Role of α7- and β4-Containing Nicotinic Acetylcholine Receptors in the Affective and Somatic Aspects of Nicotine Withdrawal: Studies in Knockout Mice

To assess which nicotinic acetylcholine receptors (nAChRs) are involved in the aversive aspects of nicotine withdrawal, brain reward function and the somatic signs of nicotine withdrawal were assessed in mice that lack α7 and β4 nAChR subunits. Brain reward function was assessed with the intracranial self-stimulation (ICSS) procedure, in which elevations in ICSS thresholds reflect an anhedonic mood state. At 3–6 h of spontaneous nicotine/saline withdrawal, thresholds were elevated in nicotine-withdrawing α7^+/+ and β4^+/+, but not α7^−/− or β4^−/−, mice compared with saline-withdrawing mice, indicating a delay in the onset of withdrawal in the knockout mice. From 8 to 100 h of withdrawal, thresholds in α7^+/+ and α7^−/− mice were equally elevated, whereas thresholds in β4^+/+ and β4^−/− mice returned to baseline levels. Somatic signs were attenuated in nicotine-withdrawing β4^−/−, but not α7^−/−, mice. Administration of a low dose of the nAChR antagonist mecamylamine induced threshold elevations in α7^−/−, but not α7^+/+, mice, whereas the highest dose tested only elevated thresholds in α7^+/+ mice. Mecamylamine-induced threshold elevations were similar in β4^−/− and β4^+/+ mice. In conclusion, null mutation of the α7 and β4 nAChR subunits resulted in a delayed onset of the anhedonic aspects of the spontaneous nicotine withdrawal syndrome. Previous findings of attenuated somatic signs of nicotine withdrawal in β4^−/−, but not α7^−/−, mice were confirmed in the present study, indicating an important role for β4-containing nAChRs in the somatic signs of nicotine withdrawal. The mecamylamine-precipitated withdrawal data suggest that compensatory adaptations may occur in constitutive α7^−/− mice or that mecamylamine may interact with other receptors besides nAChRs in these mice. In summary, the present results indicate an important role for α7 and β4-containing nAChRs in the anhedonic or somatic signs of nicotine withdrawal.     

Role of Interleukin-6 in Immune Complex Induced Models of Vascular Injury

Previous studies have suggested that Interleukin-6 (IL-6) acts as a marker of vasculitis. To determine the role of IL-6 in vasculitis we utilized two models of immune complex induced vascular injury (dermal Arthus and acute pulmonary alveolitis) in IL-6 deficient (IL-6^−/−) and IL-6 sufficient (IL-6^+/+) mice. Plasma and bronchoalveolar lavage (BAL) levels of IL-6 were elevated in the injured IL-6^+/+ mice with acute alveolitis and in the plasma of IL-6^+/+ mice with dermal Arthus vasculitis. While, IL-6 levels in IL-6^−/− mice were near or below the levels of detection. Histological examination of the intensity of vascular injury response demonstrated no significant differences between IL-6^−/− and IL6^+/+ mice. More specifically, lung permeability (total protein in the BAL) in the lung injury model in IL-6^−/− mice was the same as injured IL-6^+/+ mice. As a corollary, assessment of vascular permeability in both models was the same in the IL-6^−/− as the IL-6^+/+ mice. Quantification of leukocyte influx into the injured tissues in both models also revealed no differences between the IL-6^−/− and IL-6^+/+ mice. These data demonstrate that while IL-6 is upregulated in acute vascular injury it does not appear to be critical in the development of the vascular inflammatory response.This work is supported in part by: Pfizer Global Research & Development     

