Albumin-fused long-acting FGF21 analogue for the treatment of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) currently affects about 25% of the world's population, and the numbers continue to rise as the number of obese patients increases. However, there are currently no approved treatments for NAFLD. This study reports on the evaluation of the therapeutic effect of a recombinant human serum albumin-fibroblast growth factor 21 analogue fusion protein (HSA-FGF21) on the pathology of NAFLD that was induced by using two high-fat diets (HFD), HFD-60 and STHD-01. The HFD-60-induced NAFLD model mice with obesity, insulin resistance, dyslipidemia and hepatic lipid accumulation were treated with HSA-FGF21 three times per week for 4 weeks starting at 12 weeks after the HFD-60 feeding. The administration of HSA-FGF21 suppressed the increased body weight, improved hyperglycemia, hyperinsulinemia, and showed a decreased accumulation of plasma lipid and hepatic lipid levels. The elevation of C16:0, C18:0 and C18:1 fatty acids in the liver that were observed in the HFD-60 group was recovered by the HSA-FGF21 administration. The increased expression levels of the hepatic fatty acid uptake receptor (CD36) and fatty acid synthase (SREBP-1c, FAS, SCD-1, Elovl6) were also suppressed. In adipose tissue, HSA-FGF21 caused an improved adipocyte hypertrophy, a decrease in the levels of inflammatory cytokines and induced the expression of adiponectin and thermogenic factors. The administration of HSA-FGF21 to the STHD-01-induced NAFLD model mice resulted in suppressed plasma ALT and AST levels, oxidative stress, inflammatory cell infiltration and fibrosis. Together, HSA-FGF21 has some potential for use as a therapeutic agent for the treatment of NAFLD.

Engineering of a Long-Acting Bone Morphogenetic Protein-7 by Fusion with Albumin for the Treatment of Renal Injury.

The bone morphogenetic protein-7 (BMP7) is capable of inhibiting TGF-ß/Smad3 signaling, which subsequently results in protecting the kidney from renal fibrosis, but its lower blood retention and osteogenic activity are bottlenecks for its clinical application. We report herein on the fusion of carbohydrate-deficient human BMP7 and human serum albumin (HSA-BMP7) using albumin fusion technology and site-directed mutagenesis. When using mouse myoblast cells, no osteogenesis was observed in the glycosylated BMP7 derived from Chinese hamster ovary cells in the case of unglycosylated BMP7 derived from Escherichia coli and HSA-BMP7. On the contrary, the specific activity for the Smad1/5/8 phosphorylation of HSA-BMP7 was about 25~50-times lower than that for the glycosylated BMP7, but the phosphorylation activity of the HSA-BMP7 was retained. A pharmacokinetic profile showed that the plasma half-life of HSA-BMP7 was similar to that for HSA and was nearly 10 times longer than that of BMP7. In unilateral ureteral obstruction mice, weekly dosing of HSA-BMP7 significantly attenuated renal fibrosis, but the individual components, i.e., HSA or BMP7, did not. HSA-BMP7 also attenuated a cisplatin-induced acute kidney dysfunction model. The findings reported herein indicate that HSA-BMP7 has the potential for use in clinical applications for the treatment of renal injuries.

Recombinant Long-Acting Thioredoxin Ameliorates AKI to CKD Transition via Modulating Renal Oxidative Stress and Inflammation.

An effective strategy is highly desirable for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Thioredoxin-1 (Trx), a redox-active protein that has anti-oxidative and anti-inflammatory properties, would be a candidate for this but its short half-life limits its clinical application. In this study, we examined the renoprotective effect of long-acting Trx that is comprised of human albumin and Trx (HSA-Trx) against AKI to CKD transition. AKI to CKD mice were created by renal ischemia-reperfusion (IR). From day 1 to day 14 after renal IR, the recovery of renal function was accelerated by HSA-Trx administration. On day 14, HSA-Trx reduced renal fibrosis compared with PBS treatment. At the early phase of fibrogenesis (day 7), HSA-Trx treatment suppressed renal oxidative stress, pro-inflammatory cytokine production and macrophage infiltration, thus ameliorating tubular injury and fibrosis. In addition, HSA-Trx treatment inhibited G2/M cell cycle arrest and apoptosis in renal tubular cells. While renal Trx protein levels were decreased after renal IR, the levels were recovered by HSA-Trx treatment. Together, HSA-Trx has potential for use in the treatment of AKI to CKD transition via its effects of modulating oxidative stress and inflammation.

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.

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.

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.

A downstream molecule of 1,25-dihydroxyvitamin D3, alpha-1-acid glycoprotein, protects against mouse model of renal fibrosis.

Renal fibrosis, the characteristic feature of progressive chronic kidney disease, is associated with unremitting renal inflammation. Although it is reported that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, elicits an anti-renal fibrotic effect, its molecular mechanism is still unknown. In this study, renal fibrosis and inflammation observed in the kidney of unilateral ureteral obstruction (UUO) mice were reduced by the treatment of 1,25(OH)2D3. The plasma protein level of alpha-1-acid glycoprotein (AGP), a downstream molecule of 1,25(OH)2D3, was increased following administration of 1,25(OH)2D3. Additionally, increased mRNA expression of ORM1, an AGP gene, was observed in HepG2 cells and THP-1-derived macrophages that treated with 1,25(OH)2D3. To investigate the involvement of AGP, exogenous AGP was administered to UUO mice, resulting in attenuated renal fibrosis and inflammation. We also found the mRNA expression of CD163, a monocyte/macrophage marker with anti-inflammatory potential, was increased in THP-1-derived macrophages under stimulus from 1,25(OH)2D3 or AGP. Moreover, AGP prevented lipopolysaccharide-induced macrophage activation. Thus, AGP could be a key molecule in the protective effect of 1,25(OH)2D3 against renal fibrosis. Taken together, AGP may replace vitamin D to function as an important immune regulator, offering a novel therapeutic strategy for renal inflammation and fibrosis.