Proceedings of the National Academy of Sciences, 2001
Adenosine is a determinant of metabolic control of organ function increasing oxygen supply throug... more Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR(+/+), A1AR(+/-), and A1AR(-/-) mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR(-/-) mice (stop flow pressure response, -6.8 +/- 0.55 mmHg and -0.4 +/- 0.2 in A1AR(+/+) and A1AR(-/-) mice; early proximal flow rate response, -3.4 +/- 0.4 nl/min and +0.02 +/- 0.3 nl/min in A1AR(+/+) and A1AR(-/-) mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.
Aging is associated with progressive structural and functional deterioration of the kidney. Among... more Aging is associated with progressive structural and functional deterioration of the kidney. Among the morphological changes associated with renal aging is an accumulation of extracellular matrix (ECM) in the glomeruli and tubuloinsterstitium, which may ultimately lead to the development of renal fibrosis. The mechanisms governing the regulation of ECM metabolism during renal aging are only incompletely defined. We present data from a genome-wide mRNA expression study on renal tissue from 90 wk old male Wistar rats and 10 wk old controls using Illumina BeadArray cDNA microarray. Regulation of candidate gene products was verified by real-time PCR. Morphological changes were evaluated by routine histological methods. Activated fibroblasts were identified by their expression of alpha-smooth muscle actin and collagen I. Morphological analysis demonstrated an expansion of the tubulointerstitial compartment with increased amounts of fibrous collagen but no overt glomerular or tubular damage in the aged rats. Activated fibroblasts were readily detectable in the adventitial layer of large renal vessels in controls and were not found in the old animals. In agreement with this finding, gene expression analysis revealed significant downregulation of collagen I mRNA along with numerous other ECM components. Concomitantly, collagen-stabilizing proteins were induced, whereas matrix metalloproteinase 9, an enzyme involved in collagen breakdown, was reduced. In conclusion, our results suggest that ECM expansion during renal aging results from an augmented stabilization in conjunction with a reduced breakdown of collagen fibers. Collagen stabilizing proteins may be essential for the control of renal ECM turnover and the pathogenesis of kidney fibrosis.
Journal of the American Society of Nephrology, 2013
Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia... more Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.
It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide syntha... more It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are co-expressed in macula densa cells, and that the expression of both enzymes is stimulated in a number of high renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS -/-mice on a mixed genetic background, and in COX-2 -/-mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies we accumulated evidence to show an inhibitory influence of PGE 2 on nNOS expression. In a cultured macula densa cell line, PGE 2 significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 -/-mice, nNOS mRNA expression in the kidney, determined by real time RT-PCR, was upregulated throughout the postnatal periods ranging from postnatal day (PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 -/-mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE 2 on nNOS mRNA expression indicates a novel interaction between NO and prostaglandinsmediated pathways of renin regulation.
In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V(2)... more In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V(2) receptors (V(2)Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of V(2)Rs have been inconclusive with respect to segment- and cell-type-related V(2)R expression. Our study therefore aimed to present a high-resolution analysis of V(2)R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced V(2)R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the V(2)R agonist desmopressin. We found solid expression of V(2)R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional V(2)R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong V(2)R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced V(2)R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong V(2)R expression in macula densa may be related to tubulovascular signal transfer.
ABSTRACT Vasopressin influences salt and water transport in renal epithelia. This is coordinated ... more ABSTRACT Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na+-Cl- cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11beta-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V(2) receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT.
Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (T... more Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)β, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)β to NKCC2 regulation. Immunoreactive iPLA(2)β protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)β abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)β in Sprague-Dawley rats with FKGK 11 (0.5 μM) or in mTAL cells with FKGK 11 (10 μM) or (S)-bromoenol lactone (5 μM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)β acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)β may be a relevant step in AVP-mediated urine concentration.
We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor... more We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor in cultured cells via stabilization of hypoxia-inducible factor-1alpha. Here we investigated whether the expression of TrkB and other neurotrophin receptors is oxygen-sensitive also in vivo, and explored the functional consequences of an oxygen-regulated TrkB expression. Rats were exposed either to 21% O(2) or 8% O(2) for 6 h and TrkB was analysed by reverse transcription real-time PCR, in situ mRNA hybridization, and immunological techniques. The importance of the brain-derived neurotrophic factor (BDNF)-TrkB pathway in the control of mechanical airway function was assessed on isolated tracheal segments from normoxic and hypoxic rats. TrkB transcripts were increased approx. 15-fold in the lungs of hypoxic rats, and the respiratory epithelium was identified as the site of enhanced TrkB expression in hypoxia. The TrkB ligand, BDNF, significantly increased the contractile response to acetylcholine (ACh) of isolated tracheal segments from hypoxic but not from normoxic rats. This effect of BDNF was prevented by pre-incubation of the tissue specimens with the tyrosine kinase inhibitor K252a and by mechanical removal of the TrkB containing airway epithelium. Likewise, the nitric oxide (NO) synthase inhibitor l-NAME abrogated the influence of BDNF on ACh-induced contractions of isolated tracheal segments from hypoxic rats. These results demonstrate that systemic hypoxia stimulates expression of the TrkB neurotrophin receptor in the airway epithelium. Furthermore, activation of TrkB signalling by BDNF in hypoxia enhances mechanical airway contractility to ACh through a mechanism that requires NO.
