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Podocyte damage is commonly accompanied by destabilization of the podocalyxin (PC)/ezrin complex. Serum- and glucocorticoid-inducible kinase 3 (SGK3) plays a role in the maintenance of podocyte function, but the details of this role are poorly understood. Herein we demonstrated that SGK3 and its downstream target protein neural precursor cell expressed developmentally downregulated protein 4 subtype 2 (Nedd4-2) triggered PC and ezrin interaction. In adriamycin (ADR)-induced nephritic mice, and after puromycin aminonucleoside (PAN)-induced podocyte damage in vitro, PC and ezrin protein expression levels decreased significantly, while Nedd4-2 activity increased. Moreover, PAN treatment increased PC and ezrin ubiquitination and decreased PC/ezrin interaction in cultured mouse podocytes. The downregulation of SGK3 activity in mouse podocytes resulted in decreased PC and ezrin protein expression and increased the ubiquitin-proteasome degradation of PC and ezrin. Furthermore, upregulation of SGK3 activity mostly reversed the PAN-induced decrease in PC and ezrin protein expression. Overexpression of Nedd4-2 led to decreased ezrin protein expression via the upregulation of ezrin ubiquitination. In contrast, Nedd4-2 knockdown resulted in increased ezrin protein expression but decreased ezrin ubiquitination. In PC-transfected human embryonic kidney (HEK293T) cells, SGK3 activity downregulation and Nedd4-2 overexpression resulted in decreased PC/ezrin interaction. These results suggested that SGK3 triggers the ubiquitin-proteasome degradation of PC and ezrin, while the SGK3/Nedd4-2 signaling pathway regulates ezrin, but not PC, ubiquitination. Thus SGK3 helps to regulate podocyte function by maintaining the stability of the PC/ezrin complex.

PurposeThe purpose of this study was to investigate the hyperpolarized ketone body 13 C-acetoacetate (AcAc) and its conversion to 13 C-β-hydroxybutyrate (βOHB) in vivo, catalyzed by β-hydroxybutyrate dehydrogenase (BDH), as a novel direct marker of mitochondrial redox state.Methods[1,3-13 C2 ]AcAc was synthesized by hydrolysis of the ethyl ester, and hyperpolarized via dissolution DNP. Cold storage under basic conditions resulted in sufficient chemical stability for use in hyperpolarized (HP) MRI studies. Polarizations and relaxation times of HP [1,3-13 C2 ]AcAc were measured in a clinical 3T MRI scanner, and 8 rats were scanned by dynamic HP 13 C MR spectroscopy of a slab through the kidneys. Four rats were scanned after acute treatment with high dose metformin (125 mg/kg, intravenous), which is known to modulate mitochondrial redox via inhibition of mitochondrial complex I. An additional metformin-treated rat was scanned by abdominal 2D CSI (8 mm × 8 mm).ResultsPolarizations of 7 ± 1% and 7 ± 3%, and T1 relaxation times of 58 ± 5 s and 52 ± 3 s, were attained at the C1 and C3 positions, respectively. Rapid conversion of HP AcAc to βOHB was detected in rat kidney in vivo, via the C1 label. The product HP βOHB was resolved from closely resonating acetate. Conversion to βOHB was also detected via 2D CSI, in both kidney as well as liver regions. Metformin treatment resulted in a significant increase (40%, P = 0.01) of conversion of HP AcAc to βOHB.ConclusionRapid conversion of HP AcAc to βOHB was observed in rat kidney in vivo and is a promising new non-invasive marker of mitochondrial redox state. Magn Reson Med 79:1862-1869, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Importance:Mutations in genes traditionally associated with syndromic retinal disease are increasingly found to cause nonsyndromic inherited retinal degenerations. Mutations in CLN3 are classically associated with juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease with early retinal degeneration and progressive neurologic deterioration, but have recently also been identified in patients with nonsyndromic inherited retinal degenerations. To our knowledge, detailed clinical characterization of such cases has yet to be reported. Objective:To provide detailed clinical, electrophysiologic, structural, and molecular genetic findings in nonsyndromic inherited retinal degenerations associated with CLN3 mutations. Design, Setting, and Participants:A multi-institutional case series of 10 patients who presented with isolated nonsyndromic retinal disease and mutations in CLN3. Patient ages ranged from 16 to 70 years; duration of follow-up ranged from 3 to 29 years. Main Outcomes and Measures:Longitudinal clinical evaluation, including full ophthalmic examination, multimodal retinal imaging, perimetry, and electrophysiology. Molecular analyses were performed using whole-genome sequencing or whole-exome sequencing. Electron microscopy studies of peripheral lymphocytes and CLN3 transcript analysis with polymerase chain reaction amplification were performed in a subset of patients. Results:There were 7 females and 3 males in this case series, with a mean (range) age at last review of 37.1 (16-70) years. Of the 10 patients, 4 had a progressive late-onset rod-cone dystrophy, with a mean (range) age at onset of 29.7 (20-40) years, and 6 had an earlier onset rod-cone dystrophy, with a mean (range) age at onset of 12.1 (7-17) years. Ophthalmoscopic examination features included macular edema, mild intraretinal pigment migration, and widespread atrophy in advanced disease. Optical coherence tomography imaging demonstrated significant photoreceptor loss except in patients with late-onset disease who had a focal preservation of the ellipsoid zone and outer nuclear layer in the fovea. Electroretinography revealed a rod-cone pattern of dysfunction in 6 patients and were completely undetectable in 2 patients. Six novel CLN3 variants were identified in molecular analyses. Conclusions and Relevance:This report describes detailed clinical, imaging, and genetic features of CLN3-associated nonsyndromic retinal degeneration. The age at onset and natural progression of retinal disease differs greatly between syndromic and nonsyndromic CLN3 disease, which may be associated with genotypic differences.

