Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine

Small intestinal bacterial overgrowth (SIBO) may play an important role in the gastrointestinal complications of cystic fibrosis (CF). This work explored two potential factors in development of SIBO in the CF (cftr(tm1UNC)) mouse: impaired Paneth cell innate defenses and altered gastrointestinal motility. Postnatal differentiation of Paneth cells was followed by Defcr, Lyzs, and Ang4 gene expression, and SIBO was measured by quantitative PCR of the bacterial 16S rRNA gene. Paneth cell gene expression was low in 4-day-old CF and wild-type (WT) mice and increased similarly in both groups of mice between 12 and 16 days. Peak Paneth cell gene expression was reached by 40 days of age and was less for Defcr and Lyzs in CF mice compared with WT, whereas Ang4 levels were greater in CF mice. SIBO occurred by postnatal day 8 in CF mice, which is before Paneth cell development. With the use of gavaged rhodamine-dextran to follow motility, gastric emptying in CF mice was slightly decreased compared with WT, and small intestinal transit was dramatically less. Since antibiotics improve weight gain in CF mice, their effects on gastric emptying and small intestinal transit were determined. Antibiotics did not affect gastric emptying or transit in CF mice but did significantly slow intestinal transit in WT mice, suggesting a potential role of normal microflora in regulating transit. In conclusion, small intestinal transit was significantly slower in CF mice, and this is likely a major factor in SIBO in CF.     

Platelet-activating factor and metastasis: calcium-independent phospholipase A2β deficiency protects against breast cancer metastasis to the lung

We determined the contribution of calcium-independent phospholipase A(2)β (iPLA(2)β) to lung metastasis development following breast cancer injection into wild-type (WT) and iPLA(2)β-knockout (iPLA(2)β-KO) mice. WT and iPLA(2)β-KO mice were injected in the mammary pad with 200,000 E0771 breast cancer cells. There was no difference in primary tumor size between WT and iPLA(2)β-KO mice at 27 days postinjection. However, we observed an 11-fold greater number of breast cancer cells in the lungs of WT mice compared with iPLA(2)β-KO animals (P < 0.05). Isolated WT lung endothelial cells demonstrated a significant increase in platelet-activating factor (PAF) production when stimulated with thrombin [1 IU/ml, 10 min, 4,330 ± 555 vs. 15,227 ± 1,043 disintegrations per minute (dpm), P < 0.01] or TNF-α (10 ng/ml, 2 h, 16,532 ± 538 dpm, P < 0.01). Adherence of E0771 cells to WT endothelial cells was increased by thrombin (4.8 ± 0.3% vs. 70.9 ± 6.3, P < 0.01) or TNF-α (60.5 ± 4.3, P < 0.01). These responses were blocked by pretreatment with the iPLA(2)β-selective inhibitor (S)-bromoenol lactone and absent in lung endothelial cells from iPLA(2)β-KO mice. These data indicate that endothelial cell iPLA(2)β is responsible for PAF production and adherence of E0771 cells and may play a role in cancer cell migration to distal locations.     

Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine

The Lkb1 tumour suppressor is a multitasking kinase participating in a range of physiological processes. We have determined the impact of Lkb1 deficiency on intestinal homeostasis, particularly focussing on secretory cell differentiation and development since we observe strong expression of Lkb1 in normal small intestine Paneth and goblet cells. We crossed mice bearing an Lkb1 allele flanked with LoxP sites with those carrying a Cyp1a1-specific inducible Cre recombinase. Lkb1 was efficiently deleted from the epithelial cells of the mouse intestine after intraperitoneal injection of the inducing agent β-naphthoflavone. Bi-allelic loss of Lkb1 led to the perturbed development of Paneth and goblet cell lineages. These changes were characterised by the lack of Delta ligand expression in Lkb1-deficient secretory cells and a significant increase in the levels of the downstream Notch signalling effector Hes5 but not Hes1. Our data show that Lkb1 is required for the normal differentiation of secretory cell lineages within the intestine, and that Lkb1 deficiency modulates Notch signalling modulation in post-mitotic cells.     

