α1-Acid Glycoprotein Enhances the Immunosuppressive and Protumor Functions of Tumor-Associated Macrophages.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell-derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte-derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. SIGNIFICANCE: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

An acute phase protein α1-acid glycoprotein mitigates AKI and its progression to CKD through its anti-inflammatory action.

The molecular mechanism for acute kidney injury (AKI) and its progression to chronic kidney disease (CKD) continues to be unclear. In this study, we investigated the pathophysiological role of the acute phase protein α1-acid glycoprotein (AGP) in AKI and its progression to CKD using AGP KO mice. Plasma AGP levels in WT mice were increased by about 3.5-fold on day 1-2 after renal ischemia-reperfusion (IR), and these values then gradually decreased to the level before renal IR on day 7-14. On day 1 after renal IR, the AGP KO showed higher renal dysfunction, tubular injury and renal inflammation as compared with WT. On day 14, renal function, tubular injury and renal inflammation in WT had recovered, but the recovery was delayed, and renal fibrosis continued to progress in AGP KO. These results obtained from AGP KO were rescued by the administration of human-derived AGP (hAGP) simultaneously with renal IR. In vitro experiments using RAW264.7 cells showed hAGP treatment suppressed the LPS-induced macrophage inflammatory response. These data suggest that endogenously induced AGP in early renal IR functions as a renoprotective molecule via its anti-inflammatory action. Thus, AGP represents a potential target molecule for therapeutic development in AKI and its progression CKD.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.

A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein.

Renal fibrosis is a major factor in the progression of chronic kidney disease and the final common pathway of kidney injury. Therefore, the effective therapies against renal fibrosis are urgently needed. The objective of this study was to investigate the effect of Am80, a synthetic retinoic acid receptor (RAR) agonist, in the treatment of renal interstitial fibrosis using unilateral ureteral obstruction (UUO) mice. The findings indicate that Am80 treatment suppressed renal fibrosis and inflammation to the same degree as the naturally-occuring retinoic acid, all-trans retinoic acid (atRA). But the adverse effect of body weight loss in Am80-treated mice was lower compared to the atRA treatment. The hepatic mRNA levels of alpha-1-acid glycoprotein (AGP), a downstream molecule of RAR agonist, was increased following administration of Am80 to healthy mice. In addition, increased AGP mRNA expression was also observed in HepG2 cells and THP-1-derived macrophages that had been treated with Am80. AGP-knockout mice exacerbated renal fibrosis, inflammation and macrophage infiltration in UUO mice, indicating endogenous AGP played an anti-fibrotic and anti-inflammatory role during the development of renal fibrosis. We also found that no anti-fibrotic effect of Am80 was observed in UUO-treated AGP-knockout mice whereas atRA treatment tended to show a partial anti-fibrotic effect. These collective findings suggest that Am80 protects against renal fibrosis via being involved in AGP function.

Simple transport and cryopreservation of cold-stored mouse embryos.

The cold storage of two-cell embryos is a useful technique for transporting genetically engineered mice without the shipment of live animals. However, the developmental ability of cold-stored embryos decreases with prolonged storage periods. Therefore, the transported embryos must be readily transferred to recipient mice upon arrival. The cryopreservation of cold-transported embryos may improve the flexibility of the schedule of embryo transfer. In this paper, we examined the viability and developmental ability of vitrified-warmed mouse embryos at the two-cell stage after cold storage in refrigerated temperatures for 0, 24, 48, 72, or 96 h. The viability of vitrified-warmed embryos after cold storage was comparable to vitrified-warmed embryos without cold storage. Vitrified-warmed embryos after cold storage also developed normally to pups by embryo transfer. In addition, live pups were obtained from vitrified-warmed embryos after cold-transportation from Asahikawa Medical University. In summary, cold-stored embryos can be used for the transportation and archive of genetically engineered mice.

Advanced Oxidation Protein Products Contribute to Renal Tubulopathy via Perturbation of Renal Fatty Acids.

