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The natriuretic peptides (NPs) ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide) mediate their widespread effects by activating the natriuretic peptide receptor-A (NPR-A), while C-type natriuretic peptide (CNP) acts via natriuretic peptide receptor-B (NPR-B). NPs are removed from the circulation by internalization via the natriuretic peptide clearance receptor natriuretic peptide receptor-C (NPR-C). In addition to their well-known functions, for instance on blood pressure, all three NPs confer significant cardioprotection and renoprotection. Since neither the NP-mediated renal functions nor the renal target cells of renoprotection are completely understood, we performed systematic localization studies of NP receptors using in situ hybridization (RNAscope) in mouse kidneys. NPR-A mRNA is highly expressed in glomeruli (mainly podocytes), renal arterioles, endothelial cells of peritubular capillaries, and PDGFR-receptor β positive (PDGFR-β) interstitial cells. No NPR-A mRNA was detected by RNAscope in the tubular system. In contrast, NPR-B expression is highest in proximal tubules. NPR-C is located in glomeruli (mainly podocytes), in endothelial cells and PDGFR-β positive cells. To test for a possible regulation of NPRs in kidney diseases, their distribution was studied in adenine nephropathy. Signal intensity of NPR-A and NPR-B mRNA was reduced while their spatial distribution was unaltered compared with healthy kidneys. In contrast, NPR-C mRNA signal was markedly enhanced in cell clusters of myofibroblasts in fibrotic areas of adenine kidneys. In conclusion, the primary renal targets of ANP and BNP are glomerular, vascular, and interstitial cells but not the tubular compartment, while the CNP receptor NPR-B is highly expressed in proximal tubules. Further studies are needed to clarify the function and interplay of this specific receptor expression pattern.

The inhibition of renal SGLT2 glucose reabsorption has proven its therapeutic efficacy in chronic kidney disease. SGLT2 inhibitors (SGLTi) have been intensively studied in rodent models to identify the mechanisms of SGLT2i-mediated nephroprotection. So far, the overwhelming effects from clinical trials, could only partially be reproduced in rodent models of renal injury. However, a commonly disregarded observation from these studies, is the increase in kidney weight after SGLT2i administration. Increased kidney mass often relies on tubular growth in response to reabsorption overload during glomerular hyperfiltration. Since SGLT2i suppress hyperfiltration but concomitantly increase renal weight, it seems likely that SGLT2i have a growth promoting effect on the kidney itself, independent of GFR control. This study aimed to investigate the effect of SGLT2i on kidney growth in wildtype animals, to identify enlarged nephron segments and classify the size increase as hypertrophic/hyperplastic growth or cell swelling. SGLT2i empagliflozin increased kidney weight in wildtype mice by 13% compared to controls, while bodyweight and other organs were not affected. The enlarged nephron segments were identified as SGLT2-negative distal segments of proximal tubules and as collecting ducts by histological quantification of tubular cell area. In both segments protein/DNA ratio, a marker for hypertrophic growth, was increased by 6% and 12% respectively, while tubular nuclei number (hyperplasia) was unchanged by empagliflozin. SGLT2-inhibition in early proximal tubules induces a shift of NaCl resorption along the nephron causing compensatory NaCl and H2O reabsorption and presumably cell growth in downstream segments. Consistently, in collecting ducts of empagliflozin-treated mice, mRNA expression of the Na+-channel ENaC and the H2O-channels Aqp-2/Aqp-3 were increased. In addition, the hypoxia marker Hif1α was found increased in intercalated cells of the collecting duct together with evidence for increased proton secretion, as indicated by upregulation of carbonic anhydrases and acidified urine pH in empagliflozin-treated animals. In summary, these data show that SGLT2i induce cell enlargement by hypertrophic growth and possibly cell swelling in healthy kidneys, probably as a result of compensatory glucose, NaCl and H2O hyperreabsorption of SGLT2-negative segments. Particularly affected are the SGLT2-negative proximal tubules (S3) and the collecting duct, areas of low O2 availability.

Cardiovascular risk factors such as high glucose, LDL-cholesterol, blood pressure, and impaired kidney function are particularly frequent in old-aged individuals. However, population-based data on the extent of cardiovascular risk factor control in the old-aged population is limited. AugUR is a cohort of the mobile “70+”-year-old population of/near Regensburg, recruited via population registries. We conducted cross-sectional analyses assessing the proportion of AugUR participants with LDL-cholesterol, HbA1c, or blood pressure beyond recommended levels and their association with impaired creatinine- and cystatin-based estimated glomerular filtration rate (eGFR

The renin-angiotensin-system (RAS) plays a critical role in diabetic nephropathy, and inhibitors of the RAS are central components in its therapy. Renin circulates in the blood in its active form but also in the form of its enzymatically inactive precursor prorenin. In humans, the plasma prorenin concentration exceeds that of active renin several times. While the plasma concentration of active renin is unchanged or even reduced in diabetic patients, the prorenin concentration increases significantly and it has been shown that high prorenin levels are associated with diabetic microvascular damage. Why prorenin increases in diabetes while active renin is reduced is unclear. Previous studies suggest that there may be formation of prorenin in the tubular system of diabetic kidneys. To investigate the functional consequences of possible tubular renin formation in diabetes, we generated mice with inducible tubule-specific deletion of the renin gene (tubule-renin KO).Under control conditions, tubule-renin KO had no apparent phenotype and plasma and tissue levels of renin and prorenin were similar to those of mice with intact tubular renin (control mice). In control mice type-1 diabetes (streptozotocin, STZ) for 8 to 12 weeks stimulated tubular renin mRNA and protein expression, especially in distal nephron segments including collecting ducts. This increase in renin synthesis in renal tubules was markedly attenuated or even absent in tubule-renin KO mice. Similar to diabetic patients, plasma prorenin was markedly elevated in diabetic control mice. This stimulation of plasma prorenin was absent in mice with tubule-specific deletion of the renin gene. Moreover, the high prorenin levels in renal tissue, which were observed in diabetic control mice, were markedly reduced in tubule-renin KO. Noteworthy, plasma renin activity was not reduced in tubule-renin KO compared with controls, suggesting that renal tubules of diabetic mice mainly release prorenin. Kidneys of control mice with intact tubular renin showed classical signs of diabetic renal damage, such as albuminuria, mesangial expansion, fibrosis, inflammation and capillary rarefaction. All of these parameters were significantly ameliorated in tubule-renin KO, indicating that tubular renin, most likely in its prorenin form, significantly aggravates renal damage in diabetes.These data provide clear evidence for the first time that the tubular renin system is an additional source for circulating prorenin in diabetes, hereby providing an explanation for the paradox regulation of active renin and prorenin in diabetes. Moreover, the data show that tubular renin markedly contributes to the progression of diabetic nephropathy.

