Keiko Sekizuka

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.

Detection of Serum IL-6 in Patients with Diabetic Nephropathy

Yasuhiko Tomino. MD, Division of Nephrology, Department of Medicine, Juntentto University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113 (Japan) Dear Sir, A study on the detection of serum IL-6 in patients with non-insulin-dependent diabetes mellitus (NIDDM) with or without nephropathy is described. IL-6 is generally regarded as a multifunctional cytokine which has a variety of biological activities, including the ability to stimulate bone marrow stem cell proliferation, B cell differentiation, immuno-globulin secretion, T cell activation, and acute phase protein synthesis [1, 2], IL-6 is also produced by the renal glomerular mesan-gial cells. Cytokines are known to play an important role in autoimmunity and appear to be involved in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). However, Cavallo et al. [3] reported that detectable levels of serum IL-6 were observed in only 10% of IDDM patients. Serum samples were obtained from 9 patients with NIDDM with nephropathy (diabetic nephropathy), 9 patients with NIDDM without nephropathy and 29 patients with chronic glomerulonephritis (CGN). NIDDM was diagnosed with a 75-gram glucose tolerance test. Patients with diabetic nephropathy continuously showed more than 200 mg/24 h. Serum IL-6 levels were measured with ELISA as described previously [4]. Mouse monoclonal anti-IL-6 antibody (HH61-10) and monoclonal horse radish peroxidase-conjugated anti-IL-6 antibody (HH61-2 Fab’) were used in a double-antibody sandwich ELISA [5]. Levels of serum IL-6 of healthy controls were less than 4.0 pg/ml [5]. The mean levels of serum IL-6 in all patients with NIDDM were significantly higher than those in patients with CGN (p < 0.05). The levels of serum IL-6 in patients with diabetic nephropathy were significantly higher than those in cases of CGN or NIDDM without nephropathy (p < O.Ol and p < 0.05, respectively; table 1). It appears that the presence of IL-6 in the patients’ sera may reflect increased localized production of this cytokine at the pancreatic and/or glomerular me-sangial levels. The measurement in serum IL-6 may add

Effects of angiotensin-converting enzyme inhibitor, angiotensin II receptor antagonist and calcium antagonist on urinary podocytes in patients with IgA nephropathy.

The urinary podocyte is postulated to be a marker for estimation of the severity of active glomerular injury and a predictor of disease progression in children with glomerulonephritis. Non-dihydropyridine calcium antagonist, including verapamil, reduce proteinuria to an extent similar to that of the angiotensin-converting enzyme inhibitor (ACEI), including trandolapril, but to a greater extent than other antihypertensives. Angiotensin (Ang) II receptor antagonists, including candesartan cilexetil, show potent and long-term preventive effects against the progression of renal injury. The aim of the present study is to assess whether verapamil, trandolapril and candesartan cilexetil affect proteinuria and urinary podocytes in patients with IgA nephropathy. Thirty-two normotensive patients aged 18-54 years with biopsy-proven IgA nephropathy, nonnephrotic proteinuria (1-3 g/day), and normal renal function (creatinine clearance >80 ml/min) were studied. Twenty patients with diffuse mesangial proliferative glomerulonephritis (non-IgA PGN) and 20 healthy controls were also included in this study. The number of urinary podocytes in patients with advanced IgA nephropathy (n = 16) was significantly higher than that in patients with the disease in the mild stage (n = 16) (p < 0.01) or in patients with non-IgA PGN (p < 0.01). Urinary podocytes were not detected in healthy controls. The 32 patients with IgA nephropathy were randomly divided into four treatment groups: those treated with verapamil (120 mg/day, n = 8); those treated with trandolapril (2 mg/day, n = 8); those treated with candesartan cilexetil (8 mg/day, n = 8), and those given a placebo (n = 8). Treatment continued for 3 months. Antiproteinuric response in the trandolapril group was similar to that in the candesartan cilexetil group (-38 vs. -40%). The action of trandolapril or candesartan cilexetil was greater than that of verapamil (p < 0.01). Reduction in the number of urinary podocytes from baseline was significantly greater in patients treated with trandolapril or candesartan cilexetil than in patients treated with verapamil (p < 0.01). However, there was no difference between patients treated with trandolapril and those treated with candesartan cilexetil. Proteinuria and urinary podocytes were unaffected in the placebo group. These data suggest that urinary podocytes may be a marker of disease activity in adult patients with IgA nephropathy and that trandolapril and candesartan cilexetil are more effective than verapamil in reducing the number of podocytes.

