Contribution of the α1-Acid Glycoprotein in Drug Pharmacokinetics: The Usefulness of α1-Acid Glycoprotein-Knockout Mice.

α1-Acid glycoprotein (AGP) is a primary binding protein for many basic drugs in plasma. The number of drugs that bind to AGP, such as molecular target anticancer drugs, has been continuously increasing. Since the plasma level of AGP fluctuates under various pathological conditions such as inflammation, it is important to evaluate the contribution of AGP to drug pharmacokinetics. Here, we generated conventional AGP-knockout (AGP-KO) mice and used them to evaluate the contribution of AGP. The pharmacokinetics of drugs that bind to two AGP variants (F1*S or A variants) or albumin were evaluated. Imatinib (a F1*S-binding drug) and disopyramide (an A-binding drug) or ibuprofen (an albumin-binding drug) were administered to wild-type (WT) and AGP-KO. The plasma level of imatinib and disopyramide decreased rapidly in AGP-KO as compared to WT. In AGP-KO, AUC and t1/2 were decreased, then CLtot was increased. Compared with disopyramide, imatinib pharmacokinetics showed more marked changes in AGP-KO as compared to WT. The results seemed to be due to the difference in plasma level of each AGP variant (F1*S:A = 2-3:1). No differences were observed in ibuprofen pharmacokinetics between the WT and AGP-KO mice. In vitro experiments using plasma from WT and AGP-KO showed that unbound fractions of imatinib and disopyramide were higher in AGP-KO. These results suggest that the rapid elimination of imatinib and disopyramide in AGP-KO could be due to decreased protein binding to AGP. Taken together, the AGP-KO mouse could be a potential animal model for evaluating the contribution of AGP to the pharmacokinetics of various drugs.

Carbon monoxide-loaded cell therapy as an exercise mimetic for sarcopenia treatment.

Sarcopenia is characterized by loss of muscle strength and muscle mass with aging. The growing number of sarcopenia patients as a result of the aging population has no viable treatment. Exercise maintains muscle strength and mass by increasing peroxisome growth factor activating receptor γ-conjugating factor-1α (PGC-1α) and Akt signaling in skeletal muscle. The present study focused on the carbon monoxide (CO), endogenous activator of PGC-1α and Akt, and investigated the therapeutic potential of CO-loaded red blood cells (CO-RBCs), which is bioinspired from in vivo CO delivery system, as an exercise mimetic for the treatment of sarcopenia. Treatment of C2C12 myoblasts with the CO-donor increased the protein levels of PGC-1α which enhanced mitochondrial biogenesis and energy production. The CO-donor treatment also activated Akt, indicating that CO promotes muscle synthesis. CO levels were significantly elevated in the skeletal muscle of normal mice after intravenous administration of CO-RBCs. Furthermore, CO-RBCs restored the mRNA expression levels of PGC-1α in the skeletal muscle of two experimental sarcopenia mouse models, denervated (Den) and hindlimb unloading (HU) models. CO-RBCs also restored muscle mass in Den mice by activating Akt signaling and suppressing the muscle atrophy factors myostatin and atrogin-1, and oxidative stress. Treadmill tests further showed that the reduced running distance in HU mice was significantly restored by CO-RBC administration. These findings suggest that CO-RBCs have potential as an exercise mimetic for sarcopenia treatment.

Carbon Monoxide Alleviates Post-ischemia-reperfusion Skeletal Muscle Injury and Systemic Inflammation.