SGK1 dependence of insulin induced hypokalemia

Insulin stimulates cellular K⁺ uptake leading to hypokalemia. Cellular K⁺ uptake is accomplished by parallel stimulation of Na⁺/H⁺ exchange, Na⁺,K⁺,2Cl⁻ co-transport, and Na⁺/K⁺ ATPase and leads to cell swelling, a prerequisite for several metabolic effects of the hormone. Little is known about underlying signaling. Insulin is known to activate the serum and glucocorticoid-inducible kinase SGK1, which in turn enhances the activity of all three transport proteins. The present study thus explored the contribution of SGK1 to insulin-induced hypokalemia. To this end, gene-targeted mice lacking SGK1 (sgk1 ^−/− ) and their wild-type littermates (sgk1 ^+/+ ) have been infused with insulin (2 mU kg⁻¹ min⁻¹) and glucose at rates leaving the plasma glucose concentration constant. Moreover, isolated liver perfusion experiments have been performed to determine stimulation of cellular K⁺ uptake by insulin (100 nM). As a result, combined glucose and insulin infusion significantly decreased plasma K⁺ concentration despite a significant decrease of urinary K⁺ excretion in sgk1 ^+/+ but not in sgk1 ^−/− mice. Accordingly, the plasma K⁺ concentration was within 60 min significantly lower in sgk1 ^+/+ than in sgk1 ^−/− mice. In isolated liver perfusion experiments, cellular K⁺ uptake was stimulated by insulin (100 nM), an effect blunted by 72% in sgk1 ^−/− mice as compared to sgk1 ^+/+ mice. Accordingly, insulin-induced cell hydration was 63% lower in sgk1 ^−/− mice than in sgk1 ^+/+ mice. Moreover, volume regulatory K⁺ release was 31% smaller in sgk1 ^−/− mice than in sgk1 ^+/+ mice. In conclusion, the serum and glucocorticoid-inducible kinase SGK1 participates in the signaling mediating the hypokalemic effect of insulin.     

Potential contribution of vasoconstriction to suppression of heat loss and homeothermic regulation in UCP1-deficient mice

To investigate the thermoregulatory mechanism in mice lacking uncoupling protein 1 (UCP1) from the viewpoint of heat loss, we measured oxygen consumptions (VO2), skin-surface temperatures (Tskin, an index of heat release), blood flows in the tails, and rectal temperatures (Trectal) of mice housed in an animal room under the standard thermal condition of ∼23°C. Compared with wild-type (Ucp1 ^+/+) mice, adult UCP1-deficient (Ucp1 ^−/−) mice tended to show a reduced VO2. Thermograhic analysis of the acute response of Ucp1 ^−/− mice to a small change (a drop of 1–2°C) in the ambient temperature revealed a sustained fall in the Tskin of Ucp1 ^−/− mice; but this fall was only transient in Ucp1 ^+/+ mice. Analysis of tail blood flow under anesthesia clearly showed a stronger vasoconstrictor response in Ucp1 ^−/− mice than in Ucp1 ^+/+ mice. Administration of a vasodilator, evodiamine, transiently increased Tskin in Ucp1 ^+/+ and Ucp1 ^−/− mice similarly; whereas the induction of vasodilation caused a greater and more prolonged reduction in Trectal in Ucp1 ^−/− mice than in Ucp1 ^+/+ mice. These results indicate that Ucp1 ^−/− mice highly, or at least partly, rely on vasoconstriction for heat conservation to compensate for their UCP1 deficiency and to maintain homeothermy under the condition of normal housing temperature.     

Blunted DOCA/high salt induced albuminuria and renal tubulointerstitial damage in gene-targeted mice lacking SGK1

Mineralocorticoids stimulate renal tubular Na⁺ reabsorption, enhance salt appetite, increase blood pressure, and favor the development of renal fibrosis. The effects of mineralocorticoids on renal tubular Na⁺ reabsorption and salt appetite involve the serum- and glucocorticoid-inducible kinase 1 (SGK1). The kinase is highly expressed in fibrosing tissue. The present experiments thus explored the involvement of SGK1 in renal fibrosis. To this end, SGK1-knockout mice (sgk1 ^−/−) and their wild-type littermates (sgk1 ^+/+) were implanted with desoxycorticosterone acetate (DOCA)-release pellets and offered 1% saline as drinking water for 12 weeks. The treatment led to significant increases in fluid and Na⁺ intake and urinary output of fluid and Na⁺ in sgk1 ^+/+ mice, effects blunted in sgk1 ^−/− mice. Blood pressure increased within the first 7 weeks to a similar extent in both genotypes, but within the next 5 weeks, it increased further only in sgk1 ^+/+ mice. Creatinine clearance did not change significantly but albuminuria increased dramatically in sgk1 ^+/+ mice, an effect significantly blunted in sgk1 ^−/− mice. Histology after 12 weeks treatment revealed marked glomerular sclerosis and tubulointerstitial damage with interstitial fibrosis and inflammation in kidneys from sgk1 ^+/+ mice, but not from sgk1 ^−/− mice. In conclusion, a lack of SGK1 protects against DOCA/high-salt-induced albuminuria and renal fibrosis.     

