Yasushi Yamasaki

2-(8-Hydroxy-6-Methoxy-1-Oxo-1H-2-Benzopyran-3-yl) Propionic Acid, an Inhibitor of Angiogenesis, Ameliorates Renal Alterations in Obese Type 2 Diabetic Mice

One of the mechanisms involved in the progression of diabetic nephropathy, the most common cause of end-stage renal failure, is angiogenic phenomenon associated with the increase of angiogenic factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2, an antagonist of Ang-1. In the present study, we examined the therapeutic efficacy of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3), a small molecule isocoumarin with antiangiogenic activity, using diabetic db/db mice, a model of obese type 2 diabetes. Increases in kidney weight, glomerular volume, creatinine clearance, urinary albumin excretion, total mesangial fraction, glomerular type IV collagen, glomerular endothelial area (CD31+), and monocyte/macrophage accumulation (F4/80+) observed in control db/db mice were significantly suppressed by daily intraperitoneal injection of NM-3 (100 mg/kg, for 8 weeks). Increases in renal expression of VEGF-A, Ang-2, fibrogenic factor transforming growth factor (TGF)-β1, and chemokine monocyte chemoattractant protein-1 but not tumor necrosis factor-α were also inhibited by NM-3 in db/db mice. Furthermore, decreases of nephrin mRNA and protein levels in db/db mice were recovered by NM-3. In addition, treatment of db/db mice with NM-3 did not affect body weight, blood glucose, serum insulin, or food consumption. NM-3 significantly suppressed the increase of VEGF induced by high glucose in cultured podocytes and also suppressed the increase of VEGF and TGF-β induced by high glucose in cultured mesangial cells. Taken together, these results demonstrate the potential use of NM-3 as a novel therapeutic agent for renal alterations in type 2 diabetes.

Glomerular cell apoptosis in human lupus nephritis

Disturbed apoptosis is proposed to be involved in the pathogenesis of systemic lupus erythematosus. However, the role of renal cell apoptosis in the pathogenesis and progression of human lupus nephritis is still controversial. We have investigated glomerular cell apoptosis and the clinicopathological relationship between apoptosis and immunoserological or histological findings in 22 patients with lupus nephritis using electron microscopy and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Resident glomerular cells as well as infiltrating neutrophils undergoing apoptosis were observed in 12 of 20 patients with lupus nephritis using electron microscopy. TUNEL-positive cells were recognized in 93% of patients with diffuse proliferative lupus nephritis (class IV) in contrast to the 20% of patients with class V. The number of TUNEL-positive cells in glomeruli significantly correlated with the level of immunoserological activity of lupus, such as anti-double-stranded DNA autoantibody and consumption of plasma complement. There was a positive correlation between glomerular cell apoptosis and the degree of proliferation in lupus nephritis. These data suggest that apoptosis is increased, but not decreased in glomeruli from patients with lupus nephritis. The signals that could induce glomerular cell apoptosis in lupus nephritis will need to be identified.

Clinical Significance of Serum Oxidized Low-Density Lipoprotein/β2-Glycoprotein I Complexes in Patients with Chronic Renal Diseases

Background: Peroxidation of low-density lipoprotein (LDL) plays an important role in the development of dyslipidemias associated with the progression of chronic renal disorders. We recently reported [J Lipid Res 2001;42:697, 2002;43:1486, 2003;44:716] that oxidized LDL (oxLDL) interacts with an endogenous plasma protein, β2-glycoprotein I (β2GPI), via specific ligands. In the present study, the prevalence and clinical significance of oxLDL/β2GPI complexes were evaluated in patients with chronic renal disorders. Methods: Serum levels of oxLDL/β2GPI complexes were measured by ELISA in patients with chronic renal disease and their association with clinical manifestations was assessed. Results: The serum levels of oxLDL/β2GPI complexes were significantly higher in patients with chronic renal failure (CRF), chronic nephritis (CN) and diabetes mellitus than those in healthy individuals. The presence of complexes in patients with CN was significantly associated with high dietary protein and sodium chloride intake, but not with lipid metabolic parameters. Malondialdehyde-modified LDL was significantly associated with total cholesterol and LDL cholesterol in all patient groups, but did not correlate with renal function parameters. Conclusions: Serum oxLDL/β2GPI complexes, generated by oxidative stress and associated with high dietary protein and salt intake, might be a novel risk factor and a diagnostic marker for the development of chronic renal diseases, especially IgA nephropathy.