Significance of urinary type IV collagen in patients with diabetic nephropathy using a highly sensitive one‐step sandwich enzyme immunoassay

Urinary concentrations of type IV collagen in patients with diabetic nephropathy were measured by a highly sensitive, one‐step sandwich enzyme immunoassay. Samples from 298 patients with non‐insulin‐dependent diabetes mellitus (NIDDM) and 80 healthy controls were examined. In diabetic patients with macroalbuminuria or renal insufficiency, the concentrations of urinary type IV collagen were significantly higher than those of diabetic patients with normoalbuminuria or healthy controls (P < 0.001). Urinary type IV collagen concentration in diabetic patients with microalbuminuria was significantly higher than that in diabetic patients with normoalbuminuria or that in healthy controls (P < 0.001). In contrast, there were no significant changes in the concentration of serum type IV collagen between microalbuminuric patients and normoalbuminuric patients. The area under the receiver operating characteristic (ROD) curve for the urinary type IV collagen concentration was equivalent to that of urinary albumin. It was concluded that urinary type IV collagen concentration determined using this method might be a useful marker for the early detection of diabetic nephropathy. J. Clin. Lab. Anal. 11:110–116.

Effects of LDL apheresis and vitamin E-modified membrane on carotid atherosclerosis in hemodialyzed patients with arteriosclerosis obliterans

Background: Hemodialysis patients manifest accelerated atherosclerosis. Hemodialysis is associated with oxidative stress, which can be partially prevented with the use of a vitamin E-coated dialyzer. Adsorption of low-density lipoprotein (LDL) has been applied in the treatment of arteriosclerosis obliterans (ASO). The aim of the present study was to determine whether the vitamin E-coated dialyzer and/or LDL apheresis affects carotid atherosclerosis in hemodialysis patients with ASO. Methods: Thirty hemodialysis patients with ASO were divided into four treatment groups: treatment with conventional cellulose or synthetic membranes (group A, n = 12), treatment with vitamin E-coated membrane (group B, n = 7), treatment with conventional membrane and LDL apheresis (group C, n = 6), and treatment with vitamin E-coated membrane and LDL apheresis (group D, n = 5). Carotid artery intima-media thickness (IMT) and arterial stiffness assessed by pulse wave velocity (PWV), plasma C-reactive protein (CRP) and interleukin (IL)-6 were measured before and 10 weeks after treatment and compared between groups. All values were referred to measurements after LDL apheresis. Results: IMT and PWV, plasma CRP and IL-6 showed little change in group A throughout the experimental period. These decreased slightly from the baseline value in group B, but the change was not significant. In group C, IMT decreased from 1.12 ± 0.24 to 1.02 ± 0.18 mm (p < 0.05), and PWV decreased from 2,266 ± 380 to 1,968 ± 342 cm/s (p < 0.05). Plasma CRP and IL-6 concentrations also decreased significantly compared with baseline (p < 0.05). In group D, IMT decreased from 1.18 ± 0.26 to 0.92 ± 0.18 mm (p < 0.01), and PWV decreased from 2,284 ± 390 to 1,786 ± 284 cm/s (p < 0.01). Plasma CRP and IL-6 levels also decreased significantly compared with baseline (p < 0.01). Conclusion: These data suggest that LDL apheresis and the vitamin E-coated membrane dialysis in combination may prevent further progression of atherosclerosis in hemodialysis patients with ASO.

Follow-up study on urinary type IV collagen in patients with early stage diabetic nephropathy

Type IV collagen is a major component released from the glomerular and tubular basement membranes. To investigate the alteration of renal type IV collagen turnover in early stage diabetic nephropathy, urinary type IV collagen was measured by a highly sensitive one‐step sandwich enzyme immunoassay (EIA). Urinary samples were obtained from 94 diabetic patients without overt proteinuria. Among those patients, 61 were normoalbuminuric and 33 patients were in the microalbuminuric group. Levels of urinary type IV collagen were serially examined at the start of this study and again one year later. The levels of urinary type IV collagen in patients in the microalbuminuric group were significantly higher than those in the normoalbuminuric group (P < 0.01). There was a significant correlation between the concentration of urinary albumin and urinary type IV collagen in both groups (P < 0.05). Twenty‐eight patients (45.3%) in the normoalbuminuric group who showed an abnormal elevation of urinary type IV collagen in comparison to the reference range of normal healthy adults (normal range; less than 3.5 μg/g · Cr). Seven (25%) out of these 28 normoalbuminuric patients with increased urinary type IV collagen progressed to the microalbuminuric group one year later. The levels of urinary type IV collagen in such patients were significantly increased. In the 21 patients who stayed within the normoalbuminuric group, the urinary type IV collagen levels were significantly decreased one year later. It appears that the levels of urinary type IV collagen might reflect ongoing alteration of the extracellular matrix (ECM) turnover and might define more specifically the early stage diabetic nephropathy than the detection of microalbuminuria. It is concluded that the serial measurement of urinary type IV collagen can be a useful marker for detecting renal injury in diabetes. J. Clin. Lab. Anal. 12:378–382, 1998.

