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.

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-ß 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.

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.

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.

Connective tissue growth factor expression pattern in lung development.

The aim of this study was to investigate the expression and distribution pattern of connective tissue growth factor (CTGF) in lung development during different stages, and to compare with a model of stimulated lung growth after tracheal ligation (TL) and with the teratogen model of induced congenital diaphragmatic hernia (CDH) and lung hypoplasia after nitrofen. Sprague-Dawley rat fetuses were obtained on gestational days 14, 17, and 21 (E14, E17, E21). For the experimental CDH group, pregnant rats were given 100 mg nitrofen on gestational day 9.5 (E9.5), and delivered E21. In another group, Sprague-Dawley rat fetuses were subjected to intrauterine tracheal ligation (TL) on gestational day 19 (E19), and delivered on day 21 (E21). All fetuses were delivered by cesarean section and lungs harvested. Lungs from 1-day-old newborn healthy, nonoperated rats were also obtained. Immunohistochemical (IHC) analysis for CTGF was performed on the different lung sections. CTGF mRNA expression levels in hyperplastic lungs after TL, hypoplastic lungs and CDH after nitrofen administration, and fetal controls at E21 were analyzed with real-time polymerase chain reaction (PCR). Immunohistochemical staining for CTGF at E14 showed that it was merely localized to the epithelium of terminal bronchiole, increasing during gestation, being more abundant at E17 and at E21. In the CDH group, lungs had an immature appearance and CTGF protein expression was decreased in the epithelium of the distal airways compared to the control group at E21, and was mainly observed in the lung mesenchyme. In the TL group, CTGF expression was more abundant compared to the control group at E21, especially in the epithelium of the terminal bronchioles, with a decreasing expression pattern distally. In the newborn lungs, CTGF had a pattern of expression in the epithelium of terminal bronchiole similar to TL lungs. At the mRNA level, CTGF expression was increased after TL, and decreased in the teratogen model of CDH and lung hypoplasia after nitrofen administration. This is, to the authors' knowledge, the first report of CTGF expression pattern during lung development, and of an impaired expression in CDH lungs after nitrofen. CTGF is suggested to enhance alveologenesis and microvascular development at late stages of lung development, and a decreased expression could lead to the impaired alveologenesis and abnormal microvascular pulmonary bed observed in CDH lungs. Increased understanding of the molecular mechanisms that control lung growth could provide a key to develop novel therapeutic techniques to stimulate pre- and/or postnatal lung growth in infants with impaired lung growth and development, such in congenital diaphragmatic hernia.

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.

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.

Expression of chloride channels in trachea-occluded hyperplastic lungs and nitrofen-induced hypoplastic lungs in rats.

BACKGROUND: Congenital diaphragmatic hernia is accompanied by pulmonary hypoplasia. Fetal lung growth is dependent on the secretion of lung liquid, in which Cl(-) secretion by the pulmonary epithelium plays a crucial role. A decrease of lung liquid production during fetal development renders marked pulmonary hypoplasia, while accelerated fetal lung growth in the form of pulmonary hyperplasia can be achieved by in utero tracheal occlusion (TO). Cl(-) secretion presumably involves NKCC-1, the primary basolateral Cl(-) entry pathway in airway epithelia, coupled to an apical Cl(-) exit pathway. The chloride channels ClC-2, -3 and -5, members of the CLC gene family, are all localized to the apical membrane of fetal respiratory epithelia, which makes them possible candidates for being mediators of fetal apical Cl(-) secretion. The aim of the study was to examine the potential of ClC-2, -3 and -5 as alternative apical airway epithelial Cl(-) channels in normal lung development and their possible role in the development of hypoplastic lungs in CDH. We also wanted to examine ClC-2, -3 and -5 together with the NKCC-1 in hyperplastic lungs created by TO. METHODS: Pregnant Sprague-Dawley rat dams were given nitrofen on gestational day 9.5 to induce pulmonary hypoplasia. Controls were given only olive oil. The rat fetuses were removed on days 17, 19 and 21. Hyperplastic lungs were created by intrauterine TO of rat fetuses on day 19 and the lungs were harvested on day 21. The pulmonary expression of ClC-2, -3, -5 and NKCC-1 was then analyzed using Western blot. RESULTS: We found that the temporal expression of ClC-2 and -3 in normal fetal lungs points toward a developmental regulation. ClC-2 and -3 were also both down-regulated on day 21 in hypoplastic CDH lungs. In TO induced hyperplastic lungs, the levels of ClC-2 were found to be significantly up-regulated. NKCC-1 showed a tendency toward up-regulation in hyperplastic lungs, while ClC-3 showed a tendency to be down-regulated, but no statistically significant changes could be seen. There was no difference between controls and any of the groups for the expression of ClC-5. CONCLUSION: We show that the developmental changes in ClC-2 and ClC-3 protein expression are negatively affected in hypoplastic CDH lungs. Lung hyperplasia created by TO up-regulates the expression of ClC-2. ClC-2 is therefore an interesting potential target in the development of novel, non-invasive, therapies for CDH treatment.

