Interleukin-6 plays a protective role in development of cisplatin-induced acute renal failure through upregulation of anti-oxidative stress factors

AimsCisplatin, a major chemotherapeutic agent, accumulates in proximal tubules of the kidneys and causes acute renal failure dose-dependently. We previously reported that cisplatin induced more severe renal dysfunction in interleukin-6 (IL-6) knockout (IL-6^?/?) mice than in wild-type (WT) mice. Expression of a pro-apoptotic protein was significantly increased with cisplatin in IL-6^?/? mice compared to that in WT mice. IL-6, locally expressed in renal tubular cells after cisplatin administration, prevents the development of renal dysfunction at an early stage. In the present study, we focused on downstream signals of IL-6 and oxidative stress induced by cisplatin in order to evaluate the protective role of IL-6 in the development of acute renal failure.Main methodsWT and IL-6^?/? mice were given either cisplatin (30 mg/kg) or saline intraperitoneally. Blood and kidney samples were collected at 24 h and 72 h after cisplatin administration. The changes in expression of 4-hydroxy-2-nonenal protein (4-HNE, oxidative stress marker) and cyclooxygenase-2 (cox-2), activities of superoxide dismutases and caspase-3, and phosphorylation of extracellular signal-regulated kinase (ERK) were examined.Key findingsCisplatin increased the expression of 4-HNE and cox-2, and phosphorylation of ERK in IL-6^?/? mice than in WT mice. On the other hand, activity of superoxide dismutase, an anti-oxidative enzyme, was significantly decreased in the kidney obtained from IL-6^?/? mice after cisplatin administration.SignificanceOur findings suggest that IL-6 plays a protective role in the development of cisplatin-induced acute renal failure through upregulation of anti-oxidative stress factors.     

Serine palmitoyl-CoA transferase (SPT) deficiency and sphingolipid levels in mice

Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, and all these functions may have an impact on atherosclerotic development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme in sphingolipid biosynthesis. To evaluate in vivo SPT activity and its role in sphingolipid metabolism, we applied homologous recombination to embryonic stem cells, producing mice with long chain base 1 (Sptlc1) and long chain base 2 (Sptlc2), two subunits of SPT, gene deficiency. Homozygous Sptlc11 and Sptlc2 mice are embryonic lethal, whereas heterozygous versions of both animals (Sptlc1^+/?, Sptlc2^+/?) are healthy. Analysis showed that, compared with WT mice, Sptlc1^+/? and Sptlc2^+/? mice had: (1) decreased liver Sptlc1 and Sptlc2 mRNA by 44% and 57% (P < 0.01 and P < 0.0001, respectively); (2) decreased liver Sptlc1 mass by 50% and Sptlc2 mass by 70% (P < 0.01 and P < 0.01, respectively), moreover, Sptlc1 mass decreased by 70% in Sptlc2^+/? mouse liver, while Sptlc2 mass decreased by 53% in Sptlc1^+/? mouse liver (P < 0.001 and P < 0.01, respectively); (3) decreased liver SPT activity by 45% and 60% (P < 0.01, respectively); (4) decreased liver ceramide (22% and 39%, P < 0.05 and P < 0.01, respectively) and sphingosine levels (22% and 31%, P < 0.05 and P < 0.01, respectively); (5) decreased plasma ceramide (45% and 39%, P < 0.01, respectively), sphingosine-1-phosphate (31% and 32%, P < 0.01, respectively) and sphingosine levels (22.5% and 25%, P < 0.01, respectively); (6) dramatically decreased plasma lysosphingomyelin (17-fold and 16-fold, P < 0.0001, respectively); and (7) no change of plasma sphingomyelin, triglyceride, total cholesterol, phospholipids, and liver sphingomyelin levels. These results indicated that both Sptlc1 and Sptlc2 interactions are necessary for SPT activity in vivo, and that SPT activity directly influences plasma sphingolipid levels. Furthermore, manipulation of SPT activity might well influence the course of such diseases as atherosclerosis.     