Restoration of blood flow in skeletal muscle after a prolonged period of ischemia induces muscular ischemia-reperfusion injury, leading to local injury/dysfunction in muscles followed by systemic inflammatory responses. However, preventive/curative agents for skeletal muscle ischemia injury are unavailable in clinics to date. Increasing evidence has validated that carbon monoxide (CO) prevents the progression of ischemia-reperfusion injury in various organs owing to its versatile bioactivity. Previously, we developed a bioinspired CO donor, CO-bound red blood cells (CO-RBC), which mimics the dynamics of RBC-associated CO in the body. In the present study, we have tested the therapeutic potential of CO-RBC in muscular injury/dysfunction and secondary systemic inflammation induced by skeletal muscle ischemia-reperfusion. The results indicate that CO-RBC rather than RBC alone suppressed elevation of plasma creatine phosphokinase, a marker of muscular injury, in rats subjected to both hind limbs ischemia-reperfusion. In addition, the results of the treadmill walking test revealed a significantly decreased muscular motor function in RBC-treated rats subjected to both hind limbs ischemia-reperfusion than that in healthy rats, however, CO-RBC treatment facilitated sustained muscular motor functions after hind limbs ischemia-reperfusion. Furthermore, CO-RBC rather than RBC suppressed the production of tumour necrosis factor (TNF)-α and interleukin (IL)-6, which were upregulated by muscular ischemia-reperfusion. Interestingly, CO-RBC treatment induced higher levels of IL-10 compared to saline or RBC treatments. Based on these findings, we suggest that CO-RBC exhibits a suppressive effect against skeletal muscle injury/dysfunction and systemic inflammatory responses after skeletal muscle ischemia-reperfusion.

[Albumin-based Drug Delivery System Targeting Mannose Receptors and Its Application to Medical Treatments].

The onset and progression of liver diseases and cancer have shown to be affected by over-active macrophages and fibroblasts. Therefore, developing methods to suppress the activation of these cells has become an urgent task. Prior to this study, a mannosylated-albumin (Man-HSA) that targets mannose receptors expressed in hepatic macrophages (Kupffer cells) or fibroblasts was created. Here, we report on the development of medical treatments based on Man-HSA. To target the reactive oxygen species or inflammation derived from Kupffer cells, we developed a nano-antioxidant, i.e., polythiolated (SH)-Man-HSA, by introducing thiol groups into Man-HSA, or a nano-anti-inflammatory drug, i.e., Man-HSA-IFNα2b, by fusing Man-HSA and IFNα2b. SH-Man-HSA or Man-HSA-IFNα2b attenuated Kupffer cell-derived oxidative stress or inflammation, respectively, resulting in the suppression of liver damage and overall improvement of the survival rate in mice with acute and chronic liver injuries. Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF), both of which are present in the stroma of intractable cancers, also express mannose receptors. Thus, mono-polyethylene glycol modified Man-HSA (monoPEG-Man-HSA) was synthesized as a novel drug delivery carrier targeting TAM/CAF. A complex of monoPEG-Man-HSA with paclitaxel suppressed tumor growth by decreasing the number of TAM/CAF and the stroma area. For the present study, we focused on the mannose receptors expressed in macrophages and fibroblasts, and developed drug delivery carriers that target these cells. Considering the excellent drug-carrying capacity and high biocompatibility of HSA, it is expected that this research will pave the way for innovative pharmacotherapy to treat unmet medical needs, i.e., intractable liver diseases and cancer.

Carbon Monoxide-Loaded Red Blood Cell Prevents the Onset of Cisplatin-Induced Acute Kidney Injury.

Cisplatin-induced acute kidney injury (AKI) is an important factor that limits the clinical use of this drug for the treatment of malignancies. Oxidative stress and inflammation are considered to be the main causes of not only cisplatin-induced death of cancer cells but also cisplatin-induced AKI. Therefore, developing agents that exert antioxidant and anti-inflammatory effects without weakening the anti-tumor effects of cisplatin is highly desirable. Carbon monoxide (CO) has recently attracted interest due to its antioxidant, anti-inflammatory, and anti-tumor properties. Herein, we report that CO-loaded red blood cell (CO-RBC) exerts renoprotective effects on cisplatin-induced AKI. Cisplatin treatment was found to reduce cell viability in proximal tubular cells via oxidative stress and inflammation. Cisplatin-induced cytotoxicity, however, was suppressed by the CO-RBC treatment. The intraperitoneal administration of cisplatin caused an elevation in the blood urea nitrogen and serum creatinine levels. The administration of CO-RBC significantly suppressed these elevations. Furthermore, the administration of CO-RBC also reduced the deterioration of renal histology and tubular cell injury through its antioxidant and anti-inflammatory effects in cisplatin-induced AKI mice. Thus, our data suggest that CO-RBC has the potential to substantially prevent the onset of cisplatin-induced AKI, which, in turn, may improve the usefulness of cisplatin-based chemotherapy.

