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Jul 11, 2012

TGen Isolation Method May Help Diagnose Kidney Disease

  • Translational Genomics Research Institute (TGen) researchers today published a method of diagnosing chronic kidney disease (CKD) that entails producing, then isolating high amounts of protein-rich urinary exosomes, a process the researchers said could help in discovery of biomarkers for the disease.

    Researchers said their method—published in the journal Kidney International—was ultimately designed to offer less expensive, less invasive, and more accurate diagnoses of CKD, characterized by a progressive deterioration of the kidney's ability to filter waste from the blood. The study’s immediate goal, they said, was to identify the best isolation methods for both proteomic analysis and RNA profiling of the urinary exosomes as a first step toward biomarker discovery.

    In "Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers," the researchers evaluated six different methods of isolating exosomes, including one based on a commercially available exosome precipitation reagent called ExoQuick-TC. By itself, ExoQuick-TC did not yield high quantities or pure preparations of protein and RNA. But the TGen modification of the protocol led to the highest yields of miRNA and mRNA, which can subsequently be used in genetic profiling experiments.

    CKD is now diagnosed by detecting increased levels of urinary albumin filtered out of urine in healthy kidneys, or of serum creatinine produced through the breakdown of creatine found in muscle. Neither measurement can accurately indicate a patient’s type of kidney injury or disease, which can be present with minimal or even no change in levels of albumin or creatinine.

    “In contrast to renal biopsy, urine is an ideal source of biomarkers, particularly for diseases of the kidney and urinary tract because it can be conveniently collected in large amounts without risk to the patient, Johanna DiStefano, Ph.D., director of TGen's Diabetes, Cardiovascular and Metabolic Diseases Division, and the study's senior author, said in a statement announcing publication of the study.

    She said exosome isolation in urine is an improvement over current commonly used centrifuge processes because they are time-consuming, requires expensive equipment, and can only process low volumes and a few samples of urine at a time, and are thus unsuitable for clinical use. Another current process, ultrafiltration, retains unwanted proteins that interfere with the isolation of exosomal proteins, according to Dr. DiStefano.

    "Faster and more efficient methods of isolating exosomes are needed," concluded Lucrecia Alvarez, Ph.D., a postdoctoral fellow at TGen and the study's lead author, in the statement.

    In 2009, researchers from TGen and UCLA identified genetic markers they said could aid in treating CKD among diabetics using a DNA analysis tool they developed.

    TGen researchers identified genes that could potentially contribute to end-stage renal disease (ESRD) in people with Type 1 diabetes. The researchers tested the pooled genomic DNA from more than 500 cases of those with ESRD and compared that to more than 500 patients who had Type I diabetes for at least 20 years with no sign of ESRD. Researchers then performed a whole genome association scan to look for single nucleotide polymorphisms (SNPs) that indicated a susceptibility to ESRD.

    Experiments identified at least eight locations along the nearly three-billion-base human genome that are ripe for further investigation of their ties to ESRD, according to a paper published in the December 2010 edition of Diabetic Medicine (“Genome-wide SNP genotyping study using pooled DNA to identify candidate markers mediating susceptibility to end-stage renal disease attributed to Type 1 diabetes.”)

    Sources:

    TGen (July 11) — http://www.eurekalert.org/pub_releases/2012-07/ttgr-tmi070912.php

    TGen (Dec. 16, 2009) — http://tgen.org/news/index.cfm?newsid=1734


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