Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May;4(9):e12782.
doi: 10.14814/phy2.12782. Epub 2016 May 15.

Dietary Salt Regulates Uroguanylin Expression and Signaling Activity in the Kidney, but Not in the Intestine

Affiliations
Free PMC article

Dietary Salt Regulates Uroguanylin Expression and Signaling Activity in the Kidney, but Not in the Intestine

Robert C Fellner et al. Physiol Rep. .
Free PMC article

Abstract

The peptide uroguanylin (Ugn) is expressed at significant levels only in intestine and kidney, and is stored in both tissues primarily (perhaps exclusively) as intact prouroguanylin (proUgn). Intravascular infusion of either Ugn or proUgn evokes well-characterized natriuretic responses in rodents. Furthermore, Ugn knockout mice display hypertension and salt handling deficits, indicating that the Na(+) excretory mechanisms triggered when the peptides are infused into anesthetized animals are likely to operate under normal physiological conditions, and contribute to electrolyte homeostasis in conscious animals. Here, we provide strong corroborative evidence for this hypothesis, by demonstrating that UU gnV (the rate of urinary Ugn excretion) approximately doubled in conscious, unrestrained rats consuming a high-salt diet, and decreased by ~15% after salt restriction. These changes in UU gnV were not associated with altered plasma proUgn levels (shown here to be an accurate index of intestinal proUgn secretion). Furthermore, enteric Ugn mRNA levels were unaffected by salt intake, whereas renal Ugn mRNA levels increased sharply during periods of increased dietary salt consumption. Together, these data suggest that diet-evoked Ugn signals originate within the kidney, rather than the intestine, thus strengthening a growing body of evidence against a widely cited hypothesis that Ugn serves as the mediator of an entero-renal natriuretic signaling axis, while underscoring a likely intrarenal natriuretic role for the peptide. The data further suggest that intrarenal Ugn signaling is preferentially engaged when salt intake is elevated, and plays only a minor role when salt intake is restricted.

Keywords: Dietary salt; entero‐renal endocrine axis; natriuretic peptide; sodium homeostasis.

