Ole Skøtt

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.

Rapid Inhibition of Vasoconstriction in Renal Afferent Arterioles by Aldosterone

Abstract— Aldosterone has been suggested to elicit vessel contraction via a nongenomic mechanism. We tested this proposal in microdissected, perfused rabbit renal afferent arterioles. Aldosterone had no effect on internal diameter in concentrations from 10−10 to 10−5 mol/L, but aldosterone abolished the ability of 100 mmol/L KCl to induce vascular contraction. The inhibitory effect of aldosterone was observed from 1 pmol/L. The inhibitory effect was significant after 5 minutes and maximal after 20 minutes and was fully reversible. Actinomycin D (10−6 mol/L) prolonged the effect of aldosterone. The effect was abolished by the mineralocorticoid receptor antagonist spironolactone (10−7 mol/L) but not by the glucocorticoid receptor antagonist mifepristone (10−6 mol/L). The K+-mediated increase of intracellular calcium concentration in afferent arterioles was not affected by aldosterone. Mineralocorticoid receptor was detected by reverse transcription–polymerase chain reaction and immunohistochemistry in rat renal vasculature and rabbit endothelial cells. Inhibition of phosphatidylinositol (PI)-3 kinase with LY 294002 (3×10−6 mol/L) restored sensitivity to K+ in the presence of aldosterone, and afferent arterioles were immunopositive for PI-3 kinase subunit p110&agr;. Inhibition of NO formation by L-NAME (10−4 mol/L) or inhibition of soluble guanylyl cyclase with 1H-(1,2,4)Oxadiazolo[4,3-a]quinoxaline-1-one restored K+-induced vasoreactivity in the presence of aldosterone. Similar to aldosterone, the NO donor sodium nitroprusside inhibited K+-induced vascular contraction. Geldanamycin (10−6 mol/L), an inhibitor of heat shock protein 90, abolished aldosterone-induced vasorelaxation. We conclude that aldosterone inhibits depolarization-induced vasoconstriction in renal afferent arterioles by a rapid nongenomic mechanism that is initiated by mineralocorticoid receptor activation and involves PI-3 kinase, protein kinase B, and heat shock protein 90–mediated stimulation of NO generation.

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.

Prostaglandin E2 Induces Vascular Relaxation by E-Prostanoid 4 Receptor-Mediated Activation of Endothelial Nitric Oxide Synthase

The present experiments were designed to test the hypothesis that prostaglandin (PG) E2 causes vasodilatation through activation of endothelial NO synthase (eNOS). Aortic rings from mice with targeted deletion of eNOS and E-prostanoid (EP) receptors were used for contraction studies. Blood pressure changes in response to PGE2 were measured in conscious mice. Single doses of PGE2 caused concentration-dependent relaxations during contractions to phenylephrine (EC50=5*10−8 mol/L). Relaxation after PGE2 was absent in rings without endothelium and in rings from eNOS−/− mice and was abolished by NG-nitro-l-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H1,2,4-oxadiazolo-[4,3-a]quinoxalin-1-one. In PGE2-relaxed aortic rings, the cGMP content increased significantly. PGE2-induced relaxations were abolished by the EP4 receptor antagonist AE3–208 (10−8 mol/L) and mimicked by an EP4 agonist (AE1–329, 10−7 mol/L) in the presence of endothelium and eNOS only. Relaxations were attenuated significantly in rings from EP4−/− mice but normal in EP2−/−. Inhibitors of the cAMP-protein kinase A pathway attenuated, whereas the inhibitor of protein phosphatase 1C, calyculin (10−8 mol/L), abolished the PGE2-mediated relaxation. In aortic rings, PGE2 dephosphorylated eNOS at Thr495. Chronically catheterized eNOS−/− mice were hypertensive (137±3.6 mm Hg, n=13, versus 101±3.9 mm Hg, n=9) and exhibited a lower sensitivity of blood pressure reduction in response to PGE2 compared with wild-type mice. There was no difference in the blood pressure response to nifedipine. These findings show that PGE2 elicits EP4 receptor-mediated, endothelium-dependent stimulation of eNOS activity by dephosphorylation at Thr495 resulting in guanylyl cyclase–dependent vasorelaxation and accumulation of cGMP in aortic rings.