Angiotensin-converting enzyme 2 ameliorates renal fibrosis by blocking the activation of mTOR/ERK signaling in apolipoprotein E-deficient mice

Angiotensin-converting enzyme 2 (ACE2) has been shown to prevent atherosclerotic lesions and renal inflammation. However, little was elucidated upon the effects and mechanisms of ACE2 in atherosclerotic kidney fibrosis progression. Here, we examined regulatory roles of ACE2 in renal fibrosis in the apolipoprotein E (ApoE) knockout (KO) mice. The ApoEKO mice were randomized to daily deliver either angiotensin (Ang) II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. Downregulation of ACE2 and upregulation of phosphorylated Akt, mTOR and ERK1/2 levels were observed in ApoEKO kidneys. Ang II infusion led to increased tubulointerstitial fibrosis in the ApoEKO mice with greater activation of the mTOR/ERK1/2 signaling. The Ang II-mediated renal fibrosis and structural injury were strikingly rescued by rhACE2 supplementation, associated with reduced mRNA expression of TGF-β1 and collagen I and elevated renal Ang-(1–7) levels. In cultured mouse kidney fibroblasts, exposure with Ang II (100 nmol L⁻¹) resulted in obvious elevations in superoxide generation, phosphorylated levels of mTOR and ERK1/2 as well as mRNA levels of TGF-β1, collagen I and fibronectin 1, which were dramatically prevented by rhACE2 (1 mg mL⁻¹) or mTOR inhibitor rapamycin (10 μmol L⁻¹). These protective effects of rhACE2 were eradicated by the Ang-(1–7)/Mas receptor antagonist A779 (1 μmol L⁻¹). Our results demonstrate the importance of ACE2 in amelioration of kidney fibrosis and renal injury in the ApoE-mutant mice via modulation of the mTOR/ERK signaling and renal Ang-(1–7)/Ang II balance, thus indicating potential therapeutic strategies by enhancing ACE2 action for preventing atherosclerosis and fibrosis-associated kidney disorders.     

Glucagon receptor is required for long-term survival: A natural history study of the Mahvash disease in a murine model

Background and aimWe have described a novel Mahvash disease of hyperglucagonemia and pancreatic neuroendocrine tumors (PNETs) associated with an inactivating glucagon receptor mutation, and identified the glucagon receptor-deficient (Gcgr^?/?) mice as its murine model. We aim to elucidate the natural history of the rare Mahvash disease by long-term observation of the Gcgr^?/? mice.Materials and methodWild type (WT) (n = 52), heterozygous (n = 127), and Gcgr^?/? (n = 56) mice living under standard vivarium conditions were observed without specific treatments over 22 months. Autopsy was performed on dead animals.ResultsThe WT and heterozygous mice did not exhibit any measurable differences. The Gcgr^?/? mice became progressively lethargic and cachexic after 12 months. Random glucose levels were stable in WT and heterozygous mice but decreased with age in the Gcgr^?/? mice. At the end of observation, 28/56 Gcgr^?/?, 7/52 WT, and 24/127 heterozygous mice died. The survival curve of Gcgr^?/? mice began to separate from those of WT and heterozygous mice at 12 months and the survival difference widened with age. At 18 months, survival probability was 17% for Gcgr^?/? mice but 77% for WT and 81% for heterozygous mice. Autopsy revealed numerous PNETs up to 15 mm in diameter in most well-preserved Gcgr^?/? pancreata (17/20) but none in WT or heterozygous ones. Four Gcgr^?/? mice developed liver or subcutaneous metastasis.ConclusionThe untreated Mahvash disease may cause cachexia, severe hypoglycemia, and early death. Patients with Mahvash disease need to undergo life-long surveillance for PNETs. Functional glucagon receptor is thus required for long-term survival.     

Effects of nitric oxide on renal interstitial fibrosis in rats with unilateral ureteral obstruction

