Biomedical subjects

Armin Kurtz

Publications and source records attributed to Armin Kurtz.

At least 19 recordsLinked to original sources

PubMed2006

The calcium paradoxon of renin release: calcium suppresses renin exocytosis by inhibition of calcium-dependent adenylate cyclases AC5 and AC6.

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.

Adenylyl Cyclase Inhibitors↗

PubMed2005

Calcium inhibits renin gene expression by transcriptional and posttranscriptional mechanisms.

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 Ca2+ 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 renin1C-promoter-reporter constructs revealed that thapsigargin inhibited renin gene transcription. Analysis of deletion constructs of the renin1C 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 > or =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.

Animals↗

PubMed2005

Osmolarity-induced renin secretion from kidneys: evidence for readily releasable renin pools.

Our study aimed to characterize the influence of changes in extracellular osmolarity on renin secretion from the whole kidney. For this purpose, the osmolarity of the perfusion medium of isolated rat or mouse kidneys was either decreased by lowering the NaCl concentration by 20% or was increased up to 133% by the addition of various salts or sugars. It turned out that changes in osmolarity led to instantaneous transient changes followed by a plateau of renin secretion, in that increases in osmolarity stimulated renin secretion, whereas decreases attenuated renin secretion. The peak amplitude of changes in renin secretion was related to steady-state renin secretion rates before the osmotic challenge but was independent of the maneuver used to modulate steady-state renin secretion. Osmolarity-induced changes in renin secretion were more related to relative rather than to absolute changes in osmolarity and were not dependent on the formation of nitric oxide or of prostanoids and did not require Na-K-2Cl cotransport function or swelling-activated chloride channels. Moreover, we obtained evidence that the pool of renin secretion excitable by hyperosmolarity is exhaustible and that its complete refilling takes at least 2 min. The observed behavior of renin secretion fits the concept about exocytosis proposing the existence of different pools of committed secretory vesicles, which have not yet undergone the final modification for initiation of exocytosis. Probably, a pool of readily releasable vesicles determines steady-state secretion rates from kidneys.

Animals↗

PubMed2005

Gene expression of 5-, 12-, and 15-lipoxygenases and leukotriene receptors along the rat nephron.

The arachidonate signaling pathways comprise prostanoids formed by cyclooxygenases, EETs, and HETEs formed by cytochrome P-450 (CYP) enzymes and HETEs and leukotrienes generated by lipoxygenases. Whereas the intrarenal localization of cyclooxygenases and of some CYP enzymes along the nephron has already been determined, the localization of lipoxygenases and leukotriene-forming enzymes together with leukotriene receptors in the kidney is less clear. This study therefore aimed to determine the expression of 5-, 12-, and 15-lipoxygenases as well as the leukotriene receptors along the rat nephron. The kidneys were dissected into cortex and outer and inner medulla, and the microdissected nephron segments were collected after a collagenase digestion. mRNA abundance was determined by RT-PCR and real-time PCR. 15-LOX mRNA showed a characteristic expression pattern along the distal nephron. 12-LOX mRNA was only found in the glomerulus. Similarly, 5-LOX mRNAs together with 5-LOX-activating protein mRNAs were expressed in the glomerulus and also in the vasa recta. The leukotriene A4 hydrolase was found in all nephron segments, whereas leukotriene C4 synthase mRNA could not be found in any nephron segment. The leukotriene receptor B4 and the cysteinyl leukotriene receptor type 1 were selectively expressed in the glomerulus, whereas cysteinyl receptor type 2 was not found in any nephron segment. Our data suggest that the glomerulus is a major source and target for 5- and 12-HETE and for leukotrienes. The collecting duct system, on the other hand, appears to be a major source of 15-HETE.

Animals↗

PubMed2005

Blood pressure-dependent inhibition of Renin secretion requires A1 adenosine receptors.

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 140 mm Hg suppressed RSR to 64% or 40% of baseline. Inactivation of A1 adenosine receptors by either pharmacological blockade (DPCPX 1 micromol/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 approximately 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.

Adenosine A1 Receptor Antagonists↗

PubMed2005

Role of CREB1 and NF{kappa}B-p65 in the down-regulation of renin gene expression by tumor necrosis factor {alpha}.

