Isao Ebihara

mRNA Expression of Growth Factors in Glomeruli From Diabetic Rats

Evaluations of glomerular mRNA levels encoding for PCNA, TNF-α, PDGF-A and -B chains, TGF-β, IGF-I, bFGF, and EGF were made at 4, 12, and 24 wk after injection of STZ in Sprague-Dawley rats. The mRNA levels for PCNA, TNF-α, PDGF-B chain, TGF-β, and bFGF increased with age in STZ-induced diabetic rats. At 24 wk after STZ injection, mRNA levels for PCNA, TNF-α, PDGF-B chain, TGF-β, and bFGF were increased 3.8-fold, (P < 0.01), 4.2-fold (P < 0.01), 4.0-fold (P < 0.01), 5.2-fold (P < 0.001), and 3.6-fold (P < 0.01), respectively, in the glomeruli of diabetic rats when compared with control rats. In contrast, mRNA levels for IGF-I, PDGF-A chain, and EGF were not altered in glomeruli from diabetic and control rats throughout the experimental period. Insulin treatment partially ameliorated the increase in mRNA levels for PCNA, TNF-α, PDGF-B chain, TGF-β, and bFGF in the glomeruli of diabetic rats. These data indicate that alterations in growth factor mRNA levels in glomeruli may be a manifestation of diabetic nephropathy, and that hyperglycemia or insulin deficiency may play a role in abnormal growth factor gene regulation.

Effect of a Specific Endothelin Receptor A Antagonist on mRNA Levels for Extracellular Matrix Components and Growth Factors in Diabetic Glomeruli

We have previously reported that the mRNA levels of extracellular matrix (ECM) components including αl(I), αl(III), and αl(IV) collagen chains, laminin Bl and B2 chains, and growth factors including tumor necrosis factor (TNF)-α, platelet-derived growth factor (PDGF)-B chain, transforming growth factor (TGF)-β, and basic fibroblast growth factor (FGF) all increase with age in diabetic glomeruli. The present study was designed to assess whether glomerular expression of these mRNAs in rat diabetic glomeruli is affected by a specific endothelin receptor A antagonist, FR139317. Diabetes was produced by streptozotocin injection, and animals were divided into four groups: untreated diabetic rats, FR139317-treated diabetic rats, control nondiabetic rats, and FR139317-treated control rats. FR139317 treatment was continued for 24 weeks. FR139317 attenuated the rise in creatinine clearance (P < 0.01) and reduced urinary protein excretion (P < 0.01) in diabetic rats, but did not affect blood pressure. FR139317 attenuated the increases in glomerular mRNA levels of αl(I) (P < 0.01), α1(III) (P < 0.01), and α1(IV) (P < 0.01) collagen chains, laminin B1 (P < 0.01) and B2 (P < 0.01) chains, TNF-α (P < 0.01), PDGF-B (P < 0.01), TGF-β (P < 0.001) and basic FGF (P < 0.01) in diabetic rats. However, FR139317 did not affect these mRNA levels in glomeruli of control rats. These findings suggest that FR139317 may be useful in the treatment of diabetic glomerulopathy by reducing mRNA levels of ECM components and growth factors.

Comparative effects of pioglitazone, glibenclamide, and voglibose on urinary endothelin-1 and albumin excretion in diabetes patients.