Retinopathy of prematurity (ROP) is a retinal disease that threatens the vision of prematurely born infants. Severe visual impairment up to complete blindness is caused by neovascularization and inflammation, progressively destroying the immature retina. ROP primarily affects newborns in middle- and low-income countries with limited access to current standard treatments such as intraocular drug injections and laser- or cryotherapy. To overcome these limitations, we developed a nanotherapeutic that effectively prevents ROP development with one simple intravenous injection. Its lipid nanocapsules transport the antiangiogenic and anti-inflammatory cyclosporin A efficiently into disease-driving retinal pigment epithelium cells. In a mouse model of ROP, a single intravenous injection of the nanotherapeutic prevented ROP and led to normal retinal development by counteracting neovascularization and inflammation. This nanotherapeutic approach has the potential to bring about a change of paradigm in ROP therapy and prevent millions of preterm born infants from developing ROP.

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP–dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract A graphic abstract is available for this article.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile αVβ3 integrins with a previously concealed secondary ligand, thereby creating a stepwise particle-cell interplay of primary NP attachment and subsequent uptake. Manufactured adenovirus-mimetic NPs show great avidity for both target motifs in vitro, leading to a substantial binding as well as subsequent cell uptake into target mesangial cells. Additionally, steric shielding of secondary ligand visibility leads to a highly controllable, sequential ligand-receptor interaction, whereby hetero-functional NPs activate mesangial cell surface integrins only after a successful prior binding to the AT1r. This stepwise cell identification significantly enhances mesangial cell specificity in co-culture assays with different off-target cells. Additionally, described NPs display excellent in vivo robustness by efficiently accumulating in the mesangium upon injection, thereby opening new paths for possible drug delivery applications.

NMR spectroscopy is a widely used method for the detection and quantification of metabolites in complex biological fluids. However, the large number of metabolites present in a biological sample such as urine or plasma leads to considerable signal overlap in one-dimensional NMR spectra, which in turn hampers both signal identification and quantification. As a consequence, we have developed an easy to use R-package that allows the fully automated deconvolution of overlapping signals in the underlying Lorentzian line-shapes. We show that precise integral values are computed, which are required to obtain both relative and absolute quantitative information. The algorithm is independent of any knowledge of the corresponding metabolites, which also allows the quantitative description of features of yet unknown identity.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile α

Poor drug availability in the tissue of interest is a frequent cause of therapy failure. While nanotechnology has developed a plethora of nanocarriers for drug transport, their ability to unequivocally identify cells of interest remains moderate. Viruses are the ideal nanosized carriers as they are able to address their embedded nucleic acids with high specificity to their host cells. Here, it is reported that particles endowed with a virus‐like ability to identify cells by three consecutive checks have a superior ability to recognize mesangial cells (MCs) in vivo compared to conventional nanoparticles. Mimicking the initial viral attachment followed by a stepwise target cell recognition process leads to a 5‐ to 15‐fold higher accumulation in the kidney mesangium and extensive cell uptake compared to particles lacking one or both of the viral traits. These results highlight the relevance that the viral cell identification process has on specificity and its application on the targeting strategies of nanomaterials. More so, these findings pave the way for transporting drugs into the mesangium, a tissue that is pivotal in the development of diabetic nephropathy and for which currently no efficient pharmacotherapy exists., Nanoparticles (NPs) endowed with viral targeting strategies are presented. A triple‐check for cell identity is implemented by mimicking the initial viral cell attachment, followed by a stepwise enzyme‐activated recognition and internalization. Particles with these viral attributes are able to accumulate in mesangial cells, a therapeutic target of paramount importance due to their involvement in kidney diseases, such as diabetic nephropathy.

Background: Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone, but the target cells remain controversial. The cGMP-producing guanylyl cyclase–B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (SMCs). However, whereas endothelial cell–specific CNP knockout mice are hypertensive, mice with deletion of GC-B in vascular SMCs have unaltered blood pressure. Methods: We analyzed whether the vasodilating response to CNP changes along the vascular tree, ie, whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B ( Npr2 ) gene were interbred with Tie2-Cre or PDGF-Rβ-Cre ERT2 lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMCs and capillary pericytes. Intravital microscopy, invasive and noninvasive hemodynamics, fluorescence energy transfer studies of pericyte cAMP levels in situ, and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. Results: Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases toward small-diameter arterioles and capillaries. CNP consistently did not prevent endothelin-1–induced acute constrictions of proximal arterioles, but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, ie, in pericytes. It is notable that the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion, but abolished in mice lacking GC-B in microcirculatory SMCs and pericytes. CNP, via GC-B/cGMP signaling, modulates 2 signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator-activated phosphoprotein, and it inhibits phosphodiesterase 3A, thereby enhancing pericyte cAMP levels. These pathways ultimately prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMCs and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. Conclusions: Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMCs and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure.

Endotoxemia-related acute kidney injury (AKI) is associated with increased formation of prostaglandins, which may serve as a compensatory mechanism to maintain renal function. We hypothesized that an increase of renal EP2 or EP4 receptors and/or a downregulation of renal EP1 and EP3 receptors enhances PGE2-induced renal vasodilatation. Injection of lipopolysaccharide (LPS; 3 mg/kg i.p.) increased microsomal prostaglandin E synthase (mPGES)-1 and prostacyclin synthase expression, whereas mPGES-2 expression was unaltered. Further, LPS increased the mRNA abundance for the prostaglandin EP4 receptor, whereas the expressions of the EP1 and EP3 receptors were decreased. In isolated-perfused kidneys from control mice, PGE2 exerted a dual effect on renal vascular tone, inducing vasodilatation at lower concentrations and vasoconstriction at higher concentrations. In kidneys from endotoxemic mice, the vasodilatory component was more pronounced, whereas the vasoconstriction at higher PGE2 concentrations was absent. Similarly, prostacyclin (PGI2)-induced vasodilatation was more pronounced in endotoxemic kidneys. The enhanced vasodilatory effect was paralleled by an increase in renal vascular EP4 and prostacyclin IP receptor mRNA expression. Further, stimulation of renin secretion rate by PGE2 and PGI2 was enhanced in endotoxemic kidneys. Pretreatment with the cyclooxygenase (COX)-2 inhibitor SC-236 (10 mg/kg) did not alter the basal GFR, but augmented the LPS-induced decline in GFR, and attenuated the LPS-induced increase in plasma renin concentration in vivo. Our data suggest that an activation of the COX-2/mPGES-1 synthetic pathway is responsible for the increased renal formation of PGE2 in response to LPS and that the vasodilatory effect of PGE2 and PGI2 is enhanced during endotoxemia.