Glucocorticoid induced leucine zipper protein (GILZ) is an aldosterone-regulated protein that controls sodium transport in cultured kidney epithelial cells. Mice lacking GILZ have been reported previously to have electrolyte abnormalities. However, the mechanistic basis has not been explored. Here we provide evidence supporting a role for GILZ in modulating the balance of renal sodium and potassium excretion by regulating the sodium-chloride cotransporter (NCC) activity in the distal nephron. Gilz-/- mice have a higher plasma potassium concentration and lower fractional excretion of potassium than wild type mice. Furthermore, knockout mice are more sensitive to NCC inhibition by thiazides than are the wild type mice, and their phosphorylated NCC expression is higher. Despite increased NCC activity, knockout mice do not have higher blood pressure than wild type mice. However, during sodium deprivation, knockout mice come into sodium balance more quickly, than do the wild type, without a significant increase in plasma renin activity. Upon prolonged sodium restriction, knockout mice develop frank hyperkalemia. Finally, in HEK293T cells, exogenous GILZ inhibits NCC activity at least in part by inhibiting SPAK phosphorylation. Thus, GILZ promotes potassium secretion by inhibiting NCC and enhancing distal sodium delivery to the epithelial sodium channel. Additionally, Gilz-/- mice have features resembling familial hyperkalemic hypertension, a human disorder that manifests with hyperkalemia associated variably with hypertension.

PurposeTo develop a specialized multislice, single-acquisition approach to detect the metabolites of hyperpolarized (HP) [2-13 C]dihydroxyacetone (DHAc) to probe gluconeogenesis in vivo, which have a broad 144 ppm spectral range (∼4.6 kHz at 3T). A novel multiband radio-frequency (RF) excitation pulse was designed for independent flip angle control over five to six spectral-spatial (SPSP) excitation bands, each corrected for chemical shift misregistration effects.MethodsSpecialized multiband SPSP RF pulses were designed, tested, and applied to investigate HP [2-13 C]DHAc metabolism in kidney and liver of fasted rats with dynamic 13 C-MR spectroscopy and an optimal flip angle scheme. For comparison, experiments were also performed with narrow-band slice-selective RF pulses and a sequential change of the frequency offset to cover the five frequency bands of interest.ResultsThe SPSP pulses provided a controllable spectral profile free of baseline distortion with improved signal to noise of the metabolite peaks, allowing for quantification of the metabolic products. We observed organ-specific differences in DHAc metabolism. There was two to five times more [2-13 C]phosphoenolpyruvate and about 19 times more [2-13 C]glycerol 3-phosphate in the liver than in the kidney.ConclusionA multiband SPSP RF pulse covering a spectral range over 144 ppm enabled in vivo characterization of HP [2-13 C]DHAc metabolism in rat liver and kidney. Magn Reson Med 77:1419-1428, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

PurposeThe purpose of this study was to characterize tissue-specific alterations in metabolism of hyperpolarized (HP) gluconeogenic precursors 13 C-lactate and 13 C-pyruvate by rat liver and kidneys under conditions of fasting or insulin-deprived diabetes.MethodsSeven normal rats were studied by MR spectroscopic imaging of both HP 13 C-lactate and 13 C-pyruvate in both normal fed and 24 h fasting states, and seven additional rats were scanned after induction of diabetes by streptozotocin (STZ) with insulin withdrawal. Phosphoenolpyruvate carboxykinase (PEPCK) expression levels were also measured in liver and kidney tissues of the STZ-treated rats.ResultsMultiple sets of significant signal modulations were detected, with graded intensity in general between fasting and diabetic states. An approximate two-fold reduction in the ratio of 13 C-bicarbonate to total 13 C signal was observed in both organs in fasting. The ratio of HP lactate-to-alanine was markedly altered, ranging from a liver-specific 54% increase in fasting, to increases of 69% and 92% in liver and kidney, respectively, in diabetes. Diabetes resulted in a 40% increase in renal lactate signal. STZ resulted in 5.86-fold and 2.73-fold increases in PEPCK expression in liver and kidney, respectively.ConclusionMRI of HP 13 C gluconeogenic precursors may advance diabetes research by clarifying organ-specific roles in abnormal diabetic metabolism. Magn Reson Med 77:1429-1437, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

