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1.
Am J Physiol Renal Physiol ; 293(6): F1858-64, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17913837

ABSTRACT

Aquaporin (AQP) 1 null mice have a defect in the renal concentrating gradient because of their inability to generate a hyperosmotic medullary interstitium. To determine the effect of vasopressin on renal medullary gene expression, in the absence of high local osmolarity, we infused 1-deamino-8-d-arginine vasopressin (dDAVP), a V(2) receptor (V(2)R)-specific agonist, in AQP1 null mice for 7 days. cDNA microarray analysis was performed on the renal medullary tissue, and 5,140 genes of the possible 12,000 genes on the array were included in the analysis. In the renal medulla of AQP1 null mice, 245 transcripts were identified as increased by dDAVP infusion and 200 transcripts as decreased (1.5-fold or more). Quantitative real-time PCR measurements confirmed the increases seen for cyclin D1, early growth response gene 1, and activating transcription factor 3, genes associated with changes in cell cycle/growth. Changes in mRNA expression were correlated with changes in protein expression by semiquantitative immunoblotting; cyclin D1 and ATF3 were increased significantly in abundance following dDAVP infusion in the renal medulla of AQP1 null mice (161 and 461%, respectively). A significant increase in proliferation of medullary collecting ducts cells, following V(2)R activation, was identified by proliferating cell nuclear antigen immunohistochemistry; colocalization studies with AQP2 indicated that the increase in proliferation was primarily observed in principal cells of the inner medullary collecting duct (IMCD). V(2)R activation, via dDAVP, increased AQP2 and AQP3 protein abundance in the cortical collecting ducts of AQP1 null mice. However, V(2)R activation did not increase AQP2 protein abundance in the IMCD of AQP1 null mice.


Subject(s)
Aquaporin 1/genetics , Kidney Medulla/cytology , Receptors, Vasopressin/physiology , Animals , Antidiuretic Hormone Receptor Antagonists , Blotting, Western , Cell Proliferation/drug effects , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Deamino Arginine Vasopressin/pharmacology , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Immunohistochemistry , In Situ Hybridization , Kidney Medulla/drug effects , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/metabolism , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Osmolar Concentration , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/physiology , RNA/biosynthesis , RNA/genetics , Renal Agents/pharmacology , Reverse Transcriptase Polymerase Chain Reaction
2.
Am J Physiol Renal Physiol ; 293(1): F193-9, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17389681

ABSTRACT

Changes in the estrogen/testosterone balance at menopause may negatively influence the development of diabetic kidney disease. Furthermore, recent studies suggest that changes in hormone levels during perimenopause may influence disease development. Injection of 4-vinylcyclohexene diepoxide (VCD) in B(6)C(3)F(1) mice induces gradual ovarian failure, preserving both the perimenopausal (peri-ovarian failure) and menopausal (post-ovarian failure) periods. To address the impact of the transition into menopause on the development of diabetes and diabetic kidney damage, we used streptozotocin (STZ)-induced diabetes in the VCD model of menopause. After 6 wk of STZ-induced diabetes, blood glucose was significantly increased in post-ovarian failure (post-OF) diabetic mice compared with cycling diabetic mice. In peri-ovarian failure (peri-OF) diabetic mice, blood glucose levels trended higher but were not significantly different from cycling diabetic mice, suggesting a continuum of worsening blood glucose across the menopausal transition. Cell proliferation, an early marker of damage in the kidney, was increased in post-OF diabetic mice compared with cycling diabetic mice, as measured by PCNA immunohistochemistry. In post-OF diabetic mice, mRNA abundance of early growth response-1 (Egr-1), collagen-4alpha1, and matrix metalloproteinase-9 were increased and 3beta-hydroxysteroid dehydrogenase 4 (3beta-HSD4) and transforming growth factor-beta(2) (TGF-beta(2)) were decreased compared with cycling diabetic mice. In peri-OF diabetic mice, mRNA abundance of Egr-1 and 3beta-HSD4 were increased, and TGF-beta(2) was decreased compared with cycling diabetic mice. This study highlights the importance and utility of the VCD model of menopause, as it provides a physiologically relevant system for determining the impact of the menopausal transition on diabetes and diabetic kidney damage.


Subject(s)
Cyclohexenes/pharmacology , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/pathology , Hormones/blood , Menopause/physiology , Vinyl Compounds/pharmacology , Animals , Blood Glucose/metabolism , Cell Proliferation/drug effects , Disease Progression , Female , Humans , Immunohistochemistry , Kidney Cortex/metabolism , Menopause/drug effects , Mice , Mice, Inbred Strains , Primary Ovarian Insufficiency/chemically induced , Primary Ovarian Insufficiency/pathology , Proliferating Cell Nuclear Antigen/metabolism , RNA/biosynthesis , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Weight Gain/drug effects
3.
Am J Physiol Lung Cell Mol Physiol ; 292(4): L953-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17158601

ABSTRACT

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.


