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Background: The progression of diabetic nephropathy is closely related to disturbances in glomerular hemodynamics, such as glomerular hypertension and/or hyperperfusion. The aim of this study was to observe and to analyze glomerular hemodynamics in rats with diabetes mellitus (DM) in vivo using confocal laser scan microscopy (CLSM). We also examined the effects of candesartan cilexetil (TCV-116), a selective angiotensin II type 1 receptor blocker (ARB), on glomerular hemodynamics in DM., Methods: Munich-Wistar rats were divided into six groups: (1) four-day control; (2) four-day DM; (3) 28-day control; (4) 28-day DM; (5) DM treated with insulin; (6) DM treated with TCV-116. The kidney-to-body weight ratio, glomerular volume, and proteinuria were estimated. Glomerular hemodynamic changes were observed using CLSM and renal expression of endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) was evaluated by immunofluorescence., Results: The kidney-to-body weight ratio, glomerular volume, the diameters of afferent arterioles (AA) and efferent arterioles (EA), erythrocyte velocities within glomeruli, and volume flow in glomerular capillary loops in four-day DM were significantly higher than in control rats, and increases were even more pronounced in the 28-day DM. TCV-116 treatment ameliorated all these findings and significantly decreased proteinuria, but there was no effect on the blood glucose level. On the other hand, insulin treatment was followed by normalization of all these changes induced in DM. Enhanced renal expression of eNOS in DM was suppressed when treated with either TCV-116 or insulin, while expression of nNOS was unaltered among the four groups., Conclusion: This imaging procedure allowed us to evaluate glomerular microcirculation in vivo, including the diameters of AA and EA, erythrocyte velocity, and volume flow. DM significantly induced glomerular hemodynamic alteration and renal hypertrophy. DM treated with either insulin or ARB ameliorated these changes. This study shows that progress in imaging technology promises to make major contributions to revealing the involvement of hemodynamic changes in glomerular diseases, aiding prognosis and the monitoring of therapeutic effects, as well.

Background: It has been proposed that glomerular hemodynamic changes or glomerular growth response may promote the development of glomerulosclerosis, irrespective of its etiology. Further experimental models are needed to clarify the cellular and molecular mechanisms that lead to progressive glomerulosclerosis with an irreversible course. We designed a model of irreversible glomerulosclerosis, using anti-Thy-1.1 injection followed by uninephrectomy, and examined the role of glomerular endothelial cell responses in the process of progressive sclerotic changes., Method: Rats were injected with anti-Thy-1.1 monoclonal antibody, 1-22-3, and 30 minutes later, unilateral nephrectomy (one-kidney group) or sham operation (two-kidney group) was performed. Rats were sacrificed for histological examination on days 3, 14, 56, and 84 after injection. The density of the glomerular capillary tuft was assessed by immunofluorescent staining for endothelial specific antigens. The mRNA expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial growth factor (VEGF) also was followed up by reverse transcription-polymerase chain reaction (RT-PCR)., Results: Semiquantitative analysis revealed that the capillary density and mRNA expression of PECAM-1, VCAM-1 and VEGF were significantly lower in the one-kidney group compared to the two-kidney group on day 14. On day 84, progressive glomerulosclerotic lesions were found, followed by a decrease of the capillary density in the one-kidney group, while the glomerular architecture recovered to an almost normal state in the two-kidney group., Conclusions: Progressive glomerulosclerosis can be induced in the rat by a one shot injection of anti-Thy 1.1 monoclonal antibody followed by unilateral nephrectomy. This model shows that there is a positive association between impairment of vascular regeneration and the development of glomerulosclerosis.