Cell-penetrating albumin enhances the sublingual delivery of antigens through macropinocytosis.

Innovations in oral immunotherapy have greatly advanced the therapeutic control of allergies. However, these therapeutic effects suffer from the fact that the amount of antigen delivered to antigen-presenting cells is limited given the formulations that are currently available. We recently designed a cell-penetrating albumin and found that this modified albumin enters cells via the induction of macropinocytosis. Herein, we report on a novel system for delivering antigens based on cell-penetrating albumin-inducible macropinocytosis that allows larger amounts of antigens to be delivered to antigen-presenting cells. A treatment with cell-penetrating albumin significantly increased the permeability of ovalbumin (45 kDa) or dextran (2000 kDa) on monolayers derived from human oral squamous carcinoma cells. Flow cytometric analyses showed that the cell-penetrating albumin treatment resulted in a significant elevation in the amount of dextran that was delivered to two types of antigen-presenting cells. Finally, mice that had been sensitized by Japanese cedar pollen extract (JCPE) and cell-penetrating albumin showed a decline in the frequency of nose-rubbing against a subsequent intranasal administration of JCPE. These findings suggest that the sublingual administration of cell-penetrating albumin efficiently delivers antigens to antigen-presenting cells via the induction of macropinocytosis, resulting in an enhancement in the therapeutic effect of sublingual immunotherapy.

Indoxyl Sulfate Contributes to mTORC1-Induced Renal Fibrosis via The OAT/NADPH Oxidase/ROS Pathway.

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial-mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.

Involvement of the Parathyroid Hormone-Related Protein on Changes in the CYP3A Expression in Cancer Cachexia.

Parathyroid hormone-related protein (PTHrP), which is secreted from a tumor, contributes to the progression of cachexia, a condition that is observed in half of all cancer patients. Although drug clearance was reported to decrease in patients with cancer cachexia, the details have not been clarified. The present study reports on an investigation of whether PTHrP is involved in the alternation of drug metabolism in cases of cancer cachexia. Cancer cachexia model rats with elevated serum PTHrP levels showed a significant decrease in hepatic and intestinal CYP3A2 protein expression. When midazolam, a CYP3A substrate drug, was administered intravenously or orally to the cancer cachexia rats, its area under the curve (AUC) was increased by about 2 and 5 times, as compared to the control group. Accordingly, the bioavailability of midazolam was increased by about 3 times, thus enhancing its pharmacological effect. In vitro experiments using HepG2 cells and Caco-2 cells showed that the addition of serum from cancer cachexia rats or active PTHrP (1-34) to each cell resulted in a significant decrease in the expression of CYP3A4 mRNA. Treatment with a cell-permeable cAMP analog also resulted in a decreased CYP3A4 expression. Pretreatment with protein kinase A (PKA), protein kinase C (PKC), and nuclear factor-kappa B (NF-κB) inhibitors recovered the decrease in CYP3A4 expression that was induced by PTHrP (1-34). These results suggest that PTHrP suppresses CYP3A expression via the cAMP/PKA/PKC/NF-κB pathway. Therefore, it is likely that PTHrP would be involved in the changes in drug metabolism observed in cancer cachexia.

Advanced oxidation protein products contribute to chronic kidney disease-induced muscle atrophy by inducing oxidative stress via CD36/NADPH oxidase pathway.

