Comparison of urea reduction ratio and dialysis efficacy in evaluating haemodialysis adequacy in chronic kidney disease patients in Wangaya Hospital

Background:An increasing number of patients with chronic kidney disease (CKD) impact an increased need for hemodialysis. Inadequate hemodialysis affects morbidity in patients with CKD. Determination of the urea removal index can be accomplished by several invasive and non-invasive methods. The purpose of this study was to compare the urea reduction ratio (URR) and dialysis efficiency (Kt/V)calculated automatically by hemodialysis machines to assess the adequacy of hemodialysis in patients with CKD.Methods:A cross-sectional analysis study was conducted on 58 CKD patients with age ?18 years, conventional 5-hour hemodialysis sessions twice weekly, using single use-hollow fiber dialyzers, and who had been receiving hemodialysis for ?6 months in the hemodialysis unit atWangaya Hospital from April 2022 to May 2022. Study data were obtained from medical records then described and analyzed using the statistical package for the social sciences (SPSS)program.Results:The mean of URR was 70.74�.04, while the mean of Kt/Vdelivered by machine was 1.27�19. More hemodialysis patients received adequate hemodialysis based on URR parameters compared to Kt/V parameters (84.5% versus 1.7%). There was no significant difference between age, sex, body mass index (BMI), comorbidities, vascular access and duration of dialysis with adequacy hemodialysis. There was a significant difference between URR and Kt/V in the evaluation adequacy of hemodialysis (p=0.000). In addition, there was a positive correlation between URR and Kt/V in theevaluation adequacy of hemodialysis (r=0.592, p=0.000).Conclusions:The URR is a more accurate parameter, but the Kt/V delivered by machine can help the URR demonstrate the adequacy of haemodialysis patients with CKD.

Usefulness of Assessing Hemodialysis Adequacy Using the Stop Dialysate Flow (SDF) Method / 대한신장학회잡지

BACKGROUND: K/DOQI guidelines recommend the slow flow method as a standardized method of postdialysis blood sampling for measuring hemodialysis adequacy. However, it is not easy to adopt this method when working in busy renal units where it is often difficult to obtain repeated samples exactly at the specified time. The stop dialysis flow (SDF) method recommended by the Scottish Renal Association since 1998 has the advantage of involving 2 steps only: (1) switch off dialysate flow at the end of hemodialysis without altering the blood pump speed and (2) take a blood sample after 5 minutes from the arterial or venous port. However, there are some limitations to SDF mthod in that it does not allow for tissue rebound after the first 5 minutes postdialysis and cannot be used directly to calculate equilibrated Kt/V (Kt/Veq) using either a 30-minute postdialysis sample. We derived a formula that uses a 5-minute postdialysis BUN sample using the SDF method to estimate the BUN concentration at 30 minutes and investigated if it is useful to assess hemodialysis adequacy using this method. METHODS: A total of 51 patients who had been undergoing hemodialysis in Chonnam National University Hospital and had agreed in joining this study were involved. Patients were randomly selected to 2 groups. Blood samples were obtained immediately before dialysis and at 0, 5, and 30 minutes postdialysis. We calculated the linear relationship between the 5-minute and 30-minute postdialysis samples in group A patients (n=25) and validated this equation using the data from the other group B patients (n= 26). We predicted what the 30-minute BUN concentration would be using the measured value of BUN at 5 minutes and compared directly the value of our estimated 30-minute BUN with the measured 30- minute BUN. RESULTS: There was a tight linear correlation (R2=0.993, p<0.05), between measured 5-minute postdialysis BUN concentrations and measured 30-minute postdialysis BUN concentrations in group A patients. This relationship is described by the linear regression equation: 30-minute BUN concentration=1.05x(5-minute BUN concentraion)+1.04. We used this equation to estimate the 30-minute BUN concentration in group B patients based on the 5-minute postdialysis BUN sample from these patients. And there was a close correlation between estimated and measured 30-minute postdialysis BUN concentration (R2= 0.989, p<0.05). The sensitivity, specificity, positive, and negative predictive values of this equation were high when used to estimate 30-minute urea reduction ratio (URR) greater than 65% (88.9%, 100%, 100 %, and 94.4%, respectively) and 30-minute Kt/Vsp greater than 1.2 (100%, 100%, 100%, 100%, respectively). CONCLUSION: We could estimate 30-minute postdialysis BUN concentration, 30-minute Kt/V, and 30-minute URR exactly using SDF method and linear regression equation derived in this study. The advantage of involving 2 steps only makes SDF method a useful tool in assessing hemodialysis adequacy.

