Yoko Mizuno-Horikawa

HEPATOCYTE GROWTH FACTOR PREVENTS RENAL FIBROSIS AND DYSFUNCTION IN A MOUSE MODEL OF CHRONIC RENAL DISEASE

Chronic renal disease (CRD) is generally thought to be incurable, except through renal transplantation, and the number of patients with CRD is on the increase. Glomerulosclerosis and tubulointerstitial fibrosis represent the morphological equivalent of end-stage CRD. In this study, we demonstrated the preventive effect of hepatocyte growth factor (HGF) on the progression of renal dysfunction and fibrosis, using a spontaneous mouse model for CRD (ICGN strain). The mice progressively developed glomerular sclerotic injury, tubular atrophy, and renal dysfunction until they were 17 wk of age. When recombinant HGF was injected into these mice during a 4-wk-period (from weeks 14-17 after birth), DNA synthesis of tubular epithelial cells was found to be 4.4-fold higher than in mice without HGF injection, thereby suggesting tubular parenchymal expansion promoted by HGF. Notably, HGF suppressed the expression of transforming growth factor-beta and of platelet-derived growth factor as well as myofibroblast formation in the affected kidney. Consequently, the onset of tubulointerstitial fibrosis was almost completely inhibited by HGF, while HGF attenuated the progression of glomerulosclerosis, both leading to preventing manifestation of renal dysfunction. From our results, supplement therapy with HGF may be taken into consideration as a novel option for prevention and treatment of CRD.

Carbohydrate Binding Specificity of a Fucose-specific Lectin from Aspergillus oryzae A NOVEL PROBE FOR CORE FUCOSE

The α1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the α1,6-fucosylated chain among α1,2-, α1,3-, α1,4-, and α1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with α1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the α1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from α1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated α-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on α1-antitrypsin, α1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used α1–6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an α1–6 linkage. Interestingly, the levels of α1-antitrypsin and α1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.

Nephrotic mice (ICGN strain) : A model of diffuse mesangial sclerosis in infantile nephrotic syndrome

The ICGN mouse strain is a unique model for naturally occurring nephrotic syndrome. In the present study, we examined the onset of the clinical manifestation of nephrotic syndrome and determined the sequence of intraglomerular events associated with progression of nephrotic conditions. Laboratory analysis revealed that homozygous (nep/nep) mice showed urinary albumin excretion during the suckling stage, rapidly leading to hypoalbuminemia accompanied by body growth failure. Renal pathology demonstrated that an initial intraglomerular event in the nephrotic mice was observed 3 weeks after birth in the form of mesangiolytic lesions, characterized by microaneurysm, platelet accumulation and capillary ballooning. In 6-week-old homozygous mice, mesangial sclerosis, characterized by mesangial expansion and glomerular hypertrophy, was observed in a diffuse fashion. Immunohistochemistry revealed that the glomerular cells in the 3-week-old homozygous suckling mice were positive for α-smooth muscle actin, suggesting a phenotypic change in the mesangial cells. Mesangial expansion, confirmed by the over-deposition of type I collagen, was evident until 6 weeks after weaning, while it was of interest that fibrogenic cytokines such as platelet-derived growth factor and transforming growth factor-β were not detected in the sclerotic glomeruli throughout the observations. Furthermore, the nephrotic features were shown to be resistant to steroid therapy with a high dose of prednisolone. Our results suggest that diffuse mesangial sclerosis, a hereditary glomerular disease, may be genetically generated through early myofibroblast formation, which occurs and develops probably independently of up-regulation of these fibrogenic cytokines. In conclusion, the homozygous nephrotic mouse (ICGN strain) is believed to be a good model for investigating not only nephrotic conditions but also cellular and molecular pathogenesis of diffuse mesangial sclerosis in steroid-resistant infantile nephrotic syndrome.

