Characterization of mRNA Expression in Mouse Renal Vasculature and Glomerulus Using Laser Capture Microdissection
Authors: LoDuca Thomas , Kumar Suresh , Duris Christine , North Paula E , Tabatabai Niloofar M 
Published: November 2023

The Centers for Disease Control and Prevention estimated that 29.1 million people, or 9.3% of the population, in the United States had diabetes in 2012. Diabetic nephropathy, one form of kidney disease, accounted for 44% of the new cases of kidney disease in the United States in 2011. The relationship between diabetes and the development of nephropathy is complex and not fully understood. Understanding the underlying molecular changes that occur in a diabetic kidney is vital in understanding the pathogenesis of diabetic nephropathy. Alterations in both renal vascular function and glomerular filtration in diabetic patients have been shown previously, but a protocol to examine the molecular changes that occur specifically at the glomerulus and renal vasculature has not been well established. The aim of this study was to establish a protocol that allows for the investigation of changes in gene expression of factors involved in glomerular filtration. We specifically looked at elucidating a method for studying gene expression of renal vasculature smooth muscle and glomeruli podocyte. The developed protocol was centered upon collected high quality RNA from structure specific regions in the kidney. Our protocol begins with tissue collection. Left and right kidneys were collected from a male C57BL/6 mouse (Charles River Laboratories) and embedded in Optimal Cutting Temperature Compound (Sakura). The tissue was sectioned by cryostat into 10μm sagittal sections. The tissue sections were placed onto polyethylene naphthalate membrane slide (Zeiss) and immediately placed into −20°C‐cold acetone for 2 min. The slide is then transferred into a fresh aliquot of cold acetone for an additional 2 min. Following the acetone fixation, the slide is air dried for 5 min. The tissue then undergoes staining using 1% cresyl violet acetate prepared in 75% ethanol in diethylpyrocarbonate treated H2O. The staining procedure involves rehydrating the tissue in serial dilutions of ethanol prior to staining with the 1% cresyl violet acetate solution. After staining, the tissue is dehydrated in increasing concentrations of ethanol, followed by clearing in xylene. The stained tissue is allowed to air dry for at least 15 minutes before beginning laser capture microdissection. Laser capture microdissection was performed using the Zeiss PALM Microbeam system. Cells of interest, either renal vasculature or glomeruli, were dissected and collected into AdhesiveCap 200 clear tubes (Zeiss). The target amount of tissue area collected was between 75,000–100,000 μm2. RNA collection was performed using RNeasy Micro Kit (Qiagen) and quantification was performed using NanoDrop 2000 (Thermo Scientific). cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) and Oligo‐dT primers (Invitrogen). PCR was performed using Platinum Taq DNA Polymerase (Invitrogen). Primers for the housekeeping gene ribosomal protein S15 (S15, 361 base pairs (bp)), alpha 2 smooth muscle actin (α‐SMA, 538 bp) gene, and Wilms Tumor 1 (WT‐1, 107 bp) gene, as vascular smooth muscle and glomeruli markers, respectively. The PCR products were run on 2% agarose gel at 100V for 60 min. The results showed expression of all three genes in the respective tissue types and determined our protocol is robust for determination of gene expression in mouse renal vasculature and glomeruli. This protocol can be applied in the study of gene expression in the diabetic kidney to understand the development of diabetic nephropathy.

Support or Funding Information

The National Institutes of Health grants R01DK085031 and R25DK098104