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BACKGROUND/AIMS - Loss-of-Function (LOF) of the potassium chloride cotransporter 3 (KCC3) results in hereditary sensorimotor neuropathy with Agenesis of the Corpus Callosum (HSMN/ACC). Our KCC3 knockout mouse recapitulated axonal swelling and tissue vacuolization observed in autopsies of individuals with HSMN/ACC. We previously documented the first human case of a KCC3 gain-of-function (GOF) in which the patient also exhibited severe peripheral neuropathy. Furthermore, the GOF mouse model exhibited shrunken axons implicating the cotransporter in cell volume homeostasis. It is unclear how both KCC3 LOF and GOF lead to peripheral neuropathy. Thus, we sought to study differences in cell volume regulation of dorsal root ganglion neurons isolated from different mouse lines.
METHODS - Using wide-field microscopy, we measured calcein fluorescence intensity through pinhole measurements at the center of cells and compared cell swelling and cell volume regulation/recovery of wild-type, LOF, and GOF dorsal root ganglia neurons, as well as wild-type neurons treated with a KCC-specific inhibitor.
RESULTS - In contrast to control neurons that swell and volume regulate under a hypotonic challenge, neurons lacking KCC3 swell but fail to volume regulate. Similar data were observed in wild-type neurons treated with the KCC inhibitor. We also show that sensory neurons expressing a constitutively active KCC3 exhibited a blunted swelling phase compared to wild-type neurons, questioning the purely osmotic nature of the swelling phase.
CONCLUSION - These findings demonstrate the integral role of KCC3 in cell volume homeostasis and support the idea that cell volume homeostasis is critical to the health of peripheral nerves.
© Copyright by the Author(s). Published by Cell Physiol Biochem Press.
From early unicellular organisms that formed in salty water environments to complex organisms that live on land away from water, cells have had to protect a homeostatic internal environment favorable to the biochemical reactions necessary for life. In this chapter, we will outline what steps were necessary to conserve the water within our cells and how mechanisms have evolved to maintain and regulate our cellular and organismal volume. We will first examine whole body water homeostasis and the relationship between kidney function, regulation of blood pressure, and blood filtration in the process of producing urine. We will then discuss how the composition of the lipid-rich bilayer affects its permeability to water and salts, and how the cell uses this differential to drive physiological and biochemical cellular functions. The capacity to maintain cell volume is vital to epithelial transport, neurotransmission, cell cycle, apoptosis, and cell migration. Finally, we will wrap up the chapter by discussing in some detail specific channels, cotransporters, and exchangers that have evolved to facilitate the movement of cations and anions otherwise unable to cross the lipid-rich bilayer and that are involved in maintaining or regulating cell volume.
Copyright © 2018 Elsevier Inc. All rights reserved.
The potassium chloride cotransporter, KCC3, is an electroneutral cotransporter expressed in the peripheral and central nervous system. KCC3 is responsible for the efflux of K and Cl in neurons to help maintain cell volume and intracellular chloride levels. A loss-of-function (LOF) of KCC3 causes Hereditary Motor Sensory Neuropathy with Agenesis of the Corpus Callosum (HMSN/ACC) in a population of individuals in the Charlevoix/Lac-Saint-Jean region of Quebec, Canada. A variety of mouse models have been created to understand the physiological and deleterious effects of a KCC3 LOF. Though this KCC3 LOF in mouse models has recapitulated the peripheral neuropathy phenotype of HMSN/ACC, we still know little about the development of the disease pathophysiology. Interestingly, the most recent KCC3 mouse model that we created recapitulated a peripheral neuropathy-like phenotype originating from a KCC3 gain-of-function (GOF). Despite the past two decades of research in attempting to understand the role of KCC3 in disease, we still do not understand how dysfunction of this cotransporter can lead to the pathophysiology of peripheral neuropathy. This review focuses on the function of KCC3 in neurons and its role in human and health and disease.
Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.
