Pharmacological evaluation of adipose dysfunction via 11ß-hydroxysteroid dehydrogenase type 1 in the development of diabetes in diet-induced obese mice with cortisone pellet implantation.

Signals from intracellular glucocorticoids (GCs) via 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in adipose tissues have been reported to serve as amplifiers leading to deterioration of glucose metabolism associated with obesity. To elucidate adipose dysfunction via 11ß-HSD1 activation in the development of obesity-related diabetes, we established novel diabetic mice by implanting a cortisone pellet (CP) in diet-induced obesity (DIO) mice. Cortisone pellet-implanted DIO mice (DIO/CP mice) showed hyperglycemia, insulin resistance, hyperlipidemia, and ectopic fat accumulation, whereas cortisone pellet implantation in lean mice did not induce hyperglycemia. In DIO/CP mice, indexes of lipolysis such as plasma glycerol and nonesterified fatty acids (NEFAs) increased before hyperglycemia appeared. Furthermore, the adipose mRNA level of 11ß-HSD1 was up-regulated in DIO/CP mice compared with sham-operated DIO mice. RU486 (mifepristone, 11ß-[p-(dimethylamino)phenyl]-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), a glucocorticoid receptor antagonist, decreased adipose mRNA levels of 11ß-HSD1 as well as adipose triglyceride lipase. RU486 also improved plasma NEFA, glycerol, and glucose levels in DIO/CP mice. These results demonstrate that lipolysis in adipose tissues caused by GC activation via 11ß-HSD1 serves as a trigger for diabetes with ectopic fat accumulation. Our findings also indicate the possibility of a vicious circle of GC signals via 11ß-HSD1 up-regulation in adipose tissues, contributing to deterioration of glucose metabolism to result in diabetes. Our DIO/CP mouse could be a suitable model of type 2 diabetes to evaluate adipose dysfunction via 11ß-HSD1.

Cortactin interacts with podocalyxin and mediates morphological change of podocytes through its phosphorylation.

BACKGROUND/AIMS: Morphological change of podocytes is closely correlated with the development of proteinuria. Podocalyxin is a major sialoglycoprotein of the podocytes and is thought to be involved in the maintenance of the foot processes structure. Our aim was to examine the mechanism by which podocalyxin contributes to the morphological change of podocytes. METHODS: We searched protein(s) which coprecipitate with podocalyxin using rat glomerular lysate. Localization of podocalyxin and the coprecipitated protein, cortactin, was studied in a model of puromycin aminonucleoside (PAN) nephrosis by immunocytochemistry. Tyrosine phosphorylation of cortactin was examined. Association of the podocalyxin/cortactin complex with the actin cytoskeleton was evaluated by extraction with Triton-X and immunoblotting. RESULTS: Cortactin was found to be co-immunoprecipitated with podocalyxin. Immunocytochemical analysis revealed that these 2 proteins colocalized in the apical side of podocytes. In PAN nephrosis, localization of cortactin was altered after the onset of proteinuria, with increased tyrosine phosphorylation. Simultaneously, the dissociation of the podocalyxin/cortactin complex from the actin cytoskeleton was induced. CONCLUSIONS: These results indicate that cortactin mediates interaction between podocalyxin and actin filaments in podocytes and that alteration of this interaction may play a role in the process of morphological change of podocytes.

Genetic variations in the gene encoding ELMO1 are associated with susceptibility to diabetic nephropathy.