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.

NKCC-1 and ENaC are down-regulated in nitrofen-induced hypoplastic lungs with congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is accompanied by pulmonary hypoplasia and pulmonary hypertension. Fetal lung growth is dependent on the secretion of lung liquid, which normally is absorbed at partus. The ion channel NKCC-1 is involved in this secretory process, but has recently also been reported to be implicated in absorption. CDH patients show a disturbed transition from secretion to absorption. alpha- and beta-ENaC are essential for lung liquid absorption. Common for all transcellular ion transport is the need for Na/K-ATPase as a primary driving force. The aim of the study was first to map the normal pulmonary expression of the above proteins during late gestation and secondly to see if the expression was affected in a CDH rat model. Pregnant Sprague-Dawley rat dams were given nitrofen on gestational day 9.5 to induce CDH. The fetuses were removed on gestational days E18 and E21. In addition, newborn rats were harvested postpartum on day P2. The fetuses were put into one of two groups: hypoplastic lungs without CDH (N-CDH) and hypoplastic lungs with CDH (N+CDH). The pulmonary expression of NKCC-1, alpha-/beta-ENaC and Na/K-ATPase was then analyzed using Western blot. We found that the protein levels of NKCC-1 on gestational days E18 and E21 were significantly lower among fetuses with N+CDH as well as N-CDH compared to controls. The expression of beta-ENaC was also significantly down-regulated in both the groups on E18 and E21. The protein levels of alpha-ENaC and Na/K-ATPase were not found to be significantly decreased, but both showed a tendency towards down-regulation. The marked down-regulation of NKCC-1 in fetal hypoplastic lungs with CDH indicates a possibly decreased lung liquid production. This may be one of the mechanisms behind the disturbed pulmonary development in CDH. We also show that beta-ENaC is down-regulated. Down-regulation of beta-ENaC may result in abnormal lung liquid absorption, which could be one of the mechanisms behind the respiratory distress seen in CDH patients postpartum.

Effect of TNF-alpha on CD3-zeta and MHC-I in postnatal rat hippocampus.

The zeta subunit of the CD3 T-cell receptor complex and the major histocompatibility complex class 1 (MHC-I) are important not only for the immune response to antigens, they also function as signal molecules in the brain, where they play a role in the postnatal maturation process. The expression of these molecules can be regulated by cytokines. In situations associated with increased cytokine production, such as neonatal hypoxia, the hippocampus is particularly susceptible to permanent damage. This has prompted us to examine the MHC-I and CD3-zeta expression in hippocampus from early postnatal, weanling and adolescent rats and to record the effects of TNF-alpha and IL-1beta, cytokines commonly increased in neonatal hypoxia, on MHC-I and CD3-zeta expression in the hippocampus. We show that there is a robust postnatal up-regulation of CD3-zeta and MHC-I protein as well as of MHC-I mRNA and that TNF-alpha down-regulates the expression of CD3-zeta protein and MHC-I mRNA in early postnatal but not in weanling nor in adolescent rats. These results may offer a molecular explanation to the adverse effects of increased circulating levels of cytokines on brain in neonatal hypoxia.

Prolactin, a natriuretic hormone, interacting with the renal dopamine system.