BACKGROUND: Sarcopenia with chronic kidney disease (CKD) progression is associated with life prognosis. Oxidative stress has attracted interest as a trigger for causing CKD-related muscular atrophy. Advanced oxidation protein products (AOPPs), a uraemic toxin, are known to increase oxidative stress. However, the role of AOPPs on CKD-induced muscle atrophy remains unclear. METHODS: In a retrospective case-control clinical study, we evaluated the relationship between serum AOPPs levels and muscle strength in haemodialysis patients with sarcopenia (n = 26, mean age ± SEM: 78.5 ± 1.4 years for male patients; n = 22, mean age ± SEM: 79.1 ± 1.5 for female patients), pre-sarcopenia (n = 12, mean age ± SEM: 73.8 ± 2.0 years for male patients; n = 4, mean age ± SEM: 74.3 ± 4.1 for female patients) or without sarcopenia (n = 12, mean age ± SEM: 71.3 ± 1.6 years for male patients; n = 7, mean age ± SEM: 77.7 ± 1.6 for female ). The molecular mechanism responsible for the AOPPs-induced muscle atrophy was investigated by using 5/6-nephrectomized CKD mice, AOPPs-overloaded mice, and C2C12 mouse myoblast cells. RESULTS: The haemodialysis patients with sarcopenia showed higher serum AOPPs levels as compared with the patients without sarcopenia. The serum AOPPs levels showed a negative correlation with grip strength (P < 0.01 for male patients, P < 0.01 for female patients) and skeletal muscle index (P < 0.01 for male patients). Serum AOPPs levels showed a positive correlation with cysteinylated albumin (Cys-albumin), a marker of oxidative stress (r2  = 0.398, P < 0.01). In the gastrocnemius of CKD mice, muscle AOPPs levels were also increased, and it showed a positive correlation with atrogin-1 (r2  = 0.538, P < 0.01) and myostatin expression (r2  = 0.421, P < 0.05), but a negative correlation with PGC-1α expression (r2  = 0.405, P < 0.05). Using C2C12 cells, AOPPs increased atrogin-1 and myostatin expression through the production of reactive oxygen species via CD36/NADPH oxidase pathway, and decreased myotube formation. AOPPs also induced mitochondrial dysfunction. In the AOPPs-overloaded mice showed that decreasing running time and hanging time accompanied by increasing AOPPs levels and decreasing cross-sectional area in gastrocnemius. CONCLUSIONS: Advanced oxidation protein products contribute to CKD-induced sarcopenia, suggesting that AOPPs or its downstream signalling pathway could be a therapeutic target for the treatment of CKD-induced sarcopenia. Serum AOPPs or Cys-albumin levels could be a new diagnostic marker for sarcopenia in CKD.

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.

Systemic and sustained thioredoxin analogue prevents acute kidney injury and its-associated distant organ damage in renal ischemia reperfusion injury mice.

The mortality of patients with acute kidney injury (AKI) remains high due to AKI associated-lung injury. An effective strategy for preventing both AKI and AKI-associated lung injury is urgently needed. Thioredoxin-1 (Trx) is a redox-active protein that possesses anti-oxidative, anti-apoptotic and anti-inflammatory properties including modulation of macrophage migration inhibitory factor (MIF), but its short half-life limits its clinical application. Therefore, we examined the preventive effect of a long-acting Trx, which is a fusion protein of albumin and Trx (HSA-Trx), against AKI and AKI-associated lung injury. Recombinant HSA-Trx was expressed using a Pichia expression system. AKI-induced lung injury mice were generated by bilateral renal ischemia reperfusion injury (IRI). HSA-Trx administration attenuated renal IRI and its-associated lung injury. Both renal and pulmonary oxidative stress were suppressed by HSA-Trx. Moreover, HSA-Trx inhibited elevations of plasma IL-6 and TNF-α level, and suppressed IL-6-CXCL1/2-mediated neutrophil infiltration into lung and TNF-α-mediated pulmonary apoptosis. Additionally, HSA-Trx suppressed renal IRI-induced MIF expression in kidney and lung. Administration of HSA-Trx resulted in a significant increase in the survival rate of renal IRI mice. Collectively, HSA-Trx could have therapeutic utility in preventing both AKI and AKI-associated lung injury as a consequence of its systemic and sustained multiple biological action.

The Late-Stage Protective Effect of Mito-TEMPO against Acetaminophen-Induced Hepatotoxicity in Mouse and Three-Dimensional Cell Culture Models.