To search for a gene(s) conferring susceptibility to diabetic nephropathy (DN), we genotyped over 80,000 gene-based single nucleotide polymorphisms (SNPs) in Japanese patients and identified that the engulfment and cell motility 1 gene (ELMO1) was a likely candidate for conferring susceptibility to DN, in view of the significant association of an SNP in this gene with the disease (intron 18+9170, GG vs. GA+AA, chi(2) = 19.9, P = 0.000008; odds ratio 2.67, 95% CI 1.71-4.16). In situ hybridization (ISH) using the kidney of normal and diabetic mice revealed that ELMO1 expression was weakly detectable mainly in tubular and glomerular epithelial cells in normal mouse kidney and was clearly elevated in the kidney of diabetic mice. Subsequent in vitro analysis revealed that ELMO1 expression was elevated in cells cultured under high glucose conditions (25 mmol/l) compared with cells cultured under normal glucose conditions (5.5 mmol/l). Furthermore, we identified that the expression of extracellular matrix protein genes, such as type 1 collagen and fibronectin, were increased in cells that overexpress ELMO1, whereas the expression of matrix metalloproteinases was decreased. These results indicate that ELMO1 is a novel candidate gene that both confers susceptibility to DN and plays an important role in the development and progression of this disease.

Altered expression of matrix-related molecules in the development of chronic Thy1.1 nephritis.

BACKGROUND/AIM: Matrix production and degradation are critically important in chronic nephritis. Our aim was to investigate the precise expression of matrix-related molecules which is essential for understanding the pathogenesis of renal disease. METHODS: Chronic nephritis was induced by a single injection of anti-Thy1.1 antibody to unilaterally nephrectomized rats. RNA was extracted from renal cortex and isolated glomeruli 4, 7, and 10 weeks after the antibody injection. Matrix-related gene expressions were measured by polymerase chain reaction. The expression of alpha1(IV) and alpha3(IV) collagens was studied by immunohistochemistry. The gelatinolytic activity in the glomeruli was assayed by gelatin zymography. RESULTS: Polymerase chain reaction revealed an increase of alpha1(IV) in both glomeruli and renal cortex from nephritic rats. In contrast, the expression of alpha3(IV), normally a component of the glomerular basement membrane, was decreased in nephritic animals. Immunohistochemistry confirmed the finding that alpha1(IV) and alpha3(IV) were up- and downregulated, respectively, in the glomeruli. Gene expression and activity of matrix metalloproteinase 2 were enhanced, while those of matrix metalloproteinase 9 were clearly suppressed in nephritis. CONCLUSIONS: Downregulation of alpha3(IV) and enhancement of the matrix metalloproteinase-2 activity in the glomeruli may contribute to the glomerular damage by altering the glomerular basement membrane components. Impairment of the glomerular basement membrane integrity may possibly be implicated in irreversible renal dysfunction.

A selective peroxisome proliferator-activated receptor δ agonist PYPEP suppresses atherosclerosis in association with improvement of the serum lipoprotein profiles in human apolipoprotein B100 and cholesteryl ester transfer protein double transgenic mice.

OBJECTIVE: Although peroxisome proliferator-activated receptor (PPAR) δ agonists have been shown to improve the serum lipoprotein profiles in humans, the impact of the changes in these lipoprotein profiles on atherosclerosis remains to be elucidated. The aim of this study was to investigate the relationship between the selective PPARδ agonist-induced alterations of serum lipoprotein profiles and the development of atherosclerosis in human apolipoprotein B100 and cholesterol ester transfer protein double transgenic (hApoB100/hCETP-dTg) mice with human-like hypercholesterolemic dyslipidemia. METHODS: hApoB100/hCETP-dTg mice fed an atherogenic diet received a novel PPARδ agonist (PYPEP) or vehicle for 18 weeks, followed by evaluation of atherosclerosis. Serum samples were collected during the treatment period at least at 3-week intervals to determine the lipoprotein levels and the levels of an inflammatory marker, macrophage chemotactic protein-1 (MCP-1), and to analyze the lipoprotein profile by fast protein liquid chromatography. The cholesterol efflux capacity of high-density lipoprotein (HDL) was examined using [(3)H]-cholesterol labeled macrophages. RESULTS: Compared with vehicle treatment, PYPEP treatment caused increases in the serum levels of HDL cholesterol and apolipoprotein A-I (ApoA-I), as well as reductions in the serum non-HDL cholesterol and MCP-1 levels. The HDL fraction from the PYPEP-treated group maintained its cholesterol efflux capacity and showed an increased population of smaller HDL particles. PYPEP substantially suppressed atherosclerotic lesion progression, and the lesion areas had significant correlations with non-HDL cholesterol, HDL cholesterol, ApoA-I and MCP-1 by Pearson's correlation analysis. A multiple regression analysis revealed that non-HDL cholesterol and ApoA-I were significantly associated with the atherosclerotic lesion area. CONCLUSION: A novel PPARδ agonist, PYPEP, suppressed atherosclerotic lesion progression by improving the serum lipoprotein profiles, including increased levels of ApoA-I and functional HDL particles, as well as a reduced non-HDL cholesterol level, in hApoB100/hCETP-dTg mice with human-like hypercholesterolemic dyslipidemia.

