Urinary Sediment Cells Research Articles

Nuclear factor kappa B in urine sediment: a useful indicator to detect acute kidney injury in Plasmodium falciparum malaria

BackgroundAcute kidney injury (AKI) is one of the major complications of Plasmodium falciparum malaria, especially among non-immune adults. It has recently been revealed that activation of transcription factor nuclear factor kappa B (NF-κB) induces pro-inflammatory gene expression involved in the development of progressive renal inflammatory diseases. The aim of this study was to determine whether urinary sediment NF-κB p65 can act as a biomarker for AKI in patients with P. falciparum malaria.MethodsUrinary sediments from malaria patients, including Plasmodium vivax malaria, uncomplicated P. falciparum malaria, complicated P. falciparum malaria without AKI (serum creatinine-Cr <3 mg/dl) and complicated P. falciparum malaria with AKI (Cr ≥3 mg/dl) were used to determine NF-κB p65 level by sandwich enzyme-linked immunosorbent assay (ELISA). Urinary sediments obtained from healthy controls were used as a normal baseline. Correlations between levels of urinary sediment NF-κB p65 and pertinent clinical data were analysed.ResultsUrinary sediment NF-κB p65 levels were significantly increased on the day of admission (day 0) and on day 7 post-treatment in complicated P. falciparum malaria patients with AKI, compared with those without AKI (p = 0.001, p <0.001, respectively), P. vivax patients (all p <0.001) and healthy controls (all p <0.001). NF-κB p65 levels in urinary sediment cells showed a significant positive correlation with serum Cr (Day 0: rs = 0.792; p <0.001, Day 7: rs = 0.605; p <0.001) and blood urea nitrogen (BUN) (Day 0: rs = 0.839; p <0.001, Day 7: rs = 0.596; p <0.001).ConclusionsUrinary sediment NF-κB p65 level is a useful indicator for estimating renal tubular epithelial cell damage and subsequent development of AKI among patients with P. falciparum malaria.

Successful fabrication of a convex platform PMMA cell-counting slide using a high-precision perpendicular dual-spindle CNC machine tool

This study presents a novel approach to the fabrication of a biomedical-mold for producing convex platform PMMA (poly-methyl-meth-acrylate) slides for counting cells. These slides allow for the microscopic examination of urine sediment cells. Manufacturing of such slides incorporates three important procedures: (1) the development of a tabletop high-precision dual-spindle CNC (computerized numerical control) machine tool; (2) the formation of a boron-doped polycrystalline composite diamond (BD-PCD) wheel-tool on the machine tool developed in procedure (1); and (3) the cutting of a multi-groove-biomedical-mold array using the formed diamond wheel-tool in situ on the developed machine. The machine incorporates a hybrid working platform providing wheel-tool thinning using spark erosion to cut, polish, and deburr microgrooves on NAK80 steel directly. With consideration given for the electrical conductive properties of BD-PCD, the diamond wheel-tool is thinned to a thickness of 5 µm by rotary wire electrical discharge machining. The thinned wheel-tool can grind microgrooves 10 µm wide. An embedded design, which inserts a close fitting precision core into the biomedical-mold to create step-difference (concave inward) of 50 µm in height between the core and the mold, is also proposed and realized. The perpendicular dual-spindles and precision rotary stage are features that allow for biomedical-mold machining without the necessity of uploading and repositioning materials until all tasks are completed. A PMMA biomedical-slide with a plurality of juxtaposed counting chambers is formed and its usefulness verified.

Renal prognosis a long time after renal biopsy on patients with diabetic nephropathy

BackgroundA new classification of diabetic nephropathy was reported by Tervaert et al., but the association between pathological findings and the clinical outcomes remains unclear.MethodsAmong 310 patients with diabetes mellitus who underwent renal biopsy from March 1985 to January 2010 and were confirmed to have diabetic nephropathy according to the Tervaert's classification, 205 patients were enrolled in this study. Cox proportional hazard regression analysis was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) for death-censored renal death. Each regression analysis employed two levels of multivariate adjustment.ResultsAfter adjustment for age, gender, estimated glomerular filtration rate, type of diabetes, urinary protein excretion, systolic blood pressure, body mass index, HbA1c, diabetic retinopathy and red blood cells in urinary sediment at the time of renal biopsy, compared with glomerular class IIA, the HRs for death-censored renal death of glomerular classes I, IIB, III and IV were 0.21 (95% CI: 0.04–1.25), 2.12 (0.89–5.04), 4.23 (1.80–9.90), and 3.27 (1.32–8.10), respectively. Also, compared with an interstitial fibrosis and tubular atrophy score 1 group, HRs for score 0 group, score 2 group and score 3 group were 0.08 (0.01–0.57), 2.17 (0.96–4.91), 4.78 (1.96–11.68), respectively.ConclusionsThe progression of glomerular, tubulointerstitial and vascular lesions was associated with higher HRs for renal death. These results suggest the clinical utility of Tervaert's pathological classification.

