Government royalties on sales of biomedical products developed with substantial public funding.

This paper proposes a policy of royalties paid to the government on the sales of biomedical products developed with public funds. The proposed policy would increase the incentives to create and to transfer to the private sector useful biomedical inventions from the research done in federal laboratories and in universities. The royalties policy would also address the concern that taxpayers pay prices perceived to be unreasonable for biomedical products developed with substantial taxpayer funding.

Pragmatic statistical issues in biological research: Introduction to special series.

This is the introduction to a series of articles will be published over the next year in JMCC on statistical issues which commonly arise in types of studies published in the journal. Each article will cover a specific statistical topic and be prefaced with a typical related question that is likely to arise in laboratory and biomedical studies. There will be a discussion of the underlying statistical concepts followed by several websites which may be used to perform the relevant analysis on data.

PR65A phosphorylation regulates PP2A complex signaling.

Serine-threonine Protein phosphatase 2 A (PP2A), a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac); a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa); and one of at least 18 associated variable regulatory proteins (B subunits) classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314). Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A) and non-phosphorylated (N-PR65A) amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A), elongation factor 2, heat shock protein 60 (HSP60), NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure.

Molecular evolution of A-kinase anchoring protein (AKAP)-7: implications in comparative PKA compartmentalization.

BACKGROUND: A-Kinase Anchoring Proteins (AKAPs) are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA), directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18) are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport. RESULTS: Shown in evolutionary analyses, the formation of the AKAP7-RI/RII binding domain (required for AKAP/PKA-R interaction) corresponds to vertebrate-specific gene duplication events in the PKA-RI/RII subunits. Species analyses of AKAP7 splice variants shows the ancestral AKAP7 splice variant is AKAP7α, while the ancestral long form AKAP7 splice variant is AKAP7γ. Multi-species AKAP7 gene alignments, show the recent formation of AKAP7δ occurs with the loss of native AKAP7γ in rats and basal primates. AKAP7 gene alignments and two dimensional Western analyses indicate that AKAP7γ is produced from an internal translation-start site that is present in the AKAP7δ cDNA of mice and humans but absent in rats. Immunofluorescence analysis of AKAP7 protein localization in both rat and mouse heart suggests AKAP7γ replaces AKAP7δ at the cardiac sarcoplasmic reticulum in species other than rat. DNA sequencing identified Human AKAP7δ insertion-deletions (indels) that promote the production of AKAP7γ instead of AKAP7δ. CONCLUSIONS: This AKAP7 molecular evolution study shows that these vital scaffolding proteins developed in ancestral vertebrates and that independent mutations in the AKAP7 genes of rodents and early primates has resulted in the recent formation of AKAP7δ, a splice variant of likely lesser importance in humans than currently described.

Phosphoprotein abundance changes in hypertensive cardiac remodeling.

There is over-whelming evidence that protein phosphorylations regulate cardiac function and remodeling. A wide variety of protein kinases, e.g., phosphoinositide 3-kinase (PI3K), Akt, GSK-3, TGFß, and PKA, MAPKs, PKC, Erks, and Jaks, as well as phosphatases, e.g., phosphatase I (PP1) and calcineurin, control cardiomyocyte growth and contractility. In the present work, we used global phosphoprotein profiling to identify phosphorylated proteins associated with pressure overload (PO) cardiac hypertrophy and heart failure. Phosphoproteins from hypertrophic and systolic failing hearts from male hypertensive Dahl salt-sensitive rats, trans-aortic banded (TAC), and spontaneously hypertensive heart failure (SHHF) rats were analyzed. Profiling was performed by 2-dimensional difference in gel electrophoresis (2D-DIGE) on phospho-enriched proteins. A total of 25 common phosphoproteins with differences in abundance in (1) the 3 hypertrophic and/or (2) the 2 systolic failure heart models were identified (CI>99%) by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Mascot analysis. Among these were (1) myofilament proteins, including alpha-tropomyosin and myosin regulatory light chain 2, cap Z interacting protein (cap ZIP), and tubulin ß5; (2) mitochondrial proteins, including pyruvate dehydrogenase α, branch chain ketoacid dehydrogenase E1, and mitochondrial creatine kinase; (3) phosphatases, including protein phosphatase 2A and protein phosphatase 1 regulatory subunit; and (4) other proteins including proteosome subunits α type 3 and ß type 7, and eukaryotic translation initiation factor 1A (eIF1A). The results include previously described and novel phosphoproteins in cardiac hypertrophy and systolic failure.