An overdose of acetaminophen (APAP), the most common cause of acute liver injury, induces oxidative stress that subsequently causes mitochondrial impairment and hepatic necroptosis. N-acetyl-L-cysteine (NAC), the only recognized drug against APAP hepatotoxicity, is less effective the later it is administered. This study evaluated the protective effect of mitochondria-specific Mito-TEMPO (Mito-T) on APAP-induced acute liver injury in C57BL/6J male mice, and a three dimensional (3D)-cell culture model containing the human hepatoblastoma cell line HepG2. The administration of Mito-T (20 mg/kg, i.p.) 1 h after APAP (400 mg/kg, i.p.) injection markedly attenuated the APAP-induced elevated serum transaminase activity and hepatic necrosis. However, Mito-T treatment did not affect key factors in the development of APAP liver injury including the activation of c-jun N-terminal kinases (JNK), and expression of the transcription factor C/EBP homologous protein (CHOP) in the liver. However, Mito-T significantly reduced the APAP-induced increase in the hepatic oxidative stress marker, nitrotyrosine, and DNA fragmentation. Mito-T markedly attenuated cytotoxicity induced by APAP in the HepG2 3D-cell culture model. Moreover, liver regeneration after APAP hepatotoxicity was not affected by Mito-T, demonstrated by no changes in proliferating cell nuclear antigen formation. Therefore, Mito-T was hepatoprotective at the late-stage of APAP overdose in mice.

Indoxyl Sulfate Contributes to Adipose Tissue Inflammation through the Activation of NADPH Oxidase.

Adipose tissue inflammation appears to be a risk factor for the progression of chronic kidney disease (CKD), but the effect of CKD on adipose tissue inflammation is poorly understood. The purpose of this study was to clarify the involvement of uremic toxins (indoxyl sulfate (IS), 3-indoleacetic acid, p-cresyl sulfate and kynurenic acid) on CKD-induced adipose tissue inflammation. IS induces monocyte chemoattractant protein-1 (MCP-1) expression and reactive oxygen species (ROS) production in the differentiated 3T3L-1 adipocyte. An organic anion transporter (OAT) inhibitor, an NADPH oxidase inhibitor or an antioxidant suppresses the IS-induced MCP-1 expression and ROS production, suggesting the OAT/NADPH oxidase/ROS pathway is involved in the action of IS. Co-culturing 3T3L-1 adipocytes and mouse macrophage cells showed incubating adipocytes with IS increased macrophage infiltration. An IS-overload in healthy mice increased IS levels, oxidative stress and MCP-1 expression in epididymal adipose tissue compared to unloaded mice. Using 5/6-nephrectomized mice, the administration of AST-120 suppressed oxidative stress and the expression of MCP-1, F4/80 and TNF-α in epididymal adipose tissue. These collective data suggest IS could be a therapeutic target for the CKD-related inflammatory response in adipose tissue, and that AST-120 could be useful for the treatment of IS-induced adipose tissue inflammation.

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.

Development of a long acting FGF21 analogue-albumin fusion protein and its anti-diabetic effects.

Fibroblast growth factor 21 (FGF21) is a hormone-like protein that improves blood glucose and lipid metabolism. However, its short half-life and instability are bottlenecks to its clinical applications. In this study, to extend its pharmacological action, we created a stabilized mutant FGF21 (mFGF21:ΔHPIP, P171G, A180E, L118C-A134C, S167A) and then genetically fused it with human albumin (HSA-mFGF21) via a polypeptide linker. Physicochemical analyses suggested that HSA-mFGF21 was formed from both intact HSA and mFGF21. Pharmacokinetic findings indicated the half-life of HSA-mFGF21 was 20 times longer than that of FGF21. In addition, HSA-mFGF21 was persistently distributed in adipose tissue as a target tissue. The in vivo hypoglycemic activity of HSA-mFGF21 using streptozotocin (STZ)-induced type I diabetes model mice, in which insulin secretion was suppressed, showed that a single intravenous administration of HSA-mFGF21 rapidly alleviated hyperglycemia. At that time, HSA-mFGF21 increased GLUT1 mRNA expression in adipose tissue without having any effect on insulin secretion. A twice weekly administration of HSA-mFGF21 continuously suppressed blood glucose levels and ameliorated the abnormalities of adipose tissue induced by STZ treatment. Interestingly, HSA-mFGF21 showed no hypoglycemic effects in healthy mice. Together, HSA-mFGF21 could be a novel biotherapeutic for the treatment of metabolic disorders including diabetes mellitus.

