Plasminogen Deficiency and Amiloride Mitigate Angiotensin II-Induced Hypertension in Type 1 Diabetic Mice Suggesting Effects Through the Epithelial Sodium Channel.

Background Diabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro. Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus. Methods and Results Male plasminogen knockout (plasminogen-deficient [Plg-/-]) and wild-type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30-60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild-type, diabetic ANGII-treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24-hour urine albumin and plasminogen excretion. Diabetic Plg-/- mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild-type, diabetic wild-type, and Plg-/- control mice, ANGII did not change blood pressure in diabetic Plg-/- mice. Compared with ANGII infusion alone, wild-type ANGII-infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild-type mice evoked larger amiloride-sensitive current than urine from Plg-/- mice with or without diabetes mellitus. Full-length γ-ENaC and α-ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa γ-ENaC cleavage product was increased in diabetic versus nondiabetic mice. Conclusions Plasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.

Urokinase-type plasminogen activator contributes to amiloride-sensitive sodium retention in nephrotic range glomerular proteinuria in mice.

AIM: Activation of sodium reabsorption by urinary proteases has been implicated in sodium retention associated with nephrotic syndrome. The study was designed to test the hypothesis that nephrotic proteinuria in mice after conditional deletion of podocin leads to urokinase-dependent, amiloride-sensitive plasmin-mediated sodium and water retention. METHODS: Ten days after podocin knockout, urine and faeces were collected for 10 days in metabolic cages and analysed for electrolytes, plasminogen, protease activity and ability to activate γENaC by patch clamp and western blot. Mice were treated with amiloride (2.5 mg kg-1 for 2 days and 10 mg kg-1 for 2 days) or an anti-urokinase-type plasminogen activator (uPA) targeting antibody (120 mg kg-1 /24 h) and compared to controls. RESULTS: Twelve days after deletion, podocin-deficient mice developed significant protein and albuminuria associated with increased body wt, ascites, sodium accumulation and suppressed plasma renin. This was associated with increased urinary excretion of plasmin and plasminogen that correlated with albumin excretion, urine protease activity co-migrating with active plasmin, and the ability of urine to induce an amiloride-sensitive inward current in M1 cells in vitro. Amiloride treatment in podocin-deficient mice resulted in weight loss, increased sodium excretion, normalization of sodium balance and prevention of the activation of plasminogen to plasmin in urine in a reversible way. Administration of uPA targeting antibody abolished urine activation of plasminogen, attenuated sodium accumulation and prevented cleavage of γENaC. CONCLUSIONS: Nephrotic range glomerular proteinuria leads to urokinase-dependent intratubular plasminogen activation and γENaC cleavage which contribute to sodium accumulation.

Albuminuria in kidney transplant recipients is associated with increased urinary serine proteases and activation of the epithelial sodium channel.

Albuminuria predicts adverse renal outcome in kidney transplant recipients. The present study addressed the hypothesis that albuminuria is associated with increased urine serine proteases with the ability to activate the epithelial sodium channel (ENaC) and with greater extracellular volume and higher blood pressure. In a cross-sectional design, kidney transplant recipients with ( n = 18) and without ( n = 19) albuminuria were included for office blood pressure measurements, estimation of volume status by bioimpedance, and collection of spot urine and plasma samples. Urine was analyzed for serine proteases and for the ability to activate ENaC current in vitro. Urine exosome protein was immunoblotted for prostasin and γ-ENaC protein. In the present study, it was found that, compared with nonalbuminuria (8.8 mg/g creatinine), albuminuric (1,722 mg/g creatinine) kidney transplant recipients had a higher systolic and diastolic blood pressure, despite receiving significantly more antihypertensives, and a greater urinary total plasminogen, active plasmin, active urokinase-type plasminogen activator, and prostasin protein abundance, which correlated significantly with u-albumin. Fluid overload correlated with systolic blood pressure, urinary albumin/creatinine, and plasminogen/creatinine. Urine from albuminuric kidney transplant recipients evoked a greater amiloride- and aprotinin-sensitive inward current in single collecting duct cells (murine cell line M1). γENaC subunits at 50 and 75 kDa showed increased abundance in urine exosomes from albuminuric kidney transplant recipients when compared with controls. These findings show that albuminuria in kidney transplant recipients is associated with hypertension, ability of urine to proteolytically activate ENaC current, and increased abundance of γENaC. ENaC activity could contribute to hypertension and adverse outcome in posttransplant proteinuria.

