Guanylyl Cyclase-G Modulates Jejunal Apoptosis and Inflammation in Mice with Intestinal Ischemia and Reperfusion

Background

Membrane bound guanylyl cyclase-G (mGC-G), a novel form of GC mediates ischemia and reperfusion (IR)-induced renal injury. We investigated the roles of mGC-G in intestinal IR-induced jejunal damage, inflammation, and apoptosis.

Materials and methods

Male C57BL/6 wild-type (WT) and mGC-G gene knockout (KO) mice were treated with a sham operation or 45 min of superior mesenteric arterial obstruction followed by 3, 6, 12, or 24 h of reperfusion.

Results

Sham-operated KO mice had significantly lower plasma nitrate and nitrite (NOx) levels and jejunal villus height, crypt depth, and protein expression of phosphorylated-nuclear factor-kappa-B (NF-κB), phosphorylated-c-Jun N-terminal kinases (JNK) 2/3, phosphorylated-p38, and B-cell lymphoma-2 (Bcl-2). They had significantly greater jejunal interleukin-6 mRNA, cytochrome c protein, and apoptotic index compared with sham-operated WT mice. Intestinal IR significantly decreased plasma NOx in WT mice and increased plasma NOx in KO mice. The jejunal apoptotic index and caspase 3 activities were significantly increased, and nuclear phosphorylated-NF-κB and phosphorylated-p38 protein were significantly decreased in WT, but not KO mice with intestinal IR. After reperfusion, KO mice had an earlier decrease in jejunal cyclic GMP, and WT mice had an earlier increase in jejunal proliferation and a later increase in cytosol inhibitor of kappa-B-alpha. Intestinal IR induced greater increases in plasma and jejunal interleukin-6 protein in WT mice and a greater increase in jejunal interleukin-6 mRNA in KO mice.

Conclusions

mGC-G is involved in the maintenance of jejunal integrity and intestinal IR-induced inflammation and apoptosis. These results suggest that targeting cGMP pathway might be a potential strategy to alleviate IR-induced jejunal damages.     

Genetic disruption of soluble epoxide hydrolase is protective against streptozotocin-induced diabetic nephropathy

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important roles in regulating cardiovascular functions. The anti-inflammatory, antiapoptotic, proangiogenic, and antihypertensive properties of EETs suggest a beneficial role for EETs in diabetic nephropathy. Endogenous EET levels are maintained by a balance between synthesis by CYP epoxygenases and hydrolysis by epoxide hydrolases into physiologically less active dihydroxyeicosatrienoic acids. Genetic disruption of soluble epoxide hydrolase (sEH/EPHX2) results in increased EET levels through decreased hydrolysis. This study investigated the effects of sEH gene disruption on diabetic nephropathy in streptozotocin-induced diabetic mice. Streptozotocin-induced diabetic manifestations were attenuated in sEH-deficient mice relative to wild-type controls, with significantly decreased levels of Hb A(1c), creatinine, and blood urea nitrogen and urinary microalbumin excretion. The sEH-deficient diabetic mice also had decreased renal tubular apoptosis that coincided with increased levels of antiapoptotic Bcl-2 and Bcl-xl, and decreased levels of the proapoptotic Bax. These effects were associated with activation of the PI3K-Akt-NOS3 and AMPK signaling cascades. sEH gene inhibition and exogenous EETs significantly protected HK-2 cells from TNFα-induced apoptosis in vitro. These findings highlight the beneficial role of the CYP epoxygenase-EETs-sEH system in the pathogenesis of diabetic nephropathy and suggest that the sEH inhibitors available may be potential therapeutic agents for this condition.     

Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.     

Suppression of Rapidly Progressive Mouse Glomerulonephritis with the Non-Steroidal Mineralocorticoid Receptor Antagonist BR-4628

Background/Aim

Steroidal mineralocorticoid receptor antagonists (MRAs) are effective in the treatment of kidney disease; however, the side effect of hyperkalaemia, particularly in the context of renal impairment, is a major limitation to their clinical use. Recently developed non-steroidal MRAs have distinct characteristics suggesting that they may be superior to steroidal MRAs. Therefore, we explored the benefits of a non-steroidal MRA in a model of rapidly progressive glomerulonephritis.