WNK kinases, along with their upstream regulators (CUL3/KLHL3) and downstream targets (the SPAK/OSR1 kinases and the cation-Cl cotransporters [CCCs]), comprise a signaling cascade essential for ion homeostasis in the kidney and nervous system. Recent work has furthered our understanding of the WNKs in epithelial transport, cell volume homeostasis, and GABA signaling, and uncovered novel roles for this pathway in immune cell function and cell proliferation.
Copyright © 2017 Elsevier Inc. All rights reserved.
Two diffusion-based approaches, CG (constant gradient) and FEXI (filtered exchange imaging) methods, have been previously proposed for measuring transcytolemmal water exchange rate constant k, but their accuracy and feasibility have not been comprehensively evaluated and compared. In this work, both computer simulations and cell experiments in vitro were performed to evaluate these two methods. Simulations were done with different cell diameters (5, 10, 20μm), a broad range of k values (0.02-30s) and different SNR's, and simulated k's were directly compared with the ground truth values. Human leukemia K562 cells were cultured and treated with saponin to selectively change cell transmembrane permeability. The agreement between measured k's of both methods was also evaluated. The results suggest that, without noise, the CG method provides reasonably accurate estimation of k especially when it is smaller than 10s, which is in the typical physiological range of many biological tissues. However, although the FEXI method overestimates k even with corrections for the effects of extracellular water fraction, it provides reasonable estimates with practical SNR's and more importantly, the fitted apparent exchange rate AXR showed approximately linear dependence on the ground truth k. In conclusion, either CG or FEXI method provides a sensitive means to characterize the variations in transcytolemmal water exchange rate constant k, although the accuracy and specificity is usually compromised. The non-imaging CG method provides more accurate estimation of k, but limited to large volume-of-interest. Although the accuracy of FEXI is compromised with extracellular volume fraction, it is capable of spatially mapping k in practice.
Copyright © 2016 Elsevier Inc. All rights reserved.
PURPOSE - A temporal diffusion MRI spectroscopy based approach has been developed to quantify cancer cell size and density in vivo.
METHODS - A novel imaging microstructural parameters using limited spectrally edited diffusion (IMPULSED) method selects a specific limited diffusion spectral window for an accurate quantification of cell sizes ranging from 10 to 20 μm in common solid tumors. In practice, it is achieved by a combination of a single long diffusion time pulsed gradient spin echo (PGSE) and three low-frequency oscillating gradient spin echo (OGSE) acquisitions. To validate our approach, hematoxylin and eosin staining and immunostaining of cell membranes, in concert with whole slide imaging, were used to visualize nuclei and cell boundaries, and hence, enabled accurate estimates of cell size and cellularity.
RESULTS - Based on a two compartment model (incorporating intra- and extracellular spaces), accurate estimates of cell sizes were obtained in vivo for three types of human colon cancers. The IMPULSED-derived apparent cellularities showed a stronger correlation (r = 0.81; P < 0.0001) with histology-derived cellularities than conventional ADCs (r = -0.69; P < 0.03).
CONCLUSION - The IMPULSED approach samples a specific region of temporal diffusion spectra with enhanced sensitivity to length scales of 10-20 μm, and enables measurements of cell sizes and cellularities in solid tumors in vivo. Magn Reson Med 78:156-164, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
© 2016 International Society for Magnetic Resonance in Medicine.
During pregnancy, maternal β-cells undergo compensatory changes, including increased β-cell mass and enhanced glucose-stimulated insulin secretion. Failure of these adaptations to occur results in gestational diabetes mellitus. The secreted protein connective tissue growth factor (CTGF) is critical for normal β-cell development and promotes regeneration after partial β-cell ablation. During embryogenesis, CTGF is expressed in pancreatic ducts, vasculature, and β-cells. In adult pancreas, CTGF is expressed only in the vasculature. Here we show that pregnant mice with global Ctgf haploinsufficiency (Ctgf(LacZ/+)) have an impairment in maternal β-cell proliferation; no difference was observed in virgin Ctgf(LacZ/+) females. Using a conditional CTGF allele, we found that mice with a specific inactivation of CTGF in endocrine cells (Ctgf(ΔEndo)) develop gestational diabetes during pregnancy, but this is due to a reduction in glucose-stimulated insulin secretion rather than impaired maternal β-cell proliferation. Moreover, virgin Ctgf(ΔEndo) females also display impaired GSIS with glucose intolerance, indicating that underlying β-cell dysfunction precedes the development of gestational diabetes in this animal model. This is the first time a role for CTGF in β-cell function has been reported.