Transforming Growth Factor-β1 Induces Vascular Endothelial Growth Factor Expression in Murine Proximal Tubular Epithelial Cells

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen that promotes angiogenesis, vasculogenesis, and increases vascular permeability. VEGF is expressed in renal tubular epithelial cells and urinary VEGF excretion is increased in various glomerular disorders. However, the mechanisms underlying expression of VEGF in renal tubular epithelial cells have not been fully elucidated. In the present study, we attempted to define a predominant regulator of VEGF expression using a cultured murine renal proximal tubular epithelial cell line (mProx24). VEGF protein concentration in the culture supernatant was measured by sandwich enzyme-linked immunosorbent assay. mProx24 constitutively produced VEGF at low level. Major isoforms expressed in this cell line were VEGF164 and VEGF120 determined by reverse transcription-polymerase chain reaction method. Among various stimuli including angiotensin II, transforming growth factor-β1 (TGF-β1), lipopolysaccharides, interleukin-1β, interleukin-10 and interferon-γ, only TGF-β1 significantly increased the level of VEGF protein at 24 h in a dose-dependent manner. The steady-state mRNA level of VEGF was dose dependently increased by TGF-β1 detected by Northern blotting. Treatment with neutralizing anti-TGF-β1 antibody abolished TGF-β1-induced VEGF expression by 70%. Inhibitors of protein kinase C (PKC), Ro-31-8220 and staurosporin, significantly suppressed TGF-β1-induced VEGF protein expression. These results demonstrate the role of TGF-β1 on the expression of VEGF in proximal tubular epithelial cells mediated potentially via PKC pathway. This regulatory mechanism may be associated with the progression of tubulointerstitial lesions in renal disorders.

Amelioration of cisplatin-induced acute renal injury by renal progenitor-like cells derived from the adult rat kidney

The replacement of a necrotic tubular epithelium with functional tubular epithelial cells is required for recovery from acute renal failure (ARF). A rat renal progenitor-like (rKS56) cell line was recently established derived from the S3 segment of renal proximal tubules. The therapeutic efficacy of rKS56 cells was examined in a rat model of cisplatin-induced ARF. rKS56-lacZ cells expressing β-galactosidase were injected into SD rats either at the subcapsule of the left kidney (rKS-SC) or via the left renal artery (rKS-IA) 2 days after the injection of cisplatin. Bluo-gal(+) rKS56-lacZ cells were observed in the subcapsule in the rKS-SC group on day 5, and were further increased in number on day 9, accompanied by partial distribution in the corticomedullary junction, but not in the rKS-IA group. A portion of Bluo-gal(+) cells coexpressed Ki-67, aquaporin-1, hepatocyte growth factor (HGF), and c-Met. rKS-SC treatment significantly improved the tubular injury scores, ameliorated tubular cell apoptosis, and induced cell proliferation. The renal function also significantly improved in the rKS-SC group on day 5. These results demonstrate that locally implanted rKS56 cells could differentiate into tubular epithelial cells, thereby accelerating the recovery from tubular injury, most likely by producing tubular trophic factors. These results suggest the therapeutic potential of this novel approach for patients with end-stage renal failure.

Elevated serum sFlt-1/Ang-2 ratio in women with preeclampsia.

Background: An imbalance of angiogenesis-associated factors may predispose to preeclampsia. Here, we determined the ratio of serum concentration of soluble fms-like tyrosine kinase 1 (sFlt-1), a natural inhibitor of pro-angiogenic vascular endothelial growth factor (VEGF) relative to angiopoietin-2 (Ang-2), a natural antagonist of angiopoietin-1 (Ang-1) involved in promoting angiogenesis in the presence of VEGF, in women with preeclampsia. Methods: The levels of serum sFlt-1 and Ang-2 were measured by enzyme-linked immunosorbent assay. Results: Significant decrease of serum Ang-2 and the increase of sFlt-1 were observed in women with preeclampsia as compared to healthy pregnant women. The serum sFlt-1/Ang-2 ratio was strikingly increased in preeclamptic women in contrast to healthy pregnant women exhibiting lower value similar to non-pregnant women. The serum sFlt-1 concentrations tended to positively correlate with mean blood pressure (BP) in preeclamptic women, but not in healthy pregnant women. A cut-off value >0.25 in the serum sFlt-1/Ang-2 ratio showed 87.1% sensitivity and 82.8% specificity in differentiating preeclamptic women from healthy pregnant women. Conclusion: The serum sFlt-1/Ang-2 ratio is significantly elevated in preeclamptic women as compared to healthy pregnant women. Remarkable difference of sFlt-1/Ang-2 ratio between these two groups with excellent specificity and sensitivity suggests the clinical usefulness of the serum sFlt-1/Ang-2 ratio in diagnosing and potentially predicting the onset of preeclampsia.