Multiplex Protein Signature for the Detection of Bladder Cancer in Voided Urine Samples

Accurate urine assays for bladder cancer detection would benefit patients and health care systems. Through extensive genomic and proteomic profiling of urine components we previously identified a panel of 8 biomarkers that can facilitate the detection of bladder cancer in voided urine samples. In this study we confirmed this diagnostic molecular signature in a diverse multicenter cohort. We performed a case-control, phase II study in which we analyzed voided urine from 102 subjects with bladder cancer and 206 with varying urological disorders. The urinary concentration of 8 biomarkers (IL-8, MMP-9 and 10, PAI-1, VEGF, ANG, CA9 and APOE) was assessed by enzyme-linked immunosorbent assay. Diagnostic performance of the panel of tested biomarkers was evaluated using ROCs and descriptive statistical values, eg sensitivity and specificity. Seven of the 8 urine biomarkers were increased in subjects with bladder cancer relative to those without bladder cancer. The 7 biomarkers were assessed in a new model, which had an AUROC of 0.88 (95% CI 0.84-0.93), and 74% sensitivity and 90% specificity. In contrast, the sensitivity of voided urine cytology and the UroVysion® cytogenetic test in this cohort was 39% and 54%, respectively. Study limitations include analysis performed on banked urine samples and the lack of voided urine cytology and cytogenetic test data on controls. The study provides further evidence that the reported panel of diagnostic biomarkers can reliably achieve the noninvasive detection of bladder cancer with higher sensitivity than currently available urine based assays.

Expression and significance of respiratory chain enzyme of cells in urine sediment in MELAS syndrome

To explore the expression and significance of respiratory chain enzyme of cells in urine sediment in mitochondrial encephalopathy myopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Through enzyme histochemistry, the authors analyzed the changes of respiratory chain enzyme in urine sediment in 20 MELAS patients due to mitochondrial A3243G mutation (MELAS group) and 20 health peoples (control group). And the impact on the expression of protein encoded by nuclear DNA (A21347) and mitochondrial DNA (A6404) was detected by immunochemistry. Image pro Plus 6.0 software was used for analysis of absorbance (A) of staining images as staining intensity. The data were expressed as M (Q1, Q3) and analyzed through statistical software. The staining intensity of complexes Iin the MELAS group was lower than that in the control group (0.06(0.01, 0.12) vs 0.12(0.01, 0.62), P = 0.010). The intergroup staining intensity of complex II showed no marked difference. Increased density of blue particle and cytoplasmic gathering was found in 13 cased (65%) of the MELAS group under light microscope. The staining intensity of complexes IV was expressed at a low level in the MELAS group (0.14(0.03, 0.32) vs 0.23(0.06, 0.43), P = 0.038). The expression of protein encoded by nuclear DNA (A21347) was lower than that in the control group (0.05(0.02, 0.45) vs 0.17(0.03, 0.70), P = 0.000). The expression of protein encoded by mitochondrial DNA (A6404) was also lower than that in the control group (0.03(0.01, 0.07) vs 0.15 (0.09, 0.23), P = 0.000). Abnormal change of respiratory chain enzyme in urine sediment in MELAS due to mitochondrial A3243G mutation and a low expression of proteins encoded by two kinds of DNA in complexes IV can help to confirm the genetic diagnosis of mitochondrial encephalomyopathies so that different subtypes may be classified and its pathogenesis elucidated.