NMR spectroscopy is a widely used method for the detection and quantification of metabolites in complex biological fluids. However, the large number of metabolites present in a biological sample such as urine or plasma leads to considerable signal overlap in one-dimensional NMR spectra, which in turn hampers both signal identification and quantification. As a consequence, we have developed an easy to use R-package that allows the fully automated deconvolution of overlapping signals in the underlying Lorentzian line-shapes. We show that precise integral values are computed, which are required to obtain both relative and absolute quantitative information. The algorithm is independent of any knowledge of the corresponding metabolites, which also allows the quantitative description of features of yet unknown identity.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Synthesis of renin in renal renin-producing cells (RPCs) is controlled via the intracellular messenger cAMP. Interference with cAMP-mediated signaling by inducible knockout of Gs-alpha (Gsα) in RPCs of adult mice resulted in a complex adverse kidney phenotype. Therein, glomerular endothelial damage was most striking. In this study, we investigated whether Gsα knockout leads to a loss of RPCs, which itself may contribute to the endothelial injury. We compared the kidney phenotype of three RPC-specific conditional mouse lines during continuous induction of recombination. Mice expressing red fluorescent reporter protein tdTomato (tdT) in RPCs served as controls. tdT was also expressed in RPCs of the other two strains used, namely with RPC-specific Gsα knockout (Gsα mice) or with RPC-specific diphtheria toxin A expression (DTA mice, in which the RPCs should be diminished). Using immunohistological analysis, we found that RPCs decreased by 82% in the kidneys of Gsα mice as compared with controls. However, the number of tdT-positive cells was similar in the two strains, demonstrating that after Gsα knockout, the RPCs persist as renin-negative descendants. In contrast, both renin-positive and tdT-labeled cells decreased by 80% in DTA mice suggesting effective RPC ablation. Only Gsα mice displayed dysregulated endothelial cell marker expression indicating glomerular endothelial damage. In addition, a robust induction of genes involved in tissue remodelling with microvascular damage was identified in tdT-labeled RPCs isolated from Gsα mice. We concluded that Gsα/renin double-negative RPC progeny essentially contributes for the development of glomerular endothelial damage in our Gsα-deficient mice.

The cardiac natriuretic peptides (NPs), atrial NP and B-type NP, regulate fluid homeostasis and arterial BP through renal actions involving increased GFR and vascular and tubular effects. Guanylyl cyclase-A (GC-A), the transmembrane cGMP-producing receptor shared by these peptides, is expressed in different renal cell types, including podocytes, where its function is unclear. To study the effects of NPs on podocytes, we generated mice with a podocyte-specific knockout of GC-A (Podo-GC-A KO). Despite the marked reduction of GC-A mRNA in GC-A KO podocytes to 1% of the control level, Podo-GC-A KO mice and control littermates did not differ in BP, GFR, or natriuresis under baseline conditions. Moreover, infusion of synthetic NPs similarly increased the GFR and renal perfusion in both genotypes. Administration of the mineralocorticoid deoxycorticosterone-acetate (DOCA) in combination with high salt intake induced arterial hypertension of similar magnitude in Podo-GC-A KO mice and controls. However, only Podo-GC-A KO mice developed massive albuminuria (controls: 35-fold; KO: 5400-fold versus baseline), hypoalbuminemia, reduced GFR, and marked glomerular damage. Furthermore, DOCA treatment led to decreased expression of the slit diaphragm-associated proteins podocin, nephrin, and synaptopodin and to enhanced transient receptor potential canonical 6 (TRPC6) channel expression and ATP-induced calcium influx in podocytes of Podo-GC-A KO mice. Concomitant treatment of Podo-GC-A KO mice with the TRPC channel blocker SKF96365 markedly ameliorated albuminuria and glomerular damage in response to DOCA. In conclusion, the physiologic effects of NPs on GFR and natriuresis do not involve podocytes. However, NP/GC-A/cGMP signaling protects podocyte integrity under pathologic conditions, most likely by suppression of TRPC channels.

The angiotensin II receptor type 1 (AT1R), which is expressed in blood vessels of the posterior eye, is of paramount significance in the pathogenesis of severe ocular diseases such as diabetic retinopathy and age-related macular degeneration. However, small molecule angiotensin receptor blockers (ARBs) have not proven to be a significant therapeutic success. We report here on a nanoparticle system consisting of ARB molecules presented in a multivalent fashion on the surface of quantum dots (Qdots). As a result of the multivalent receptor binding, nanoparticles targeted cells with high AT1R expression and inhibited their angiotensin receptor signaling with an IC50 of 3.8 nM while showing only minor association to cells with low AT1R expression. After intravenous injection into the tail vein of mice, multivalent nanoparticles accumulated in retinal and choroidal blood vessels of the posterior eye. At the same time, multivalent ligand display doubled the Qdot concentration in the blood vessels compared to non-targeted Qdots. Remarkably, ARB-targeted Qdots showed no pronounced accumulation in AT1R-expressing off-target tissues such as the kidney. Following systemic application, this multivalent targeting approach has the potential to amplify AT1R blockade in the eye and concomitantly deliver a therapeutic payload into ocular lesions.

Aims Arterial hypertension is a major risk factor for cardiovascular diseases. The kidney and its natriuretic function are in the centre of the prevailing models to explain the pathogenesis of hypertension; however, the mechanisms underlying blood pressure elevation remain unclear in most patients. Development of hypertension is strongly correlated with age, and this blood pressure increase typically accelerates in the fourth decade of life. The cause of age-dependent blood pressure elevation is poorly understood. This study aims to understand the role of procontractile G-protein-mediated signalling pathways in vascular smooth muscle in age-dependent hypertension. Methods and results Similar to humans at mid-life, we observed in 1-year-old mice elevated blood pressure levels without any evidence for increased vessel stiffness, impaired renal function, or endocrine abnormalities. Hypertensive aged mice showed signs of endothelial dysfunction and had an increased vascular formation of reactive oxygen species (ROS) and elevated endothelial ET-1 expression. Age-dependent hypertension could be normalized by ETA receptor blockade, smooth muscle-specific inactivation of the gene encoding the ETA receptor, as well as by acute disruption of downstream signalling via induction of smooth muscle-specific Gα12/Gα13, Gαq/Gα11, or LARG deficiency using tamoxifen-inducible smooth muscle-specific conditional mouse knock-out models. Induction of smooth muscle-specific ETA receptor deficiency normalized the blood pressure in aged mice despite the continuous presence of signs of endothelial dysfunction. Conclusion Age-dependent blood pressure elevation is due to a highly reversible activation of procontractile signalling in vascular smooth muscle cells indicating that increased vascular tone can be a primary factor in the development of hypertension.