Methods

Accelerated anti-glomerular basement membrane (GBM) glomerulonephritis was induced in groups of C57BL/6J mice which received no treatment, vehicle or a non-steroidal MRA (BR-4628, 5mg/kg/bid) from day 0 until being killed on day 15 of disease. Mice were examined for renal injury.

Results

Mice with anti-GBM glomerulonephritis which received no treatment or vehicle developed similar disease with severe albuminuria, impaired renal function, glomerular tuft damage and crescents in 40% of glomeruli. In comparison, mice which received BR-4628 displayed similar albuminuria, but had improved renal function, reduced severity of glomerular tuft lesions and a 50% reduction in crescents. The protection seen in BR-4628 treated mice was associated with a marked reduction in glomerular macrophages and T-cells and reduced kidney gene expression of proinflammatory (CCL2, TNF-α, IFN-γ) and profibrotic molecules (collagen I, fibronectin). In addition, treatment with BR-4626 did not cause hyperkalaemia or increase urine Na+/K+ excretion (a marker of tubular dysfunction).

Conclusions

The non-steroidal MRA (BR-4628) provided substantial suppression of mouse crescentic glomerulonephritis without causing tubular dysfunction. This finding warrants further investigation of non-steroidal MRAs as a therapy for inflammatory kidney diseases.     

Magnetic Resonance Imaging Is Sensitive to Pathological Amelioration in a Model for Laminin-Deficient Congenital Muscular Dystrophy (MDC1A)

Purpose

To elucidate the reliability of MRI as a non-invasive tool for assessing in vivo muscle health and pathological amelioration in response to Losartan (Angiotensin II Type 1 receptor blocker) in DyW mice (mouse model for Laminin-deficient Congenital Muscular Dystrophy Type 1A).

Methods

Multiparametric MR quantifications along with histological/biochemical analyses were utilized to measure muscle volume and composition in untreated and Losartan-treated 7-week old DyW mice.

Results

MRI shows that DyW mice have significantly less hind limb muscle volume and areas of hyperintensity that are absent in WT muscle. DyW mice also have significantly elevated muscle levels (suggestive of inflammation and edema). Muscle T₂ returned to WT levels in response to Losartan treatment. When considering only muscle pixels without T₂ elevation, DyW T₂ levels are significantly lower than WT (suggestive of fibrosis) whereas Losartan-treated animals do not demonstrate this decrease in muscle T₂. MRI measurements suggestive of elevated inflammation and fibrosis corroborate with increased Mac-1 positive cells as well as increased Picrosirius red staining/COL1a gene expression that is returned to WT levels in response to Losartan.

Conclusions

MRI is sensitive to and tightly corresponds with pathological changes in DyW mice and thus is a viable and effective non-invasive tool for assessing pathological changes.     

Protective Effect of Creatine Elevation against Ischaemia Reperfusion Injury Is Retained in the Presence of Co-Morbidities and during Cardioplegia

Aims

Ischaemic heart disease is most prevalent in the ageing population and often exists with other comorbidities; however the majority of laboratory research uses young, healthy animal models. Several recent workshops and focus meetings have highlighted the importance of using clinically relevant models to help aid translation to realistic patient populations. We have previously shown that mice over-expressing the creatine transporter (CrT-OE) have elevated intracellular creatine levels and are protected against ischaemia-reperfusion injury. Here we test whether elevating intracellular creatine levels retains a cardioprotective effect in the presence of common comorbidities and whether it is additive to protection afforded by hypothermic cardioplegia.

Methods and Results

CrT-OE mice and wild-type controls were subjected to transverse aortic constriction for two weeks to induce compensated left ventricular hypertrophy (LVH). Hearts were retrogradely perfused in Langendorff mode for 15 minutes, followed by 20 minutes ischaemia and 30 minutes reperfusion. CrT-OE hearts exhibited significantly improved functional recovery (Rate pressure product) during reperfusion compared to WT littermates (76% of baseline vs. 59%, respectively, P = 0.02). Aged CrT-OE mouse hearts (78±5 weeks) also had enhanced recovery following 15 minutes ischaemia (104% of baseline vs. 67%, P = 0.0007). The cardioprotective effect of hypothermic high K⁺ cardioplegic arrest, as used during cardiac surgery and donor heart transplant, was further enhanced in prolonged ischaemia (90 minutes) in CrT-OE Langendorff perfused mouse hearts (76% of baseline vs. 55% of baseline as seen in WT hearts, P = 0.02).