Tumor suppressor gene methylation in tissue and urine specimens of bladder transitional cell carcinoma

Objective To investigate abnormal promoter methylation status of death-associated protein kinase(DAPK),retinoic acid receptorβ(RARβ),Hyperplastic polyps protein(HPP1) and integrin alpha-4 precursor (ITGA4) genes,and the diagnostic value for bladder transitional cell carcinoma (BTCC).Methods Methylation-specific polymerase chain reaction (MSP) was used to detect the methylation status of DAPK,RARβ,HPP1 and ITGA4 genes in tissue and urine specimens of 112 patients with BTCC.Results The methylation rate of DAPK,RARβ,HPP1 and ITGA4 in BTCC tissue was 29.4％,91.0％,75.0％ and 48.2％ respectively,and that in urine sediment cells was 26.7％,87.5％,69.6％ and 27.6％ respectively.Aberrant methylation rate of DAPK,HPP1 and RARβ genes in BTCC was significantly different from that in the glandular cystitis (P ＜ 0.05).There was no significant difference in the methylation rate of ITGA4 between BTCC and and cystitis glandularis (P ＞ 0.05).Conclusion Aberrant methylation status of HPP1,DAPK1,and RARβ is closely related with incidence of bladder cancer. Key words: Bladder carcinoma; Urine sediment cells; Tumor suppressor gene; Methylation

Acidosis with hyperuricemia and renal tubular damage in viral gastroenteritis

Acidosis with hyperuricemia and renal tubular damage in viral gastroenteritis

Molecular and immunohistochemical detection of rotavirus in urinary sediment cells of children with rotavirus gastroenteritis

This is the first report showing that rotavirus infects the urinary sediment cells in immunocompetent children with rotavirus gastroenteritis. We found that inclusion-bearing cells were frequently detected in the urine samples of patients with rotavirus gastroenteritis. These cells were positive for cytokeratin, which was sometimes coexpressed with rotavirus antigen, in our immunohistochemical analysis. Moreover, in nested RT-PCR experiments, we detected rotavirus double-stranded RNA in some urine samples of patients with rotavirus gastroenteritis. We concluded that rotavirus could lead to infection of the urinary sediment cells concomitantly with rotavirus gastroenteritis.

The significance of loss of 3q26. 1 small fragment in urothelial carcinoma of th bladder

Objective To investigate the copy number changes on chromosome 3q26. 1 in urothelial carcinoma of the bladder, and to explore its potential clinical significance. Methods The microarray-based comparative genomic hybridization (Array-CGH) approach was used to analyze the genome-wide copy number changes of 35 tumor tissue samples of bladder cancer. To confirm the loss of a small fragment in 3q26. 1 detected by Array-CGH, real-time fluorescent quantitative polymerase chain reaction (real-time PCR) was performed with 57 frozen tumor tissue samples and 34 formalinfixed paraffin-embedded (FFPE) tumor tissue samples. The urine sediment cells collected from 15 healthy volunteers and 29 bladder cancer patients were checked as above. Results The Array-CGH data showed that the copy number loss of a small fragment in 3q26. 1 was detected in 77.1% (27/35)of the tumor tissue samples investigated. Real-time PCR analysis validated this loss of a small fragment of 3q26.1 with high frequencies in both 57 frozen tumor samples and 34 FFPE tumor samples.The percentage of samples exhibiting loss was 78.9% (45/57) and 100. 0% (34/34) respectively.Furthermore, the relative copy number of the 3q26.1 small fragment was significantly lower in the urinary sediment cells of the patients (median=0. 0020), comparing with that of healthy controls (median=0. 0030) (P＜0.01). Conclusions Loss of the small fragment in 3q26.1 could be a characteristic genetic change of urothelial carcinoma of the bladder. It may serve as a potential molecular marker for bladder cancer. Key words: Urinary bladder neoplasms; Carcinoma; DNA probes; Tumor markers, biological; DNA copy number

Development of a novel custom micro-tool for effective cutting of a precision microgroove array on a microscope slide

This study presents a novel, economical and efficient fabrication technique for precisely generating multiple microgrooves on a microscope slide to allow for microscopic examination of urine sediment cells. This study incorporates two important phases: a precision wheel-tool array is fabricated and then the developed tool is used in fast on-line grinding of multiple microgrooves. The wheel-tool blank is made of diamond grit of 0–2 µm grade via co-deposition. Subsequently, it is trued, sliced and sharpened by means of micro wire electro discharge dressing. The finished wheel-tool is utilized on-line to grind multiple microgrooves using ‘high-speed and fast-shallow grinding’. A ductile grinding regime is established to obtain a nano-metric surface finish for the multiple microgrooves generated on the microscope slide. The depth and width of the grooves in the array are both 10 µm and a surface finish of Ra equal to 0.010 µm is simultaneously achieved. This multiple microgrooving technique can supply high-quality fast grinding in the fabrication of bio-medical devices, such as those used for stationing and counting urine sediment cells.