Cardiac dysfunction and impaired compensatory response to pressure overload in mice deficient in stem cell antigen-1

Stem cell antigen-1 (Sca-1) has been used to identify cardiac stem cells in the mouse heart. To investigate the function of Sca-1 in aging and during the cardiac adaptation to stress, we used Sca-1-deficient mice. These mice developed dilated cardiomyopathy [end-diastolic left ventricular diameter at 18 wk of age: wild-type (WT) mice, 4.2 mm ± 0.3; Sca-1-knockout (Sca-1-KO) mice, 4.6 mm ± 0.1; ejection fraction: WT mice, 51.1 ± 2.7%; Sca-1-KO mice, 42.9 ± 2.7%]. Furthermore, the hearts of mice lacking Sca-1 demonstrated exacerbated susceptibility to pressure overload [ejection fraction after transaortic constriction (TAC): WT mice, 43.5 ± 3.2%; Sca-1-KO mice, 30.8% ± 4.0] and increased apoptosis, as shown by the 2.5-fold increase in TUNEL(+) cells in Sca-1-deficient hearts under stress. Sca-1 deficiency affected primarily the nonmyocyte cell fraction. Indeed, the number of Nkx2.5(+) nonmyocyte cells, which represent a population of cardiac precursor cells (CPCs), was 2-fold smaller in Sca-1 deficient neonatal hearts. In vitro, the ability of CPCs to differentiate into cardiomyocytes was not affected by Sca-1 deletion. In contrast, these cells demonstrated unrestricted differentiation into cardiomyocytes. Interestingly, proliferation of cardiac nonmyocyte cells in response to stress, as judged by BrdU incorporation, was higher in mice lacking Sca-1 (percentages of BrdU(+) cells in the heart after TAC: WT mice, 4.4 ± 2.1%; Sca-1-KO mice, 19.3 ± 4.2%). These data demonstrate the crucial role of Sca-1 in the maintenance of cardiac integrity and suggest that Sca-1 restrains spontaneous differentiation in the precursor population. The absence of Sca-1 results in uncontrolled precursor recruitment, exhaustion of the precursor pool, and cardiac dysfunction.     

Heat shock factor 1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of bone marrow stem/progenitor cells

Bone marrow (BM)-derived stem/progenitor cells play an important role in ischemia-induced angiogenesis in cardiovascular diseases. Heat shock factor 1 (HSF1) is known to be induced in response to hypoxia and ischemia. We examined whether HSF1 contributes to ischemia-induced angiogenesis through the mobilization and recruitment of BM-derived stem/progenitor cells using HSF1-knockout (KO) mice. After the induction of ischemia, blood flow and microvessel density in the ischemic hindlimb were significantly lower in the HSF1-KO mice than in the wild-type (WT) mice. The mobilization of BM-derived Sca-1- and c-kit-positive cells in peripheral blood after ischemia was significantly lower in the HSF1-KO mice than in the WT mice. BM stem/progenitor cells from HSF1-KO mice showed a significant decrease in their recruitment to ischemic tissue and in migration, adhesion, and survival when compared with WT mice. Blood flow recovery in the ischemic hindlimb significantly decreased in WT mice receiving BM reconstitution with donor cells from HSF1-KO mice. Conversely, blood flow recovery in the ischemic hindlimb significantly increased in HSF1-KO mice receiving BM reconstitution with donor cells from WT mice. These findings suggest that HSF1 contributes to ischemia-induced angiogenesis by regulating the mobilization and recruitment of BM-derived stem/progenitor cells.     

Caspr3-Deficient Mice Exhibit Low Motor Learning during the Early Phase of the Accelerated Rotarod Task

Caspr3 (Contactin-associated protein-like 3, Cntnap3) is a neural cell adhesion molecule belonging to the Caspr family. We have recently shown that Caspr3 is expressed abundantly between the first and second postnatal weeks in the mouse basal ganglia, including the striatum, external segment of the globus pallidus, subthalamic nucleus, and substantia nigra. However, its physiological role remains largely unknown. In this study, we conducted a series of behavioral analyses on Capsr3-knockout (KO) mice and equivalent wild-type (WT) mice to investigate the role of Caspr3 in brain function. No significant differences were observed in most behavioral traits between Caspr3-KO and WT mice, but we found that Caspr3-KO mice performed poorly during the early phase of the accelerated rotarod task in which latency to falling off a rod rotating with increasing velocity was examined. In the late phase, the performance of the Caspr3-KO mice caught up to the level of WT mice, suggesting that the deletion of Caspr3 caused a delay in motor learning. We then examined changes in neural activity after training on the accelerated rotarod by conducting immunohistochemistry using antibody to c-Fos, an indirect marker for neuronal activity. Experience of the accelerated rotarod task caused increases in the number of c-Fos-positive cells in the dorsal striatum, cerebellum, and motor cortex in both Caspr3-KO and WT mice, but the number of c-Fos-positive cells was significantly lower in the dorsal striatum of Caspr3-KO mice than in that of WT mice. The expression of c-Fos in the ventral striatum of Caspr3-KO and WT mice was not altered by the training. Our findings suggest that reduced activation of neural cells in the dorsal striatum in Caspr3-KO mice leads to a decline in motor learning in the accelerated rotarod task.     