Effects of treatment with angiotensin-converting enzyme inhibitor (ACEI) or angiotensin II receptor antagonist (AIIRA) on renal function and glomerular injury in subtotal nephrectomized rats

The aim of this study was to determine if treatment with angiotensin‐converting enzyme inhibitors (ACEI) or angiotensin II receptor antagonists (AIIRA) might decrease urinary albumin excretion and prevent glomerular enlargement and glomerulosclerosis in subtotal (5/6) nephrectomized rats. Morphometric image analysis of glomeruli was also performed in the subtotal nephrectomized rats. The nephrectomized rats were treated with ACEI (enalapril 100mg/l), AIIRA (L‐158,809 10 mg/l) or TRX (reserpine 5 mg/l, hydralazine 80 mg/l, and hydrochlorothiazide 25 mg/l) and euthanized at 16 weeks after renal ablation. Treatments were started at 2 weeks (early treatment: Group I) or 8 weeks (later treatment: Group II) after the ablation. ACEI and AIIRA treatments were equally and significantly effective in limiting albuminuria and progression of glomerular sclerosis. TRX was also as effective in decreasing urinary albumin excretion and preserving the renal function as ACEI or AIIRA in Group I. The improvement of albuminuria, glomerular enlargement and sclerosis after these treatments in Group II was significantly less than that in Group I. It appears that the early treatment with angiotensin converting enzyme inhibitor, angiotensin II receptor antagonist or reserpine, hydralazine and hydrochlorothiazide (TRX) may prevent glomerular injury in human patients with renal hypertension. J. Clin. Lab. Anal. 11:53–62.

Detection of Urinary MCP-1 in Patients withDiabetic Nephropathy

Accessible online at: http://BioMedNet.com/karger Dear Sir, A study of the detection of urinary monocyte chemoattractant protein-1 (MCP-1) in patients with diabetic nephropathy is described. MCP-1 is generally considered to be chemotactic for monocytes and T lymphocytes. A variety of cell types, including glomerular endothelial cells, mesangial cells and monocytes, produce MCP-1 in response to inflammatory signals, including cytokines (IL-1, TNF-· and INF-Á) and immune complexes [1, 2]. Urinary samples were obtained from 5 patients in the macroalbuminuric stage of diabetic nephropathy and 6 patients in the microalbuminuric stage of this disease. Patients in the macroalbuminuric stage of diabetic nephropathy continuously showed more than 300 mg/g WCr of urinary albumin. Urinary MCP-1 levels were measured by a quantitative sandwich ELISA as described previously [3]. A murine monoclonal antibody against MCP-1 and MCP-1 conjugate was used in this method. Mean levels of urinary MCP-1 in patients with the macroalbuminuric stage of diabetic nephropathy or all patients with diabetic nephropathy were significantly higher than in healthy controls (p ! 0.01 and p ! 0.05, respectively). The levels of urinary MCP-1 in the macroalbuminuric stage were slightly higher than those in the microalbuminuric stage, but there was no significant difference. The levels of BUN, s-Cr, HbA1c and FBS in the macroalbuminuric stage were slightly higher than those in the microalbuminuric stage, but there was no significant difference (table 1). Patients who showed high levels of FBS and HbA1c had an increase in urinary MCP-1 levels. We already reported that IL-6 is produced by the glomerular mesangial cells, and it appears that the presence of IL-6 in the patients’ sera may reflect an increase in localized production of this cytokine in the pancreatic and/or glomerular mesangial cells [4]. It is speculated that the high glucose and/or some cytokines, i.e. IL-6, might stimulate MCP-1 production from renal cells, such as glomerular mesangial cells and/or tubular epithelial cells. It was thus suggested that measurement of urinary MCP-1 is useful in evaluating the degree of renal injuries and/or prognosis in patients with diabetic nephropathy. OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Comparison between the Angiotensin II Receptor Antagonist Candesartan Cilexetil and the Angiotensin-Converting Enzyme Inhibitor Trandolapril in Microalbuminuria of Patients with Early Diabetic Nephropathy

