| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Abstracts of Oak Ridge Posters |
Departments of1 Chemical Pathology and 2 Nephrology, Guy’s & St. Thomas’ Hospital, London, United Kingdom;
aaddress correspondence to this author at: Department of Chemical Pathology, 5th Floor, North Wing, St. Thomas’ Hospital, London SE1 7EH, UK; fax 44-207-9284226, e-mail r.swaminathan{at}kcl.ac.uk)
Kidney disease affects more than 20 million individuals in the United States alone. Although the causes of kidney failure are diverse, the glomerular filtration barrier is often the target of injury. Identification of the type of disease is important for targeting therapy. One area of particular interest is the ability to predict which patients with nephrotic syndrome might respond to steroids or cytotoxic therapies and which will not (1). Often this depends on renal biopsy, an invasive procedure with some risk of complications. A noninvasive test to determine the type of renal disease would be advantageous.
During the past decade, nucleic acids have been detected in the circulation, and measurements of tissue-specific DNA and mRNA offer enormous potential for diagnosis and prognosis (2). Increased amounts of nucleic acids have been reported in disease, including lung cancer (3), thyroid cancer (4)(5), stroke(6), and trauma (7). We have recently shown that measurement of total DNA in plasma is a good prognostic marker of outcome in critically ill patients (8). In another study we observed that mRNA for rhodopsin is increased in diabetic retinopathy, and it may be a useful and inexpensive way to detect and monitor diabetic patients for retinopathy (9).
Nephrin, a transmembrane protein with a large extracellular portion including 8 immunoglobulin-like domains, is expressed by visceral epithelial cells (podocytes) in the slit diaphragm of the glomerulus (10). A product of the NPHS1 gene located on chromosome 19, this protein is crucial for the integrity of the slit diaphragm, and abnormalities in this protein can lead to proteinuria and nephrotic syndrome. Measurement of nephrin mRNA in peripheral blood by real-time quantitative PCR (qPCR) may provide a clue to the etiology of the renal disease. In the present study, we developed a real-time qPCR assay for nephrin mRNA and used this assay to measure the concentrations in healthy individuals and in renal transplantation patients to determine the factors that may be associated with the observed nephrin mRNA concentrations in peripheral blood.
Blood samples were collected from healthy volunteers (n = 14) with no known disease and from patients, after kidney transplantation (n = 53), from the transplant clinic at Guy’s Hospital, London. The protocol for the study was approved by the Guy’s Hospital Local Research Ethics Committee. All patients had stable renal function, and the period after transplantation ranged from months to >10 years. Informed consent was obtained from each participant before blood collection. Peripheral vein blood (2.5 mL) was drawn directly into PAXgeneTM Blood RNA tubes specifically designed for the collection and stabilization of RNA from whole blood (PreAnalytiX). Whole-blood RNA was extracted with the PAXgene Blood RNA Kit and treated with the supplied DNase to prevent genomic DNA contamination, strictly according to manufacturer’s instructions. Extracted RNA was stored at –80 °C until required for cDNA synthesis. Reverse transcription was carried out with SuperScript IITM reverse transcriptase according to the manufacturer’s instructions (Invitrogen Life Sciences). The cDNA generated was stored at –80 °C until required for quantification. Separately, samples were also subjected to the above procedure with the exception that SuperScript II was replaced with water (negative control).
cDNA was amplified and PCR products were detected by use of sequence-specific oligonucleotide probes and intron-spanning specific primers on the ABI 7000 Sequence Detection System (PE Applied Biosystems). ß-Actin cDNA was amplified by the manufacturer’s method (Pre-Developed Assay Reagents for the TaqMan® assay; PE Applied Biosystems). For the TaqMan nephrin assay, 300 nM each of the forward and reverse primers, 100 nM probe, 25 µL of 2x TaqMan Universal Master Mix, and 10 µL of cDNA sample were used for each reaction. For both assays, calibrators and samples were analyzed in duplicate in a final reaction volume of 50 µL. Calibration curves were prepared from serial dilutions of cDNA (Ambion Corp.) obtained from healthy human kidney. A water blank was also incorporated in each run for the respective assays. Both assays were run simultaneously on 96-well optical reaction plates. PCR amplification included an initial phase of 2 min at 50 °C, followed by 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at 60 °C.
