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

AIM: The basolateral chloride channel ClC-Kb facilitates Cl reabsorption in the distal nephron of the human kidney. Functional mutations in CLCNKB are associated with Bartter's syndrome type 3, a hereditary salt-losing nephropathy. To address the function of ClC-K2 in vivo, we generated ClC-K2-deficient mice. METHODS: ClC-K2-deficient mice were generated using TALEN technology. RESULTS: ClC-K2-deficient mice were viable and born in a Mendelian ratio. ClC-K2-/- mice showed no gross anatomical abnormalities, but they were growth retarded. The 24-h urine volume was increased in ClC-K2-/- mice (4.4 ± 0.6 compared with 0.9 ± 0.2 mL per 24 h in wild-type littermates; P = 0.001). Accordingly, ambient urine osmolarity was markedly reduced (590 ± 39 vs. 2216 ± 132 mosmol L-1 in wild types; P < 0.0001). During water restriction (24 h), urinary osmolarity increased to 1633 ± 153 and 3769 ± 129 mosmol L-1 in ClC-K2-/- and wild-type mice (n = 12; P < 0.0001), accompanied by a loss of body weight of 12 ± 0.4 and 8 ± 0.2% respectively (P < 0.0001). ClC-K2-/- mice showed an increased renal sodium excretion and compromised salt conservation during a salt-restricted diet. The salt-losing phenotype of ClC-K2-/- mice was associated with a reduced plasma volume, hypotension, a slightly reduced glomerular filtration rate, an increased renal prostaglandin E2 generation and a massively stimulated renin-angiotensin system. Clckb-/- mice showed a reduced sensitivity to furosemide and were completely resistant to thiazides. CONCLUSION: Loss of ClC-K2 compromises TAL function and abolishes salt reabsorption in the distal convoluted tubule. Our data suggest that ClC-K2 is crucial for renal salt reabsorption and concentrating ability. ClC-K2-deficient mice in most aspects mimic patients with Bartter's syndrome type 3.

Desmoplakin is involved in organization of an adhesion complex in peripheral nerve regeneration after injury.

Peripheral nerves have the unique capability to regenerate after injury. Insights into regeneration of peripheral nerves after injury may have implications for neurodegenerative diseases of the nervous system. In this study, we analyzed the expression and function of desmoplakin in peripheral nerve regeneration. Desmoplakin was upregulated in spinal cord motoneurons after sciatic nerve injury. Conditional ablation of desmoplakin in motoneurons demonstrated that desmoplakin is necessary for normal motor regeneration. SiRNA and desmoplakin deletion-constructs revealed a role of desmoplakin in neurite extension in vitro. A complex of N-cadherin, plakoglobin, desmoplakin and vimentin was shown in motoneuronal cell cultures and peripheral nerves after injury in vivo. Motor nerve fiber regeneration and localization of N-cadherin and vimentin to axonal growth fronts were reduced in conditionally desmoplakin-ablated mice. These data indicate a function of desmoplakin in motor nerve regeneration by linking N-cadherin to intermediate filaments in regenerating motor axons.

[Genetics of neuropathies]. / Genetik der Neuropathien.

Hereditary neuropathies belong to the most common neurogenetic disorders. They appear mostly as sensory and motor neuropathies but there are also pure sensory, pure motor as well as sensory and autonomic hereditary neuropathies. In clinical practice, knowledge of hereditary neuropathies is important in order to recognize them among polyneuropathies and achieve a successful genetic diagnosis. The molecular genetics of hereditary neuropathies are very heterogeneous with currently more than 40 known disease-causing genes. The 4 most common genes account for almost 90% of the genetically diagnosed hereditary neuropathies. In this review article we provide an overview of the currently known genes and propose a rational genetic work-up protocol of the most common genes.

[Clinical relevance of normal and enlarged Virchow-Robin spaces]. / Klinische Bedeutung normaler und erweiterter Virchow-Robin-Räume.

Virchow-Robin spaces ensheathe the penetrating vessels of the brain. They communicate with the subpial space, are filled with interstitial fluid and contain a specific population of macrophages.Virchow-Robin spaces are a common finding in both CT and MR imaging. Recent radiologic studies have led to a concise definition of Virchow-Robin spaces.Virchow-Robin spaces appear isointense to cerebrospinal fluid on all imaging sequences. They are typically localised in the basal ganglia, subcortically or in the midbrain and pons. Enlarged Virchow-Robin spaces may appear as a single or multiple lesion(s). They may cause hydrocephalus in rare cases. Some studies indicate that enlarged Virchow-Robin spaces occur more frequently in elderly patients, in patients with arterial hypertension or CADASIL.In this review we illustrate the diagnostic criteria of normal and enlarged Virchow-Robin spaces and discuss their clinical relevance. Furthermore, we present an overview of the current knowledge on the anatomy, physiology and pathology of Virchow-Robin spaces.

