Analysis of segmental renal gene expression by laser capture microdissection.
Analysis of segmental renal gene expression by laser capture microdissection.


BACKGROUND
The study of normal renal physiology has been greatly aided by microdissection techniques that have delineated the exceptional functional and cellular heterogeneity both along the nephron and between different nephron populations. These techniques are not widely used to study renal injury as microdissection is difficult because of tissue necrosis or fibrosis. We developed a procedure to detect specific gene expression in specific locations of the kidney in histologic sections.


METHODS
The anatomic specificity of laser capture microdissection (LCM) was employed with the sensitivity of reverse transcriptase-polymerase chain reaction (RT-PCR).


RESULTS
LCM/RT-PCR detected mRNA for podoplanin in 2% of a single glomerulus, rat basic amino acid transporter in 6% of a single cross-section of proximal straight tubule, and renin in eight proximal convoluted tubule cross-sections. LCM/RT-PCR could isolate pure populations of proximal convoluted tubules, proximal straight tubules, and thick ascending limbs from renal histologic sections, although pure collecting ducts could not be isolated. LCM/RT-PCR localized ischemia-reperfusion-induced induction of KC/interleukin-8 primarily to the medullary thick ascending limb, and detected transforming growth factor-beta (TGF-beta) mRNA in glomeruli of a patient with membranous glomerulonephropathy.


CONCLUSIONS
When used with an appropriate laser spot size, LCM/RT-PCR can measure gene expression in glomeruli or specific parts of the nephron and can study alterations in steady-state mRNA levels in animal models of renal disease. The applications, limitations, and refinements of this approach are discussed.
Kohda
Y
Y
University of Texas Southwestern Medical Center, Veterans Administration Medical Center, Dallas, Texas, USA.
Murakami
H
H
Moe
O W
OW
Star
R A
RA
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D015870:Gene Expression; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics; D007678:Kidney Glomerulus / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism*; D007834:Lasers; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000235:genetics; D000098853:Podoplanin
D017931:DNA Primers; D016209:Interleukin-8; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C117468:PDPN protein, human; D000098853:Podoplanin; C092025:Pdpn protein, rat