Conclusions

These observations in clinically relevant models further support the development of modulators of intracellular creatine content as a translatable strategy for cardiac protection against ischaemia-reperfusion injury.     

Over-Expression of Calpastatin Inhibits Calpain Activation and Attenuates Post-Infarction Myocardial Remodeling

Background

Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.

Method

We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.

Results

The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.

Conclusions

Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.     

PD-L1 Deficiency within Islets Reduces Allograft Survival in Mice

Background

Islet transplantation may potentially cure type 1 diabetes mellitus (T1DM). However, immune rejection, especially that induced by the alloreactive T-cell response, remains a restraining factor for the long-term survival of grafted islets. Programmed death ligand-1 (PD-L1) is a negative costimulatory molecule. PD-L1 deficiency within the donor heart accelerates allograft rejection. Here, we investigate whether PD-L1 deficiency in donor islets reduces allograft survival time.

Methods

Glucose Stimulation Assays were performed to evaluate whether PD-L1 deficiency has detrimental effects on islet function. Islets isolated from PDL1-deficient mice or wild- type (WT) mice (C57BL/6j) were implanted beneath the renal capsule of streptozotocin (STZ)-induced diabetic BALB/c mice. Blood glucose levels and graft survival time after transplantation were monitored. Moreover, we analyzed the residual islets, infiltrating immune cells and alloreactive cells from the recipients.

Results

PD-L1 deficiency within islets does not affect islet function. However, islet PD-L1 deficiency increased allograft rejection and was associated with enhanced inflammatory cell infiltration and recipient T-cell alloreactivity.

Conclusions

This is the first report to demonstrate that PD-L1 deficiency accelerated islet allograft rejection and regulated recipient alloimmune responses.     

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice

Background

Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia.

Methods

Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression.

Results

A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups.

Conclusions

These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.     

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice

Introduction

Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown.

Objective

We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na⁺ channel (βENaC).

Methods

βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured.

Results

Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements.

Conclusions

We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.     

Over-Expression of CD200 Protects Mice from Dextran Sodium Sulfate Induced Colitis

Background and aim

CD200:CD200 receptor (CD200R) interactions lead to potent immunosuppression and inhibition of autoimmune inflammation. We investigated the effect of "knockout"of CD200 or CD200R, or over-expression of CD200, on susceptibility to dextran sodium sulfate (DSS)—induced colitis, a mouse model of inflammatory bowel disease (IBD).

Methods

Acute or chronic colitis was induced by administration of dextran sodium sulfate (DSS) in four groups of age-matched C57BL/6 female mice: (1) CD200-transgenic mice (CD200^tg); (2) wild-type (WT) mice; (3) CD200 receptor 1-deficient (CD200R1KO) mice; and (4) CD200-deficient (CD200KO) mice. The extent of colitis was determined using a histological scoring system. Colon tissues were collected for quantitative RT-PCR and Immunohistochemical staining. Supernatants from colonic explant cultures and mononuclear cells isolated from colonic tissue were used for ELISA.

Results

CD200KO and CD200R1KO mice showed greater sensitivity to acute colitis than WT mice, with accelerated loss of body weight, significantly higher histological scores, more severe infiltration of macrophages, neutrophils and CD3⁺ cells, and greater expression of macrophage-derived inflammatory cytokines, whose production was inhibited in vitro (in WT/CD200KO mouse cells) by CD200. In contrast, CD200^tg mice showed less sensitivity to DSS compared with WT mice, with attenuation of all of the features seen in other groups. In a chronic colitis model, greater infiltration of Foxp3⁺ regulatory T (Treg) cells was seen in the colon of CD200^tg mice compared to WT mice, and anti-CD25 mAb given to these mice attenuated protection.