Urinary endothelin (ET)-1 excretion is present in non-insulin dependent diabetes (NIDDM) patients with microalbuminuria, and an increase in circulating ET-1 precedes the microalbuminuric phase of renal injury related to diabetes. The aim of the present study was to determine whether various drugs alter urinary ET-1 levels and urinary albumin excretion (UAE) in NIDDM patients with microalbuminuria. Forty-five NIDDM patients with microalbuminuria were randomly assigned to three groups: those treated with pioglitazone at 30 mg/day (n=15), those treated with glibenclamide at 5 mg/day (n=15), and those treated with voglibose at 0.6 mg/day (n=15). Patients received these drugs for 3 months. UAE, urinary ET-1, and plasma ET-1 levels were measured in these patients before and after treatment. Before treatment, UAE, urinary ET-1, and plasma ET-1 levels differed little among the three groups. UAE in the 45 NIDDM patients (156.2+/-42.8 microg/min) was greater than that in 30 healthy controls (8.2+/-2.6 microg/min) (P<.001). Urinary ET-1 levels in the NIDDM patients (8.7+/-1.3 ng/g urinary creatinine (UC)) were significantly higher than that in the controls (2.4+/-0.2 ng/g UC) (P<.01). Plasma ET-1 levels, however, in the NIDDM patients (1.3+/-0.4 pg/ml) did not differ significantly from the levels in healthy controls (1.0+/-0.6 pg/ml). Pioglitazone but no glibenclamide or voglibose reduced UAE from 142.8+/-42.2 to 48. 4+/-18.2 microg/min (P<.01) and urinary ET-1 levels from 8.6+/-1.3 to 3.4+/-0.5 ng/g UC (P<.01). These data suggest pioglitazone to be effective in reducing UAE and urinary ET-1 concentrations in NIDDM patients with microalbuminuria.

ECM Gene Expression and Its Modulation by Insulin in Diabetic Rats

The steady-state levels of mRNA encoding for the α1(IV) collagen chain, laminin B1 and B2 chains, basement membrane HSPG, and α1(I) and a1(III) collagen chains were examined in rat glomeruli at 4, 12, and 24 wk after injection of STZ. The mRNA levels for the α1(IV) collagen chain, laminin B1 and B2 chains, and α1 (I) and α1(I) and α1(III) collagen chains increased significantly with age in the STZ-induced diabetic rats before morphological thickening of basement membrane occurred. In contrast, the mRNA levels for HSPG decreased markedly 4 wk after STZ injection and then increased with age compared with those for control rats. The mRNA levels for these ECM components showed a continuous decline with age in controls. Treating the diabetic rats with insulin for 4 wk ameliorated the abnormally regulated ECM gene expression in the glomeruli. These data suggest that the abnormal regulation of ECM gene expression in the glomeruli may contribute to the expansion of mesangial matrix and basement membrane thickening in diabetic rats, and that hyperglycemia may play a role in the abnormal ECM gene expression.

Increased plasma metalloproteinase-9 concentrations precede development of microalbuminuria in non-insulin-dependent diabetes mellitus

We determined plasma metalloproteinase-9 (MMP-9) concentrations in 30 patients with non-insulin-dependent diabetes mellitus (NIDDM) at an initial examination (baseline) and on three separate occasions during a 48-month follow-up period. All patients had normal urinary albumin excretion (<20 microg/min) at the first three examinations. At the fourth examination (48 months after the first examination), 22 patients had normal urinary albumin excretion and eight had microalbuminuria (median, 36.4 microg/min; range, 20.2 to 46.6 microg/min). Compared with patients with normal urinary albumin excretion, patients with microalbuminuria had significantly higher plasma levels of MMP-9 at the second (56+/-14 microg/L v36+/-12 microg/L; P < 0.05), third (88+/-23 microg/L v 39+/-14 microg/L; P < 0.01), and fourth (117+/-30 microg/L v 44+/-16 microg/L; P < 0.01) examinations, but not at the first examination (34+/-12 microg/L v 33+/-14 microg/L; P=NS). An increase in plasma MMP-9 concentrations preceded the occurrence of microalbuminuria within 4 years. The groups did not differ with regard to age, sex, duration of NIDDM, blood pressure, or mean glycated hemoglobin. In addition, the eight patients with microalbuminuria were treated with an angiotensin-converting enzyme inhibitor (cilazapril; 0.5 mg once daily) for 6 months. Microalbuminuria was reduced to within the normal range, and plasma MMP-9 concentrations were significantly decreased with the cilazapril treatment (52+/-18 microg/L; P < 0.01). However, serum MMP-1 and tissue inhibitor of MMP-1 showed no change during the study period. These data suggest that plasma MMP-9 concentrations preceded and may predict the development of microalbuminuria in NIDDM.

