Ann-Christine Eklöf

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.

Transepidermal Water Loss in Developing Rats: Role of Aquaporins in the Immature Skin

In the extremely preterm infant, high transepidermal water loss (TEWL) can result in severe dehydration. TEWL has been attributed to the structural properties of the epidermis but might also be influenced by mechanisms that facilitate water transport. To investigate whether aquaporins (AQP) may be involved in the extreme losses of water through immature skin, we examined the presence and cellular distributions of AQP-1 and AQP-3 in embryonic and adult rat skin by immunohistochemistry. The expression of AQP mRNA in skin was analyzed with the use of semiquantitative reverse transcription-PCR. In rat pups of different embryonic (E) and postnatal (P) ages (days), TEWL and skin hydration were measured. AQP-1 was detected in dermal capillaries, and AQP-3 was abundant in basal epidermal layers. Both AQP displayed several times higher expression in embryonic than in adult skin. TEWL was highest at embryonic day 18 (E18) (133 ± 18 g/m2h) and lower at E20 (25 ± 1 g/m2h) and P4 (9 ± 2 g/m2h). Skin hydration measured as skin electrical capacitance paralleled TEWL, being highest in fetal skin (794 ± 15 pF at E18) and decreasing to 109 ± 11 pF at E20 and to 0 ± 0 pF at P4. We conclude that, as in infants, water loss through the skin of rats decreases markedly with maturation during the perinatal period. The expression and cellular localization of the AQP are such that they might influence skin hydration and water transport and contribute to the high losses of water through the immature skin.

Binding of Losartan to Angiotensin AT1 Receptors Increases Dopamine D1 Receptor Activation

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling.

Prolactin and dopamine 1-like receptor interaction in renal proximal tubular cells

Prolactin is a natriuretic hormone and acts by inhibiting the activity of renal tubular Na(+)-K(+)-ATPase activity. These effects require an intact renal dopamine system. Here, we have studied by which mechanism prolactin and dopamine interact in Sprague-Dawley rat renal tissue. Na(+)-K(+)-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in microdissected renal proximal tubular segments. Intracellular signaling pathways were studied by a variety of different techniques, including Western blotting using phosphospecific antibodies, immunoprecipitation, and biotinylation assays. We found that dopamine and prolactin regulated Na(+)-K(+)-ATPase activity via similar signaling pathways, including protein kinase A, protein kinase C, and phosphoinositide 3-kinase activation. The cross talk between prolactin and dopamine 1-like receptors was explained by a heterologous recruitment of dopamine 1-like receptors to the plasma membrane in renal proximal tubular cells. Prolactin had no effect on Na(+)-K(+)-ATPase activity in spontaneously hypertensive rats, a rat strain with a blunted response to dopamine. These results further emphasize the central role of the renal dopamine system in the interactive regulation of renal tubular salt balance.

Gene expression analysis after prenatal tracheal ligation in fetal rat as a model of stimulated lung growth

PURPOSE Prenatal tracheal occlusion or ligation (TL) has been proven to accelerate lung growth, but the mechanism of this is poorly understood. To increase understanding of the biological mechanisms involved in growth stimulation after TL in the fetal lung, we performed Global gene expression analysis using microarray technology. MATERIAL AND METHODS Sprague-Dawley rats underwent surgery on gestational day 19. After a small hysterotomy, the trachea was mobilized and tied. As controls, we used littermates to manipulated fetuses. On day 21, fetuses were removed and lungs harvested. Global gene expression analysis was performed using Affymetrix Platform and the RAE 230 set arrays (Affymetrix Inc, Santa Clara, Calif). For validation of microarray data, we performed real time polymerase chain reaction (PCR) of the most significant upregulated or downregulated genes, combined with immunohistochemical (IHC) analysis of lung sections. RESULTS In the group that underwent TL, several growth factors had an increased expression including connective tissue growth factor (CTGF), insulin-like growth factor 1 (IGF-1), and fibroblast growth factor 18 (FGF-18). Some of the genes that were downregulated in the group that underwent TL compared with controls were surfactant protein A (SP-A), apolipoprotein E (Apo-E), and phospholipase group II A2 (plg2a2). These results could be confirmed with real time PCR and IHC studies. DISCUSSION Tracheal occlusion or ligation is a well-documented stimulator of fetal lung growth, and the present study provides novel insights into the underlying molecular mechanisms, with increased expression of genes and proteins with growth factor activity. One of these growth factors, CTGF, has never been previously described in this model. Also, decreased levels of genes involved in surfactant metabolism were observed, providing molecular insights into the decreased surfactant production that is known to occur in TL. Increased understanding of the molecular mechanisms that control lung growth may be the key to develop novel therapeutic techniques to stimulate prenatal and/or postnatal lung growth.