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis,gating pyruvate generation,and increasing pyruvate flux into the TCA cycle

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.     

CARM1 deficiency inhibits osteoblastic differentiation of bone marrow mesenchymal stem cells and delays osteogenesis in mice

Bone repair remains a clinical challenge due to low osteogenic capacity. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that mediates arginine methylation and endochondral ossification. However, the roles of CARM1 in osteoblastic differentiation and bone remodeling have not been explored. In our study, heterozygous CARM1-knockout (KO) mice were generated using the CRISPR-Cas9 system and a model of femoral defect was created. At day 7 postsurgery, CARM1-KO mice exhibited obvious bone loss compared with wild type (WT) mice, as evidenced by reduced bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Deletion of CARM1 in mice lowered synthesis and accumulation of collagen at the injury sites. The alkaline phosphatase (ALP) activity and osteogenic-related gene expression were declined in CARM1-KO mice. To further understand the role of CARM1 in osteoblastic differentiation, bone marrow mesenchymal stem cells (BMSCs) were isolated from the tibia and femur of WT or CARM1-KO mice. CARM1 deletion decreased histone arginine methylation and inhibited osteoblastic differentiation and mineralization. The mRNA sequencing of CARM1-KO BMSCs revealed the possible regulatory molecules by CARM1, which could deepen our understanding of CARM1 regulatory mechanisms. These data could be of interest to basic researchers and provide the direction for future research into bone-related disorders.     

Induction of Paneth cell degranulation by orally administered Toll-like receptor ligands

The secretory activity of Paneth cells is related to the bacterial milieu in the small intestine; however, the molecules involved in inducing Paneth cell secretion of enzymes and antimicrobial peptides are not well-defined. Mice treated orally with CpG-oligodeoxynucleotide (ODN), an agonist of Toll-like receptor (TLR) 9, showed rapid and massive Paneth cell degranulation. CpG-ODN-induced degranulation was not observed in TLR9(-/-) mice or in chimeric TLR9(-/-) mice reconstituted with wild-type (WT) bone marrow, but was observed in WT mice reconstituted with TLR9(-/-) bone marrow, indicating a role for TLR9-expressing gastrointestinal cells in CpG recognition. The TLR3 agonist polyinosinic-polycytidylic acid also induced rapid degranulation, whereas the TLR4 and TLR5 agonists LPS and flagellin, respectively, induced late degranulation mediated by TNF-α. Our evidence that TLR9 and TLR3 agonists induce Paneth cell degranulation points to the need for further studies of the mechanisms underlying Paneth cell function as an avenue toward preventing infection and treating inflammatory bowel diseases.     

Effect of targeted deletions of beta1- and beta2-adrenergic-receptor subtypes on heart rate variability

Beta-adrenergic receptors (beta-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (beta1, beta2, and beta3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of beta-ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, beta1-KO, beta2-KO, and beta1/beta2 double KO (DL-KO), to examine the role that beta-AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in beta1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] mice compared with wild-type [WT; 638 (SD 30)] or beta2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking beta1-ARs (beta1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF powers compared with mice with intact beta1-ARs (WT and beta2-KO). These results demonstrate the differential role of beta-AR subtypes in regulating autonomic signaling.     

An SOD1 deficiency aggravates proteasome inhibitor bortezomib-induced testicular damage in mice

Proteasomes play a key role in maintaining cellular homeostasis by the proteolytic removal of proteins, including ubiquitinated proteins and/or oxidatively-damaged proteins. The proteasome inhibitor bortezomib (BTZ) has been reported to exert testicular toxicity in mice. In the current study, we treated SOD1-knockout (KO) mice with BTZ and investigated the issue of whether oxidative stress is involved in the development of testicular toxicity. The BTZ treatment significantly increased superoxide production and cell death in the testes of SOD1-KO mice compared to wild-type (WT) mice. We also found that high levels of both ubiquitinated proteins and p62 accumulated and underwent aggregation in the seminiferous tubules of BTZ-injected SOD1-KO mice. Furthermore, the proteolytic activities of proteasomes were significantly decreased in the testes of BTZ-injected SOD1-KO mice compared to their WT counterparts. These results suggest that a combination of oxidative stress caused by an SOD1 deficiency and proteasome inhibition by BTZ accelerates the impairment of proteasomes, which results in severe testicular damage in SOD1-KO mice.     