Tumor necrosis factor-alpha (TNFalpha) is a potent inhibitor of renin gene expression in renal juxtaglomerular cells. We have found that TNFalpha suppresses renin transcription via transcription factor NFkappaB, which targets a cAMP responsive element (CRE) in the renin promoter. Here we aimed to further clarify the role of NFkappaB and the canonical CRE-binding proteins of the CRE-binding protein/activating transcription factor (CREB/ATF) family in the inhibition of renin gene expression by TNFalpha in the juxtaglomerular cell line As4.1. TNFalpha caused a moderate decrease in the binding of CREB1 to its cognate CRE DNA binding site. On the other hand, NFkappaB-p65 transcriptional activity was substantially reduced by TNFalpha, which targeted a trans-activation domain at the very C terminus of the p65 molecule. Our results suggest that TNFalpha inhibits renin gene expression by decreasing the transactivating capacity of NFkappaB-p65 and partially by attenuating CREB1 binding to CRE.

Animals↗

PubMed2005

Gene delivery with low molecular weight linear polyethylenimines.

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.

Animals↗

PubMed2004

Stimulation of renin release by prostaglandin E2 is mediated by EP2 and EP4 receptors in mouse kidneys.

PGE(2) is a potent stimulator of renin release. So far, the contribution of each of the four PGE(2) receptor subtypes (EP(1)-EP(4)) in the regulation of renin release has not been characterized. Therefore, we investigated the effects PGE(2) on renin secretion rates (RSR) from isolated, perfused kidneys of EP(1)-/-, EP(2)-/-, EP(3)-/-, EP(4)-/-, and wild-type mice. PGE(2) concentration dependently stimulated RSR from kidneys of all four knockout strains with a threshold concentration of 1 nM in EP(1)-/-, EP(2)-/-, EP(3)-/-, and wild-type mice, whereas the threshold concentration was shifted to 10 nM in EP(4)-/- mice. Moreover, the maximum stimulation of RSR by PGE(2) at 1 microM was significantly reduced in EP(4)-/- (12.8-fold of control) and EP(2)-/- (15.9-fold) compared with wild-type (20.7-fold), EP(1)-/- (23.8-fold), and EP(3)-/- (20.1-fold). In contrast, stimulation of RSR by either the loop diuretic bumetanide or the beta-adrenoceptor agonist isoproterenol was similar in all strains. PGE(2) exerted 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 EP(2)-/- as well as EP(4)-/- mice, vasodilatation at low PGE(2) concentrations was prevented, whereas vasoconstriction at higher concentrations was augmented. In contrast, the vasodilatory component was pronounced in kidneys of EP(1) and EP(3) knockout mice, whereas in both genotypes the vasoconstriction at higher PGE(2) concentrations was markedly blunted. Our data provide evidence that PGE(2) stimulates renin release via activation of EP(2) and EP(4) receptors, whereas EP(1) and EP(3) receptors appear to be without functional relevance in juxtaglomerular cells. In contrast, all four receptor subtypes are involved in the control of renal vascular tone, EP(1) and EP(3) receptors increasing, and EP(2) as well as EP(4) receptors, decreasing it.

Animals↗

PubMed2004

Hypoxia up-regulates triosephosphate isomerase expression via a HIF-dependent pathway.

The glycolytic enzyme triosephosphate isomerase (TPI) catalyses the reversible conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We report here that the expression of TPI at both the mRNA and protein levels is increased by hypoxia in vivo and in vitro. The temporal pattern of hypoxic TPI induction is very similar to that of genes triggered by the hypoxia-inducible transcription factor (HIF) and is mimicked characteristically by cobalt and by deferoxamine, but is absent in cells with a defective aryl hydrocarbon receptor nuclear translocator (ARNT, here HIF-1beta) and in cells lacking HIF-1alpha protein. We conclude from these findings that the expression of TPI is regulated via the HIF pathway and thus belongs to the family of classic oxygen-regulated genes. The physiological meaning of an increased expression of TPI in hypoxygenated tissues is probably to increase the flow of triosephosphates through the glycolytic cascade thus leading to an increase of anaerobic energy generation.

Actins↗

PubMed2004

Permissive role of nitric oxide in macula densa control of renin secretion.

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 N(omega)-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.

Adrenergic beta-Antagonists↗

PubMed2004

Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis.