Antenatal imatinib treatment reduces pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia

The pathophysiology of congenital diaphragmatic hernia (CDH) is constituted by pulmonary hypoplasia and pulmonary hypertension (PH). We previously reported successful treatment with imatinib of a patient with CDH. This study examines the effect of antenatal imatinib administration on the pulmonary vasculature in a rat model of CDH. Pregnant rats were given nitrofen to induce CDH. Controls were given olive oil. Half of the CDH fetuses and half of the controls were treated with imatinib antenatally E17-E21, rendering four groups: Control, Control+Imatinib, CDH, and CDH+Imatinib. Lung sections were obtained for morphometry and immunohistochemistry, and protein was purified for Western blot. Effects of nitrofen and imatinib on Ki-67, caspase-3, PDGF-B, and PDGF receptors were analyzed. Imatinib significantly reduced medial wall thickness in pulmonary arteries of rats with CDH. It also normalized lumen area and reduced the proportion of fully muscularized arteries. Imatinib also caused medial thinning in the control group. Cell proliferation was increased in CDH, and this proliferation was significantly reduced by imatinib. PDGF-B and PDGFR-β were upregulated in CDH, and imatinib treatment resulted in a downregulation. PDGFR-α remained unchanged in CDH but was significantly downregulated by imatinib. Antenatal imatinib treatment reduces development of medial wall thickness and restores lumen area in pulmonary arteries in nitrofen-induced CDH. The mechanism is reduced cell proliferation. Imatinib is an interesting candidate for antenatal therapy for PH in CDH, but potential side effects need to be investigated and more specific targeting of PDGF signaling is needed.

Gene expression analysis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia

BACKGROUND Pulmonary hypoplasia and persistent pulmonary hypertension are the main causes of mortality and morbidity in newborns with congenital diaphragmatic hernia (CDH). Nitrofen is well known to induce CDH and lung hypoplasia in a rat model, but the mechanism remains unknown. To increase the understanding of the underlying pathogenesis of CDH, we performed a global gene expression analysis using microarray technology. METHODS Pregnant rats were given 100 mg nitrofen on gestational day 9.5 to create CDH. On day 21, fetuses after nitrofen administration and control fetuses were removed; and lungs were harvested. Global gene expression analysis was performed using Affymetrix Platform and the RAE 230 set arrays. For validation of microarray data, we performed real-time polymerase chain reaction and Western blot analysis. RESULTS Significantly decreased genes after nitrofen administration included several growth factors and growth factors receptors involved in lung development, transcription factors, water and ion channels, and genes involved in angiogenesis and extracellular matrix. These results could be confirmed with real-time polymerase chain reaction and protein expression studies. CONCLUSIONS The pathogenesis of lung hypoplasia and CDH in the nitrofen model includes alteration at a molecular level of several pathways involved in lung development. The complexity of the nitrofen mechanism of action reminds of human CDH; and the picture is consistent with lung hypoplasia and vascular disease, both important contributors to the high mortality and morbidity in CDH. Increased understanding of the molecular mechanisms that control lung growth may be the key to develop novel therapeutic techniques to stimulate pre- and postnatal lung growth.

Development of colonic fluid and electrolyte transport: influence of weaning pattern.

The purpose of this study was to investigate the normal postnatal development of electrolyte and water transport in the large intestines of rats aged 10-20 days, to focus on the events taking place around the time of weaning, and to study the influence of different feeding patterns at the time of weaning on the normal development of the transport mechanisms in the large intestine. The net absorption of sodium, potassium, and water was investigated by using in vivo luminal isotonic perfusion in rats aged 12-40 days. The activity of the active transport enzyme Na-KATPase in the colonic mucosa of rats aged 10-40 days was determined. We found a significant decrease in the net absorption of electrolytes and water that was greatest around the time of weaning. Since the NaKATPase activity was lower in 10-16-day-old rats than in 40-day-old rats, we suggest that the high sodium and water uptake in the colon of suckling rats is independent of the sodium pump. Changes in the feeding patterns around the time of weaning, such as prolonged suckling, were associated with a significantly higher net absorption of electrolytes and water than in rats that were normally weaned at 20 days of age. The Na-KATPase activity was not significantly influenced by the weaning pattern.

Mechanisms for colonic sodium transport during ontogeny: loss of an amiloride-sensitive sodium pathway

ABSTRACT: Net colonic sodium and fluid absorption is higher in suckling and weanling rats than in adult rats. This study was undertaken to investigate the mechanisms behind these differences. In vivo perfusion of the colon was performed in 14− to 80-day-old rats. Net Na and water uptake decreased exponentially from 14 to 80 days of age. Na uptake was 402 ± 73 μgEq/min/g DT in 20-day-old rats and 116 ± 6 μEq/min/g DT in 40-day-old rats. After amiloride treatment, net Na transport was reversed to net secretion (-79 ± 36 μEq/min/g DT) in 20-day-old rats. Amiloride had no effect on the net Na transport in 40-day-old rats. cAMP stimulation significantly increased the net Na uptake in 20-day-old rats and significantly reduced the net Na uptake in 80-day-old rats. cAMP did not increase the net uptake in amiloride-treated 20-day-old rats. We conclude that there are both quantitative and regulatory changes in the pathways for colonic Na transport during development, and we speculate that the large amiloride-sensitive sodium uptake in the young colon is adaptive and contributes to the sodium accretion necessary for rapid growth during late postnatal development

Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease

There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time- and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.