Biosensor cell assay for measuring real-time aldosterone-induced release of histamine from mesenteric arteries.

AIMS: The aims were to develop a method for real-time detection of histamine release and to test whether incubation with aldosterone induces histamine release from isolated, perfused mice mesenteric arteries. METHODS: Fura-2-loaded HEK-293 cells transfected with the histamine H1 receptor was used as a sensitive biosensor assay for histamine release from isolated mouse mesenteric arteries. Activation of the H1 receptor by histamine was measured as an increased number of intracellular Ca2+ transient peaks using fluorescence imaging. RESULTS: The developed biosensor was sensitive to histamine in physiological relevant concentrations and responded to substances released by the artery preparation. Aldosterone treatment of mesenteric arteries from wild-type mice for 50 min resulted in an increased number of intracellular Ca2+ transient peaks in the biosensor cells, which was significantly inhibited by the histamine H1 blocker pyrilamine. Mesenteric arteries from mast cell-deficient SASH mice induced similar pyrilamine-sensitive Ca2+ transient response in the biosensor cells. Mesenteric arteries from wild-type and SASH mice expressed histamine decarboxylase mRNA, indicating that mast cells are not the only source of histamine release. CONCLUSION: The developed biosensor assay can measure release of substances from vascular preparations. Histamine is released from the vessel preparation in response to aldosterone treatment independently of mast cells. The assay enables us to study a new signaling mechanism for vascular responses induced by aldosterone.

TMEM16A is a Ca(2+) -activated Cl(-) channel expressed in the renal collecting duct.

AIM: In the renal collecting ducts, ATP stimulates a Ca(2+) -activated chloride current. The identity of the channel responsible for the current under physiological conditions is not known and it was hypothesized that TMEM16a is a relevant candidate in the renal collecting duct. METHODS: The cortical collecting duct cell line M-1 was used as a model of the collecting duct. The ATP induced Ca(2+) signalling was imaged in cells loaded with Ca(2+) -sensitive fluorescent probes using confocal laser-scanning fluorescence microscopy. Chloride current was determined by mounting M-1 cell layers in Ussing chamber. The expression of TMEM16a in human kidney was tested by immunohistochemistry. RESULTS: M-1 cells displayed a transient increase in intracellular Ca(2+) concentration in response to 100 nm ATP. This response was completely blocked by addition of 100 µm suramin, indicating that ATP signals through purinergic P2 receptors. Apical addition of 100 nm ATP induced a Cl(-) current, which was blocked by suramin, DPC and the cysteine-modifying compound MTSET. M-1 cells were found to express TMEM16a at the mRNA and protein level. Functionally, it was found that knock-down of TMEM16a expression in M-1 cells inhibited the ATP induced Cl(-) -current. In human and mouse kidney sections, TMEM16a protein expression was localized to the collecting duct, and TMEM16a was found to be excreted in human urinary exosomes. CONCLUSION: TMEM16a is a Ca(2+) -activated Cl(-) channel expressed in the collecting ducts.

Mechanisms of renal NaCl retention in proteinuric disease.

In diseases with proteinuria, for example nephrotic syndrome and pre-eclampsia, there often are suppression of plasma renin-angiotensin-aldosterone system components, expansion of extracellular volume and avid renal sodium retention. Mechanisms of sodium retention in proteinuria are reviewed. In animal models of nephrotic syndrome, the amiloride-sensitive epithelial sodium channel ENaC is activated while more proximal renal Na(+) transporters are down-regulated. With suppressed plasma aldosterone concentration and little change in ENaC abundance in nephrotic syndrome, the alternative modality of proteolytic activation of ENaC has been explored. Proteolysis leads to putative release of an inhibitory peptide from the extracellular domain of the γ ENaC subunit. This leads to full activation of the channel. Plasminogen has been demonstrated in urine from patients with nephrotic syndrome and pre-eclampsia. Urine plasminogen correlates with urine albumin and is activated to plasmin within the urinary space by urokinase-type plasminogen activator. This agrees with aberrant filtration across an injured glomerular barrier independent of the primary disease. Pure plasmin and urine samples containing plasmin activate inward current in single murine collecting duct cells. In this study, it is shown that human lymphocytes may be used to uncover the effect of urine plasmin on amiloride- and aprotinin-sensitive inward currents. Data from hypertensive rat models show that protease inhibitors may attenuate blood pressure. Aberrant filtration of plasminogen and conversion within the urinary space to plasmin may activate γ ENaC proteolytically and contribute to inappropriate NaCl retention and oedema in acute proteinuric conditions and to hypertension in diseases with chronic microalbuminuria/proteinuria.