To investigate regional aspects of hypoxic regulation of adrenomedullin (AM) in kidneys, we mapped the distribution of AM in the rat kidney after hypoxia (normobaric hypoxic hypoxia, carbon monoxide, and CoCl(2) for 6 h), anemia (hematocrit lowered by bleeding) and after global transient ischemia for 1 h (unilateral renal artery occlusion and reperfusion for 6 and 24 h) and segmental infarct (6 and 24 h). AM expression and localization was determined in normal human kidneys and in kidneys with arterial stenosis. Hypoxia stimulated AM mRNA expression significantly in rat inner medulla (CO 13 times, 8% O(2) 6 times, and CoCl(2) 8 times), followed by the outer medulla and cortex. AM mRNA level was significantly elevated in response to anemia and occlusion-reperfusion. Immunoreactive AM was associated with the thin limbs of Henle's loop, distal convoluted tubule, collecting ducts, papilla surface epithelium, and urothelium. AM labeling was prominent in the inner medulla after CO and in the outer medulla after occlusion-reperfusion. The infarct border zone was strongly labeled for AM. In cultured inner medullary collecting duct cells, AM mRNA was significantly increased by hypoxia. AM mRNA was equally distributed in human kidney and AM was localized as in the rat kidney. In human kidneys with artery stenosis, AM mRNA was not significantly enhanced compared with controls, but AM immunoreactivity was observed in tubules, vessels, and glomerular cells. In summary, AM expression was increased in the rat kidney in response to hypoxic and ischemic hypoxia in keeping with oxygen gradients. AM was widely distributed in the human kidney with arterial stenosis. AM may play a significant role to counteract hypoxia in the kidney.

Adrenomedullin↗

PubMed2004

Chronic cyclooxygenase-2 inhibition does not alter blood pressure and kidney function in renovascular hypertensive rats.

BACKGROUND: It has been shown that the macula densa participates in the regulation of increased renin expression in two-kidney one-clip (2K1C) renovascular hypertension. Prostaglandins might be one of the mediators of macula densa function, because the cyclooxygenase-2 (COX-2), one of the rate-limiting enzymes of the prostaglandin pathway, is upregulated in 2K1C renovascular hypertensive rats. We tested the effect of chronic COX-2 inhibition on blood pressure, urinary aldosterone excretion and kidney morphology, as well as kidney function. METHODS: Four groups were established: two groups of 2K1C renovascular hypertensive rats treated with the specific COX-2 inhibitor Celecoxib (cele) (15 mg/kg per day) or placebo immediately after operation, and two sham-operated control groups fed with Celecoxib or placebo. RESULTS: Long-term COX-2 inhibition in 2K1C renovascular hypertensive rats did not alter blood pressure at any point of time. Urinary aldosterone excretion was elevated by clipping the renal artery (2K1C, 8.1 +/- 1.9, versus controls, 3.6 +/- 0.5 ng/24 h; P = 0.05) but was not influenced by treatment with Celecoxib. Also, Celecoxib treatment did not alter glomerular filtration rate (GFR), serum sodium, serum creatinine, serum urea or proteinuria in 2K1C renovascular hypertensive rats. Interstitial fibrosis of the left clipped kidney was markedly reduced (2K1C, 6.19 +/- 0.83% versus 2K1C + cele 3.00 +/- 0.68% of total area; P = 0.012), whereas the interstitial fibrosis of the non-clipped kidney or the glomerulosclerosis of both kidneys were not affected by Celecoxib treatment. CONCLUSIONS: Celecoxib reduces the interstitial fibrosis of the clipped kidney. Blood pressure, urinary aldosterone excretion or whole kidney function were not affected in renal hypertensive rats.

Aldosterone↗

PubMed2004

Gene expression of adenosine receptors along the nephron.

BACKGROUND: In view of the multiple effects of adenosine on kidney function, this study aimed to determine the expression of adenosine receptors (AR) along the rat and mouse nephron. METHODS: For this purpose, we semiquantified mRNA abundance for adenosine A1-, A2A-, A2B-, and A3 receptors by RNAse protection and by reverse transcription-polymerase chain reaction (RT-PCR) in the kidney zones and in the different nephron segments of mice and rats. RESULTS: We found very similar expression patterns for rat and mice. For the kidney zones A1-AR mRNA and A2A-AR mRNA abundance displayed a marked difference, with an increase from cortex to the inner medulla. This was not seen for A2B receptors, which showed in general a rather weak expression. Along the nephron, A1-AR was strongly expressed in the thin limbs of Henle and in the collecting duct system and to a lesser extent in the medullary thick ascending limb. A2A-AR mRNA was clearly detected in glomeruli but not in other nephron segments. A2B-AR mRNA was strongly expressed in the cortical thick ascending limb of Henle and in the distal convoluted tubule. A3-AR mRNA was not found in any nephron segment. CONCLUSION: Our data demonstrate a distinct mutual expression of the AR subtypes along the nephron. A1 receptors are expressed in medullary tubular structures, while A2B receptors are predominant in cortical tubular structures. A2A receptor expression in the kidney appears to be restricted to vascular cells.