Conclusions

The CD200:CD200R axis plays an immunoregulatory role in control of DSS induced colitis in mice.     

Mechanisms Underpinning Increased Plasma Creatinine Levels in Patients Receiving Vemurafenib for Advanced Melanoma

Context

Serum creatinine has been reported to increase in patients receiving Vemurafenib, yet neither the prevalence nor the mechanism of this adverse event are known.

Objective

We aimed to evaluate the frequency and the mechanisms of increases in plasma creatinine level in patients receiving Vemurafenib for advanced melanoma.

Methods

We performed a retrospective monocentric study including consecutive patients treated with Vemurafenib for an advanced melanoma. We collected clinical and biological data concerning renal function before introduction of Vemurafenib and in the course of monthly follow-up visits from March 2013 to December 2014. Cystatin C-derived glomerular filtration rate was evaluated before and after Vemurafenib initiation, as increase in serum cystatin C is specific to a decrease in the glomerular filtration rate. We also performed thorough renal explorations in 3 patients, with measurement of tubular secretion of creatinine before and after Vemurafenib initiation and a renal biopsy in 2 patients.

Results

70 patients were included: 97% of them displayed an immediate, and thereafter stable, increase in creatinine (+22.8%) after Vemurafenib initiation. In 44/52 patients in whom Vemurafenib was discontinued, creatinine levels returned to baseline. Serum cystatin C increased, although proportionally less than serum creatinine, showing that creatinine increase under vemurafenib was indeed partly due to a renal function impairment. In addition, renal explorations demonstrated that Vemurafenib induced an inhibition of creatinine tubular secretion.

Conclusion

Thus, Vemurafenib induces a dual mechanism of increase in plasma creatinine with both an inhibition of creatinine tubular secretion and slight renal function impairment. However, this side effect is mostly reversible when Vemurafenib is discontinued, and should not lead physicians to discontinue the treatment if it is effective.     

Aggravation of Allergic Airway Inflammation by Cigarette Smoke in Mice Is CD44-Dependent

Background

Although epidemiological studies reveal that cigarette smoke (CS) facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation.

Methods

Wild type (WT) and CD44 knock-out (KO) mice were exposed simultaneously to house dust mite (HDM) extract and CS. Inflammatory cells, hyaluronic acid (HA) and osteopontin (OPN) levels were measured in bronchoalveolar lavage fluid (BALF). Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th) 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures.

Results

In WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS)/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice.

Conclusion

We demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics.     

Action of epoxyeicosatrienoic acids on cellular function

Epoxyeicosatrienoic acids (EETs), which function primarily as autocrine and paracrine mediators in the cardiovascular and renal systems, are synthesized from arachidonic acid by cytochrome P-450 epoxygenases. They activate smooth muscle large-conductance Ca²⁺-activated K⁺ channels, producing hyperpolarization and vasorelaxation. EETs also have anti-inflammatory effects in the vasculature and kidney, stimulate angiogenesis, and have mitogenic effects in the kidney. Many of the functional effects of EETs occur through activation of signal transduction pathways and modulation of gene expression, events probably initiated by binding to a putative cell surface EET receptor. However, EETs are rapidly taken up by cells and are incorporated into and released from phospholipids, suggesting that some functional effects may occur through a direct interaction between the EET and an intracellular effector system. In this regard, EETs and several of their metabolites activate peroxisome proliferator-activated receptor (PPAR) and PPAR, suggesting that some functional effects may result from PPAR activation. EETs are metabolized primarily by conversion to dihydroxyeicosatrienoic acids (DHETs), a reaction catalyzed by soluble epoxide hydrolase (sEH). Many potentially beneficial actions of EETs are attenuated upon conversion to DHETs, which do not appear to be essential under routine conditions. Therefore, sEH is considered a potential therapeutic target for enhancing the beneficial functions of EETs. soluble epoxide hydrolase; eicosanoids; dihydroxyeicosatrienoic acids; cytochrome P-450; peroxisome proliferator-activated receptor     

Role of 20-hydroxyeicosatetraenoic acid in mediating hypertension in response to chronic renal medullary endothelin type B receptor blockade

Background

The renal medullary endothelin (ET-1) system plays an important role in the control of sodium excretion and arterial pressure (AP) through the activation of renal medullary ET-B receptors. We have previously shown that blockade of endothelin type B receptors (ET-B) leads to salt-sensitive hypertension through mechanisms that are not fully understood. One possible mechanism is through a reduction in renal medullary production of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE, a metabolite of arachidonic acid, has natriuretic properties similar to ET-B activation. While these findings suggest a possible interaction between ET-B receptor activation and 20-HETE production, it is unknown whether blockade of medullary ET-B receptors in rats maintained on a high sodium intake leads to reductions in 20-HETE production.