BACKGROUND: Although prolactin affects sodium and water transport across the plasma membrane and interacts with dopamine in the brain, its role in the kidney is unclear. Here we examined the effect of prolactin and its possible interaction with the intrarenal natriuretic hormone dopamine, on proximal tubular Na(+), K(+)-ATPase activity in vitro and renal function in anesthetized rats. METHODS: Na(+), K(+)-ATPase activity was measured as ouabain-sensitive adenosine triphosphate (ATP) hydrolysis in microdissected proximal tubular segments. Renal function was studied during euvolemic conditions by conventional clearance techniques. RESULTS: Prolactin induced a dose-dependent inhibition of proximal tubular Na(+), K(+)-ATPase activity. A maximal inhibitory effect of 48% of control was observed at an in vitro prolactin concentration of 1 microg/mL. This effect was completely abolished by a dopamine D1 receptor antagonist. In tubules preincubated with inhibitors of aromatic amino acid decarboxylase (AADC), the rate-limiting enzyme in renal dopamine formation, prolactin had no effect on Na(+), K(+)-ATPase activity. In rats, prolactin infusion resulted in an increase in urinary sodium, potassium, and water excretion. These effects were also completely abolished by the D1 receptor antagonist. Prolactin had no significant effects on glomerular filtration rate (GFR) or mean arterial blood pressure. CONCLUSION: We conclude that prolactin is a natriuretic hormone which interacts with the renal dopamine system for its effects. The natriuretic response is associated with inhibition of proximal tubular Na(+), K(+)-ATPase activity.

Insulinlike growth factor I gene expression is increased in the fetal lung after tracheal ligation.

BACKGROUND/PURPOSE: The mortality and morbidity in congenital diaphragmatic hernia are mainly caused by pulmonary hypoplasia. To improve clinical results, further methods inducing lung growth may have to be used. The aim of this report was to evaluate the expression of insulinlike growth factor I (IGF-I), estrogen receptor alpha, estrogen receptor beta, growth hormone receptor, and thioredoxin in a rat model of hypoplastic, hyperplastic, and normal fetal lungs to improve understanding of lung growth. METHODS: Hypoplastic diaphragmatic hernia lungs were created by giving nitrofen by gavage to pregnant rats on day 9.5. Hyperplastic lungs were achieved by intrauterine tracheal ligation of rat fetuses on day 19. All lungs were harvested on gestational day 21. Total nucleic acids were extracted by proteinase K digestion and extraction in phenol/chloroform. The total nucleic acids mixture was hybridized with radioactively labeled RNA probes, and the radioactivity of the hybrids was compared with the respective standard curve of known amounts of in vitro synthesized mRNA. Immunohistochemistry staining was performed for IGF-I. RESULTS: The IGF-I mRNA was significantly (P < .01) higher in hyperplastic lungs compared with control and hypoplastic lungs. The latter 2 did not differ. No difference was found between the other mRNA levels in the study groups. CONCLUSIONS: IGF-I is involved in the accelerated lung growth seen after intrauterine tracheal ligation.

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.

Role of oxidative stress in advanced glycation end product-induced mesangial cell activation.

BACKGROUND: Levels of advanced glycation end products (AGE) are elevated in individuals with advancing age, renal failure, and diabetes, and accumulation of these molecules may contribute to disease progression. The mechanism by which AGE proteins alter glomerular mesangial cell function, however, is not completely understood. The present study assessed the involvement of oxidative stress in AGE-dependent mesangial cell signaling events. METHODS: Primary cultures of rat renal mesangial cells were exposed to in vitro AGE-BSA and H2O2. Nuclear factor-kappaB (NF-kappaB) and protein kinase C (PKC) isoform activation were studied using confocal microscopy and Western blotting. Quantitative polymerase chain reaction (PCR) was used to measure transforming growth factor-beta1 (TGF-beta1) levels. The involvement of oxidative stress was assessed by supplementing or compromising cellular antioxidant capacity. RESULTS: NF-kappaB was dose-dependently activated by AGE. PKC activation was not involved in this response, but analysis of PKC-beta1 activation showed a stimulatory effect of AGE proteins on this isoform. Transcription of TGF-beta1 was stimulated by AGE and was prevented by PKC inhibition. Challenge with H2O2 had similar downstream effects on mesangial cell signaling. Antioxidants, vitamin E and nitecapone, prevented AGE-dependent NF-kappaB activation and normalized PKC activity and associated TGF-beta1 transcription. Depletion of the intracellular antioxidant, glutathione, effectively lowered the AGE concentration needed for mesangial cell activation of NF-kappaB and PKC-beta1. Treatment with a suboptimal AGE dose, under glutathione-depleted conditions, revealed a synergistic effect on both parameters. CONCLUSION: The results support a central role for oxidative stress in AGE-dependent mesangial cell signaling and emphasize the importance of ROS in determining cell responsiveness.