Ouabain protects against adverse developmental programming of the kidney

The kidney is extraordinarily sensitive to adverse fetal programming. Malnutrition, the most common form of developmental challenge, retards the formation of functional units, the nephrons. The resulting low nephron endowment increases susceptibility to renal injury and disease. Using explanted rat embryonic kidneys, we found that ouabain, the Na,K-ATPase ligand, triggers a calcium–nuclear factor-κB signal, which protects kidney development from adverse effects of malnutrition. To mimic malnutrition, kidneys were serum deprived for 24 h. This resulted in severe retardation of nephron formation and a robust increase in apoptosis. In ouabain-exposed kidneys, no adverse effects of serum deprivation were observed. Proof of principle that ouabain rescues development of embryonic kidneys exposed to malnutrition was obtained from studies on pregnant rats given a low-protein diet and treated with ouabain or vehicle throughout pregnancy. Thus, we have identified a survival signal and a feasible therapeutic tool to prevent adverse programming of kidney development

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM

Effects of intestinal ischemia-reperfusion on major conduit arteries

Intestinal ischemia-reperfusion (I-R) is a common and serious clinical condition associated with simultaneous remote organ dysfunction. The purpose of this study was to investigate the effects of intestinal I-R on the vasomotor functions of major conduit arteries. Anesthetized rabbits were randomly assigned to one of three groups: sham-operated controls (Group I), and one-hour intestinal ischemia with two-hour reperfusion (Group II) or four-hour reperfusion (Group III). The following mechanisms of vasomotor functions were studied in abdominal aorta, superior mesenteric, renal, pulmonary, and carotid arterial rings: (1) endothelial-dependent vasodilation response to acetylcholine, (2) endothelial-independent vasodilation response to nitroprusside, (3) beta-adrenergic vasodilation response to isoproterenol, and (4) phenylephrine-induced vasoconstriction. Intestinal injury was quantified using malondialdehyde (MDA) concentration and wet-to-dry intestine weight ratio. Intestinal I-R did not affect the maximal responsiveness or the sensitivity to acetylcholine, nitroprusside, and isoproterenol in all the vessels studied. The maximal contractile response to phenylephrine increased significantly in mesenteric artery in Group II, (227.1 +/- 15.1% vs 152.8 +/- 11.7% in controls) (p < 0.05). Intestinal MDA concentration, a marker of oxidant injury, increased from 39.87 +/- 9.41 nmol/g to 67.8 +/- 8.8 nmol/g in group II (p < 0.01), and to 94.8 +/- 7.56 nmol/g in Group III (p < 0.001). Wet-to-dry intestine weight ratio increased from 3.62 +/- 0.12 to 4.28 +/- 0.17 in Group II (p < 0.01), to 4.62 +/- 0.14 in Group III (p < 0.001). These data indicate that although the intestines of the animals subjected to intestinal I-R are seriously injured, the smooth muscle relaxation of major conduit arteries was not affected

Effects of Electrical Stimulation and Insulin on Na+–K+-ATPase ([3H]Ouabain Binding) in Rat Skeletal Muscle

Exercise has been reported to increase the Na+–K+-ATPase (Na+–K+ pump) α2 isoform in the plasma membrane 1.2- to 1.9-fold, purportedly reflecting Na+–K+ pump translocation from an undefined intracellular pool. We examined whether Na+–K+ pump stimulation, elicited by muscle contraction or insulin, increases the plasma membrane Na+–K+ pump content ([3H]ouabain binding) in muscles from young rats. Stimulation of isolated soleus muscle for 10 s at 120 Hz caused a rapid rise in intracellular Na+ content, followed by an 18-fold increase in the Na+ re-extrusion rate (80 % of theoretical maximum). Muscles frozen immediately or 120 s after 10–120 s stimulation showed 10–22 % decrease in [3H]ouabain binding expressed per gram wet weight, but with no significant change expressed per gram dry weight. In soleus muscles from adult rats, [3H]ouabain binding was unaltered after 10 s stimulation at 120 Hz. Extensor digitorum longus (EDL) muscles stimulated for 10–60 s at 120 Hz showed no significant change in [3H]ouabain binding. Insulin (100 mU ml−1) decreased intracellular Na+ content by 27 % and increased 86Rb uptake by 23 % soleus muscles, but [3H]ouabain binding was unchanged. After stimulation for 30 s at 60 Hz soleus muscle showed a 30% decrease in intracellular Na+ content, demonstrating increased Na+–K+ pump activity, but [3H]ouabain binding measured 5 to 120 min after stimulation was unchanged. Stimulation of soleus or EDL muscles for 120–240 min at 1 Hz (continuously) or 10 Hz (intermittently) produced no change in [3H]ouabain binding per gram dry weight. In conclusion, the stimulating effects of electrical stimulation or insulin on active Na+, K+-transport in rat skeletal muscle could not be even partially accounted for by an acute increase in the content of functional Na+–K+ pumps in the plasma membrane

