Bernhard Gess

Atrap Deficiency Increases Arterial Blood Pressure and Plasma Volume

The angiotensin receptor-associated protein (Atrap) interacts with angiotensin II (AngII) type 1 (AT1) receptors and facilitates their internalization in vitro, but little is known about the function of Atrap in vivo. Here, we detected Atrap expression in several organs of wild-type mice; the highest expression was in the kidney where it localized to the proximal tubule, particularly the brush border. There was no Atrap expression in the renal vasculature or juxtaglomerular cells. We generated Atrap-deficient (Atrap-/-) mice, which were viable and seemed grossly normal. Mean systolic BP was significantly higher in Atrap-/- mice compared with wild-type mice. Dose-response relationships of arterial BP after acute AngII infusion were similar in both genotypes. Plasma volume was significantly higher and plasma renin concentration was markedly lower in Atrap-/- mice compared with wild-type mice. (125)I-AngII binding showed enhanced surface expression of AT1 receptors in the renal cortex of Atrap-/- mice, accompanied by increased carboanhydrase-sensitive proximal tubular function. In summary, Atrap-/- mice have increased arterial pressure and plasma volume. Atrap seems to modulate volume status by acting as a negative regulator of AT1 receptors in the renal tubules.

Hypoxia and cobalt stimulate vascular endothelial growth factor receptor gene expression in rats

Abstract This study aimed to examine the influence of acute tissue hypo-oxygenation on the expression of the vascular endothelial growth factor (VEGF) receptor genes. To this end male Sprague-Dawley rats were exposed to different hypoxic conditions such as 10% or 8% oxygen, 0.1% carbon monoxide and cobalt chloride (60 mg/kg) for 6 h and the abundance of flt-1, flt-4 and flk-1 mRNA in lungs and livers was determined by RNase protection assay. The relative proportions of flt-1, flt-4 and flk-1 were 10 : 2.5 : 1 and 10 : 10 : 2 in normoxic lungs and livers, respectively. It was found that 8% but not 10% oxygen increased flt-1 mRNA two- to threefold in both organs, whilst flt-4 and flk-1 mRNA were not changed by acute inspiratory hypoxia. Carbon monoxide inhalation also increased flt-1 mRNA but not flt-4 or flk-1 mRNA in both organs. Subcutaneous cobalt administration increased flt-1 mRNA in the livers only, whilst flt-4 and flk-1 mRNA remained unchanged. These findings show that acute tissue hypo-oxygenation is a rather selective stimulus for flt-1 gene expression. The efficiency of the different manoeuvres applied to stimulate flt-1 gene expression is rather similar to the stimulation of erythropoietin gene expression. It is not unreasonable to assume, therefore, that the oxygen-dependent regulation of both genes at the cellular level has significant similarities.

Divergent regulation of vascular endothelial growth factor and of erythropoietin gene expression in vivo

There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor (VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt. Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O2 ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF-188, -164, and -120 amino acid isoforms in lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF mRNA abundance of lung ≫ heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and 3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys. Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence, the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the VEGF and EPO genes may not follow the same essential mechanisms in vivo.

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-1β) and in cells lacking HIF-1α 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.

Intravital Imaging Reveals Angiotensin II–Induced Transcytosis of Albumin by Podocytes

Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (P<0.001), and 239.4±34.6 µm(3) (P<0.001) albumin-containing vesicles per glomerulus, respectively, compared with none at baseline or 10 ng/kg per minute Ang II. Immunostaining of Ang II-infused kidneys confirmed the presence of albumin-containing vesicles, which colocalized with megalin, in podocin-positive cells. Furthermore, podocyte endocytosis of albumin was markedly reduced in the presence of gentamicin, a competitive inhibitor of megalin-dependent endocytosis. Ang II infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and gentamicin further increased this concentration. Some endocytic vesicles were acidified and colocalized with LysoTracker. Most vesicles migrated from the capillary to the apical aspect of the podocyte and were eventually released into the urinary space. This transcytosis accounted for approximately 10% of total albumin filtration. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. Ang II enhances the endocytosis and transcytosis of plasma albumin by podocytes, which may eventually impair podocyte function.

Renin gene and angiotensin II AT1 receptor gene expression in the kidneys of normal and of two-kidney/one-clip rats

This study aimed to investigate the inter-relation between the angiotensin II (ANG II) AT1 receptor and renin gene expression in rat kidneys. To this end, renin mRNA levels and mRNA levels for AT1a and AT1b were assayed by RNase protection in the kidneys of normal rats, in animals treated with the AT1 antagonist losartan and in rats bearing 0.2-mm left renal artery clips for 2 days. In normal rats, we found a negative correlation between renin mRNA levels and AT1a receptor mRNA levels. Losartan led to a fourfold increase in renin mRNA levels without changing AT1 receptor mRNA levels. Unilateral renal artery clipping increased renin mRNA levels fourfold in the clipped kidney and suppressed renin mRNA levels in the contralateral kidneys. AT1 receptor mRNA levels were not changed in the contralateral intact kidneys, but were significantly decreased by 15–25% in the clipped kidneys. Renin mRNA levels were inversely correlated to AT1a mRNA levels in the clipped, but not in the contralateral, kidneys. Our findings suggest that the systemic activity of the renin angiotensin system has no regulatory influence on renal AT1 receptor gene expression. Renin mRNA levels in normal and in clipped kidneys appear to be negatively determined by the level of AT1a receptor gene expression. Thus modulation of AT1a receptor gene expression could be a pathway for indirect modulation of renin gene expression by ANG II. This conclusion is in agreement with the observation that AT1 receptor antagonists are powerful stimulators of the renin system.

Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes.