Subject(s)
Calcium/metabolism , Fetal Diseases/metabolism , Hypertension, Pulmonary/metabolism , Muscle, Smooth, Vascular/metabolism , Pulmonary Artery/metabolism , Animals , Blotting, Western , Calcium Channels/metabolism , Calcium Signaling/physiology , Disease Models, Animal , Female , Humans , Hypertension, Pulmonary/embryology , Infant, Newborn , Muscle, Smooth, Vascular/cytology , Persistent Fetal Circulation Syndrome/metabolism , Pregnancy , Pulmonary Artery/cytology , Sheep , Transient Receptor Potential Channels/physiology
4.
Am J Physiol Lung Cell Mol Physiol ; 290(3): L426-33, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16467248

ABSTRACT

Pulmonary artery smooth muscle cell (PASMC) relaxation at birth results from an increase in cytosolic cGMP, cGMP-dependent and kinase-mediated activation of the Ca2+-sensitive K+ channel (KCa), and closure of voltage-operated Ca2+ channels (VOCC). How chronic intrauterine pulmonary hypertension compromises perinatal pulmonary vasodilation remains unknown. We tested the hypothesis that chronic intrauterine pulmonary hypertension selectively modifies gene expression to mitigate perinatal pulmonary vasodilation mediated by the cGMP kinase-KCa-VOCC pathway. PASMC were isolated from late-gestation fetal lambs that had undergone either ligation of the ductus arteriosus (hypertensive) or sham operation (control) at 127 days of gestation and were maintained under either hypoxic (approximately 25 Torr) or normoxic (approximately 120 Torr) conditions in primary culture. We studied mRNA levels for cGMP kinase Ialpha (PKG-1alpha), the alpha-chain of VOCC (Cav1.2), and the alpha-subunit of the KCa channel. Compared with control PASMC, hypertensive PASMC had decreased VOCC, KCa, and PKG-1alpha expression. In response to sustained normoxia, expression of VOCC and KCa channel decreased and expression of PKG-1alpha increased. In contrast, sustained normoxia had no effect on PKG-1alpha levels and an attenuated effect on VOCC and KCa channel expression in hypertensive PASMC. Protein expression of PKG-1alpha was consistent with the mRNA data. We conclude that chronic intrauterine pulmonary hypertension decreases PKG expression and mitigates the genetic effects of sustained normoxia on pulmonary vasodilation, because gene expression remains compromised even after sustained exposure to normoxia.


Subject(s)
Fetal Diseases/physiopathology , Gene Expression , Hypertension, Pulmonary/physiopathology , Muscle, Smooth, Vascular/physiology , Pulmonary Artery/physiology , Animals , Calcium/metabolism , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Calcium-Transporting ATPases , Cells, Cultured , Chronic Disease , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Female , Fetal Diseases/metabolism , Fetus , Hypertension, Pulmonary/metabolism , Hypoxia/metabolism , Hypoxia/physiopathology , Muscle, Smooth, Vascular/cytology , Oxygen/pharmacology , Potassium Channels/genetics , Potassium Channels/metabolism , Pregnancy , Pregnancy, Animal , Pulmonary Artery/cytology , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sarcoplasmic Reticulum Calcium-Transporting ATPases , Sheep , Vasodilation/physiology
5.
Am J Physiol Renal Physiol ; 291(1): F218-24, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16478974

ABSTRACT

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.


Subject(s)
3-Hydroxysteroid Dehydrogenases/analysis , 3-Hydroxysteroid Dehydrogenases/genetics , Gene Expression Regulation, Enzymologic/physiology , Kidney Medulla/chemistry , Kidney Tubules, Collecting/chemistry , Water Deprivation/physiology , Animals , Aquaporin 2/analysis , Aquaporin 2/genetics , Aquaporin 2/physiology , Aquaporin 3/analysis , Aquaporin 3/genetics , Aquaporin 3/physiology , DNA, Complementary/analysis , Epithelial Sodium Channels , Fructose-Bisphosphate Aldolase/analysis , Fructose-Bisphosphate Aldolase/genetics , Fructose-Bisphosphate Aldolase/physiology , Kidney Medulla/physiology , Kidney Tubules, Collecting/physiology , Mice , Mice, Inbred ICR , Oligonucleotide Array Sequence Analysis , PAX8 Transcription Factor , Paired Box Transcription Factors/analysis , Paired Box Transcription Factors/genetics , Paired Box Transcription Factors/physiology , RNA, Messenger/analysis , Receptors, Vasopressin/analysis , Receptors, Vasopressin/genetics , Receptors, Vasopressin/physiology , Reverse Transcriptase Polymerase Chain Reaction , Sodium Channels/analysis , Sodium Channels/genetics , Sodium Channels/physiology , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/physiology , Vasopressins/blood , Vasopressins/physiology
6.
Am J Physiol Lung Cell Mol Physiol ; 288(5): L917-23, 2005 May.
Article in English | MEDLINE | ID: mdl-15695541

ABSTRACT

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca(2+)](i)) through inactivation of a K(+) channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca(2+)](i) by 11.7 +/- 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca(2+)](i) was mimicked by pharmacological blockade of the voltage-sensitive K(+) channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca(2+)](i). Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca(2+)](i). Endothelin increased [Ca(2+)](i) in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca(2+)](i) via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.


Subject(s)
Calcium/metabolism , Ductus Arteriosus/metabolism , Inositol Phosphates/metabolism , Muscle, Smooth/metabolism , Oxygen/metabolism , Animals , Cells, Cultured , Cytosol/metabolism , Ductus Arteriosus/cytology , Extracellular Space/metabolism , Female , Fetal Hypoxia/metabolism , Hypoxia/metabolism , Oxygen/pharmacology , Partial Pressure , Potassium Channel Blockers/pharmacology , Potassium Channels/metabolism , Pregnancy , Sheep
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