell and molecular changes that occur in hydrocephalus will help find better pharmacological targets for this disease. Funding: United States Department of Defense Congressionally Directed Medical Research Program Award; Hydrocephalus Association. 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.

Aquaporin expression in cryopreserved human sperm: exploring the capabilities of natural deep eutectic solvents (NADES)

BACKGROUND: Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryotolerance of sperm cells. OBJECTIVE: This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection. MATERIALS AND METHODS: From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes. RESULTS: The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group. CONCLUSION: These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters.

Lack of Pkd1 enhances intestinal integrity and motility prior to accelerated kidney cyst growth

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder, primarily caused by PKD1 germline mutations. Polycystin-1, PKD1’s encoded protein, is essential to maintain kidney epithelial cell integrity, but its role in the gut has yet to be determined. Considering macrophage infiltration precedes accelerated cyst growth in Pkd1 knockout ( Pkd1KO) mice, and the gut is the body’s largest reservoir of inflammatory cells, we hypothesized that loss of Pkd1 would weaken the intestinal epithelial cell barrier and mount an inflammatory response, potentially preceding kidney damage. Plasma, urine, kidney, and intestines were collected from 11-12 week old male and female conditional Pkd1KO (CAGG-creER; tamoxifen-induced at 5-6 weeks) and flox (control) mice. Plasma was used to measure intestinal permeability as well as monocyte chemoattractant protein-1 (MCP-1) and lipocalin-2 (LCN-2) concentration. Urine was utilized to assess kidney injury, while tissues were used for RT-PCR and histology. Pkd1KO mice had significantly elevated kidney-to-body weight ratio, cyst ratio, and expression of tubular injury markers kidney injury molecule 1 (Kim1) and Lcn2 compared to flox mice. Despite this cellular injury, kidney inflammatory gene expression and urine osmolality did not differ between genotypes. No overt morphological changes were observed in the ileum or colon, nor were there differences in expression of proinflammatory chemokines or cytokines in either tissue. Plasma abundance of MCP-1 and LCN-2 was similar between genotypes. Epithelial permeability in the small and large intestines was significantly decreased in Pkd1KO versus flox mice. Moreover, Pkd1KO mice had a significantly reduced whole gut transit time compared to flox mice. This corresponded with increases in colonic expression of epithelial integrity marker occludin and water channels aquaporin 4 and aquaporin 8 in Pkd1KO mice. Together, these data insinuate that loss of Pkd1 in the intestines leads to a tighter intestinal barrier accompanied by increased water channel expression and gut motility. These findings appear unrelated to inflammatory involvement in early PKD. These studies were supported by NIH T32 DK007545 to RS, R01 DK132028 to TS, and R03 DK119717 to TS. 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.

Deletion of AT1a Receptors Selectively in Renomedullary Interstitial Cells Attenuates Basal, Angiotensin II-, or Vasopressin-induced Vasoactive Factors' mRNA Expression in The Mouse Kidney Medulla

