Detection of Bcl-2 mRNA and its product in the glomerular podocytes of the normal rat kidney

Podocyte apoptosis underlies podocytepenia leading to glomerulosclerosis. An apoptosis inhibitory protein Bcl-2 is expressed in the podocytes in the early stage of nephrogenesis and downregulated in the maturing stage of human fetal kidneys. Recent studies reported changed localization and expression of Bcl-2 in the renal glomeruli under the pathologic conditions. This study aimed to confirm in situ localization of Bcl-2 mRNA and its product in the glomeruli, and to demonstrate the local expression of Bcl-2 mRNA in normal rat glomeruli. Paraffin sections of the kidneys from normal male Wistar rats were immunostained by anti-Bcl-2 monoclonal antibody. The localization of Bcl-2 mRNA in the glomeruli was evaluated by in situ hybridization. The glomeruli were dissected from frozen sections of the kidneys with the laser microdissection (LMD) system. Total RNA extracted from 10, 100 or 200 dissected glomeruli was used for reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Bcl-2 mRNA and its product were detected in the podocytes but barely in the mesangial cells. In RT-PCR, the specific-sized bands of Bcl-2 from 100 or 200 dissected glomeruli were clearly observed. Real-time PCR for Bcl-2 showed that cDNA from 100 or 200 dissected glomeruli became amplified at 36 or 33cycles, respectively. Bcl-2 is expressed in the glomerular podocytes of the normal rat kidney and quantitative analysis of Bcl-2 mRNA in the renal glomeruli is possible using the LMD technique.