Description: Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by massive enlargement of fluid-filled cysts in the kidney. However, there is no effective therapy yet for this disease. To examine whether ginkgolide B, a natural compound, inhibits cyst development, a Madin-Darby canine kidney (MDCK) cyst model, an embryonic kidney cyst model, and a PKD mouse model were used. Interestingly, ginkgolide B significantly inhibited MDCK cyst formation dose dependently, with up to 69% reduction by 2 μM ginkgolide B. Ginkgolide B also significantly inhibited cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model. To determine the underlying mechanisms, the effect of ginkgolide B on MDCK cell viability, proliferation, apoptosis, chloride transporter CFTR activity, and intracellular signaling pathways were also studied. Ginkgolide B did not affect cell viability, proliferation, and expression and activity of the chloride transporter CFTR that mediates cyst fluid secretion. Ginkgolide B induced cyst cell differentiation and altered the Ras/MAPK signaling pathway. Taken together, our results demonstrate that ginkgolide B inhibits renal cyst formation and enlargement, suggesting that ginkgolide B might be developed into a novel candidate drug for ADPKD.

Description: Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of Notch has yet to be explored. We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. MI was induced by coronary artery ligation. A high level of Notch intracellular domain was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl- l -alanyl)-S-phenylglycine-t-butyl ester (DAPT) (intravenously 30 min before MI and then daily until death) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages and attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase. At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1β were also detected. In vitro studies revealed that LPS/IFN- upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. We concluded that Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation.

Description: Bisphenol A diglycidyl ether (BADGE), a PPAR2 antagonist, has been shown to inhibit marrow adipogenesis and promote bone formation in intact animals. We investigated the impact of BADGE on a new and more clinically relevant physiological model, the ovariectomized (OVX) rat model. Forty female Wistar rats were divided into four treatment groups for 12 wk ( n = 10/group): sham+vehicle, sham+BADGE, OVX+vehicle, and OVX+BADGE. Postmortem analyses included MRI, micro-CT, serological test, histomorphometry, biomechanical tests, RT-PCR, and Western blot. Overall, OVX induced a sequential marrow fat expansion accompanied by bone deterioration. Compared with OVX controls, BADGE reduced fat fraction of the distal femur by 36.3%, adipocyte density by 33.0%, adipocyte size by 28.6%, adipocyte volume percentage by 57.8%, and adipogenic markers PPAR2 and C/EBPα by ~50% in OVX rats. Similar results were observed in sham rats vs. vehicle. BADGE could promote bone quality in sham rats; however, BADGE did not significantly improve trabecular microarchitecture, biomechanical strength, and dynamic histomorphometric parameters except for trabecular separation in OVX rats. We concluded that early BADGE treatment at a dose of 30 mg/kg attenuates marrow adiposity in ovary-intact and OVX rats and stimulates bone formation in ovary-intact rats but does not significantly rescue bone quality in OVX rats.

Description: Glucagon-like peptides (GLP-1/2) are cosecreted from endocrine L cells in the gut and preproglucagonergic neurons in the brain. Peripheral GLP-2 action is essential for maintaining intestinal homeostasis, improving absorption efficiency and blood flow, promoting immune defense, and producing efficacy in treatment of gastrointestinal diseases. However, it is unknown if CNS GLP-2 plays a physiological role in the control of energy homeostasis. Since GLP-1/2 are cotranslated from preproglucagongene and coproduced by prohormone convertase-1, it is challenging to knockout GLP-2 only. Instead, our laboratory has generated a Glp2r -floxed mouse line to dissect cell-specific GLP-2 receptor GLP-2R) action in the regulation of energy balance. Our objective was to determine if GLP-2R in the hypothalamus modulates feeding behavior and gastric emptying. We show that Glp2r mRNA and protein are highly expressed in the arcuate nucleus and dorsomedial nucleus of the mouse hypothalamus. Using the Cre-LoxP system, we generated mice that lack Glp2r expression in POMC neurons (KO; mainly in the hypothalamus). The KO mice showed hyperphagic behavior (such as increases in food intake and meal frequency), accelerated gastric emptying (assessed by [ 13 C]octanoic acid breath test), and late-onset obesity, yet there was no decrease in basal metabolic rate. Infusion of GLP-2 (2.5 nmol into the 4th ventricle) suppressed food intake and gastric emptying, while GLP-2-mediated effects were abolished in the melanocortin receptor-4 (MC4R) KO mice. We conclude that Glp2r deletion in POMC neurons enhances feeding behavior and gastric motility, whereas icv GLP-2R activation suppresses food intake and gastric emptying through the MC4R signaling pathway. This study indicates that CNS GLP-2R plays a physiological role in the control of feeding behavior and gastric emptying and that this is mediated probably through the melanocortin system.