Methodology/Principal Findings

The effect of increasing sodium intake from low (NS = .8%) to high (HS = 8%) on renal medullary production of 20-HETE in the presence and absence of renal medullary ET-B receptor antagonism was examined. Renal medullary blockade of ET-B receptors resulted in salt sensitive hypertension. In control rats, blood pressure rose from 112.8±2.4 mmHg (NS) to 120.7±9.3 mmHg (HS). In contrast, when treated with an ET-B receptor blocker, blood pressure was significantly elevated from 123.7±3.2 (NS) to 164.2±7.1 (HS). Furthermore, increasing sodium intake was associated with elevated medullary 20-HETE (5.6±.8 in NS vs. 14.3±3.7 pg/mg in HS), an effect that was completely abolished by renal medullary ET-B receptor blockade (4.9±.8 for NS and 4.5±.6 pg/mg for HS). Finally, the hypertensive response to intramedullary ET-B receptor blockade was blunted in rats pretreated with a specific 20-HETE synthesis inhibitor.

Conclusion

These data suggest that increases in renal medullary production of 20-HETE associated with elevating salt intake may be, in part, due to ET-B receptor activation within the renal medulla.     

Disturbed ratio of renal 20-HETE/EETs is involved in androgen-induced hypertension in cytochrome P450 4F2 transgenic mice

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22 mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.     

Severe Nephrotoxic Nephritis following Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

Background

Inflammation is the hallmark of nephrotoxic nephritis. Stanniocalcin-1 (STC1), a pro-survival factor, inhibits macrophages, stabilizes endothelial barrier function, and diminishes trans-endothelial migration of leukocytes; consistently, transgenic (Tg) overexpression of STC1 protects from nephrotoxic nephritis. Herein, we sought to determine the phenotype of nephrotoxic nephritis after conditional and kidney-specific knockdown of STC1.

Methods

We used Tg mice that, express either STC1 shRNA (70% knockdown of STC1 within 4d) or scrambled shRNA (control) upon delivery of Cre-expressing plasmid to the kidney using ultrasound microbubble technique. Sheep anti-mouse GBM antibody was administered 4d after shRNA activation; and mice were euthanized 10 days later for analysis.

Results

Serum creatinine, proteinuria, albuminuria and urine output were similar 10 days after anti-GBM delivery in both groups; however, anti-GBM antibody delivery to mice with kidney-specific knockdown of STC1 produced severe nephrotoxic nephritis, characterized by severe tubular necrosis, glomerular hyalinosis/necrosis and massive cast formation, while control mice manifested mild tubular injury and crescentic glomerulonephritis. Surprisingly, the expression of cytokines/chemokines and infiltration with T-cells and macrophages were also diminished in STC1 knockdown kidneys. Staining for sheep anti-mouse GBM antibody, deposition of mouse C₃ and IgG in the kidney, and antibody response to sheep IgG were equal.

Conclusions

nephrotoxic nephritis after kidney-specific knockdown of STC1 is characterized by severe tubular and glomerular necrosis, possibly due to loss of STC1-mediated pro-survival factors, and we attribute the paucity of inflammation to diminished release of cytokines/chemokines/growth factors from the necrotic epithelium.     

Estrogen Deficiency Leads to Further Bone Loss in the Mandible of CKD Mice

Background

Chronic kidney disease (CKD) has been regarded as a grave public health problem. Estrogen is a critical factor for both renal protection and bone remodeling. Our previous study demonstrated that CKD impairs the healing of titanium implants. The aim of this study was to investigate the effects of estrogen deficiency on the mandibular bone in CKD mice.

