Aquaporin 2 promotes cell migration and epithelial morphogenesis

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin β1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin β1 by mutating the RGD motif led to reduced endocytosis, retention of integrin β1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin β1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.     

原发性肾病综合征患者尿水通道蛋白异常及其意义

目的：探讨原发性肾病综合征患者尿液水通道蛋白含量变化与其在肾组织中表达量的相关性.方法：54例经肾活检病理证实为原发性肾小球疾病的患者分为3组,即非NS组（A组）、NS不伴水肿组（B组）、NS伴水肿组（C组）,应用ELISA法检测患者尿液AQP1及AQP2含量,免疫组化法检测3组患者肾组织AQP1及AQP2的表达.结果：（1）C组尿液AQP1含量为（43.078±17.923）μg,较A组的（39.189±12.448）μg及B组的（41.492±14.766）μg有升高,但差异无统计学意义;C组（45.309±16.921）μg、B组（38.621±13.187）μg尿AQP2含量显著高于A组的（30.320±9.528）μg;（2）AQP1主要表达于近端小管,C组AQP1阳性面积百分比为（0.414±0.201）%,显著低于A组的（0.683±0.311）%及B组的（0.652±0.300）%,A、B组间表达差异无统计学意义;AQP2主要表达于集合管,C组AQP2阳性面积百分比为（0.823±0.002）%,显著高于A组的（0.512±0.213）%及B组的（0.665±0.228）%,A、B两组间表达也差异有统计学意义;（3）尿AQP1含量与AQP1阳性面积百分比无显著相关性;B、C两组尿AQP2含量与AQP2阳性面积百分比呈显著正相关.结论：尿AQP2含量能反映其在肾组织中表达的多寡,但尿AQP1含量与其在肾组织的表达无明显相关.     

Cyclosporine Interacts with Mycophenolic Acid by Inhibiting the Multidrug Resistance-Associated Protein 2

In mycophenolate mofetil (MMF)-treated organ transplant recipients, lower mycophenolic acid (MPA) plasma concentrations have been found in cyclosporine (CsA) compared with tacrolimus (Tac)-based immunosuppressive regimens. We previously demonstrated that CsA decreases exposure to MPA and increases exposure to its metabolite MPA-glucuronide (MPAG), possibly by interfering with the biliary excretion of MPAG. To elucidate the role of the multidrug resistance-associated protein (Mrp)-2 in the interaction between MMF and CsA, we treated three groups of 10 Mrp2-deficient rats (TR- rat) for 6 days with either vehicle, CsA (8 mg/kg) or Tac (4 mg/kg) by oral gavage. Hereafter, co-administration with MMF (20 mg/kg) was started in all groups and continued through day 14. The 24-h MPA/MPAG area under the concentration-time curve (AUC) was determined after single (day 7) and multiple MMF doses (day 14). On both study days, there were no significant differences in the mean MPA and MPAG AUC between CsA and Tac-treated animals. We conclude that the pharmacokinetics of MMF are comparable in Mrp2-deficient rats receiving either CsA or Tac as co-medication. This finding suggests that CsA-mediated inhibition of the biliary excretion of MPAG by the Mrp2 transporter is the mechanism responsible for the interaction between CsA and MMF.     

Aquaporin 2 and apical calcium-sensing receptor: new players in polyuric disorders associated with hypercalciuria

The kidney plays a critical role in regulating water homeostasis through specific proteins highly expressed in the kidney, called aquaporins, allowing water permeation at a high rate. This brief review focuses on some nephropathies associated with impaired urinary concentrating ability and in particular analyzes the role of aquaporin 2 in hypercalciuria, the most common metabolic abnormality in patients with nephrolithiasis. Specifically, this review discusses the relationship between hypercalciuria and impaired aquaporin 2-mediated water handling in both acquired and inherited disorders characterized by hypercalciuria, including those affecting the sensor of extracellular calcium concentration, the calcium-sensing receptor, which represents the principal target for extracellular calcium regulation of several tissues including parathyroid glands and kidney. In the kidney, the calcium-sensing receptor regulates renal calcium excretion and influences the transepithelial movement of water and other electrolytes. Understanding the molecular basis of alteration of kidney concentrating ability found in hypercalciuria will help for devising strategies for reducing the risk of nephrocalcinosis, nephrolithiasis, and renal insufficiency.     