Duffy Antigen Receptor for Chemokines Regulates Post-Fracture Inflammation

There is now considerable experimental data to suggest that inflammatory cells collaborate in the healing of skeletal fractures. In terms of mechanisms that contribute to the recruitment of inflammatory cells to the fracture site, chemokines and their receptors have received considerable attention. Our previous findings have shown that Duffy antigen receptor for chemokines (Darc), the non-classical chemokine receptor that does not signal, but rather acts as a scavenger of chemokines that regulate cell migration, is a negative regulator of peak bone density in mice. Furthermore, because Darc is expressed by inflammatory and endothelial cells, we hypothesized that disruption of Darc action will affect post-fracture inflammation and consequently will affect fracture healing. To test this hypothesis, we evaluated fracture healing in mice with targeted disruption of Darc and corresponding wild type (WT) control mice. We found that fracture callus cartilage formation was significantly greater (33%) at 7 days post-surgery in Darc-KO compared to WT mice. The increased cartilage was associated with greater Collagen (Col) II expression at 3 days post-fracture and Col-X at 7 days post-fracture compared to WT mice, suggesting that Darc deficiency led to early fracture cartilage formation and differentiation. We then compared the expression of cytokine and chemokine genes known to be induced during inflammation. Interleukin (Il)-1β, Il-6, and monocyte chemotactic protein 1 were all down regulated in the fractures derived from Darc-KO mice at one day post-fracture, consistent with an altered inflammatory response. Furthermore, the number of macrophages was significantly reduced around the fractures in Darc-KO compared to WT mice. Based on these data, we concluded that Darc plays a role in modulating the early inflammatory response to bone fracture and subsequent cartilage formation. However, the early cartilage formation was not translated with an early bone formation at the fracture site in Darc-KO compared to WT mice.     

A genetic study of the role of the Wnt/beta-catenin signalling in Paneth cell differentiation

Wnt/β-catenin signalling plays a key role in the homeostasis of the intestinal epithelium. Whereas its role in the maintenance of the stem cell compartment has been clearly demonstrated, its role in the Paneth cell fate remains unclear. We performed genetic studies to elucidate the functions of the Wnt/β-catenin pathway in Paneth cell differentiation. We analysed mice with inducible gain-of-function mutations in the Wnt/β-catenin pathway and mice with a hypomorphic β-catenin allele that have not been previously described. We demonstrated that acute activation of Wnt/β-catenin signalling induces de novo specification of Paneth cells in both the small intestine and colon and that colon cancers resulting from Apc mutations expressed many genes involved in Paneth cell differentiation. This suggests a key role for the Wnt/β-catenin pathway in Paneth cell differentiation. We also showed that a slight decrease in β-catenin gene dosage induced a major defect in Paneth cell differentiation, but only a modest effect on crypt morphogenesis. Overall, our findings show that a high level of β-catenin activation is required to determine Paneth cell fate and that fine tuning of β-catenin signalling is critical for correct Paneth cell lineage.     

Molecular Characterization of Notch1 Positive Progenitor Cells in the Developing Retina