Figures

Figure 1
Figure 1
Characterization of the antibody used for the proUgn western blot assay. (A) Experiments with recombinant propeptides demonstrate the selectivity of anti‐proUgn antibody 6910. The two left lanes were loaded with identical samples of purified recombinant proGn that were run side‐by‐side on the same gel, then separated for incubation with different primary antibodies, as indicated. Anti‐proGn antibody 2538 recognizes proGn (the minor immunopositive band of lower molecular weight presumably represents small amounts of a proGn degradation product), but antibody 6910 does not. In contrast, antibody 6910 selectively recognizes purified recombinant proUgn (right lane). (B) Rat plasma (50 μL) was fractionated by C‐18 reverse‐phase HPLC, and then 500 μL of each 1 mL fraction was dried, resuspended in electrophoresis sample buffer, and analyzed by immunoblotting. Antibody 6910 should not recognize Ugn or proGn, and the blot confirms a complete lack of crossreactivity at retention times where Ugn or proGn would elute from the column. Here, and in all other relevant figures, retention times for recombinant proUgn, proGn, and Ugn standards were established in parallel column runs, and are indicated by the arrows. The left‐most lane of the gel was loaded with a rat proUgn standard. (C) Western blot analysis of urine samples obtained from three normal rats (control) and three animals previously subjected to surgical ablation of five‐sixths of their renal mass (renal failure). Each urine sample (100 μL) was prefractionated on a Superdex column, as described in the Methods, and then fractions 31–34 (where proUgn elutes from this column) were pooled, dried, resuspended in electrophoresis sample buffer, and half of this final sample was analyzed by immunoblotting. The left‐hand lanes of the gel were loaded with a dilution series of recombinant rat proUgn, as a standard curve.
Figure 2
Figure 2
Validation of a novel Ugn binding assay. (A) Ability of synthetic proUgn, Ugn, and Gn to displace radioligand in the competitive binding assay. (B) The analyte responsible for the binding displacement activity of urine coelutes with authentic Ugn from a high‐resolution C‐18 HPLC column. A 200 μL sample of urine (collected at 3 am on day 2 of a high‐salt diet) was loaded on the column; this is four times the volume that is loaded in a typical assay. Retention times of Gn, Ugn, proGn, and proUgn standards are indicated by the arrows. (C) Displacements of radioligand by urine samples from a Ugn knockout mouse and a wild‐type mouse are interpolated in a standard curve generated with a rat Ugn standard. (D) Endogenous Ugn is not detected in rat plasma (black symbols), whereas a strong peak is readily observed when 2 nmol exogenous Ugn are added to a plasma sample prior to chromatography (white symbols – retention times of Gn, Ugn, proGn, and proUgn standards are indicated by the arrows).
Figure 3
Figure 3
Urinary excretion of Ugn and Na+ as a function of diet. Longitudinal determination of Na+ consumption and urinary Na+ and Ugn excretion for rats fed sequentially on normal (N‐1), low (L), high (H), and normal (N‐2) Na+ diets. Vertical dotted lines correspond to 12 am (midnight), and the small black/white boxes along the horizontal axis indicate night/day status, respectively. (A) In the upper panel, the gray symbols indicate daily Na+ ingestion in mEq/24 h, the white symbols indicate daily urinary Na+ excretion in mEq/24 h, and the black symbols show excretion as a percentage of ingestion (= 5). (B) The lower panel shows urinary Ugn excretion rate in fmol/min, as determined for each 6‐h collection period (black and white symbols indicate samples that were obtained during the dark and light periods, respectively, = 4 or 5).
Figure 4
Figure 4
Urinary Na+ excretion as a function of urinary Ugn excretion under different dietary conditions. Each data point represents urinary salt excretion plotted as a function of contemporaneous urinary Ugn excretion for an individual animal at a specific time point. Data were obtained from nocturnal urine samples collected during different salt feeding phases (see text and Fig. 3; N‐1 = initial period of normal salt consumption, L = low salt consumption, H = high salt consumption). Lines were fit by linear regression. For high salt, = 0.0799x + 6.2673, R² = 0.27; for normal salt, = 0.0068x + 0.334, R² = 0.17; for low salt, = 0.0009x + 0.02, R² = 0.39.
Figure 5
Figure 5
Western blot analysis of plasma proUgn. The quantitative western blot assay is described in the Methods and in Figure 1. The number of replicates (n) is given in parentheses for each measurement. (A) Time course of plasma levels of proUgn (black circles) and inulin (white circles) after ligation of splanchnic or renal arteries, as indicated. (B) Circadian fluctuations in circulating plasma levels of proUgn are illustrated for a subset of the animals included in the diet study. Data were acquired from rats fed normal chow (N), after 5 days on low‐salt chow (L5), and after 5 days on high‐salt chow (H5). Plasma samples (100 μL) were taken every 6 h via an indwelling carotid cannula, and assayed for proUgn content using the quantitative western blot assay. Data points are plotted against the time of sample acquisition. The dashed line indicates 12 am (midnight). Asterisk indicates < 0.05. (C) Diurnal (white bars) and nocturnal (black bars) plasma levels of proUgn in animals fed ad libitum (left panel) or animals fasted (right panel) for 12 h (diurnal fasting values) or 24 h (nocturnal fasting values). (D) Representative western blots, showing a subset of the raw data used to generate the graphs in panels e and f. For quantitation, the infrared signal intensity of each sample was interpolated in a standard curve derived from known amounts of recombinant rat proUgn, as shown at the right for gel 2 (black symbols = standards, white symbols = plasma samples). Values from multiple gels could be combined to generate the final graphs in panels e and f, because every gel was loaded with an identical standard curve. (E) Circulating levels of proUgn at 3 am under defined dietary conditions. (F) Average rates of nocturnal urinary Ugn excretion (UU gnV) plotted as a function of average nocturnal plasma proUgn concentration for animals consuming normal salt (NS), low salt for 5 days (LS5), or high salt for 1 (HS1), 2 (HS2) or 5 (HS5) days (R² = 0.009 for a linear fit to the data).
Figure 6
Figure 6
Ugn mRNA expression in the kidneys and small intestines of animals consuming normal, low, and high salt diets. All tissues for these mRNA measurements were collected from animals killed at 3 am (n is given in parentheses; asterisk indicates < 0.05 vs. control). Expression of Ugn mRNA transcript is given as a percent of control (normal salt) levels in either kidney or small intestine when animals were fed a low‐salt diet for 5 days (L5), 24 h after switching to a high‐salt diet (H1), or 5 days after switching to a high‐salt diet (H5). The 2ΔΔCt method was used to determine relative expression, as described in Methods.

Similar articles

See all similar articles

References

    1. Carey R. M. 1978. Evidence for a splanchnic sodium input monitor regulating renal sodium excretion in man. Lack of dependence upon aldosterone. Circ. Res. 43:19–23. - PubMed
    1. Carrithers S. L., Hill M. J., Johnson B. R., O'Hara S. M., Jackson B. A., Ott C. E., et al. 1999. Renal effects of uroguanylin and guanylin in vivo. Braz. J. Med. Biol. Res. 32:1337–1344. - PubMed
    1. Carrithers S. L., Eber S. L., Forte L. R., and Greenberg R. N.. 2000a. Increased urinary excretion of uroguanylin in patients with congestive heart failure. Am. J. Physiol. Heart Circ. Physiol. 278:H538–H547. - PubMed
    1. Carrithers S. L., Taylor B., Cai W. Y., Johnson B. R., Ott C. E., Greenberg R. N., et al. 2000b. Guanylyl cyclase‐C receptor mRNA distribution along the rat nephron. Regul. Pept. 95:65–74. - PubMed
    1. Carrithers S. L., Jackson B. A., Cai W. Y., Greenberg R. N., and Ott C. E.. 2002. Site‐specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine. Regul. Pept. 107:87–95. - PubMed

Publication types

Feedback