Meshwork Structures in Bovine Glomerular and Tubular Basement Membrane as Revealed by Ultra-High Resolution Scanning Electron Microscopy

We examined the ultrastructure of the bovine glomerular basement membrane (GBM) and tubular basement membrane (TBM) using ultra-high-resolution scanning electron microscopy after conductive staining without metal coating. Purified basement membranes (BMs) were obtained by sonication and acellular BMs by detergent treatments. Purified GBMs (PGBMs) and acellular GBMs (AGBMs) showed similar meshwork structures composed of regular round or oval pores and branching strands. Pore diameters were 10.2 +/- 2.4 nm (mean +/- SD) in PGBMs and 9.8 +/- 3.1 nm in AGBMs. Purified TBMs (PTBMs) and acellular TBMs (ATBMs) exhibited heterogeneous meshwork structures in which compact meshes in the cristae were combined with coarse meshes in the invaginations on the epithelial surfaces. Pore diameters were 12.6 +/- 5.2 nm in PTBMs and 11.4 +/- 4.0 nm in ATBMs which were significantly larger than those in GBMs. The width of the strands ranged from 3 to 15 nm in all BMs. A 4 M guanidine hydrochloride extraction of the acellular BMs revealed large polygonal networks in the invaginations of the TBM and twisted strands which were considered to be type IV collagen fibrils. Ultra-high-resolution scanning electron microscopy and conductive staining were useful for the study of three-dimensional architectures of BMs and revealed heterogeneous meshwork structures in the GBM and TBM which were probably caused by a different ratio of the major components.

Mesangial cell Fas ligand: upregulation in human lupus nephritis and NF-κB-mediated expression in cultured human mesangial cells

BackgroundFas ligand (FasL) is a well-known death factor; however, the role of FasL in the regulation of human glomerulonephritis remains unclear.MethodsWe investigated the renal expression and localization of FasL in various forms of human glomerulonephritis by immunohistochemistry, utilizing confocal laser scanning microscopy. We further evaluated cytokine-induced FasL expression via nuclear factor (NF)κB in cultured human mesangial cells (HMC). The level of soluble FasL was measured by a specific enzyme-linked immunosorbent assay (ELISA).ResultsThe frequency of glomerular FasL-positive cases was higher in lupus nephritis (37.9%) as compared with other forms of glomerulonephritis (8.7%). The glomerular FasL score in proliferative lupus nephritis was significantly higher than that in nonproliferative forms. Patients with a high apoptosis score, severe microhematuria, proteinuria, or decreased renal function had a high FasL score. Double immunolabelling demonstrated that the most prevalent phenotypes of FasL-positive cells were mesangial cells. In cultured HMC, interleukin (IL)1β, lipopolysaccharide (LPS), or γ interferon (IFN) upregulated membrane-bound FasL. IL1β significantly, and LPS or γIFN weakly activated NFκB, but none of these agents activated NFκB/Rel-related nuclear factor of activated T cells (NFATc) or IFN regulatory factor-1. IL1β-mediated NFκB was completely inhibited in the presence of lactacystin, a potent inhibitor of NFκB. Lactacystin-mediated inhibition of NFκB reduced FasL protein levels. Matrix metalloproteinase (MMP)-7, but not other MMPs (1, 2, 3, 8, or 9), significantly sensitized HMC to release soluble FasL after IL1β stimulation.ConclusionsThe results suggest that: (1) upregulation of mesangial FasL may contribute to the glomerular inflammation in proliferative lupus nephritis in vivo; (2) proinflammatory cytokines, in particular IL1β, produced in nephritis can upregulate FasL via the transcription factor NFκB in HMC; and (3) MMP-7-mediated release of soluble FasL could control the mesangial inflammation.

Minimal Change Nephrotic Syndrome Developing during Postoperative Interferon-Beta Therapy for Malignant Melanoma

A 64-year-old man presented with proteinuria during postoperative interferon (IFN)-β therapy against malignant melanoma. Renal pathologic findings were consistent with minimal change nephrotic syndrome (MCNS) showing extensive foot process effacement of visceral glomerular epithelial cells (podocyte). Nephrotic range proteinuria gradually regressed after stoppage of local injection of IFN-β without glucocorticoid treatment. To our knowledge this is the first report that demonstrates histological abnormalities of the glomerulus associated with postoperative IFN-β therapy for the malignant melanoma.

Renal involvement in rheumatoid arthritis: analysis of renal biopsy specimens from 100 patients

Abstract  We analyzed renal biopsy specimens from 100 patients to evaluate the characteristics of renal involvement in patients with rheumatoid arthritis (RA). Membranous nephropathy (MN) was the most common renal histological pattern (31%). Mesangial proliferative glomerulonephritis (GN) was found in 21% of cases (IgA nephropathy 12%, non-IgA GN 9%), minor changes in 17%, renal amyloidosis in 11%, interstitial nephritis in 9%, sclerotic GN in 4%, and crescentic GN in 2%. MN was relatively more frequent in men than in women, and most developed nephrotic syndrome, while a few developed renal failure. Disease-modifying antirheumatic drugs (DMARDs) correlated with MN in 26 of 31 cases. Mesangial proliferative GN showed high-grade hematuria. Amyloidosis correlated with long duration of RA; approximately half of the cases with amyloidosis also had nephrotic syndrome, and 82% developed renal failure. Of the 100 patients, 82% showed some tubulo-interstitial changes, which might be related to non-steroidal anti-inflammatory drugs. Because renal lesions in RA are very diverse, and early stage cases of MN and amyloidosis can be detected only by histological examinations, renal biopsy should be performed in cases with continuous urinary abnormalities or progressive renal failure.