Chronic treatment with vitamin D lowers arterial blood pressure and reduces endothelium-dependent contractions in the aorta of the spontaneously hypertensive rat.

Vitamin D has cardiovascular protective effects besides regulating calcium homeostasis. To examine the chronic in vivo effect of a physiological dose of 1,25-dihydroxyvitamin D(3) on the occurrence of endothelium-dependent contractions, spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were treated with the vitamin D derivative for 6 wk. The serum 1,25-dihydroxyvitamin D(3) level of both treated WKY and SHR was significantly higher than in untreated rats while the mean arterial blood pressure of the treated SHR was significantly lower than that of control SHR. Aortic rings with or without endothelium were studied in conventional organ chambers for isometric force measurement. Confocal microscopy was used to measure the cytosolic free calcium concentration (with the fluorescent dye fluo 4) and reactive oxygen species (ROS; with dichlorodihydrofluorescein diacetate). Reverse transcription PCR and Western blotting were used to determine the mRNA and protein expression level of cyclooxygenase-1 (COX-1), prostacyclin synthase, and thromboxane synthase. The endothelium-dependent concentration-contraction curves to both acetylcholine- and A-23187-induced contractions were shifted to the right in aortas from treated SHR but not from treated WKY. The chronic treatment normalized the relaxations of contracted preparations to acetylcholine. There were no significant differences in the increases in cytosolic free calcium concentration evoked by acetylcholine and A-23187 between control and treated groups. The endothelial ROS level was higher in SHR than WKY aortas and reduced by the chronic treatment. The gene and protein expression studies indicated that the overexpression of COX-1 observed in SHR aorta was reduced by the chronic treatment. These results demonstrate that chronic treatment with 1,25-dihydroxyvitamin D(3) modulates vascular tone and this modulation is accompanied by a lowered blood pressure, reduced expression of COX-1 mRNA and protein, and reduced ROS level in SHR. The reduction in endothelium-dependent contractions does not involve the surge in endothelial cytosolic calcium concentration that initiates the contractions.

Apical serine protease activity is necessary for assembly of a high-resistance renal collecting duct epithelium.

AIM: We hypothesized that the serine protease prostasin is necessary for differentiation of a high-resistance renal collecting duct epithelium governed by glucocorticoid. METHODS: Postnatal rat kidney and adult human kidney was used to study the expression and localization of prostasin. The murine collecting duct cell line (M-1) was cultured in Snapwell inserts to investigate the significance of prostasin for the development of transepithelial electrical resistance (TER). RESULTS: In the cortex and medulla of rat kidney, prostasin mRNA and protein increased significantly between birth and weaning (day 21) and was detected in collecting ducts. Immunoreactive prostasin was associated with collecting ducts and loops of Henle in human kidney. In rat, adrenalectomy at day 10 had no effect on prostasin mRNA level in kidney at day 20. Cultured M-1 cells exhibited parallel increases in prostasin mRNA, protein and TER 5 days after seeding. Apical addition of the serine protease inhibitor aprotinin to M-1 cell cultures inhibited development of TER and led to aberrant localization of E-cadherin. This effect was mimicked by the protease inhibitor nafamostat. Apical addition of phospholipase C to cleave glycosylphosphatidylinositol (GPI) anchors released prostasin to the medium and attenuated development of TER with time of culture. Disruption of lipid rafts by methyl-ß-cyclodextrin attenuated development of TER in M-1 cells. Omission of dexamethasone impaired development of TER in M-1 cells, while prostasin protein abundance and E-cadherin distribution did not change. CONCLUSION: Apical, GPI-anchored, lipid raft-associated serine protease activity, compatible with prostasin, is necessary for the development of a high-resistance collecting duct epithelium.