Animals↗

PubMed2004

Cyclosporine A attenuates the natriuretic action of loop diuretics by inhibition of renal COX-2 expression.

BACKGROUND: It is known that inhibition of cyclooxygenase (COX) impairs the renal actions of loop diuretics. Recently, we found that cyclosporine A (CsA) inhibits renal COX-2 expression. Therefore, we examined the interferences of CsA with the renal actions of loop diuretics. METHOD: We investigated the renal effects of furosemide administration (12 mg/day subcutaneously) in male Sprague-Dawley rats receiving in addition vehicle, CsA (15 mg/kg x day), rofecoxib (10 mg/kg x day), or a combination of both. RESULTS: CsA, rofecoxib, and their combination lowered the furosemide-induced increase of prostaglandin E(2) (PGE(2)) and of 6-keto prostaglandin F(1 alpha) (6-keto PGF(1 alpha)) excretion by 55% and by 70%. They also lowered furosemide stimulated renal excretion of sodium and water by about 65% and 60%. Basal as well as furosemide-induced stimulation of plasma renin activity (PRA) and of renal renin mRNA was further enhanced by CsA. In contrast, rofecoxib attenuated the furosemide-induced rise of PRA and of renin mRNA, both in the absence and in the presence of CsA. In addition, the increase in plasma 6-keto PGF(1 alpha) levels by furosemide was further enhanced by CsA and was attenuated by rofecoxib. CONCLUSION: Taken together, our data suggest that CsA acts as an antinatriuretic, likely by the inhibition of COX-2-mediated renal prostanoid formation. Since the furosemide-induced stimulation of the renin system is not attenuated by CsA but by COX-2 inhibition, we speculate that extrarenal COX-2-derived prostanoids may be involved in the stimulation of the renin system by CsA and by loop diuretics.

6-Ketoprostaglandin F1 alpha↗

PubMed2004

Upregulation of macula densa cyclooxygenase-2 expression is not dependent on glomerular filtration.

Although the regulation of cyclooxygenase-2 (COX-2) expression in the kidney cortex has been extensively characterized, the physiological control mechanisms of COX-2 expression at the level of the kidney and at the level of the tubular cells are not well understood. Based on the current hypothesis that tubular salt transport might be a crucial regulator of COX-2 expression, this study aimed to determine the impact of salt delivery to the tubules (glomerular filtration) for the regulation of COX-2 in the kidney cortex in vivo. To this end, glomerular filtration of the right kidney was abrogated by the ligation of the right ureter of male Sprague-Dawley rats. After 1 wk of ligation, the animals were treated with subcutaneous infusions of furosemide (12 mg x kg(-1) x day(-1)) or with a low-salt or a high-salt diet (0.02% wt/wt; 8% wt/wt), and COX-2 as well as renin mRNA expression were determined in the ligated and the nonligated contralateral kidney. During ureteral ligation, hydronephrosis developed with a reduction of medullary mass, while the cortex was preserved. Expressions of the Na-K-2Cl cotransporter isoforms A and B were both reduced in the hydronephrotic cortex to 70 and 35% of the corresponding contralateral intact kidney. Despite the abrogation of glomerular filtration, detected by inulin clearance measurements, renocortical COX-2 mRNA abundance was stimulated by furosemide treatment (3.2-fold) or low-salt diet (2.9-fold) to similar degrees compared with the intact contralateral kidney (2.7-fold for both treatments), whereas a high-salt diet did not significantly suppress COX-2 mRNA in the macula densa region of either kidney. Renin mRNA expression was regulated strictly in parallel in both kidneys, a low-salt diet or furosemide treatment stimulating and a high-salt diet suppressing it. We conclude from these findings that salt delivery to the tubules is not an essential requirement for the upregulation of COX-2 by salt deficiency or by loop diuretics in the rat kidney cortex nor is it for chronic stimulation of renin mRNA expression.

