EXPRESSION AND LOCALIZATION OF THE RETINOBLASTOMA GENE DURING RADIATION-INDUCED APOPTOSIS IN NEONATAL RAT-KIDNEY

An in vivo neonatal rat kidney model was used to study an association between expression and localization of the retinoblastoma tumor-suppre ssor gene (Rb), or its protein product (pRb), and localization of radi ation-induced apoptosis. The rat kidney has two distinct zones of diff erentiation at birth-an outer nephrogenic zone, in which cells are und ifferentiated and new nephrons are forming, and a differentiated zone internal to this zone that has essentially the adult kidney form. At 6 h after radiation (5 Gy), high levels of relatively synchronous apopt osis are induced in the nephrogenic zone, with little effect on the di fferentiated zone, and proliferation in the nephrogenic zone is almost totally inhibited by radiation treatment, again with little effect in the differentiated area. We have used our knowledge of this model to analyze control (sham-treated) surd irradiated renal tissue for Rb mRN A transcript levels and localization (Northern blot and in situ hybrid ization (ISH)), pRb expression (Western blot and immunolocalization), apoptosis and mitosis (light and electron microscopy, and DNA gel elec trophoresis for apoptosis), and cells in S-phase ([H-3]thymidine uptak e and autoradiography). Northern blots showed no detectable alteration in RX, transcript levels between control and irradiated tissues, wher eas Western blots indicated increased expression of pRb in protein ext racted fr om irradiated kidney compared with controls. ISH confirmed t hat Rb transcripts were not substantially altered in the nephrogenic a nd differentiated zones in control versus irradiated renal tissue. Imm unolocalization of pRb demonstrated little effect in the differentiate d zone, but in the nephrogenic zone pRb expression was increased, espe cially the S-shaped prenephrons, and was also found in many, but not a ll, apoptotic cells in this zone. The results link radiation-induced a poptosis and increased pRb expression in a zone of the neonatal kidney having a low level of cell differentiation. (C) 1997 Academic Press.