Diabetic nephropathy is associated with increased urine excretion of proteases plasmin, prostasin and urokinase and activation of amiloride-sensitive current in collecting duct cells.

BACKGROUND: Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na(+) excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the γ subunit. METHODS: In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n = 19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract. RESULTS: Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride-sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly. CONCLUSION: DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na(+) excretion and hypertension.

Plasmin in urine from patients with type 2 diabetes and treatment-resistant hypertension activates ENaC in vitro.

BACKGROUND: Aberrant filtration of plasminogen from plasma and subsequent activation to plasmin in the urinary space may activate proteolytically the epithelial sodium channel, ENaC. In conditions with chronic albuminuria, this may cause hypertension. It was hypothesized that patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension excrete plasmin(ogen) in urine in proportion to albumin and that plasmin confers to urine the ability to activate ENaC. METHOD: Patients (n = 113) with T2DM and resistant hypertension, defined as systolic blood pressure (SBP) more than 130 mmHg and/or diastolic blood pressure (DBP) more than 80 mmHg despite use of at least three drugs with one diuretic and one renin-angiotensin system inhibitor, were included. Urine was analyzed for albumin, creatinine, plasmin(ogen), protease activity, and ability to activate inward current in single collecting duct cells. RESULTS: Mean ambulatory SBP/DBP was 143 ±â€Š1/77 ±â€Š0.7 mmHg; HbA1c 7.35%; and eGFR 81.0 ml/min per 1.73 m (geometric means). Patients with microalbuminuria (39%) and macroalbuminuria (13%) displayed significantly elevated levels of urinary plasmin(ogen) normalized to urine creatinine compared with patients with normal excretion of albumin (48%). Urinary plasminogen correlated significantly to urine albumin. Western immunoblotting and gelatine zymography confirmed active plasmin in urine samples from patients with microalbuminuria and macroalbuminuria. Single collecting duct cells displayed significantly increased, amiloride-sensitive, inward current when superfused with urine from albuminuric patients compared with patients with normal albumin excretion. Urinary plasminogen/creatinine ratio correlated significantly with 24-h ambulatory blood pressure. CONCLUSION: Aberrant presence of plasmin in preurine may inappropriately activate ENaC in patients with type 2 diabetes and microalbuminuria. This may contribute to treatment-resistant hypertension.

Remission of nephrotic syndrome diminishes urinary plasmin content and abolishes activation of ENaC.

BACKGROUND: Urinary plasmin activates the epithelial Na(+) channel (ENaC) in vitro and may possibly be a mechanism of sodium retention in nephrotic syndrome (NS). This study used a paired design to test the hypothesis that remission of NS is associated with a decreased content of urinary plasmin and reduced ability of patients' urine to activate ENaC. METHODS: Samples were collected during active NS and at stable remission from 20 patients with idiopathic NS, aged 9.1 ± 3.2 years. Plasminogen-plasmin concentration was measured with an enzyme-linked immunosorbent assay. Western immunoblotting for plasminogen-plasmin was performed in paired urine samples. The patch clamp technique was used to test the ability of urine to evoke an inward current on collecting duct cells and human lymphocytes. RESULTS: The urinary plasminogen-plasmin/creatinine ratio was 226 [95 % confidence interval (CI) 130-503] µg/mmol in nephrotic urine versus 9.5 (95 % CI 8-12) µg/mmol at remission (p < 0.001). Western immunoblotting confirmed the presence of active plasmin in urine collected during active NS, while samples collected at remission were negative. Nephrotic urine generated an inward amiloride- and α2-anti-plasmin- sensitive current, whereas the observed increase in current in urine collected at remission was significantly lower (201 ± 31 vs. 29 ± 10 %; p = 0.005). CONCLUSIONS: These findings support the hypothesis that aberrantly filtered plasminogen-plasmin may contribute to ENaC activation and mediate primary renal sodium retention during active childhood NS.

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 E(2)-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.

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 γ-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 µmol/L; P<0.001), α(2)-antiplasmin (1 µmol/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.

Erythropoietin increases expression and function of transient receptor potential canonical 5 channels.