AimsIt is well recognized that microvascular injury is a major determinant of renal fibrosis. Mounting evidence shows that nitric oxide (NO) plays an important role in angiogenesis. Therefore, we investigated to the effects of NO on kidney angiogenesis and renal fibrosis.MethodsIn the present study, a unilateral ureteral obstruction (UUO) model was established with l-arginine (l-Arg, 1 g/dl) and N-nitro-l-arginine methyl ester (L-NAME, 5 mg/dl) serving as interference factors. We investigated the alteration of NO concentration with spectrophotometry, peritubular capillary (PTC) density with aminopeptidase P (JG12) immunohistochemical staining, and the expression of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) with immunohistochemical staining and Western blotting at weeks 2, 3 and 4.Key findingsOur findings showed that the expressions of VEGF, eNOS and PTC density were significantly decreased in rats with UUO, which was accompanied by a progressive increase in HIF-1α, TGF-β1 and an area of renal interstitial fibrosis. The administration of l-Arg promoted the synthesis of NO and significantly elevated the expressions of VEGF, eNOS and PTC density with the conspicuous loss of HIF-1α and TGF-β1 expressions and ultimately ameliorated renal fibrosis, which was markedly aggravated by L-NAME administration.SignificanceThese findings demonstrate that NO appears to play an important role in kidney angiogenesis and in slowing the progression of renal interstitial fibrosis, which suggests that NO may serve as a novel therapeutic strategy for preventing renal fibrosis as well as fibrosis in other organs.     

Reduced NOV/CCN3 Expression Limits Inflammation and Interstitial Renal Fibrosis after Obstructive Nephropathy in Mice

The main hallmark of chronic kidney disease (CKD) is excessive inflammation leading to interstitial tissue fibrosis. It has been recently reported that NOV/CCN3 could be involved in kidney damage but its role in the progression of nephropathies is poorly known. NOV/CCN3 is a secreted multifunctional protein belonging to the CCN family involved in different physiological and pathological processes such as angiogenesis, inflammation and cancers. The purpose of our study was to determine the role of NOV/CCN3 in renal inflammation and fibrosis related to primitive tubulointerstitial injury. After unilateral ureteral obstruction (UUO), renal histology and real-time PCR were performed in NOV/CCN3-/- and wild type mice. NOV/CCN3 mRNA expression was increased in the obstructed kidneys in the early stages of the obstructive nephropathy. Interestingly, plasmatic levels of NOV/CCN3 were strongly induced after 7 days of UUO and the injection of recombinant NOV/CCN3 protein in healthy mice significantly increased CCL2 mRNA levels. Furthermore, after 7 days of UUO NOV/CCN3-/- mice displayed reduced proinflammatory cytokines and adhesion markers expression leading to restricted accumulation of interstitial monocytes, in comparison with their wild type littermates. Consequently, in NOV/CCN3-/- mice interstitial renal fibrosis was blunted after 15 days of UUO. In agreement with our experimental data, NOV/CCN3 expression was highly increased in biopsies of patients with tubulointerstitial nephritis. Thus, the inhibition of NOV/CCN3 may represent a novel target for the progression of renal diseases.     

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.     

Activation of cardiac hypertrophic signaling pathways in a transgenic mouse with the human PRKAG2 Thr400Asn mutation

Human mutations in PRKAG2, the gene encoding the γ2 subunit of AMP activated protein kinase (AMPK), cause a glycogen storage cardiomyopathy. In a transgenic mouse with cardiac specific expression of the Thr400Asn mutation in PRKAG2 (TG^T400N), we previously reported initial cardiac hypertrophy (ages 2–8 weeks) followed by dilation and failure (ages 12–20 weeks). We sought to elucidate the molecular mechanisms of cardiac hypertrophy. TG^T400N mice showed significantly increased cardiac mass/body mass ratios up to ～ 3-fold beginning at age 2 weeks. Cardiac expression of ANP and BNP were ～ 2- and ～ 5-fold higher, respectively, in TG^T400N relative to wildtype (WT) mice at age 2 weeks. NF-κB activity and nuclear translocation of the p50 subunit were increased ～ 2- to 3-fold in TG^T400N hearts relative to WT during the hypertrophic phase. Phosphorylated Akt and p70S6K were elevated ～ 2-fold as early as age 2 weeks. To ascertain whether these changes in TG^T400N mice were a consequence of increased AMPK activity, we crossbred TG^T400N with TG^α2DN mice, which express a dominant negative, kinase dead mutant of the AMPK α2 catalytic subunit and have low myocardial AMPK activity. Genetic reversal of AMPK overactivity led to a reduction in hypertrophy, nuclear translocation of NF-κB, phosphorylated Akt, and p70S6K. We conclude that inappropriate activation of AMPK secondary to the T400N PRKAG2 mutation is associated with the early activation of NF-κB and Akt signaling pathway, which mediates cardiac hypertrophy.     