Results for nephrin mRNA are reported as its ratio to total blood ß-actin mRNA. Statistical analysis was performed with SPSS 11 (SPSS Inc.). Differences in nephrin mRNA between groups were analyzed by the Mann–Whitney U-test, and a P value <0.05 was considered statistically significant.
The slopes of 10 consecutive calibration curves (10-fold serial dilutions of kidney cDNA to obtain a 5-point curve) showed good reproducibility for both the target and housekeeping genes (CV for slopes, 4% for nephrin and 6% for ß-actin). Nephrin mRNA was detected in peripheral blood from all healthy and transplant patients. In the healthy controls, the median nephrin mRNA concentration was 4.99 x 10–3 (n = 14), higher in females than in males (Table 1
). In transplant patients, the median nephrin mRNA concentration was 1.29 x 10–3 (n = 53); in this group also, females had concentrations higher than males (Table 1
). When healthy individuals and transplant patients of the same gender were compared, significant differences were observed (Table 1
). Because there were few individuals in the healthy group, it was not possible examine the effect of age. In male transplant patients, an age effect was seen: Patients <30 years of age had higher nephrin mRNA (2.41 x 10–3; n = 10) than patients >50 years of age (1.07 x 10–3; n = 11; P = 0.02). In a multiple regression analysis of nephrin as the dependent variable and gender, age group, and patient group as independent variables, only gender (P = 0.019) and age group (0.045) were significant contributors to the variation in nephrin mRNA.
|
To our knowledge, this is the first study to measure nephrin mRNA in blood by real-time qPCR. This method does not distinguish between the possible sources of RNA, i.e., renal cells, blood cells, or other cells. It is also possible that the nephrin mRNA detected by our assay may arise from the presence of "illegitimate transcription", i.e., basal transcription of tissue-specific genes outside of the tissues where they are typically active. Although this phenomenon is known to occur for certain genes, the illegitimate transcripts are thought to be of very low abundance (11), and this may account, in part, for some of the nephrin mRNA detected in our study. Extensive searches of available genomic databases nevertheless seem to suggest that nephrin expression is overwhelmingly kidney specific.
This study shows that nephrin mRNA in circulation is influenced by age and transplantation, both of which are associated with reduced renal mass: Renal function decreases with age, and transplant patients have lower renal mass. The mean serum creatinine concentration in the transplant group in this study was 178 mol/L. Serum creatinine and renal function are lower in females than in males, and nephrin mRNA would be expected to be lower in females. In our study, however, mRNA was higher in females, both in healthy individuals and in transplant patients. This finding suggests that the nephrin mRNA expression is influenced by estrogens or other female hormones. There were not enough female participants, particularly in the postmenopausal age group, to test this hypothesis, and further studies are necessary to confirm our findings.
We conclude that nephrin mRNA in peripheral blood measured by real-time qPCR is higher in females and in individuals with higher renal mass. These factors must be taken into account in interpreting the results of nephrin mRNA.
Acknowledgments
We thank the staff of the Molecular Working Group at St. Thomas’ Hospital for the use of the facilities in the Molecular Diagnostic Laboratory and the staff in the renal transplant unit for helping with the study.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  G. Wang, F. M.-M. Lai, K.-B. Lai, K.-M. Chow, B. C.-H. Kwan, P. K.-T. Li, and C.-C. Szeto Urinary messenger RNA expression of podocyte-associated molecules in patients with diabetic nephropathy treated by angiotensin-converting enzyme inhibitor and angiotensin receptor blocker Eur. J. Endocrinol., March 1, 2008; 158(3): 317 - 322. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
