Abstract 342: RNA-seq Profiling of Individually Microdissected Renal Tubule Segments

Abstract

Control of renal tubule NaCl transport plays an important role in blood pressure regulation. In the past, transport studies and biochemical assays have been conducted in microdissected renal tubule segments to discover mechanisms of transport regulation. Here, we extend the use of microdissected renal tubules to transcriptomic analysis using the RNA-seq method with small sample amplification. The feasibility of the approach was tested in microdissected cortical thick ascending limbs (cTAL) isolated by hand dissection from collagenase-incubated rat renal cortex. After lysis of isolated cTAL cells, poly(A)-targeted reverse transcription and library preparation were performed ( Nature Protocols 5:516-535). Paired-end sequencing of the cDNA library was done using an Illumina HiSeq 2000 sequencer. The generated 50-bp reads were aligned to the UCSC rat reference genome ( Bioinformatics 25, 1754-60). Reads per kilobase exon model normalized by million mapped reads (RPKM) were calculated. In an initial sample of 5 mm cTALs, 451,694,588 total reads were obtained of which 235,735,784 reads (52%) were mapped and properly paired. In this experiment, 5410 RefSeq genes were identified. To assess accuracy, we surveyed G-protein coupled receptors (GPCRs) and transport proteins that were identified. Among the GPCRs known to be expressed in cTAL ( Avpr2, Pth1r, Gcgr, Calcr, Casr , and Ptger3 ) only the calcitonin receptor was not found. Among the transporter proteins generally accepted to be expressed in cTAL ( Slc12a1, Kcnj1, Clcnkb, Cldn16, Slc9a3, Slc2a4 and Atp1a1 ) only Slc9a3 was not found. In contrast, transporter proteins characteristic of neighboring tubule segments were not found. In addition, gene-set analysis showed overrepresentation of mitochondrial genes among transcripts with high RPKM . When longer lengths of cTAL were used, the number of mapped transcripts increased, but not linearly (15 mm, 8455 genes; 25 mm, 12,110 genes). Experiments with a second segment, medullary TALs, gave a profile very similar to that of the cTAL. These studies establish the feasibility of RNA-seq-based transcriptomic profiling in microdissected renal tubule segments and ‘open the door’ for profiling of gene expression along the entire renal tubule.