Ulla G. Friis

Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC gamma subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

Vascular Smooth Muscle Cells Express the α1A Subunit of a P-/Q-Type Voltage-Dependent Ca2+Channel, and It Is Functionally Important in Renal Afferent Arterioles

In the present study, we tested whether the &agr;1A subunit, which encodes a neuronal isoform of voltage-dependent Ca2+ channels (VDCCs) (P-/Q-type), was present and functional in vascular smooth muscle and renal resistance vessels. By reverse transcription–polymerase chain reaction and Southern blotting analysis, mRNA encoding the &agr;1A subunit was detected in microdissected rat preglomerular vessels and vasa recta, in cultures of rat preglomerular vascular smooth muscle cells (VSMCs), and in cultured rat mesangial cells. With immunoblots, &agr;1A subunit protein was demonstrated in rat aorta, brain, aortic smooth muscle cells (A7r5), VSMCs, and mesangial cells. Immunolabeling with an anti-&agr;1A antibody was positive in acid-macerated, microdissected preglomerular vessels and in A7r5 cells. Patch-clamp experiments on aortic A7r5 cells showed 22±4% (n=6) inhibition of inward Ca2+ current by &ohgr;-Agatoxin IVA (10–8 mol/L), which in this concentration is a specific inhibitor of P-type VDCCs. Measurements of intracellular Ca2+ in afferent arterioles with fluorescence-imaging microscopy showed 32±9% (n=10) inhibition of the K+-induced rise in Ca2+ in the presence of 10–8 mol/L &ohgr;-Agatoxin IVA. In microperfused rabbit afferent arterioles, &ohgr;-Agatoxin IVA inhibited depolarization-mediated contraction with an EC50 of 10–17 mol/L and complete blockade at 10–14 mol/L. We conclude that the &agr;1A subunit is expressed in VSMCs from renal preglomerular resistance vessels and aorta, as well as mesangial cells, and that P-type VDCCs contribute to Ca2+ influx in aortic and renal VSMCs and are involved in depolarization-mediated contraction in renal afferent arterioles.

Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases.

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (Cm) with a marked increase in Cm at 1 &mgr;mol/L, no net change at 10 &mgr;mol/L, and a decrease at 100 &mgr;mol/L cAMP. cGMP also had a dual effect on Cm at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on Cm. Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on Cm and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

Prostaglandin F2alpha elevates blood pressure and promotes atherosclerosis.

Little is known about prostaglandin F2α in cardiovascular homeostasis. Prostaglandin F2α dose-dependently elevates blood pressure in WT mice via activation of the F prostanoid (FP) receptor. The FP is expressed in preglomerular arterioles, renal collecting ducts, and the hypothalamus. Deletion of the FP reduces blood pressure, coincident with a reduction in plasma renin concentration, angiotensin, and aldosterone, despite a compensatory up-regulation of AT1 receptors and an augmented hypertensive response to infused angiotensin II. Plasma and urinary osmolality are decreased in FP KOs that exhibit mild polyuria and polydipsia. Atherogenesis is retarded by deletion of the FP, despite the absence of detectable receptor expression in aorta or in atherosclerotic lesions in Ldlr KOs. Although vascular TNFα, inducible nitric oxide enzyme and TGFβ are reduced and lesional macrophages are depleted in the FP/Ldlr double KOs, this result reflects the reduction in lesion burden, as the FP is not expressed on macrophages and its deletion does not alter macrophage cytokine generation. Blockade of the FP offers an approach to the treatment of hypertension and its attendant systemic vascular disease.