Aims: Pathological cardiac hypertrophy is a major predictor for the development of cardiac diseases. It is associated with chronic neurohumoral stimulation and with altered cardiac Ca2+ signalling in cardiomyocytes. TRPC proteins form agonist-induced cation channels, but their functional role for Ca2+ homeostasis in cardiomyocytes during fast cytosolic Ca2+ cycling and neurohumoral stimulation leading to hypertrophy is unknown. Methods and results: In a systematic analysis of multiple knockout mice using fluorescence imaging of electrically paced adult ventricular cardiomyocytes and Mn2+-quench microfluorimetry, we identified a background Ca2+ entry (BGCE) pathway that critically depends on TRPC1/C4 proteins but not others such as TRPC3/C6. Reduction of BGCE in TRPC1/C4-deficient cardiomyocytes lowers diastolic and systolic Ca2+ concentrations both, under basal conditions and under neurohumoral stimulation without affecting cardiac contractility measured in isolated hearts and in vivo. Neurohumoral-induced cardiac hypertrophy as well as the expression of foetal genes (ANP, BNP) and genes regulated by Ca2+-dependent signalling (RCAN1-4, myomaxin) was reduced in TRPC1/C4 knockout (DKO), but not in TRPC1- or TRPC4-single knockout mice. Pressure overload-induced hypertrophy and interstitial fibrosis were both ameliorated in TRPC1/C4-DKO mice, whereas they did not show alterations in other cardiovascular parameters contributing to systemic neurohumoral-induced hypertrophy such as renin secretion and blood pressure. Conclusions: The constitutively active TRPC1/C4-dependent BGCE fine-tunes Ca2+ cycling in beating adult cardiomyocytes. TRPC1/C4-gene inactivation protects against development of maladaptive cardiac remodelling without altering cardiac or extracardiac functions contributing to this pathogenesis. Fil: Camacho Londoño, Juan E.. Pharmakologisches Institut; Alemania. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania. German Centre for Cardiovascular Research; Alemania Fil: Tian, Qinghai. Institut fur Molekulare Zellbiologie; Alemania Fil: Hammer, Karin. Institut fur Molekulare Zellbiologie; Alemania Fil: Schröder, Laura. Institut fur Molekulare Zellbiologie; Alemania Fil: Camacho Londoño, Julia. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Reil, Jan C.. Universitat des Saarlandes; Alemania Fil: He, Tao. German Centre for Cardiovascular Research; Alemania. Research Unit Cardiac Epigenetics; Alemania. Tongji Hospital; China Fil: Oberhofer, Martin. Institut fur Molekulare Zellbiologie; Alemania Fil: Mannebach, Stefanie. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Mathar, Ilka. Pharmakologisches Institut; Alemania. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Philipp, Stephan E.. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Tabellion, Wiebke. Institut fur Molekulare Zellbiologie; Alemania Fil: Schweda, Frank. Universitat Regensburg; Alemania Fil: Dietrich, Alexander. Walther-Straub-Institut fur Pharmakologie und Toxikologie; Alemania Fil: Kaestner, Lars. Institut fur Molekulare Zellbiologie; Alemania Fil: Laufs, Ulrich. Universitat des Saarlandes; Alemania Fil: Birnbaumer, Lutz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Transmembrane Signaling Group; Alemania Fil: Flockerzi, Veit. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania Fil: Freichel, Marc. Pharmakologisches Institut; Alemania. Experimentelle und Klinische Pharmakologie und Toxikologie; Alemania. German Centre for Cardiovascular Research; Alemania Fil: Lipp, Peter. Institut fur Molekulare Zellbiologie; Alemania

The renin-angiotensin-aldosterone system (RAAS) is a central element in the control of the salt and water balance of the body and arterial blood pressure. The activity of the RAAS is controlled by the protease renin, which is released from renal juxtaglomerular epithelioid cells (JGE cells) into the circulation. Renin release is regulated by a complex interplay of several locally acting hormones or mechanisms and longer feedback loops one of which involves salt intake. Acute NaCl loads or longer lasting high salt intakes suppress plasma renin activity, whereas reductions in NaCl intake stimulate it. Because the activation of the RAAS conserves the salt content of the body, a classical feedback loop between salt intake/body salt content and renin is established. Despite of its important role for body fluid homeostasis, the precise signaling pathways connecting salt intake with the synthesis and release of renin are only incompletely understood. Four putative controllers of the salt-dependent regulation of the RAAS have been suggested: (1) the macula densa mechanism which adjusts renin release in response to changes in the renal tubular salt concentration; (2) salt-dependent changes in the arterial blood pressure; (3) circulating salt-dependent hormones, particularly the atrial natriuretic peptide (ANP); and (4) renal sympathetic nervous activity, which is regulated by extracellular volume and arterial blood pressure. In this review, the role of these known controllers of the RAAS will be discussed with special emphasis on their relative contributions to the salt-dependent regulation of the RAAS at different time frames.

The renin-angiotensin-aldosterone system and cardiac natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are opposing control mechanisms for arterial blood pressure. Accordingly, an inverse relationship between plasma renin concentration (PRC) and ANP exists in most circumstances. However, PRC and ANP levels are both elevated in renovascular hypertension. Because ANP can directly suppress renin release, we used ANP knockout (ANP−/−) mice to investigate whether high ANP levels attenuate the increase in PRC in response to renal hypoperfusion, thus buffering renovascular hypertension. ANP−/− mice were hypertensive and had reduced PRC compared with that in wild-type ANP+/+ mice under control conditions. Unilateral renal artery stenosis (2-kidney, 1-clip) for 1 wk induced similar increases in blood pressure and PRC in both genotypes. Unexpectedly, plasma BNP concentrations in ANP−/− mice significantly increased in response to two-kidney, one-clip treatment, potentially compensating for the lack of ANP. In fact, in mice lacking guanylyl cyclase A (GC-A−/− mice), which is the common receptor for both ANP and BNP, renovascular hypertension was markedly augmented compared with that in wild-type GC-A+/+ mice. However, the higher blood pressure in GC-A−/− mice was not caused by disinhibition of the renin system because PRC and renal renin synthesis were significantly lower in GC-A−/− mice than in GC-A+/+ mice. Thus, natriuretic peptides buffer renal vascular hypertension via renin-independent effects, such as vasorelaxation. The latter possibility is supported by experiments in isolated perfused mouse kidneys, in which physiological concentrations of ANP and BNP elicited renal vasodilatation and attenuated renal vasoconstriction in response to angiotensin II.

