Localization of erythropoietin mRNA in the rat kidney by polymerase chain reaction

Abstract

Erythropoietin (Epo) is a glycoprotein secreted by kidney cells which plays an important role in the regulation of erythropoiesis. Localization of the Epo production by immunohistochemical studies and in situ hybridization has not been definitively established and is still a matter of controversy. Epo and glyceraldehyde 3-dehydrogenase (GAPDH) mRNA levels were determined in total RNA isolated from control and CoCl2-treated rats using a coupled reverse transcriptase/polymerase chain reaction method (RT/PCR). As indicated by the amount of amplification product, Epo mRNA levels were several-fold higher in CoCl2-treated rat kidney. In contrast, GAPDH mRNA levels were similar in control and CoCl2-treated rats. This RT/PCR method was also used to assess the level of Epo and GAPDH mRNA in microdissected nephron segments. All nephron segments tested lacked any detectable levels of Epo mRNA in either control or CoCl2-treated rats. On the other hand, peritubular cells (capillary fraction: afferent/efferent arteriole, vasa recta) were the only cells where the Epo mRNA was detected. Using a specific primer for GAPDH, the RT/PCR method could identify GAPDH mRNA in all microdissected nephron segments where the Epo mRNA was not expressed. Thus, a combination of microdissected nephron segments and RT/PCR enabled us to detect GAPDH mRNA populations in all nephron segments, whereas the failure to detect Epo mRNA in all segments but the capillary fraction, is due to the specific and localized expression of the Epo gene to this fraction.