We recently shown that deletion of the vasoactive peptide angiotensin II (Ang II) AT1a receptors selectively in renomedullary interstitial cells (RMICs) of the kidney medulla significantly lowered basal blood pressure and impaired urine concentration in mice. However, the underlying signaling mechanisms involved remains poorly understood. In the present study, AT1a receptors were selectively deleted in the RMICs of the mouse kidney medulla using tamoxifen-inducible Tenascin-C-CreER2/Agtr1af/f approach. Three groups of adult male wild-type (WT) and RMIC- Agtr1a−/− mice (n=6-10) were pretreated with or without tamoxifen (150 mg/kg, i.p., for 5 days) before Ang II was infused to induce Ang II-dependent hypertension (~0.75 mg/kg/day, i.p., for 2 weeks). Systolic, diastolic, and mean arterial blood pressure and urine osmolality were determined before and after Ang II treatment. At the end of the experiment, the kidneys were harvested, and the inner and outer medulla were dissected out for the measurement of the mRNA expression of key vasoactive factors that are associated with the regulation of blood pressure and urine concentration using digital droplet PCR. Deletion of AT1a receptors in RMICs significantly decreased AT1a receptor mRNA expression by 41% (WT: 554 ± 49 vs. RMIC- Agtr1a−/−: 327 ± 10 copies/ng RNA; P<0.01) and bradykinin B2 receptor mRNA expression by 52% (WT: 10.10 ± 0.75 vs. RMIC- Agtr1a−/−: 4.90 ± 0.56 copies/ng RNA; P<0.01), without altering the expression of endothelin ETB (WT: 466 ± 21 vs. RMIC- Agtr1a−/−: 393 ± 24 copies/ng RNA; n.s.) and vasopressin V2 receptor mRNAs in the kidney medulla (WT: 580 ± 60 vs. RMIC- Agtr1a−/−: 586 ± 61 copies/ng RNA; n.s.). Deletion of AT1a receptors in RMICs also significantly decreased the expression of eNOS (WT: 87.0 ± 3.0 vs. RMIC- Agtr1a−/−: 61.2 ± 2.8 copies/ng RNA; P<0.01) and aquaporin 2 (AQP2) mRNAs (WT: 12463 ± 2015 vs. RMIC- Agtr1a−/−: 6613 ± 692 copies/ng RNA; P<0.01). Interestingly, deletion of AT1a receptors in RMICs did not alter the mRNA expression of cyclooxygenase-2 (COX-2, n.s.), prostaglandin E2 (PGE2, n.s.), epithelial sodium channel (ENaC, n.s.) or prorenin receptors (PRR, n.s.) in the kidney medulla. In response to Ang II infusion, deletion of AT1a receptors in RMICs significantly attenuated Ang II-induced expression of bradykinin B2 receptors, COX-2, eNOS, and AQP2 mRNAs ( P<0.01), respectively. Furthermore, deletion of AT1a receptors in RMICs also significantly attenuated vasopressin-induced bradykinin B2 receptor, eNOS, and AQP2 mRNA expression in the kidney medulla ( P<0.01). We concluded that AT1a receptors in RMICs in the kidney medulla play an important role in maintaining physiological blood pressure homeostasis and urine concentration process likely via AT1a receptor/bradykinin B2 receptor/eNOS/AQP2-mediated signaling pathways. 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.

Oxidative stress induced by menadione (vitamin K3) results in non-canonical AQP2 membrane traffcking in LLC-AQP2 cells

Oxidative stress affects a wide variety of cellular processes including morphology, protein traffcking, and cell death. Several studies show that reactive oxygen species (ROS) are able to pass through some water channels, and that ROS may modulate aquaporin expression and traffcking. However, a relationship between ROS status and the traffcking of aquaporin-2 (AQP2) has yet to be well-defined. To address this question, we first treated LLC-PK1 cells expressing cmyc-tagged AQP2 (LLC-AQP2 cells) for 30 min with menadione (MK3, 20 μM), an agent widely used to generate ROS in cell culture. Similar to vasopressin (VP), MK3 induced significant AQP2 accumulation in the plasma membrane of these cells. Using LLC-AQP2 cells expressing secreted soluble green fluorescent protein (ss-GFP), we revealed significantly increased vesicle exocytosis upon treatment with MK3. Surprisingly, MK3 did not affect rhodamine-labeled dextran endocytosis, suggesting that the MK3 effect is mainly due an increase in AQP2 exocytosis into the plasma membrane. Next, we studied the role of AQP2 c-terminal serine 256 phosphorylation in this process. MK3 treatment resulted in significant plasma membrane accumulation of the non-phosphorylatable AQP2 S256A mutant, whereas VP had no effect, as previously reported. To understand the contribution of protein kinase A (PKA) to MK3-mediated AQP2 traffcking, we used CRISPR-generated LLC-AQP2,koPKA cells that lack PKA isoforms. Interestingly, an increase in AQP2 membrane accumulation was still observed, suggesting that the MK3 effect is PKA-independent. By western blotting with anti-phospho-AQP2 antibodies, we found that MK3 did not affect phosphorylation of the critical S256 residue, but in contrast to VP, MK3 actually increased the phosphorylation level of S261 instead of reducing it. Finally, we showed that AQP2 accumulation in both LLC-AQP2 and LLC-AQP2 S256A cells was inhibited in the presence of glutathione (5 mM), an anti-oxidant or reducing agent. Interestingly, western blot analysis showed that glutathione abolished the MK3-induced increase of S261 AQP2 phosphorylation. In summary, our study shows that menadione (MK3) induces AQP2 membrane accumulation in LLC-AQP2 cells, suggesting a role of ROS in this process. This effect is largely due a change in AQP2 vesicle exocytosis. It does not require phosphorylation of the c-terminal S256 residue of AQP2, but MK3 does cause increased S261 phosphorylation. While incompletely understood at present, these data reveal that alternative, probably ROS-related, regulatory mechanisms can be involved in AQP2 traffcking and recycling. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). 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.

