Jennifer Hawkins

Uroguanylin knockout mice have increased blood pressure and impaired natriuretic response to enteral NaCl load

Guanylin and uroguanylin, peptides synthesized in the intestine and kidney, have been postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of uroguanylin in the regulation of sodium excretion, we created gene-targeted mice in which uroguanylin gene expression had been ablated. Northern and Western analysis confirmed the absence of uroguanylin message and protein in knockout mice, and cGMP levels were decreased in the mucosa of the small intestine. Ussing chamber analysis of jejunum revealed that Na+/H+ exchanger-mediated Na+ absorption and tissue conductance was not altered in the knockout animals, but short-circuit current, an index of electrogenic anion secretion, was reduced. Renal clearance measurements showed that uroguanylin deficiency results in impaired ability to excrete an enteral load of NaCl, primarily due to an inappropriate increase in renal Na+ reabsorption. Finally, telemetric recordings of blood pressure demonstrated increased mean arterial pressure in uroguanylin knockout animals that was independent of the level of dietary salt intake. Together, these findings establish a role for uroguanylin in an enteric-renal communication axis as well as a fundamental principle of this axis in the maintenance of salt homeostasis in vivo.

Randomized, Controlled Human Challenge Study of the Safety, Immunogenicity, and Protective Efficacy of a Single Dose of Peru-15, a Live Attenuated Oral Cholera Vaccine

ABSTRACT Peru-15 is a live attenuated oral vaccine derived from a Vibrio cholerae O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 × 108 CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 105 CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (≥3,000 g of diarrheal stool) (P = 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (P < 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool V. cholerae excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.

The proximal convoluted tubule is a target for the uroguanylin-regulated natriuretic response.

Objectives and Methods: Guanylin and uroguanylin are peptides synthesized in the intestine and kidney that are postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of guanylin and uroguanylin in the regulation of sodium excretion, we used gene-targeted mice in which the uroguanylin, guanylin or the peptide receptor guanylate cyclase C gene expression had been ablated. Results: Metabolic balance studies demonstrated that there was impaired excretion of a sodium load in uroguanylin (but not in guanylin or guanylate cyclase C) knockout mice. Uroguanylin-dependent natriuresis occurred without an increase in circulating prouroguanylin. A distinct morphological phenotype was present in the proximal convoluted tubules of uroguanylin knockout animals after an enteral salt loading. Marked vacuolization of the proximal convoluted tubule epithelial cells was observed by using light and electron microscopy. There was also a change in the distribution of the sodium hydrogen exchanger 3 (NHE3) after an enteral salt loading. In wild-type animals, there was a partial redistribution of NHE3 from the villus fraction to the less accessible submicrovillus membrane compartment, but this effect was less apparent in uroguanylin knockout animals, presumably resulting in greater Na+/H+ exchange. Conclusions: Together, these findings further establish a role for uroguanylin in fluid homeostasis and support a role for uroguanylin as an integral component of a signaling mechanism that mediates changes in Na+ excretion in response to an enteral salt loading. Proximal tubular NHE3 activity is a possible target for uroguanylin-mediated changes in Na+ excretion.

Safety and Immunogenicity of Escalating Dosages of a Single Oral Administration of Peru-15 pCTB, a Candidate Live, Attenuated Vaccine against Enterotoxigenic Escherichia coli and Vibrio cholerae

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) organisms are a leading cause of infectious diarrhea in developing countries. A live, attenuated cholera strain that expresses high levels of the nontoxic B subunit of cholera toxin, which might also serve as an ETEC protective antigen, was evaluated for safety, excretion, and immunogenicity in healthy volunteers. We enrolled four inpatient dose-escalation cohorts of 15 to 16 eligible subjects to randomly (3:1) receive a single oral dose of vaccine or placebo (buffer alone), evaluating 1 ×107, 1 ×108, 1 ×109, and 1 ×1010 CFU of the vaccine. The vaccine was well tolerated, although some subjects experienced moderate diarrhea. The serum Inaba vibriocidal antibody response appeared to display a dose-response relationship with increasing dosages of vaccine, plateauing at the 109-CFU dosage. The serum antitoxin (cholera toxin and heat-labile enterotoxin) antibody seroconversion rate (4-fold increase over baseline) also appeared to display a dose-response relationship. The vaccine strain was excreted in stool cultures, displaying a dose-response relationship. A single oral dose of Peru-15 pCTB at dosages up to 1 ×1010 CFU was safe and immunogenic in this first-in-human trial. These encouraging data support the ongoing clinical development of this candidate combined cholera and ETEC vaccine. (This study has been registered at ClinicalTrials.gov under registration no. NCT00654108.)