Modulation of Plasma Metalloproteinase-9 Concentrations and Peripheral Blood Monocyte mRNA Levels in Patients with Septic Shock: Effect of Fiber-Immobilized Polymyxin B Treatment

The authors measured plasma metalloproteinase (MMP)-9 and corresponding monocyte mRNA in 20 patients with septic shock. Plasma MMP-9 concentrations and monocyte MMP-9 mRNA levels were significantly higher in the 10 nonsurviving patients with septic shock than in 10 surviving patients and 25 normal controls. Hemoperfusion using polymyxin B immobilized on fibers (PMX-F), a reportedly effective treatment for septic shock, was studied for effects on MMP-9 in the patients. Increases in plasma MMP-9 concentrations and corresponding monocyte mRNA levels were attenuated significantly by PMX-F treatment in both nonsurviving and surviving patients. These data suggest that plasma MMP-9 concentrations and monocyte MMP-9 mRNA levels may be useful prognostic markers in septic shock, and that PMX-F treatment affects MMP-9.

Urinary Podocytes for the Assessment of Disease Activity in Lupus Nephritis

BackgroundDetection of podocytes in the urine indicates that severe injury of podocytes occurred in the glomerulus in children. MethodsThe pathological significance of podocytes in the urine was determined in patients with lupus nephritis. Podocytes were detected by immunofluorescence using a monoclonal antibody against podocalyxin present on the surface of podocytes. Subjects who participated in the present study were of the following types: patients with systemic lupus erythematosus with stable renal function (group A, n = 8; WHO classes IIIa, b, IVb, and IVc at the time of biopsy); patients with clinically active lupus nephritis (group B, n = 8; WHO classes IVb and IVc); and healthy control subjects (group C, n = 10). ResultsPodocytes were absent in the urine of subjects in groups A and C. However, podocytes were present in the urine of group B subjects. Patients in group B were examined monthly for urinary podocytes and were treated with methylprednisolone followed by prednisolone. Urinary podocytes were absent in all patients in group B after treatment. ConclusionsThese data indicate that urinary podocytes may be markers of the severity of lupus nephritis and that steroid therapy may be effective for podocyte injury in lupus nephritis.

Oral ADSORBENT AST-120 decreases carotid intima-media thickness and arterial stiffness in patients with chronic renal failure

Background/Aim: Intima media thickness (IMT) and stiffness of the carotid arteries is related to coronary artery disease, and chronic renal failure patients are at high risk for such diseases. An oral adsorbent, AST-120 (Kremezin; Kureha Chemical Industry, Tokyo, Japan), can delay the progression of chronic renal failure in undialyzed uremic patients. The aim of the present study was to determine whether AST-120 affects carotid artery IMT and pulse wave velocity (PWV) in patients with chronic renal failure not undergoing dialysis. Methods: Fifty patients with non-diabetic chronic renal failure were randomly divided into two groups: 30 patients (18 men and 12 women; mean age 53.5 years; mean serum creatinine 3.2 mg/dl) who were given AST-120 (6.0 g/day) and 20 patients (12 men and 8 women; mean age 52.0 years; mean serum creatinine 3.5 mg/dl) who were not given AST-120. Thirty healthy age-matched subjects (18 men and 12 women; mean age 51.5 years; mean serum creatinine 0.9 mg/dl) were also included. The treatment period was 24 months. IMT and arterial stiffness were measured before and after treatment. Results: The slope of the reciprocal serum creatinine concentration over time became significantly less steep in the AST-120 group than in the non-AST-120 group (p < 0.001). Before treatment, carotid artery IMT differed little between the AST-120 group (0.90 ± 0.22 mm) and the non-AST-120 group (0.88 ± 0.20 mm). IMT in these two groups was significantly greater than IMT in the control group (0.64 ± 0.14 mm) (p < 0.01). Carotid IMT in the AST-120 group decreased slightly but not significantly to 0.84 ± 0.20 mm after 12 months and then significantly after 24 months to 0.78 ± 0.18 mm (p < 0.05). Carotid IMT in the non-AST group showed little change throughout the experimental period. PWV differed little between the AST-120 group (1,980 ± 330 cm/s) and the non-AST group (1,940 ± 360 cm/s) before treatment. PWV values in these two groups were significantly greater than PWV in the control group (1,280 ± 240 cm/s) (p < 0.01). After 12 and 24 months, PWV in the AST-120 group decreased significantly to 1,840 ± 280 cm/s (p < 0.05) and to 1,780 ± 260 cm/s (p < 0.05), respectively; however, PWV in the non-AST group showed a slight increase during the experimental period. Conclusion: The data suggest that AST-120 may reduce arterial stiffness and IMT in non-diabetic chronic renal failure patients before dialysis.