Renoprotective action of a matrix metalloproteinase inhibitor in progressive mesangioproliferative nephritis.

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) play pivotal roles in extracellular matrix turnover and are involved in chronic kidney disease. The renoprotective action of a synthetic MMP inhibitor, compound A, was investigated in chronic nephritis. METHODS: Nephritis was induced by a single injection of anti-Thy1.1 antibody to unilaterally nephrectomized rats. The effects of compound A on proteinuria, blood urea nitrogen, and matrix-related gene expressions were evaluated. Collagen accumulation, as assessed by periodic acid-Schiff staining and hydroxyproline content, was determined. The integrity of glomerular epithelial cells and glomerular basement membrane was evaluated with desmin immunohistochemistry and electron microscopic detection of anionic charge sites, respectively. RESULTS: Treatment with compound A notably attenuated proteinuria, ameliorated blood urea nitrogen, and prevented glomerulosclerosis. Gene upregulation of collagen and transforming growth factor ß1 in the cortex was prevented in the treated animals. Glomerular epithelial cell injury was milder, and glomerular basement membrane anionic sites were protected with the treatment. CONCLUSION: A novel MMP inhibitor, compound A, exerts protective effects in progressive glomerulonephritis. Compound A ameliorates various aspects of renal injuries and may have therapeutic potential toward kidney diseases.

Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells.

Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement.

Involvement of dipeptidyl peptidase IV in immune complex-mediated glomerulonephritis.

Dipeptidyl peptidase IV (DPPIV) is widely expressed in many tissues; however, its precise biological function is poorly understood. One of its possible physiologic roles is an involvement in the immune system, which plays a pivotal role in the pathogenesis of glomerulonephritis. The present study focused on the involvement of DPPIV in immune complex-mediated glomerulonephritis. Experimental nephritis was induced by anti-Thy-1.1 monoclonal antibody E30 using Wistar or F344 rats. The application of a new monoclonal antibody against DPPIV, F16, completely suppressed E30-induced proteinuria and mesangial proliferation in Wistar rats, whereas these preventive effects of F16 were not observed in F344 rats, which spontaneously lack DPPIV protein. Treatment with F16 inhibited glomerular deposition of complement C3 and complement C4 after the binding of E30 to the mesangial cell surface. Because the preventive effect of F16 was attributable to suppression of the complement cascade, we examined its influences on complement-dependent mesangial cell lysis in vitro. We discovered that the complement cascade was markedly inactivated in F16-treated Wistar rat serum but not in F16-treated F344 rats. These results indicate that DPPIV may play a somewhat crucial role in regulating the complement cascade and that inhibition of DPPIV may serve as a new target for preventing complement-dependent tissue injury.

Intussusceptive capillary growth is required for glomerular repair in rat Thy-1.1 nephritis.