Description: A balance between alveolar liquid absorption and secretion is critical for maintaining optimal alveolar subphase liquid height and facilitating gas exchange in the alveolar space. However, the role of cystic fibrosis transmembrane regulator protein (CFTR) in this homeostatic process has remained elusive. Using a newly developed porcine model of cystic fibrosis, in which CFTR is absent, we investigated ion transport properties and alveolar liquid transport in isolated type II alveolar epithelial cells (T2AECs) cultured at the air-liquid interface. CFTR was distributed exclusively to the apical surface of cultured T2AECs. Alveolar epithelia from CFTR –/– pigs failed to increase liquid absorption in response to agents that increase cAMP, whereas cAMP-stimulated liquid absorption in CFTR +/– epithelia was similar to that in CFTR +/+ epithelia. Expression of recombinant CFTR restored stimulated liquid absorption in CFTR –/– T2AECs but had no effect on CFTR +/+ epithelia. In ex vivo studies of nonperfused lungs, stimulated liquid absorption was defective in CFTR –/– alveolar epithelia but similar between CFTR +/+ and CFTR +/– epithelia. When epithelia were studied at the air-liquid interface, elevating cAMP levels increased subphase liquid height in CFTR +/+ but not in CFTR –/– T2AECs. Our findings demonstrate that CFTR is required for maximal liquid absorption under cAMP stimulation, but it is not the rate-limiting factor. Furthermore, our data define a role for CFTR in liquid secretion by T2AECs. These insights may help to develop new treatment strategies for pulmonary edema and respiratory distress syndrome, diseases in which lung liquid transport is disrupted.

Description: Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to β-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca 2+ channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca 2+ channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca 2+ current ( I Ca,L ) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α 1C subunit of voltage-gated Ca 2+ channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α 1C were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I Ca,L in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I Ca,L stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted β-adrenergic stimulation of I CaL in failing hearts results from hyperphosphorylation of Ca 2+ channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca 2+ channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca 2+ channel remodeling of the failing heart.

Description: Apolipoprotein A-IV (apoA-IV) is synthesized by the intestine and secreted when dietary fat is absorbed and transported into lymph associated with chylomicrons. We have recently demonstrated that loss of apoA-IV increases chylomicron size and delays its clearance from the blood. There is still uncertainty, however, about the precise role of apoA-IV on the transport of dietary fat from the intestine into the lymph. ApoA-IV knockout (KO) mice do not have a gross defect in dietary lipid absorption, as measured by oral fat tolerance and fecal fat measurements. Here, using the in vivo lymph fistula mouse model, we show that the cumulative secretion of triglyceride (TG) into lymph in apoA-IV KO mice is very similar to that of wild-type (WT) mice. However, the apoA-IV KO mice do have subtle changes in TG accumulation in the intestinal mucosa during a 6-h continuous, but not bolus, infusion of lipid. There are no changes in the ratio of esterified to free fatty acids in the intestinal mucosa of the apoA-IV KO, however. When we extended these findings, by giving a higher dose of lipid (6 μmol/h) and for a longer infusion period (8 h), we found no effect of apoA-IV KO on intestinal TG absorption. This higher lipid infusion most certainly stresses the intestine, as we see a drastically lower absorption of TG (in both WT and KO mice); however, the loss of A-IV does not exacerbate this effect. This supports our hypothesis that apoA-IV is not required for TG absorption in the intestine. Our data suggest that the mechanisms by which the apoA-IV KO intestine responds to intestinal lipid may not be different from their WT counterparts. We conclude that apoA-IV is not required for normal lymphatic transport of TG.