The Notch γ-secretase inhibitor ameliorates kidney fibrosis via inhibition of TGF-β/Smad2/3 signaling pathway activation

Kidney fibrosis is a common feature of chronic kidney disease (CKD). A recent study suggests that abnormal Notch signaling activation contributes to the development of renal fibrosis. However, the molecular mechanism that regulates this process remains unexplored. Unilateral ureteral obstruction (UUO) or sham-operated C57BL6 mice (aged 10 weeks) were randomly assigned to receive dibenzazepine (DBZ, 250 μg/100 g/d) or vehicle for 7 days. Histologic examinations were performed on the kidneys using Masson's trichrome staining and immunohistochemistry. Real-time PCR and western blot analysis were used for detection of mRNA expression and protein phosphorylation. The expression of Notch 1, 3, and 4, Notch intracellular domain (NICD), and its target genes Hes1 and HeyL were upregulated in UUO mice, while the increase in NICD protein was significantly attenuated by DBZ. After 7 days, the severity of renal fibrosis and expression of fibrotic markers, including collagen 1α1/3α1, fibronectin, and α-smooth muscle actin, were markedly increased in UUO compared with sham mice. In contrast, administration of DBZ markedly attenuated these effects. Furthermore, DBZ significantly inhibited UUO-induced expression of transforming growth factor (TGF)-β, phosphorylated Smad 2, and Smad 3. Mechanistically, Notch signaling activation in tubular epithelial cells enhanced fibroblast proliferation and activation in a coculture experiment. Our study provides evidence that Notch signaling is implicated in renal fibrogenesis. The Notch inhibitor DBZ can ameliorate this process via inhibition of the TGF-β/Smad2/3 signaling pathway, and might be a novel drug for preventing chronic kidney disease.     

Endogenous osteopontin induces myocardial CCL5 and MMP-2 activation that contributes to inflammation and cardiac remodeling in a mouse model of chronic Chagas heart disease

Cardiac dysfunction with progressive inflammation and fibrosis is a hallmark of Chagas disease caused by persistent Trypanosoma cruzi infection. Osteopontin (OPN) is a pro-inflammatory cytokine that orchestrates mechanisms controlling cell recruitment and cardiac architecture. Our main goal was to study the role of endogenous OPN as a modulator of myocardial CCL5 chemokine and MMP-2 metalloproteinase, and its pathological impact in a murine model of Chagas heart disease. Wild-type (WT) and OPN-deficient (spp1 ?/?) mice were parasite-infected (Brazil strain) for 100 days. Both groups developed chronic myocarditis with similar parasite burden and survival rates. However, spp1 ?/? infection showed lower heart-to-body ratio (P < 0.01) as well as reduced inflammatory pathology (P < 0.05), CCL5 expression (P < 0.05), myocyte size (P < 0.05) and fibrosis (P < 0.01) in cardiac tissues. Intense OPN labeling was observed in inflammatory cells recruited to infected heart (P < 0.05). Plasma concentration of MMP-2 was higher (P < 0.05) in infected WT than in spp1 ?/? mice. Coincidently, specific immunostaining revealed increased gelatinase expression (P < 0.01) and activity (P < 0.05) in the inflamed hearts from T. cruzi WT mice, but not in their spp1 ?/? littermates. CCL5 and MMP-2 induction occurred preferentially (P < 0.01) in WT heart-invading CD8⁺ T cells and was mediated via phospho-JNK MAPK signaling. Heart levels of OPN, CCL5 and MMP-2 correlated (P < 0.01) with collagen accumulation in the infected WT group only. Endogenous OPN emerges as a key player in the pathogenesis of chronic Chagas heart disease, through the upregulation of myocardial CCL5/MMP-2 expression and activities resulting in pro-inflammatory and pro-hypertrophic events, cardiac remodeling and interstitial fibrosis.     