Endogenous nitric oxide attenuates erythropoietin gene expression in vivo.

This study aimed to investigate the role of endogenous nitric oxide (NO) in erythropoietin (EPO) gene expression in mice in vivo. For this purpose EPO mRNA was semiquantitated by ribonuclease protection assay in livers and kidneys of three groups of mice: wild-type (wt), endothelial NO-synthase (NOS) knockout mice (eNOS-/-), and wt treated with the NOS inhibitor N(G)-nitro-L-arginine methyl ester (50 mg x kg(-1) x day(-1)) for 4 days (wt+L-NAME). EPO gene expression was stimulated by normobaric hypoxia (8% O2) or by 0.1% carbon monoxide (CO) inhalation for 4 h each, or by intraperitoneal injection of 60 mg/kg cobaltous chloride (CoCl2) for 6 h. Renal EPO mRNA in wt increased 12-, 40-, and 13-fold over normoxic levels in response to hypoxia, CO and CoCl2 respectively. EPO mRNA was detectable in the livers only after CO exposure. Renal and hepatic EPO gene expression in wt+L-NAME appeared moderately increased relative to wt with a maximal 2.5-fold enhancement after CO exposure. EPO mRNA levels in eNOS-/- mirrored those of wt+L-NAME, but the effects were less prominent. Our data suggest that endogenous NO attenuates EPO gene expression in mice. This effect is dependent on the rate of EPO gene induction.

Acute hypoxia upregulates NOS gene expression in rats.

This study aimed to investigate the influence of acute tissue hypoxygenation on the expression of NO synthase (NOS) genes in vivo. To this end, male Sprague-Dawley rats were exposed either to 9% oxygen or to 0.1% carbon monoxide for 6 h, and mRNA levels of NOS-I, -II, and -III in kidneys, livers, lungs, and left and right heart ventricles were assayed by ribonuclease protection. For comparison, mRNA levels of erythropoietin were also measured in these tissues. NOS-III mRNA was highly abundant in all organs investigated. NOS-II mRNA was detected in lungs and hearts but not in kidneys and livers. NOS-I mRNA was found in kidneys, lungs, and hearts but not in livers. NOS-III mRNA levels were upregulated by hypoxia in all tissues examined, with the least effect (1.2-fold) in the left ventricle and the greatest effect (2.6-fold) in the lung. NOS-II mRNA was substantially downregulated in the ventricles by both treatments but not changed in the lung. NOS-I mRNA was upregulated by carbon monoxide in kidneys and lungs and by 9% oxygen in the lung. These findings suggest that NOS-III and possibly also NOS-I gene expression behave like oxygen-regulated genes, whereas the general effect of tissue hypoxygenation on NOS-II gene expression is less clear. Because NOS-III is primarily expressed in endothelial cells, a general upregulation of NOS in these cells may be of relevance for the regulation and maintenance of blood flow through hypoxic tissues.

In vivo carbon monoxide exposure and hypoxic hypoxia stimulate immediate early gene expression.

This study aimed to examine the influence of acute tissue hypoxygenation on the expression of immediate early genes in different rat tissues. To this end male Sprague-Dawley rats were exposed to 0.1% carbon monoxide for 0.5, 1 and 6 h or to 9% oxygen for 6 h and mRNA levels for c-jun, c-fos, c-myc and EGR-1 were assayed by RNase protection in hearts, kidneys, livers and lungs. We found that hypoxia increased c-jun mRNA levels between twofold (lung) and eightfold (liver) in all organs examined; c-fos mRNA increased between three-fold (lung) and 20-fold (heart); c-myc mRNA increased between twofold (lung) and sixfold (heart); and EGR-1 mRNA increased between twofold (lung) and sixfold (heart). Our findings suggest that acute tissue hypoxygenation is a general stimulus of the expression of immediate early genes in vivo. With regard to the sensitivity to hypoxia, organ differences appear to exist in that the lung is rather insensitive, whilst the heart is rather sensitive.

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

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 three-fold 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.