The oscillatory expression of Notch signaling in neural progenitors suggests that both repressors and activators of neural fate specification are expressed in the same progenitors. Since Notch1 regulates photoreceptor differentiation and contributes (together with Notch3) to ganglion cell fate specification, we hypothesized that genes encoding photoreceptor and ganglion cell fate activators would be highly expressed in Notch1 receptor-bearing (Notch1⁺) progenitors, directing these cells to differentiate into photoreceptors or into ganglion cells when Notch1 activity is diminished. To identify these genes, we used microarray analysis to study expression profiles of whole retinas and isolated from them Notch1⁺ cells at embryonic day 14 (E14) and postnatal day 0 (P0). To isolate Notch1⁺ cells, we utilized immunomagnetic cell separation. We also used Notch3 knockout (Notch3KO) animals to evaluate the contribution of Notch3 signaling in ganglion cell differentiation. Hierarchical clustering of 6,301 differentially expressed genes showed that Notch1⁺ cells grouped near the same developmental stage retina cluster. At E14, we found higher expression of repressors (Notch1, Hes5) and activators (Dll3, Atoh7, Otx2) of neuronal differentiation in Notch1⁺ cells compared to whole retinal cell populations. At P0, Notch1, Hes5, and Dll1 expression was significantly higher in Notch1⁺ cells than in whole retinas. Otx2 expression was more than thirty times higher than Atoh7 expression in Notch1⁺ cells at P0. We also observed that retinas of wild type animals had only 14% (P < 0.05) more ganglion cells compared to Notch3KO mice. Since this number is relatively small and Notch1 has been shown to contribute to ganglion cell fate specification, we suggested that Notch1 signaling may play a more significant role in RGC development than the Notch3 signaling cascade. Finally, our findings suggest that Notch1⁺ progenitors—since they heavily express both pro-ganglion cell (Atoh7) and pro-photoreceptor cell (Otx2) activators—can differentiate into either ganglion cells or photoreceptors.     

The nuclear form of glutathione peroxidase 4 is associated with sperm nuclear matrix and is required for proper paternal chromatin decondensation at fertilization

The nuclear isoform of the selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase (nGPx4) is expressed in haploid male germ cells, contains several cysteines and is able to oxidize protein thiols, besides glutathione. In this study we have investigated the subnuclear localization of this isoform in isolated mouse male germ cells at different steps of maturation. Immunoblotting and confocal microscopy analyses of subnuclear fractions showed that nGPx4 is localized to the nuclear matrix together with well known markers of this subnuclear compartment like lamin B and topoisomerase IIβ at all stages of germ cell differentiation. The peculiar nGPx4 distribution was confirmed by both biochemical and morphological analyses of COS-1 cells overexpressing Flag-tagged nGPx4. To test the functional role of nGPx4 in the process of chromatin assembly, sperm isolated from the caput and the cauda epididymides of wild-type (WT) and genetically deficient in nGPx4 (nGPx4-KO) mice were analyzed in an in vitro chromatin decondensation assay. Results showed that sperm from nGPx4-KO mice were more prone to decondense than those from WT mice at all stages of epididymal maturation, providing conclusive evidence that nGPx4 is required for a correct sperm chromatin compaction. We next addressed the issue of whether the lack of nGPx4 impacts on early events occurring at fertilization. Indeed, in vitro fertilization experiments showed an acceleration of sperm chromatin dispersion in oocytes fertilized by nGpx4-KO sperm compared with control. Overall these data indicate that the absence of nGPx4 leads to sperm nuclear matrix/chromatin instability that may negatively affect the embryo development.     

Deletion of Glutathione Peroxidase-2 Inhibits Azoxymethane-Induced Colon Cancer Development

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (−Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the −Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to −Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.     

Aquaporin 5 knockout mouse lens develops hyperglycemic cataract

The scope of this investigation was to understand the role of aquaporin 5 (AQP5) for maintaining lens transparency and homeostasis. Studies were conducted using lenses of wild-type (WT) and AQP5 knockout (AQP5-KO) mice. Immunofluorescent staining verified AQP5 expression in WT lens sections and lack of expression in the knockout. In vivo and ex vivo, AQP5-KO lenses resembled WT lenses in morphology and transparency. Therefore, we subjected the lenses ex vivo under normal (5.6 mM glucose) and hyperglycemic (55.6 mM glucose) conditions to test for cataract formation. Twenty-four hours after incubation in hyperglycemic culture medium, AQP5-KO lenses showed mild opacification which was accelerated several fold at 48 h; in contrast, WT lenses remained clear even after 48 h of hyperglycemic treatment. AQP5-KO lenses displayed osmotic swelling due to increase in water content. Cellular contents began to leak into the culture medium after 48 h. We reason that water influx through glucose transporters and glucose cotransporters into the cells could mainly be responsible for creating hyperglycemic osmotic swelling; absence of AQP5 in fiber cells appears to cause lack of required water efflux, challenging cell volume regulation and adding to osmotic swelling. This study reveals that AQP5 could play a critical role in lens microcirculation for maintaining transparency and homeostasis, especially by providing protection under stressful conditions. To the best of our knowledge, this is the first report providing evidence that AQP5 facilitates maintenance of lens transparency and homeostasis by regulating osmotic swelling caused by glucose transporters and cotransporters under hyperglycemic stressful conditions.