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells.

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.

Non-histone lysine acetylated proteins in heart failure.

Both histone-acetylations and histone deacetylases have been shown to play a key role in cardiac remodeling. Recently, it has become abundantly clear that many non-histone proteins are modified by post-translational lysine acetylations and that these acetylations regulate protein activity, conformation, and binding. In the present study, non-histone acetylated proteins associated with heart failure were identified. Global screening for lysine acetylated proteins was performed using 2-dimensional gel electrophoresis coupled with immunoblotting with a primary monoclonal anti-acetyl-lysine antibody. Lysine acetylated proteins were compared in two rodent models of hypertensive heart failure, the Dahl salt-sensitive (SS) and spontaneously hypertensive heart failure prone (SHHF) rats with those in corresponding controls, i.e., the Dahl salt-resistant (SR) and W (W) rat strains, respectively. Forty-one and 66 acetylated proteins were detected in SS and SHHF failing hearts, respectively, but either not detected or detected with less abundance in corresponding control hearts. Twelve of these acetylated proteins were common to both models of heart failure. These were identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) mass spectrometry followed by Mascot Analysis and included mitochondrial enzymes: ATP synthase, long-chain acyl-CoA dehydrogenase, creatine kinase, malate dehydrogenase, and pyruvate dehydrogenase. The abundance of NAD-dependent deacetylase sirtuin-3 (Sirt3), a mitochondrial deacetylase was reduced in SS and SHHF failing hearts. This is the first description of non-histone protein acetylations associated with heart failure and raises the prospect that acetylations of mitochondrial proteins linked to reduced Sirt3 mediate, in part, metabolic changes in heart failure.

Implication of microRNAs in atrial natriuretic peptide and nitric oxide signaling in vascular smooth muscle cells.

MicroRNAs (miRs) are endogenous small RNA molecules that suppress gene expression by binding to complementary sequences in the 3' untranslated regions of their target genes. miRs have been implicated in many diseases, including heart failure, ischemic heart disease, hypertension, cardiac hypertrophy, and cancers. Nitric oxide (NO) and atrial natriuretic peptide (ANP) are potent vasorelaxants whose actions are mediated through receptor guanylyl cyclases and cGMP-dependent protein kinase. The present study examines miRs in signaling by ANP and NO in vascular smooth muscle cells. miR microarray analysis was performed on human vascular smooth muscle cells (HVSMC) treated with ANP (10 nM, 4 h) and S-nitroso-N-acetylpenicillamine (SNAP) (100 µM, 4 h), a NO donor. Twenty-two shared miRs were upregulated, and 21 shared miRs were downregulated, by both ANP and SNAP (P < 0.05). Expression levels of four miRs (miRs-21, -26b, -98, and -1826), which had the greatest change in expression, as determined by microarray analysis, were confirmed by quantitative RT-PCR. Rp-8-Br-PET-cGMPS, a cGMP-dependent protein kinase-specific inhibitor, blocked the regulation of these miRs by ANP and SNAP. 8-bromo-cGMP mimicked the effect of ANP and SNAP on their expression. miR-21 was shown to inhibit HVSMC contraction in collagen gel lattice contraction assays. We also identified by computational algorithms and confirmed by Western blot analysis new intracellular targets of miR-21, i.e., cofilin-2 and myosin phosphatase and Rho interacting protein. Transfection with pre-miR-21 contracted cells and ANP and SNAP blocked miR-21-induced HVSMC contraction. Transfection with anti-miR-21 inhibitor reduced contractility of HVSMC (P < 0.05). The present results implicate miRs in NO and ANP signaling in general and miR-21 in particular in cGMP signaling and vascular smooth muscle cell relaxation.