In the present study, we aimed to elucidate the effects of chronic vasopressin administration on ... more In the present study, we aimed to elucidate the effects of chronic vasopressin administration on renal medullary oxygen levels. Adult Sprague Dawley or vasopressin-deficient Brattleboro rats were treated with the vasopressin V2 receptor agonist, desmopressin (5 ng/h; 3d), or its vehicle via osmotic minipumps. Immunostaining for pimonidazole and the transcription factor HIF-1α (hypoxia-inducible factor-1α) were used to identify hypoxic areas. Activation of HIF-target gene expression following desmopressin treatment was studied by microarray analysis. Pimonidazole staining was detected in the outer and inner medulla of desmopressin-treated rats, whereas staining in control animals was weak or absent. HIF-1α immunostaining demonstrated nuclear accumulation in the papilla of desmopressin-treated animals, whereas no staining was observed in the controls. Gene expression analysis revealed significant enrichment of HIF-target genes in the group of desmopressin-regulated gene products (P = 2.6*10(-21) ). Regulated products included insulin-like growth factor binding proteins 1 and 3, angiopoietin 2, fibronectin, cathepsin D, hexokinase 2 and cyclooxygenase 2. Our results demonstrate that an activation of the renal urine concentrating mechanism by desmopressin causes renal medullary hypoxia and an upregulation of hypoxia-inducible gene expression.
In the adult rodent kidney cortex, cyclooxygenase-2 (COX-2), NO synthase (NOS1), and renin synthe... more In the adult rodent kidney cortex, cyclooxygenase-2 (COX-2), NO synthase (NOS1), and renin synthesis change in parallel on alterations in distal tubular NaCl concentration, and their products in part may mutually determine synthesis and activity of these enzymes. Epithelial NO synthesis has been postulated to exert a stimulatory role on COX-2 expression. Changes in COX-2 and NOS1 may be assessed
Proceedings of the National Academy of Sciences, 2001
Adenosine is a determinant of metabolic control of organ function increasing oxygen supply throug... more Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR(+/+), A1AR(+/-), and A1AR(-/-) mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR(-/-) mice (stop flow pressure response, -6.8 +/- 0.55 mmHg and -0.4 +/- 0.2 in A1AR(+/+) and A1AR(-/-) mice; early proximal flow rate response, -3.4 +/- 0.4 nl/min and +0.02 +/- 0.3 nl/min in A1AR(+/+) and A1AR(-/-) mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.
Aging is associated with progressive structural and functional deterioration of the kidney. Among... more Aging is associated with progressive structural and functional deterioration of the kidney. Among the morphological changes associated with renal aging is an accumulation of extracellular matrix (ECM) in the glomeruli and tubuloinsterstitium, which may ultimately lead to the development of renal fibrosis. The mechanisms governing the regulation of ECM metabolism during renal aging are only incompletely defined. We present data from a genome-wide mRNA expression study on renal tissue from 90 wk old male Wistar rats and 10 wk old controls using Illumina BeadArray cDNA microarray. Regulation of candidate gene products was verified by real-time PCR. Morphological changes were evaluated by routine histological methods. Activated fibroblasts were identified by their expression of alpha-smooth muscle actin and collagen I. Morphological analysis demonstrated an expansion of the tubulointerstitial compartment with increased amounts of fibrous collagen but no overt glomerular or tubular damage in the aged rats. Activated fibroblasts were readily detectable in the adventitial layer of large renal vessels in controls and were not found in the old animals. In agreement with this finding, gene expression analysis revealed significant downregulation of collagen I mRNA along with numerous other ECM components. Concomitantly, collagen-stabilizing proteins were induced, whereas matrix metalloproteinase 9, an enzyme involved in collagen breakdown, was reduced. In conclusion, our results suggest that ECM expansion during renal aging results from an augmented stabilization in conjunction with a reduced breakdown of collagen fibers. Collagen stabilizing proteins may be essential for the control of renal ECM turnover and the pathogenesis of kidney fibrosis.
Journal of the American Society of Nephrology, 2013
Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia... more Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.