Detection of loss of heterozygosity in patients with urinary bladder carcinoma: neoplastic tissue vs. urine sediment cells

IntroductionLoss of heterozygosity (LOH) is frequently observed in urinary bladder neoplasms. In the reported study, an attempt was undertaken to determine the loss of heterozygosity of TP53(17p13), RB1(13q14), CDKN2A/ ARF(9p21) genes in DNA from neoplastic tissue, collected from patients with diagnosed urinary bladder carcinoma, and to compare the results with those of LOH evaluation in DNA isolated from urine sediment cells.Material and methodsAfter isolation, DNA was amplified (PCR) by means of primers to five polymorphic microsatellite markers, the products being then separated on agarose gel. Following the method, a total of 125 DNA samples were obtained, isolated from neoplastic cells, together with 125 corresponding DNA samples, isolated from urine sediment cells.ResultsThe loss of heterozygosity in at least one marker was identified in 39.2%. (49/125) of DNA from studied tumors and in 34.3% (43/125) of DNA samples, isolated from urine sediment cells. An analysis of LOH from the DNA, isolated from urine sediment cells, allowed for identification of 81.8% of neoplastic tumors with 99.7% specificity.ConclusionsOur observations have demonstrated that LOH within 13q14, 17p13 and 9p21 loci is more often observed in clinically more advanced neoplasms. LOH in 17p13 locus is more frequently found in tumors at high histopathological stage, while in low-stage neoplasms, LOH is most often observed on chromosome 9. The high sensitivity (81.8%) and specificity (99.7%) of LOH studies in DNA, isolated from urine sediment cells, make this technique an advantageous, non-invasive method for detection and screening of bladder cancer.

Detection of BK virus infection in renal transplant recipients and clinical application

To study the detection methods of BK virus infection in kidney transplant recipients, and to explore the clinical application. 132 cases of renal transplant recipients were undertaken BK virus detection including presence of decoy cells in urinary sediment, urine and serum BKV-DNA to demonstrate the BK virus replication. Among 132 cases of renal transplant recipients, urinary decoy cell was found in 37 (28.0%) patients and the median time was 12 months after surgery. 32 (24.2%) patients were diagnosed as BK viruria at a median of 11 months after surgery, and 16 (12.1%) recipients were diagnosed as BK viremia at a median of 15 months after surgery, 5 patients with BK viruria were diagnosed as BK virus associated nephropathy according to allograft biopsy. To make early diagnosis of BK virus infection, detection of urine decoy cells and BKV-DNA in urine and plasma sample is important,which provides an important basis for the prevention of BK virus associated nephropathy.

Fluorescent Microsatellite Analysis of Urine Sediment in Patients with Urothelial Carcinoma

Aims: To detect microsatellite loci alterations by fluorescent multiplex PCR in urine sediment cell of urothelial carcinoma, and to determine if they can be used as genetic markers for diagnosis of urothelial carcinoma. Materials and Methods: Microsatellite alteration analysis was conducted using fluorescent multiplex PCR with samples from 64 cases of urothelial carcinomas of the bladder. Three microsatellites spanning the 3p14 and two additional microsatellites in 9q33 and 9p22 were analyzed. Microsatellite alterations (microsatellite instability and/or loss of heterozygosity) in urine sediment cells of urothelial carcinoma patients matched for peripheral blood and tumor tissue were all analyzed. Results: The frequency of microsatellite alterations in urothelial carcinoma was chromosome 3p (D3S1234: 14.6% (7/48), D3S1300: 16.7% (8/48), D3S1313: 8.35% (4/48)); 9q (D9S242: 33.3% (16/48)), and 9p (D9S162: 27.1% (13/48)). Microsatellite alterations happened in 62.5% (40/64) of the patients when combined with all five markers. Our study showed a significant correlation between the microsatellite alteration of the five-locus panel and recurrence (p = 0.010) and smoking habit (p = 0.006). Conclusions: The results suggest that these microsatellite loci alterations may have an important role in the recurrence of urothelial carcinomas. Further studies are needed to better determine the effect of microsatellite loci alterations on prognosis.