L-Endoglin Overexpression Increases Renal Fibrosis after Unilateral Ureteral Obstruction

Transforming growth factor-β (TGF-β) plays a pivotal role in renal fibrosis. Endoglin, a 180 KDa membrane glycoprotein, is a TGF-β co-receptor overexpressed in several models of chronic kidney disease, but its function in renal fibrosis remains uncertain. Two membrane isoforms generated by alternative splicing have been described, L-Endoglin (long) and S-Endoglin (short) that differ from each other in their cytoplasmic tails, being L-Endoglin the most abundant isoform. The aim of this study was to assess the effect of L-Endoglin overexpression in renal tubulo-interstitial fibrosis. For this purpose, a transgenic mouse which ubiquitously overexpresses human L-Endoglin (L-ENG⁺) was generated and unilateral ureteral obstruction (UUO) was performed in L-ENG⁺ mice and their wild type (WT) littermates. Obstructed kidneys from L-ENG⁺ mice showed higher amounts of type I collagen and fibronectin but similar levels of α-smooth muscle actin (α-SMA) than obstructed kidneys from WT mice. Smad1 and Smad3 phosphorylation were significantly higher in obstructed kidneys from L-ENG⁺ than in WT mice. Our results suggest that the higher increase of renal fibrosis observed in L-ENG⁺ mice is not due to a major abundance of myofibroblasts, as similar levels of α-SMA were observed in both L-ENG⁺ and WT mice, but to the higher collagen and fibronectin synthesis by these fibroblasts. Furthermore, in vivo L-Endoglin overexpression potentiates Smad1 and Smad3 pathways and this effect is associated with higher renal fibrosis development.     

Sphingomyelin synthase 2 over-expression induces expression of aortic inflammatory biomarkers and decreases circulating EPCs in ApoE KO mice

AimsThis study sought to assess the effect of sphingomyelin synthase 2 (SMS2) over-expression on plaque component and endothelial dysfunction in atherosclerosis.Main methodsWe generated recombinant adenovirus vectors containing human SMS2 cDNA (AdV-SMS2) or control gene GFP cDNA (AdV-GFP). Both AdVs were injected (i.v.) into ApoE KO mice to establish SMS2 over-expressing and control mice models, respectively. The mice were fed a high fat diet for 30 days. We then examined their plasma lipid levels, expression levels of aortic inflammatory biomarkers critical for the plaque's stability, and numbers of peripheral endothelial progenitor cells (EPC).Key findingsCompared with the control mice, SMS2 over-expression had significantly (1) increased aortic matrix metalloproteinase-2 (MMP-2), monocyte chemoattractant protein-1 (MCP-1), tissue factor (TF) and cyclooxygenase-2 (COX-2) mRNA levels (1.9-fold, 2.2-fold, 2.6-fold and 3.2-fold, respectively, P < 0.01) and protein levels (2.2-fold, 1.9-fold, 1.9-fold and 2.1-fold, respectively, P < 0.01); (2) increased MMP-2, COX-2 in situ expression in aortic root (2.6-fold and 2.3-fold, respectively, P < 0.01); (3) decreased aortic COX-1 mRNA levels (65%, P < 0.01) and protein levels (64%, P < 0.01); and (4) decreased CD34/KDR-positive cells (33%, P < 0.01), circulating angiogenic cells (CACs) (50%, P < 0.05), and colony forming units (CFUs) (40%, P < 0.05) in circulation.SignificanceSMS2 over-expression was probably associated with increased expression of aortic inflammatory biomarkers, as well as decreased numbers of CD34/KDR-positive cells, CACs and CFUs in circulation. Therefore, SMS2 over-expression might correlate with endothelial dysfunction and aggravate atherosclerotic plaque instability in ApoE KO mice.     

Estrogen dependence of the renal vasodilatory effect of nicotine in rats: role of α7 nicotinic cholinergic receptor/eNOS signaling

