Urinary WT1-positive cells as a non-invasive biomarker of crescent formation.

OBJECTIVE: The purpose of this study was to assess the relationship between urinary WT1-positive cells (podocytes and active parietal epithelial cells) and WT1-positive cells in renal biopsy to investigate whether urinary WT1-positive cells are useful for detection of crescent formation. METHODS: Fifty-two patients with kidney disease were investigated (15 cases with crescentic lesions and 37 cases with non-crescentic lesions) for immunoenzyme staining using anti-WT1 antibody for urine cytology and renal biopsy. Numbers of WT1-positive cells in urine and renal biopsy were counted. RESULTS: There was no correlation between urinary WT1-positive cells and WT1-positive cells in renal biopsy. However, the number of urinary WT1-positive cells in patients with crescentic lesions was significantly higher than in patients with non-crescentic lesions. In addition, the best cut-off value to detect patients with crescentic lesions using urinary was 5 cells/10-mL (area under the concentration-time curve=0.735). CONCLUSIONS: The results of our study suggest urinary WT1-positive cells can be used to detect patients with crescent formation using 5 cells/10-mL cutoff value. WT1-positive glomerular podocytes and parietal epithelial cells may be shed into urine in active glomerular disease. This study, investigating the relationship between WT1-positive cells in urine and in the renal biopsy found no correlation; however, the results do suggest that, using a cutoff value of 5 cells/10 mL, WT1 positive urinary cells can be used to detect patients with crescent formation.

WT1 immunoenzyme staining using SurePath(™) processed urine cytology helps to detect kidney disease.

OBJECTIVES: Damage and detachment of podocytes and loss into the urine have been implicated in the progression of kidney diseases. The purpose of this study was to investigate the potential role of urine cytology based on SurePath(™) combined with immunoenzyme staining using Wilms' tumour 1 (WT1) antibody as a podocyte marker in the discrimination of normality and non-renal urinary tract disease from kidney disease. METHODS: Sixty-six patients with kidney disease, 45 patients with lower urinary tract disease and 30 healthy volunteers were examined. Urine cytology slides were prepared using the SurePath method and immunoenzyme stained with WT1 antibody, and the number of WT1-positive cells was counted. RESULTS: In kidney disease, WT1-positive cells were found in 33 (50%) of 66 samples. No WT1-positive cells were found in 45 patients with lower urinary tract disease or in 30 healthy volunteers. The positive rates for WT1 varied with disease type, but not significantly: immunoglobulin A (IgA) nephropathy, (14/23); membranous glomerulonephritis, (4/10); Henoch-Schönlein purpura nephritis, (3/5); diabetic glomerulopathy, (5/5); minor glomerular abnormality/minimal change nephrotic syndrome (0/4). CONCLUSIONS: The results suggest that WT1 immunoenzyme staining of urine cytology can be used to detect some types of kidney disease.

Comparison of formaldehyde and methanol fixatives used in the detection of ion channel proteins in isolated rat ventricular myocytes by immunofluorescence labelling and confocal microscopy.

In this study, a fixation protocol using a 10% neutral buffered formalin (FA) solution and another protocol using a methanol (MeOH) solution were compared for detection of ion channels, Kv1.5, Kv4.2, Cav1.2, Kir6.2, Nav1.5 and Nav1.1 in rat myocytes by immunolabelling. Kv1.5 and Kv4.2 at intercalated discs and Cav1.2 at transverse tubules were not detected by FA but were detected by MeOH. Kir6.2 at transverse tubules and Nav1.5 at sarcolemma were detected by FA but not by MeOH. It is suggested that both FA and MeOH fixation protocols should be used for the detection of cardiac ion channels by immunolabelling.

Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath™ liquid-based cytology compared with fresh urine sediment examination.

OBJECTIVE: To assess whether the morphology of urine erythrocytes can be an effective tool for distinguishing glomerular disease from lower urinary tract disease in SurePath™ liquid-based cytology (SP-LBC). METHODS: We examined four morphological parameters of erythrocytes: (1) irregular erythrocytes (of all types including fragmented forms) comprising greater than or equal to 20% of erythrocytes; (2) uniform erythrocytes (>80%); (3) doughnut or target-like shaped (D/T) erythrocytes (≥1%); and (4) acanthocytes (≥1%) in glomerular disease (n = 32) and lower urinary tract disease (n = 20) with SP-LBC slides in cases that had also been assessed by fresh urine sediment examination. RESULTS: Sensitivity of D/T erythrocytes and acanthocytes (dysmorphic erythrocytes) for glomerular disease were 100% and 87.5%, respectively, with urine sediment examination, and 81.3% and 46.9%, respectively, in SP-LBC slides. Specificity was 100% for D/T erythrocytes and acanthocytes using either procedure. While irregular erythrocytes were specific for glomerular disease using urine sediment examination, they were seen in 70% of those with lower urinary tract disease using SP-LBC slides as a result of the deformation of erythrocytes by the fixative. CONCLUSIONS: Although the sensitivity of D/T erythrocytes and acanthocytes for glomerular disease was lower in SP-LBC slides than fresh urine sediment examination, their specificity was equally high. Therefore, urine erythrocyte morphology is useful in the detection of glomerular disease with the SP-LBC slides. However, morphological features apart from D/T erythrocytes and acanthocytes are not useful in SP-LBC slides.