Some Studies Suggest Antirejection Drugs Not Always Needed to Protect Kidney Graft

RESEARCHERS ARE EXPLORING THE tantalizing prospect of identifying patients with kidney transplants who do not need to continue to take antirejection medicines to preserve their donated organ. At the very least, this line of research might enable physicians to recognize those patients with renal allografts who could take less immunosuppressive medicine, reducing the risks of infection and cancer associated with such drugs. Such hopes were raised by researchers in the United States and Europe who identified biological signatures of a small minority of patients who had stopped taking antirejection medicines for a variety of reasons, such as adverse effects or cost, yet still preserved their allograft function. The researchers found that renal allograft tolerance was strongly associated with a distinct pattern of genes expressed by B lymphocytes (B cells). If more rigorous studies replicate these findings, it might be possible to design drug-weaning protocols for certain patients with a favorable B cell signature. In the US-based study, researchers enrolled 25 patients who had stable graft function despite receiving no immunosuppression for at least 1 year and as long as 32 years and compared them with persons who maintained stable graft function while receiving immunosuppression therapy and with nontransplanted controls. In tolerant patients, the B cell signature was associated with upregulation of CD20 messenger RNA in urine sediment cells and elevated numbers of peripheral blood-naive and transitional B cells compared with the other cohorts (Newell KA et al. J Clin Invest. 2010; 120[6]:1836-1847). The European-based researchers studied 24 renal transplant recipients and compared them with individuals with stable graft function while receiving immunosuppression therapy, those with biopsy-proven and immunologically driven chronic rejection, and nontransplanted controls. Compared with the other cohorts, the tolerant patients displayed, among a variety of factors, an expansion of peripheral blood

Identification of a B cell signature associated with renal transplant tolerance in humans

Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.

Loss of Heterozygosis on IFN-α Locus is a Prognostic Indicator of Bacillus Calmette-Guerin Response for Nonmuscle Invasive Bladder Cancer

We evaluated the role of loss of heterozygosity on the interferon-alpha locus to predict the response to bacillus Calmette-Guerin therapy in patients with nonmuscle invasive bladder cancer. A total of 117 consecutive patients were selected, including 77 with nonmuscle invasive bladder cancer and 40 controls. Loss of heterozygosity on the interferon-alpha locus (chromosome 9p21) was assessed in blood and urine samples before transurethral resection. All patients underwent transurethral resection and then 6 weekly bacillus Calmette-Guerin instillations. Those with nonmuscle invasive bladder cancer were assigned to groups 1 and 2 with and without loss of heterozygosity on the interferon-alpha locus, respectively. Of the 77 patients with nonmuscle invasive bladder cancer 39 (50.6%) had loss of heterozygosity on the interferon-alpha locus (group 1) and 38 (49.4%) had no alteration (group 2). Only 1 of 40 controls showed loss of heterozygosity on the interferon-alpha locus. At the end of followup 13 patients in group 1 and 27 in group 2 were alive without recurrence. We noted a significant difference between loss of heterozygosity on interferon-alpha and followup status (dF 01, LR 11.252, p = 0.003). Kaplan-Meier analysis revealed a significant difference in recurrence probability (response to bacillus Calmette-Guerin) and loss of heterozygosity on interferon-alpha (p <0.0001). On multivariate analysis loss of heterozygosity (HR 4.09, 95% CI 2.59-6.28, p = 0.002), grade (grade 3 HR 3.31, 95% CI 1.38-3.35, p = 0.03) and the number of lesions (3 or greater HR 2.31, 95% CI 1.38-3.25, p = 0.03) were independent predictors of the bacillus Calmette-Guerin response. This study highlights the predictive value of loss of heterozygosity analysis on interferon-alpha in patients with nonmuscle invasive bladder cancer treated with bacillus Calmette-Guerin.