Pituitary Adenylate Cyclase–Activating Polypeptide Stimulates Renin Secretion via Activation of PAC1 Receptors

Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because the kidney expresses both PACAP and PACAP-binding sites, it was speculated that PACAP might regulate cardiovascular function by direct vascular effects and indirectly by regulating renin release from the kidneys. PACAP (1-27) stimulated renin secretion from isolated perfused kidneys of rats 4.9-fold with a half-maximum concentration of 1.9 nmol/L. In addition, PACAP stimulated renin release and enhanced membrane capacitance of isolated juxtaglomerular cells, indicating a direct stimulation of exocytotic events. The effect of PACAP on renin release was mediated by the specific PACAP receptors (PAC1), because PACAP (1-27) applied in concentrations in the physiologic range (10 and 100 pmol/L) did not enhance renin release from isolated kidneys of PAC1 receptor knockout mice (PAC1-/-), whereas it stimulated renin release 1.38- and 2.5-fold in kidneys from wild-type mice. Moreover, plasma renin concentration was significantly lower in PAC1-/- compared with their wild-type littermates under control conditions as well as under a low- or high-salt diet and under treatment with the angiotensin-converting enzyme inhibitor ramipril, whereas no differences in plasma renin concentration between the genotypes were detectable after water deprivation. These data show that PACAP acting on PAC1 receptors potently stimulates renin release, serving as a tonic enhancer of the renin system in vivo.

Molecular and Functional Identification of Cyclic AMP-Sensitive BKCa Potassium Channels (ZERO Variant) and L-Type Voltage-Dependent Calcium Channels in Single Rat Juxtaglomerular Cells

Abstract— This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Cav) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between −60 and −10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was −32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Cav blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Cav 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

Coexpression of Voltage-Dependent Calcium Channels Cav1.2, 2.1a, and 2.1b in Vascular Myocytes

Voltage-dependent Ca2+ channels Cav1.2 (L type) and Cav2.1 (P/Q type) are expressed in vascular smooth muscle cells (VSMCs) and are important for the contraction of renal resistance vessels. In the present study we examined whether native renal VSMCs coexpress L-, P-, and Q-type Ca2+ currents. The expression of both Cav2.1a (P-type) and Cav2.1b (Q-type) mRNA was demonstrated by RT-PCR in renal preglomerular vessels from rats and mice. Immunolabeling was performed on A7r5 cells, renal cryosections, and freshly isolated renal VSMCs with anti-Cav1.2 and anti-Cav2.1 antibodies. Conventional and confocal microscopy revealed expression of both channels in all of the smooth muscle cells. Whole-cell patch clamp on single preglomerular VSMCs from mice showed L-, P-, and Q-type currents. Blockade of the L-type currents by calciseptine (20 nmol/L) inhibited 35.6±3.9% of the voltage-dependent Ca2+ current, and blocking P-type currents (&ohgr;-agatoxin IVA 10 nmol/L) led to 20.2±3.0% inhibition, whereas 300 nmol/L of &ohgr; agatoxin IVA (blocking P/Q-type) inhibited 45.0±7.3%. In rat aortic smooth muscle cells (A7r5), blockade of L-type channels resulted in 28.5±6.1% inhibition, simultaneous blockade of L-type and P-type channels inhibited 58.0±11.8%, and simultaneous inhibition of L-, P-, and Q-type channels led to blockade (88.7±5.6%) of the Ca2+ current. We conclude that aortic and renal preglomerular smooth muscle cells express L-, P-, and Q-type voltage-dependent Ca2+ channels in the rat and mouse.

Urinary Plasmin Activates Collecting Duct ENaC Current in Preeclampsia

In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the &ggr;-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability of tubular fluid to activate ENaC. Urine was sampled from 16 preeclamptic (PE) patients and 17 normotensive pregnant women (Ctrl). Urine was analyzed for plasmin(ogen), creatinine, albumin, aldosterone, Na+, K+, proteolytic activity, and for its effect on inward current in cortical collecting duct cells (M1 cells) by whole-cell patch clamp. In PE, urine plasmin(ogen): creatinine ratio was elevated 40-fold (geometric mean, 160 versus 4 µg/g; P<0.0001) and urine aldosterone: creatinine ratio was suppressed to 25% of Ctrl (geometric mean, 27 versus 109 µg/g; P<0.001). A significant negative correlation was found in PE between urinary plasmin(ogen) and aldosterone (P<0.05). In PE, proteolytic activity was detected at 90 to 75 kD by gelatin zymography in 14 of 16 patients and confirmed by serine protease assay. Immunoblotting showed active plasmin in PE urine. Whole-cell inward current increased in M1 cells on exposure to urine from PE (173±21%; n=6; P<0.001). The increase in current was abolished by amiloride (2 &mgr;mol/L; P<0.001), &agr;2-antiplasmin (1 &mgr;mol/L; P<0.001), and heat denaturation (P<0.001). Preeclampsia is associated with urinary excretion of plasmin(ogen) and plasmin-dependent activation of ENaC by urine. Proteolytic activation of ENaC by plasmin may contribute to Na+ retention and hypertension in preeclampsia.