Study on the Skincare Effects of Red Rice Fermented by Aspergillus oryzae In Vitro.

Red rice, a variety of pigmented grain, serves dual purposes as both a food and medicinal resource. In recent years, we have witnessed an increasing interest in the dermatological benefits of fermented rice extracts, particularly their whitening and hydrating effects. However, data on the skincare advantages derived from fermenting red rice with Aspergillus oryzae remain sparse. This study utilized red rice as a substrate for fermentation by Aspergillus oryzae, producing a substance known as red rice Aspergillus oryzae fermentation (RRFA). We conducted a preliminary analysis of RRFA's composition followed by an evaluation of its skincare potential through various in vitro tests. Our objective was to develop a safe and highly effective skincare component for potential cosmetic applications. RRFA's constituents were assessed using high-performance liquid chromatography (HPLC), Kjeldahl nitrogen determination, the phenol-sulfuric acid method, and enzyme-linked immunosorbent assay (ELISA). We employed human dermal fibroblasts (FB) to assess RRFA's anti-aging and antioxidative properties, immortalized keratinocytes (HaCaT cells) and 3D epidermal models to examine its moisturizing and reparative capabilities, and human primary melanocytes (MCs) to study its effects on skin lightening. Our findings revealed that RRFA encompasses several bioactive compounds beneficial for skin health. RRFA can significantly promote the proliferation of FB cells. And it markedly enhances the mRNA expression of ECM-related anti-aging genes and reduces reactive oxygen species production. Furthermore, RRFA significantly boosts the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), and Hyaluronan Synthase 1 (HAS1) mRNA, alongside elevating moisture levels in a 3D epidermal model. Increases were also observed in the mRNA expression of Claudin 1 (CLDN1), Involucrin (IVL), and Zonula Occludens-1 (ZO-1) in keratinocytes. Additionally, RRFA demonstrated an inhibitory effect on melanin synthesis. Collectively, RRFA contains diverse ingredients which are beneficial for skin health and showcases multifaceted skincare effects in terms of anti-aging, antioxidant, moisturizing, repairing, and whitening capabilities in vitro, highlighting its potential for future cosmetic applications.

Aquaporins: Navigating the channels of cellular hydration

Aquaporin’s are water channel proteins found in all plasma membrane of various cells in all forms of life from bacteria to mammals. Around 17 mammalian aquaporin’s have been identified till date, which are classified into 3 subcategories based on their permeability. In normal physiology, AQPs serve as essential modulators of fluid transport and homeostasis in multiple organs and tissues. Altered expression of aquaporin’s is linked to numerous pathologies including fluid dysregulation, tumor metastasis and traumatic injury. Although some potential AQP modulators have been identified, challenges associated with the development of better modulators, include the suitability of the assay methods used to identify modulators and the drug ability of the target. The present review focus on aquaporin types and their altered conditions along with role of aquaporin’s in cancer and other diseases.

The relation between autophagy modulation by intermittent fasting and aquaporin 2 expression in experimentally induced diabetic nephropathy in albino rat

Polyuria is an early sign of diabetic nephropathy (DN) that produces dehydration in diabetic patients. This could be caused by alteration of renal aquaporin 2 (AQP2) expression. This study aimed to describe the relation between autophagy modulation via intermittent fasting (IF) and renal AQP2 expression and polyuria in case of DN. We divided the rats into control, DN and IF groups. After 2 and 4 weeks of diabetes induction, blood glucose (BG), serum creatinine (Scr), urine volume, and 24 hours urine protein (UP) were examined. Diabetic nephropathy histopathological index (DNHI) was calculated to evaluate histopathological changes. Immunohistochemistry and real-time PCR were performed to measure the levels of AQP2 and the autophagy marker; LC3 in kidney tissue. DNHI was correlated to the PCR and immunoexpression of AQP2 and LC3. Intermittent fasting significantly decreased the BG, Scr, urine volume, 24 hours UP, and DNHI as compared diabetes. Diabetes significantly elevated the immunoreactivity and mRNA expression levels of AQP2 and LC3 as compared to the control. However, the IF decreased AQP2 and stimulated autophagy in cyclic fashion. Our data revealed significant positive correlations between AQP2 and LC3 at the level of immunoexpression and mRNA at 2nd weeks. Taken together, these data showed that autophagy stimulation didn’t regulate AQP2 expression in case of diabetic nephropathy, however IF decreased polyuria through improvement of glycemic state.