Hypertension is a common complication in hemodialysis patients during erythropoietin (EPO) treatment. The underlying mechanisms of EPO-induced hypertension still remain to be determined. Increased transient receptor potential canonical (TRPC) channels have been associated with hypertension. Now, TRPC gene expression was investigated using quantitative real-time RT-PCR and immunoblotting in cultured human endothelial cells and in monocytes from hemodialysis patients. EPO dose-dependently increased TRPC5 mRNA in endothelial cells. EPO increased TRPC5 mRNA stability, that is, EPO prolonged the half-life period for TRPC5 mRNA from 16 hours (control) to 24 hours (P<0.05). The poly(A) tail length was measured by rapid amplification of cDNA ends-poly(A) test. Increased TRPC5 mRNA stability was attributed to longer 3' poly(A) tail lengths after EPO administration. EPO also significantly increased TRPC5 channel protein abundance by 70% (P<0.05). Whole-cell patch clamp showed that angiotensin II-induced, TRPC5-mediated currents were dramatically increased in endothelial cells treated with EPO. Fluorescent dye techniques confirmed that increased calcium influx after EPO treatment was abolished after TRPC5 knockdown (P<0.05). EPO also significantly increased intracellular reactive oxygen species production. Knockdown of TRPC5 alleviated EPO-induced reactive oxygen species generation in endothelial cells (P<0.05). In vivo, EPO-treated hemodialysis patients showed significantly increased amounts of TRPC5 mRNA in monocytes compared with EPO-free hemodialysis patients (6.0±2.4 [n=12] versus 1.0±0.5 [n=9]; P<0.01). Patients undergoing EPO treatment also showed significantly elevated systolic blood pressure (160±7 versus 139±6 mm Hg; P<0.05). Our findings suggest that upregulated functional TRPC5 gene may be one cause of EPO-induced hypertension in patients with chronic kidney disease.

Physiological regulation of epithelial sodium channel by proteolysis.

PURPOSE OF REVIEW: Activation of epithelial sodium channel (ENaC) by proteolysis appears to be relevant for day-to-day physiological regulation of channel activity in kidney and other epithelial tissues. Pathophysiogical, proteolytic activation of ENaC in kidney has been demonstrated in proteinuric disease. RECENT FINDINGS: A variation in sodium and potassium intake or plasma aldosterone changes the number of cleaved α and γ-ENaC subunits and is associated with changes in ENaC currents. The protease furin mediates intracellular cleavage, whereas the channel-activating protease prostasin (CAP-1), which is glycophosphatidylinositol-anchored to the apical cell surface, mediates important extracellular cleavage. Soluble protease activity is very low in urine under physiological conditions but rises in proteinuria. In nephrotic syndrome, the dominant soluble protease activity is plasmin, which is formed from filtered plasminogen via urokinase-type plasminogen activator. Plasmin activates ENaC directly at high concentrations and through prostasin at lower concentrations. SUMMARY: The discovery of serine protease-mediated activation of renal ENaC in physiological and pathophysiological conditions opens the way for new understanding of the pathogenesis of proteinuric sodium retention, which may involve plasmin and present several potential new drug targets.

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 ß-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 ß-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.

Teaching acid/base physiology in the laboratory.

Acid/base homeostasis is one of the most difficult subdisciplines of physiology for medical students to master. A different approach, where theory and practice are linked, might help students develop a deeper understanding of acid/base homeostasis. We therefore set out to develop a laboratory exercise in acid/base physiology that would provide students with unambiguous and reproducible data that clearly would illustrate the theory in practice. The laboratory exercise was developed to include both metabolic acidosis and respiratory alkalosis. Data were collected from 56 groups of medical students that had participated in this laboratory exercise. The acquired data showed very consistent and solid findings after the development of both metabolic acidosis and respiratory alkalosis. All results were consistent with the appropriate diagnosis of the acid/base disorder. Not one single group failed to obtain data that were compatible with the diagnosis; it was only the degree of acidosis/alkalosis and compensation that varied.

Increased renin production in mice with deletion of peroxisome proliferator-activated receptor-gamma in juxtaglomerular cells.