An evolutionary analysis of cAMP-specific Phosphodiesterase 4 alternative splicing.

BACKGROUND: Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP). The cAMP-specific PDE family 4 (PDE4) is widely expressed in vertebrates. Each of the four PDE4 gene isoforms (PDE4 A-D) undergo extensive alternative splicing via alternative transcription initiation sites, producing unique amino termini and yielding multiple splice variant forms from each gene isoform termed long, short, super-short and truncated super-short. Many species across the vertebrate lineage contain multiple splice variants of each gene type, which are characterized by length and amino termini. RESULTS: A phylogenetic approach was used to visualize splice variant form genesis and identify conserved splice variants (genome conservation with EST support) across the vertebrate taxa. Bayesian and maximum likelihood phylogenetic inference indicated PDE4 gene duplication occurred at the base of the vertebrate lineage and reveals additional gene duplications specific to the teleost lineage. Phylogenetic inference and PDE4 splice variant presence, or absence as determined by EST screens, were further supported by the genomic analysis of select vertebrate taxa. Two conserved PDE4 long form splice variants were found in each of the PDE4A, PDE4B, and PDE4C genes, and eight conserved long forms from the PDE4 D gene. Conserved short and super-short splice variants were found from each of the PDE4A, PDE4B, and PDE4 D genes, while truncated super-short variants were found from the PDE4C and PDE4 D genes. PDE4 long form splice variants were found in all taxa sampled (invertebrate through mammals); short, super-short, and truncated super-short are detected primarily in tetrapods and mammals, indicating an increasing complexity in both alternative splicing and cAMP metabolism through vertebrate evolution. CONCLUSIONS: There was a progressive independent incorporation of multiple PDE4 splice variant forms and amino termini, increasing PDE4 proteome complexity from primitive vertebrates to humans. While PDE4 gene isoform duplicates with limited alternative splicing were found in teleosts, an expansion of both PDE4 splice variant forms, and alternatively spliced amino termini predominantly occurs in mammals. Since amino termini have been linked to intracellular targeting of the PDE4 enzymes, the conservation of amino termini in PDE4 splice variants in evolution highlights the importance of compartmentalization of PDE4-mediated cAMP hydrolysis.

Evidence for cross-talk between atrial natriuretic peptide and nitric oxide receptors.

Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC's (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (alpha/beta) beta1 isoform of the beta subunit of NOGC (NOGCbeta1) was specifically focused. NOGCbeta1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCbeta1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCbeta1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 +/- 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 +/- 3.4 vs. 1.2 +/- 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCbeta1 abundance was reduced using specific siRNA to NOGCbeta1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCbeta1 (530.2 +/- 141.4 vs. 26.1 +/- 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCbeta1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 +/- 22 vs. 92 +/- 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCbeta1 and GC-A interact and that NOGCbeta1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.

Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates.

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.

Renal phosphodiesterase 4B is activated in the Dahl salt-sensitive rat.

Reduced beta-adrenoreceptor signaling is associated with increased sympathoadrenal activity in hypertensive patients and animal models of hypertension. However, the mechanism that accounts for this characteristic decline in beta-adrenergic signaling is unclear. In the present study, we investigated renal phosphodiesterase 4B, which metabolizes cAMP. Immunoblot analysis detected only the phosphodiesterase 4B4 isoform present in kidney tissue from spontaneously hypertensive rats, hypertensive Dahl salt-sensitive (SS) rats, and Dahl salt-resistant rats. The phosphorylated (activated) form of the protein was present at 2-fold greater levels in Dahl SS rats than in spontaneously hypertensive rats and Dahl salt-resistant rats, whereas the unphosphorylated form of the protein was reduced by approximately one half in SS animals. In accord with immunoblot data, rolipram-inhibitable cAMP hydrolyzing activity, a measure of PDE4 activity, was approximately 3-fold greater in kidney cytosolic extracts from SS rats than in extracts from spontaneously hypertensive rats and salt-resistant rats. Phosphodiesterase 4B expression was detected by immunohistochemistry in the renal vasculature, proximal tubules, and distal tubules. These results raise the possibility that increased PDE4 activity, specifically phosphodiesterase 4B4 activity, reduces beta-adrenergic signaling in the kidney and contributes to salt-sensitive hypertension in the Dahl SS rat.