White matter hyperintensities and the surrounding normal appearing white matter are associated with water channel disruption in the oldest old.

Age-related magnetic resonance imaging (MRI) T2 white matter hyperintensities (WMHs) are common and associated with neurological decline. We investigated the histopathological underpinnings of MRI WMH and surrounding normal appearing white matter (NAWM), with a focus on astroglial phenotypes. Brain samples from 51 oldest old Oregon Alzheimer's Disease Research Center participants who came to autopsy underwent post mortem (PM) 7 tesla MRI with targeted histopathological sampling of WMHs and NAWM. Stained slides were digitized and quantified. Mixed-effects models determined differences in molecular characteristics between WMHs and the NAWM and across NAWM. PM MRI-targeted WMHs are characterized by demyelination, microglial activation, and prominent astrocytic alterations, including disrupted aquaporin (AQP) expression. Similar changes occur within the surrounding NAWM in a pattern of decreasing severity with increased distance from WMHs. Decreased AQP expression within WMH and proximal NAWM suggest an overwhelmed system wherein water homeostasis is no longer maintained, contributing to WM damage in older individuals. Post mortem magnetic resonance imaging (MRI) was used to characterize the pathology of white matter hyperintensities (WMHs) and surrounding normal appearing white matter (NAWM). Stained immunohistochemical (IHC) slides from targeted WMH and NAWM samples were digitized and quantified. WMHs and NAWM were associated with inflammation, demyelination, and gliosis. WMHs and NAWM astrocytic changes included decreased AQP1 and AQP4 expression. Abnormal NAWM pathology diminished in severity with increasing distance from WMH.

Immunohistochemical Analysis of Sperm Aquaporin-3 Expression in Men with Varicocele

Objective: The primary objective of this study is to detect potential alterations in sperm cells in cases of varicocele using immunohistochemical methods. Materials and Methods: In this study, semen samples obtained with consent from infertile males aged between 25-40 years were analyzed for the presence of varicocele and other determined factors, and were divided into two groups: a normozoospermic control group and a group diagnosed with varicocele. Direct swim-up techniques were utilized to separate spermatozoa with high motility. Immunohistochemical staining methods were employed to visualize protein expression, and the results were photographed at high magnification for documentation. Results: This research was conducted on sperm samples collected from males diagnosed with varicocele and from a healthy control group. The findings suggest that the presence of aquaporin-3 protein is associated with low or negative expression in sperm cells in the presence of varicocele. Immunohistochemical analyses have revealed a reduced or absent expression of aquaporin 3 in patients with varicocele compared to healthy individuals. Conclusion: The results of this study clearly demonstrate the adverse effects of varicocele on the structural and functional integrity of spermatozoa. Given that aquaporin-3 protein is critical for the motility of spermatozoa, the reduced expression or absence of this protein sheds light on the molecular mechanisms of male infertility. Keywords: Sperm DNA fragmentation, immunohistochemistry, Varicocele, protein expression, male infertility, aquaporin-3.

Consumption of sucrose in maternal and postnatal stages leads to kidney affectation in adult male rats

High sugar-sweetened beverage intake has been related to human kidney disease and metabolic alterations. We determine the impact of high sucrose intake from pregnancy until early postnatal days and post-weaning on kidneys from adult male offspring rats. Wistar female rats were mated and assigned into two groups: one control drinking tap water (CM) and another drinking 5 % sucrose diluted in water (SM). Two offspring per mother were randomly allocated into two experimental groups at weaning. One had free access to simple water (CO) and the other to 5 % sucrose (SO) for 14 weeks. After treatment, levels of relative aquaporin-2 (AQP2), glomerulosclerosis index (GI), collecting tube area, perirenal fat, blood creatinine, and blood ureic nitrogen concentration (BUN) were determined. Two-way ANOVA followed by Bonferroni post-hoc test was used, considering P ≤ 0.05 as a significant statistical difference. Sucrose consumption during gestation/lactation and interaction increased AQP2 expression in the renal cortex and BUN concentration. In contrast, gestation/lactation consumption increased collecting tube area, post-weaning consumption favored perirenal fat, and finally, gestation/lactation, post-weaning, and the interaction caused glomerulosclerosis. Our results suggest that the consumption of sucrose water during gestation/lactation or post-weaning or combination triggers pathological changes in the kidneys of adult rats.