PurposeTo investigate acute changes in glucose metabolism in liver and kidneys in vivo after a bolus injection of either fructose or glucose, using hyperpolarized [2-13 C]dihydroxyacetone.MethodsSpatially registered, dynamic, multislice MR spectroscopy was acquired for the metabolic products of [2-13 C]dihydroxyacetone in liver and kidneys. Metabolism was probed in 13 fasted rats at three time points: 0, 70, and 140 min. At 60 min, rats were injected intravenously with fructose (n = 5) or glucose (n = 4) at 0.8 g/kg to initiate acute response. Controls (n = 4) did not receive a carbohydrate challenge.ResultsTen minutes after fructose infusion, levels of [2-13 C]phosphoenolpyruvate and [2-13 C]glycerol-3-phosphate halved in liver: 51% (P = 0.0010) and 47% (P = 0.0001) of baseline, respectively. Seventy minutes later, levels returned to baseline. The glucose challenge did not alter the signals significantly, nor did repeated administration of the dihydroxyacetone imaging bolus. In kidneys, no statistically significant changes were detected after sugar infusion other than a 20% increase of the glycerol-3-phosphate signal between 10 and 80 min after fructose injection (P = 0.0028).ConclusionHyperpolarized [2-13 C]dihydroxyacetone detects a real-time, transient metabolic response of the liver to an acute fructose challenge. Observed effects possibly include ATP depletion and changes in the unlabeled pool sizes of glycolytic intermediates. Magn Reson Med 77:65-73, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

The epithelial Na(+) channel (ENaC) is decisive for sodium reabsorption by the aldosterone-sensitive distal nephron (ASDN) of the kidney. ENaC is regulated by the serum- and glucocorticoid-inducible kinase 1 (SGK1), a kinase genomically upregulated by several hormones including glucocorticoids and mineralocorticoids. SGK1 is activated by the serine/threonine kinase mammalian target of rapamycin (mTOR) isoform mTORC2. SGK1 knockout (sgk1(-/-) mice) impairs renal Na(+) retention during salt depletion. The mTOR catalytic site inhibitor, PP242, but not mTORC1 inhibitor rapamycin, inhibits ENaC, decreases Na(+) flux in isolated perfused tubules and induces natriuresis in wild-type mice. PP242 does not lead to further impairment of Na(+) reabsorption in sgk1(-/-) mice. The mTORC2/SGK1 sensitive renal Na(+) retention leads to extracellular volume expansion with increase of blood pressure. A SGK1 gene variant (prevalence ∼ 3-5% in Caucasians, ∼ 10% in Africans) predisposes to hypertension, stroke, obesity and type 2 diabetes. Future studies will be required to define the role of mTORC2 in the regulation of further SGK1 sensitive transport proteins, such as further ion channels, carriers and the Na(+)/K(+)-ATPase. Moreover, studies are required disclosing the impact of mTORC2 on SGK1 sensitive disorders, such as hypertension, obesity, diabetes, thrombosis, stroke, inflammation, autoimmune disease, fibrosis and tumour growth.

The epithelial Na+ channel (ENaC) is essential for Na+ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na+ and K+ transport in WT and Sgk1-/- mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na+ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K+ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na+ homeostasis through SGK1-dependent modulation of ENaC activity.

The principal cell of the kidney collecting duct is one of the most highly regulated epithelial cell types in vertebrates. The effects of hormonal, autocrine, and paracrine factors to regulate principal cell transport processes are central to the maintenance of fluid and electrolyte balance in the face of wide variations in food and water intake. In marked contrast with the epithelial cells lining the proximal tubule, the collecting duct is electrically tight, and ion and osmotic gradients can be very high. The central role of principal cells in salt and water transport is reflected by their defining transporters-the epithelial Na(+) channel (ENaC), the renal outer medullary K(+) channel, and the aquaporin 2 (AQP2) water channel. The coordinated regulation of ENaC by aldosterone, and AQP2 by arginine vasopressin (AVP) in principal cells is essential for the control of plasma Na(+) and K(+) concentrations, extracellular fluid volume, and BP. In addition to these essential hormones, additional neuronal, physical, and chemical factors influence Na(+), K(+), and water homeostasis. Notably, a variety of secreted paracrine and autocrine agents such as bradykinin, ATP, endothelin, nitric oxide, and prostaglandin E2 counterbalance and limit the natriferic effects of aldosterone and the water-retaining effects of AVP. Considerable recent progress has improved our understanding of the transporters, receptors, second messengers, and signaling events that mediate principal cell responses to changing environments in health and disease. This review primarily addresses the structure and function of the key transporters and the complex interplay of regulatory factors that modulate principal cell ion and water transport.

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains—two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus. [ABSTRACT FROM AUTHOR]