Yasunori Kitamoto

Effect of Captopril on Heavy Proteinuria in Azotemic Diabetics

We investigated whether captopril, an angiotensin-converting-enzyme inhibitor, would reduce proteinuria in patients with advanced diabetic nephropathy. Captopril (37.5 mg given in divided doses three times daily) was administered to 10 azotemic diabetics with heavy proteinuria. Urinary protein decreased promptly within two weeks (from 10.6 +/- 2.2 to 6.1 +/- 1.4 g per day [mean +/- S.E.M.]; P less than 0.01). The decrease in proteinuria did not coincide with a fall in systemic blood pressure or in the blood glucose concentration. Serum creatinine and potassium values did not change in any of the patients except one. We suggest that captopril caused a decrease in intrarenal hypertension, which contributed to the reduction of urinary protein excretion. The therapeutic value of this intervention remains to be established.

Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis.

Homeostasis of body fluid is maintained by the kidneys, which contain two million glomeruli for blood filtration. A glomerulus is formed by growth of Bowmans capsule harmonized with a capillary during kidney development. The vascular endothelial growth factor (VEGF) is an essential angiogenic cytokine, and VEGF deficiency is known to be fatal in mice in early embryonic stages. As secretions of VEGF from cultured kidneys vary according to developmental stages, the role of VEGF in kidney development was studied in vivo by blocking the endogenous VEGF activity with antibody in newborn mice, in which most organs are already developed but kidneys are still developing. The antibody-treated animals showed normal growth but systemic edema. Vessel formation in the superficial renal cortex was disturbed, nephrogenic areas were diminished, and the number of developing nephrons decreased significantly. Many abnormal glomeruli, lacking capillary tufts, were observed in the antibody-treated mice, and VEGF expression in their Bowmans capsule showed a compensatory increase. These results suggest that VEGF mediates communication between the Bowmans capsule and capillary endothelial cells for developing a glomerulus as well as promoting nephrogenesis. In conclusion, VEGF is likely to be an essential molecule for kidney development, and especially for glomerulogenesis.

Differentiation of hematuria using a uniquely shaped red cell.

Although variously shaped urinary red cells have been reported in glomerulonephritic hematuria, no specific shapes with concrete definition have been proposed. This made morphological differentiation of hematuria vague and caused different results among different observers. To solve these problems and improve the diagnostic rate, we employed a uniquely shaped red cell, which only appeared in glomerulonephritic hematuria, as a probe for diagnosis. We studied 182 hematuria cases from 90 glomerulonephritic patients and 95 hematuria cases from 68 urological disease patients. Fresh urine was collected and observed by differential interference microscopy. The red cell, referred to as G1, has a distinctive doughnut-like shape with blebs and was highly specific for glomerulonephritic hematuria. Occurrence of G1 cells increased at lower pH an higher osmolality of urine. A presence of 5% or more G1 cells could be an indicator of glomerulonephritic hematuria. Specificity and sensitivity of this criterion were 100 and 73%. However, when only acidic concentrated urine (pH < or = 6.4, osmolality > or = 400 mosm/kg H2O) was used, the specificity and sensitivity increased to 100 and 99.2%, respectively. Glomerulonephritic and urological hematuria were correctly diagnosed by counting the urinary red cells with doughnut-like shape in acidic and concentrated urine. This method seems to be superior to others in diagnostic rate, simplicity and clarity.

Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1–4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1–4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.

Assessment of thrombin in the urine of glomerulonephritic patients by enzyme-linked immunosorbent assay

Background: Accumulating evidence suggests that blood clotting occurs in inflamed glomeruli, although its role in the pathophysiology of glomerulonephritis remains to be elucidated. To address this issue, a simple and reliable method for evaluating clotting in glomeruli is necessary. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for thrombin in urine to evaluate the degree of clotting activation in diseased glomeruli. Methods: Monoclonal antibodies against human α-thrombin were raised and used for sandwich ELISA to measure thrombin. Thrombin was measured in urine samples from normal volunteers and from patients with glomerulonephritis or disseminated intravascular coagulation (DIC). Results: Thrombin antigen was not detected in the urine of healthy volunteers or of patients with DIC, but was detected in the urine from two-thirds of glomerulonephritic patients. The average concentration in positive samples was 3.79 µg/L. Urinary thrombin concentrations measured by ELISA correlated well with thrombin activities measured by hydrolysis of a synthetic substrate. Conclusion: We suggest that thrombin antigen in urine measured by ELISA is not affected by systemic thrombin production in the vessels, and reflects blood clotting activation in glomerulonephritic lesions. A close relationship between urinary thrombin and glomerulonephritis indicates a possible involvement of clotting in disease development, and measurement of urinary thrombin may provide a real-time marker for monitoring renal diseases.