AQP1在骨巨细胞瘤中的表达和意义

目的研究水通道蛋白1（aquaporin 1,AQP1）在骨巨细胞瘤中的分布及在骨巨细胞瘤组和对照组中的表达差别,并确定AQP1在骨巨细胞瘤各级中的表达差别,从而确定AQP1在骨巨细胞瘤中的作用。方法对42例骨巨细胞瘤标本和24例对照组标本,采用免疫组织化学方法,镜下观察AQP1在组织中的分布,应用自动图像分析检测AQP1在两组中表达情况。结果a）本免疫组化染色显示：AQP1表达于所有标本的肿瘤微血管和小血管内皮细胞上;AQP1表达于大多数骨巨细胞瘤的肿瘤细胞上,AQP1在对照组中无表达或表达很微弱;b）自动图像分析系统所得平均光密度在两组中差别显著（P〈0.01）,AQP1在骨巨细胞瘤中高表达;c）AQP1的表达在骨巨细胞瘤级和骨巨细胞瘤级差别显著（P〈0.01）,而AQP1的表达在骨巨细胞瘤其他各级之间无差别（P〉0.05）。结论大多数骨巨细胞瘤的肿瘤细胞高表达AQP1的原因不明;AQP1在骨巨细胞瘤中高表达,预示其在骨巨细胞瘤的生物学行为中的作用;AQP1的表达在骨巨细胞瘤级和骨巨细胞瘤级差别显著,提示AQP1可能有区分骨巨细胞瘤级和骨巨细胞瘤级的作用。     

Lysosomal Ca2+ Signaling is Essential for Osteoclastogenesis and Bone Remodeling

Lysosomal Ca²⁺ emerges as a critical component of receptor‐evoked Ca²⁺ signaling and plays a crucial role in many lysosomal and physiological functions. Lysosomal Ca²⁺ release is mediated by the transient receptor potential (TRP) family member TRPML1, mutations that cause the lysosomal storage disease mucolipidosis type 4. Lysosomes play a key role in osteoclast function. However, nothing is known about the role of lysosomal Ca²⁺ signaling in osteoclastogenesis and bone metabolism. In this study, we addressed this knowledge gap by studying the role of lysosomal Ca²⁺ signaling in osteoclastogenesis, osteoclast and osteoblast functions, and bone homeostasis in vivo. We manipulated lysosomal Ca²⁺ signaling by acute knockdown of TRPML1, deletion of TRPML1 in mice, pharmacological inhibition of lysosomal Ca²⁺ influx, and depletion of lysosomal Ca²⁺ storage using the TRPML agonist ML‐SA1. We found that knockdown and deletion of TRPML1, although it did not have an apparent effect on osteoblast differentiation and bone formation, markedly attenuated osteoclast function, RANKL‐induced cytosolic Ca²⁺ oscillations, inhibited activation of NFATc1 and osteoclastogenesis‐controlling genes, suppressed the formation of tartrate‐resistant acid phosphatase (TRAP)‐positive multinucleated cells (MNCs), and markedly reduced the differentiation of bone marrow–derived macrophages into osteoclasts. Moreover, deletion of TRPML1 resulted in enlarged lysosomes, inhibition of lysosomal secretion, and attenuated the resorptive activity of mature osteoclasts. Notably, depletion of lysosomal Ca²⁺ with ML‐SA1 similarly abrogated RANKL‐induced Ca²⁺ oscillations and MNC formation. Deletion of TRPML1 in mice reduced the TRAP‐positive bone surfaces and impaired bone remodeling, resulting in prominent osteopetrosis. These findings demonstrate the essential role of lysosomal Ca²⁺ signaling in osteoclast differentiation and mature osteoclast function, which play key roles in bone homeostasis. © 2016 American Society for Bone and Mineral Research.     

Dysregulation of Principal Cell miRNAs Facilitates Epigenetic Regulation of AQP2 and Results in Nephrogenic Diabetes Insipidus

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Nitrogen at Raised Pressure Interacts with the GABAA Receptor to Produce Its Narcotic Pharmacological Effect in the Rat

Background: Strong evidence supports the concept that conventional anesthetics, including inhalational agents and inert gases, such as xenon and nitrous oxide, interact directly with ion channel neurotransmitter receptors. However, there is no evidence that nitrogen, which only exhibits narcotic potency at increased pressure, may act by a similar mechanism.

Methods: We compared the inhibitory and sedative effects of [gamma]-aminobutyric acid (GABA) and nitrogen pressure on locomotor activity and striatal dopamine release in freely moving rats and investigated the pharmacologic properties of the GABA-induced and nitrogen pressure-induced narcotic action using the highly selective competitive GABAA receptor antagonist bicuculline.

Results: Intracerebroventricular GABA infusion up to 60 [mu]mol or exposure to nitrogen pressure up to 3 MPa decreased to a similar extent striatal dopamine release (r2= 0.899, df = 4, P < 0.01) and locomotor activity (r2 = 0.996, df = 28, P < 0.001). However, both agents only showed small effects on striatal dopamine release, reducing dopamine currents by only 12-13% at sedative concentrations. Pretreatment with bicuculline at 0.5, 1, and 2.5 pmol reduced the sedative action of GABA on locomotor activity by 10, 20, and 41%, respectively. Bicuculline in the nanomole range at 1, 2.5, and 5 nmol but not in the picomole range reduced the sedative action of nitrogen pressure by 5, 37, and 73%, respectively. Schild plot analysis is consistent with the fact that bicuculline is a competitive antagonist of both GABA and nitrogen at pressure.     