Expression of vimentin and high-molecular-weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low-grade urothelial carcinoma cells in voided urine.

OBJECTIVE: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low-grade urothelial carcinoma (LG-UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high-molecular-weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG-UC. METHODS: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG-UC. All slides were stained with vimentin and 34ßE12. RESULTS: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG-UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG-UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. CONCLUSIONS: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG-UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.

Value of computer-assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low-grade urothelial carcinoma.

OBJECTIVE: To assess whether computer-assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low-grade urothelial carcinoma cells (LG-UCs) in voided urine. METHODS: Nuclear morphometry was performed by a computer-assisted image analyser system on Papanicolaou-stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG-UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. RESULTS: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG-UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG-UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG-UCs. On the other hand, the linear factor showed a mean higher value among LG-UCs than reactive renal tubular cells (P = 0.023). CONCLUSIONS: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG-UCs.

Can cytological features differentiate reactive renal tubular cells from low-grade urothelial carcinoma cells?

OBJECTIVE: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low-grade urothelial carcinoma cells (LG-UCs). METHODS: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG-UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette-like arrangement, (iv) hobnail-shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann-Whitney U-test and chi-square test, with differences considered significant at P < 0.05. RESULTS: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG-UCs. In LG-UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. CONCLUSIONS: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette-like arrangement, hobnail-shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG-UCs.

'Cannibalism' (cell phagocytosis) does not differentiate reactive renal tubular cells from urothelial carcinoma cells.

OBJECTIVE: Cannibalism of one cell by another in voided urine cytology has been considered a cytological feature for differentiating urothelial carcinoma (UC) from benign lesions. Recently, however, we observed cannibalism in voided urine obtained from patients with renal glomerular disease (RGD). The purpose of this study was to determine the cytomorphological and immunocytochemical characteristics of cannibalism in voided urine from RGD. METHODS: Seventy cytology specimens of voided urine were examined and the findings were compared with the histological findings. In addition, we compared the cytomorphological and immunocytochemical differences in cannibalism found in RGD and cases of UC selected as showing cannabilism. RESULTS: Cannibalism in voided urine was found in three (5.5%) of 55 RGD cases. The finding was measured as (1+) < 5 cells, (2+) 5-20 cells, and (3+) > 20 cells and was (1+) in all three RGD cases, compared with 6.7%, 60% and 33.3% respectively in 15 UC cases. Differences in low cellularity cases (1+) and moderate to high cellularity cases (2+ or 3+) were statistically significant between RGD (3 and 0) and UC (1 and 14) (P=0.005). The maximum diameter of cannibalized cells in RGD was 24.3-33.0 microm (mean 29.8 microm) versus 18.0-30.4 microm (mean 23.3 microm) in UC (P=0.004). Necrosis and isomorphic erythrocytes were absent in RGD, but were found in 46.7% and 86.7%, respectively, of UC cases (P=0.245 and P=0.012). Dysmorphic erythrocytes were identified in all three cases with RGD and 13.3% of UC (P=0.012). Vimentin reactivity was found in all cases with cannibalism in RGD, but never in UC (P=0.001). CONCLUSIONS: Our results demonstrated that cannibalism in voided urine is present not only in UC but also in RGD. Furthermore, we showed that cellularity of cannibalism, vimentin reactivity and background differed significantly and can be used for differential diagnosis between the two groups.

Thymidine kinase-1/CD31 double immunostaining for identifying activated tumor vessels.

Although angiogenesis plays a crucial role in cancer growth and progression, no reliable method for assessing angiogenesis in tumor tissue sections currently is available. Using biomarkers with high specificity for proliferating endothelial cells could help quantify angiogenic activity. Thymidine kinase-1 (TK1) is an enzyme involved in the salvage pathway of DNA synthesis and its activity is correlated with cell proliferation. We investigated the use of double immunostaining for TK1 and CD31 for identifying activated tumor vessels. Differences in TK1/CD31 positive vessel rates (PVRs) between tumor and adjacent normal tissues were evaluated in 39 colorectal carcinoma (CRC) samples and compared with those of Ki67/CD31 double stained tissues. Mean TK1/CD31 PVR (23.6%) in CRCs was 13.9 fold greater than in adjacent normal tissues (1.7%)). By comparison, mean Ki67/CD31 PVR in CRCs was 20.0%, i.e. only 4.8 fold greater than in normal tissues (4.2%). Also, mean TK1/CD31 PVR in normal tissues was significantly less than mean Ki67/CD31 PVR. Our findings indicate that double immunostaining for TK1/CD31 can detect activated tumor vessels more accurately than staining for Ki67/CD31 and potentially could identify tumors that will respond to anti-angiogenic therapy.

