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.

Mouse Type-I Interferon-Mannosylated Albumin Fusion Protein for the Treatment of Chronic Hepatitis.

Although a lot of effort has been put into creating drugs and combination therapies against chronic hepatitis, no effective treatment has been established. Type-I interferon is a promising therapeutic for chronic hepatitis due to its excellent anti-inflammatory effects through interferon receptors on hepatic macrophages. To develop a type-I IFN equipped with the ability to target hepatic macrophages through the macrophage mannose receptor, the present study designed a mouse type-I interferon-mannosylated albumin fusion protein using site-specific mutagenesis and albumin fusion technology. This fusion protein exhibited the induction of anti-inflammatory molecules, such as IL-10, IL-1Ra, and PD-1, in RAW264.7 cells, or hepatoprotective effects on carbon tetrachloride-induced chronic hepatitis mice. As expected, such biological and hepatoprotective actions were significantly superior to those of human fusion proteins. Furthermore, the repeated administration of mouse fusion protein to carbon tetrachloride-induced chronic hepatitis mice clearly suppressed the area of liver fibrosis and hepatic hydroxyproline contents, not only with a reduction in the levels of inflammatory cytokine (TNF-α) and fibrosis-related genes (TGF-ß, Fibronectin, Snail, and Collagen 1α2), but also with a shift in the hepatic macrophage phenotype from inflammatory to anti-inflammatory. Therefore, type-I interferon-mannosylated albumin fusion protein has the potential as a new therapeutic agent for chronic hepatitis.

Possible Involvement of Protein Binding Inhibition in Changes in Dexmedetomidine Concentration in Extracorporeal Circuits during Midazolam Use.

It was recently reported that the dexmedetomidine concentration within the extracorporeal circuit decreases with co-administration of midazolam. In this study, we investigated whether displacement of dexmedetomidine by midazolam from the binding site of major plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AAG), would increase levels of free dexmedetomidine that could be adsorbed to the circuit. Equilibrium dialysis experiments indicated that dexmedetomidine binds to a single site on both HSA and AAG with four times greater affinity than midazolam. Midazolam-mediated inhibition of the binding of dexmedetomidine to HSA and AAG was also examined. The binding of dexmedetomidine to these proteins decreased in the presence of midazolam. Competitive binding experiments suggested that the inhibition of binding by midazolam was due to competitive displacement at site II of HSA and due to non-competitive displacement at the site of AAG. Thus, our present data indicate that free dexmedetomidine displaced by midazolam from site II of HSA or from AAG is adsorbed onto extracorporeal circuits, resulting in a change in the dexmedetomidine concentration within the circuit.

Acute Kidney Injury Caused by Rhabdomyolysis Is Ameliorated by Serum Albumin-Based Supersulfide Donors through Antioxidative Pathways.

Oxidative stress is responsible for the onset and progression of various kinds of diseases including rhabdomyolysis-induced acute kidney injury (AKI). Antioxidants are, therefore, thought to aid in the recovery of illnesses linked to oxidative stress. Supersulfide species have been shown to have substantial antioxidative activity; however, due to their limited bioavailability, few supersulfide donors have had their actions evaluated in vivo. In this study, human serum albumin (HSA) and N-acetyl-L-cysteine polysulfides (NACSn), which have polysulfides in an oxidized form, were conjugated to create a supersulfide donor. HSA is chosen to be a carrier of NACSn because of its extended blood circulation and high level of biocompatibility. In contrast to a supersulfide donor containing reduced polysulfide in HSA, the NACSn-conjugated HSAs exhibited stronger antioxidant activity than HSA and free NACSn without being uptaken by the cells in vitro. The supersulfide donor reduced the levels of blood urea nitrogen and serum creatinine significantly in a mouse model of rhabdomyolysis-induced AKI. Supersulfide donors significantly reduced the expression of oxidative stress markers in the kidney. These results indicate that the developed supersulfide donor has the therapeutic effect on rhabdomyolysis-induced AKI.

Development of α-Cyclodextrin-Based Orally Disintegrating Tablets for 4-Phenylbutyrate.

Despite major improvements brought about by the introduction of taste-masked formulations of 4-phenylbutyrate (PB), poor compliance remains a significant drawback to treatment for some pediatric and dysphagic patients with urea cycle disorders (UCDs). This study reports on the development of a cyclodextrin (CD)-based orally disintegrating tablet (ODT) formulation for PB as an alternative to existing formulations. This is based on previous reports of the PB taste-masking potential of CDs and the suitability of ODTs for improving compliance in pediatric and dysphagic populations. In preliminary studies, the interactions of PB with α and ßCD in the solid state were characterized using X-ray diffraction, scanning electron microscopy, dissolution, and accelerated stability studies. Based on these studies, lyophilized PB-CD solid systems were formulated into ODTs after wet granulation. Evaluation of the ODTs showed that they had adequate physical characteristics, including hardness and friability and good storage stability. Notably, the developed αCD-based ODT for PB had a disintegration time of 28 s and achieved a slightly acidic and agreeable pH (≈5.5) in solution, which is suitable for effective PB-CD complexation and taste masking. The developed formulation could be helpful as an alternative to existing PB formulations, especially for pediatric and dysphagic UCD patients.