c-Fos expression in ouabain-treated vascular smooth muscle cells from rat aorta: evidence for an intracellular-sodium-mediated, calcium-independent mechanism

In this study, we examined the effect of Na+–K+ pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain-treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c-Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c-Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca2+ response element binding protein (CREB)-1 and c-Myc was not altered. Markedly augmented c-Fos expression was also observed under Na+–K+ pump inhibition in potassium-depleted medium. Na+–K+ pump inhibition triggered c-Fos expression via elevation of the [Na+]i/[K+]i ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c-Fos expression in high-potassium-low-sodium medium and from the comparison of dose responses of Na+–K+ pump activity, [Na+]i and [K+]i content and c-Fos expression to ouabain. A fourfold increment of c-Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na+]i but did not affect [K+]i. Augmented c-Fos expression was also observed under VSMC depolarization in high-potassium medium. Increments in both c-Fos expression and 45Ca uptake in depolarized VSMC were abolished under inhibition of L-type Ca2+ channels with 0.1 μM nicardipine. Ouabain did not affect the free [Ca2+]i or the content of exchangeable [Ca2+]i. Ouabain-induced c-Fos expression was also insensitive to the presence of nicardipine and [Ca2+]o, as well as chelators of [Ca2+]o (EGTA) and [Ca2+]i (BAPTA). The effect of ouabain and serum on c-Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk-1, serum response factor, CREB and activator protein-1 transcription factors identified within the c-Fos promoter. These results suggest that Na+–K+ pump inhibition triggers c-Fos expression via [Na+]i-sensitive [Ca2+]i-independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter

Transplanted Induced Pluripotent Stem Cells Mitigate Oxidative Stress and Improve Cardiac Function through the Akt Cell Survival Pathway in Diabetic Cardiomyopathy

Recent evidence suggests transplanted stem cells improve left ventricular function in diabetic induced cardiomyopathy (DICM). However, little is known about the mechanisms by which induced pluripotent stem (iPS) cells or factors released from these cells inhibit adverse cardiac remodeling in DICM. The present study was designed to determine molecular mediators and pathways regulated by transplanted iPS cells and their conditioned media (CM) in DICM. Animals were divided into four experimental groups such as control, streptozotocin (STZ), STZ+iPS-CM, and STZ+iPS cells. Experimental diabetes was induced in C57BL/6 mice by intraperitoneal STZ injections (100 mg/kg body weight for 2 consecutive days). Following STZ injections, iPS cells or CM was given intravenously for 3 consecutive days. Animals were humanely killed, and hearts were harvested at D14. Animals transplanted with iPS cells or CM demonstrated a significant reduction in apoptosis, mediated by Akt upregulation and ERK1/2 downregulation, and inhibition of interstitial fibrosis via MMP-9 suppression compared with the STZ group. Oxidative stress was significantly hindered in iPS cell and CM groups as evidenced by diminished pro-oxidant expression and enhanced antioxidant (catalase and MnSOD) concentration. Echocardiography data suggest a significant improvement in cardiac function in cells and CM groups in comparison to STZ. In conclusion, our data strongly suggest that iPS cells and CM attenuate oxidative stress and associated apoptosis and fibrosis. Moreover, we also suggest that increased antioxidant levels, decreased adverse cardiac remodeling, and improved cardiac function is mediated by iPS CM and cells in DICM through multiple autocrine and paracrine mechanisms. © 2013 American Chemical Society

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis

Recent studies demonstrated that in addition to Na(+),K(+)-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na(+)](i)/[K(+)](i)-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K(+)](i) and gain of [Na(+)](i). Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na(+)](i)/[K(+)](i) ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na(+)](i)/[K(+)](i) ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na(+)](i)/[K(+)](i) ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na(+)](i)/[K(+)](i) ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na(+)](i)/[K(+)](i) sensors involved in transcription regulation should be identified in forthcoming studies