Accessible online at: www.karger.com/journals/nef Dear Sir, Experimental and clinical studies show that in various kinds of glomerular diseases, angiotensin II (AngII) contributes to the appearance of proteinuria and that angiotensin-converting enzyme inhibitors (ACEIs) significantly reduce proteinuria [1]. Intervention trials have demonstrated that ACEIs have a particular beneficial effect over other antihypertensive drugs in retarding the development and progression of diabetic nephropathy, implicating the renin-angiotensin system in its pathogenesis [2]. AngII receptor antagonists have recently been introduced as a new class of therapeutic drugs for the treatment of hypertension. AngII receptor antagonists have a more direct mechanism of action than other drugs affecting the renin-angiotensin system [3]. Recently, Russo et al. [1] reported that an ACEI (enalapril) and an AngII receptor antagonist (losartan), when administered alone, reduced proteinuria to the same extent in normotensive patients with IgA nephropathy. Candesartan cilexetil is a potent, highly selective AngII receptor antagonist devoid of agonist activity. It has a distinctive receptorbinding property characterized, in current pharmacologic terms, as being insurmountable [4]. Noda et al. [5] have reported that candesartan cilexetil shows potent and longterm preventive effects against the progression of renal injury in rats. Trandolapril is a fairly new, orally active, long-acting nonsulfhydryl ACEI, suitable for a once-daily regimen [6]. We recently compared the antimicroalbuminuric effects of candesartan cilexetil and trandolapril in patients with early diabetic nephropathy. Thirty patients with microalbuminuria were divided into two groups at random. Group A patients (10 men and 5 women, age 52.6 B 10.4 years) were treated with 8 mg candesartan cilexetil once daily. Group B patients (9 men and 6 women, age 51.6 B 9.8 years) were treated with 2 mg trandolapril once daily. After 8 weeks, microalbuminuria was reduced to the same extent in each group: from 124.6 B 66.2 to 38.2 B 16.4 Ìg/min (p ! 0.01) in the candesartan cilexetil group and from 132.2 B 70.8 to 40.6 B 18.4 Ìg/min (p ! 0.01) in the trandolapril group. A significant change in blood pressure was not seen in either group (from 124.6 B 12.8 to 116.8 B 10.8 mm Hg in group A and from 122.8 B 13.6 to 117.6 B 12.2 mm Hg in group B). Candesartan cilexetil may have a renoprotective effect similar to that of trandolapril and not related to the decrease in blood pressure in normotensive microalbuminuric patients with early diabetic nephropathy. It would be of interest to evaluate whether combination therapies have an additional renoprotective effect in patients with diabetic nephropathy. References

Effect of Polymyxin B-Immobilized Fiber Hemoperfusion on Sepsis-Induced Rhabdomyolysis with Acute Renal Failure

Accessible online at: www.karger.com/journals/nef Dear Sir, Rhabdomyolysis is a clinical and laboratory syndrome resulting from leakage of muscle cell contents into the plasma [1]. Gabow et al. [2] reported that infection, mainly viral and, to a lesser extent, bacterial, accounts for 5% of reported cases of rhabdomyolysis. The mechanism of bacterial sepsis remains unclear, but it is likely to be related to multiple factors: anoxia, impaired metabolism, hyperosmolality, various mediators including cytokines and toxins, and direct bacterial invasion to muscle fibers [3, 4]. Streptococcus pneumonia and Staphylococcus aureus are the most common gram-positive bacteria known to induce rhabdomyolysis [5, 6]. Betrosian et al. [1] recently reported gram-negative bacteria in patients with sepsis-induced rhabdomyolysis. Acute renal failure is a common complication of severe rhabdomyolysis [2]; direct toxic effects of myoglobin or its decomposition products on renal tubules, renal ischemia due to release of vasoconstrictive mediators, and a decreased glomerular perfusion rate have been proposed as causative factors [1]. An endotoxin removal cartridge composed of polymyxin B-immobilized fiber (PMX-F) has been developed in Japan, and PMX-F therapy has been effective in improving mortality and morbidity among septic patients [7, 8]. We studied whether PMXF affects survival associated with acute renal failure and sepsis-induced rhabdomyolysis in 22 such patients. All 22 septic patients had a blood culture positive for gram-negative bacteria and elevated serum creatine phosphokinase levels (above 2,500 IU/l) [1], either on admission or during their hospital stay. All patients were treated conventionally for sepsis in one of our intensive care units by intravenous fluid infusion, antibiotics, and Á-globulins. Rhabdomyolysis was treated with adequate hydration and forced diuresis with frusemide. The 22 patients were randomly divided into 4 groups: group 1, conventional treatment only (n = 5); group 2, conventional treatment + hemodialysis (n = 5); group 3, conventional treatment + PMX-F (n = 6); group 4, conventional treatment + hemodialysis + PMX-F (n = 6). The groups were similar with regard to age, APACHE II score, mean blood pressure, and serum creatine phosphokinase levels. PMX-F and the hemoperfusion column have been described previously [7, 8]. PMXF treatment was repeated twice in all group 3 and group 4 patients as previously described [9]. Survival rate was 20% in group 1, 20% in group 2, 83% in group 3, and 83% in group 4. These data suggest that the hemodialysis itself did not affect the survival rate in these acute renal failure patients with bacterial sepsis-induced rhabdomyolysis and that the PMX-F was the agent responsible for the improved survival in these patients. References