Quantification of Low Abundance Natriuretic Peptide Receptor mRNA in Rat Tissues

Abstract

Natriuretic peptides are important regulators of vascular resistance and volume and electrolyte homeostasis. The quantification of natriuretic peptide receptor (NPR) mRNA is important for the understanding of the regulation of this humoral system, but is difficult due to low expression of the NPR mRNA. We report here on the evaluation of a polymerase chain reaction (PCR)-aided transcript titration assay for quantification of all three NPR subtypes (NPR-A, NPR-B, and NPR-C) mRNA. A multispecific internal standard RNA with parts of NPR-A, NPR-B, NPR-C and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nucleotide sequences was constructed and reverse transcription of standard and sample RNA (400 ng) was performed in parallel for all three NPRs and GAPDH. The specific PCR yielded differently sized products, which were quantified by high performance liquid chromatography (HPLC). The determination of specific mRNA concentrations was not influenced by cDNA input and did not depend on the PCR cycle number. Linearity between sample RNA input and mRNA concentration was demonstrated. Application of the evaluated method showed that the NPR-A mRNA expression was the most abundant of the three natriuretic peptide receptor mRNAs in rat lungs, glomeruli and left ventricles, followed by the NPR-C mRNA and the NPR-B mRNA expression. Thus, the described method allows the reliable quantification of the specific mRNA expression of all three NPRs with small amounts of RNA. The presented method might foster future research on the regulation of this humoral system in cardiovascular and kidney diseases.

C-type natriuretic peptide as a podocyte hormone and modulation of its cGMP production by glucose and mechanical stress

“…Apart from NPR-B, also A-and C-type receptors for NPs were identified in podocytes [6]. However, the B-type receptor has been reported as the least abundant of all three NP receptors, not only in the whole kidney [24] but also in the glomeruli [25,26]. Furthermore, its ligand, CNP, has been suggested to preferentially act as a paracrine hormone, exerting weaker renal [48] and glomerular [49] effects as compared to ANP.…”

“…Three types of natriuretic peptide receptor (NPR) are found in the glomeruli: NPR-A and NPR-B, which are linked to the particulate guanylyl cyclase, and NPR-C, a clearance receptor devoid of the GC activity [24][25][26]. Despite the lack of absolute specificity of NPs for particular receptors, ANP predominantly binds to NPR-A, whereas C-type natriuretic peptide (CNP) binds practically selectively to NPR-B [27,28].…”

“…Results of quantification studies on the NPR distribution show that the NPR-B is the least abundant type of NP receptor in glomeruli [25,26]. Also, the biologic role of its ligand, CNP, is in the kidney less apparent than the role of other NPs.…”

Activation of Glomerular Basement Membrane–Specific B Cells in the Renal Draining Lymph Node after T Cell–Mediated Glomerular Injury

“…PCR was used to detect B lymphocyte-induced maturation protein (blimp) mRNA using a pair of primers (5Ј-CCTTGCTACATGTGTGTCAGTTGG-3Ј, 5Ј-TGCTAG-CATGTGTGGAATCTCTGG-3Ј), which resulted in a 400-bp product, under the following conditions: Preheating at 94°C for 3 min followed by 30 cycles (94°C for 60 s, 64°C for 30 s, 72°C for 60 s; GeneAmp9700; Applied Biosystems) (21). Expression of a housekeeper gene (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control following a previous published RT-PCR method, which resulted in a 238-bp product (22). The products were separated by electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light illumination.…”