Description: Elevated levels of intracellular Ca 2+ ([Ca 2+ ] i ) inhibit Na + /H + exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC- directly binds NHE3, an interaction that is necessary for [Ca 2+ ] i inhibition of NHE3 activity, and that PLC- Src homology 2 (SH2) domains may scaffold Ca 2+ signaling proteins necessary for regulation of NHE3 activity. [Ca 2+ ] i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC- might link c-Src to NHE3-containing complexes to mediate [Ca 2+ ] i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ~40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y 416 phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC- and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC- SH2 domains, an interaction that was prevented by blocking the PLC- SH2 domain. This study demonstrated that c-Src 1 ) activity is necessary for [Ca 2+ ] i inhibition of NHE3 activity, 2 ) activation occurs rapidly (~1 min) after CCh treatment, 3 ) directly binds PLC- SH2 domains and associates dynamically with PLC- under elevated [Ca 2+ ] i conditions, and 4 ) does not directly bind NHE3. Under elevated [Ca 2+ ] i conditions, PLC- scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC- from NHE3 and subsequent endocytosis of NHE3.

Description: We used mouse cortical collecting duct principal cells (mpkCCD c14 cell line) as a model to determine whether statins reduce the harmful effects of cyclosporine A (CsA) on the distal nephron. The data showed that treatment of cells with CsA increased transepithelial resistance and that the effect of CsA was abolished by lovastatin. Scanning ion conductance microscopy showed that CsA significantly increased the height of cellular protrusions near tight junctions. In contrast, lovastatin eliminated the protrusions and even caused a modest depression between cells. Western blot analysis and confocal microscopy showed that lovastatin also abolished CsA-induced elevation of both zonula occludens-1 and cholesterol in tight junctions. In contrast, a high concentration of CsA induced apoptosis, which was also attenuated by lovastatin, elevated intracellular ROS via activation of NADPH oxidase, and increased the expression of p47 phox . Sustained treatment of cells with lovastatin also induced significant apoptosis, which was attenuated by CsA, but did not elevate intracellular ROS. These results indicate that both CsA and lovastatin are harmful to principal cells of the distal tubule, but via ROS-dependent and ROS-independent apoptotic pathways, respectively, and that they counteract probably via mobilization of cellular cholesterol levels.

Description: Klebsiella pneumoniae (Kp) is a Gram-negative bacterium that can cause serious infections in humans. Autophagy-related gene 7 (Atg7) has been implicated in certain bacterial infections; however, the role of Atg7 in macrophage-mediated immunity against Kp infection has not been elucidated. Here we showed that Atg7 expression was significantly increased in murine alveolar macrophages (MH-S) upon Kp infection, indicating that Atg7 participated in host defense. Knocking down Atg7 with small-interfering RNA increased bacterial burdens in MH-S cells. Using cell biology assays and whole animal imaging analysis, we found that compared with wild-type mice atg7 knockout (KO) mice exhibited increased susceptibility to Kp infection, with decreased survival rates, decreased bacterial clearance, and intensified lung injury. Moreover, Kp infection induced excessive proinflammatory cytokines and superoxide in the lung of atg7 KO mice. Similarly, silencing Atg7 in MH-S cells markedly increased expression levels of proinflammatory cytokines. Collectively, these findings reveal that Atg7 offers critical resistance to Kp infection by modulating both systemic and local production of proinflammatory cytokines.