Stromal cell-derived factor-1 (SDF1)-dependent recruitment of bone marrow-derived renal endothelium-like cells in a mouse model of acute kidney injury

Ischemic acute kidney injury (AKI) is the most key pathological event for accelerating progression to chronic kidney disease through vascular endothelial injury or dysfunction. Thus, it is critical to elucidate the molecular mechanism of endothelial protection and regeneration. Emerging evidence indicates that bone marrow-derived cells (BMCs) contribute to tissue reconstitution in several types of organs post-injury, but little is known whether and how BMCs contribute to renal endothelial reconstitution, especially in an early-stage of AKI. Using a mouse model of ischemic AKI, we provide evidence that incorporation of BMCs in vascular components (such as endothelial and smooth muscle cells) becomes evident within four days after renal ischemia and reperfusion, associated with an increase in stromal cell-derived factor-1 (SDF1) in endothelium and that in CXCR4/SDF1-receptor in BMCs. Notably, anti-CXCR4 antibody decreased the numbers of infiltrated BMCs and BMC-derived endothelium-like cells, but not of BMC-derived smooth muscle cell-like cells. These results suggest that reconstitution of renal endothelium post-ischemia partially depends on a paracrine loop of SDF1-CXCR4 between resident endothelium and BMCs. Such a chemokine ligand-receptor system may be attributable for selecting a cellular lineage (s), required for renal vascular protection, repair and homeostasis, even in an earlier phase of AKI.

Protein restriction ameliorates renal tubulointerstitial nephritis and reduces renal transforming growth factor-beta expression in unilateral ureteral obstruction.

In contrast to the substantial evidence for attenuation of the glomerular lesions by a low-protein (LP) diet, it remains to be determined whether and how such a diet lessens the progression of tubulointerstitial lesions, which show the strongest correlation with renal function. Chronic unilateral ureteral obstruction (UUO) results in interstitial fibrosis of the affected kidney. We investigated the therapeutic effects of an LP diet on the progression of interstitial fibrosis in UUO mice. Sixty ICR mice underwent UUO or sham operation; half of these mice were fed a normal-protein (NP) and the other half a LP diet. They were sacrificed at 3, 7 and 14 days postoperatively. The degree of tubular lesion, the distribution of transforming growth factor-β (TGF-β), α-smooth muscle actin and fibronectin and the activated TGF-β1 level were determined. The LP diet significantly reduced the progression of tubular injury, depositions of fibronectin, tubulointerstitial myofibroblast formation, the interstitial expression of TGF-β-positive cells (at 14 days; NP = 6.91 ± 3.35 vs. LP = 1.67 ± 0.41; p < 0.005), and renal active TGF-β1 concentration (at 14 days; NP = 5.72 ± 2.03 vs. LP = 2.96 ± 0.72; p < 0.01). We conclude that protein restriction may aid the attenuation of progression of tubulointerstitial fibrosis through the reduction in tubulointerstitial expression of TGF-β.

Transforming Growth Factor-β(beta) Targeting Strategies for Prevention of Coronary Arterial Restenosis Post-angioplasty

Restenosis often occurs after placement of bare metal stents in arteries, and is histologically characterized by neointimal formation. In this process, endothelial cells (ECs) are injured and replaced with migrated vascular smooth muscle cells (VSMCs), along with collagen deposition. Transforming growth factor- β(beta) (TGF-β[beta]) has been recognized as a key contributor for triggering tissue fibrosis in various types of organs, such as liver and kidneys. Emerging evidence indicates that TGF-β(beta) plays an essential role for the establishment of restenosis post-angioplasties. Indeed, TGF-β(beta) production is locally upregulated near the neointimal areas in patients, while forced expression of TGF-β(beta) in arterial walls leads to neointima-like lesions in healthy animals, hence identifying TGF-β(beta) as a major contributor for atherogenic events. TGF-β(beta) induces apoptosis or mesenchymal differentiation in culture of ECs. Furthermore, TGF-β(beta) enhances migration and collagen production in VSMCs. There is now ample evidence that several types of TGF-β(beta) targeting agents produce therapeutic effects in animal models. In this review, we discuss the pathogenesis-based anti-restenotic strategy to prevent (or reverse) the neointimal formation and thickening, with a focus on TGF-β(beta)-mediated signaling cascades.