Apoptosis Inhibitor Expressed by Macrophages Tempers Autoimmune Colitis and the Risk of Colitis-Based Carcinogenesis in TCRα<Superscript>−/−</Superscript> Mice

Background Crohn’s disease and ulcerative colitis (UC) are the two main entities involved in human inflammatory bowel disease (IBD). However, their precise etiologies remain unclear. To study the development of mucosal inflammation, and chronic inflammation-based dysplasia and carcinoma formation, we examined possible roles of the apoptosis inhibitor expressed by macrophages (AIM) in an experimental IBD model. Methods In this study, we used T cell receptor α deficient (TCRα^−/−) mice, a known UC-like colitis model. We generated TCRα^−/− × AIM^−/− double knockout mice by crossbreeding TCRα^−/− with AIM^−/− mice. At 24 weeks of age, mice were killed to obtain colon tissues for pathological examinations. TCRα^−/− × AIM^+/− mice, heterozygous littermates of TCRα^−/− × AIM^−/− mice, were used as controls. Results Severe colitis was observed in TCRα^−/− × AIM^−/− mice, when compared with TCRα^−/− × AIM^+/− mice. Dysplasia was detected in TCRα^−/− × AIM^−/− mice, but not in TCRα^−/− × AIM^+/− mice. Adenocarcinoma formation was observed from dysplasia only in TCRα^−/− × AIM^−/− mice. Conclusion Not only a high incidence of severe colitis but also dysplasia and adenocarcinoma formation were observed in TCRα^−/− × AIM^−/− mice only. AIM have some regulatory roles in inflammation and progression of dysplasia to carcinoma in TCRα^−/− mice.     

CB1 and TRPV1 receptors mediate protective effects on colonic electrophysiological properties in mice

CB1 and TRPV1 receptors modulate enteric neurotransmission and colonic inflammation. This study investigates early electrophysiological changes in distal colon of wild-type and receptor deficient mice after an inflammatory insult set by dinitrobenzene sulfonic acid (DNBS). Colitis was induced by DNBS in CB1^−/− mice, TRPV1^−/− mice, and their respective wild-type littermates. Electrophysiological properties consisting of membrane potentials and electrically induced inhibitory junction potentials (IJP) of circular smooth muscle cells were evaluated at different time points. Additionally a histological colitis severity score was evaluated in CB1^+/+ and CB1^−/− mice 24 h after DNBS. Inflammation caused spontaneous atropine insensitive rhythmic action potentials in CB1^−/− and TRPV1^−/− mice but not in wild-type animals. This indicates that membrane stability is disturbed, which in turn indicates a lack of protective mechanisms. Focal electrical neuronal stimulation of the myenteric plexus induced IJP in the smooth muscle cells. Twenty-four hours after initiation of inflammation, the duration of the IJP is prolonged in all animals, indicating disturbances within neuromuscular interaction. In CB1^−/− mice, it is interesting that the duration of IJP was significantly extended, as compared to CB1^+/+ mice pointing toward missing protective mechanisms in the CB1^−/− mice. Inflammatory insults in the mouse colon induce reproducible changes in the electrophysiological properties and such changes correlate with duration of colitis. In mutants, these electrophysiological changes display different patterns, suggesting the lack of protective properties for neuromuscular interactions and membrane stability.     

