Abstract
To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH).
RNA was extracted from normal human glomeruli, and the cDNA library was constructed by plasmid cloning. Out of 5 x 10(3) clones from the library, 91 UniGene clusters of more than three clones were identified as 'glomerular-abundant genes'. All these genes were referred to the SMD, and 18 genes were defined as 'glomerular preferential genes'. Four unknown genes - IFI27, CRHBP, FLJ10154 and SEMA5B - were selected for RT-PCR to compare expression in the glomerulus with that in the cortex and medulla, and for ISH to examine glomerular localization. Also, three unknown genes that were glomerular abundant but not listed in the SMD - DDX5, HSPC138, and MGC10940 - were selected for RT-PCR and ISH. Finally, a kidney biopsy specimen of crescentic glomerulonephritis was used for ISH to examine glomerular expression for CRHBP mRNA.
Among the selected seven glomerular-abundant genes, six were confirmed as 'glomerular preferential genes' by RT-PCR. By ISH, all these genes were demonstrated in podocytes. The expression of CRHBP mRNA in a single living podocyte was not changed between normal and crescentic glomerulus.
Glomerular preferential expression and podocyte localization of these novel genes have been demonstrated for the first time. Because some of these genes were not listed in SMD, our database can be a useful tool to find novel human glomerular genes.