Role of Trichoderma asperelloides and Trichoderma brevicompactum in improving drought tolerance in rice

Drought is considered as one of the major abiotic stresses affecting rice production under rainfed ecologies of Bangladesh hampering the country's self-sufficiency in rice production through reduced crop yield and quality. In the present study, we studied the effect of two species of plant growth promoting Trichoderma v iz; Trichoderma asperelloides and Trichoderma brevicompactum on rice plant development under drought stress and their role in inducing tolerance to drought at physiological and biochemical level. Results reveal that both species are capable of inducing plant tolerance to drought stress when tested on drought tolerant (BRRI dhan56) and drought susceptible (IR64) varieties. As the number of drought stress days increased, there was an increase in root length of both the varieties but increase in root length was more in BRRI dhan56 as compared to IR 64 when seeds of both varieties were primed with Trichoderma. There was significant change in proline, malondialdehyde (MDA), total Phenolic content (TFC), H2O2 content in Trichoderma treated versus non-treated plants for both varieties. These biochemical changes were verified with the expression of aquaporin (AQU), dehydrin (DHN) and DREB genes in both rice varieties treated with both Trichoderma species. Interaction studies of Trichoderma species with two rice genotypes suggested that AQU-2 expressed more in tolerant variety (BRRI dhan56) when primed with Trichoderma asperelloides whereas, both Trichoderma spp. up regulated the expression of DHN-4 and DREB genes in both rice genotypes under drought conditions. The present findings suggest that the both Trichoderma asperelloides and Trichoderma brevicompactum are most effective in improving drought tolerance in rice, and its prospective application will contribute to the advancement of rice genotypes to sustain crop productivity under drought stress.

Effects of Zinc Combined with Metformin on Zinc Homeostasis, Blood-Epididymal Barrier, and Epididymal Absorption in Male Diabetic Mice.

Diabetes increases the likelihood of germ cell damage, hypogonadism, and male infertility. Diabetes leads to lower zinc (Zn) levels, an important micronutrient for maintaining male fertility, and zinc deficiency can lead to decreased male fertility through multiple mechanisms. The aim of this study was to investigate the effect of combined metformin and zinc administration on epididymis in diabetic mice; 10 of 50 male mice were randomly selected as the control group (group C), and the remaining 40 mice were randomly divided into untreated diabetes group (group D), diabetes + zinc group (group Z), diabetes + metformin group (group M), and diabetes + metformin + zinc group (group ZM) with 10 mice each. Diabetic mice in group Z received oral zinc (10 mg/kg) once daily for 4 weeks; diabetic mice in group M received oral metformin (200 mg/kg) once daily for 4 weeks; diabetic mice in group ZM received oral metformin and zinc once daily for 4 weeks; and groups C and D received the same amount of sterile water by gavage. Overnight fasted mice were sacrificed, and blood samples, mouse epididymides, and sperm were collected for further experiments. In group D, fasting blood glucose and insulin resistance index increased significantly, semen quality, serum insulin, and testosterone decreased, and epididymal structure was disordered. In group D, epididymal tissue zinc, free zinc ions in the caput, and cauda of epididymis and zinc transporter (ZnT2) decreased significantly, while ZIP12, metallothionein (MT), and metal transcription factor (MTF1) increased significantly. In addition, the expressions of blood-epididymal barrier (BEB)-related molecules (including ZO-1 β-catenin and N-cadherin) and aquaporins (AQPs, including AQP3, AQP9, and AQP11) in the epididymis of mice in group D were significantly decreased. In addition, compared with groups D, Z, and M, in the ZM group, the expression of BEB-related molecules (including ZO-1, β-catenin, and N-cadherin) and aquaporins (AQP3, AQP9, and AQP11) in epididymis tissue were significantly increased, and sperm motility and serum testosterone were significantly increased. It was concluded that male diabetic mice have a disturbed epididymal structure and decreased semen quality by causing an imbalance in epididymal zinc homeostasis, BEB, and impaired absorptive function. The combination of zinc and metformin is an effective and safe alternative treatment and provides additional benefits over metformin alone.