MP-20.03: Prognostic Significance of Epidermal Growth Factor Receptor (EGFR) and Survivin Expression in Bladder Cancer Tissue and Urine Cytology Sediment of Patients with Transitional Cell Carcinoma of the Urinary Bladder

To assess whether epidermal growth factor receptor (EGFR) and survivin immunostaining of tumour cells in urinary sediment and tissue of patients with bladder cancer has prognostic significance.

Urinary CD4+ effector memory T cells reflect renal disease activity in antineutrophil cytoplasmic antibody–associated vasculitis

Numbers of circulating CD4+ effector memory T cells are proportionally increased in patients with proteinase 3 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) whose disease is in remission and are decreased during active disease, which presumably reflects their migration toward sites of inflammation. Since renal infiltrating cells may appear in urine, we investigated the presence of CD4+ effector memory T cells in urinary sediment as a reflection of renal disease activity in AAV. CD4+ effector memory (CD45RO+CCR7-CD3+CD4+) T cells were quantitated in the urine and peripheral blood of patients with AAV with renal involvement (n = 33), patients with AAV without renal involvement (n = 18), patients with AAV whose disease was in remission (n = 29), and patients with active disease (n = 22), using 4-color flow cytometric analysis. Numbers and percentages of urine CD4+ effector memory T cells in 12 patients with AAV with active renal disease were obtained over several weeks of followup during remission induction. A notable increase in urine CD4+ effector memory T cell numbers was observed in patients with active renal AAV compared with patients whose disease was in remission and patients with active disease without renal involvement. The increase in these cells in the urine of patients with active renal AAV was accompanied by a reciprocal decrease in these cells in peripheral blood. Results from followup analysis showed a clear reduction in urine CD4+ effector memory T cells following treatment. Moreover, a negative correlation was observed between percentages of circulating and urine CD4+ effector memory T cells, consistent with their migration toward sites of inflammation. Our findings indicate that the presence of CD4+ effector memory T cells in urine reflects renal involvement in AAV. Flow cytometric analysis of these cells in urine may contribute to assessing renal disease activity in patients with AAV.

Prediction of diagnosis of immunoglobulin a nephropathy prior to renal biopsy and correlation with urinary sediment findings and prognostic grading

Several clinical markers correlate well with the diagnosis and prognosis of IgA nephropathy (IgAN). In the present study, we re-evaluated the usefulness of these four clinical markers for prediction of the diagnosis of patients with IgAN through a comparison between many more patients with IgAN and those with other types of renal diseases. 364 patients with IgAN and 289 with other types of renal disease were examined. An analysis was performed prior to renal biopsy, using clinical markers including, serum IgA, serum IgA/C3 ratio, number of red blood cells in urinary sediments, and urinary protein. Patients with IgAN were divided into four groups according to histopathological findings. Presence of microscopic hematuria, persistent proteinuria, high serum IgA levels, and the serum IgA/C3 ratios are useful for prediction of diagnosis of IgAN and distinguishing it from other renal diseases. Blood pressure, urinary protein, serum uric acid, renal function, and urinary sediment findings may be useful for prediction of prognostic grading in patients with IgAN.

Detection of a transcript abnormality in mRNA of the SLC12A3 gene extracted from urinary sediment cells of a patient with Gitelman's syndrome.

To date, many mutations, including intronic nucleotide changes, in the SLC12A3 gene encoding the thiazide-sensitive sodium-chloride cotransporter (NCCT) have been reported in Gitelman's syndrome (GS) patients. However, it has not been clarified whether intronic nucleotide changes affect mRNA content. Since mRNA analysis is possible only after obtaining renal biopsy specimens, no studies have been conducted to identify transcript abnormalities in GS. In the study reported here, we investigated such transcript abnormalities for the first time by using mRNA expressed in a patient's urinary sediment cells. Direct sequencing analysis of leukocyte DNA disclosed one known missense mutation (R399C) and one known nucleotide change of the splicing acceptor site of intron 13 (1670-1 g > t). mRNA extracted from the urinary sediment cells was analyzed by RT-PCR to determine the pathogenic role of the intron mutation. A fragment encompassing exon 13 to 15 was amplified as two products, one consisting of all three exons and the other lacking only exon 14 in its entirety. Our investigation was the first to demonstrate exon 14 skipping in an NCCT transcript in renal cells. This methodology thus constitutes a potential noninvasive analytical tool for every inherited kidney disease.