AimsWe recently reported that acute exposure to nicotine vasodilates the renal vasculature of male rats via facilitation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether this effect of nicotine is sexually dimorphic and the role of estrogen in modulating the nicotine effect.Main methodsNicotine-evoked vasodilation was evaluated in phenylephrine-preconstricted perfused kidneys obtained from male, proestrus female, ovariectomized (OVX) and estrogen-replaced OVX (OVXE₂) rats.Key findingsNicotine infusion (5 × 10^? 5, 1 × 10^? 4, and 5 × 10^? 4 M) produced greater concentration-dependent reductions in the renal perfusion pressure (RPP) in an isolated kidney from proestrus females than from males. Inhibition of NOS by N^G-nitro-l-arginine abolished the nicotine-evoked reduction in RPP and abolished the gender difference in the nicotine effect. Nicotine vasodilation was also attenuated in kidneys isolated from OVX and diestrus rats, models characterized by reduced estrogen levels. Further, estrogen or l-arginine supplementation in OVX rats largely restored the renal vasodilatory response to nicotine. Estrogen receptor blockade by tamoxifen abrogated the enhanced nicotine-evoked vasodilation elicited by E₂ in OVX rats. The nitrite/nitrate levels and protein expressions of eNOS and α₇ nicotinic cholinergic receptor (α₇ nAChRs) were significantly higher in renal tissues of OVXE₂ compared with OVX rats, suggesting a facilitatory effect for E₂ on α₇ nAChRs/eNOS signaling.SignificanceEstrogen-dependent facilitation of NOS signaling mediates the enhanced vasodilator capacity of nicotine in the renal vasculature of female rats. Preliminary evidence also suggests a potential role for α₇ nAChRs in this estrogen-dependent phenomenon.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Ablation of Gadd45β ameliorates the inflammation and renal fibrosis caused by unilateral ureteral obstruction

The growth arrest and DNA damage‐inducible beta (Gadd45β) protein have been associated with various cellular functions, but its role in progressive renal disease is currently unknown. Here, we examined the effect of Gadd45β deletion on cell proliferation and apoptosis, inflammation, and renal fibrosis in an early chronic kidney disease (CKD) mouse model following unilateral ureteral obstruction (UUO). Wild‐type (WT) and Gadd45β‐knockout (KO) mice underwent either a sham operation or UUO and the kidneys were sampled eight days later. A histological assay revealed that ablation of Gadd45β ameliorated UUO‐induced renal injury. Cell proliferation was higher in Gadd45β KO mouse kidneys, but apoptosis was similar in both genotypes after UUO. Expression of pro‐inflammatory cytokines after UUO was down‐regulated in the kidneys from Gadd45β KO mice, whereas UUO‐mediated immune cell infiltration remained unchanged. The expression of pro‐inflammatory cytokines in response to LPS stimulation decreased in bone marrow‐derived macrophages from Gadd45β KO mice compared with that in WT mice. Importantly, UUO‐induced renal fibrosis was ameliorated in Gadd45β KO mice unlike in WT mice. Gadd45β was involved in TGF‐β signalling pathway regulation in kidney fibroblasts. Our findings demonstrate that Gadd45β plays a crucial role in renal injury and may be a therapeutic target for the treatment of CKD.     

Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

BackgroundClinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases.MethodsAntibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones.ResultsCross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5α-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R² = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice.ConclusionWe describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia.     

Atg5 deficiency-mediated mitophagy aggravates cardiac inflammation and injury in response to angiotensin II

ObjectiveHypertension induces end-organ damage through inflammation, and autophagy plays a crucial role in the regulation of cellular homeostasis. In the present study, we aimed to define the role of autophagy in the development of inflammation and cardiac injury induced by angiotensin II (Ang II).Methods and ResultsAutophagy protein 5 (Atg5) haplodeficiency (Atg5^+/−) and age-matched wild-type (WT) C57BL/6 J mice were infused with Ang II (1500 ng/kg/min) or saline for 7 days. Heart sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical stains. Cytokine and LC3 levels were measured using real-time PCR or western blot analysis. After Ang II infusion, the WT mice exhibited marked macrophage accumulation, cytokine expression, and reactive oxygen species (ROS) production compared with saline-infused controls. However, these effects induced by Ang II infusion were aggravated in Atg5^+/− mice. These effects were associated with Atg5-mediated impaired autophagy, accompanied by increased production of ROS and activation of nuclear factor-κB (NF-κB) in macrophages. Finally, increased cardiac inflammation in Atg5 haplodeficient mice was associated with increased cardiac fibrosis.ConclusionAtg5 deficiency-mediated autophagy increases ROS production and NF-κB activity in macrophages, thereby contributing to cardiac inflammation and injury. Thus, improving autophagy may be a novel therapeutic strategy to ameliorate hypertension-induced inflammation and organ damage.