Cycloxygenase-2 Is Expressed in Vasculature of Normal and Ischemic Adult Human Kidney and Is Colocalized with Vascular Prostaglandin E2 EP4 Receptors

The study was performed to elucidate the distribution and cellular localization of cyclooxygenase (COX)-2 in human kidney and to address localization of downstream targets for COX-derived prostanoids. Cortex and outer and inner medulla tissue were obtained from control kidneys (cancer specimens), kidneys with arterial stenosis, and kidneys of patients who received angiotensin II inhibition or acetylsalicylic acid. Ribonuclease protection assay and Western blot test revealed that COX-1 and -2 mRNA and protein were expressed in all regions of human kidney (mRNA ratio, cortex:outer medulla:inner medulla COX-1 1:3:20 and COX-2 1:1:3). In adult kidney, immunohistochemical labeling for COX-2 was associated with smooth muscle cells in pre- and postglomerular vessels and with endothelium, particularly in vasa recta and medullary capillaries. Western blot test confirmed COX-2 expression in renal artery. COX-2 had a similar localization in fetal kidney and was additionally observed in Henles loop and macula densa. Human tissue arrays displayed COX-2 labeling of vascular smooth muscle in multiple extrarenal tissues. Vascular COX-2 expression was significantly increased in kidneys with arterial stenosis. COX-1 was colocalized with microsomal prostaglandin E(2) synthase (PGES) in collecting ducts, and PGES was also detected in macula densa cells. Vascular COX-2 was colocalized with prostaglandin E(2) EP4 receptors but not with EP2 receptors. Thus, renovascular COX-2 expression was a constitutive feature encountered in human kidneys at all ages, whereas COX-2 was seen in macula densa only in fetal kidney. Vascular COX-2 activity in human kidney and extrarenal tissues may support blood flow and affect vascular wall-blood interaction.

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.

Prostasin-dependent activation of epithelial Na+ channels by low plasmin concentrations

Several pathophysiological conditions, including nephrotic syndrome, are characterized by increased renal activity of the epithelial Na(+) channel (ENaC). We recently identified plasmin in nephrotic urine as a stimulator of ENaC activity and undertook this study to investigate the mechanism by which plasmin stimulates ENaC activity. Cy3-labeled plasmin was found to bind to the surface of the mouse cortical collecting duct cell line, M-1. Binding depended on a glycosylphosphatidylinositol (GPI)-anchored protein. Biotin-label transfer showed that plasmin interacted with the GPI-anchored protein prostasin on M-1 cells and that plasmin cleaved prostasin. Prostasin activates ENaC by cleavage of the gamma-subunit, which releases an inhibitory peptide from the extracellular domain. Removal of GPI-anchored proteins from the M-1 cells with phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited plasmin-stimulated ENaC current in monolayers of M-1 cells at low plasmin concentration (1-4 microg/ml). At a high plasmin concentration of 30 microg/ml, there was no difference between cell layers treated with or without PI-PLC. Knockdown of prostasin attenuated binding of plasmin to M1 cells and blocked plasmin-stimulated ENaC current in single M-1 cells, as measured by whole-cell patch clamp. In M-1 cells expressing heterologous FLAG-tagged prostasin, gammaENaC and prostasin were colocalized. A monoclonal antibody directed against the inhibitory peptide of gammaENaC produced specific immunofluorescence labeling of M-1 cells. Pretreatment with plasmin abolished labeling of M-1 cells in a prostasin-dependent way. We conclude that, at low concentrations, plasmin interacts with GPI-anchored prostasin, which leads to cleavage of the gamma-subunit and activation of ENaC, while at higher concentrations, plasmin directly activates ENaC.

