Abstract 1553 Neonatal Immunology & Hematology Poster Symposium, Monday, 5/3

Infants born prematurely lack a normal erythropoietin (Epo) response to anemia, and fail to increase Epo production despite an apparent need for improved tissue oxygenation. We hypothesized that the anemia of prematurity might involve a delay or a defect in the premature kidney to produce Epo. Using qualitative PCR and immunocytochemistry, we previously showed that Epo mRNA and protein production is present in mid-trimester human kidneys. In this study we compared the quantity of Epo mRNA produced by mid-trimester human kidney versus the liver, using reverse transcription and the ABI PRISM sequence detection system. This system uses a reported fluorescent dye and a quencher dye attached to an oligonucleotide probe which is added to the PCR reaction. While the probe is intact, the proximity of the reported and quencher dyes greatly reduces the fluorescence of the reported dye. During annealing of the probe to the target sequence, the reported dye is cleaved by the 5' nuclease activity of Taq DNA polymerase, separating the reporter from the quencher dye and causing an increase in fluorescence proportional to the amount of amplicon produced. Data are collected with every cycle. Fetal liver and kidney samples were obtained at 12 through 22 weeks gestation. Total RNA was isolated and quantified by spectrophotometric analysis. Hep3B cells, known to produce Epo message, were used as a positive control. Following reverse transcription, Epo cDNA was quantified in each sample, and as an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also quantified. Epo cDNA quantity was standardized to the quantity of GAPDH present in each sample. All samples were run in duplicate. Epo cDNA concentrations were low, and did not change from 12 to 22 weeks in kidney samples, but increased in liver samples as gestation increased (p<0.01, 12-14 week liver samples versus 20-22 week liver samples, and 17-19 week liver samples versus 20-22 week liver samples; see table below). Epo cDNA concentrations in 12-16 week gestation kidney samples were 6.6±1.8 fg/µg GAPDH cDNA, and were 3.4±0.9 in 17-22 week gestation samples. Epo cDNA concentrations were nearly twenty-fold greater in fetal liver samples, averaging 49.2±17.9 fg/µg GAPDH in 12-16 week gestation samples and 156.3±40.5 in 17-22 week gestation samples (p<0.05, early versus late gestation liver samples; p<0.01, liver versus kidney samples). For comparison, Epo cDNA concentrations in Hep3B cells were 178.5±15.7 fg/µg GAPDH. We conclude that the fetal human kidney produces approximately 1/20th the amount of Epo message during the early mid-trimester that the fetal liver produces; this ratio decreases as production in the fetal liver increases during the late mid-trimester. It remains to be determined under what hypoxic conditions these tissues produce Epo protein, and how these conditions are affected by premature birth. (Table)

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