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

After oral administration of 100 mg/kg b. w. (235.8 µmol/kg) salicortin to Wistar rats, peak serum concentrations of 1.43 mg/L (13.0 µM) catechol were detected after 0.5 h in addition to salicylic acid by HPLC-DAD after serum processing with β-glucuronidase and sulphatase. Both metabolites could also be detected in the serum of healthy volunteers following oral administration of a willow bark extract (Salicis cortex, Salix spec., Salicaceae) corresponding to 240 mg of salicin after processing with both enzymes. In humans, the cmax (1.46 mg/L, 13.3 µM) of catechol was reached after 1.2 h. The predominant phase-II metabolite in humans and rats was catechol sulphate, determined by HPLC analysis of serum samples processed with only one kind of enzyme. Without serum processing with glucuronidase and sulphatase, no unconjugated catechol could be detected in human and animal serum samples. As catechol is described as an anti-inflammatory compound, these results may contribute to the elucidation of the mechanism of the action of willow bark extract.

Chronic alterations in calcium (Ca2+) signalling in podocytes have been shown to cause proteinuria and progressive glomerular diseases. However, it is unclear whether short Ca2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca2+ peaks contribute to podocyte disease.

High dietary potassium (K(+) ) intake dephosphorylates and inactivates the NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Using several ex vivo models, we show that physiological changes in extracellular K(+) , similar to those occurring after a K(+) rich diet, are sufficient to promote a very rapid dephosphorylation of NCC in native DCT cells. Although the increase of NCC phosphorylation upon decreased extracellular K(+) appears to depend on cellular Cl(-) fluxes, the rapid NCC dephosphorylation in response to increased extracellular K(+) is not Cl(-) -dependent. The Cl(-) -dependent pathway involves the SPAK/OSR1 kinases, whereas the Cl(-) independent pathway may include additional signalling cascades. A high dietary potassium (K(+) ) intake causes a rapid dephosphorylation, and hence inactivation, of the thiazide-sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT). Based on experiments in heterologous expression systems, it was proposed that changes in extracellular K(+) concentration ([K(+) ]ex ) modulate NCC phosphorylation via a Cl(-) -dependent modulation of the with no lysine (K) kinases (WNK)-STE20/SPS-1-44 related proline-alanine-rich protein kinase (SPAK)/oxidative stress-related kinase (OSR1) kinase pathway. We used the isolated perfused mouse kidney technique and ex vivo preparations of mouse kidney slices to test the physiological relevance of this model on native DCT. We demonstrate that NCC phosphorylation inversely correlates with [K(+) ]ex , with the most prominent effects occurring around physiological plasma [K(+) ]. Cellular Cl(-) conductances and the kinases SPAK/OSR1 are involved in the phosphorylation of NCC under low [K(+) ]ex . However, NCC dephosphorylation triggered by high [K(+) ]ex is neither blocked by removing extracellular Cl(-) , nor by the Cl(-) channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid. The response to [K(+) ]ex on a low extracellular chloride concentration is also independent of significant changes in SPAK/OSR1 phosphorylation. Thus, in the native DCT, [K(+) ]ex directly and rapidly controls NCC phosphorylation by Cl(-) -dependent and independent pathways that involve the kinases SPAK/OSR1 and a yet unidentified additional signalling mechanism.

Vasopeptidase inhibition (VPI), a therapeutic strategy by dual inhibition of both ACE and neutral endopeptidase 24.11, has not shown a prognostic benefit over ACE inhibition in chronic severe heart failure (CHF). Nevertheless, the effects of early treatment by VPI on cardiac remodelling have not been well assessed. We analysed the effects of early chronic VPI (50 mg/kg/day Omapatrilat) on cardiac remodelling and neurohumoral function during the progression of rapid ventricular pacing-induced heart failure in rabbits (early left ventricular dysfunction [ELVD]: 10 days at 330 bpm, CHF: further 10 days at 360 bpm). VPI-treated animals (ELVD-VPI n = 6; CHF-VPI n = 8) and placebo treated animals (ELVD n = 6; CHF n = 7) were compared with control rabbits (CTRL n = 5). LV fractional shortening (FS) and enddiastolic diameter (LVEDD) were assessed by echocardiography (12 MHz probe). LV BNP- and LV IL-6 gene expression was analysed quantitatively by real time PCR. Neurohumoral function was assessed by ANP, cGMP, plasma renin activity (PRA) and Aldosterone. In ELVD, LVEDD and atrial mass were significantly increased (both p

Severe sepsis is often accompanied by acute renal failure with renal tubular dysfunction. Albuminuria is a common finding in septic patients and we studied whether it was due to an impairment of proximal tubular endocytosis of filtered albumin. We studied the regulation of megalin and cubilin, the two critical multiligand receptors responsible for albumin absorption, during severe experimental endotoxemia. Lipopolysaccharide (LPS) caused a time- and dose-dependent suppression of megalin and cubilin expression that was paralleled by a decrease in plasma albumin levels and an increase in the urine concentration of albumin in mice. Incubation of rat renal cortical slices with LPS also reduced the mRNA expression of megalin and cubilin. Further, LPS suppressed megalin and cubilin mRNA expression in murine primary proximal tubule cells and decreased the uptake of FITC albumin in these cells. In addition, the increase in urine levels of albumin in response to ischemia/reperfusion-induced acute renal failure was paralleled by a decrease in the expression of megalin and cubilin. Thus, our data indicate that the expression of megalin and cubilin is decreased during experimental endotoxemia and in response to renal ischemia/reperfusion injury. This downregulation may contribute, in part, to an increase in urine levels of albumin during acute renal failure.

Deletion of the gap-junction-forming protein connexin40 leads to renin-dependent hypertension in mice, but whether observed human variants in connexin40, such as A96S, promote hypertension is unknown. Here, we generated mice with the A96S variant in the mouse connexin40 gene. Although mice homozygous for the A96S mutations had normal expression patterns of connexin40 in the kidney, they were hypertensive, had sixfold higher plasma renin concentrations, and had 40% higher levels of renin mRNA than controls. Renin-expressing cells were aberrantly located outside the media layer of afferent arterioles, and increased renal perfusion pressure did not inhibit renin secretion from kidneys isolated from homozygous A96S mice. Treatment with a low-salt diet in combination with an ACE inhibitor increased renin mRNA levels, plasma renin concentrations, and the number of aberrantly localized renin-producing cells. Taken together, these findings suggest that the A96S mutation in connexin40 leads to renin-dependent hypertension in mice. Modulation of renin secretion by BP critically depends on functional connexin40; with the A96S mutation, the aberrant extravascular localization of renin-secreting cells in the kidney likely impairs the pressure-mediated inhibition of renin secretion.