This study aimed to investigate the sequence of events involved in the stimulation of erythropoietin (EPO) gene expression by hypoxia in hepatocytes. To this end, primary cultures of rat hepatocytes were kept at either high (40% O2) or low (3% O2) oxygen tensions for 2.5 h. Hypoxia increased EPO mRNA about fifteen‐fold, whilst the divalent cation cobalt (50–100 μM) or the iron chelator desferrioxamine (10–200 μM) did not increase EPO mRNA levels. Addition of hydrogen peroxide (100–500 μM) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50–200 μg/ml) to the culture medium resulted in a lower level of hypoxia‐induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 μM) completely abolished the increase of EPO mRNA in response to hypoxia. Hypoxia but not cobalt increased the appearance of the hypoxia‐inducible factor 1 (HIF‐1), and this increase was blunted by cycloheximide. Taken together, these findings suggest that a classic heme protein and a related oxygen‐dependent production of oxygen radicals is less likely to be involved in the regulation of the EPO gene by oxygen in hepatocytes. On the other hand, intact protein synthesis is an absolute requirement for the hypoxia‐induced appearance of HIF‐1 and for hypoxia‐stimulated expression of the EPO gene in hepatocytes.

Differential effects of kinase inhibitors on erythropoietin and vascular endothelial growth factor gene expression in rat hepatocytes

This study sought to investigate whether a common protein kinase activity is involved in the sequence of events by which oxygen controls the expression of the genes for erythropoietin (EPO) and for vascular endothelial growth factor (VEGF) in rat hepatocytes. To this end we examined the influence of the non-specific kinase inhibitor staurosporine and of the tyrosine kinase inhibitor genistein on EPO and VEGF mRNA levels in primary cultures of rat hepatocytes kept at either high (20% O2) or low (1% O2) oxygen tension. We found that 3 h of exposure to the low O2 tension increased EPO mRNA levels about 20-fold and the three VEGF (−180, −164, −120) mRNA levels, on average, about fourfold. Staurosporine did not change EPO and VEGF mRNA levels at 20% O2, but in a concentration-dependent manner, decreased EPO and VEGF mRNA at 1% O2 with IC50 values of 30 nM and 1000 nM, respectively. In the presence of 1 % O2, genistein decreased EPO mRNA and VEGF mRNA levels with IC50 values of about 36 and 360 μM, respectively. Although mRNA levels for glycerine aldehyde phosphatehydrogenase (GAPDH) were not changed, staurosporine and genistein inhibited uridine incorporation into total RNA with IC50 values of about 1 μM and 100 μM, respectively. Comparison with the transcription inhibitor actinomycin D suggested that the effects of both kinase inhibitors on VEGF mRNA but not on EPO mRNA levels could be attributed to the non-specific inhibition of transcription in hepatocytes. These findings suggest that a kinase activity is specifically involved in the O2-dependent control of EPO gene expression but not of VEGF gene expression in hepatocytes.

Salt-losing nephropathy in mice with a null mutation of the Clcnk2 gene.

The basolateral chloride channel ClC‐Kb facilitates Cl reabsorption in the distal nephron of the human kidney. Functional mutations in CLCNKB are associated with Bartters syndrome type 3, a hereditary salt‐losing nephropathy. To address the function of ClC‐K2 in vivo, we generated ClC‐K2‐deficient mice.

Hypoxia-induced accumulation of erythropoietin mRNA in isolated hepatocytes is inhibited by protein kinase C

To define the role of protein kinase C (PKC) in oxygen-dependent production of erythropoietin (EPO) in the liver, we have determined EPO messenger ribonucleic acid (mRNA) expression in primary cultures of juvenile rat hepatocytes incubated at different oxygen tensions in the absence and presence of phorbol esters, vasopressin, and structurally different kinase inhibitors. Upon reduction of oxygen concentrations from 40% to 3% EPO mRNA in cultured hepatocytes increased markedly within 1.25 h, reached maximal values after 2.5 h and remained elevated for up to 72 h. Treatment of hepatocytes during 1.25–5 h of hypoxic exposure with phorbol 12-myristate-13 acetate (PMA) attenuated hypoxia-induced EPO mRNA levels dose-dependently by a maximum of approximately 50%. This inhibitory effect of PMA disappeared upon treatment for more than 5 h and was completely lost after incubation for 9 and 18 h in the presence of 10−6 M and 10−7 M PMA, respectively. Phorbol 12,13-dibutyrate and vasopressin also inhibited EPO mRNA accumulation, whereas 4 alpha-phorbol 12,13-didecanoate was ineffective. Western blot analysis of PKC isozymes revealed the presence of PKC alpha, beta II, delta, epsilon and zeta and provided no evidence that the PMA-induced inhibition of EPO expression was associated with depletion of any of these isozymes. Conversely, PMA-induced inhibition of EPO mRNA accumulation was paralleled by translocation of PKC alpha from cytosol to membranes and the time- and dose-dependent attenuation of the inhibitory effect of PMA on EPO mRNA levels was paralleled by down-regulation of PKC alpha. A dose-dependent inhibition of EPO mRNA formation, independent of effects on total RNA synthesis, as determined by [3H]uridine incorporation, was also found in the presence of the kinase inhibitor staurosporine (ED50 ∼2×10−8 M) and three structurally related derivatives with increased selectivity for PKC (RO 317549, ED50 ∼1×10−6 M; RO 318220, ED50 ∼1× 10−6 M and CGP 41251, ED50 ∼4×10−6 M). The markedly lower potency of the latter three compounds as compared to staurosporine suggests that this suppression of EPO gene induction was not mediated by inhibition of PKC. In summary the data indicate that PKC alpha is a negative modulator of EPO gene expression in hepatocytes. A kinase other than PKC, however, appears to be an essential element of hypoxic signalling.