The urinary podocyte as a marker for the differential diagnosis of idiopathic focal glomerulosclerosis and minimal-change nephrotic syndrome.

Background/Aims: Detection of podocytes in the urine sediment of children indicates that severe podocyte injury occurred in the glomerulus. Focal glomerulosclerosis (FGS) and minimal-change nephrotic syndrome (MCNS) are kidney diseases characterized by massive proteinuria. The aim of the present study was to determine whether urinary podocytes can be detected in patients with idiopathic FGS or MCNS and whether immunosuppression therapy alters these cells. Methods: Twenty patients with MCNS (nephrotic stage, n = 12; remission stage, n = 8), 15 patients with FGS and 20 healthy controls were included in the present study. Urinary podocytes were stained by immunofluorescence. All patients with MCNS at the nephrotic stage received prednisolone for 6 months, and all patients with FGS received some form of immunosuppression therapy including prednisolone, cyclophosphamide or mizoribine for 12 months. Results: The 12 nephrotic-stage MCNS patients achieved remission after treatment. Seven of the 15 FGS patients also achieved remission, but the other 8 remained in the nephrotic stage. Urinary podocytes were not detected in any patient with MCNS nor were they detected in healthy controls. Urinary podocytes were detected in all FGS patients (mean, 4.2 cells/ml) before treatment and the number of cells decreased in the 7 patients who achieved remission. The number of podocytes was unchanged in the other 8 patients even after treatment. Conclusion: Urinary podocytes may be a useful diagnostic indicator for differentiation between FGS and MCNS. These cells may also mark disease progression in cases of FGS.

Treatment with polymyxin B-immobilized fiber reduces platelet activation in septic shock patients: Decrease in plasma levels of soluble P-selectin, platelet factor 4 and β-thromboglobulin

Objective: To clarify whether plasma concentrations of soluble P-selectin, platelet factor-4 (PF-4) and β-thromboglobulin (βTG) are altered in patients with septic shock and whether polymyxin B-immobilized fiber (PMX-F) treatment affects these changes.¶Subjects: Thirty patients with septic shock who were treated with PMX-F (group A), 20 such patients who received conventional therapies (group B) and 20 healthy control subjects (group C).¶Methods: ELISA using commercial kits. Endotoxin elimination by direct hemoperfusion using PMX-F.¶Results: Blood endotoxin levels decreased significantly from 49.4 ± 8.8 pg/ml to 13.0 ± 4.5 pg/ml after PMX-F treatment. The pretreatment plasma concentrations of soluble P-selectin, PF-4 and βTG in patients in groups A and B were significantly higher than those in group C (p < 0.001). Plasma concentrations of these factors decreased significantly in group A after PMX-F treatment (p < 0.01); however, the concentrations in group B were not altered after conventional treatment. The survival rate of group A (60%) was higher than that of group B (30%).¶Conclusions: Our findings suggest that soluble P-selectin, PF-4 and βTG may be associated with septic shock and that PMX-F is effective in reducing these markers in patients with septic shock.