Renin-producing juxtaglomerular cells are connected to each other and to endothelial cells of afferent arterioles by gap junctions containing Connexin 40 (Cx40), abundantly expressed by these two cell types. Here, we generated mice with cell-specific deletion of Cx40 in endothelial and in renin-producing cells, as its global deletion caused local dissociation of renin-producing cells from endothelial cells, renin hypersecretion, and hypertension. In mice lacking endothelial Cx40, the blood pressure, renin-producing cell distribution, and the control of renin secretion were similar to wild-type mice. In contrast, mice deficient for Cx40 in renin-producing cells were hypertensive and these cells were ectopically localized. Although plasma renin activity and kidney renin mRNA levels of these mice were not different from controls, the negative regulation of renin secretion by pressure was inverted to a positive feedback in kidneys lacking Cx40 in renin-producing cells. Thus, our findings show that endothelial Cx40 is not essential for the control of renin expression and/or release. Cx40 in renin-producing cells is required for their correct positioning in the juxtaglomerular area and the control of renin secretion by pressure.

Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4-/- and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4-/- mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

The protease renin is the key enzyme of the renin-angiotensin-aldosterone cascade, which is relevant under both physiological and pathophysiological settings. The kidney is the only organ capable of releasing enzymatically active renin. Although the characteristic juxtaglomerular position is the best known site of renin generation, renin-producing cells in the kidney can vary in number and localization. (Pro)renin gene transcription in these cells is controlled by a number of transcription factors, among which CREB is the best characterized. Pro-renin is stored in vesicles, activated to renin, and then released upon demand. The release of renin is under the control of the cAMP (stimulatory) and Ca2+(inhibitory) signaling pathways. Meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control the renin secretion directly at the level of renin-producing cells, by activating either of the signaling pathways mentioned above. The broad spectrum of biological actions of (pro)renin is mediated by receptors for (pro)renin, angiotensin II and angiotensin-( 1 – 7 ).

The influence of pantoprazole 40 mg twice daily on the bioavailability of a single dose of mycophenolate mofetil 1000 mg or enteric-coated mycophenolate sodium is investigated in healthy volunteers. The plasma concentrations of mycophenolic acid and of the inactive metabolite mycophenolic acid glucuronide are measured by high-performance liquid chromatography. The pharmacokinetic parameters following sole administration are similar for mycophenolate mofetil and enteric-coated mycophenolate sodium except for the time to peak concentration, which is longer in the enteric-coated mycophenolate sodium group. Concomitant treatment with pantoprazole significantly (P < .001) lowers the mycophenolic acid exposure following administration of mycophenolate mofetil. The peak concentrations drop by 57%, and area under the curve decreases from 0 to 12 hours by 27%. In contrast, pantoprazole does not change the pharmacokinetics of enteric-coated mycophenolate sodium. Given that mycophenolic acid exposure correlates with the incidence of biopsy-proven acute rejections in renal transplant recipients, these findings may have clinical implications. Administration of pantoprazole in combination with mycophenolate mofetil could possibly result in an insufficient mycophenolic acid exposure, increasing the risk of treatment failure.

Within the juxtaglomerular apparatus, renin-producing cells and endothelial cells of the afferent arterioles express connexin (Cx)37 and Cx40, which form abundant gap junctions among these cells. Deletion of Cx40 leads to strong hyperreninemia and ectopic localization of renin-producing cells; however, the relevance of Cx37 for the renin system in the kidney has not been investigated. We therefore studied renin expression and renin secretion in kidneys from Cx37-deficient mice, both on normal salt diet and during chronic challenge of the renin system by pretreatment of mice with a low-salt diet in combination with an angiotensin I-converting enzyme inhibitor. This treatment procedure strongly enhances renin gene expression and renin secretion. We found that renal renin mRNA abundance and plasma renin concentration did not differ between wild-type and Cx37-/- mice under normal conditions. The stimulation of renin gene expression and renin secretion by salt depletion was even more pronounced in Cx37-/- as compared to wild-type mice. The regulation of renin secretion from isolated perfused kidneys by perfusion pressure and by angiotensin II was normal in Cx37-/- mice. In addition, the localization of renin-expressing cells was also regular in Cx37-/- kidneys. Finally, the expression pattern of other vascular Cxs such as Cx40, Cx43, and Cx45 was not altered in Cx37-/- kidneys. Our findings suggest that Cx37 is not essential for normal development and function of renin-producing cells. As a consequence, it appears unlikely that Cx40 exerts its important function in renin-producing cells via Cx37/Cx40 heteromeric gap junctions.

Connexins (Cxs) are a family of transmembrane proteins that form gap junctions with unique and redundant biophysical functions. Juxtaglomerular cells express Cx40, which is crucial to the control of renin secretion by blood pressure and angiotensin II, and mice that lack Cx40 have high plasma renin and hypertension. To examine whether normal juxtaglomerular cell function depends on the unique properties of Cx40, we measured renin release in mice where the coding sequence for Cx40 was replaced by that for Cx45, using the knock-in method. We first found that the knock-in strategy indeed resulted in expression of Cx45 but not Cx40 in the juxtaglomerular cells of these mice. The plasma renin concentration of the knock-in mice was similar to that in wild-type mice. The high blood pressure of the Cx40 knockout mice was significantly reduced when Cx45 was knocked into the locus but remained mildly elevated compared to wild-type mice. Blockade of angiotensin II formation by enalapril increased the plasma renin concentration in wild-type and the Cx45 knock-in mice but not in the Cx40 knockout mice. Infusion of angiotensin II into isolated perfused kidneys results in decreased renin release, a phenomenon that was attenuated in the Cx40 knockout mice. However, in the Cx45 knock-in mice, angiotensin II suppressed renin release similar to its effect in wild type mice. Unilateral renal artery stenosis increased the plasma renin concentration and blood pressure in both the wild-type and the Cx45 knock-in mice but not in the Cx40 knockout mice. Since Cx40 can be replaced by Cx45, a connexin with a significantly lower conductivity, we suggest that the regulation of renin release is not dependent on the unique electrical properties of these channel proteins.

Increased activity of the renin angiotensin system with enhanced levels of angiotensin II leads to oxidative stress with endothelial dysfunction, hypertension, and atherosclerosis. Epidemiologic studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. Because elevated angiotensin II levels might contribute to carcinogenesis, we tested whether angiotensin II induces DNA damage in the kidney. In isolated perfused mouse kidneys, as little as 1 nmol/L angiotensin II caused a significant increase in DNA strand breaks, measured with the comet assay. This damage was independent of the hemodynamic effect of angiotensin II and mediated by the angiotensin II type 1 receptor. Angiotensin II also caused double-strand breaks in the cells of the isolated perfused kidney, detected with an antibody against the double-strand break marker γ-H2AX. Studies in cell culture allowed further characterization of the DNA damage induced by angiotensin II. Single- and double-strand breaks, abasic sites, and 7,8-dihydro-8-oxo-guanine, all types of oxidative DNA lesions, were detected in angiotensin II–treated renal cells. The majority of detected strand breaks was repaired within 1 hour, but double-strand breaks increased and persisted for at least 24 hours. [Cancer Res 2008;68(22):9239–46]

According to the vascular and tubular effects of the natriuretic peptides ANP and BNP, their common receptor guanylyl cyclase A (GC-A) is expressed in renal vessels and tubular cells. Additionally ...