Bile acids modulate glucocorticoid metabolism and the hypothalamic–pituitary–adrenal axis in obstructive jaundice ☆

Background & Aims Suppression of the hypothalamic–pituitary–adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5β-reductase. Methods The effect of bile acids on glucocorticoid metabolism was studied in vitro in hepatic subcellular fractions and hepatoma cells, allowing quantitation of the kinetics and transcript abundance of 5β-reductase. Metabolism was subsequently examined in vivo in rats following dietary manipulation or bile duct ligation. Finally, glucocorticoid metabolism was assessed in humans with obstructive jaundice. Results In rat hepatic cytosol, chenodeoxycholic acid competitively inhibited 5β-reductase (Ki 9.19 ± 0.40 μM) and reduced its transcript abundance (in H4iiE cells) and promoter activity (reporter system, HepG2 cells). In Wistar rats, dietary chenodeoxycholic acid (1% w/w chow) inhibited hepatic 5β-reductase activity, reduced urinary excretion of 3α,5β-tetrahydrocorticosterone and reduced adrenal weight. Conversely, a fat-free diet suppressed bile acid levels and increased hepatic 5β-reductase activity, supplementation of the fat-free diet with CDCA reduced 5β-reductase activity, and urinary 3α,5β-reduced corticosterone. Cholestasis in rats suppressed hepatic 5β-reductase activity and transcript abundance. In eight women with obstructive jaundice, relative urinary excretion of 3α,5β-tetrahydrocortisol was significantly lower than in healthy controls. Conclusion These data suggest a novel role for bile acids in inhibiting hepatic glucocorticoid clearance, of sufficient magnitude to suppress hypothalamic–pituitary–adrenal axis activity. Elevated hepatic bile acids may account for adrenal insufficiency in liver disease.

Uninephrectomy in Young Age or Chronic Salt Loading Causes Salt-Sensitive Hypertension in Adult Rats

The importance of nephron endowment and salt intake for the development of hypertension is under debate. The present study was designed to investigate whether reduced nephron number, after completion of nephrogenesis, or chronic salt loading causes renal injury and salt-sensitive hypertension in adulthood. Rats were operated at 3 weeks of age (after completed nephrogenesis) and then subjected to either normal or high-salt diets for 6 to 8 weeks. Four different experimental groups were used: sham-operated animals raised with normal-salt diet (controls) or high-salt diet (HS) and uninephrectomized animals raised with normal-salt diet (UNX) or high-salt diet (UNX+HS). In the adult animals, renal and cardiovascular functions were evaluated and blood pressure recorded telemetrically under different sodium conditions (normal, high, and low). Hypertension was present in UNX+HS (122±9 mm Hg), UNX (101±3 mm Hg), and HS (96±1 mm Hg) groups on normal-salt diets compared with the controls (84±2 mm Hg), and the blood pressure was salt sensitive (high- versus normal-salt diet; 23±3, 9±2, 7±2, and 1±1 mm Hg, respectively). The hypertensive groups (UNX+HS, UNX, and HS) had increased diuresis and reduced ability to concentrate urine. The glomerular filtration rate (milliliters per minute) in anesthetized rats was reduced in the UNX+HS (2.36±0.30) and UNX animals (2.00±0.31) compared with both HS animals (3.55±0.45) and controls (3.01±0.35). Hypertensive groups displayed reduced plasma renin concentrations during high sodium conditions and hypertrophic kidneys and hearts with various degrees of histopathologic changes. In conclusion, at a young age after completed nephrogenesis, uninephrectomy or chronic salt loading causes renal and cardiovascular injury with salt-sensitive hypertension.

Episodic release of renin from single isolated superfused rat afferent arterioles

Doubts have been raised about the involvement of an exocytotic event in the renin release process. This motivated the development of a technique which permitted the study of renin release from one single superfused rat afferent arteriole with a time resolution of 20 seconds.By using this technique it is shown in 43 experiments that the undisturbed renin release is episodic with a renin discharge of 45.2±3.3 (SEM) nano Goldblatt units per episode (n=114) and a frequency of one episode per 5 min. The total renin content of one arteriole was about 30 μGU. The renin discharge and frequency correspond to calculated values for the renin content of single juxtaglomerular cell granules and the release rates in vivo, respectively. Release activity could be stimulated by an acute decrease in the osmolality of the superfusion medium (−20 mOsm sucrose,n=14) indicating that an osmotic water movement is involved in the secretory process.This study provides functional evidence that renin release is exocytotic. In addition it reports what appears to be the first direct measurement of release of secretory material compatible with secretion of single granules from any secretory system.