SGK1 is not required for regulation of colonic ENaC activity

The serum and glucocorticoid-inducible kinase SGK1 is known to be upregulated by mineralocorticoids and to enhance ENaC activity in several expression systems. Moreover, the amiloride-sensitive transepithelial potential difference in the collecting duct is lower in gene-targeted mice lacking SGK1 (sgk1 ^−/−) than in their wild-type littermates (sgk1 ^+/+). Accordingly, the ability of sgk1 ^−/− mice to decrease urinary sodium output during salt depletion is impaired. These observations highlight the importance of SGK1 in the stimulation of renal ENaC activity. In colonic epithelium, ENaC activity and, thus, transepithelial potential difference (V _te) are similarly upregulated by mineralocorticoids. The present study thus explored V _te and the apparent amiloride-sensitive equivalent short circuit current (I _amil) in the colon from sgk1 ^−/− and sgk1 ^+/+ mice before and after treatment with low salt diet, the glucocorticoid dexamethasone [DEXA, 10 μg/g body weight (BW)], or the mineralocorticoid deoxycorticosterone acetate (DOCA, 1.5 mg/day). Surprisingly, V _te and I _amil were both significantly (p<0.05) higher in sgk1 ^−/− than in sgk1 ^+/+ untreated mice. A 7-day exposure to low salt diet increased V _te and I _amil in both genotypes, but did not abrogate the differences of V _te and I _amil between sgk1 ^−/− and sgk1 ^+/+ mice. Plasma aldosterone levels were significantly higher in sgk1 ^−/− than in sgk1 ^+/+ mice both under control conditions and under low salt diet, which may explain the enhanced V _te in sgk1 ^−/− mice. Treatment with DEXA or DOCA both significantly increased V _te and I _amil in sgk1 ^+/+ mice and tended to increase V _te and I _amil in sgk1 ^−/− mice. Under treatment with DEXA or DOCA, V _te and I _amil were similar in sgk1 ^−/− and sgk1 ^+/+ mice. Fecal Na⁺ excretion was similar in sgk1 ^+/+ mice and in sgk1 ^−/− mice and was similarly decreased by low Na⁺ diet in both genotypes. In conclusion, transepithelial potential and amiloride-sensitive short circuit current are enhanced in the colonic epithelium of SGK1-deficient mice. Thus, lack of SGK1 does not disrupt colonic ENaC activity and its regulation by salt depletion.This article has a shared first authorship between Rexhep Rexhepaj and Ferruh Artunc.     

Regulation of Na+/H+ exchanger in dendritic cells by Akt2

Dendritic cells (DCs) are antigen-presenting cells decisive in primary immune responses and establishment of immunological memory. They are activated by bacterial lipopolysaccharides (LPS), which lead to activation of Na⁺/H⁺ exchanger activity, cell swelling, reactive oxygen species (ROS) formation, and migration. The effects require functional phosphoinositide 3 kinase and are paralleled by Akt phosphorylation. The present study explored the putative involvement of the Akt isoform Akt2. To this end, experiments were performed in DCs isolated from bone marrow of mice lacking functional Akt2/PKB? (akt2 ^−/−) and respective wild-type animals (akt2 ^+/+). Based on BCECF fluorescence, cytosolic pH (pH_i) was significantly lower in akt2 ^−/− than in akt2 ^+/+ DCs. Transient exposure to NH₄Cl was followed by profound cytosolic acidification in both genotypes. Subsequent re-alkalinization was largely dependent on Na⁺ thus reflecting Na⁺/H⁺ exchanger activity and was significantly lower in akt2 ^−/− than in akt2 ^+/+ DCs. According to forward scatter in FACS analysis, cell volume was significantly lower in akt2 ^−/− than in akt2 ^+/+ DCs. Exposure of DCs to LPS led within 4 h to significant increases of Na⁺/H⁺ exchanger activity, cell volume, ROS production, and migration in akt2 ^+/+ mice, and its effects were significantly blunted in akt2 ^−/− DCs. The present observations disclose a role of Akt2 in the regulation of pH_i, cell volume, ROS production, and migration in dendritic cells.     

Melanocortin-5 Receptor Deficiency in Mice Blocks a Novel Pathway Influencing Pheromone-Induced Aggression

The rodent preputial gland secretes aggression-promoting pheromones and expresses melanocortin-5 receptor (MC5R), but the functional relationship is poorly understood. We investigated whether MC5R deficiency in male mice alters stimulatory melanocortin influences on preputial growth and pheromone-induced aggression. In wild-type (MC5R^+/+) pairs, repeated NDP-MSH injection decreased attack latency and increased aggression in initial attackers. Similar NDP-MSH treatment in MC5R-deficient (MC5R^−/−) pairs failed to alter attack latency or aggression frequency, but aggression increased in vehicle-injected opponents. NDP-MSH treatment promoted preputial hypertrophy, and in MC5R^+/+ mice paired against non-aggressive stimulus opponents it decreased attack latency and increased aggression. MC5R^−/− mice were insensitive to behavioral and physiological effects of NDP-MSH, and preputialectomized mice were insensitive to behavioral effects of NDP-MSH. The results suggest that MC5R inactivation reduced a pheromonal signal for aggression that acts on donors, rather than their opponents.     

