Analysis of sodium pump gene expression in microdissected nephrons using competitive RT-PCR and a novel HPLC technique
Hayward-Lester, Amanda L
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Hypertension is determined by the interaction of multiple genetic and environmental factors, making it difficult to elucidate any single genetic determinant. Biochemical markers such as intraerythrocytic sodium concentration, erythrocytic ouabain binding-site density and passive sodium leak indicate that abnormal membrane cation flux segregates with some hypertension subtypes. The sodium pump, Na+, K+ ATP-ase (NKA) may therefore participate in the development of hypertension. It is a multi-subunit cell membrane protein which translocates sodium and potassium ions with hydrolysis of ATP. It is inhibited by ouabain and regulated by phosphorylation. The alpha subunit (of which there are four isoforms encoded by different genes) is currently ascribed all catalytic function, while the beta and gamma subunits may have regulatory roles. We examined NBCA alpha and gamma subunit gene expression in spontaneously hypertensive rats (SHR) and Wistar Kyoto controls (WKY). Both prehypertensive and adult SHR exhibit abnormal renal sodium retention. Solution hybridization studies in adult SHR revealed a decrease in alpha 1 expression in kidney. Kidney is a heterogeneous tissue whose functional unit, the nephron, may be divided into 12 distinct segments. To examine NKA expression in individual segments requires an assay allowing quantitation of NKA alpha and gamma isoform RNA in microdissected tissue samples. We combined competitive RT-PCR with a novel ion-paired reversed phase HPLC to produce rapid, accurate and precise measurement of gene expression in a single-tube assay. The ability of HPLC to resolve heteroduplex molecules formed between native and competitor products proved essential. Assay validation confirmed absolute quantification is possible if competitors have identical reverse-transcription efficiency to the native RNA. We used the assay to examine qualitative and quantitative expression of NKA subunits in normotensive Sprague-Dawley, prehypertensive and adult SHR and WKY. Qualitative analysis revealed alpha 1 and gamma expression in all segments examined. Expression of the other alpha isoforms was not detected. Quantitative analysis in the prehypertensive SHR revealed that a selective alteration in alpha 1 expression in proximal convoluted tubule may explain the results obtained in whole kidney and suggest an attempted feedback response by SHR to reduce sodium reabsorption.