We recently found that endogenous (free fatty acids) and pharmacological (thiazolidinediones) agonists of nuclear receptor Peroxisome proliferator-activated receptor (PPAR)gamma stimulate renin transcription. In addition, the renin gene was identified as a direct target of PPARgamma. The mouse renin gene is regulated by PPARgamma through a distal enhancer direct repeat closely related to consensus PPAR response element (PPRE). In vitro studies demonstrated that PPARgamma knockdown stimulated PPRE-driven transcription. These data predicted that deficiency of PPARgamma would upregulate mouse renin expression. Consistent with these observations knockdown of PPARgamma increased the transcription of a reporter gene driven by the mouse renin PPRE-like motif in vitro. To study the impact of PPARgamma on renin production in vivo, we used a cre/lox system to generate double-transgenic mice with disrupted PPARgamma locus in renin-producing juxtaglomerular (JG) cells of the kidney (RC-PPARgamma(fl/fl) mice). We provide evidence that PPARgamma expression was effectively reduced in JG cells of RC-PPARgamma(fl/fl) mice. Fluorescent immunohistochemistry showed stronger renin signal in RC-PPARgamma(fl/fl) than in littermate control RC-PPARgamma(wt/wt) mice. Renin mRNA levels and plasma renin concentration in RC-PPARgamma(fl/fl) mice were almost 2-fold higher than in littermate controls. Arterial blood pressure and pressure control of renal vascular resistance, which play decisive roles in the regulation of renin production were indistinguishable between RC-PPARgamma(wt/wt) and RC-PPARgamma(fl/fl) mice. These data demonstrate that the JG-specific PPARgamma deficiency results in increased mouse renin expression in vivo thus corroborating earlier in vitro results. PPARgamma appears to be a relevant transcription factor for the control of renin gene in JG cells.

Inhibition of calcineurin phosphatase promotes exocytosis of renin from juxtaglomerular cells.

To examine the role of the calcium/calmodulin-dependent phosphatase calcineurin in regulation of renin release, we assayed exocytosis using whole-cell patch clamp of single juxtaglomerular cells in culture. The calcineurin inhibitor, cyclosporine A (CsA), significantly increased juxtaglomerular cell membrane capacitance, an index of cell surface area and an established measure of exocytosis in single-cell assays. This effect was mimicked by intracellular delivery of a calcineurin inhibitory peptide, the calcium chelator ethylene glycol tetraacetic acid (EGTA), or the calmodulin inhibitor W-13. Simultaneous exposure to EGTA and CsA had no additive effect. The protein kinase A (PKA) blocker RpcAMPs had no effect on the CsA-induced increase in membrane capacitance. Intra- and extracellular application of tacrolimus did not alter membrane capacitance. A calmodulin antagonist (calmidazolium) and CsA, but not tacrolimus, significantly stimulated renin release from cultured juxtaglomerular cells. Juxtaglomerular cells expressed the calcineurin isoforms A-beta and A-gamma but not A-alpha. Plasma renin concentrations (PRCs) were not different in wild-type, calcineurin A-alpha, or A-beta knockout mice but increased after CsA treatment of the A-alpha knockout, while renin mRNA was suppressed. We conclude that calcineurin and calcium/calmodulin suppress exocytosis of renin from juxtaglomerular cells independent of PKA.

Hypotonicity-induced Renin exocytosis from juxtaglomerular cells requires aquaporin-1 and cyclooxygenase-2.

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C(m)) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C(m) significantly and enhanced outward current. Indomethacin, PLA(2) inhibition, and an antagonist of prostaglandin transport impaired the C(m) and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2(-/ -) and AQP1(-/ -) mice, hypotonicity increased neither C(m) nor outward current, but 0.1-muM PGE(2) increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C(m) and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA(2)/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.

Prostaglandin F2alpha elevates blood pressure and promotes atherosclerosis.

Little is known about prostaglandin F(2alpha) in cardiovascular homeostasis. Prostaglandin F(2alpha) 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 TNF(alpha), inducible nitric oxide enzyme and TGF(beta) 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.

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.

Renin release.

The aspartyl-protease renin is the key regulator of the renin-angiotensin-aldosterone system, which is critically involved in salt, volume, and blood pressure homeostasis of the body. Renin is mainly produced and released into circulation by the so-called juxtaglomerular epithelioid cells, located in the walls of renal afferent arterioles at the entrance of the glomerular capillary network. It has been known for a long time that renin synthesis and secretion are stimulated by the sympathetic nerves and the prostaglandins and are inhibited in negative feedback loops by angiotensin II, high blood pressure, salt, and volume overload. In contrast, the events controlling the function of renin-secreting cells at the organ and cellular level are markedly less clear and remain mysterious in certain aspects. The unravelling of these mysteries has led to new and interesting insights into the process of renin release.

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.