Aminopeptidase N in arterial hypertension.

Aminopeptidase N (APN) or CD13 is a conserved type II integral membrane zinc-dependent metalloprotease in the M1 family of ectoenzymes. APN is abundant in the kidneys and central nervous system. Identified substrates include Angiotensin III (Ang III); neuropeptides, including enkephalins and endorphins; and homones, including kallidan and somatostatin. It is developmentally expressed, a myelomonocytic marker for leukemias, and a receptor for coronovirus. There is evolving support for APN in the regulation of arterial blood pressure and the pathogenesis of hypertension. In rodent strains, intracerebraventricular (i.c.v.) infusions of APN reduces, while inhibitors of APN activity have a pressor effect on blood pressure. Dysregulation of central APN has been linked to the pathogenesis of hypertension in the spontaneously hypertensive rat. There is evidence that renal tubule APN inhibits Na flux and plays a mechanistic role in salt-adaptation. A functional polymorphism of the ANP gene has been identified in the Dahl salt-sensitive rat. Signaling by APN impacting on blood pressure is likely mediated by regulation of the metabolism of Ang III to Ang IV. Whether APN regulates arterial blood pressure in humans or is a therapeutic target for hypertension are subjects for future exploration.

Aminopeptidase N reduces basolateral Na+ -K+ -ATPase in proximal tubule cells.

Aminopeptidase N/CD13 (Anpep) is a membrane-bound protein that catalyzes the formation of natriuretic hexapeptide angiotensin IV (ANG IV) from ANG III. We previously reported that Anpep is more highly expressed in the kidneys of Dahl salt-resistant (SR/Jr) than salt-sensitive (SS/Jr) rats, Anpep maps to a quantitative trait locus for hypertension, and that the Dahl SR/Jr rat contains a functional polymorphism of the gene. This suggests that renal Anpep may be linked to salt sensitivity; however, its effect on renal Na handling has not been determined. Here, we examined regulation of basolateral Na(+)-K(+)-ATPase, a preeminent basolateral Na(+) transporter in proximal tubule cells, by Anpep in LLC-PK1 cells. Treatment of the cells with Anpep siRNA increased total cellular Na(+)-K(+)-ATPase activity and basolateral Na(+)-K(+)-ATPase abundance by approximately twofold. Conversely, Anpep overexpression reduced Na(+)-K(+)-ATPase activity and basolateral abundance by approximately 50%. Similar effects were observed after treatment with ANG IV (10 nM, x30 min and 12 h). ANG IV receptor (AGTRIV) knockdown via specific siRNA relieved the decreases in basolateral Na(+)-K(+)-ATPase levels and activity induced by Anpep overexpression. In sum, these results demonstrate that Anpep reduces basolateral Na(+)-K(+)-ATPase levels via ANG IV/AGTRIV signaling. This novel pathway may be important in renal adaptation to high salt.

Functional polymorphism of the Anpep gene increases promoter activity in the Dahl salt-resistant rat.

We have reported that aminopeptidase N/CD13, which metabolizes angiotensin III to angiotensin IV, exhibits greater renal tubular expression in the Dahl salt-resistant (SR/Jr) rat than its salt-sensitive (SS/Jr) counterpart. In this work, aminopeptidase N (Anpep) genes from SS/Jr and SR/Jr strains were compared. The coding regions contained only silent single nucleotide polymorphisms between strains. The 5' flanking regions also contained multiple single nucleotide polymorphisms, which were analyzed by electrophoretic mobility-shift assay using renal epithelial cell (HK-2) nuclear extracts and oligonucleotides corresponding with single nucleotide polymorphism-containing regions. A unique single nucleotide polymorphism 4 nucleotides upstream of a putative CCAAT/enhancer binding protein motif (nucleotides -2256 to -2267) in the 5' flanking region of the SR/Jr Anpep gene was associated with DNA-protein complex formation, whereas the corresponding sequences in SS rats were not. A chimeric reporter gene containing approximately 4.4 Kb of Anpep 5' flank from the Dahl SR/Jr rat exhibited 2.5- to 3-fold greater expression in HK-2 cells than the corresponding construct derived from the SS strain (P<0.05). Replacing the CCAAT/enhancer binding protein cis-acting element from the SS rat with that from the SR strain increased reporter gene expression by 2.5-fold (P<0.05) and abolished this difference. CCAAT/enhancer binding protein association was confirmed by chromatin immunoprecipitation and correlated with expression, suggesting selection for a functional CCAAT/enhancer binding protein polymorphism in the 5' flank of Anpep in the Dahl SR/Jr rat. These results highlight a possible association of the Anpep gene with hypertension in Dahl rat and raise the prospect that increased Anpep may play a mechanistic role in adaptation to high salt.