Suppression of thrombin formation during hemodialysis with triglyceride.

Effect of blood-air contact in the venous line air-trap chamber on blood clotting was studied. Fourteen chronic hemodialysis patients (six men, eight women; mean age, 49 years) with elevation of thrombin-antithrombin III were studied. To prevent blood-air contact, triglyceride (NOF-005) was floated over the blood in the chamber. Control hemodialysis was performed for 4 weeks and hemodialysis using NOF-005 followed for the next 4 weeks. Clot formation in the circuit was examined after each hemodialysis and clotting factors including thrombin-antithrombin III, FXII Antigen, vWF Antigen, PF4 and beta-thromboglobulin were measured before and after the last hemodialysis of each control and NOF-005 hemodialysis. Clotting in the chamber was improved when NOF-005 was used. Thrombin-antithrombin III increase during hemodialysis was suppressed to about 30% of control values by using NOF-005. blood-air contact seems to promote thrombin generation and accelerate clot formation.

Dermal Patch Anesthesia: Pain-Free Puncture of Blood Access in Hemodialysis Patients

Clinical application of dermal patch anesthesia to relieve pain at venous cannulation of blood-access was studied in hemodialysis patients. Aqueous gel of 10% lidocaine base with 3% glycyrrhetinic acid monohemiphthalate disodium (GA MHPh 2Na) was applied for 60 minutes to the skin of the patients. Degree of pain was expressed as a pain score. Analgesic effect of the lidocaine gel was evaluated in 16 patients in a placebo-controlled, double-blind, cross-over design by comparing the gel with lidocaine with a placebo gel without lidocaine. The mean pin-prick pain score (1.0 +/- 0.5) in the lidocaine gel patch (n = 16) was significantly lower than that (2.3 +/- 0.3) in the placebo gel patch (P < 0.01). In 8.8% of the patients, blood pressure was elevated after venous cannulation, but this tendency was modified by dermal patch anesthesia with the lidocaine gel. Plasma concentration of lidocaine was under the detection limit of assay (< 0.05 micrograms/mL) after dermal patch anesthesia in six subsequent dialysis treatments.

Urinary thrombin: A novel marker of glomerular inflammation for the diagnosis of Crescentic glomerulonephritis (prospective observational study)

Background Crescentic glomerulonephritis (CresGN), an uncommon rapidly progressive disease, is characterized by severe glomerular inflammation with fibrin deposition. The lack of specific CresGN biomarkers delays diagnosis and threatens life. Because fibrin deposits in CresGN glomeruli indicate thrombin generation, we hypothesized that thrombin is excreted in urine and is a specific CresGN biomarker. Methods We measured urinary thrombin activity in 200 untreated patients (17 with CresGN, 183 with primary glomerulonephritis) and controls (8 patients with healed CresGN, 11 with nephrosclerosis, and 10 with tubulointerstitial nephritis, and 66 healthy volunteers). CresGN types included 15 pauci-immune and 2 immune complex. We assessed the diagnostic accuracy of thrombinuria in 169 patients with hematuria and proteinuria. Renal biopsy tissues were immunostained for tissue factor and fibrin. We analyzed the relationship of thrombinuria to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, glomerular fibrin deposition, antineutrophil cytoplasmic antibodies (ANCAs), and C-reactive protein (CRP). We studied changes in thrombin activities after glucocorticoid treatment in 12 patients with thrombinuria. Results The highest thrombinuria occurrence was in CresGN (70.6%), followed by membranoproliferative glomerulonephritis (41.7%), IgA nephropathy (9.2%), and acute glomerulonephritis (0%). More than 75% of patients with nonproliferative glomerulonephritis manifested no thrombinuria. No controls had thrombinuria. Thrombinuria showed high CresGN specificity (90.1%) and moderate sensitivity (70.6%) and was detected in 4 of 7 patients with ANCA-negative CresGN. In CresGN, thrombinuria was associated with fibrin deposition in glomerular extracapillary tissue, where monocytes/macrophages expressed tissue factor. Thrombinuria in CresGN was unrelated to plasma thrombin-antithrombin complex, hematuria, proteinuria, glomerular filtration rate, and CRP. After glucocorticoid treatment, thrombinuria in patients with CresGN rapidly disappeared but proteinuria and hematuria persisted. Conclusions Thrombinuria was specific for glomerular inflammation, was unaffected by systemic inflammation or coagulation, and demonstrated good diagnostic accuracy for CresGN including ANCA-negative cases. Thrombinuria measurement may provide risk-free diagnosis and screening for CresGN.