[Characterization of Supersulfide in Serum Albumin and Its Therapeutic Application].

Recent studies have shown that proteins already possess supersulfides during the translation. However, the distribution and the role of supersulfides are not fully understood. In this review, we focus on supersulfides in biological fluids, especially in serum. Various methods for measuring supersulfides have been developed, and these methods have elucidated the presence of supersulfides in serum proteins including serum albumin. Since the levels of supersulfides in serum and serum albumin of patients with chronic kidney disease were lower than those in healthy subjects and recovered by hemodialysis, the levels of supersulfides in serum would be an indicator reflecting oxidative stress. In addition, it has long been known that serum albumin is responsible for sulfur transference. We have applied this phenomenon to the synthesis of sulfur-added albumin (Sn-HSA) by the reaction of serum albumin with sodium polysulfide (Na2Sn). Sn-HSA suppressed the melanin production via scavenging oxidative stress. As described above, studies on the characterization of supersulfides in serum albumin may contribute to the monitoring of redox balance and prevention of oxidative stress-related diseases.

Preparation and in Vitro Characterization of Fatty-Acid Modified Pirarubicin Nanosuspensions Stabilized by Albumin.

Pirarubicin (THP) shows more rapid intracellular uptake, more effective antitumor activity, and less cardiac toxicity, compared to doxorubicin. However, THP is distributed to both tumor and normal tissues indiscriminately. This study aimed to develop a nanosuspension to deliver THP to tumor tissues more efficiently. Fatty-acid-modified THPs (FA-THPs; octanoic acid, dodecanoic acid, palmitic acid-THPs) were synthesized to increase the hydrophobicity of THP. Nanosuspensions of these FA-THPs were then prepared using an antisolvent precipitation technique. Among the FA-THPs, the most efficiently drug-loaded nanosuspension was obtained from palmitic acid-THP (pal-THP) using an aqueous antisolvent containing bovine serum albumin as a stabilizer. The pal-THP nanoparticles in the nanosuspension were confirmed to be of optimal size (100-125 nm) for delivery to tumor tissues using dynamic light scattering and transmission electron microscopy. The pal-THP nanosuspension showed cytotoxicity in colon 26 cells. The nanosuspension was shown to disintegrate in the presence of surfactants such as lecithin, liberating pal-THP, which was converted to free THP in acidic media. It is therefore proposed that pal-THP nanoparticles that reach tumor cells after intravenous administration would exert antitumor effect by liberating pal-THP (i.e., disintegration of nanoparticles by the interaction with cell membrane), followed by the release of free THP in the acidic milieu of tumor cells. These findings indicate that FA-THP nanosuspensions, particularly pal-THP nanosuspension, hold promise as a candidate for cancer treatment. However, further in vivo studies are necessary.

Study of the Structural Chemistry of the Inclusion Complexation of 4-Phenylbutyrate and Related Compounds with Cyclodextrins in Solution: Differences in Inclusion Mode with Cavity Size Dependency.

4-phenylbutyrate (PB) and structurally related compounds hold promise for treating many diseases, including cancers. However, pharmaceutical limitations, such as an unpleasant taste or poor aqueous solubility, impede their evaluation and clinical use. This study explores cyclodextrin (CD) complexation as a strategy to address these limitations. The structural chemistry of the CD complexes of these compounds was analyzed using phase solubility, nuclear magnetic resonance (NMR) spectroscopic techniques, and molecular modeling to inform the choice of CD for such application. The study revealed that PB and its shorter-chain derivative form 1:1 αCD complexes, while the longer-chain derivatives form 1:2 (guest:host) complexes. αCD includes the alkyl chain of the shorter-chain compounds, depositing the phenyl ring around its secondary rim, whereas two αCD molecules sandwich the phenyl ring in a secondary-to-secondary rim orientation for the longer-chain derivatives. ßCD includes each compound to form 1:1 complexes, with their alkyl chains bent to varying degrees within the CD cavity. γCD includes two molecules of each compound to form 2:1 complexes, with both parallel and antiparallel orientations plausible. The study found that αCD is more suitable for overcoming the pharmaceutical drawbacks of PB and its shorter-chain derivative, while ßCD is better for the longer-chain derivatives.

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.

Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticles for the Treatment of Hepatitis.

Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.