Saturation transfer ratio imaging in invasive ductal carcinomas of the breast.

A prospective study was performed to investigate the correlations between saturation transfer ratio (STR) and histologic parameters of invasive ductal carcinomas in human breast. The histologic parameters investigated were the extent of fibrosis in the intercellular matrix, dysplastic changes of nuclei, and mitotic index. Twenty-seven patients with breast carcinoma were examined using an off-resonance saturation pulse in conjunction with conventional field-echo T(1)-weighted imaging at frequency offsets of 448 Hz and 1200 Hz from water resonance. The values of STR at frequency offset of 1200 Hz (STR(1200)) increased from non-scirrhous carcinoma to scirrhous carcinoma. Although STR(1200) showed correlation with the extent of fibrosis in the intercellular matrix (p<0.01, n = 27), they did not correlate with the dysplastic changes of nuclei or mitotic index. On the other hand, the values of STR at frequency offset of 448 Hz (STR(448)) demonstrated close correlation to dysplastic changes of nuclei and mitotic index (p<0.01, n = 27). STR(1200) correlates with the structural characteristics and STR(448) correlates with the nature of malignant cells with regard to nuclear dysplasia and mitotic potential.

Comparison of several methods for the measurement of urinary hippuric acid as an index of toluene exposure.

The levels of hippuric acid in the urine of people exposed to toluene vapour were measured by paper chromatography, direct colorimetry, high performance liquid chromatography and gas chromatography. The control was a similar group not exposed to toluene vapour. The values were analyzed statistically, conversion equations calculated, and the propriety of these equation discussed. Since the three chromatographic methods gave similar values, the measurement of urinary hippuric acid by these methods can be used as an index of toluene exposure. The colorimetric method gave higher levels the chromatographic methods, especially for the urine of people not exposed to toluene. This may have been due to glycine conjugates (other than hippuric acid) developing a similar color, resulting in elevated values for hippuric acid. This colorimetric method should be used with caution for biological evaluation of workers with low toluene exposure.

Stearoyl-CoA desaturase mRNA expression during bovine adipocyte differentiation in primary culture derived from Japanese Black and Holstein cattle.

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids (MUFAs). In cattle, the MUFAs are related to softness and flavor of meat. In order to investigate gene expression profile during bovine preadipocyte differentiation, we isolated stromal-vascular cells from perirenal adipose tissues of Japanese Black and Holstein steers. Gene expression level of adipocyte type fatty acid binding protein (FABP4), SCD, sterol regulatory element binding protein 1 (SREBP1) and CCAAT/enhancer binding protein alpha (C/EBP-alpha) were elucidated by real-time PCR assay. The levels of SCD mRNA expression were significantly increased to 10.8 and 6.3-fold in Japanese Black and Holstein, respectively, on day 1 of the culture. The difference in SCD expression between the two breeds may reflect differences in the fat development characteristics of the cattle breeds. Although transcription factors SREBP1 and C/EBP-alpha are supposed to regulate SCD expression, expression levels of the two factors were not completely consistent with that of SCD.

Role of MLC serum inhibitory factors in high MLC-reactive kidney transplant recipients pretreated with donor-specific blood transfusion (DST).

In order to clarify the beneficial effect of donor-specific blood transfusions (DST) on kidney allograft survival, sera from 16 patients treated with DST were studied using the mixed lymphocyte culture (MLC) serum inhibition test. The results demonstrate that MLC inhibitory factors could be induced in the serum of the recipients after the completion of DST, and that these factors are directed against cells of the recipient but not against cell from the donor. Regarding the correlation with rejection episodes and clinical outcome, a significant improvement in renal transplant survival and reduction in rejection episodes was observed when MLC inhibitory factors were present in post-DST sera. These data suggest that such factors may contain antibodies directed against recognition sites on T lymphocytes, e.g., anti-idiotypic antibodies, and be associated with prolonged graft survival of living-related, high MLC-reactive one-haplotype-mismatched kidney.

A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats.

In view of a cytoprotective effect of elastase inhibitor on chemokine-mediated tissue injury, we examined the neuroprotective effect of ONO-5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine-induced neutrophil chemo-attractant (CINC)-1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC-1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO-5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC-1 protein and mRNA expression. ONO-5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC-1 contributed to inflammation in rat spinal cord injury and ONO-5046 attenuated neurologic damage partly by blocking CINC-1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.