It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide syntha... more It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are co-expressed in macula densa cells, and that the expression of both enzymes is stimulated in a number of high renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS -/-mice on a mixed genetic background, and in COX-2 -/-mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies we accumulated evidence to show an inhibitory influence of PGE 2 on nNOS expression. In a cultured macula densa cell line, PGE 2 significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 -/-mice, nNOS mRNA expression in the kidney, determined by real time RT-PCR, was upregulated throughout the postnatal periods ranging from postnatal day (PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 -/-mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE 2 on nNOS mRNA expression indicates a novel interaction between NO and prostaglandinsmediated pathways of renin regulation.
In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V(2)... more In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V(2) receptors (V(2)Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of V(2)Rs have been inconclusive with respect to segment- and cell-type-related V(2)R expression. Our study therefore aimed to present a high-resolution analysis of V(2)R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced V(2)R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the V(2)R agonist desmopressin. We found solid expression of V(2)R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional V(2)R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong V(2)R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced V(2)R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong V(2)R expression in macula densa may be related to tubulovascular signal transfer.
ABSTRACT Vasopressin influences salt and water transport in renal epithelia. This is coordinated ... more ABSTRACT Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na+-Cl- cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11beta-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V(2) receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT.
Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (T... more Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)β, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)β to NKCC2 regulation. Immunoreactive iPLA(2)β protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)β abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)β in Sprague-Dawley rats with FKGK 11 (0.5 μM) or in mTAL cells with FKGK 11 (10 μM) or (S)-bromoenol lactone (5 μM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)β acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)β may be a relevant step in AVP-mediated urine concentration.
We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor... more We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor in cultured cells via stabilization of hypoxia-inducible factor-1alpha. Here we investigated whether the expression of TrkB and other neurotrophin receptors is oxygen-sensitive also in vivo, and explored the functional consequences of an oxygen-regulated TrkB expression. Rats were exposed either to 21% O(2) or 8% O(2) for 6 h and TrkB was analysed by reverse transcription real-time PCR, in situ mRNA hybridization, and immunological techniques. The importance of the brain-derived neurotrophic factor (BDNF)-TrkB pathway in the control of mechanical airway function was assessed on isolated tracheal segments from normoxic and hypoxic rats. TrkB transcripts were increased approx. 15-fold in the lungs of hypoxic rats, and the respiratory epithelium was identified as the site of enhanced TrkB expression in hypoxia. The TrkB ligand, BDNF, significantly increased the contractile response to acetylcholine (ACh) of isolated tracheal segments from hypoxic but not from normoxic rats. This effect of BDNF was prevented by pre-incubation of the tissue specimens with the tyrosine kinase inhibitor K252a and by mechanical removal of the TrkB containing airway epithelium. Likewise, the nitric oxide (NO) synthase inhibitor l-NAME abrogated the influence of BDNF on ACh-induced contractions of isolated tracheal segments from hypoxic rats. These results demonstrate that systemic hypoxia stimulates expression of the TrkB neurotrophin receptor in the airway epithelium. Furthermore, activation of TrkB signalling by BDNF in hypoxia enhances mechanical airway contractility to ACh through a mechanism that requires NO.
In the present study, we aimed to elucidate the effects of chronic vasopressin administration on ... more In the present study, we aimed to elucidate the effects of chronic vasopressin administration on renal medullary oxygen levels. Adult Sprague Dawley or vasopressin-deficient Brattleboro rats were treated with the vasopressin V2 receptor agonist, desmopressin (5 ng/h; 3d), or its vehicle via osmotic minipumps. Immunostaining for pimonidazole and the transcription factor HIF-1α (hypoxia-inducible factor-1α) were used to identify hypoxic areas. Activation of HIF-target gene expression following desmopressin treatment was studied by microarray analysis. Pimonidazole staining was detected in the outer and inner medulla of desmopressin-treated rats, whereas staining in control animals was weak or absent. HIF-1α immunostaining demonstrated nuclear accumulation in the papilla of desmopressin-treated animals, whereas no staining was observed in the controls. Gene expression analysis revealed significant enrichment of HIF-target genes in the group of desmopressin-regulated gene products (P = 2.6*10(-21) ). Regulated products included insulin-like growth factor binding proteins 1 and 3, angiopoietin 2, fibronectin, cathepsin D, hexokinase 2 and cyclooxygenase 2. Our results demonstrate that an activation of the renal urine concentrating mechanism by desmopressin causes renal medullary hypoxia and an upregulation of hypoxia-inducible gene expression.
In the adult rodent kidney cortex, cyclooxygenase-2 (COX-2), NO synthase (NOS1), and renin synthe... more In the adult rodent kidney cortex, cyclooxygenase-2 (COX-2), NO synthase (NOS1), and renin synthesis change in parallel on alterations in distal tubular NaCl concentration, and their products in part may mutually determine synthesis and activity of these enzymes. Epithelial NO synthesis has been postulated to exert a stimulatory role on COX-2 expression. Changes in COX-2 and NOS1 may be assessed
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