Evaluation of the Safety and Gastrointestinal Migration of Guanidinylated Chitosan after Oral Administration to Rats.

Arginine-rich membrane-permeable peptides (APPs) can be delivered to cells by forming complexes with various membrane-impermeable bioactive molecules such as proteins. We recently reported on the preparation of guanidinylated chitosan (GCS) that mimics arginine peptides, using chitosan, a naturally occurring cationic polysaccharide, and confirmed that it enhances protein permeability in an in vitro cell system. However, studies on the in vivo safety of GCS are not available. To address this, we evaluated the in vivo safety of GCS and its translocation into the gastrointestinal tract in rats after a single oral administration of an excessive dose (500 mg/kg) and observed changes in body weight, major organ weights, and organ tissue sections for periods of up to 2 weeks. The results indicated that GCS causes no deleterious effects. The results of an oral administration of rhodamine-labeled chitosan and an evaluation of its migration in the gastrointestinal tract suggested that the disappearance of rhodamine-labeled GCS from the body appeared to be slower than that of the non-dose group and pre-guanidinylated chitosan due to its mucoadhesive properties. In the future, we plan to investigate the use of GCS to improve absorption using Class III and IV drugs, which are poorly water-soluble as well as poorly membrane-permeable.

Structural Basis of the Change in the Interaction Between Mycophenolic Acid and Subdomain IIA of Human Serum Albumin During Renal Failure.

Mycophenolic acid (MP) is an active metabolite of mycophenolate mofetil, a widely used immunosuppressive drug. MP normally exhibits high plasma protein binding (97-99%), but its binding rate is decreased in patients with renal insufficiency. This decreased protein binding is thought to be associated with leukopenia, a side effect of MP. In this study, we characterized the change in protein binding of MP in renal failure patients. Our findings indicate that MP binds strongly to subdomain IIA of human serum albumin. X-ray crystallographic data indicated that the isobenzofuran group of MP forms a stacking interaction with Trp214, and the carboxyl group of MP is located at a position that allows the formation of hydrogen bonds with Tyr150, His242, or Arg257. Due to the specific binding of MP to subdomain IIA, MP is thought to be displaced by uremic toxin (3-carboxy-4-methyl-5-propyl-2-furan-propionic acid) and fatty acids (oleate or myristate) that can bind to subdomain IIA, resulting in the decreased plasma protein binding of MP in renal failure.

Study of the inclusion complexes formed between 4-phenylbutyrate and α-, ß- and γ-cyclodextrin in solution and evaluation on their taste-masking properties.

OBJECTIVES: 4-Phenylbutyrate (PB), which is used in the management of urea cycle disorders, has an unpleasant taste leading to poor patient compliance. Existing PB formulations though helpful, have some limitations in their use. This study reports on attempts to mask this unpleasant taste by complexing PB with cyclodextrins (CDs) to improve patient compliance. METHODS: α, ß and γCD were used as CDs. Phase solubility studies, circular dichroism, 1H-NMR spectroscopy, including ROESY, and molecular modelling were used to investigate and characterize the PB-CD interactions in solution. The taste-masking effect of the CDs was evaluated using in vitro taste sensor measurements. KEY FINDINGS: PB interacts with α, ß and γCD in solution to form 1:1, 1:1 and 1:2 CD: PB inclusion complexes, respectively, with stability constants in the order αCD > ßCD > γCD. Taste evaluation revealed that the CDs significantly mask the taste of PB through the formation of the inclusion complexes. Notably, αCD masked the bitter taste of PB to 30% of the initial taste at a 1:1 molar ratio. CONCLUSION: αCD significantly masks the unpleasant taste of PB in solution and can be used to formulate PB to address the limitations of existing formulations and improve patient compliance and quality of life.

Chlorine Atoms of an Aripiprazole Molecule Control the Geometry and Motion of Aripiprazole and Deschloro-aripiprazole in Subdomain IIIA of Human Serum Albumin.

Aripiprazole (ARP), an antipsychotic drug, binds more strongly to human serum albumin (HSA) than the other ARP derivatives. In addition, the signs for the extrinsic Cotton effects for HSA complexed with ARP or deschloro-ARP are reversed. In this study, we report on a structural-chemical approach using circular dichroism (CD) spectroscopic analysis, X-ray crystallographic analysis, and molecular dynamics simulations. The objective was to examine the relationship between the induced CD spectra and the structural features of the HSA complexes with ARP or deschloro-ARP. The intensity of the induced CD spectra of the HSA complexes with ARP or deschloro-ARP was reduced with increasing temperature. We determined the crystal structure of the HSA complexed with deschloro-ARP in this study and compared it to HSA complexed with ARP that we reported previously. The comparison of these structures revealed that both ARP and deschloro-ARP were bound at the site II pocket in HSA and that the orientation of the molecules was nearly identical. Molecular dynamics simulations indicated that the molecular motions of ARP and deschloro-ARP within the site II pocket were different from one another and the proportion of stacking interaction formations of Tyr411 with the dihydroquinoline rings of ARP and deschloro-ARP was also different. These findings indicate that the induced CD spectra are related to the molecular motions and dynamic interactions of ARP and deschloro-ARP in HSA and may help to understand the molecular recognition and motion that occurs within the binding site for the other HSA ligands more clearly.