Background: Blood flow in the microvasculature plays a pivotal role in determining the outcome of injury and repair in inflamed tissue. Real-time observation of the kidney microvasculature, including the glomerular capillary tufts, is extremely difficult because of the methodological limitations of currently available microscope optics. In the present study, we attempted to analyze hemodynamic events that occurred in vivo during microvascular regeneration following destruction of the glomerular capillary tuft, functionally and quantitatively by the use of a real-time confocal laser-scanning microscope (CLSM) system., Methods: A polyethylene catheter was inserted into the carotid artery to allow blood pressure measurement. Mesangiolytic lesions producing microaneurysms were induced by the injection of anti-Thy-1.1 antibody. On days 3 and 7 after antibody injection, we examined hemodynamic changes under an intravital microscope equipped with real-time CLSM in combination with a high-speed CCD video camera. To measure vessel diameter and erythrocyte velocity, rats were injected with fluorescein isothiocyanate (FITC)-labeled dextran and FITC-labeled red blood cells (RBCs)., Results: On day 3 of the disease, mean arterial blood pressure was 112 +/- 5 mm Hg, which was significantly higher than that of normal rat or of rats on day 7 (93 +/- 1 and 101 +/- 9 mm Hg, respectively). Within mircroaneurysms on day 3, RBC velocity was greatly suppressed. By day 7, RBC velocity, in glomeruli with normal appearances, recovered to about half of the level seen in normal controls (430.6 +/- 284.7 microm/sec), while in narrowed glomerular tufts, it was still only 104.6 +/- 35.1 microm/sec., Conclusions: The noninvasive procedure, using CLSM in combination with a high-speed video camera, allowed us to examine hemodynamic events quantitatively and to analyze microvascular architecture three dimensionally in the kidney. It is useful for estimating hemodynamic response and vascular regeneration in vivo and may be promising for clinical application.

Background/aims: Fibronectin is a multifunctional glycoprotein and plays important roles in cell-to-cell or cell-to-matrix interaction. The molecular and functional diversity of fibronectin arises from alternative splicing of pre-mRNA at three variable regions, termed ED-A, ED-B and IIICS. Cellular fibronectin with ED-A and ED-B regions has different biological activities from plasma fibronectin lacking these regions. This study was aimed at investigating the type-specific expression of fibronectin in human liver diseases., Methods: Immunohistochemistry with anti-total and anti-cellular fibronectin monoclonal antibodies, in situ hybridization with cDNA probes detecting common and ED-A regions and RT-PCR to amplify each variable region were performed in 35 specimens, including 4 control, 16 chronic hepatitis, 7 liver cirrhosis and 8 hepatocelular carcinoma., Results: In control liver, there were slight deposits of cellular fibronectin [ED-A(+)fibronectin] in portal areas. In chronic hepatitis, it was strongly deposited at the margin of the fibrously enlarged portal areas where new collagen fibers were formed. Cellular fibronectin was evenly and abundantly accumulated in fibrotic septa in liver cirrhosis, and in fibrotic septa and capsules of tumor nodules in hepatocellular carcinoma. In control liver, cellular fibronectin mRNA was localized in a few hepatocytes and non-parenchymal cells around central veins, and was increased in the same cell populations near fibrously enlarged portal areas as hepatic fibrosis progressed. In hepatocellular carcinoma, it was expressed in most hepatoma cells. Fibronectin mRNA with three variable regions was detectable by RT-PCR in control liver as well as in each disease group., Conclusions: The expression of cellular fibronectin was increased in fibrotic human liver and hepatocellular carcinoma. In human liver, both non-parenchymal cells and hepatocytes participated together in cellular fibronectin production. In hepatocellular carcinoma, hepatoma cells were the main producer. Our results indicate that, in human liver, cellular fibronectin may participate in the hepatic fibrogenesis and in the malignant phenotypes of hepatocellular carcinoma.

Immunolocalization of a fibronectin-binding proteoglycan (PG-P1) in relation to fibronectin, type IV collagen and laminin, in normal and fibrotic human liver was investigated by light and electron microscopy. HS-42, which is a monoclonal antibody to PG-P1 and is reported to recognize a heparan sulfate proteoglycan named HSPG2/perlecan, was used for this purpose. Light microscopy in the human liver with minimal changes revealed that PG-P1 was present along the hepatic sinusoids as well as fibronectin and type IV collagen, whereas laminin was only weakly detected. In portal areas, PG-P1 was only localized on basement membranes around bile duct systems and blood vessels, as well as laminin and type IV collagen, while fibronectin was scarcely detected in basement membranes. In the fibrotic liver, fibronectin was abundant in necrotic and/or newly fibrosing areas, while PG-P1 was absent in these regions. Using immunoelectron microscopy, PG-P1 was localized in the space of Disse in nearly normal livers and was only detected on basement membranes in portal tracts. In fibrotic livers, PG-P1 in the space of Disse occasionally showed a basement-membrane-like deposition in parallel with the increased light microscopical deposition of laminin in this area, suggesting the positive participation of PG-P1 in the sinusoidal capillarization. Most capillary and sinusoidal endothelial cells, and rarely bile epithelial cells revealed the reaction products of PG-P1 in their rough endoplasmic reticulum and small vesicles. Thus, it was suggested that these cell types are mainly, if not wholly, responsible for PG-P1 production.(ABSTRACT TRUNCATED AT 250 WORDS)