cAMP-dependent fluid secretion in rat inner medullary collecting ducts.

We used an unambiguous in vitro method to determine if inner medullary collecting ducts (IMCD) have intrinsic capacities to absorb and secrete solutes and fluid in an isotonic medium. IMCD(1), IMCD(2), and IMCD(3) were dissected from kidneys of young Sprague-Dawley rats. 8-Bromo-3',5'-cyclic monophosphate (8-BrcAMP) stimulated lumen formation and progressive dilation in all IMCD subsegments; lumen formation was greatest in IMCD(1.) Benzamil potentiated the rate of lumen expansion in response to 8-BrcAMP. Fluid entered tubule lumens by transcellular secretion rather than simple translocation of intracellular fluid. Secreted lumen solutes were osmometrically active. Inhibition of protein kinase A with H-89 and Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate blocked fluid secretion. The rate of lumen expansion was reduced by the selective addition of ouabain, barium, diphenyl-2-carboxylate, bumetanide, glybenclamide, or DIDS, or reduction of extracellular Cl(-). We conclude that IMCD absorb and secrete electrolytes and fluid in vitro and that secretion is accelerated by cAMP. We suggest that salt and fluid secretion by the terminal portions of the renal collecting system may have a role in modulating the composition and volume of the final urine.

Prevalence of cocaine use and its impact on asthma exacerbation in an urban population.

OBJECTIVES: To assess the prevalence of cocaine use, and its impact on severity of presentation, among adults presenting to the emergency department (ED) with asthma. A secondary aim was to assess the use of various asthma treatment modalities, with reference to the 1997 National Asthma Education and Prevention Program (NAEPP) guidelines. METHODS: All adults aged 18 to 55 years who presented to the ED of this institution with an asthma attack, were approached about participating in the study, which required giving informed consent, answering a facilitated questionnaire, and giving a urine sample for drug screening. RESULTS: Patients were enrolled during a 7-month period. A total of 163 patients were approached to enter the study; 116 patients consented to participate in the study, with 103 submitting complete urine samples. Thirty-seven patients refused to participate, and 10 were excluded. Sixty-eight percent of the patients were women, with a mean age of 33 years. African-Americans made up 89% of the total group. Thirty-five percent were cigarette smokers. Urine cocaine tests were positive in 13 of 103 (13%); 6 of 103 (5.8%) were positive for opiates. In the cocaine-positive group, 5 of 13 patients (38%) were admitted to the hospital, including two patients requiring intubation and mechanical ventilation. Of the total group, 23 of 103 patients (22%) were admitted, and 5 of those 23 admitted patients (22%) were cocaine-positive. Length of stay was significantly longer (5 vs 2.5 days, p < 0.05) in the cocaine-positive admitted patients. Forty-six percent of all patients reported using inhaled corticosteroids (ICS), with 39% of admitted patients using them. Thirty-two percent of all patients had obtained three or more refills of their beta(2)-agonist inhaler in the previous month. CONCLUSIONS: The prevalence of cocaine use may be much higher than the 13% shown in this study, because of patients' refusal to participate in the study. Second, the severity of exacerbation appears to be worse in the cocaine-positive group. Finally, the majority of patients presenting did not use ICS in accordance with the NAEPP guidelines.

cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway.

BACKGROUND: : Cellular proliferation is a key factor in the enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). We determined the extent to which adenosine 3':5'-cyclic monophosphate (cAMP) may regulate the in vitro proliferation of cyst epithelial cells derived from human ADPKD cysts. METHODS: : Epithelial cells from cysts of individuals with ADPKD and from normal human kidney cortex (HKC) of individuals without ADPKD were cultured. The effects of agonists and inhibitors on the rate of cellular proliferation and the activation of extracellular signal-regulated kinase (ERK1/2) were determined. RESULTS: : 8-Br-cAMP (100 micromol/L) stimulated the proliferation of cells from eight different ADPKD subjects to 99.0% above baseline; proliferation was inhibited by protein kinase A (PKA) antagonists H-89 (97%) and Rp-cAMP (90%). Forskolin (10 micromol/L), which activates adenylyl cyclase, increased proliferation 124%, and receptor-mediated agonists arginine vasopressin, desmopressin, secretin, vasoactive intestinal polypeptide, and prostaglandin E2 stimulated proliferation 54.2, 56.3, 46.7, 37.1, and 48.3%, respectively. The mitogen extracellular kinase (MEK) inhibitor PD98059 completely inhibited ADPKD cell proliferation in response to cAMP agonists, but genistein, a receptor tyrosine kinase inhibitor, did not block cAMP-dependent proliferation. cAMP agonists increased the activity of ERK above control levels within five minutes. In contrast to ADPKD, proliferation and ERK activity of cells derived from normal HKC were not stimulated by cAMP agonists, although electrogenic Cl- secretion was increased by these agonists in both ADPKD and HKC cell monolayers. CONCLUSIONS: : We conclude that cAMP agonists stimulate the proliferation of ADPKD but not HKC epithelial cells through PKA activation of the ERK pathway at a locus distal to receptor tyrosine kinase. We suggest that the adenylyl cyclase signaling pathway may have a unique role in determining the rate of cyst enlargement in ADPKD through its actions to stimulate cellular proliferation and transepithelial solute and fluid secretion.

