Based on this recovery, and the detection limit of the assay, we calculate that a single rat kidney must contain 100 fmol of free Ugn (= 7). For comparison, we Ellipticine then measured the propeptide content of the kidney, Ellipticine using a well-validated quantitative Western blot assay (58). of propeptide greatly exceeds that of Ugn. However, immunocytochemical analysis reveals that renal proUgn is found exclusively in distal tubular segments, sites previously shown not to accumulate radiolabeled proUgn after intravascular infusions. Thus proUgn appears to be synthesized within the kidney, but the factors that determine its abundance (rates of transcription, translation, processing, and secretion) must be balanced quite differently than in the gut. Surprisingly, we also find negligible expression of GC-C in the rat kidney, a result confirmed both by RT-PCR and by functional assays that measure Ugn-activated cGMP synthesis. Taken together, these data provide evidence for an intrarenal Ugn system that differs from the well-described intestinal system in its regulatory mechanisms and in the receptor targeted by the peptide. and shows an aligned Western blot analysis of individual fractions. The retention time of recombinant rat proUgn is indicated by the arrowheads in each chromatogram. for 30 min at 4C, and the supernatant fraction was collected and stored at ?80C before analysis. For the RT-PCR studies reported in Fig. 8, tissues were rapidly cut into small pieces (1 mm3) and immersed overnight at room temperature in RNAlater (Qiagen, Valencia, CA). RNA was subsequently extracted and purified using an RNEasy Mini kit (Qiagen), then treated with DNase I to minimize contamination by genomic DNA. Open in a separate window Fig. 1. Relative preprouroguanylin (preproUgn) mRNA and proUgn polypeptide levels in rat small intestine and kidney. Eng represent mean results from 7 independent column runs, each based on an extract from an independent kidney. Authenticated peptide standards elute at the points indicated by the black arrowheads. half of the gel contains samples from 5 independent kidney extracts (half of the gel contains a standard curve constructed with known amounts of recombinant rat proUgn, as indicated. Western blot procedures and quantitative proUgn assay. Immunoblots were performed as described previously (45, 58), using antibodies 6910, 6911, 6912, or 1C11 (described above). Ellipticine Immunoblots in Fig. 1were treated with chemiluminescence reagent (Roche Diagnostics) and exposed to film. For quantitation, films were digitized at a resolution of 600 dpi (LaCie Silverscanner IV) and analyzed densitometrically using NIH Image software (available at http://rsb.info.nih.gov/nih-image/download.html). All other immunoblots were performed with an infrared-emitting secondary antibody (IRDye 800-coupled goat anti-rabbit from Rockland, Gilbertsville, PA, diluted 1:2,000) and analyzed with an Odyssey infrared gel imaging system (LI-COR Biosciences, Lincoln, NE). The quantitative Western blot-based assay is described in detail in a previous publication (58). Northern blot procedures. For Northern blots, isolated RNA was denatured by glyoxal, size-fractionated by electrophoresis on 1% agarose gels, pressure-transferred to positively charged nylon membranes (ICN), and allowed to hybridize with an antisense GC-C probe, as previously described (43). RT-PCR amplifications. Random hexamer-primed cDNA was prepared from isolated RNA, using the SuperScript III First-Strand Synthesis System (Invitrogen) according to the supplier’s protocol. Subsequent RT-PCR analysis employed primer pairs and probes, as specified in Table 1. Table 1. Primers used for RT-PCR (GC-C)(GC-C)(-actin)(cyclophilin A)and were used for traditional endpoint RT-PCR. Reactions were performed in a DNA thermal cycler (Ericorp, San Diego, CA), using a GeneAmp DNA amplification kit (PerkinElmer Cetus) and standard Ellipticine ionic conditions. The cycler was programmed to heat to 94C for 2 min, followed by up to 45 thermal cycles (94C for 1 min, 60C for 1 min, 72C for 2 min), and a final incubation at 72C for 10 min. PCR products were stained with ethidium bromide, electrophoresed on agarose gels, and visualized under UV light. Primer and were used for real-time RT-PCR. Each sample included a fluorescent probe with a reporter dye (fluorescein) at the 5 end.