Animals↗

PubMed2004

Polyplexes of polyethylenimine and per-N-methylated polyethylenimine-cytotoxicity and transfection efficiency.

For non-viral gene delivery, the carriers for DNA transfer into cells must be vastly improved. The branched cationic polymer polyethylenimine has been described as an efficient gene carrier. However, polyethylenimine was demonstrated to mediate substantial cytotoxicity. Therefore, this study is aimed at investigating per-N-methylated polyethylenimine, which is thought to have a much lower cytotoxicity due to its lower charge density. Results from a gel retardation assay and laser light scattering indicated that per-N-methylated polyethylenimine condenses DNA into small and compact nanoparticles with a mean diameter <150 nm. Furthermore, polyplexes of polyethylenimine and per-N-methylated polyethylenimine with DNA had a positive zeta potential and the polymers protected DNA from nuclease-mediated digestion. The transfection efficiency of polyethylenimine and per-N-methylated polyethylenimine was tested in CHO-K1 cells. Using green fluorescent protein as reporter gene and flow cytometry analysis, we demonstrated that per-N-methylated polyethylenimine has a lower cytotoxicity, but also a significantly lower transfection efficiency. Using propidium iodide staining, we could additionally distinguish between viable and dead cells. At NP > or = 12, per-N-methylated polyethylenimine showed a much higher cell viability and the ratio of viable and transfected cells to dead and transfected cells was about 1.5 to 1.7 fold higher than for polyethylenimine. The results of cell viability from flow cytometry analysis were confirmed by the MTS assay. Using luciferase reporter gene for transfection experiments, the gene expression of per-N-methylated polyethylenimine was lower at NP 6, 12 and 18 as compared to polyethylenimine, but at NP 24 it yielded similar levels.

Animals↗

PubMed2003

Aldosterone enhances renin gene expression in juxtaglomerular cells.

The secretion and synthesis of renin as the key regulator of the renin-angiotensin-aldosterone system are directly controlled by ANG II in the sense of a negative feedback. Because we found that renal afferent arterioles including the juxtaglomerular portion express the mineralocorticoid receptor, we aimed to characterize a possible direct effect of aldosterone on renin synthesis and renin secretion at the level of renal juxtaglomerular cells. Aldosterone (100 nM) clearly enhanced renin mRNA levels in primary cultures of mouse juxtaglomerular cells prestimulated with isoproterenol (100 nM) but had no effect on the exocytosis of stored renin. Similarly, in the mouse juxtaglomerular cell line As4.1, aldosterone time and concentration dependently increased renin mRNA abundance and prorenin secretion up to 2.5-fold. Moreover, aldosterone potentiated cAMP-induced renin gene expression in As4.1 cells. The effect of aldosterone was inhibited by spironolactone and was mimicked by corticosteroid hormones but not by sex steroids. Aldosterone had no influence on basal renin promoter activity but increased the renin mRNA half-life about threefold. In summary, these data suggest that aldosterone exerts a direct positive effect on renin gene expression at the cellular level probably by stabilizing renin mRNA.

Aldosterone↗

PubMed2003

Tumor necrosis factor-alpha activates NFkappaB to inhibit renin transcription by targeting cAMP-responsive element.

Tumor necrosis factor-alpha (TNFalpha) is known to inhibit renin gene expression in juxtaglomerular cells, which are the main source of renin in vivo. In the present study we aimed to characterize the intracellular mechanisms of TNFalpha signaling to renin gene in the mouse juxtaglomerular cell line As4.1. TNFalpha was found to activate NFkappaB, which is one of the principal intracellular mediators of TNFalpha signal transduction. Constitutive activation of NFkappaB suppressed renin gene transcription, but NFkappaB appeared not to target the NFkappaB binding sites in the renin promoter. Thus, NFkappaB, but not the canonical NFkappaB binding sequences in the renin promoter, seemed to be involved in the suppression of renin transcription by TNFalpha. Deletion/mutation analysis revealed that the effect of TNFalpha on renin gene is transmitted by a cAMP-responsive element (CRE) located at -2697 to -2690. Mobility shift/supershift assays evidenced for the presence of NFkappaB proteins in the complex that binds to mouse renin CRE. Our results strongly suggest that NFkappaB mediates the effect of TNFalpha on renin transcription targeting a CRE in the mouse renin promoter.

Animals↗