The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro.

Transepithelial fluid secretion promotes the progressive enlargement of cysts in autosomal dominant polycystic kidney disease (ADPKD). Recent indirect evidence indicated that active chloride transport may drive net fluid secretion in cultures of epithelia derived from ADPKD cysts. We now report that forskolin, which stimulates adenylate cyclase, increased the efflux rate constant for 36Cl in monolayers of ADPKD cells in vitro from 0.23 +/- 0.02 min-1 to 0.44 +/- 0.05 min-1 (N = 4) and that diphenylamine 2-carboxylate (DPC), which blocks chloride channels, eliminated the forskolin-stimulated chloride efflux from these cells. To establish whether the cAMP-regulated chloride transporter, cystic fibrosis transmembrane conductance regulator (CFTR), may potentially be involved in the chloride transport and fluid secretion of ADPKD epithelia, we examined CFTR mRNA and protein in these cultures. Northern blot hybridization using a human (h) CFTR cDNA probe demonstrated the presence of an approximately 6.5 kb transcript in total RNA from polarized cultures of ADPKD, normal human kidney cortex (HKC), and T84 cells. Utilizing several antibodies to hCFTR, immunocytochemistry and confocal fluorescence microscopy localized an immunoreactive protein primarily in the apical region of forskolin-stimulated ADPKD cells grown on permeable supports. This immunoreactivity could be eliminated by preincubation of antibody with immunizing peptide. To determine the effect of CFTR abundance on the magnitude of net fluid secretion, polarized ADPKD cultures were treated with deoxyoligonucleotides that were either complementary (antisense), homologous (sense), or partially complementary (misantisense) to a sequence near the translation initiation site in hCFTR mRNA. Treatment with 5.0 microM antisense oligonucleotide resulted in a 73% reduction in forskolin-stimulated fluid secretion and a comparable reduction in the abundance of CFTR as detected by immunocytochemistry. By contrast, treatment with 5.0 microM sense oligonucleotide reduced fluid secretion by only 34% and had less of an effect on CFTR abundance, while the effects of 5.0 microM misantisense oligonucleotide on both fluid secretion and CFTR abundance were insignificant. On the basis of these results we suggest that CFTR is a major mediator of forskolin-stimulated chloride and fluid secretion by epithelial cells of human polycystic kidneys in vitro.

Effects of sodium and chloride on potassium transport by the bullfrog kidney.

We have studied the effect on K transport of reductions in the Na and Cl concentrations of solutions perfusing the isolated bullfrog kidney. We used recently developed techniques for estimating the unidirectional reabsorptive and secretory K fluxes. Reduction of Na and Cl concentrations in the arterial perfusate from 112 and 100 mM to 22 and 10 mM, respectively, inhibited K secretion 82% and K reabsorption 97%. Reduction of only the Na concentration inhibited K secretion 42% but did not affect K reabsorption. Arterial and portal perfusion with 37 mM Na, 23 mM Cl reduced urine Na concentration to 6 mM and Na reabsorption by 59%. However, K secretion rose 88% and reabsorption fell 76%. Arterial and portal perfusates with 37 mM Na, 100 mM Cl reduced urine Na concentration to 2 mM and Na reabsorption by 46%. Still, K secretion was elevated 57% by an increase in urine flow rate. K reabsorption was not reduced. Arterial and portal perfusates with 112 mM Na, 23 mM Cl, and containing SO4 also stimulated K secretion 26% and inhibited K reabsorption 91%. Thus, reduction of perfusate Na concentration to 22 mM inhibited secretion but 37 mM was sufficient to permit stimulation of secretion by low Cl concentrations and by increased tubular fluid flow rate. Reduction of the perfusate Cl concentration stimulated secretion and inhibited reabsorption. We conclude that a minimum level of Na reabsorption is required to maintain K secretion, but above that minimum level changes in the rate of Na reabsorption do not affect the rate of K secretion. The tubular fluid Cl concentration or the rate of Cl reabsorption affects both reabsorption and secretion of K and, therefore, may be an important regulator of the rate of K excretion.