Spontaneous recovery of injured Achilles tendon in inducible nitric oxide synthase gene knockout mice

Objective and Design: To determine if inducible nitric oxide synthase (iNOS) gene could affect Achilles tendon healing using iNOS gene knockout mice. Methods: 21 iNOS knockout (iNOS^−/−) mice and 8 of the wild type (iNOS^+/+) mice were utilized in this study. Group 1: iNOS^+/+ mice (n = 8), group 2: iNOS^−/− mice (n = 11) and group 3: iNOS^−/− with a NOS inhibitor, (aminoguanidine, 500 mg/kg/day, via an intraperitoneal mini-osmotic pump for 7 days, n = 10). The right Achilles tendon was transected in all mice and harvested on day 7 for cross-sectional area and biomechanical properties. Serum nitrate concentration of the mice was measured by gas chromatography mass spectrometry (GC/MS). Results: A significant reduction in cross-sectional area of the healing Achilles tendon was observed in group 3 mice compared to group 2 mice (p < 0.01). The serum nitrate concentration in both group 2 and group 3 mice was lower than that in group 1 mice (p < 0.01) iNOS gene deletion and inhibition of NOS did not affect the biomechanical properties of the healing tendons. Conclusions: iNOS gene is not solely responsible for the beneficial effects of nitric oxide (NO) on tendon healing. Received: 30 March 2005; returned for revision 4 August 2005; returned for final revision 27 September 2005; accepted by M. Parnham 28 September 2005     

Accelerated suicidal erythrocyte death in Klotho-deficient mice

Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho ^−/− ) than in their wild-type littermates (klotho ^+/+ ). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho ^−/− mice. In a variety of cells, 1,25(OH)₂D₃ stimulates Ca²⁺ entry. In erythrocytes, increased Ca²⁺ entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca²⁺ concentration was significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Reticulocyte number was significantly increased in klotho ^−/− mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca²⁺ entry and annexin V-binding of klotho ^−/− erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca²⁺ activity in and suicidal death of erythrocytes.     

Major histocompatibility complex class I- and II-deficient knock-out mice are resistant to primary but susceptible to secondary Eimeria papillata infections

Two distinct mechanisms seem to function in reducing oocyst output during Eimeria papillata infections in mice. For naive mice, immunity was afforded␣by␣a T-cell-independent gamma-interferon (IFN-γ) response mediated by natural killer (NK) cells. On reinfection, resistance was associated with T-cells and, to a lesser extent, perforin. To determine if antigen presentation with major histocompatibility complex␣(MHC) molecules was required to control oocyst production by NK cells during primary infection or by T-cells during secondary infection, mutant mice that lacked H2-IAβb (Aβb^−/−) or β₂-microglobulin (β2m^−/−) were used. Since MHC molecules are required for the maturation of αβ T-cells, Aβb^−/− and β2m^−/− mutant mice are also deficient in functional αβ⁺CD4⁺ or αβ⁺CD8⁺ T-cells, respectively. As compared with wild-type control mice, oocyst output by mutant mice was not significantly affected during primary infection, suggesting that the ability of NK cells to control parasite replication is not dependent on the expression of MHC molecules. On reinfection, differences were observed for mutant mice as compared with controls. Aβb^−/− mice were found to be more susceptible than β2m^−/− mice, suggesting that the αβ⁺CD4⁺ T-cell subset plays a greater role in resistance to reinfection than does the αβ⁺CD8⁺ T-cell subset. The mechanism of resistance depends on the immune status of the host and requires the coordinated interaction of both αβ⁺ T-cell subsets for optimal parasite control during subsequent infections. Received: 6 August 1997 / Accepted: 24 October 1997     

Sustained expression of NADPH oxidase 4 by p38 MAPK-Akt signaling potentiates radiation-induced differentiation of lung fibroblasts

Radiation-induced fibrosis (RIF) is a long-term adverse effect of curative radiotherapy; however, the distinct molecular mechanisms of RIF in neighboring normal tissue are not fully understood. We investigated the mechanisms underlying radiation-induced fibroblast differentiation into myofibroblasts. Lung fibroblasts produced reactive oxygen species (ROS) immediately after irradiation, the level of which remained increased for 24 h. The NADPH oxidase inhibitor, diphenyleneiodonium (DPI), suppressed ROS production and significantly decreased the radiation-induced expression of α-smooth muscle actin (α-SMA) and fibronectin (FN). The mRNA and protein expression of Nox4 was increased by radiation, and siRNA knockdown of Nox4 reduced α-SMA and FN levels. Increased phosphorylation of p38MAPK, Erk, and PI3k/Akt was observed after irradiation. Inhibitors of p38 MAPK and Akt, but not of Erk, reduced radiation-induced fibroblast differentiation and Nox4 expression. Notably, DPI partially decreased phosphorylation of p38MAPK and Akt, suggesting that p38MAPK, Akt, and Nox4 may cooperate in a positive feedback loop. Nox4 expression was also increased during bleomycin-induced fibroblast differentiation, and downregulation of Nox4 reduced α-SMA levels and extracellular matrix (ECM) accumulation. These results demonstrate that interfering Nox4 activation can be a potential strategy to disrupt fibrotic process.     