Glomerular hyperfiltration, as seen for instance in diabetic nephropathy or in response to loss of kidney tissue, induces hypertrophy of proximal tubular cells. In our study we tested whether the i...

An increase in the free intracellular calcium concentration promotes exocytosis in most secretory cells. In contrast, renin release from juxtaglomerular (JG) cells is suppressed by calcium. The further downstream signaling cascades of this so called “calcium paradoxon” of renin secretion have been incompletely defined. Because cAMP is the main intracellular stimulator of renin release, we hypothesized that calcium might exert its suppressive effects on renin secretion via the inhibition of the calcium-regulated adenylate cyclases AC5 and AC6. In primary cultures of JG cells, calcium-dependent inhibitors of renin release (angiotensin II, endothelin-1, thapsigargin) suppressed renin secretion, which was paralleled by decreases in intracellular cAMP levels [cAMP]. When [cAMP] was clamped by membrane permeable cAMP derivates, renin release was not suppressed by any of the calcium liberators. Additionally, both endothelin and thapsigargin suppressed cAMP levels and renin release in isoproterenol or forskolin-pretreated As4.1 cells, a renin-producing cell line that expresses AC5 and AC6. The calcium-dependent inhibition of intracellular cAMP levels and renin release was prevented by small interfering RNA-mediated knockdown of AC5 and/or AC6 expression, underlining the functional significance of these AC isoforms in renin-producing cells. Finally, in isolated perfused mouse kidneys, angiotensin II completely inhibited the stimulation of renin secretion induced by adenylate cyclase activation (isoproterenol) but not by membrane permeable cAMP analogs, supporting the conclusion that the suppressive effect of calcium liberators on renin release is mediated by inhibition of adenylate cyclase activity.

The aim of this study was to compare the bioavailability of cyclosporine (CyA) from the generic dispersion formulation Cicloral (CIC) with the microemulsion formulation Neoral (NEO) and the original Sandimmune (SIM) capsules after single doses of 100, 300, or 600 mg of drug, respectively. The study was performed according to an open 3-period cross-over design with 12 young healthy male volunteers for each dosage. The concentrations of CyA and its main metabolites were determined by high performance liquid chromatography in whole blood and urine up to 48 hours postdosing. Peak concentrations and area under the time-concentration curve were greater for the NEO and CIC formulations compared with SIM, and the mean bioavailability of CIC was significantly (P

Both the processing and release of secretory granules involve water movement across granule membranes. It was hypothesized that the water channel aquaporin (AQP)1 directly contributes to the recruitment of renin-positive cells in the afferent arteriole. AQP1−/− and AQP1+/+ mice were fed a low-salt (LS) diet [0.004% (wt/wt) NaCl] for 7 days and given enalapril [angiotensin-converting enzyme inhibitor (ACEI), 0.1 mg/ml] in drinking water for 3 days. There were no differences in plasma renin concentration at baseline. After LS-ACEI, plasma renin concentrations increased markedly in both genotypes but was significantly lower in AQP1−/− mice compared with AQP1+/+ mice. Tissue renin concentrations were higher in AQP1−/− mice, and renin mRNA levels were not different between genotypes. Mean arterial blood pressure was not different at baseline and during LS diet but decreased significantly in both genotypes after the addition of ACEI; the response was faster in AQP1−/− mice but then stabilized at a similar level. Renin release after 200 μl blood withdrawal was not different. Isoprenaline-stimulated renin release from isolated perfused kidneys did not differ between genotypes. Cortical tissue norepinephrine concentrations were lower after LS-ACEI compared with baseline with no difference between genotypes. Plasma nitrite/nitrate concentrations were unaffected by genotype and LS-ACEI. In AQP1−/− mice, the number of afferent arterioles with recruitment was significantly lower compared with AQP1+/+ mice after LS-ACEI. We conclude that AQP1 is not necessary for acutely stimulated renin secretion in vivo and from isolated perfused kidneys, whereas recruitment of renin-positive cells in response to chronic stimulation is attenuated or delayed in AQP1−/− mice.

The aim of this study was to investigate the role of cytosolic calcium for renin gene expression in juxtaglomerular cells. For this purpose, we used the immortalized juxtaglomerular mouse cell line As4.1. To increase cytosolic calcium concentration, we treated the cells with thapsigargin and cyclopiazonic acid, inhibitors of the endoplasmatic reticulum Ca− ATPase. Thapsigargin and cyclopiazonic acid inhibited renin gene expression in a characteristic time and concentration-dependent manner. This effect was concentration-dependently blocked by BAPTA-AM, an intracellular Ca 2+ chelator. Pharmacological blocking of protein kinase C activity by calphostin, Gö6976, and Gö6983 did not change the effect of thapsigargin on renin gene expression. Experiments with renin1 C –promoter–reporter constructs revealed that thapsigargin inhibited renin gene transcription. Analysis of deletion constructs of the renin1 C promoter indicated that regulatory elements involved in the calcium-mediated inhibition of renin gene transcription are located in the enhancer region of the renin gene and that ≥3 transcription factor-binding sites are involved in this process. In addition, thapsigargin reduced the renin mRNA half-life from 10 hours (control conditions) to 4 hours. Knockdown studies with small interfering RNA directed to dynamin-1 mRNA revealed that dynamin-1 is likely to be involved in the calcium-mediated destabilization of renin mRNA. These data suggest that calcium inhibits renin gene expression in juxtaglomerular cells via a concerted action of inhibition of renin gene transcription and destabilization of renin mRNA.