Regulation of renin secretion by renal juxtaglomerular cells

A major rate-limiting step in the renin–angiotensin–aldosterone system is the release of active renin from endocrine cells (juxtaglomerular (JG) cells) in the media layer of the afferent glomerular arterioles. The number and distribution of JG cells vary with age and the physiological level of stimulation; fetal life and chronic stimulation by extracellular volume contraction is associated with recruitment of renin-producing cells. Upon stimulation of renin release, labeled renin granules “disappear;” the number of granules decrease; cell membrane surface area increases in single cells, and release is quantal. Together, this indicates exocytosis as the predominant mode of release. JG cells release few percent of total renin content by physiological stimulation, and recruitment of renin cells is preferred to recruitment of granules during prolonged stimulation. Several endocrine and paracrine agonists, neurotransmitters, and cell swelling converge on the stimulatory cyclic AMP (cAMP) pathway. Renin secretion is attenuated in mice deficient in beta-adrenoceptors, prostaglandin E2–EP4 receptors, Gsα protein, and adenylyl cyclases 5 and 6. Phosphodiesterases (PDE) 3 and 4 degrade cAMP in JG cells, and PDE3 is inhibited by cyclic GMP (cGMP) and couples the cGMP pathway to the cAMP pathway. Cyclic AMP enhances K+-current in JG cells and is permissive for secretion by stabilizing membrane potential far from threshold that activates L-type voltage-gated calcium channels. Intracellular calcium paradoxically inhibits renin secretion likely through attenuated formation and enhanced degradation of cAMP; by activation of chloride currents and interaction with calcineurin. Connexin 40 is necessary for localization of JG cells in the vascular wall and for pressure- and macula densa-dependent suppression of renin release.

Stimulation of Renin Secretion by Catecholamines Is Dependent on Adenylyl Cyclases 5 and 6

The sympathetic nervous system stimulates renin release from juxtaglomerular cells via the &bgr;-adrenoreceptor-cAMP pathway. Recent in vitro studies have suggested that the calcium-inhibited adenylyl cyclases (ACs) 5 and 6 possess key roles in the control of renin exocytosis. To investigate the relative contribution of AC5 and AC6 to the regulation of renin release in vivo we performed experiments using AC5 and AC6 knockout mice. Male AC5−/− mice exhibited normal plasma renin concentrations, renal renin synthesis (mRNA and renin content), urinary volume, and systolic blood pressure. In male AC6−/− mice, plasma renin concentration (AC6−/−: 732±119; AC6 +/+: 436±78 ng of angiotensin I per hour*mL−1; P<0.05), and renin synthesis were stimulated associated with an increased excretion of dilute urine (1.55-fold; P<0.05) and reduced blood pressure (−10.6 mm Hg; P<0.001). Stimulation of plasma renin concentration by a single injection of the &bgr;-adrenoreceptor agonist isoproterenol (10 mg/kg IP) was significantly attenuated in AC5−/− (male: −20%; female: −33%) compared with wild-type mice in vivo. The mitigation of the plasma renin concentration response to isoproterenol was even more pronounced in AC6−/− (male: −63%; female: −50% versus AC6+/+). Similarly, the effects of isoproterenol, prostaglandin E2, and pituitary adenylyl cyclase-activating polypeptide on renin release from isolated perfused kidneys were attenuated to a higher extent in AC6−/− (−51% to −98% versus AC6+/+) than in AC5−/− (−31% to 46% versus AC5+/+). In conclusion, both AC5 and AC6 are involved in the stimulation of renin secretion in vivo, and AC6 is the dominant isoforms in this process.