Osaka University, Osaka Japan

Laser-manipulated microdissection is a method to cut out a limited region from a histological specimen by a strong laser beam under a microscope.

Microdissected samples can be collected. To study mRNA expression in glomeruli or tubules isolated from a histological specimen, we applied the laser-based techniques and quantified the mRNA expression in isolated samples using real-time PCR, which is a technique for sensitive and precise quantification of PCR products. First, we established the system that only glomeruli were isolated randomly by the methods from experimental rat glomerulonephritis model in which TGF-b1 mRNA was known to increase in a week. Total mRNA from 10 glomeruli isolated from six-micrometer thick histological specimens of control and disease-induced kidneys were extracted. TGF-b1 mRNA on day 0 was under detectable levels in isolated glomeruli by real-time PCR. TGF-b1/G3PDH mRNA ratio was increased significantly 7 day after the disease induction (Kidney Int. 57, 2000).

Next, we applied the laser-based microdissection and real-time PCR procedures to determine mRNA expression in proximal tubules (PT) of human renal biopsy specimens. PTs were successfully microdissected and collected from histological specimens. We focused on osteopontin (OPN) mRNA expression in tubules in kidney disease. Ten PTs were isolated from each specimen of renal disease patients and used for the analysis. The amount of OPN mRNA expression of an IgA nephropathy patient (virtually no pathological findings) defined as 0.05, while focal glomerulo sclerosis patient who has more severe histological changes showed value of 1.5 OPN mRNA expression. It suggested that OPN mRNA expression in proximal tubules may be increased along with the severity of pathological findings.

In conclusion, we established methods to isolate proximal tubules from histological specimens of renal biopsy under microscopic observation by a laser based technique, and succeeded in the selective evaluation of a particular mRNA.