PURPOSE - A novel quantitative magnetic resonance imaging (MRI) method, namely, temporal diffusion spectroscopy (TDS), was used to detect the response of tumor cells (notably, mitotic arrest) to a specific antimitotic treatment (Nab-paclitaxel) in culture and human ovarian xenografts and evaluated as an early imaging biomarker of tumor responsiveness.
METHODS - TDS measures a series of apparent diffusion coefficients (ADCs) of tissue water over a range of effective diffusion times, which may correspond to diffusion distances ranging from subcellular to cellular levels (~3-20 μm). By fitting the measured ADC data to a tissue model, parameters reflecting structural properties such as restriction size in solid tumors can be extracted. Two types of human ovarian cell lines (OVCAR-8 as a responder to Nab-paclitaxel and NCI/ADR-RES as a resistant type) were treated with either vehicle (PBS) or Nab-paclitaxel, and treatment responses of both in vitro and in vivo cases were investigated using TDS.
RESULTS - Acute cell size increases induced by Nab-paclitaxel in responding tumors were confirmed by flow cytometry and light microscopy in cell culture. Nab-paclitaxel-induced mitotic arrest in treated tumors/cells was quantified histologically by measuring the mitotic index in vivo using a mitosis-specific marker (anti-phosphohistone H3). Changes in the fitted restriction size, one of the parameters obtained from TDS, were able to detect and quantify increases in tumor cell sizes. All the MR results had a high degree of consistency with other flow, microscopy, and histological data. Moreover, with an appropriate analysis, the Nab-paclitaxel-responsive tumors in vivo could be easily distinguished from all the other vehicle-treated and Nab-paclitaxel-resistant tumors.
CONCLUSION - TDS detects increases in cell sizes associated with antimitotic-therapy-induced mitotic arrest in solid tumors in vivo which occur before changes in tissue cellularity or conventional diffusion MRI metrics. By quantifying changes in cell size, TDS has the potential to improve the specificity of MRI methods in the evaluation of therapeutic response and enable a mechanistic understanding of therapy-induced changes in tumors.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Mice carrying a targeted disruption of the prostaglandin E2 (PGE2) E-prostanoid receptor 3 (EP3) gene, Ptger3, were fed a high-fat diet (HFD), or a micronutrient matched control diet, to investigate the effects of disrupted PGE2-EP3 signaling on diabetes in a setting of diet-induced obesity. Although no differences in body weight were seen in mice fed the control diet, when fed a HFD, EP3(-/-) mice gained more weight relative to EP3(+/+) mice. Overall, EP3(-/-) mice had increased epididymal fat mass and adipocyte size; paradoxically, a relative decrease in both epididymal fat pad mass and adipocyte size was observed in the heaviest EP3(-/-) mice. The EP3(-/-) mice had increased macrophage infiltration, TNF-α, monocyte chemoattractant protein-1, IL-6 expression, and necrosis in their epididymal fat pads as compared with EP3(+/+) animals. Adipocytes isolated from EP3(+/+) or EP3(-/-) mice were assayed for the effect of PGE2-evoked inhibition of lipolysis. Adipocytes isolated from EP3(-/-) mice lacked PGE2-evoked inhibition of isoproterenol stimulated lipolysis compared with EP3(+/+). EP3(-/-) mice fed HFD had exaggerated ectopic lipid accumulation in skeletal muscle and liver, with evidence of hepatic steatosis. Both blood glucose and plasma insulin levels were similar between genotypes on a control diet, but when fed HFD, EP3(-/-) mice became hyperglycemic and hyperinsulinemic when compared with EP3(+/+) fed HFD, demonstrating a more severe insulin resistance phenotype in EP3(-/-). These results demonstrate that when fed a HFD, EP3(-/-) mice have abnormal lipid distribution, developing excessive ectopic lipid accumulation and associated insulin resistance.