Repeated Administration of Kupffer Cells-Targeting Nanoantioxidant Ameliorates Liver Fibrosis in an Experimental Mouse Model.

Kupffer cells are a major producer of reactive oxygen species and have been implicated in the development of liver fibrosis during chronic hepatitis in non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH). We recently reported on the development of a polythiolated and mannosylated human serum albumin (SH-Man-HSA) that functions as a Kupffer cell-targeting nanoantioxidant. In this material, the albumin is mannosylated, which permits it to be taken up by mannose receptor C type 1 expressed on Kupffer cells, and is also polythiolated to have antioxidant activity. To clarify the anti-fibrotic property of this nanoantioxidant, we repeatedly administered SH-Man-HSA to a liver fibrosis mouse model that was induced by the repeated treatment of the concanavalin-A, which mimics the liver fibrosis observed in NASH and ASH. SH-Man-HSA dramatically improved the survival rate and suppressed liver fibrosis in the experimental model. In addition, SH-Man-HSA suppressed hepatic oxidative stress levels, thereby decreasing the numbers of apoptotic cells. In contrast, N-acetylcysteine, which contains the same thiol content as the SH-Man-HSA, failed to show a substantial therapeutic effect in these mice. The expression levels of inflammatory genes including epidermal growth factor module-containing mucin-like receptor (Emr-1/F4/80), Toll-like receptor-4 (TLR-4), high mobility group box-1 (HMGB-1), CC chemokine ligand-5 (CCL-5), tumor necrosis factor-α (TNF-α), CCL-2, interleukin-6 (IL-6), and IL-1ß, as well as fibrotic (α-smooth muscle actin (α-SMA), transforming growth factor-ß (TGF-ß), and Snail) and extracellular matrix genes (collagen, type Iα2 (Col1α2), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1)), showed some decreasing trends by the SH-Man-HSA administration. These findings suggest that the repeated administration of the Kupffer cell-targeting nanoantioxidant, SH-Man-HSA, ameliorates liver fibrosis in mice by suppressing the level of oxidative stress and a portion of the inflammation, and has a potential therapeutic effect against NASH and ASH.

α1-Acid Glycoprotein Attenuates Adriamycin-Induced Nephropathy via CD163 Expressing Macrophage Induction.

Background: Recent clinical studies have shown that proteinuria is a critical factor in the progression of CKD and onset of cardiovascular disease. Inflammation and infiltration of macrophages into renal tissue are implicated as causes of proteinuria. α1-Acid glycoprotein (AGP), an acute-phase plasma protein, is leaked into the urine in patients with proteinuria. However, the relationship between urinary leakage of AGP, renal inflammation, and proteinuria remains unclear. Methods: Human AGP (hAGP) was exogenously administrated for 5 consecutive days to adriamycin-induced nephropathy model mice. Results: Adriamycin treatment increased urinary AGP, accompanied by decreased plasma AGP in mice. Exogenous hAGP administration to adriamycin-treated mice suppressed proteinuria, renal histologic injury, and inflammation. hAGP administration increased renal CD163 expression, a marker of anti-inflammatory macrophages. Similar changes were observed in PMA-differentiated THP-1 cells treated with hAGP. Even in the presence of LPS, hAGP treatment increased CD163/IL-10 expression in differentiated THP-1 cells. Conclusions: AGP alleviates proteinuria and renal injury in mice with proteinuric kidney disease via induction of CD163-expressing macrophages with anti-inflammatory function. The results demonstrate that endogenous AGP could work to protect against glomerular disease. Thus, AGP supplementation could be a possible new therapeutic intervention for patients with glomerular disease.

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.