Methods

Forty eleven-week-old female C57BL mice were used in this study. Uremia and estrogen deficiency were induced by 5/6 nephrectomy and ovariectomy (OVX), respectively. After 8 weeks, the mice were sacrificed, and their mandibles were collected for micro-CT analysis and histological examination.

Results

All the mice survived the experimental period. Serum measurements confirmed a significant increase in BUN in the CKD group that was further increased by OVX. OVX led to significant decreases in both the BV/TV and cortical thickness of the mandibular bone in CKD mice.

Conclusion

In summary, our findings indicate that estrogen deficiency leads to further mandibular bone loss in CKD mice.     

Enhanced p122RhoGAP/DLC-1 Expression Can Be a Cause of Coronary Spasm

Background

We previously showed that phospholipase C (PLC)-δ1 activity was enhanced by 3-fold in patients with coronary spastic angina (CSA). We also reported that p122Rho GTPase-activating protein/deleted in liver cancer-1 (p122RhoGAP/DLC-1) protein, which was discovered as a PLC-δ1 stimulator, was upregulated in CSA patients. We tested the hypothesis that p122RhoGAP/DLC-1 overexpression causes coronary spasm.

Methods and Results

We generated transgenic (TG) mice with vascular smooth muscle (VSM)-specific overexpression of p122RhoGAP/DLC-1. The gene and protein expressions of p122RhoGAP/DLC-1 were markedly increased in the aorta of homozygous TG mice. Stronger staining with anti-p122RhoGAP/DLC-1 in the coronary artery was found in TG than in WT mice. PLC activities in the plasma membrane fraction and the whole cell were enhanced by 1.43 and 2.38 times, respectively, in cultured aortic vascular smooth muscle cells from homozygous TG compared with those from WT mice. Immediately after ergometrine injection, ST-segment elevation was observed in 1 of 7 WT (14%), 6 of 7 heterozygous TG (84%), and 7 of 7 homozygous TG mice (100%) (p<0.05, WT versus TGs). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in TG, but not in WT mice, despite of the similar response to prostaglandin F_2α between TG and WT mice (n = 5). Focal narrowing of the coronary artery after ergometrine was documented only in TG mice.

Conclusions

VSM-specific overexpression of p122RhoGAP/DLC-1 enhanced coronary vasomotility after ergometrine injection in mice, which is relevant to human CSA.     

Preconditioning with physiological levels of ethanol protect kidney against ischemia/reperfusion injury by modulating oxidative stress

Background

Oxidative stress due to excessive production of reactive oxygen species (ROS) and subsequent lipid peroxidation plays a critical role in renal ischemia/reperfusion (IR) injury. The purpose of current study is to demonstrate the effect of antecedent ethanol exposure on IR-induced renal injury by modulation of oxidative stress.

Materials and Methods

Bilateral renal warm IR was induced in male C57BL/6 mice after ethanol or saline administration. Blood ethanol concentration, kidney function, histological damage, inflammatory infiltration, cytokine production, oxidative stress, antioxidant capacity and Aldehyde dehydrogenase (ALDH) enzymatic activity were assessed to evaluate the impact of antecedent ethanol exposure on IR-induced renal injury.

Results

After bilateral kidney ischemia, mice preconditioned with physiological levels of ethanol displayed significantly preserved renal function along with less histological tubular damage as manifested by the reduced inflammatory infiltration and cytokine production. Mechanistic studies revealed that precondition of mice with physiological levels of ethanol 3 h before IR induction enhanced antioxidant capacity characterized by significantly higher superoxidase dismutase (SOD) activities. Our studies further demonstrated that ethanol pretreatment specifically increased ALDH2 activity, which then suppressed lipid peroxidation by promoting the detoxification of Malondialdehyde (MDA) and 4-hydroxynonenal (HNE).

Conclusions

Our results provide first line of evidence indicating that antecedent ethanol exposure can provide protection for kidneys against IR-induced injury by enhancing antioxidant capacity and preventing lipid peroxidation. Therefore, ethanol precondition and ectopic ALDH2 activation could be potential therapeutic approaches to prevent renal IR injury relevant to various clinical conditions.