Renal perfusion pressure (RPP) regulates renin release with a reduction of RPP stimulating and an elevation inhibiting renin secretion. The precise sensing and effector mechanisms by which changes in arterial pressure are linked to the exocytosis of renin are not well-defined. The present experiments were designed to study the potential role of adenosine as a mediator of this renal baroreceptor mechanism. In isolated perfused mouse kidneys a stepwise reduction of RPP from 90 mm Hg to 65 and 40 mm Hg stimulated renin secretion rates (RSR) 1.4-fold and 3.6-fold, whereas stepwise elevations of RPP from 90 mm Hg to 115 and 140mm Hg suppressed RSR to 64% or 40% of baseline. Inactivation of A1 adenosine receptors by either pharmacological blockade (DPCPX 1μmol/L) or genetic deletion (A1AR −/− mice) did not modify the stimulation of renin release by a low RPP, but completely prevented the suppression of renin secretion by higher perfusion pressures. In vivo, the induction of arterial hypertension by either acute (single subcutaneous injection) or chronic (osmotic minipump for 72 hours) application of phenylephrine significantly reduced plasma renin concentration (PRC) in wild-type mice to ≈40% of control, whereas it did not significantly affect PRC in A1AR −/− mice. Together these data demonstrate that A1 adenosine receptors are indispensable for the inhibition of renin secretion by an increase in blood pressure, suggesting that formation and action of adenosine is responsible for baroreceptor-mediated inhibition of renin release. In contrast, the stimulation of the renin system by a low blood pressure appears to follow different pathways.

The soluble epoxide hydrolase (sEH) metabolizes vasodilatory epoxyeicosatrienoic acids (EETs) to their di-hydroxy derivatives. We hypothesized that the metabolism of EETs by the sEH contributes to angiotensin II–induced hypertension and tested the effects of a water-soluble sEH inhibitor, 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) on blood pressure. AUDA (130 μg/mL in drinking water) did not affect blood pressure in normotensive animals but markedly lowered it in mice with angiotensin II–induced hypertension (1 mg/kg per day). The effect of AUDA was accompanied by an increase in urinary salt and water excretion. Intravenous application of AUDA (8 mg/kg) acutely lowered blood pressure and heart rate in animals with angiotensin II–induced hypertension but failed to affect blood pressure in animals with phenylephrine-induced hypertension (29 mg/kg per day). AUDA (0.1 μmol/L) selectively lowered vascular resistance in an isolated perfused kidney preparation from angiotensin II–pretreated mice but not from control mice. In the perfused hind limb and in isolated carotid arteries from angiotensin II–treated mice, AUDA was without effect. The ω-hydroxylase inhibitor N- methylsulfonyl-12,12-dibromododec-11-enamide, which attenuates formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid, decreased tone in carotid arteries from angiotensin II–treated but not from control mice. These data demonstrate that the decrease in blood pressure observed after sEH inhibition in angiotensin II–induced hypertension can be attributed to an initial reduction in heart rate followed by pressure diuresis resulting from increased perfusion of the kidney. Direct vasodilatation of resistance arteries in skeletal muscles does not appear to contribute to the antihypertensive effects of sEH inhibition in mice.

PGE2is a potent stimulator of renin release. So far, the contribution of each of the four PGE2receptor subtypes (EP1–EP4) in the regulation of renin release has not been characterized. Therefore, we investigated the effects PGE2on renin secretion rates (RSR) from isolated, perfused kidneys of EP1−/−, EP2−/−, EP3−/−, EP4−/−, and wild-type mice. PGE2concentration dependently stimulated RSR from kidneys of all four knockout strains with a threshold concentration of 1 nM in EP1−/−, EP2−/−, EP3−/−, and wild-type mice, whereas the threshold concentration was shifted to 10 nM in EP4−/− mice. Moreover, the maximum stimulation of RSR by PGE2at 1 μM was significantly reduced in EP4−/− (12.8-fold of control) and EP2−/− (15.9-fold) compared with wild-type (20.7-fold), EP1−/− (23.8-fold), and EP3−/− (20.1-fold). In contrast, stimulation of RSR by either the loop diuretic bumetanide or the β-adrenoceptor agonist isoproterenol was similar in all strains. PGE2exerted a dual effect on renal vascular tone, inducing vasodilatation at low concentrations (1 nmol/) and vasoconstriction at higher concentrations (100 nmol/) in kidneys of wild-type mice. In kidneys of EP2−/− as well as EP4−/− mice, vasodilatation at low PGE2concentrations was prevented, whereas vasoconstriction at higher concentrations was augmented. In contrast, the vasodilatatory component was pronounced in kidneys of EP1and EP3knockout mice, whereas in both genotypes the vasoconstriction at higher PGE2concentrations was markedly blunted. Our data provide evidence that PGE2stimulates renin release via activation of EP2and EP4receptors, whereas EP1and EP3receptors appear to be without functional relevance in juxtaglomerular cells. In contrast, all four receptor subtypes are involved in the control of renal vascular tone, EP1and EP3receptors increasing, and EP2as well as EP4receptors, decreasing it.

In this review we aim to give a comprehensive overview over the current knowledge of the cellular control of renin release. We hereby focus on the inhibitory effects of calcium on the exocytosis of renin. After a short introduction into general aspects of the regulation of renin release, including a brief summary on the role of the second messengers cAMP and cGMP, we will discuss parts of the literature on the effects of calcium on the renin system together with recent studies from our laboratory, investigating putative calcium influx and extrusion pathways of juxtaglomerular cells. Finally, as the precise mechanisms by which calcium inhibits the exocytosis of renin are far from being understood, we will present some hypotheses on the intracellular events being involved in the suppression of renin release by calcium.

Experiments were performed in neuronal (nNOS)- and endothelial nitric oxide synthase (eNOS)-deficient mice to study the role of nitric oxide (NO) in macula densa control of renin secretion in vivo and in the isolated, perfused mouse kidney. Acute and chronic administration of loop diuretics was used as a method to stimulate macula densa-mediated renin secretion. Increases in plasma renin concentration (PRC) in response to a 3-day infusion of bumetanide (50 mg·kg-1·day-1) or an acute injection of furosemide (50 mg/kg ip) were not markedly altered in nNOS-/- mice. Responses to furosemide were also maintained in eNOS-/- mice, but the administration of Nω-nitro-l-arginine methyl ester (l-NAME) markedly attenuated the PRC response to furosemide in these mice. In the isolated kidney preparation, bumetanide caused similar relative increases in renin secretion in kidneys of wild-type, nNOS-/-, and eNOS-/- mice. Bumetanide only marginally increased renin secretion in l-NAME-treated kidneys, but the bumetanide effect was normalized by S-nitroso- N-acetyl-penicillamine. Basal PRC was significantly reduced in male nNOS-/- mice compared with nNOS+/+ (189 ± 28 vs. 355 ± 57 ng ANG I ·ml-1·h-1; P = 0.017). There was no significant difference in PRC between eNOS+/+ and eNOS-/- mice. Basal renin secretion rates in perfused kidneys isolated from nNOS-/- or eNOS-/- mice were markedly reduced compared with wild-type controls. Our data suggest that NO generated by macula densa nNOS does not play a specific mediator role in macula densa-dependent renin secretion. However, NO independent of its exact source permits the macula densa pathway of renin secretion to function normally.