Combined Coinhibitory and Costimulatory Modulation with Anti-BTLA and CTLA4Ig Facilitates Tolerance in Murine Islet Allografts

Complex interactions between positive and negative cosignaling receptors ultimately determine the fate of the immune response. The recently identified coinhibitory receptor, B and T lymphocyte attenuator (BTLA), contributes to regulation of autoimmune and potentially alloimmune responses. We investigated the role of BTLA in a fully major histocompatibility complex-mismatched mouse islet transplant model. We report that anti-BTLA mAb (6F7) alone does not accelerate graft rejection. Rather, while CTLA4Ig alone improved allograft survival, the addition of anti-BTLA mAb to CTLA4Ig led to indefinite (>100 days) allograft survival. Immediately after treatment with anti-BTLA mAb and CTLA4Ig, islet allografts showed intact islets and insulin production despite a host cellular response, with local accumulation of Foxp3+ cells. We clearly demonstrate that combined therapy with anti-BTLA mAb and CTLA4Ig mice induced donor-specific tolerance, since mice accepted a second donor-specific islet graft without further treatment and rejected third party grafts. CTLA4Ig and anti-BTLA mAb limited the initial in vivo proliferation of CFSE-labeled allogeneic lymphocytes, and anti-BTLA mAb enhanced the proportion of PD-1 expressing T cells while depleting pathogenic BTLA+ lymphocytes. We conclude that targeting the BTLA pathway in conjunction with CTLA4Ig costimulatory blockade may be a useful strategy for promoting immunological tolerance in murine islet allografts.     

Oral Gabapentin Activates Spinal Cholinergic Circuits to Reduce Hypersensitivity after Peripheral Nerve Injury and Interacts Synergistically with Oral Donepezil

Background: Gabapentin administration into the brain of mice reduces nerve injury-induced hypersensitivity and is blocked by intrathecal atropine and enhanced by intrathecal neostigmine. The authors tested the relevance of these findings to oral therapy by examining the efficacy of oral gabapentin to reduce hypersensitivity after nerve injury in rats and its interaction with the clinically used cholinesterase inhibitor, donepezil.

Methods: Male rats with hypersensitivity after spinal nerve ligation received gabapentin orally, intrathecally, and intracerebroventricularly with or without intrathecal atropine, and withdrawal threshold to paw pressure was determined. The effects of oral gabapentin and donepezil alone and in combination on withdrawal threshold were determined in an isobolographic design.

Results: Gabapentin reduced hypersensitivity to paw pressure by all routes of administration, and was more potent and with a quicker onset after intracerebroventricular than intrathecal injection. Intrathecal atropine reversed the effect of intracerebroventricular and oral gabapentin. Oral gabapentin and donepezil interacted in a strongly synergistic manner, with an observed efficacy at one tenth the predicted dose of an additive interaction. The gabapentin-donepezil combination was reversed by intrathecal atropine.     

SRD5A2 and HSD3B2 polymorphisms are associated with prostate cancer risk and aggressiveness

BACKGROUND: Dihydrotestosterone (DHT) is believed to play an important role in prostate carcinogenesis. Five alpha reductase type II (SRD5A2) and 3 beta-hydroxysteroid dehydrogenase type II (HSD3B2) are responsible for the biosynthesis and degradation of DHT in the prostate. Two polymorphisms, a valine (V) for leucine (L) substitution at the 89 codon of the SRD5A2 gene and a (TG)n,(TA)n,(CA)n repeat polymorphism within the third intron of the HSD3B2 gene were evaluated with regard to prostate cancer risk. METHODS: Blood samples were collected for 637 prostate cancer cases and 244 age and race frequency matched controls. In analysis, the SRD5A2 VL and LL genotypes were combined into one group and the HSD3B2 repeat polymorphism was dichotomized into short (<283) and long (> or =283) alleles. RESULTS: The SRD5A2 V89L polymorphism was not independently associated with prostate cancer risk. Carriage of at least one HSD3B2 intron 3 intron 3 short allele was associated with a significant increased risk for prostate cancer among all subjects (OR = 2.07, 95% CI = 1.08-3.95, P = 0.03) and Caucasians (OR = 2.80, CI = 2.80-7.43, P = 0.04), but not in African Americans (OR = 1.50, CI = 0.62-3.60, P = 0.37). Stratified analyses revealed that most of the prostate cancer risk associated with the intron 3 HSD3B2 short allele was confined to the SRD5A2 89L variant subgroup and indicated that in combination these polymorphisms may be associated with increased risk of aggressive (Gleason >7) disease (Gleason >7). CONCLUSIONS: In Caucasians, the HSD3B2 (TG)n,(TA)n,(CA)n intron 3 length polymorphism is associated with both prostate cancer risk and aggressiveness and the SRD5A2 V89L polymorphism may modify the risk conferred by this polymorphism.