Dietary NaCl regulates renal aminopeptidase N: relevance to hypertension in the Dahl rat.

Aminopeptidase N (APN) is an abundant metallohydrolase in the brush border of kidney proximal tubule cells that degrades angiotensin III (Ang III) to angiotensin IV (Ang IV) and, along with dipeptidylaminopeptidase, degrades Ang IV. We examined the impact of a high-salt diet on renal APN activity and transcript abundance in the Sprague-Dawley and Dahl salt-sensitive (SS/Jr) rat strains. APN transcript abundance and protein abundance were approximately 2-fold greater (P<0.05; n=6) in the kidneys of Sprague-Dawley and Lewis rats ingesting 8% versus 0.3% salt diets, suggesting that increased aminopeptidase activity may contribute to decreased renal sodium uptake during adaptation to a high-salt diet. In contrast, renal APN transcript abundance and activity were the same in Dahl SS/Jr rats ingesting 8.0% versus 0.3% salt diets. The APN gene was mapped, using a radiation-hybrid panel, to known quantitative loci on chromosome 1 for blood pressure in the Dahl SS/Jr rat. The results suggest that the APN gene is a good candidate for salt-sensitivity in the Dahl SS/Jr rat.

Dietary salt regulates renal SGK1 abundance: relevance to salt sensitivity in the Dahl rat.

Serum and glucocorticoid-induced kinase 1 (SGK1) activates the epithelial sodium channel (eNaC) in tubules. We examined renal SGK1 abundance in salt-adaptation and in salt-sensitive hypertension. Sprague-Dawley and Dahl salt-sensitive rats were placed on either 8% or 0.3% NaCl diets for 10 days. Plasma aldosterone levels were approximately 2.5-fold greater on 0.3% versus 8% NaCl diets in both rat strains. Both serum and glucocorticoid-induced kinase 1 transcript and protein abundance were less (P<0.01) in Sprague-Dawley rats and greater (P<0.01) in Dahl salt-sensitive rats on 8% versus 0.3% NaCl diets. The cDNA sequences of serum and glucocorticoid-induced kinase 1 in both strains of rat were the same. The present results provide evidence that the abundance of serum and glucocorticoid-induced kinase 1 in rat kidney may play a role in salt adaptation and the pathogenesis of hypertension and suggests that aldosterone is not the primary inducer of SGK1 in the Sprague-Dawley rat.

Aquaporin-2 transcript is differentially regulated by dietary salt in Sprague-Dawley and Dahl SS/Jr rats.

Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel in the kidney collecting duct. AQP-2 transcript has been identified by transcriptional profiling of rat kidneys as being regulated by dietary salt. We compared renal AQP-2 transcript expression in Sprague-Dawley and Dahl salt-sensitive (SS/Jr) rats using real-time RT-PCR. Expression of AQP-2 transcript is 5-fold less (P<0.01) in the Sprague-Dawley and 3-fold greater in Dahl SS/Jr rats (P<0.01) on high versus basal NaCl diets. The AQP-2 coded sequence was identical in Sprague-Dawley and Dahl SS/Jr rats. The present results provide evidence that: (1)AQP-2 plays a role in salt adaptation and (2) regulation of aquaporin transcript expression by salt is altered in the Dahl SS/Jr rat.