Deletion of galectin-3 in the host attenuates metastasis of murine melanoma by modulating tumor adhesion and NK cell activity

Galectin-3, a β galactoside–binding lectin, plays an important role in the processes relevant to tumorigenesis such as malignant cell transformation, invasion and metastasis. We have investigated whether deletion of Galectin-3 in the host affects the metastasis of B16F1 malignant melanoma. Galectin-3-deficient (Gal-3^−/−) mice are more resistant to metastatic malignant melanoma as evaluated by number and size of metastatic colonies in the lung. In vitro assays showed lower number of attached malignant cells in the tissue section derived from Gal-3^−/− mice. Furthermore, lack of Galectin-3 correlates with higher serum levels of IFN-γ and IL-17 in tumor bearing hosts. Interestingly, spleens of Gal-3^−/− mice have lower number of Foxp3⁺ T cells after injection of B16F1 melanoma cells. Finally, we found that while CD8⁺ T cell and adherent cell cytotoxicity were similar, there was greater cytotoxic activity of splenic NK cells of Gal-3^−/− mice compared with “wild-type” (Gal-3 ^+/+ ) mice. Despite the reduction in total number of CD3ε⁻NK1.1⁺, Gal-3^−/− mice constitutively have a significantly higher percentage of effective cytotoxic CD27^highCD11b^high NK cells as well as the percentage of immature CD27^highCD11b^low NK cells. In contrast, CD27^lowCD11b^high less functionally exhausted NK cells and NK cells bearing inhibitory KLRG1 receptor were more numerous in Gal-3 ^+/+ mice. It appears that lack of Galectin-3 affects tumor metastasis by at least two independent mechanisms: by a decrease in binding of melanoma cells onto target tissue and by enhanced NK-mediated anti-tumor response suggesting that Galectin-3 may be considered as therapeutic target.     

Acid sphingomyelinase gene deficiency ameliorates the hyperhomocysteinemia-induced glomerular injury in mice

Hyperhomocysteinemia (hHcys) enhances ceramide production, leading to the activation of NADPH oxidase and consequent glomerular oxidative stress and sclerosis. The present study was performed to determine whether acid sphingomyelinase (Asm), a ceramide-producing enzyme, is implicated in the development of hHcys-induced glomerular oxidative stress and injury. Uninephrectomized Asm-knockout (Asm(-/-)) and wild-type (Asm(+/+)) mice, with or without Asm short hairpin RNA (shRNA) transfection, were fed a folate-free (FF) diet for 8 weeks, which significantly elevated the plasma Hcys level compared with mice fed normal chow. By using in vivo molecular imaging, we found that transfected shRNAs were expressed in the renal cortex starting on day 3 and continued for 24 days. The FF diet significantly increased renal ceramide production, Asm mRNA and activity, urinary total protein and albumin excretion, glomerular damage index, and NADPH-dependent superoxide production in the renal cortex from Asm(+/+) mice compared with that from Asm(-/-) or Asm shRNA-transfected wild-type mice. Immunofluorescence analysis showed that the FF diet decreased the expression of podocin but increased desmin and ceramide levels in glomeruli from Asm(+/+) mice but not in those from Asm(-/-) and Asm shRNA-transfected wild-type mice. In conclusion, our observations reveal that Asm plays a pivotal role in mediating podocyte injury and glomerular sclerosis associated with NADPH oxidase-associated local oxidative stress during hHcys.     