Anti-VEGF therapy for crescentic glomerulonephritis?

rom 0.7 to 4.8 mg/dL (62-424 mol/L), but there was no vidence of nonlinearity in the calibration (r-square of 0.9994 n Fig 3 of with slopes of 0.900 and 0.907 for values below nd above 2.0 mg/dL [177 mol/L], respectively; P value or the interaction, 0.5). The points near 1 mg/dL (88.4 mol/L) appear closer to the line of identity but not more so han one would expect with a 0 intercept and constant slope f 0.9, which implies a distance of 0.1 mg/dL at 1.0 mg/dL nd 0.2 mg/dL at 2.0 mg/dL. Therefore, application of the alibration to a more limited range of serum creatinine alues is appropriate. In contrast, our NHANES paper finds different slope in the association between a kinetic rate affé assay and the Roche enzymatic assay. However, it hould be noted that across the NHANES datasets, the lopes (standard error) showed substantial variation, at 0.939 0.015), 0.997 (0.012), 1.010 (0.013), and 0.976 (0.013) Table 1 of). Thus, re-expression of the MDRD Study quation was based on data specifically comparing the DRD Study creatinine results to an assay traceable to old-standard methods, with all assays done in the Cleveand Clinic Research Laboratory. We do not think it would e helpful to combine data from other laboratories in rexpressing the MDRD Study equation. The MDRD Study quation is less useful with low serum creatinine values ecause at high values of estimated GFR, the formula nderestimates measured GFR. Josef Coresh, MD, PhD Elizabeth Selvin, PhD Lesley A. Stevens, MD, MS Andrew S. Levey, MD Fredrick Van Lente, PhD Johns Hopkins Medical Institutions Baltimore, Maryland Tufts-New England Medical Center Boston, Massachusetts Cleveland Clinic Foundation Cleveland, Ohio

Flow cytometric analysis of hematuria using fluorescent antihemoglobin antibody

To identify the morphological changes of urinary erythrocytes in hematuria objectively, flow cytometrical analysis of fluorescence-labeled erythrocytes was performed. Fifty-one fresh urine samples from 33 hematuric patients (16 with glomerulonephritis and 17 with urological disease) were obtained. Urine erythrocytes were stained with FITC-labeled antihemoglobin antibody, and distinguished from other particles with similar size. Forward scattered light intensity (FW-SC) was used as an indicator of red cell size and right orthogonally scattered light intensity (RT-SC) divided by forward scattering (RT-SC/FW-SC) was used as a marker of cell surface irregularity. The size of erythrocytes expressed by FW-SC was significantly smaller in glomerulonephritic hematuria (101.6 +/- 41.8) than that in urological hematuria (123.5 +/- 44.7). RT-SC/FW-SC was smaller in urological hematuria (1.22 +/- 0.18) in comparison with glomerulonephritic hematuria (1.33 +/- 0.12). These results suggest that erythrocytes in glomerulonephritic hematuria had smaller size and more complex surface structure in comparison with urological hematuria. When cutoff was set at 110 of FW-SC for the criteria of glomerulonephritic erythrocytes, a correct diagnosis was made in 73.3% (22/30) of glomerulonephritic hematuria and in 76.2% (16/21) of nonglomerular hematuria. We clarified more complex morphological changes of glomerulonephritic urinary erythrocytes objectively.