Background: Renal proximal tubulopathy plays a crucial role in kidney disease, but its molecular mechanism is incompletely understood. Because proximal tubular cells consume a lot of energy during reabsorption, the relationship between fatty acids (FAs) and proximal tubulopathy has been attracting attention. The purpose of this study is to investigate the association between change in renal FA composition and tubulopathy. Methods: Mice with cisplatin-induced nephrotoxicity were used as a model of AKI and 5/6-nephrectomized mice were used as a model of CKD. Renal FA composition in mice was measured by GC-MS. Human tubular epithelial cells (HK-2 cells) were used for in vitro studies. Results: In kidneys of AKI mice, increased stearic acid (C18:0) and decreased palmitic acid (C16:0) were observed, accompanied by increased expression of the long-chain FA elongase Elovl6. Similar results were also obtained in CKD mice. We show that C18:0 has higher tubular toxicity than C16:0 via induction of ER stress. Using adenovirus-expressing Elovl6 or siRNA for Elovl6 in HK-2 cells, we demonstrated that increased Elovl6 expression contributes to tubulopathy via increasing C18:0. Elovl6 knockout suppressed the increased serum creatinine levels, renal ER stress, and inflammation that would usually result after 5/6 nephrectomy. Advanced oxidation protein products (AOPPs), specifically an oxidized albumin, was found to induce Elovl6 via the mTORC1/SREBP1 pathway. Conclusions: AOPPs may contribute to renal tubulopathy via perturbation of renal FAs through induction of Elovl6. The perturbation of renal FAs induced by the AOPPs-Elovl6 system could be a potential target for the treatment of tubulopathy.

N-acetyl cysteine restores the fertility of vitrified-warmed mouse oocytes derived through ultrasuperovulation.

Oocyte cryopreservation is useful for preserving fertility and storing genetic resources. However, the small number of oocytes acquired using conventional treatment to induce superovulation and the reduction of fertility due to cryopreservation represent significant problems. Herein, we vitrified the oocytes derived through high-yield superovulation using inhibin antiserum and equine chorionic gonadotropin (IAS + eCG: IASe) and examined the yield of cryopreserved oocytes and survival rates relative to those of vitrified-warmed mouse oocytes derived through conventional superovulation using equine chorionic gonadotropin (eCG). Furthermore, we investigated the effects of N-acetyl cysteine on the fertility and developmental potential of vitrified-warmed oocytes derived using IASe. Compared with eCG, IASe increased the yield of cryopreserved oocytes and achieved equivalent survival rates. N-acetyl cysteine (0.5 mM) increased the fertilization rate of vitrified-warmed oocytes derived using IASe. Vitrification decreased thiol levels in the zona pellucida (ZP), while warming followed by N-acetyl cysteine treatment increased free thiol levels in ZP. Moreover, N-acetyl cysteine treatment recovered zona hardening by cleaving disulfide bonds and promoting the expansion of ZP. Two-cell embryos derived via in vitro fertilization using N-acetyl cysteine developed into normal pups through embryo transfer. Therefore, we developed an efficient technique for the production of cryopreserved oocytes using IASe through superovulation and found that N-acetyl cysteine improves the fertility of vitrified-warmed oocytes by cleaving the disulfide bonds and promoting the expansion of ZP.

Ovulation of juvenile, mature, and aged female C57BL/6 mice following coadministration of inhibin antiserum and equine chorionic gonadotropin.

Superovulation technique is important to improve the efficiency of oocyte and animal production and reduce the number of oocyte donors. Previously, we have reported that the coadministration of inhibin antiserum (IAS) and equine chorionic gonadotropin (eCG) results in the production of >100 oocytes in a 4-week-old female C57BL/6 mice. It is well established that superovulation depends on the age of the female mice. However, detailed data regarding the ovulation of juvenile, mature, and aged female mice following the administration of IAS and eCG as well as the performance of reproductive technologies using oocytes have not yet been investigated. In the present study, we examined the effect of the age of female mice (3-50 weeks old) on the number of ovulated oocytes via the coadministration of IAS and eCG or eCG alone. Treatment with IAS plus eCG produced the maximum number of oocytes at 4 weeks of age. Moreover, IAS plus eCG produced more oocytes than eCG alone in mice aged between 3 and 5 weeks or 7 and 30 weeks. The fertilization and birth rates were similar between the two treatments at any age. Moreover, after vitrifying and warming the embryos, the survival and birth rates of two-cell embryos were similar between the two treatments. Subsequently, we examined the optimal ages of female mice (between 24 and 34 days) to obtain a high and stable number of oocytes. In mice aged between 24 and 32 days, IAS plus eCG induced the production of more eggs than eCG alone. Notably, the coadministration of IAS and eCG in mice aged between 25 and 31 days resulted in stable ovulation and high number of oocytes. Using the tip of the optimal female aged between 25 and 31 days old, we demonstrated an efficient production of embryos and offspring between homozygous knockout males and few females aged 26-28 days via in-vitro fertilization and embryo transfer. In summary, the coadministration of IAS and eCG resulted in a higher number of oocytes in juvenile, mature, and aged female mice. This treatment may be useful for the efficient production of homozygous mutant mice from a limited number of female mice.