The mineralization phenotype in Abcc6 −/− mice is affected by Ggcx gene deficiency and genetic background—a model for pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder characterized by ectopic mineralization of connective tissues and shows considerable intra- and inter-familial phenotypic variability. PXE is caused by mutations in the ABCC6 gene, and targeted ablation of Abcc6 in mouse recapitulates PXE. In this study, we examined the hypothesis that the GGCX gene encoding γ-glutamyl carboxylase may interfere with the mineralization process in Abcc6 ^−/− mice. Thus, Abcc6 ^−/− and Ggcx ^+/− mice were generated on 129S1;C57 and 129S1;129X1;C57 genetic backgrounds, respectively, and backcrossed with C57BL/6J for five generations. Thus, these strains differ by the 129X1 contribution to the background of the mice. We then generated Abcc6 ^−/− ;Ggcx ^+/+ and Abcc6 ^−/− ;Ggcx ^+/− mice by crossing Abcc6 ^−/− and Ggcx ^+/− mice. The degree of mineralization of connective capsule of vibrissae, a biomarker of the mineralization process in PXE, was evaluated by computerized morphometric analysis and quantified colorimetrically by calcium and phosphate levels in tissues. The mineralization of the vibrissae in Abcc6 ^−/− mice takes place at ∼5–6 weeks of age and is significantly enhanced at 3 months of age in comparison to wild-type mice (>10-fold, p < 0.001). However, the onset of mineralization in Abcc6 ^−/− ;Ggcx ^+/+ mice was delayed until between 3 and 4 months of age, suggesting that the genetic background plays a role in modifying the mineralization process. The mineralization in the Abcc6 ^−/− ;Ggcx ^+/− mice was accelerated in comparison with age-matched Abcc6 ^−/− ;Ggcx ^+/+ mice, with ∼3-fold difference at 3, 4, and 9 months of age (p < 0.01). The mineralization process was also accelerated in these mice by a special custom-designed diet with mineral modifications. These findings suggest a role for both the GGCX gene and the genetic background as well as dietary factors in modulating the phenotypic severity of PXE caused by loss-of-function mutations in ABCC6.     

保护素D1 通过抑制肾脏炎症及足突细胞上皮-间充质转化而抑制早期 糖尿病肾病小鼠肾纤维化

 目的：保护素D1 (PD1) 是一个潜在的抗炎症脂蛋白分子,本实验探讨其治疗早期糖尿病肾病（DN）肾纤维化的作用及机制。方法：用链脲佐菌素125 mg/kg 2次腹腔注射C57BL/6J雌性小鼠,建立早期DN小鼠模型。糖尿病模型成功后,用PD1（0.08 mg·kg-1·d-1）腹腔注射治疗,设正常鼠及DN鼠为对照。治疗8周后检测各组小鼠24 h尿蛋白及尿白蛋白定量、体重、肾重、肾重/体重比、血清及尿肌酐和肌酐清除率;用PAS染色法检测肾小球系膜区基质/肾小球面积比,用免疫荧光染色法检测肾皮质中巨噬细胞的数量,用Western blotting检测肾小球纤连蛋白（FN）和α-平滑肌肌动蛋白（α-SMA）的表达,以及肾小球足突细胞特异性上皮标志蛋白zonula occludens-1(ZO-1)和P-cadherin的表达;同时,体外用高糖刺激小鼠巨噬细胞株RAW264.7,检测PD1对其分泌肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）的抑制作用。体外用转化生长因子β1（TGF-β1）刺激小鼠足突细胞株,用Western blotting检测PD1对其诱导足突细胞上皮-间充质转化(EMT)中上皮细胞标志蛋白P-cadherin 和ZO-1减少的恢复作用,及间充质细胞标志蛋白成纤维细胞特异性蛋白1（FSP1） 和α-SMA过表达的抑制作用。结果：PD1能减少DN小鼠肾小球系膜基质的积聚、24 h尿蛋白及尿白蛋白定量、体重、肾重和肾重/体重比,抑制异常增高的肌酐清除率。PD1能减少DN小鼠肾皮质中巨噬细胞的数量,抑制DN小鼠肾小球FN和α-SMA的表达,恢复足突细胞特异性上皮标志蛋白ZO-1和P-cadherin的表达。PD1能抑制高糖诱导RAW264.7分泌TNF-α和IL-1β,能抑制TGF-β1诱导足突细胞FSP1和α-SMA表达的增加以及ZO-1和P-cadherin表达的减少。结论：PD1能减轻早期DN小鼠肾纤维化,其部分机制可能通过抑制肾脏的炎症及足突细胞EMT。     

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin–angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of α actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of α actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.