BACKGROUND: Injection of anti-Thy-1.1 antibodies to rats causes mesangiolysis, with subsequent capillary loss. This dramatic event is followed by almost complete recovery of glomerular architecture. However, the precise cellular mechanisms of revascularization are not fully understood. METHODS: Glomerulonephritis was induced by the injection of monoclonal anti-Thy-1.1 antibody to rats. Structural changes in the glomerular vasculature, with special emphasis on the repair phase, were studied with corrosion casting technique, light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). RESULTS: Specifically during the extensive revascularization period, numerous tiny holes of about 1.5 microm in diameter were found on the glomerular cast. The presence of transluminal tissue pillar was confirmed with LM and SEM. These findings indicate the involvement of intussusceptive capillary growth. TEM study demonstrated that some tissue pillars were composed of endothelial cells only and others had cytoplasmic process of mesangial cells in their cores sandwiched both sides by endothelial walls. CONCLUSION: Intussusceptive capillary growth, or nonsprouting angiogenesis is involved in the postinjury angiogenesis in the glomeruli, in which process mesangial cells as well as endothelial cells may play important roles.

Mechanisms underlying the ameliorative property of lisinopril in progressive mesangioproliferative nephritis.

The present study was performed to clarify the mechanism underlying the beneficial effects of lisinopril on chronic glomerulonephritis. Chronic glomerulonephritis was induced by a single injection of E30 monoclonal antibody (E30) recognizing Thy-1.1 antigen to unilaterally nephrectomized rats. E30 injection resulted in persistent massive proteinuria with a decrease in anionic charge sites on the glomerular basement membrane (GBM) at 8 weeks. Also, renal tissue from rats treated with E30 showed typical glomerulosclerosis and tubulointerstitial fibrosis. Lisinopril exerted a potent antiproteinuric effect and suppressed the progression of both glomerulosclerosis and tubulointerstitial fibrosis. Lisinopril recovered the reduced number of anionic charge sites on GBM, accounting for the positive action against massive proteinuria. Immunostaining for desmin revealed that lisinopril treatment prevented the injury of glomerular epithelial cells (GECs) occurring in the chronic nephritic stage. Also, the level of gene expression of transforming growth factor-beta (TGF-beta) and plasminogen activator inhibitor-1 (PAI-1) in the renal cortex were reduced, suggesting that lisinopril improved extracellular matrix (ECM) metabolism. These results indicated that proteinuria in Thy-1.1 antibody-induced chronic nephritis is associated with a decrease in anionic charge sites on GBM, and that the antiproteinuric effect of lisinopril is attributable to protection against GEC damage. Suppression of TGF-beta and PAI-1 expression contributed to the preventive effect of lisinopril on ECM deposition in renal tissue.

Glomerular epithelial cell injury accelerates the progression of antibody-induced mesangial proliferative nephritis.

BACKGROUND: Anti-Thy-1.1 antibody induces mesangioproliferative glomerulonephritis; however, the mesangial lesion is spontaneously recovered to the normal feature. Glomerular epithelial cells (GECs) play a crucial role in the glomerular function. Very little is known about the involvement of GECs in this disease model. This study is designed to investigate whether GEC injury prolongs the mesangial lesion. METHODS: The effects of GEC damage on mesangioproliferative nephritis were studied with combined treatment using puromycin aminonucleoside (PAN) and monoclonal antibody (MAb) against rat mesangial Thy-1.1. Urinary protein, BUN, Pcr and Ccr were measured. To clarify the underlying mechanisms, morphological study and immunohistochemistry for alpha-SMA, FGF-2 and PCNA were carried out. RESULTS: Simultaneous administration of PAN plus MAb induced progressive mesangioproliferative nephritis compared to PAN or MAb alone. Rats with combined treatment displayed renal dysfunction with massive proteinuria. Morphological studies showed that the glomeruli in combined group had features resembling those of progressive mesangial proliferative glomerulonephritis in humans. Morphologic lesions of GECs in acute nephritic phase were severer than those in other groups. Immunohistochemistry revealed that glomeruli of combined treatment exhibited persistent overexpression of alpha-SMA and FGF-2. CONCLUSIONS: Simultaneous dysfunction of GECs and mesangial cells can lead to persistent glomerular perturbations with prolonged phenotypic change of mesangial cells, resulting in end-stage renal deficiency. GEC damage during the acute nephritic phase contributes to the progression of irreversible renal disease.