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.

Novel nitric oxide donor, nitrated phenylbutyrate, induces cell death of human pancreatic cancer cells and suppresses tumor growth of cancer xenografts.

Pancreatic cancer has a low response rate to chemotherapy due to the low drug transferability caused by the low blood flow around the tumor. In the present study, focusing on nitric oxide (NO) for its vasodilatory and antitumor effects, a novel NO donor, a nitrated form of phenylbutyrate (NPB) was synthesized and the antitumor effect on human pancreatic cancer cells (AsPC1 and BxPC3) and xenografts was examined. Using Annexin V, NPB was confirmed to induce cell death against AsPC1 and BxPC3 in a time­ and concentration­dependent manner. In NPB­exposed cells, DAF­FM DA (a probe to detect intracellular NO) derived fluorescence was observed. Release of nitrite and nitrate from NPB in aqueous solution was very gradual until even 72 h after dissolution. Phenylbutyrate (PB) and hydroxy PB in which the nitro group of NPB was replaced with a hydroxyl group did not have the cell death­inducing effect as observed in NPB. These results suggest that the effect of NPB was dependent on NO release form NPB. Apoptosis inhibitor, Z­VAD FMK, had no effect on the cell death­inducing effect of NPB, and NPB did not show significant activation of caspase­3/7. In addition, NPB significantly decreased cellular ATP levels, suggesting that necrosis is involved in the effect of NPB. NPB also accumulated cells specifically at the S phase of the cell cycle. A single dose of NPB (10 mg/kg) into mice with established BxPC3 xenografts significantly suppressed tumor growth for at least 7 weeks without apparent toxicity. The findings of the present study indicate that NPB has potential as a novel therapeutic agent for NO­based therapy of pancreatic cancer.

Liposomal methemoglobin as a potent antidote for hydrogen sulfide poisoning.

Hydrogen sulfide (H2S) induces acute and lethal toxicity at high concentrations. However, no specific antidotes for H2S poisoning have been approved. Liposomal methemoglobin (metHb@Lipo) was developed as an antidote for cyanide poisoning. As the toxic mechanism of H2S poisoning is the same as that of cyanide poisoning, metHb@Lipo could potentially be used as an antidote for H2S poisoning. In this study, we evaluated the antidotal efficacy of metHb@Lipo against H2S poisoning. Stopped-flow rapid-scan spectrophotometry clearly showed that metHb@Lipo scavenged H2S rapidly. Additionally, metHb@Lipo showed cytoprotective effects against H2S exposure in H9c2 cells by maintaining mitochondrial function. MetHb@Lipo treatment also improved the survival rate after H2S exposure in vivo, with the maintenance of cytochrome c oxidase activity and suppression of metabolic acidosis. Moreover, metHb@Lipo therapy maintained significant antidotal efficacy even after 1-year-storage at 4-37 °C. In conclusion, metHb@Lipo is a candidate antidote for H2S poisoning.

Effect of Fatty Acids and Uremic Toxins on the Binding of Nateglinide, an Insulin Secretagogue, to Site II on Human Serum Albumin.

Nateglinide (NAT) is used to treat diabetes, stimulating pancreatic islet ß-cells with residual insulin secretory capacity to increase insulin secretion. NAT has been reported to bind to human serum albumin (HSA), but the detail is still unclear. In the current study, we investigated the location and the affinity for the binding of NAT to HSA. Quantitative analysis data from the ultrafiltration experiment indicated that NAT binds strongly to a primary site on HSA with a high affinity. The presence of diazepam (DZP) or ibuprofen (IB), the specific site II ligands of HSA, decreased the binding constants of NAT respectively, without the significant changes in the number of binding sites. Whereas warfarin (WF), a site I specific ligand, did not affect the binding of NAT. Fluorescent replacement experiment showed that NAT replaced dansylsarcosine (DNSS), a site II probe of HSA, but not WF. An increasing level of myristate and uremic toxins, indoxyl sulphate (IS), indoxyl acetate (IA) and p-cresyl sulphate (PCS), during renal disease significantly increased the concentration of unbound NAT. These findings suggest that NAT specifically binds to site II of HSA and the binding capacity and pharmacokinetics of NAT change in renal diseases.

A bioinspired carbon monoxide delivery system prevents acute kidney injury and the progression to chronic kidney disease.

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-ß1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.