Cartilage protective and anti-edema effects of JTF in osteoarthritis via inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation

BackgroundPreventing joint edema is crucial in halting osteoarthritis (OA) progression. Growing clinical evidence indicate that Jianpi-Tongluo Formula (JTF) may have a promising anti-edema effect. However, the therapeutic properties of JTF and the underlying mechanisms remains unclear. Materials and methodsAn OA rat model was established and employed to evaluate pharmacological effects of JTF in vivo based on dynamic histopathologic assessments and micro-CT observations. Then, OA-related genes and potential targets of JTF were identified through clinical transcriptomic data analysis and "disease gene-drug target" network analysis, which were verified by a series of in vivo experiments. ResultsJTF administration effectively reduced pain and joint edema, inhibited matrix degradation, chondrocyte apoptosis, and aquaporin expression in OA rats. Notably, JTF dose-dependently reversed damage-associated molecular patterns and inflammatory factor upregulation. Mechanically, our "disease gene-drug target" network analysis indicated that the NCOA4-HMGB1-GSK3B-AQPs axis, implicated in ferroptosis and aquaporin dysregulation, may be potentially served as a target of JTF against OA. Accordingly, JTF mitigated NCOA4, HMGB1, and GSK3B expression, oxidative stress, and iron metabolism aberrations in OA rats. Furthermore, JTF treatment significantly attenuated the aberrant upregulation of AQP1, AQP3, and AQP4 proteins observed in cartilage tissues of OA rats. ConclusionOur data reveal for the first time that JTF may exert cartilage protective and anti-edema effects in osteoarthritis therapy by inhibiting NCOA4-HMGB1-driven ferroptosis and aquaporin dysregulation.

Multi-hormonal analysis and aquaporins regulation reveal new insights on drought tolerance in grapevine

Disentangling the factors that foster the tolerance to water stress in plants could provide great benefits to crop productions. In a two-year experiment, two new PIWI (fungus resistant) grapevine varieties, namely Merlot Kanthus and Sauvignon Kretos (Vitis hybrids), grown in the field, were subjected to two different water regimes: weekly irrigated (IR) or not irrigated (NIR) for two months during the summer. The two varieties exhibited large differences in terms of performance under water-limiting conditions. In particular, Merlot Kanthus strongly decreased stem water potential (Ψs) under water shortage and Sauvignon Kretos maintained higher Ψs values accompanied by generally high stomatal conductance and net carbon assimilation, regardless of the treatment. We hypothesized differences in the hormonal profile that mediate most of the plant responses to stresses or in the regulation of the aquaporins that control the water transport in the leaves. In general, substantial differences were found in the abundance of different hormonal classes, with Merlot Kanthus reporting higher concentrations of cytokinins while Sauvignon Kretos higher concentrations of auxins, jasmonate and salicylic acid. Interestingly, under water stress conditions ABA modulation appeared similar between the two cultivars, while other hormones were differently modulated between the two varieties. Regarding the expression of aquaporin encoding genes, Merlot Kanthus showed a significant downregulation of VvPIP2;1 and VvTIP2;1 in leaves exposed to water stress. Both genes have probably a role in influencing leaf conductance, and VvTIP2;1 has been correlated with stomatal conductance values. This evidence suggests that the two PIWI varieties are characterized by different behaviour in response to drought. Furthermore, the findings of the study may be generalized, suggesting the involvement of a complex hormonal cross-talk and aquaporins in effectively influencing plant performance under water shortage.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis

Sjogren's syndrome (SS) is an autoimmune disease characterized by dysfunction of exocrine glands, such as salivary glands. However, the molecular mechanism of salivary secretion dysfunction in SS is still unclear. Given the significance of glutathione peroxidase 4 (GPX4) in cellular redox homeostasis, we hypothesized that dysregulation of GPX4 may play a pivotal role in the pathogenesis of salivary secretion dysfunction observed in SS. The salivary gland of SS patients and the SS mouse model exhibited reduced expression of the ferroptosis inhibitor GPX4 and the important protein aquaporin 5 (AQP5), which is involved in salivary secretion. GPX4 overexpression upregulated and GPX4 knockdown downregulated AQP5 expression in salivary gland epithelial cells (SGECs) and salivary secretion. Bioinformatics analysis of GSE databases from SS patients' salivary glands revealed STAT4 as a key intermediary regulator between GPX4 and AQP5. A higher level of nuclear pSTAT4 was observed in the salivary gland of the SS mouse model. GPX4 overexpression inhibited and GPX4 knockdown promoted STAT4 phosphorylation and nuclear translocation in SGECs. CHIP assay confirmed the binding of pSTAT4 within the promoter of AQP5 inhibiting AQP5 transcription. GPX4 downregulation accumulates intracellular lipid ROS in SGECs. Lipid ROS inhibitor ferrostatin-1 treatment during in vitro and in vivo studies confirmed that lipid ROS activates STAT4 phosphorylation and nuclear translocation in SGECs. In summary, the downregulated GPX4 in SGECs contributes to salivary secretion dysfunction in SS via the lipid ROS/pSTAT4/AQP5 axis. This study unraveled novel targets to revitalize the salivary secretion function in SS patients.

Effects of angiopoietin (Ang) 1/endothelial-enriched Tunica interna endothelial cell kinase 2 (Tie2) signaling pathway on autophagy-based neuroprotection and expression of aquaporin 4

We investigated Ang-1/Tie2 signaling’s role in cellular autophagy-based neuroprotection and aquaporins expression. 40 mice were randomized into sham surgery group, model group, Tie2-antagonist group, and Ang-1+Tie2 agonist group, followed by observing brain pathological changes, and mRNA levels of LC3-I, LC3-II and aquaporin 4 (AQP4), together with levels of TNF-α and IL-1β. A certain regularity of brain tissue structure was observed in sham surgery group but not in model group. In Ang-1+Tie2 agonist group, the brain tissue structure was more disordered, which was ameliorated in Tie2-antagonist group, along with a proportion of intact cells. The neurological score of mice in Tie2 antagonist group was lower than Ang-1+Tie2 agonist and model groups. Levels of LC3-I and AQP4 in Tie2 antagonist group were also lower, along with higher levels of LC3-I and AQP4. LC3-II levels were higher in Tie2 antagonist group compared with those in Ang-1+Tie2 agonist and model groups. Whereas LC3-II and IL-1β/TNF-α levels in Ang-1+Tie2 agonist group were lower with higher levels of IL-1β and TNF-α. Inhibition of Ang-1/Tie2 signaling could have a restorative effect on neural function in stroke mice.

Long-Term Sodium Deficiency Reduces Sodium Excretion but Impairs Renal Function and Increases Stone Formation in Hyperoxaluric Calcium Oxalate Rats.

Excessive sodium intake is associated with nephrolithiasis, but the impact of sodium-deficient (SD) diets is unknown. Hence, we investigated the effects of short- and long-term SD diets on the expression of renal aquaporins and sodium transporters, and thus calcium oxalate (CaOx) crystal formation in hyperoxaluria rats. In a short-term sodium balance study, six male rats received drinking water and six received 0.75% ethylene glycol (EG) to induce hyperoxaluria. After a 30-day period of feeding on normal chow, both groups were treated with a normal-sodium diet for 5 days, followed by a sodium-free diet for the next 5 days. In a long-term SD study (42 days), four groups, induced with EG or not, were treated with normal-sodium water and sodium-free drinking water, alternately. Short-term sodium restriction in EG rats reversed the daily positive sodium balance, but progressively caused a negative cumulative water balance. In the long-term study, the abundant levels of of Na/H exchanger, thiazide-sensitive Na-Cl cotransporter, Na-K-ATPase, and aquaporins-1 from SD + EG rats were markedly reduced, corresponding to a decrease in Uosm, as compared to SD rats. Increased urine calcium, AP(CaOx)index, and renal CaOx deposition were also noted in SD + EG rats. Although the SD treatment reduced sodium excretion, it also increased urinary calcium and impaired renal function, ultimately causing the formation of more CaOx crystals.

Adjudin protects blood–brain barrier integrity and attenuates neuroinflammation following intracerebral hemorrhage in mice

Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood–brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1β. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.