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 nep hron populations. These techniques are not widely used to study renal injur y as microdissection is difficult because of tissue necrosis or fibrosis. W e developed a procedure to detect specific gene expression in specific loca tions of the kidney in histologic sections. Methods. The anatomic specificity of laser capture microdissection (LCM) wa s employed with the sensitivity of reverse transcriptase-polymerase chain r eaction (RT-PCR). Results. LCM/RT-PCR detected mRNA for podoplanin in 2% of a single glomerul us, rat basic amino acid transporter in 6% of a single cross-section of pro ximal 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 hi stologic sections, although pure collecting ducts could not be isolated. LC M/RT-PCR localized ischemia reperfusion-induced induction of KC/interleukin -8 primarily to the medullary thick ascending limb, and detected transformi ng growth factor-beta (TGF-beta) mRNA in glomeruli of a patient with membra nous 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 c an study alterations in steady-state mRNA levels in animal models of renal disease. The applications, limitations, and refinements of this approach ar e discussed.