Lack of control by immediate early response genes in the oxygen regulation of erythropoietin gene expression.

This study sought to investigate the role of immediate early genes in the stimulation of erythropoietin (EPO) gene expression by hypoxia. To this end freshly isolated rat hepatocytes were exposed to either normoxia (20% oxygen) or to hypoxia (1% oxygen) and the mRNA levels of the early genes c-fos, c-jun, c-myc and EGR-1 were monitored together with EPO mRNA. Isolation of the cells from the livers strongly stimulated the expression of c-fos, c-jun, c-myc and of EGR-1, whilst EPO gene expression remained unchanged. Exposure of the isolated hepatocytes to hypoxia did not further change early gene expression when compared with cells kept under normoxic conditions. EPO mRNA increased time dependently with a delay of 1 h after onset of hypoxia. These findings suggest that even strong activation of early gene expression has no influence on EPO gene expression, whilst activation of EPO gene expression during hypoxia can happen without change of early gene expression. It appears, therefore, as if immediate early genes are not causally involved in the sequence of events by which hypoxia stimulates EPO gene expression.

Induction of VEGF and VEGF receptor gene expression by hypoxia: divergent regulation in vivo and in vitro.

This study examined the expression of EPO, VEGF and VEGF receptor gene under conditions of reduced oxygen supply in primary cultures of rat hepatocytes, and compared it with the expression of these genes in hypoxic rat livers in vivo. To this end we exposed male Sprague-Dawley rats to hypoxia (10% and 8% O2), carbon monoxide (0.1% CO) or injected cobalt chloride (60 mg/kg CoCl2) subcutaneously. For the in vitro experiments we used primary cultures of rat hepatocytes which were kept at high (20% O2) and low (1% O2) oxygen tensions for three hours. The EPO mRNA was up-regulated by hypoxia in vitro and in vivo about 10-fold. The VEGF mRNA was up-regulated fivefold in the hepatocytes only, whereas the in vivo mRNA levels remained unchanged. The mRNA levels of flt-1 were up-regulated threefold by 8% O2 in livers, dependent on the strength of hypoxia (10% caused no changes in flt-1 gene expression) and on the kind of hypoxic stimulus (8% O2 was as effective as 0.1% CO and more effective than cobalt). The mRNA levels of flk-1/KDR and flt-4 remained unchanged in the liver. In vitro there were no changes in the mRNA levels of flt-1, flt-4 and flk-1/KDR. Consequently, the in vivo regulation of VEGF, which might be modulated by induction of flt-1 receptor gene expression, differs from the in vitro cell culture situation and might be different from the EPO regulation in vivo.

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 microM) or the iron chelator desferrioxamine (10-200 microM) did not increase EPO mRNA levels. Addition of hydrogen peroxide (100-500 microM) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50-200 micrograms/ml) to the culture medium resulted in a lower level of hypoxia-induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 microM) 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 microM, 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 microM and 100 microM, 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.

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.

Cobalt exerts opposite effects on erythropoietin gene expression in rat hepatocytes in vivo and in vitro.

This study investigates the effects of hypoxia and of cobalt on erythropoietin (EPO) gene expression in hepatocytes in vivo and in vitro in neonatal, juvenile, and adult rats. With the use of the ribonuclease protection assay to quantify RNA, both hypoxia (0.1% CO or 9% O2) and cobalt (60 mg/kg) elicit production of increased amounts of EPO mRNA in neonatal and juvenile rat liver in vivo. In vitro hepatocyte EPO gene expression could be reproducibly stimulated by hypoxia (3% O2) but not by cobaltous chloride (50-150 microM) within 2-20 h. Conversely, cobalt substantially attenuated the rise of EPO mRNA levels in response to hypoxia. This inhibitory effect of cobalt was mimicked by zinc but not by other metals. CO attenuated the rise of EPO mRNA levels in vitro in response to hypoxia; this inhibitory effect coincided with an inhibition of total RNA synthesis as determined by [3H]uridine incorporation. The lack of specific inhibitory effects of CO and of specific stimulatory effects of cobalt on hepatocyte EPO gene expression in vitro suggests that a specific heme oxygen sensor may be less important than in hepatoma cells.

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-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 approximately 2 x 10(-8) M) and three structurally related derivatives with increased selectivity for PKC (RO 317549, ED50 approximately 1 x 10(-6) M; RO 318220, ED50 approximately 1 x 10(-6) M and CGP 41251, ED50 approximately 4 x 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.