Accelerated Induction of Dysplastic Lesion by TPA in HPV18 URR E6/E7 Gene Expressing Transgenic Mice / 대한암학회지

PURPOSE: The research of HPV has been severely hampered by the inability to propagate HPVs in culture, particularly those of the mucosotrophic types which produce few virions in vivo. In order to study the regulation of HPV-18 expression in vivo, we constructed transgenic mice and caused cervical neoplasia. MATERIALS AND METHODS: We investigated whether tetradecanoyl phorbol acetate (TPA) increase the transcriptional activity of the URR in the C33A cervical carcinoma cells or not. And we asked whether chronic exposure of female HPV-18 URR E6/E7 transgenic mice to TPA could render the reproductive tract squamous epithelium permissive for HPV neoplasia. RESULTS: It was confirmed by RT-PCR that transgene was specifically expressed in epithelial tissues. TPAupregulated the transcriptional activity of the URR in the C33A cervical carcinoma cells. There were diffuse changes on the squamous epithelium in the cervix of the transgenic mice at fifth month following TPA treatment. CONCLUSION: We established the transgenic mice model which have the ability to reproduce the development of cervical dysplasias. Moreover this animal model will allow preclinical testing of compounds designed to interfere with the actions of the HPV oncogenes or other critical aspects of the cancer phenotype.

Usefulness of PCR-SSCP for tracing the pathogenesis of human papillomavirus-associated malignancy / 대한부인종양콜포스코피학회잡지

PURPOSE: To set up more simplified detection method for human papillomavirus (HPV) sequence polymorphism which could be used for the study of HPV-related pathogenesis, route of infection, and many other epidemiologic studies. MATERIALS AND METHODS: One hundred and thirteen cases of uterine cervical tissues containing HPV 16 DNA confirmed by polymerase chain reaction (PCR) from Korean women were subjected to investigate the URR gene mutations. PCR-amplified products were sequenced by the fluorescent dideoxy termination method and opposite strand sequencing was performed as required. The results obtained from sequencing were analysed to find the most hypervariable segment which contains the greatest number of variants and subjected to PCR-single strand conformation polymorphism (SSCP) analysis. RESULTS: Among the length of nucleotide position (np) from 7175 to 24, we found 60 sites (60/815=7.36%) of base substitutions. Segment from np 7743 to 24 was the most hypervariable and contain 20 kinds of variants. In this segment, C-to-T mutation at position 24, G-to-A at 7842, and T-to-C at 7781 were more frequently found than other sites. By comparing sequencing results with PCR-SSCP, we found 15 patterns distinguishable each other. All of the mobility shift occurred in the PCR-SSCP pattern could be accounted for by the base substitutions and nearly all of the DNA sequencing results observed were reflected as alterations in the PCR-SSCP patterns. CONCLUSIONS: We have assigned the hypervariable segment in one portion of URR which could be used as PCR-SSCP analysis. Identification of HPV polymorphism by PCR-SSCP is potentially useful for elucidating a number of epidemiologic questions such as the pathway of viral spread and so on.

Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells / 대한부인종양콜포스코피학회잡지

BACKGROUNDS: Human papillomavirus (HPV) infection is known as the major causative phenomenon in the development of cervical cancer. E6 and E7 proteins of oncogenic HPV types can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, long term use of oral contraceptives may be one of the risk factor for cervical cancer. PURPOSE: Investigation of estrogenic and anti-estrogenic effects on the proliferation of cervical cancer cells and gene expression of HPV under the regulation of HPV upstream regulatory region (URR) would help to explain the role of estradiol in HPV-associated pathogenesis of cervical cancer. METHODS: Cervical cancer cells (HeLa, CaSki and C33A) were cultured in vitro in the presence of 17 beta-estradiol or tamoxifen and the numbers of cells were directly counted to observe the growth stimulatory or suppressive effect of the treatment. The correlation between the growth regulatory effect and HPV E6/E7 gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient co-transfection assays, which were conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. Supplemental effect of estrogen receptor on the URR promoter activity was also evaluated. To analyze the growth suppressive function at the higher concentration of estradiol or tamoxifen in HeLa cells, DNA fragmentation assay was performed. RESULTS: The proliferation of HeLa and CaSki cells was stimulated by estradiol at the concentration of physiological level (< or =1 X 10-6M), reaching maximal growth at 0.5 X 10-6M. At concentration of 0.1 X 10-6M, tamoxifen also stimulated the proliferation of HeLa and CaSki cells. In contrast to HPV-positive cervical cells, C33A cells were not influenced to cell proliferation by addition of estradiol at the physiological level, indicating that HPV might play role in growth stimulatory effect of estrogen or tamoxifen. Interestingly, the proliferation of HeLa cells was totally suppressed by estradiol and tamoxifen at the higher concentration (5 and 10 X 10-6M), whereas those of CaSki and C33A cellswere not responded and little suppressed at the concentration, respectively. The levels of HPV-18 E6 and E7 mRNA were significantly increased after treatment of 0.5 X 10-6M estradiol as determined by RT-PCR. Furthermore, transient transfection experiments using the URR-CAT reporter plasmid indicated that the increased expression of HPV E6/E7 genes was related with the growth stimulatory effect of estradiol and tamoxifen. In addition, co-transfection of estrogen receptor (ER) leads to an over 4-fold increase in CAT activity after treatment of estradiol or tamoxifen with 0.5 X 10-6M. When estradiol or tamoxifen was treated at the concentration over 5 X 10-6M for 96 hr, a typical DNA ladder, a indicative of apoptosis, was observed in HeLa cells. However, DNA ladder was not detected in C33A cells of which growth was some suppressed under same concentration of estradiol. CONCLUSION: At the physiological levels, estradiol stimulated the growth of HPV-positive cervical cancer cells and tamoxifen also did at the concentration of 0.1 X 10-6M. The increased expression of HPV E6/E7 at the physiologic levels appeared to be related with the growth stimulation of HPV-positive cervical cancer cells. Growth suppression observed at the higher concentration (5 and 10 X 10-6M) might be a indicative of apoptosis shown by DNA fragmentation assay in HeLa cells. Taken together, these data suggested that the concentration of estradiol (< or =1 X 10-6M) could be a risk-factor in HPV-mediated cerivcal carcinogenesis.

Increased transcriptional activity by mutation of HPV-16URR in cervical cancers carrying episomal HPV-16 DNA / 대한부인종양콜포스코피학회잡지

HPV E2 protein is known to act as a negative regulator of transcription and the disruption of E2 open reading frame by HPV integration can release suppression of E6 and E7 mRNA expression, resulting in uncontrolled cellular growth and malignant transformation by inactivating tumor suppressor gene products (p53, pRb). YY1 mutation of HPV URR has been suggested as one of indicator that explains development of cervical neoplasia by episomal type of HPV. To extend this hypothesis, we examined whether mutation(s) in specific sites of HPV URR is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients, cloned into pUC18 for sequencing, and into pBLCAT8+ for functional CAT assay. Our previous data classified HPV-infected patients into three groups: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women: GA transition at nt 7520 (100%, 27/27), AC transition at nt 7729 (70%; 19/27), and GA transition at nt 7841 (78%; 21/27). Selective mutations were observed at the YY1-binding sites of HPV-16 URR in the 3 patients with invasive cervical cancer, who having the episomal forms of HPV-16 DNA: AC transition at nt 7484 and GA transition at nt 7488 (YY1-binding site 2; from 7481 to 7489). Additionally, CT transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) was found from 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine whether these mutations have effect on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episomal HPV-16 DNA of cervical cancers were 2- to 4-fold higher than that of HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in CIN, where no mutation of the YY1-binding site was detected, showed similar levels of promoter activity to that of URR prototype.Our results support the hypothesis that the mutation at YY1 binding site is functionally related to the development of cervical neoplasia caused by episomal HPV-16 DNA in Korean cervical cancer patient. Thus, mutation in YY1 site of episomal HPV-16 URR may play a role of HPV integration in the progression of cervical cancer.